Background Information and Scientific Rationale

Alternative Pneumococcal Vaccination Schedules for Infants in Fiji and Pneumococcal Epidemiology

Dr Fiona Mary Russell

Submitted in total fulfillment of the requirements of the degree of Doctor of Philosophy

11th October, 2010

Department of Paediatrics Faculty of Medicine, Dentistry, and Health Sciences

The University of Melbourne

ABSTRACT

This thesis documents the pneumococcal disease burden and the results of a Phase II pneumococcal vaccine trial in the low middle income country, Fiji. The overall objective was to gather sufficient evidence for the Fiji Ministry of Health to decide whether to introduce the pneumococcal vaccination into its national schedule and define an appropriate and affordable vaccination strategy.

The nasopharynx is the main reservoir for pneumococci and plays an important role in the spread of the organism. Studies of nasopharyngeal carriage offer insights into the pneumococcal disease burden in a community, particularly for potential serotypes which may cause pneumonia, and are a convenient way of determining the level of antibiotic resistance among pneumococcal isolates circulating in a population. The first study, a cross- sectional pneumococcal nasopharyngeal carriage survey of healthy children aged 3–13 months, was undertaken to document the prevalence of pneumococcal nasopharyngeal carriage, risk factors for carriage, serotypes and antimicrobial susceptibility patterns of carried pneumococci in healthy young children in Fiji (Chapter 3). Pneumococcal nasopharyngeal carriage was common in Fijian children. Penicillin resistance was documented for the first time, and, as a result, first-line treatment for meningitis was altered. A low proportion of carriage serotypes were included in the 7-valent pneumococcal conjugate vaccine.

Invasive pneumococcal disease is an important cause of morbidity and mortality, particularly in the very young and the elderly. The introduction of the 7-valent pneumococcal conjugate vaccine in the national immunisation schedule in the USA has resulted in an impressive reduction in infant invasive pneumococcal disease. In addition, the vaccine has had a more than expected herd immunity effect on invasive pneumococcal disease in the elderly and other age groups. Chapter 4 reports on a study that aimed to document age-specific burden of invasive pneumococcal disease including clinical syndromes, underlying conditions, serotype distribution, and the potential protection against invasive pneumococcal disease and chest X-ray confirmed pneumonia by 7-valent pneumococcal conjugate vaccine in Fiji. The annual invasive pneumococcal disease incidence was comparable to countries of similar socioeconomic status. Being indigenous Fijian was an independent risk factor for disease. Underlying conditions were common and the case fatality rate was high particularly in the elderly population. For every 1,930 and 128 infants vaccinated, one death and one case ii respectively, would be prevented in those <5 years, by introduction of universal immunisation with the 7-valent conjugate vaccine.

A Phase II vaccine trial was undertaken to document the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination regimens combining one, 2, or 3 doses of 7-valent pneumococcal conjugate vaccine in infancy (Chapters 5 to 10). In order to broaden the serotype coverage, the additional benefit of a booster of 23-valent pneumococcal polysaccharide vaccine at 12 months of age was also assessed. To address the theoretical concerns of hyporesponsiveness to 23-valent pneumococcal polysaccharide vaccine following re-challenge, the immunological responses at 17 months of age to a small challenge dose of 20% of 23-valent pneumococcal polysaccharide vaccine (mPPS) in children who had or had not received the 23-valent pneumococcal polysaccharide vaccine at 12 months of age was undertaken.

The immunogenicity following a 2 or 3 dose 7-valent pneumococcal conjugate vaccine primary series was similar for many serotypes. A single 7-valent pneumococcal conjugate vaccine dose would offer protection in the first 12 months of life for many serotypes. The one or 2 dose 7-valent pneumococcal conjugate vaccine schedules induced immunologic memory, with memory responses following 23-valent pneumococcal polysaccharide vaccine being most profound for children who had received only a single dose of 7-valent pneumococcal conjugate vaccine previously, compared with the 2 or 3 dose groups. Following the 23-valent pneumococcal polysaccharide vaccine booster, there were significant responses for all 23 serotypes which persisted for at least 5 months following vaccination. However despite higher antibody concentrations at 17 months in children who had received 23-valent pneumococcal polysaccharide vaccine at 12 months, the response to a re-challenge was poor to all 23 serotypes compared to children who did not receive the 12 month 23-valent pneumococcal polysaccharide vaccine. This indicates immunological hyporesponsiveness or non-responsiveness. This effect occurred regardless of pre-mPPS antibody levels and prior 7-valent pneumococcal conjugate vaccine exposure. The addition of 23-valent pneumococcal polysaccharide vaccine at 12 months had no impact on carriage, despite the substantial boosts in antibody levels observed and despite significantly higher opsonophagocytic activity and antibody avidity comparing pre- and post-levels.

In summary, a substantial burden of pneumococcal disease in Fiji was found. The 7-valent pneumococcal conjugate vaccine would provide limited coverage of invasive disease compared to its use in affluent countries. Two doses of 7-valent pneumococcal conjugate

iii vaccine have similar immunogenicity as 3 doses although a single dose still provides some protection. The 23-valent polysaccharide vaccine booster was found to be immunogenic but re-challenge resulted in hyporesponsiveness. Further research evaluating the potential of reduced dose schedules using the newer conjugate vaccines with an early conjugate booster would be recommended.

DECLARATION

This is to certify that 1. the thesis comprises only my original work towards the PhD except where indicated in the Preface,

2. due acknowledgement has been made in the text to all other materials used,

3. the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies, and appendices.

Name: Fiona Russell

Signature:

Date: 11th October, 2010

PREFACE

Abstract: This chapter is entirely my work under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis.

Chapter 1: Literature Review This chapter is entirely my work under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis.

Chapter 2: Extended Materials and Methods section I was responsible for writing the bulk of this chapter under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. Prof Kim Mulholland outlined the initial concept of the design of the study. Expert advice was sought from Porter Anderson, Brian Greenwood, George Siber, and DMID NIH personnel for various aspects of the study. The fieldwork SOPs were written by Sam Colquhoun, Jane Nelson, and Vanessa Johnson, under my direction and supervision. The laboratory ELISA and avidity SOPs and methods were written by Anne Balloch and Paul Licciardi. The OPA methods were written by Rob Burton and Moon Nahm, University of Alabama at Birmingham, Birmingham, Alabama, USA. The microbiology laboratory SOPs were written by Chris Pearce (formerly from the Royal Children’s Hospital, Melbourne) and Shirley Warren (Westmead Hospital). The statistics section, including sample size calculations were written by Graham Byrnes.

Chapter 3: Pneumococcal nasopharyngeal carriage and patterns of penicillin resistance in young children in Fiji I was responsible for the design of the study under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the protocol, submitted the study to the ethics committee for approval, and supervised the fieldwork. Senibua Ketawai, the laboratory technician, processed the specimens. Dr Viema Kunabuli and Mabel Taoi collected the specimens. Sharon Biribo and Anna Seduadua performed the serotyping. I analysed the data, wrote the first draft of the manuscript with input from the co-authors on the published paper: JR Carapetis, S Ketawai, V Kunabuli, M Taoi, S Biribo, A Seduadua, EK Mulholland.

Chapter 4: Epidemiology of Invasive Pneumococcal Disease in Fiji: the potential impact of pneumococcal conjugate vaccine I was responsible for the design of the study under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the protocol, submitted the study to the ethics committee for approval, and reviewed all the medical records. Anna Seduadua and Reginald Chand processed and stored the isolates. Catherine Satzke processed the isolates for shipment for serotyping. Shahin Oftadeh and Prof Lyn Gilbert were responsible for serotyping the isolates. I analysed the data, wrote the first draft of the manuscript with input from the co-authors on the published paper: JR Carapetis, L Tikoduadua, R Chandra, A Seduadua, C Satzke, J Pryor, E Buadromo, L Waqatakirewa, EK Mulholland.

Chapter 5: Immunogenicity Following One, Two, or Three Doses of the 7-valent Pneumococcal Conjugate Vaccine

Chapter 6: Safety and Immunogenicity of the 23-Valent Pneumococcal Polysaccharide Vaccine at 12 months of age, following One, Two, or Threes Doses of the 7-valent Pneumococcal Conjugate Vaccine in Infancy

Chapter 7: Hyporesponsiveness to Re-challenge Dose Following Pneumococcal Polysaccharide Vaccine at 12 Months of Age, a Randomized Controlled Trial

Chapter 8: Serotype-specific avidity is achieved following a single dose of the 7-valent pneumococcal conjugate vaccine, and is enhanced by 23-valent pneumococcal polysaccharide booster at 12 months For Chapters 5-8 I was responsible for writing the original protocol under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the ethics submission, was responsible for the annual ethics report submissions, and I supervised the fieldwork. The assays were performed by Anne Balloch, Paul Licciardi, and Mimi Tang. I was responsible for cleaning and analysing the data under the supervision of Yin Bun Cheung and Prof Kim Mulholland. I wrote the first draft of the manuscripts with input from the co-authors:

PV Licciardi PV, A Balloch A , V Biaukula V, L Tikoduadua L, JR Carapetis JR, J Nelson, AWJ Jenney, L Waqatakirewa, S Colquhoun, YB Cheung, MLK Tang, EK Mulholland.

Funding was provided by the U.S. NIAID (grant number R01 AI 52337) and the Australian National Health and Medical Research Council. PneumovaxTM was kindly donated by CSL Biotherapies, Australia. The co-administered TritanrixTM-HepBTM and HiberixTM vaccines were kindly donated by GlaxoSmithKline. Clinicaltrials.gov number NCT00170612.

vii

Chapter 9: Opsonophagocytic Activity Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Infant Primary Series and 23-valent Pneumococcal Polysaccharide Vaccine at 12 Months of Age I was responsible for writing the original protocol under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the ethics submission, was responsible for the annual ethics report submissions, and I supervised the fieldwork. The assays were performed by Rob Burton and Moon Nahm. I was responsible for cleaning and analysing the data under the supervision of Yin Bun Cheung and Prof Kim Mulholland. I wrote the first draft of the manuscripts with input from the co-authors:

JR Carapetis, RL Burton, J Lin J, PV Licciardi, A Balloch, L Tikoduadua, L Waqatakirewa, YB Cheung, MLK Tang, MH Nahm, EK Mulholland.

In addition to the funding stated above, funding was also provided by U.S. NIAID grant number N01 AI-30021.

Chapter 10: Pneumococcal Nasopharyngeal Carriage Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Schedule in Infancy and a 23-valent Pneumococcal Polysaccharide Vaccine Booster, a Randomised Controlled Trial I was responsible for writing the original protocol under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis. I wrote the ethics submission, was responsible for the annual ethics report submissions, and I supervised the fieldwork. The assays were performed by Anna Seduadua, Reginald Chandra, Catherine Satzke and the laboratory staff at the Centre for International Child Health (Murdoch Children’s Research Institute, Royal Children’s Hospital and Department of Paediatrics, University of Melbourne), Shahin Oftadeh, and Prof Lyn Gilbert. I was responsible for cleaning and analysing the data under the supervision of Yin Bun Cheung and Prof Kim Mulholland. I wrote the first draft of the manuscripts with input from the co-authors:

JR Carapetis, C Satzke, L Tikoduadua, L Waqatakirewa, R Chandra R, A Seduadua, S Oftadeh, YB Cheung, GL Gilbert, EK Mulholland.

Chapter 11: Conclusion This chapter is entirely my work under the supervision of Prof Kim Mulholland and Prof Jonathan Carapetis.

viii

ACKNOWLEDGEMENTS

Study Participants

I am indebted to all the parents in the Nausori, Valelevu, Makoi, and CWMH catchment who consented for their babies to participate in the studies and came to the clinics so many times for visits and procedures for 2 years. Without these willing parents these studies would not have been possible and for which I am eternally grateful.

The FiPP Team

I wish to thank all the dedicated FiPP team who tirelessly worked for up to 6 years, enthusiastically undertook what was requested of them, worked so well as a team, and were keen to learn as much as they could whilst on the project. The many FiPP study nurses who enthusiastically dedicated themselves to the study and cared for the parents and children so well: Tania Ah Kee, Felisita Tupou Ratu, Elina, Priya Frances, Liti, Agnes Rounds, Amelia Wara, and Mabel Taoi. It had been a pleasure to work closely with Dr Viema Biuakula who worked so hard and graciously, completed her own study, a Masters in Public Health, married and started her own family, and launched her own career in public health surveillance in the Ministry of Health. For Mere Vakacegu who was our first employee, starting as the cleaner and within a short time became an expert in databases and database design. For Robert Cabemaiwasa, who for a long time tolerated being our only male employee and excelled in transporting specimens, entered countless CRFs year after year without a complaint, and occasionally indulged us with his magnificent muffins. For Simi Sokiqele and his dedication to detail, precision, his delightful letters, and interesting insights into Fiji life and culture. For Senibua Kataiwai, Anna Seduadua and Reginald Chandra who worked ad naseum, tucked away in the lab processing and storing the many thousands of specimens. For Taraifina Saro and Sharonika Chand who smoothed the way through immigration, managed the office, and made sure we never ran out of pens and that the bills were paid.

I have been extremely fortunate to have 3 study co-ordinators who I am very grateful for their hard work, expertise and tireless work over 6 years. I thank Sam Colquhoun whose organisation, hard work, expertise in GCP and setting up clinical trials provided the foundation for a successful study. Jane Nelson whose uncompromising attention to detail, organisation, seemingly non-stop ability to take on more work, ability and willingness to sort out field work issues, and ongoing supervision provided such an excellent basis for all the

ix quality research undertaken. For Kathryn Bright who quickly stepped into such big shoes and co-ordinated the fieldwork to such high quality and saw the successful completion for the study with cheer and diplomacy. I thank her for her friendship. For Beth Temple who provided expert advice in database management and introduced me to the efficient magical stata “loops” for analysing the voluminous amount of data.

Fiji Ministry of Health

We have had a very close relationship with the Ministry of Health and wish to thank them enormously for collaborating and hosting our project, providing valuable space for us in the Ministry of Health clinics, providing advice on logistical issues, smoothing the way for administrative issues, and for the acceptance we have had for undertaking the first clinical trial in Fiji. Dr Lepani Waqatakirewa, the former Permanent Secretary the Fiji Ministry of Health, was an investigator on the project, was involved from the project’s inception, and I have appreciated his support and wise counsel.

I had the pleasure of working closely with Dr Lisi Tikoduadua, former Head of Paediatrics at the Colonial War Memorial Hospital, a tremendous advocate for child health for Fiji and the region. Lisi was involved in our project from its very inception and has always offered support for me personally and for our project. I have enjoyed her humour and philosophical way of dealing with delays and obstacles. She embraced research, made us welcome, and generously made office space available for us within her department. To Dr Joe Kado, the current Head of Paediatrics, who also provided support, showed much interest, and valued our work. To Dr Sala Saketa, the current Permanent Secretary for Health and Dr Josaia Samuela, Family and Reproductive Health at the Fiji Ministry of Health have been very supportive and interested the project.

Dr Eka Buadromo, Head of the Department of Pathology at Colonial War Memorial Hospital, generously provided space within the microbiology laboratory and was always supportive and involved in our work. I wish to thank all of the staff members within the microbiology laboratory particularly Shalini Singh and Parmod Kumar. I wish to thank Senibua Ketawai for establishing the lab work and joining the quest in search of a reliable source of animal blood. I wish to thank the serology laboratory staff for being so accommodating. I would like to thank the clinical staff on the paediatric wards of the hospital. In particular I would like to thank Matron Balawa who helped point me in the right direction when all we started with were 2 empty offices.

I would also like to thank the staff in the Medical Records department, particularly Niumia Hicks, the General Manager of Community Health within the CentEast Health Division, Dr Solomone Qaranivalu, and Idrish Khan, Chief Finance Officer at the Ministry of Health, who was always so accommodating regarding the financial arrangements of the project.

Fiji School of Medicine

I wish to thank Dr Jan Pryor for ongoing support and being a tremendous advocate for the value of our research. I wish to thank Prof Rob Moulds for making himself available and being interested in our work. I wish to thank Sharon Biribo for explaining the intricacies of microbiology to a novice, joining the quest to find a reliable source of animal blood, assisting the establishment of our pneumococcal lab work, and undertaking some of the serotyping. I wish to thank Vicki Chand whose energy, enthusiasm, and positive attitude rubbed off on those around her and for establishing our databases, and Dr Rosa Sa’aga who was involved in our project from its inception and was always very supportive.

Koronivia Veterinary Research Station

In search of a reliable source of animal blood to make blood agar I am indebted to the kind assistance of Fiji’s former chief veterinarian (Fiji’s own James Herriot) and the staff of Koronivia Veterinary Research Station who took me on a tour of the farms in Fiji, bled horses and sheep for us, allowed us to experiment with different blood collection techniques before arranging for the purchase of our own FiPP sheep, which they housed, took care, and bled as required.

Pneumococcal Laboratory, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne

I am grateful to Anne Balloch for establishing a first class Pneumococcal laboratory and Dr Paul Licciardi for completing the extraordinary job of churning through tens of thousands of assays over the many years of the project with such good humour, willingness, and efficiency. I am also grateful for their technical immunological insights with the write up. I wish to thank Prof Mimi Tang for her technical oversight and helpful comments, and the laboratory support from Amy Bin Chen and Timothy Gemetzis.

Pneumococcal Reference Laboratory, Centre for Infectious Diseases & Microbiology, ICPMR, Westmead, NSW

I am grateful to Prof Lyn Gilbert and Shahin Oftadeh for undertaking the task of serotyping all our isolates and solving the technical issues surrounding the serotyping.

Bacterial Respiratory Pathogen Reference Laboratory, University of Alabama at Birmingham, USA

I wish to thank Prof Moon Nahm and Rob Burton for performing all the opsonophagocytic assays so efficiently.

Centre for International Child Health

I am grateful to Prof Trevor Duke for allowing me to stay on in Fiji to continue my writing, keeping me funded, and allowing for me to get involved in other activities whilst writing up my thesis. I am grateful to Amanda O’Brien for her groundbreaking perseverance in overcoming the many administrative challenges and to Amy Auge, Kathryn Gilbert, Evan Willis, Eleanor Neal, Joelle Milne, and Caitlyn Henry for all their help over the years.

I am grateful to Dr Catherine Satzke who took charge of a large number of specimens, organised the serotyping, sorted out the many technical issues surrounding the microbiology, and agreed to inherit and finish a number of outstanding microbiology manuscripts.

I am very thankful to Dr Adam Jenney who innocently agreed to do my job as on-site PI whilst I went on maternity leave to enjoy my family. I was grateful to be able to hand over the project to his capable hands (during a coup) and that he was able to continue to lead some aspects of our work on my return. In addition, I am extremely grateful that he offered (without duress) to read my thesis, correct my poor grammar, and help structure many aspects of it.

Clinical Epidemiology and Biostatistics Unit

I wish to thank Philip Greenwood for designing the database and providing training, supervision, and ongoing support for our many databases. Thanks to Suzanna Vidmar for devising the randomisation lists and envelopes and answering other statistical questions.

xii

Royal Children’s Hospital

I am grateful to Chris Pearce who came to Fiji, assessed the microbiology lab, wrote many of the microbiology SOPs, helped with our supplies list, and answered my silly questions. I thank Gena Gonis for doing our sensitivity testing QC free of charge.

Others

I am very thankful for the many people who helped with many technical aspects of the project: Lorraine Kelpie for her expertise in performing venipuncture with a smile, Dr Vanessa Johnston, Dr Loretta Thorn, and Elizabeth Hamilton. I wish to thank Dr Graham Byrnes for his statistical input at the trial’s inception and re-design stage. I am thankful to Prof Yin Bun Cheung who made himself available to provide oversight of the statistical analysis, his incredible ease and efficiency at sorting out my analytical problems, and explaining logistic regression in a language that I could understand. I thank Shirley Warren for providing training in microbiology and serotyping, and getting involved in the many aspects of the lab particularly in helping the lab identify resistant organisms during a nosocomial outbreak.

I wish to thank Prof Porter Anderson, Prof Brian Greenwood, Dr George Siber, and Dr David Klein who had inputs to the study design at various stages. I wish to thank the members of the Data Safety and Monitoring Board whose guidance, support, and expertise were appreciated. I wish to thank CSL Biotherapies, Australia in particular Phillipe Ludekins, who arranged for the donation of PneumovaxTM . I thank GlaxoSmithKline who donated the co- administered TritanrixTM-HepBTM and HiberixTM vaccines and 2 vaccine fridges.

NIH

I am thankful to the staff at NIH particularly Elizabeth Horigan who we came to know when the study was being redesigned. Her expertise, encouragement, and support were always valuable. I wish to thank Farukh Khambaty who was always very supportive of our work and I am appreciative of all the assistance he has provided.

Funding Bodies

Funding was provided by NIAID (grant number R01 AI 52337) and the National Health and Medical Research Council. For 18 months the funding to write up my thesis was made possible by a National Health and Medical Research Council Public Health postgraduate scholarship.

xiii

Supervisors

I wish to thank Prof Jonathan Carapetis who has always been very positive, encouraging, supportive, and clear thinking, over the many years this work has developed and evolved. I have valued his advice, knowledge, and continued interest in the work. None of this work would have been possible without my supervisor Prof Kim Mulholland. I have learnt much from Kim over the years. His perseverance, when others would have given up, is extraordinary, and the belief in a better life for the world’s children has always been forefront in his decision making. His encouragement and openness to get involved in other interesting research activities has been a blessing and a welcome distraction at times, and hence the long evolution of my thesis. My professional life has been enriched by the many opportunities that Kim has provided for me, for which I am truly grateful.

Family and friends

I am grateful to our nanny Ateca and the many Fijian nannies who welcomed my children into their hearts with love and grace, and created such a wonderful, memorable childhood for my children whilst I was at work and writing. For the support of my friends in Fiji who I could always call upon, and the delight we had in watching each others’ children grow.

Most importantly, I would like to thank my husband, Alberto, who embarked on a Pacific journey, which was meant to be 2 years and lasted over 6 years. His patience, compromise, support, and understanding throughout times of chaos have been extraordinary and I am truly grateful to him. My two adorable children, Elian and Lucia, who think they are Fijian, and will miss Fiji and their close friends they have grown up with. I thank them for putting up with a working mother.

Thank you and Vinaka Vakalevu

Fiona Russell

xiv

PUBLICATIONS

1. Russell FM, Mulholland EK. Recent advances in pneumococcal vaccination of children. Ann Trop Paediatr 2004;24(4):283-94. 2. Magree HC, Russell FM, Sa'aga R, Greenwood P, Tikoduadua L, Pryor J, Waqatakirewa L, Carapetis JR, Mulholland EK. Chest X-ray-confirmed pneumonia in children in Fiji. Bull World Health Organ. 2005;83(6):427-33. 3. Russell FM, Biribo S, Selvaraj G, Oppedisano F, Warren S, Seduadua A, Mulholland EK, Carapetis JR. Citrated sheep blood agar is a practical bacterial culture medium to replace citrated human blood agar in developing country laboratories. J Clin Microbiol 2006;44(9):3346-51. 4. Russell FM, Carapetis JR, Ketawai S, Kunabuli V, Taoi M, Biribo S, Seduadua A, Mulholland EK. Pneumococcal nasopharyngeal carriage and penicillin resistance patterns in young children in Fiji. Ann Trop Paediatr 2006;26(3):187-97. 5. Russell FM, Balloch A, Tang MLK, Carapetis JR, Licciardi P, Nelson J, Jenney AWJ, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Cheung YB, Mulholland EK. Immunogenicity Following One, Two, or Three Doses of the 7-valent Pneumococcal Conjugate Vaccine. Vaccine 2009;27(41):5685-91.

6. Jin P, Kong F, Xiao M, Oftadeh S, Zhou F, Liu C, Russell F, Gilbert GL. First report of putative Streptococcus pneumoniae “serotype 6D” among nasopharyngeal isolates from Fijian children. J Infect Dis 2009;200(9):1375-80.

7. Russell FM, Carapetis JR, Balloch A, Licciardi PV, Jenney AWJ, Tikoduadua L, Waqatakirewa L, Pryor J, Nelson J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Hyporesponsiveness to Re- challenge Dose Following Pneumococcal Polysaccharide Vaccine at 12 Months of Age in a Randomised Controlled Trial. Vaccine 2010;28(19):3341-9. 8. Russell FM, Carapetis JR, Satzke C, Tikoduadua L, Waqatakirewa L, Chandra R, Seduadua A, Oftadeh S, Cheung YB, Gilbert GL, Mulholland EK. Pneumococcal Nasopharyngeal Carriage Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Schedule in Infancy and a 23-valent Pneumococcal Polysaccharide Vaccine Booster, a Randomized Controlled Trial. Accepted Clin Vaccine Immun 2010. 9. Russell FM, Carapetis JR, Tikoduadua L, Chandra R, Seduadua A, Satzke C, Pryor J, Buadromo E,

Waqatakirewa L, Mulholland EK. Invasive Pneumococcal Disease in Fiji: clinical syndromes, epidemiology, and the potential impact of pneumococcal conjugate vaccine. Pediatr Infect Dis J. 2010 Sep;29(9):870-2. 10. Satzke C, Seduadua A, Carapetis JR, Chandra, Mulholland EK, Russell FM. Comparison of citrated human, citrated sheep and defibrinated sheep blood Mueller Hinton agar for antimicrobial sensitivity testing of Streptococcus pneumoniae isolates. J Clin Microbiol. 2010 Jul 28. 11. Balloch A, Licciardi P, Russell FM, Burton R, Lin J, Nahm M, Mulholland EK, Tang MLK. 23-valent pneumococcal polysaccahruide is immunogenic in children at one year of age. J Allergy Clin Immunol. 2010 Aug;126(2):395-7. 12. Russell FM, Licciardi PV, Balloch A, Biaukula V, Tikoduadua L, Carapetis JR, Nelson J, Jenney AWJ, Waqatakirewa L, Colquhoun S, Cheung YB, Tang MLK, Mulholland EK. Safety and Immunogenicity of the 23-Valent Pneumococcal Polysaccharide Vaccine at 12 months of age, following One, Two, or Threes Doses of the 7-valent Pneumococcal Conjugate Vaccine in Infancy. Vaccine 2010 28(18):3086-94. 13. Licciardi PV, Balloch A, Russell FM, Mulholland EK, Tang ML. Antibodies to serotype 9V exhibit novel serogroup cross-reactivity following infant pneumococcal immunization. Vaccine 2010;28(22):3793-800. 14. Satzke C, Ortika BD, Oftadeh S, Russell FM, Robins-Browne R, Mulholland EK, Gilbert GL. Molecular epidemiology of Streptococcus pneumoniae serogroup 6 isolates from Fijian children, including newly identified serotypes 6C and 6D. J Clin Microbiol. 2010 Sep 1.

xvi

POSTERS AND CONFERENCE PRESENTATIONS

ORAL PRESENTATIONS

Public Health Association of Australia Conference on Immunisation, August 2006

(1) Russell FM, Carapetis JR, Colquhoun S, Kunabuli V, Magree H, Seduadua A, Pryor J, Tikoduadua L, Waqatakirewa L, Mulholland EK. High ethnic disparity in invasive pneumococcal disease and pneumonia in Fiji.

(2) Russell FM, Carapetis JR, Tang M, Balloch A, Colquhoun S, Nelson J, Pryor J, Tikoduadua L, Waqatakirewa L, Byrnes G, Mulholland EK. Immunogenicity following 1-3 doses of the 7-valent pneumococcal conjugate vaccine followed by the 23-valent pneumococcal polysaccharide vaccine booster.

NIH DMID international investigator’s meeting, Bethesda, USA, May 2007

(1) Russell F, Carapetis J, Tang M, Balloch A, Nelson J, Jenney A, Waqatakirewa L, Pryor J, Tikoduadua L, Byrnes G, Mulholland EK Pneumococcal opsonophagocytic assay results following a primary series of 0-3 doses of pneumococcal conjugate vaccine in infancy followed by a 12 month booster and 17 month microdose of the 23-valent pneumococcal polysaccharide vaccine.

Fiji Medical Association Conference, Suva, Fiji, September 2007

(1) Russell, FM. Pneumococcal Disease and Treatment Costs

(2) Russell, FM. Penicillin Resistance Surveillance

(3) Russell, FM. FiPP update

6th International Symposium on Pneumococci and Pneumococcal Diseases, Reykajek, June 2008

(1) Mulholland EK, Russell FM. Hyporesponsiveness and pneumococcal polysaccharide vaccine in

xvii

Fijian infants.

Public Health Association of Australia Conference on Immunisation, September 2008

(1) Russell FM, Tang MLK, Balloch A, Licciardi P, Carapetis JR, Nelson J, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Immunogenicity Following One, Two, or Three Doses of the 7-valent Pneumococcal Conjugate Vaccine.

(2) Russell FM, Tang MLK, Balloch A, Licciardi P, Carapetis JR, Nelson J, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Booster response of Pneumovax at 12 months of age following one, two, or three doses of Prevenar and impact on carriage.

7th International Symposium on Pneumococci and Pneumococcal Diseases, Tel Aviv, March 2010

(1) Russell FM, Carapetis JR, Balloch A, Licciardi PV, Jenney AWJ, Tikoduadua L, Waqatakirewa L, Pryor J, Nelson J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Hyporesponsiveness to Re- challenge Dose Following Pneumococcal Polysaccharide Vaccine at 12 Months of Age.

(2) Russell FM, Carapetis JR, Burton RL, Lin J, Licciardi PV, Balloch A, Tikoduadua L, Waqatakirewa L, Cheung YB, Tang MLK, Nahm MH, Mulholland EK. Opsonophagocytic Activity Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Infant Primary Series and 23-valent Pneumococcal Polysaccharide Vaccine at 12 Months of Age.

Australasian Society of Infectious Diseases, Darwin, May 2010

Mulholland K, Russell FM. Hyporesponsiveness to re-challenge dose following pneumococcal polysaccharide vaccine at 12 months of age, a randomized controlled trial.

Victorian Infection and Immunity Network student symposium. Walter and Eliza Institute, Melbourne, June 2010. Russell, FM. Hyporesponsiveness to re-challenge dose following pneumococcal polysaccharide

xviii vaccine at 12 months of age, a randomized controlled trial.

Public Health Association of Australia Conference on Immunisation, August 2010

(2) Russell FM, Carapetis JR, Satzke C, Tikoduadua L, Waqatakirewa L, Chandra R, Seduadua A, Oftadeh S, Cheung YB, Gilbert GL, Mulholland EK. Pneumococcal Nasopharyngeal Carriage Following a Reduced Dose 7-valent Pneumococcal Conjugate Vaccine Schedule in Infancy and a 23- valent Pneumococcal Polysaccharide Vaccine Booster, a Randomized Controlled Trial.

POSTER PRESENTATIONS

9th Annual Conference on Vaccine Research, Baltimore, USA, May 2006

(1) Kunabuli VL, Mulholland EK, Tikoduadua L, Seduadua A, Pryor J, Russell FM. Prospective meningitis burden of disease study and rapid assessment of neurological outcomes in children in Fiji.

5th International Symposium on Pneumococci and Pneumococcal Diseases, Alice Springs, April 2006 (1) Russell FM, Carapetis JR, Tang M, Balloch A, Colquhoun S, Nelson J, Pryor J, Tikoduadua L, Waqatikirewa L, Byrnes G, Mulholland EK. Immunogenicity following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine and booster response of the 23-valent pneumococcal polysaccharide vaccine at 12 months of age.

(2) Russell FM, Biribo S, Selvaraj G, Oppedisano F, Warren S, Seduadua A, Mulholland EK, Carapetis JR. Comparison of citrated sheep and human blood with defibrinated horse and sheep blood as culture media supplements for the isolation and antibiotic susceptibility testing of Streptococcus

xix pneumoniae.

(3) Russell FM, Carapetis JR , Ketawai S, Kunabuli V, Taoi M, Biribo S, Seduadua A, Mulholland EK. Pneumococcal Nasopharyngeal Carriage and Penicillin Resistance Patterns in Young Children in Fiji.

(4) Kunabuli VL, Mulholland EK, Tikoduadua L, Seduadua A, Pryor J, Russell FM. Prospective meningitis burden of disease study and rapid assessment of neurological outcomes in children in Fiji.

(5) Seduadua AN, Mulholland EK, Carapetis JR, Buadromo E, Russell FM. Comparison of antimicrobial sensitivity results on citrated sheep blood Mueller Hinton and citrated human blood Mueller Hinton for invasive Streptococcus pneumoniae clinical isolates.

(6) Biribo S, Russell FM, Carapetis JR, Kataiwai S, Mulholland EK. Multiserotype nasopharyngeal carriage of Streptococcus pneumoniae in infants in Fiji.

(7) Colquhoun SM, Russell FM, Carapetis JR, Tikoduadua LV, Pryor J, Waqatakirewa L, Mulholland EK. Ethnic disparity in the burden of invasive pneumococcal disease in children aged less than 5 years in Fiji.

(8) Colquhoun SM, Russell FM, Carapetis JR, Tikoduadua L, Pryor J, Wake M, Mulholland, EK. A cohort study to assess quality of life in young Fijian children who have a history of bacterial meningitis.

6th International Symposium on Pneumococci and Pneumococcal Diseases, Reykajek, June 2008

(3) Russell FM, Carapetis JR, Chandra R, Tikoduadua L, Waqatakirewa L, Seduadua A, Pryor J, Satzke C, Gosling D, Mulholland EK. Nasopharyngeal Carriage of Pneumococcal Vaccine Types Following 0, 1, 2, or 3 doses of 7-valent Pneumococcal Conjugate Vaccine.

(4) Russell FM, Carapetis JR, Kunabuli V, Nelson J, Jenney A, Bright K, Tang MLK, Tikoduadua L, Waqatakirewa L, Pryor J, Byrnes GB, Mulholland EK. Safety of the 23-valent pneumococcal polysaccharide vaccine at 12 months of age following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine.

(5) Russell FM, Chandra R, Carapetis JR, Seduadua A, Tikoduadua L, Buadromo E, Waqatakirewa L, Pryor J, Mulholland EK. Epidemiology and Serotypes of Invasive Pneumococcal Disease in all ages in Fiji.

(6) Russell FM, Carapetis JR, Tikoduadua L, Waqatakirewa L, Pryor J, Mulholland EK. Estimated Impact of the 7-valent Pneumococcal Conjugate Vaccine in Fiji.

(7) Kunabuli V, Mulholland EK, Tikoduadua LV, Azzopardi K, Robins-Browne R, Wake M, Seduadua A, Chandra R, Richmond P, Pryor J, Russell FM. Aetiology and Outcomes of Meningitis in Children in Fiji.

(8) Chandra R, Mulholland K, Buadromo E, Seduadua A, Carapetis JR, Russell FM. Pneumococcal antimicrobial resistance patterns in clinical invasive isolates in Fiji.

(9) Russell FM, Kunabuli V, Griffiths UK, Tikoduadua L, Carapetis JR, Waqatakirewa L, Pryor J, Magree H, Mulholland EK. Cost of Pneumococcal Disease in Fiji.

(10) Temple B, Tikoduadua LV, Mulholland EK, Griffiths UK, Russell FM. Cost of Outpatient Pneumonia in Children Less than 5yrs of Age in Fiji.

(11) Balloch A, Licciardi PV, Russell FM, Mulholland EK, Tang MLK. The ability of infants to respond to 23-valent unconjugated Pneumovax at 12 months in the absence of prior 7-valent conjugated Prevenar immunisation.

(12) Balloch A, Licciardi PV, Russell FM, Burton R, Nahm M, Mulholland EK, Tang MLK. Does serotype specific IgG and avidity to Streptococcus pneumoniae following infant immunisation correlate to functional opsonophagocytic activity?

(13) Licciardi PV, Balloch A, Russell FM, Mulholland EK, Tang MLK. Cross reactive anti- pneumococcal antibodies.

xxi

Public Health Association of Australia Conference on Immunisation, September 2008

(1) Russell FM, Chandra R, Carapetis JR, Seduadua A, Tikoduadua L, Buadromo E, Waqatakirewa L, Pryor J, Mulholland EK.

Epidemiology and Serotypes of Invasive Pneumococcal Disease in all ages in Fiji.

Physicians Week 2009, Sydney (accepted but withdrawn as unable to attend)

1) Russell FM, Balloch A, Biaukula V, Tikoduadua L, Carapetis JR, Licciardi P, Nelson J, Jenney AWJ, Waqatakirewa L, Pryor J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Immunogenicity Following Reduced Doses of Pneumococcal Conjugate Vaccine and 12 month Pneumococcal Polysaccharide Vaccine Booster.

2) Russell FM, Carapetis JR, Balloch A, Licciardi P, Jenney A, Tikoduadua L, Waqatakirewa L, Pryor J, Nelson J, Byrnes GB, Cheung YB, Tang MLK, Mulholland EK. Hyporesponsiveness to challenge dose following pneumococcal polysaccharide at 12 months of age.

7th International Symposium on Pneumococci and Pneumococcal Diseases, Tel Aviv, March 2010:

(1) Russell FM, Balloch A, Licciardi PV, Carapetis JR, Tikoduadua L, Waqatakirewa L, Cheung YB, Mulholland EK, Tang MLK. Serotype-specific avidity is achieved following a single dose of the 7- valent pneumococcal conjugate vaccine, and is enhanced by 23-valent pneumococcal polysaccharide booster at 12 months.

xxii

AWARDS

I was fortunate to receive the following awards/fellowships during the course of my PhD:

Quintiles Fellowship, Royal Australasian College of Physicians, 2007 in which I undertook a 10 day Advanced Course in Epidemiological Analysis at the London School of Hygiene and Tropical Medicine.

National Health and Medical Research Council Scholarship, 2008-9 to write up my PhD.

Early in Career Public Health Award in Immunisation, Public Health Association of Australia, 2008 for the presentation: “Booster response of Pneumovax at 12 months of age following one, two, or three doses of Prevenar and impact on carriage” at the Public Health Association of Australia Conference on Immunisation, September 2008.

xxiii

TABLE OF CONTENTS

1 LITERATURE REVIEW ...... 1

1.1 Background Information...... 1 1.2 Pneumococcal Disease ...... 1 1.3 Bacteriology ...... 2 1.4 Pathogenesis ...... 3 1.5 Pneumococcal Disease Burden ...... 5 1.5.1 Global Pneumococcal Serotype Distribution ...... 6 1.5.2 Pneumococcal Pneumonia ...... 7 1.5.3 Burden of Pneumococcal Disease in Fiji ...... 8 1.6 Protection Against Pneumococcal Disease ...... 11 1.6.1 Host Defense Mechanisms ...... 11 1.6.2 Pneumococcal Vaccines ...... 13 1.7 Rationale ...... 41 1.7.1 Access to Vaccines ...... 41 1.7.2 Evaluation of Alternative Pneumococcal Vaccine Schedules is a Research Priority ...... 41 1.7.3 Immunological Basis to the Vaccine Trial Design ...... 42 1.7.4 Knowledge to be Gained ...... 43 1.7.5 Evaluation of Alternative Pneumococcal Schedules in Fiji ...... 43 1.8 Objectives ...... 43 1.8.1 Change to the Original Objectives ...... 45 1.8.2 Potential Risks ...... 46 1.8.3 Known Potential Benefits ...... 47 1.9 Hypothesis ...... 47

2 MATERIALS AND METHODS ...... 49

2.1 Setting ...... 49 2.1.1 Health Infrastructure ...... 51 2.1.2 Drug Licensing Procedure ...... 51 2.1.3 Study Sites ...... 51 2.1.4 Suva Study Team ...... 52 2.1.5 Ethical Procedures ...... 52 2.2 Study Design ...... 53 2.2.1 Selection of Study Participants ...... 53 2.2.2 Informed Consent ...... 54 2.2.3 Eligibility Criteria ...... 54 2.2.4 Enrollment, Randomisation and Masking Procedures ...... 55 2.2.5 Study Vaccines, Vaccine Storage and Administration ...... 56 2.2.6 Study Visits ...... 58

xxiv

2.2.7 Follow-up ...... 60 2.2.8 Withdrawal of a Participant From the Study ...... 61 2.2.9 Assessment of Safety ...... 62 2.2.10 Sample Collection ...... 66 2.2.11 Sample Transportation - International ...... 68 2.2.12 Antibody Assays ...... 68 2.3 Data Management...... 70 2.3.1 Data capture methods ...... 70 2.4 Clinical Monitoring Plan ...... 71 2.4.1 Source Documents ...... 71 2.4.2 Protocol Deviations ...... 71 2.4.3 Quality Control and Quality Assurance ...... 72 2.5 Statistical Methods ...... 72 2.5.1 Background to Original Study Protocol ...... 72 2.5.2 Sample Size Calculation ...... 73 2.5.3 Definition of Outcome Measures ...... 76 2.5.4 Statistical Analysis of Primary Objective ...... 76 2.5.5 Statistical Analysis of Secondary Objectives ...... 77 2.5.6 Statistical Analysis of Tertiary Objectives ...... 78

3 PNEUMOCOCCAL NASOPHARYNGEAL CARRIAGE AND PATTERNS OF PENICILLIN RESISTANCE IN YOUNG CHILDREN IN FIJI ...... 80

3.1 Abstract ...... 80 3.2 Introduction ...... 80 3.3 Methods ...... 81 3.3.1 Study Site ...... 81 3.3.2 Study Design ...... 81 3.3.3 Risk Factor Evaluation ...... 82 3.3.4 Laboratory Methods ...... 82 3.3.5 Statistical Analysis ...... 83 3.3.6 Ethics Approval ...... 83 3.4 Results ...... 83 3.5 Discussion ...... 91

4 EPIDEMIOLOGY OF INVASIVE PNEUMOCOCCAL DISEASE IN FIJI: THE POTENTIAL IMPACT OF PNEUMOCOCCAL CONJUGATE VACCINE ...... 94

4.1 Abstract ...... 94 4.2 Introduction ...... 94 4.3 Methods ...... 94 4.4 Results ...... 96

xxv

4.5 Discussion ...... 101

5 IMMUNOGENICITY FOLLOWING ONE, TWO, OR THREE DOSES OF THE 7- VALENT PNEUMOCOCCAL CONJUGATE VACCINE ...... 103

5.1 Abstract ...... 103 5.2 Introduction ...... 103 5.3 Methods ...... 105 5.3.1 Study Participants ...... 105 5.3.2 Study Procedures and Vaccines ...... 105 5.3.3 Laboratory Procedures...... 106 5.3.4 Statistical Analysis ...... 107 5.4 Results ...... 107 5.5 Discussion ...... 114

6 SAFETY AND IMMUNOGENICITY OF THE 23-VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE, FOLLOWING ONE, TWO, OR THREES DOSES OF THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE IN INFANCY ...... 118

6.1 Abstract ...... 118 6.2 Introduction ...... 118 6.3 Methods ...... 120 6.3.1 Study Participants ...... 120 6.3.2 Study Procedures and Vaccines ...... 120 6.3.3 Laboratory Procedures...... 121 6.3.4 Statistical Analysis ...... 122 6.4 Results ...... 122 6.4.1 Immunogenicity to PCV Serotypes ...... 123 6.4.2 Immunogenicity to Non-PCV Serotypes ...... 129 6.4.3 Adverse Events ...... 132 6.5 Discussion ...... 133

7 HYPORESPONSIVENESS TO RE-CHALLENGE DOSE FOLLOWING PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE, A RANDOMIZED CONTROLLED TRIAL ...... 136

7.1 Abstract ...... 136 7.2 Introduction ...... 136 7.3 Methods ...... 138 7.3.1 Study Participants ...... 138 7.3.2 Study Procedures and Vaccines ...... 138 7.3.3 Laboratory Procedures...... 139

xxvi

7.3.4 Data Management and Statistical Analysis ...... 139 7.3.5 Ethical Approval ...... 140 7.4 Results ...... 140 7.5 Discussion ...... 149

8 OPSONOPHAGOCYTIC ACTIVITY FOLLOWING A REDUCED DOSE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE INFANT PRIMARY SERIES AND 23- VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE ...... 153

8.1 Abstract ...... 153 8.2 Introduction ...... 153 8.3 Materials and Methods ...... 154 8.3.1 Study Participants ...... 154 8.3.2 Study Procedures and Vaccines ...... 155 8.3.3 Laboratory Assays ...... 155 8.3.4 Statistical Analysis ...... 156 8.4 Results ...... 157 8.4.1 PCV Serotypes ...... 157 8.4.2 Non-PCV and PCV related serotypes ...... 167 8.5 Discussion ...... 170

9 SEROTYPE-SPECIFIC AVIDITY IS ACHIEVED FOLLOWING A SINGLE DOSE OF THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE, AND IS ENHANCED BY 23- VALENT PNEUMOCOCCAL POLYSACCHARIDE BOOSTER AT 12 MONTHS ...... 174

9.1 Abstract ...... 174 9.2 Introduction ...... 175 9.3 Methods ...... 176 9.3.1 Study Participants ...... 176 9.3.2 Study Procedures and Vaccines ...... 177 9.3.3 Laboratory Procedures...... 177 9.3.4 Statistical Analysis ...... 179 9.4 Results ...... 180 9.5 Discussion ...... 197

10 PNEUMOCOCCAL NASOPHARYNGEAL CARRIAGE FOLLOWING REDUCED DOSES OF 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE AND A 23-VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE BOOSTER ...... 201

10.1 Abstract ...... 201 10.2 Introduction ...... 202 10.3 Methods ...... 203

xxvii

10.3.1 Study Design ...... 203 10.3.2 Nasopharyngeal Swabs ...... 203 10.3.3 Questionnaires ...... 204 10.3.4 Statistical Analysis...... 204 10.4 Results ...... 205 10.5 Discussion ...... 212

11 CONCLUSIONS ...... 216

11.1 Implications for Pneumococcal Vaccine Policy in Fiji and Other Countries ...... 217

REFERENCES ...... 219

APPENDICES Appendix 1 Appendix 2 Appendix 3 Appendix 4

xxviii

LIST OF TABLES

Table 1: Summary of serotype-specific GMC data from trials of CRM197-conjugated pneumococcal vaccines, one month post primary series ...... 21 Table 2: Summary of serotype-specific GMC data from trials of reduced dose pneumococcal conjugate vaccines ...... 30 Table 3: Outline of study visits ...... 59 Table 4: Characteristics of the study children (n=440) ...... 86 Table5: Antimicrobial resistance patterns of Streptococcus pneumoniae isolates (n=246) ...... 87 Table 6: Level of antimicrobial resistance of Streptococcus pneumoniae isolates (n=246) ...... 87 Table7: Serogroups/types of Streptococcus pneumoniae isolates from study children (n=239) ...... 89 Table8: Risk factors for pneumococcal nasopharyngeal carriage ...... 90 Table 9: Clinical manifestations of IPD cases by age (n=83) ...... 98 Table 10: Estimated annual number of IPD and hospitalized chest X-ray confirmed pneumonia cases and deaths in <5 year olds in Fiji, and the estimated number of cases and deaths averted if PCV were introduced ...... 101 Table 11: Baseline characteristics of infants at enrolment and randomised to the different PCV groups ...... 109 Table 12: Geometric mean concentrations (GMC) of serotype-specific IgG titres taken 4 weeks following the PCV primary series, and at 9 and 12 months of age, by number of PCV doses administered in the primary series ...... 110 Table 13: Proportion of infants with antibody concentrations ≥0.35 and ≥1μg/mL at 4 weeks post primary series, and at 9 and 12 months of age, by number of PCV doses administered in the primary series ...... 112 Table 14: Serotype-specific IgG geometric mean concentrations (GMC and 95% confidence intervals) to PCV serotypes before and 14 days following the 12 month 23vPPS and by number of PCV doses administered in the primary series ...... 125 Table 15: Proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to PCV serotypes before and 14 days post-12 month 23vPPS and by number of PCV doses administered in the primary series ...... 126 Table 16: Serotype-specific IgG GMC (and 95% confidence intervals) and proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to PCV serotypes at 17 months in those who did or did not receive the 12 month 23vPPS and by number of PCV doses in the primary series…………………………………………………………………...... 127

xxix

Table 17: Serotype-specific IgG GMC (and 95%CI) and proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to non-PCV serotypes before and 14 days post-12 month 23vPPS ...... 130 Table 18: Serotype-specific IgG GMC (and 95% confidence intervals) and proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to non-PCV serotypes at 17 months of age in those that did or did not receive the 12 month 23vPPS ...... 131 Table 19: Non-serious adverse events1 in those children who received 23vPPS at 12 months of age (n=245) ...... 132 Table 20: Baseline characteristics of infants at enrolment and on randomisation to one of eight groups ...... 143 Table 21: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of infants with OI ≥8 to 6 PCV serotypes 4 weeks post primary series, and at 9 and 12 months of age, by number of PCV administered in the primary series ...... 158 Table 22: OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI ≥8 to 6 PCV serotypes pre- and 14 days post-23VPPS at 12 months of age and by number of PCV administered in the primary series ...... 161 Table23: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI)1, OPA Geometric Mean Titers2 (GMT and 95%CI) and proportions of infants with OI ≥8 pre-mPPS at 17 months of age and one month post-mPPS in those that have or have not received the 12 month 23VPPS and by number of PCV doses in the primary series ...... 162 Table 24: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI ≥8 to the non-PCV serotypes 1 and 5 pre- and 14 days post-23VPPS at 12 months of age ...... 168 Table 25: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI ≥8 to 7 non-PCV serotypes at 17 months and one month post-mPPS ...... 169 Table 26: Timing of vaccination and blood draws for each of the 8 groups ...... 178 Table27: Geometric mean (GM) concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of serotype-specific that is avid) for the PCV serotypes, taken 4 weeks following the PCV primary series, and at 9 and 12 months of age ...... 181 Table 28: Geometric mean concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of serotype-specific IgG that is avid (1) to PCV serotypes before and 14 days post 12 month 23vPPS, by number of PCV doses administered in the primary series in children randomized to receive 12 month 23vPPS ...... 186 Table29: Geometric mean concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of antibody that is avid) to PCV serotypes pre-mPPS at

xxx

17 months and one month post-mPPS in those who did or did not receive the 12 month 23vPPS shown by number of PCV doses in the primary series ...... 188 Table30: Geometric mean concentrations of serotype-specific IgG and median AI (MAI1, percentage of antibody that is avid) to non-PCV serotypes before and 14 days post 12 month 23vPPS (n=218) in infants randomized to receive 12 month 23vPPS ...... 193 Table31: Geometric mean concentrations of serotype-specific IgG and median AI (MAI1, percentage of serotype specific IgG that is avid) to non-PCV serotypes at 17 months of age and one month post mPPS, in those that did or did not receive the 12 month 23vPPS ...... 194 Table32: Timing of vaccination and blood draws for each of the 8 groups ...... 205 Table 33 Characteristics of infants by group allocation at enrolment and at each of the 4 nasopharyngeal swab visits (%, unless otherwise stated) ...... 206 Table 34: Nasopharyngeal (NP) carriage of all pneumococcal, 7-valent pneumococcal conjugate vaccine (PCV) serotypes (VT), and non-PCV serotypes (NVT) at 6, 9, and 12 months (m) of age following administration of 0, 1, 2, or 3 doses of PCV as a primary series ...... 209 Table 35: Nasopharyngeal (NP) carriage of all pneumococcal and non-PCV serotypes (NVT) at 17 months of age in those who did or did not receive the 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months of age...... 212

xxxi

LIST OF FIGURES

Figure 1: The annual incidence of hospitalised pneumonia (ICD10: J12-18) for all ages, 2006-2007 for Viti Levu ...... 10 Figure 2: Map of the Republic of the Fiji islands showing the 4 medical divisions ...... 50 Figure 3 : Flowchart of nasopharyngeal swabs and sensitivity and serotype of pneumococcal isolates ...... 84 Figure 4: Annual incidence of IPD by age group in the Central Medical Division, Fiji from 1st July 2004 to 31st October, 2007 ...... 96 Figure 5: IPD case fatality rates in the Central Medical Division, Fiji, by age group ...... 99 Figure 6: Serotype distribution amongst IPD cases (n=78) ...... 100 Figure 7: Proportion of IPD isolates, by age, potentially covered by the 7, 10, and 13-valent pneumococcal conjugate vaccine, and the 23-valent pneumococcal polysaccharide vaccine ...... 100 Figure 8: CONSORT chart of the screened and enrolled children to 12 months of age ...... 10108 Figure 9: CONSORT chart of the screened and enrolled children to 17 months of age ...... 12424 Figure 10: CONSORT chart of the screened and enrolled children to 18 months of age, showing the number having the pre-mPPS blood test1, mPPS at 17 months of age2, and blood test one month post-mPPS3 ...... 14242 Figure 11: Serotype-specific IgG GMC (μg/mL) to PCV serotypes at 17 months of age pre-mPPS ...... 143 Figure 12: Serotype-specific IgG GMC (μg/mL) to PCV serotypes at 17 months of age post-mPPS in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs...... 144 Figure 13: Serotype-specific IgG GMC (μg/mL) to non-PCV serotypes at 17 months of age pre-mPPS in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs...... 144 Figure 14: Serotype-specific IgG GMC (μg/mL) to non-PCV serotypes one month post-mPPS in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs...... 145

xxxii

Figure 15: Pre- and one month post-mPPS log antibody concentrations for non-PCV serotypes 1, 5, 7F, and 19A in those that did (+) and did not (o) receive 23vPPS at 12 months of age. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs...... 146 Figure 16: Pre- and one month post-mPPS log antibody concentrations for serotype 4 for children who did (+) or did not (o) receive 23vPPS at 12 months of age ...... 147 Figure 17: Pre- and one month post-mPPS log antibody concentrations for serotype 6B for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series ...... 147 Figure 18: Pre- and one month post-mPPS log antibody concentrations for serotype 4 for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series ...... 148 Figure 19: Pre- and one month post-mPPS log antibody concentrations for serotype 6B for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series ...... 148 Figure 20: Pre- and one month post-mPPS OPA titer for serotypes 4 and 6B, in those that did (+) and did not (o) receive 23VPPS at 12 months of age. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs...... 166 Figure 21: Median AI (MAI, percentage of serotype-specific-IgG that is avid) for the PCV serotypes taken 4 weeks following the PCV primary series, and at 9 and 12 months of age ...... 183 Figure 22: Median percentage of serotype-specific IgG that is avid for the PCV serotypes pre and post 12 month 23vPPS and pre and post 18 month mPPS in those that did or did not receive the 12 month 23vPPS ...... 190 Figure 23: Median AI (percentage of serotype-specific IgG that is avid) for selected non-PCV serotypes in those that did or did not receive the 12 month 23vPPS ...... 196 Figure 24: Cumulative proportion of infants carrying a 7-valent pneumococcal conjugate vaccine (PCV) type at 6, 9, 12, and 17 months of age by PCV and 23-valent pneumococcal polysaccharide vaccine (23vPPS) group allocation...... 210 Figure 25: Nasopharyngeal (NP) carriage rates of all pneumococcal and 7-valent pneumococcal conjugate vaccine (PCV) serotypes (VT) at 17 months of age following 0, 1, 2, or 3 PCV doses of PCV in infancy with or without 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months ...... 211

xxxiii

ABBREVIATIONS

AE Adverse event AI Avidity index ARI Acute respiratory infection CFR Case fatality rate CI Confidence interval C-PS Cell wall polysaccharide CRF Case reporting form CRP C-reactive protein CSF Cerebrospinal fluid CWMH Colonial War Memorial Hospital CXR Chest X-ray DMID Department of Microbiology and Infectious Diseases DSMB Data safety and monitoring board ELISA Enzyme-linked immunosorbent assay FiPP Fiji pneumococcal project FNRC Fiji National Research Committee GAVI Global Alliance for Vaccines and Immunisation GCP Good clinical practice GMC Geometric mean concentration GMT Geometric mean titre Hib Haemophilus influenzae type b HIV Human immunodeficiency virus ICD International classification of diseases ICH International Conference on Harmonisation Ig Immunoglobulin ILN Interleukin IMR Infant mortality rate IPD Invasive pneumococcal disease IQR Interquartile range

xxxiv

IRR Incidence rate ratio IU International units LRTI Lower respiratory tract infection MCH Maternal and Child Health MoH Ministry of Health NHMRC National Health and Medical Research Council NIAID National Institute of Allergy and Infectious Diseases NIH National Institutes of Health NK Natural killer NP Nasopharyngeal NVT Pneumococcal non-vaccine type OI Opsonization index OPA Opsonophagocytic activity PCV 7-valent pneumococcal conjugate vaccine PedsQL Pediatric quality of life questionnaire PneumoADIP Pneumococcal Vaccines Accelerated Development and Introduction Plan PI Principal investigator PPS Pneumococcal polysaccharide vaccine RR Relative risk SAE Serious adverse event TLR Toll like receptor TNF Tumour necrosis factor UK United Kingdom US United States of America VT Pneumococcal vaccine type WC White cell count WHO World Health Organization 23vPPS 23-valent pneumococcal polysaccharide vaccine

1 LITERATURE REVIEW

1.1 Background Information

Streptococcus pneumoniae (pneumococcus) was first identified by Sternberg in the US [1] and Pasteur in France in 1880 [2]. It is the most common cause of bacterial pneumonia in children worldwide and is the leading vaccine preventable cause of serious infection in infants [3]. An estimated 1.6 million deaths are attributable to pneumococcal disease each year with the majority of these deaths occurring in low income countries primarily in children and the elderly [4]. The case fatality rate (CFR) is particularly high in infants <6 months old [5].

Vaccines to prevent pneumococcal disease were first developed after the recognition of the high morbidity and mortality in South African gold miners in the early 20th century [6]. Several trials of polyvalent pneumococcal vaccines were undertaken. However the choice of controls potentially leading to bias and overestimating the vaccine efficacy, and the lack of accompanying bacteriology cast doubt over the findings [7]. The introduction of sulphonamide antibiotics in the late 1930s put a hold on further vaccine development. However, more recently, antibiotic resistance and the growing awareness of the pneumococcal burden, had become significant issues in many parts of the world necessitating the development of effective vaccines to prevent pneumococcal disease [8].

1.2 Pneumococcal Disease

S.pneumoniae colonises the mucosal surfaces of the human nasopharynx and upper airway. Through a combination of virulence-factor activity and an ability to evade the early components of the innate host immune response, S.pneumoniae can spread locally to the middle ear to cause acute otitis media (OM), to the paranasal sinuses to cause sinusitis, or can spread from the upper respiratory tract to the sterile regions of the lower respiratory tract, resulting in pneumonia. In addition, pneumococci may cause systemic infections often associated with considerable mortality, including bacteraemia and meningitis, or in rare cases, septic arthritis, peritonitis, osteomyelitis, and soft tissue infections. Invasive pneumococcal disease (IPD) refers to any pneumococcal infection occurring in a normally sterile site. Bacteraemic infection occurs as a complication of either pneumonia or direct spread from the pharynx. Thus all pneumococcal disease commences with colonisation [9- 11]. Host and bacterial factors contribute to IPD pathogenicity. Ethnicity, extremes of age,

2 co-morbidities, alcoholism, and immunosuppression including HIV are well known risk factors of IPD [12, 13] .

Secretions from colonised individuals are thought to be responsible for person-to-person spread of the organism. An individual strain can be carried for weeks to months before its eventual clearance. Colonisation is most common in early childhood and acquisition of one or more strains occurs sequentially or simultaneously [14]. Nasopharyngeal (NP) carriage rates vary by age, ethnicity, and geographical location. In developing countries, NP colonisation rates can be >60% by 2 months of age [15, 16]. In addition the rate of NP carriage by capsular serotype varies. The factors that influence these differences are not well understood. The importance of the reduction or prevention of NP carriage in children on the spread of the organism has recently been demonstrated in the US whereby the widespread use of infant 7-valent pneumococcal conjugate vaccine (PCV, Prevenar , Pfizer Inc.) resulted in substantial indirect effects [17-19]. Indirect benefits of PCV exceeded the direct effects, with more than twice the number of cases of vaccine type (VT) IPD prevented in unvaccinated persons compared with the number of cases prevented in vaccinated children [19]. The reduction in pneumococcal VT carriage in children interrupted the transmission of pneumococci to close unvaccinated contacts [17, 18].

1.3 Bacteriology

S.pneumoniae is a Gram-positive diplococcus. It is a fastidious, facultative anaerobic organism that grows in short chains. Serotypes 3 and 37 grow as large mucoid colonies but other serotypes produce smooth colonies. Pneumococci lack catalase or peroxidase. As hydrogen peroxide is one of the end products of pnumococci’s metabolism, by adding red blood cells to the growth medium the inactivation of hydrogen peroxide enhances pneumococci’s viability. S.pneumoniae alters haemoglobin under aerobic conditions, producing a greenish discolouration of the surrounding blood-containing medium. The sensitivity of pneumococci to ethylhydrocupreine (Optochin) is widely used for laboratory diagnosis. However some isolates are Optochin resistant [20] and bile solubility is usually regarded as definitive for routine diagnostic purposes.

Culture of pneumococcus from a normally sterile site still remains the most specific diagnostic gold standard. Despite the high specificity of bacterial culture, the diagnosis of pneumococcal infection from bacterial culture has numerous limitations. Prior treatment with antibiotics, delays in specimen transport to the laboratory, and other laboratory factors

3 such as the use of human blood agar to culture the organism may reduce isolation rates. Defibrinated sheep, horse, pig or goat blood agar is recommended for the isolation of S. pneumoniae [21-23]. Agar prepared using human blood is not recommended, partly because of the safety risk to laboratory personnel, but mainly because of poor bacterial isolation rates. A study evaluating the growth of S.pneumoniae on human blood agar compared with the gold standard horse or sheep blood agar found smaller colony size and absent or minimal hemolysis on human blood agar [24]. Despite this, it is common practice in many low income countries to prepare bacterial culture media using expired human blood obtained from donors for blood transfusions, because it is convenient and inexpensive [25]. In addition, most cases of pneumococcal pneumonia are non-bacteraemic so this test is less helpful for diagnosing the most common disease manifestation of this organism.

1.4 Pathogenesis

S.pneumoniae has a plethora of virulence factors. An array of virulence factors needs to be expressed in a co-ordinated fashion for tissue invasion to be successful. Important virulence factors include the polysaccharide capsule, the cell wall, choline-binding proteins, pneumococcal surface proteins A and C, the LPTXG-anchored neuroaminidase proteins, hyaluronate lyase, pneumococcal adhesion and virulence A, enolase, pneumolysin, autolysin, and the metal-binding proteins pneumococcal surface antigen A, pneumococcal iron acquisition A, and pneumococcal iron uptake A [14].

Pneumococcal capsular polysaccharide is the most important virulence factor of S.pneumoniae by virtue of its anti-phagocytic properties [26]. In addition, the expression of a capsule allows access to the epithelial surface and prevents entrapment in nasal mucus [27]. Capsule production is vital for pneumococcal virulence and the thickness of the capsule in a particular strain and serotype is related to virulence [28]. The capsule physically protects the bacterium from antibodies and complement. The potential for pneumococci to become invasive seems to be related to the polysaccharide composition [29-31] and serotype/group has been shown to be independently associated with IPD severity in adults [32-35]. This is likely to reflect their relative ability to resist phagocytosis and differences in eliciting a humoral immune response. The pneumococcus secretes a protease that cleaves the hinge region of IgA1, the most abundant immunoglobulin expressed at the host site [36]. As a result, the organism is left coated with fragments lacking Fc domains and therefore is able to evade recognition by Fc receptors or complement. A switch in the expression of important virulence factors is required for the transition from NP colonisation to the development of

4 invasive disease. In addition to capsular expression, the thickness of the pneumococcal capsule is an important factor in the degree to which pneumococci are exposed to other important pneumococcal surface structures.

Several studies have evaluated the relationship between pneumococcal serotypes (capsule) and IPD. A Swedish study of 494 adults with IPD showed that certain pneumococcal serotypes with low IPD potential behave as opportunistic pathogens causing disease in fragile persons, whereas serotypes 1 and 7F, known to have a high IPD potential, acted as primary pathogens, causing infections in previously healthy people [37]. A retrospective study of 464 IPD cases among adults in Denmark showed that after adjusting for other markers of severity, infection with serotype 3 was associated with a higher CFR (RR 2.54; 95%CI, 1.22-5.27) whereas infection with serotype 1 was associated with a decreased CFR (RR 0.23; 95%CI ,0.06-0.97) [32]. In a large study in the US of 5,579 adults aged ≥50 with IPD, serotypes 3, 11A, 19F, and 23F were found to be associated with significantly higher CFR than was serotype 14 [19]. A retrospective cohort study from the Netherlands including 1,075 hospitalised IPD cases found serotypes 3, 19F, 23A, 16F, 6B, 9N, and 18C were associated with increased CFR (group adjusted OR, 2.6; 95% CI, 1.5-4.7) indicating that serotype was independently associated with IPD severity in adults [38]. A retrospective study in German children aged <16 years with hospitalised IPD found that serotype 7F accounted for a higher risk of severe and fatal outcomes than other serotypes [35]. In contrast, a prospective multi-site study of 796 IPD cases assessed the association of serotype and host related factors with disease severity and mortality after adjusting for age and the presence of underlying conditions [12]. The results found that host factors were more important than isolate serotype in determining the severity and outcome of IPD [12]. However the most comprehensive study to date is a population-based study from Denmark of 18,858 IPD cases for those ≥5 years. Serotypes 31, 11A, 35F, 17F, 3, 16F, 19F, 15B, and 10A were associated with a higher CFR compared to serotype 1 after adjusting for age, IPD focus, underlying conditions, and other potential confounders (adjusted OR ≥3, p<0.001) suggesting specific serotypes independently affect IPD mortality [34].

Molecular epidemiological analysis has demonstrated that clonal type (based on profiles of housekeeping genes) in addition to capsular type, influences the potential of S.pneumoniae to cause invasive disease [39]. In one study from the UK, multilocus sequence typing was performed on a number of carriage and IPD isolates and the odds ratio of the invasiveness of isolates was calculated. The findings suggested that capsular serotype may be more

5 important than genotype [29]. However this study did not adjust for age and the presence of underlying conditions. In a study from Sweden involving 273 IPD isolates (mainly from adults) and 246 NP carriage isolates, clones that belonged to the same serotypes but had different abilities to cause IPD were found. In addition, isolates belonging to the same clone had different capsules because of serotype switch, and were found to have the same disease potential. These findings suggest that there are other factors, apart from capsular polysaccharide, that may be important in the ability of pneumococci to cause invasive disease [31].

Cell wall polysaccharide (C-PS) is unique to S.pneumoniae and is present in all isolates. It reacts with C-reactive protein (CRP) and activates the alternative complement pathway. Antibody to C-PS does not protect against pneumococcal infection, although it can be detected in virtually all children and adults [40].

1.5 Pneumococcal Disease Burden

In 2005, the World Health Organization (WHO) estimated that 1.6 million people die of pneumococcal disease annually [4]. Children under 5 years of age account for 0.7 to 1 million of these deaths each year, with most from low income countries [4]. A recent review estimated over 14 million episodes of serious pneumococcal disease worldwide, with over 800,000 deaths in children <5 years [41]. The burden of pneumococcal disease in the elderly in low income countries is unknown. In industrialised countries, young children and the elderly have the highest burden of pneumococcal disease [4].

Comparing disease burden rates between geographical sites is difficult due to underreporting, differences in surveillance and reporting methods, antibiotic prescribing practices, and disparities in blood culture practice and laboratory techniques. In Western Europe, the reported IPD rates in Sweden and Spain were 4.2 and 56.2 per 100,000 children aged <5 prior to vaccine introduction respectively [42]. In the UK, the IPD incidence rate pre- vaccine introduction was 24.3 per 100,000 <5 year olds [43]. In the US, incidence rates prior to vaccine introduction were many times higher than in Western Europe which probably reflected different clinical practice with a greater number of blood cultures being drawn particularly from outpatients [44]. In New Zealand, the rate of IPD in <5 year olds was 54.2 per 100,000 [45]. In Chile, a middle income country, the incidence rate for IPD was 32 per 100,000 children aged <5 years [46]. In Africa, which is comprised of mostly low income countries with high infant mortality rates (IMR), the incidence of IPD in children <5 years was

6 estimated to be 111 to 436 per 100,000 [47]. Recently WHO and the PneumoADIP standardised case definitions and supported a co-ordinated multi-site surveillance project of pneumococcal disease in Asia and Africa [48]. Where IPD incidence rates were calculated, Vietnam a low income country with low IMR, the incidence rate was 48.7 per 100,000 children aged <5 years [49]. In rural Thailand, a middle income country with low IMR had an IPD rate of 10.6-28.9 per 100,000 <5s [50]. Thus IPD is a significant burden to communities.

Similar to <5 year old data, rates of IPD in adults vary by geographical region. In 1998 prior to PCV introduction, the national incidence of IPD in the elderly population in the US was estimated to be 60 per 100,000 but rates varied between different regions in the US primarily due to blood culture practices [51]. Since the national introduction of PCV into the US infant schedule the vaccine has had a larger than expected herd immunity effect on IPD in the unvaccinated elderly and other non-vaccinated age groups [19, 52]. The rates of IPD significantly declined in all age groups: by 32% in 20 to 39 year olds, by 8% in 40 to 64 year olds, and by 18% for those aged 65 years or more [52].

Disparate IPD rates between different ethnic groups living within the same geographical region have been described. In an Auckland-based study, the incidence of IPD among Pacific Island children was nearly four times, and that among Maori over twice the rate in other ethnic groups [53]. The incidence of IPD in Australian Indigenous children in the pre-vaccine era was 3.2 times the rate compared to non-Indigenous Australian children [54]. Similarly in Israel, the IPD incidence was 2-fold higher in Bedouin children compared with Jewish children [55]. White Mountain Apache persons had an 8-fold greater risk of IPD than did the general US population pre-vaccine [56]. These different IPD rates in different ethnic groups may be related to genetic susceptibility, poorer living conditions, or other unknown factors.

1.5.1 Global Pneumococcal Serotype Distribution

The pneumococcus has a capsule composed of polysaccharide which completely envelops the pneumococcal cells. Ninety-two different capsular types of pneumococci have been identified and form the basis of antigenic serotyping of the organism [57] including the newly identified 6D discovered from our Fiji isolates [58]. Within serogroups, serotypes cross-react immunologically, but only in some cases does this appear to translate into cross- protection (eg 6B with 6A but not 19F with 19A). The association of particular serotypes with disease varies according to age, geography, and clinical site.

Serotype distributions change with age with a narrower range of serotypes causing disease in young children compared to older children and adults [59, 60]. For children <5 years old, serotype 14 is the commonest serotype causing IPD worldwide, and serotype 1 and 5 are ranked in the top 3 serotypes in the Global Alliance for Vaccines and Immunisation (GAVI)- eligible low income countries [61]. In contrast, serotypes 18C, 4, and 9V are more common causes of IPD in North America and Oceania in <5 year olds than other regions of the world [61]. In all regions of the world, 3 serotypes (1, 5, 14) account for 28-43% of IPD in <5 year olds [61]. These are the same 3 serotypes found in lung aspirate study on pneumonia cases in the US in 1941 [62]. Seven serotypes (1, 5, 6A, 6B, 14, 19F, 23F) account for approximately 58-66% of all IPD worldwide [61]. Differences in blood culture practice may affect the observed difference in geographical distribution of serotypes. As serotype/group has been shown to be independently associated with IPD severity in adults [32, 34, 35, 38, 63] those countries that perform blood cultures only on severe hospitalised cases would have higher apparent prevalence for certain serotypes than regions that perform blood cultures on both febrile inpatients and outpatients. For example, serogroup 1 has similar, low isolation rates (0.9 per 100,000 person-years) in the US and Europe [44]. However serotype 1 causes only 0.5% of IPD in the US (where blood cultures are performed routinely on febrile inpatients and outpatients) and 5% of reported IPD in western Europe where outpatient blood cultures are performed uncommonly suggesting this serotype is associated with more severe disease [63]. These differences in blood culturing pratice between countries may also account for the differences in serotype 1 incidence between industrialised and developing countries.

1.5.2 Pneumococcal Pneumonia

Pneumococcal pneumonia is estimated to cause one million deaths in children <5 years each year [64, 65]. However, this estimate is based on the contribution of S. pneumoniae to acute respiratory infections (ARI) in studies using children who mostly survived, which does not account for the role of pneumococcus in fatal pneumonia [66]. Determining the aetiology of pneumonia is difficult, and thus studies that calculate an incidence rate for pneumococcal pneumonia are fraught with inaccuracy [67]. There are only a few studies, mostly from developed countries, that have documented pneumococcal pneumonia and are largely recognised as underestimates due to the insensitivity of diagnostic methods used, which are usually blood cultures [65]. The studies vary in terms of sample size, case definitions, methods used to determine aetiology, and the age of subjects included. One of the best studies was from the Gambia where a randomised placebo-controlled trial in the Gambia was undertaken randomising infants to receiving either 3 doses of the 9-valent

8 pneumococcal conjugate vaccine or placebo. When using the vaccein as a vaccine probe, the preventable burden of clinical and radiological pneumonia was found to be about 15 episodes per 1,000 child-years [68]. This figure is likely to be an underestimate as many chest X-rays were not acquired in children seen in the first year of the study [68]. Two other studies were from countries that could be classified as ‘developed’. Israel is classified by UNICEF as both developed and developing [69], and Chile is considered to be ‘newly industrialising’ [70]. As a result, the rates in both of these countries were closer to those from developed countries, rather than those from the other developing countries.

The rates are likely to be underestimates as blood cultures remain a highly insensitive method of diagnosing pneumococcal pneumonia [71-81]. Even with the most sophisticated laboratory procedures, only a small proportion of pneumonia infections are bacteraemic [74]. Children may present to private general practitioners and receive antibiotics prior to hospital presentation. Poor laboratory facilities, inadequate transportation of specimens to the laboratory, and poorer access to health care affect bacterial isolation rates [70, 82].

It is very difficult to estimate CFR for pneumonia. In countries with good access to health care, CFRs are usually lower because children are readily given antibiotic therapy to which they generally respond rapidly [67]. Where access to health care is poor, many children die at home without receiving medical attention and their deaths often go unrecorded. Vaccine probe studies, which compare mortality caused by pneumonia in groups who do and do not receive pneumococcal vaccination for the purposes of the study, are the most accurate way to demonstrate the proportion of pneumonia deaths associated with pneumococcal infection. There is only one randomised controlled trial of the 9-valent pneumococcal conjugate vaccine from the Gambia assessing the impact of the vaccine on all-cause mortality in children. After the trial was redesigned, the impact on all-cause mortality became a secondary objective of the study and the study had become insufficiently powered to address this. Nevertheless, the study found that vaccinated children had a 16% (95% CI, 3- 28%) reduction in all-cause mortality [68].

1.5.3 Burden of Pneumococcal Disease in Fiji

Our Fiji Pneumococcal Project (FiPP) has studied the burden of pneumococcal disease since 2002. This included a cross sectional study of OM in young children, a retrospective study of the burden of chest-X-ray (CXR) confirmed hospitalised pneumonia in children <5 years of

9 age, a study of the burden of meningitis in children <5 years, and a study of the outcome of pneumococcal and other bacterial meningitis assessing the quality of life in survivors.

1.5.3.1 Burden of OM A cross sectional survey of healthy children aged 3 to 13 months attending maternal and child health centres for immunisations was performed from a selection of 8 urban and 11 rural health centres. Risk factors associated for NP carriage and OM were documented. Immunisation status, birth-weight, and current weight were confirmed by examining the child’s health card. Children had otoscopy, video otoscopy, and tympanometry performed by 2 trained staff. Seven hundered and seventy-four children (69% Indigenous Fijian and 25% Indo-Fijian, 6% other) were enrolled in the study (median age 7.7 months, range 2.5-13.6 months). The prevalence of OM in either ear was: acute OM 14%, OM with effusion 26%, and chronic suppurtive OM 0.4%. Independent risk factors of OM included being Indigenous Fijian, malnourished, or having symptoms of an ARI.

1.5.3.2 Burden of Pneumonia To calculate the incidence and document the clinical features of CXR confirmed pneumonia in children aged between one month and 5 years living in Greater Suva, Fiji a retrospective review was undertaken of children aged between one month and 5 years with a discharge diagnosis suggesting a lower respiratory tract infection (LRTI) admitted to the only admitting hospital, Colonial War Memorial Hospital (CWMH) in Suva, Fiji in the first 10 days of each month from 1st January 2001 to 31st December 2002 [83]. Clinical data were collected and CXRs were re-read and classified according to WHO standardised criteria for CXR-confirmed pneumonia [84]. Two hundred and forty-eight children with LRTI met the inclusion criteria. CXRs were obtained for 174 (70%) of these cases, of which 59 (34%) had CXR-confirmed pneumonia. The annual incidence of CXR-confirmed pneumonia was 428 cases per 100,000 children aged between one month and 5 years living in Greater Suva. If a similar proportion of the children for whom CXRs were unavailable were assumed to have CXR-confirmed pneumonia, the incidence was 607 per 100,000. The incidence rate ratio for Melanesian Fijian compared to Indo-Fijian children for all LRTI was 2.5 (95% CI, 1.9–3.4) and for CXR-confirmed pneumonia was 29.4 (95% CI, 9.8–143.2). This difference in incidence rate ratio may be due to Indo- Fijians seeking health care earlier by private pactitioners and hence negating a hospital admission. The CFR was 2.8% in all children with LRTI, and 6.8% in those with CXR-confirmed pneumonia [83].

A retrospective review of pneumonia admissions (ICD10 J12-18) in all ages was undertaken covering the main island of Viti Levu using the Ministry of Health’s (MoH) computerised discharge data for the years 2006 and 2007 (Figure 1). The denominator was taken from the 2007 Census. Data showed a peak age-specific annual incidence of hospitalised pneumonia at both extremes of age (unpublished data). Data from a different retrospective review for the years 2000-2002 showed hospitalisation rates for pneumonia to be estimated at 525 per 100,000 for the population overall, and 2,226 per 100,000 in children <5 years of age (J. Passmore, unpublished).

Figure 1: The annual incidence of hospitalised pneumonia (ICD10: J12-18) for all ages, 2006- 2007 for Viti Levu

1800 1600 1400 1200 1000 800 600 400 200

0 Annual incidence per 100,000 per Annualincidence 0-4 5y-14 15-24 25-34 35-44 45-54 55-64 65+ Age groups (years)

1.5.3.3 Burden of Meningitis A retrospective bacterial meningitis study was undertaken including all children admitted to a single hospital, CWMH, aged from one month to <5 years old with a permanent residential address within a defined catchment area over a 3-year period [85]. Cases of confirmed bacterial meningitis were those with any of: positive bacterial culture from cerebrospinal fluid (CSF); CSF pleocytosis > 100 white cell (WC), or WC 10-100 with glucose <4.0 mmol/L and protein >100 mg/dL; or latex antigen positive in the CSF and/or Gram stain positive CSF. Individual medical and laboratory records were examined to identify cases. There were 50 cases of culture proven IPD giving an annual incidence of 47.5 per 100,000 <5 years of age. The CFR was 20%. There were 28 cases of pneumococcal meningitis giving an annual incidence of 24.2 per 100,000 <5 years of age. The case fatality rate was 27% [85].

The quality of life in meningitis survivors was also assessed [86]. There were 37 meningitis cases and 148 healthy controls. Twenty-four percent (n=9) had a history of S.pneumoniae meningitis and 24% had another pathogenic organism. The remaining 52% (n=19) had sterile but purulent CSF. The quality of life score (using a quality of life questionnaire, the PedsQL) was lower in the cases (median 86, IQR 71-97) compared to the controls (median 97, IQR 91- 100). The children with a laboratory proven history of pneumococcal meningitis showed a median score of 71 (IQR 55-81). Children with a history of pneumococcal meningitis had a median score more than 10 points lower than children with non-pneumococcal meningitis [86].

1.6 Protection Against Pneumococcal Disease

1.6.1 Host Defense Mechanisms

The immune response to S.pneumoniae requires both innate and adaptive components. Once pneumococci have crossed the first natural barrier of the host, they immediately trigger the activation of some components of innate immunity in addition to the deposition of opsonins on the surface of pneumococci. Initiation of the innate response is largely mediated by Toll-like receptors (TLRs) [87, 88]. Early TLR-mediated signaling results in immune cell activation that drives the development of subsequent adaptive immunity, mediated by B and T cells. Complement plays a key role but CRP, lectins, and IgM anti- carbohydrate antibodies are responsible for opsonisation. CRP binds phosphorylcholine in the pneumococcal cell wall and activates complement. The killing of S.pneumoniae requires opsonisation by a serotype-specific antibody together with complement, followed by phagocytosis by neutrophils and macrophages. Neutrophil chemotaxis is mediated by the complement activating properties of pneumococcal components such as pneumolysin, C-PS, and cytokines. Extracellular pneumococci induce the release of pro-inflammatory cytokines (interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, IL-12, and interferon-γ (IFN- γ), and anti-inflammatory cytokines IL-4 and IL-10 [89]. Pro-inflammatory cytokines increase rapidly in response to the release of pneumococcal cell wall components during autolysis. Cytokines trigger a complex cascade of inflammatory mediators that regulate the various arms of the inflammatory response. Phagocytic cells as well as NK cells and T-cells play an important role in the elimination of pneumococci from alveoli via the production of chemotactic and regulatory cytokines.

Infants’ elicit weak immunological responses to the encapsulated pneumococcus due to the thymus independent (TI) nature of this bacteria. TI antigens do not require T-cells to induce

12 an immune response [90]. TI antigens do not or poorly induce immunologic memory. The antibodies that are produced are primarily of the IgM isotype and are produced in lesser quantities IgG2 [91]. The TI antigens are further divided into 2 categories based on their interaction with B cells: type 1 (TI-1) and type 2 (TI-2) antigens [90-92]. TI-1 antigens induce proliferation and differentiation of B cells in adults but also neonates. In contrast, TI-2 antigens induce a limited immune response in children <2 years of age, but older children and adults repond to TI-2 antigens with the formation of antibodies by activated B cells. Pneumococcal polysaccharide is a TI-2 antigen.

The spleen is important in the immune response to pneumococci as it contains both antibody-producing B cells and phagocytes [93]. The splenic marginal zone at the junction of the red and white pulp is an important site of host defense against bacterial infection. The marginal zone contains macrophages, dendritic cells, and B cells and provides the first line of defense against blood-borne pathogens. Polysaccharide antigens preferentially localise in the marginal-zone B cells found only in the spleen. These B cells are present in low numbers at birth [94] and only appear with adult features after 2 years of age. Children <2 years of age have quantitative defects in IgG2 and IgG4 isotypes [95] with IgG2 isotype being considered as the most effective immunoglobulin against some polysaccharides [96].

Humoral responses are currently considered the major adaptive mechanism for bacterial clearance [97] although there is growing evidence of the role of cell-mediated immunity in the protection from NP colonisation [98-100]. It has been well demonstrated that antibodies to the pneumococcal capsule are sufficient to protect against IPD, it is less clear whether they are necessary or they constitute the primary mechanism of natural resistance to pneumococcal infection [101]. In the absence of PCV in a population, IPD incidence naturally declines approximately 2 years prior to the age at which unimmunised children show a rise in serum anti-capsular antibody. In the pre-PCV era anti-capsular antibody was shown not to be above the putative protective level of 0.35µg/mL by 36 months of age in US children, yet disease from the common serotypes, 6 and 14, are almost 10 fold lower than at 12 months of age [101-103]. This indicates that there are other protective mechanisms apart from serum anti-capsular antibody that confer protection. NP carriage can lead to serotype- specific acquired immunity to pneumococcal carriage for some serotypes [104]. However a recent longitudinal study in Bangladeshi infants showed a reduced rate of NP pneumococcal acquisition in a manner consistent with the induction of serotype-independent protective immunity [105]. Protection may be derived from the development of CD4+ T cells of the IL-

17A lineage that recognise pneumococcal antigens that are expressed during colonisation [106, 107]. Secretion of IL-17A from these cells may recruit phagocytes to the colonisation site and help reduce the duration of carriage [101].

The critical role of pneumococcal-specific memory B cells in the first line of defense against pneumococcal infection has recently become an important area of research. IgM+CD27+ and ‘switched’ IgG+CD27+ memory B cells are involved in the immune response to the 23-valent pneumococcal polysaccharide vaccine (23vPPS) since these cell populations are deficient in patients with primary immunodeficiency syndromes who are susceptible to recurrent infections with encapsulated bacteria [108, 109]. Infant B cells are unable to recruit cognate CD4+ T-cell help through T-cell receptor recognition of peptide-major histocompatibility complex class II complexes on the surface of antigen presenting cells [110]. CD4+ cells contribute to the early host resistance to infection as shown by an early rapid T-cell infiltration to areas that are subject to increased pneumococcal invasion. Pneumolysin seems to be responsible for the pattern of T-cell infiltration [111]. Data suggests that pneumolysin induces the production of IFN- γ and TNF-α in peripheral blood mononuclear cells and IFN- γ and IL-10 in adenoidal mononuclear cells [112]. CD4+ T-cell proliferative responses to pneumolysin are significantly higher in children who do not have detectable pneumococcal NP carriage which suggests that natural CD4+ T-cell immunity to pneumococcal protein antigens could modulate NP carriage [112]. However it is unclear whether this T-cell immunity cleared existing carriage or prevented new colonisation.

1.6.2 Pneumococcal Vaccines

In 1911, Wright et al developed a crude whole-cell pneumococcal vaccine to immunise South African miners [113], a group with a very high pneumococcal burden and mortality. A number of other clinical trials were conducted in South Africa by Spencer Lister and others abroad on the safety and efficacy of polysaccharide vaccines [114-116]. However the validity of the results were questioned due to various methodological flaws in study design such as the lack of randomisation and inadequate clinical follow up. Polyvalent vaccine trials were continued in the 1940s and 2 hexavalent vaccines were commercially produced and licensed in the late 1940s. However effective antibiotics, sulphonamides from 1937 and penicillin in the mid 1940s, were soon available and with subsequent improvements in patient outcomes the interest in pneumococcal vaccine development waned. Data from the US in 1964 told a different story and it was estimated that despite effective antibiotic availability the CFR of pneumococcal bacteraemia was up to 25% [117]. This led to the relatively rapid

14 development of a polyvalent pneumococcal vaccine in the 1970s which was evaluated in South African gold miners and found a protective efficacy of 76-92% [118, 119]. These findings led to the licensure of the 14-valent pneumococcal polysaccharide vaccine (PPS) in the US in 1977 which was replaced by the 23vPPS in 1983. PCV was the first pneumococcal conjugate vaccine to be licensed. Vaccine manufacturers are developing different formulations containing more than 7 capsular polysaccharides and the newly developed 10- valent pneumococcal conjugate vaccine (with the addition of serotypes 1, 5 and 7F) and the 13-valent pneumococcal conjugate vaccine (with the addition of serotypes 1, 3, 5, 6A, 7F, and 19A) have recently been licensed in a number of countries in 2009/2010. In addition, Merck Inc. has a 15-valent pneumococcal conjugate vaccine under advanced stages of development.

1.6.2.1 Measurement of Immune Responses to Pneumococcal Vaccines Given the difficulties related to performing new vaccine efficacy trials where a product is already licensed and in widespread use, WHO has developed a number of serological criteria based on demonstrating non-inferiority compared with the licensed PCV, to assist regulatory agencies in evaluating new pneumococcal conjugate vaccine formulations. The primary criteria for the evaluation of new pneumococcal conjugate vaccines are [102]:

1. The primary endpoint should be the serotype-specific IgG antibody concentration as measured by enzyme-linked immunosorbent assay (ELISA), using 22F pre- absorption, 4 weeks following a 3 dose primary series for all pneumococcal serotypes; and

2. The proportion of vaccinees with antibody concentrations ≥0.35μg/mL for all pneumococcal serotypes;

The secondary supportive criteria are the demonstration in at least a subset of vaccinees of:

3. Functional activity: Opsonophagocytic activity (OPA) as measure by opsonophagocytic assay following a 3 dose primary series compared ideally to unvaccinated age-matched controls; and

4. Immunologic memory: Evidence of memory shown by administration of a booster dose of 23vPPS compared with unvaccinated controls; and/or antibody avidity. A recent review has recommended that conjugate vaccine as the booster be used instead of 23vPPS due to the issue of hyporesponsiveness [120].

1.6.2.1.1. Measurement of pneumococcal antibody concentrations As clinical protection against pneumococcal infection is mediated by antibodies to the capsular polysaccharide, an ELISA that measures serotype-specific IgG antibody concentrations is the accepted measure of immunogenicity. The third-generation ELISA assay was adopted in a meeting convened by WHO in 2000 [121] following the finding that the second-generation ELISA using the single absorption method with C-PS, had insufficient specificity [122]. Some sera contain polyreactive antibodies that recognise pneumococcal polysaccharides of different serotypes resulting in cross reactivity [123, 124]. The ELISA specificity has been improved by pre-absorption not only with C-PS but also with pneumococcal 22F capsular polysaccharide [123, 125]. In addition, this additional step has increased the correlation between anti-capsular polysaccharide antibody concentration and OPA [123, 126]. GlaxoSmithKline believe overnight incubation with 22F as opposed to 30 minute incubation as per the WHO method produces more accurate data at lower levels and 0.2μg/mL is equivalent to 0.35μg/mL [127].

The anti-pneumococcal antibody concentrations required for protection for an individual are not known. A concentration of IgG anti-capsular polysaccharide antibodies ≥0.35μg/mL (without using the 22F absorption step) measured 4 weeks following a primary series is recommended as the protective level at the population level [102]. This pooled estimated is based on the IPD efficacy data from 3 double-blind randomised controlled trials undertaken in Northern California [103], American Indians [128], and South African infants [129]. If a high proportion of the vaccinated population achieves antibody concentrations ≥0.35μg/mL then a high level of protection from IPD can be predicted in that population [130]. A re- analysis using the double absorption method (with C-PS and 22F reabsorption) found the protective concentration to be marginally lower at 0.32μg/mL [130].

1.6.2.1.2. Functional activity The primary mechanism for protection against IPD is mediated by the presence of opsonophagocytic antibodies [131]. Antibodies to pneumococcal polysaccharide offer protection by acting as an opsonic factor and activating complement thereby promoting phagocytosis of pneumococci. The ability of anti- pneumococcal antibodies to induce phagocytosis and killing is believed to be a surrogate for indicating protective activity. As both antibody concentration and avidity contribute to OPA and confer protection in a mouse challenge [132-135], a positive OPA is considered to indicate good vaccine derived protection. While it is recommended that antibody concentration, as measured by a standardised third generation ELISA, be the primary

16 measure when licensing new pneumococcal conjugate vaccines [102], OPA has been accepted as a necessary additional measure and the reference method for measuring the protective capacity of pneumococcal antibodies [126].

The killing OPA method, which measures the killing of live bacteria is regarded as the most biologically relevant method [126, 136]. There are a number of different methods available and there has been extensive efforts to standardise the OPA assay [120]. OPA assays are labour-intensive and difficult to perform with large numbers of samples. A number of multiplexed assays have been developed using fluorescent dye to quantify killing [137], using bacteria or latex beads coated with different polysaccharides [138], or the use of pneumococcal strains resistant to clinically irrelevant antibiotics to quantify killing [137, 139, 140]. In general, the correlation between antibody concentration and OPA seems to be good in infants who have been immunised with conjugate vaccines [141, 142]. Correlates have been high for vaccine serotypes (VT), but lower for cross-reacting serotypes like 6A and particularly 19A [143].

1.6.2.1.3. Immunologic memory Antibody concentrations alone failed to accurately predict the success of Haemophilus influenzae type b conjugate vaccines (Hib) [144] as conjugate vaccines not only induce antibodies but also prime the immune system for later protective memory responses. As this is also true with pneumococcal conjugate vaccines, memory may also play an important role in protection from pneumococcal disease. Immunologic memory to a polysaccharide antigen can be defined as a response that is present in an otherwise non-responsive individual such as infants, characterised by a higher antibody response that is dominated by IgG on exposure to an antigen, and characterised by antibodies with increased avidity as a result of affinity maturation [145]. Antibody affinity describes the strength with which an antibody binds to a complex antigen. The antigen binding capacity of polyclonal antibodies with different affinities can be measured as antibody avidity. Antibody avidity is an expression of the functional antibody affinity and may affect the protective efficacy of antibodies. In vitro, higher avidity antibody is associated with greater opsonophagocytic capacity [146-148]. Furthermore, findings from a study assessing the contribution of avidity, antibody concentration, and IgG subclass to opsonophagocytic activity demonstrated that lower amounts of high avidity antibody were sufficient for killing of bacteria whereas higher amounts of low avidity antibody were required for effective killing activity [147]. While the importance of avidity in determining protection from disease is unclear, studies of Haemophilus influenzae type b (Hib) conjugate

17 vaccine have demonstrated that antibody avidity is strongly associated with functional activity of anti-Hib polysaccharide antibodies [149, 150], and anti-Hib polysaccharide antibodies of high avidity have been found to have protective efficacy in experimental Hib infection [151].

Immunologic memory needs to be demonstrated for the licensure of new pneumococcal conjugate vaccine formulations by either the administration of a booster dose of 23vPPS and the measurement of the increase in IgG concentration, and/or assessment of antibody avidity [102]. The measurement of antibody avidity following PCV immunisation provides information on both the development of B cell memory [152] and the functional activity of antibodies [132, 153]. One method of demonstrating immunological priming is to assess the ability of 23vPPS to boost the immune response and elicit antibodies with increased affinity. Differences in affinity maturation have been used to demonstrate differences in priming capacity of some pneumococcal conjugate vaccines [147, 154-156] and in reduced dose schedules [157]. Although qualitative changes in antibody response have been demonstrated following conjugate vaccine administration, the clinical relevance of these changes is unclear.

In ELISA techniques, the binding of antibody to the coated antigen may be prevented by competitive inhibition using decreasing concentrations of free antigen by a dissociating agent such as thiocyanate. Thiocyanate interferes with the antibody-antigen binding. The elution assays are based on the dissociation of antigen-antibody complexes of low avidity. Avidity assays have not been standardised and their value in predicting protection remains to be determined

1.6.2.2 Pneumococcal conjugate vaccines Infant B cells are immunologically immature, and respond poorly to TI polysaccharide antigens. However, in the presence of activated T cells, they can be stimulated to produce both antibody-producing plasma cells and memory cells [158]. By covalently conjugating a protein carrier to bacterial capsular polysaccharide, this antigen is able to induce a T cell-dependent antibody response. This proved effective in developing vaccines for Hib, and has now proven safe and effective for pneumococcal conjugate vaccines.

Currently, PCV is marketed internationally and is included in over 70 countries national immunisation programmes and on the private market in many more. WHO considers PCV as

18 a priority in national immunisation programmes, particularly in countries where mortality is high (>50/1,000 live births) or where >50,000 children die annually [4].

Seven serotypes (1, 5, 6A, 6B, 14, 19F, 23F) account for approximately 58-66% of all IPD worldwide [61]. The PCV coverage rate of IPD serotypes in young children in industrialised countries tends to be higher (65-80%) [4]. The US, New Zealand, and Australia reported similar coverage by PCV (80%, 81%, and 84% respectively) for IPD isolates in young children pre-vaccine introduction [45, 51, 54]. Rural Thailand and Nigeria have similar but lower PCV coverage rates for IPD (51% and 55% respectively) [50, 159]. In general, the range of serotypes causing disease in affluent countries like the United States and in Europe is relatively narrow and largely confined to the serotypes found in PCV (4, 6B, 9V, 14, 18C, 19F, 23F). A recent review found that if a pneumococcal conjugate vaccine with as few as 6-7 serotypes (including serotypes 1, 5, and 14, and assuming that 6B provides cross protections against 6A disease) were developed it would cover at least 60% of all IPD worldwide. Furthermore, this review stipulated that vaccines should include serotypes that account for at least 60% of the IPD isolates among children in the region for which they are proposed [61].

Each 0.5mL dose of PCV contains 2 μg of polysaccharide for serotypes 4, 9V, 14, 19F, and 23F; 2 μg of oligosaccharide 18C, and 4 μg of polysaccharide type 6B. These saccharides are individually conjugated to diphtheria CRM197 protein. CRM197 is a nontoxic variant of diphtheria toxin. WHO recommends 3 intramuscular doses given at 6, 10, and 14 weeks of age at least 4 weeks apart. A booster dose administered after 12 months of age will improve the immune response [4]. Some European countries including the UK have introduced a “2+1” schedule with 2 doses given during infancy with the third dose given towards the end of the first year of life.

1.6.2.2.1. Safety PCV is safe and well tolerated [4]. More than 198 million doses have been distributed worldwide [160]. The PCV safety profile was evaluated in clinical trials prior to licensure in more than 18,000 infants in the US [103]. Local injection site reactions are not uncommon but are generally mild and self-limiting [103, 161-165]. Two years following licensure and national introduction into the US, post-marketing surveillance further evaluated the safety profile of PCV [166]. The majority of reports in the first 2 years after licensure of PCV described generally minor adverse events previously identified in clinical trials [166]. A retrospective cohort study of adverse events requiring medical attention found a slight increase in reactive airways disease among infants vaccinated with PCV

19 compared with a historical control of infants vaccinated with Hib vaccine [167]. A similar study design found children with Kawasaki disease having a 2 fold increased probability of having received PCV which was not statistically significant when adjusted for sex, age, race, and other factors [168]. Further evaluation showed no association between Kawasaki disease and PCV [169].

1.6.2.2.2. Immunogenicity The standard PCV immunisation schedule that has been studied has been a 3 dose primary series with or without a booster in the second year of life. The geometric mean concentrations (GMC) one month following a 3 dose primary series in infancy from the trials using PCV or the 5 or 9-valent pneumococcal conjugate vaccine (with the same carrier protein as PCV and the additions of serotypes 1 and 5) are shown in Table 1. Direct comparisons between studies are difficult as the vaccination schedules and the laboratories and techniques were different in different settings. In general, a low antibody response was elicited after the first dose, substantial antibody responses were seen after subsequent doses and a clear booster type response to a dose in the second year of life. For all serotypes good post-primary series antibody responses were elicited and serotypes 6B, 9V, and 23F tend to be less immunogenic overall [163, 170-173]. However for serotypes 6B and 23F following a booster in the second year of life the antibody concentrations were high. Antibody responses in Finnish infants were approximately 2-3 times higher than infants in the US. In South African infants, antibody concentrations against serotype 6B were approximately 4 times higher than either Finnish or US children. Differences in serotype- specific responses have been found in other populations, and this has been postulated to be due to the priming effect of tetanus toxoid given to women in pregnancy in developing countries (when tetanus-conjugated polysaccharide vaccines were administered to infants), a nonspecific BCG vaccine effect, early pneumococcal NP acquisition, and genetic differences amongst the populations [174].

An important characteristic of the pneumococcal conjugate vaccines are their ability to create immunologic memory. Antibody concentrations achieved after primary vaccination usually decline during subsequent months. However booster immunisation with either 23vPPS or conjugate induces a marked increase in antibody concentrations in children who have been primed with conjugate vaccine in infancy. Another feature of the anamnestic response produced by conjugate vaccines is the rapidity of the antibody response within 7 to 10 days after the re-vaccination [173, 175-184]. IgG antibodies tend to dominate the response. These findings indicate that conjugate vaccines prime the infant immune system

20 to elicit a robust response on subsequent exposure to either polysaccharide or conjugate vaccine.

There have been several studies involving children in a number of countries using different pneumococcal conjugate formulations and schedules, comparing the immunogenicity of a 23vPPS or PCV booster following a pneumococcal conjugate vaccine primary series. The majority of studies have shown that serotype-specific antibody concentrations are generally higher following 23vPPS than PCV booster [157, 172, 185-188]. The higher response may be due to the higher dose of pneumococcal polysaccharide in the 23vPPS, compared to PCV, enhancing the stimulation of memory B cells or by stimulating a greater number of B cells overall [189]. Despite higher antibodies generated post PCV/23vPPS a study comparing PCV/23vPPS with a PCV/PCV schedule found similar vaccine efficacy results against acute OM [188].

Studies have found that a conjugate vaccine booster is better at increasing antibody avidity than a PPS booster [147, 154, 157, 190]. Infants primed with PCV and who received a PCV booster, but not PPS booster, had an increase in avidity of anti-pneumococcal polysaccharide [154] which suggested the response to PCV was T cell dependent, but the T cell-independent PPS only triggered existing memory B cells. A study in infants immunized at 2, 4, and 6 months of age with one of 4 different conjugate vaccines, and boosted at 14 months with the homologous conjugate or PPS found that the avidity of serotypes 6B, 14, 19F and 23F increased with age, but only a booster dose of conjugate further increased avidity compared to a PPS booster [147]. Similarly, a reduced dose study in the UK found that a booster with PCV gave significantly higher avidity for the 3 serotypes tested (6B, 14, 23F) compared to a 23vPPS booster [157]. Avidity for 3 serotypes tested in Ghanaian children was higher in those who had received PCV rather than 23vPPS [190]. This phenomenon may be due to the fact that conjugate boosters promote affinity maturation by T cell dependent mechanisms. An alternative explanation for the differences in avidity is that the amount of antigen in the PPS is high enough to induce both high and low avidity B cell clones to produce antibodies, whereas only high avidity clones are induced by lower concentrations of polysaccharides in conjugate vaccines [191].

Table 1: Summary of serotype-specific GMC data from trials of CRM197-conjugated pneumococcal vaccines, one month post primary series

Co-administered GMC (μg/mL) Ref. Country PCV Schedule parenteral n vaccines 1 4 5 6B 9V 14 18C 19F 23F

[163] Canada 7v 2,4,6m DTaP-IPV-Hib, HBV 123 3.84 3.35 2.07 6.37 3.01 3.3 1.83

[192] Finland 7v 2,4,6m DTwP-Hib 60 2.9 1.4 1.8 5.2 2.6 7.1 4.2

[193] France 7v 2,3,4m DTwP-IPV/Hib 53-54 4.0 2.91 1.92 5.6 2.0 4.22 1.6

[162] Germany 7v 2,3,4m DTaP-HBV-IPV/Hib 115 5.19 3.20 2.60 6.88 2.38 4.44 2.00

[165] Germany 7v 2,3,4m DTaP-HBV-IPV/Hib 141 3.97 0.91 3.21 4.65 3.21 3.72 2.10

[164] Germany 7v 2,3,4m DTaP-IPV-Hib (GSK) 83 3.46 2.5 2.29 4.68 2.55 4.1 1.9

9v- [194] Iceland 3,4,5m DTaP-IPV/Hib 110 3.34 2.97 1.52 1.94 1.99 6.95 1.83 4.19 1.77 MCC

[195] UK 9v-MCC 2,3,4m DTPwP/Hib 100 1.43 1.2 0.77 1.2 0.94 2.48 0.97 1.83 1.15

[157] UK 9v 2,3,4m DTaP-Hib, MCC 73 2.77 1.82 1.49 1.12 1.60 4.78 1.68 3.00 1.52

[172] UK 7v 2,3,4m DTwP, Hib 112-115 2.40 1.11 1.50 2.23 1.42 2.45 1.52

[196] US 7v 2,4,6m DTaP-HBV-IPV, Hib 138-168 1.74 0.80 1.55 4.68 2.63 1.09 1.48

[103] US 7v 2,4,6m DTaP 2 75 1.37 2.14 1.23 5.04 1.88 1.52 1.21

Co-administered GMC (μg/mL) Ref. Country PCV Schedule parenteral n vaccines 1 4 5 6B 9V 14 18C 19F 23F

[173] US 7v 2,4,6m DTwP-Hib, HBV 156 1.30 1.22 1.01 3.72 1.44 1.95 2.54

[182] US 7v 2,4,6m DTwP/Hib 90 1.36 1.37 0.98 3.48 1.24 3.45 1.8

US Navajo/White [197] 7v 6w,4,6m DTaP, Hib, IPV, HBV1 223 3.21 8.25 2.47 6.81 2.61 2.74 2.59 Mountain Apache [177] Bangladesh 7v 18,24,28w DTwP-Hib3 106 8.61 13.34 3.68 10.23 5.14 5.75 6.65

199- [198] South Africa 9v 6,10,14w DTwP-Hib, HBV 5.30 4.02 6.18 5.87 3.24 3.61 4.78 2.99 2.73 206 South Africa [199] 9v 6,10,14w DTwP, Hib, HBV 63 7.55 4.09 5.79 1.76 3.35 3.62 4.55 6.02 3.15 (HIV-) [200] The Gambia 9v 2,3,4m DTwP-Hib, HBV4 60 3.52 1.92 4.21 2.48 1.25 3.04 1.63 2.03 1.13

[179] The Gambia 9v 2,3,4m DTwP, HBV4 83-89 6.94 4.90 5.84 4.93 4.07 4.45 4.89 2.91 2.85

[201] The Gambia 5v 2,3,4m DTwP, HBV4 30 3.61 7.59 3.76 2.49 4.29

[202] The Gambia 9v 6,10,14w DTwP-Hib, HBV 88-101 5.79 4.88 4.67 7.08 2.61 11.09 3.0 6.41 3.2

1 IPV and Hib co-administered with 1st and 2nd dose of PCV only; HBV co-administered with 1st dose of PCV only 2 Details of other co-administered vaccines and vaccine combinations unknown 3 DTwP-Hib and OPV co-administered with 1st dose of PCV only 4 HBV co-administered with 1st and 3rd dose of PCV only MCC Meningococcal conjugate vaccine

1.6.2.2.3. Efficacy In a large randomised controlled trial in the US, PCV given at 2, 4, and 6 months of age, with a fourth dose in the second year of life, was immunogenic and provided 97.4% (95% CI, 82.7-99.9%) protection against VT IPD in infants who received at least 3 doses of the vaccine [103]. In the intent-to treat analysis, for children who had received at least one dose of the vaccine there was an 89.1% (95% CI, 73.7-95.85%) reduction against VT IPD overall [103]. In the Gambia, a randomised controlled trial of 3 doses of the 9-valent pneumococcal conjugate vaccine (with the addition of serotypes 1 and 5) in infancy had an efficacy of 77% (95% CI, 51-90%) against VT IPD and 45% (95% CI, 19- 62%) efficacy against all IPD serotypes [68]. In South Africa, the 9-valent pneumococcal conjugate vaccine given at 6, 10, and 14 weeks of age and had 83% (95% CI, 39-97%) protective efficacy against VT IPD in HIV-negative children and 65% (95% CI, 24-86%) efficacy in HIV-positive children [129]. There was 35% (95% CI, -31-68%) vaccine efficacy against all IPD serotypes [129]. In a group-randomised study in Navajo and White Mountain Apache Indian children younger than 2 years, the per protocol primary efficacy of PCV against VT IPD was 76.8% (95% CI, -9.4-95.1%) and the intention-to-treat efficacy was 82.6% (95% CI, 21.4- 96.1%), after group randomisation had been controlled for [128].

As most cases of pneumococcal pneumonia are non-bacteraemic, the lack of a sensitive and specific endpoint to determine vaccine efficacy for pneumococcal pneumonia has resulted in the use of radiologically defined pneumonia as an endpoint in clinical trials [84]. However this endpoint may under-estimate the true public health benefit of pneumococcal conjugate vaccines [203, 204]. Using the WHO method, the efficacy against first episode of radiographically confirmed pneumonia adjusting for age, gender, and year of vaccination for children from the US who received 3 doses of PCV in infancy followed by a fourth dose at 12- 15 months of age, was 25.5% (95% CI, 6.5-40.7%) for intent-to-treat and 30.3% (95% CI, 10.7-45.7%) for the per protocol analysis [205]. The 9-valent pneumococcal vaccine trials in the Gambia and South Africa described previously, documented a vaccine efficacy against the first episode of radiologically confirmed pneumonia in the per protocol analysis of 37% (95%CI, 27-45%) and 25% (95%CI, 4-41%) respectively [68, 129]. In the South African and Gambian trial, the efficacy estimate against all clinically diagnosed pneumonia was similar at 7% (95% CI, -1-14) and 7% (95% CI, 1-12) respectively [68, 129]. In addition, vaccinated Gambian children showed a 16% (95% CI, 3-28%) reduction in all-cause mortality [68]. In the Philippines, children who had received 3 doses of an 11-valent pneumococcal conjugate vaccine (with the addition of serotypes 1, 3, 5, and 7F) showed a vaccine efficacy of 22.9%

(95% CI, -1.1-41.2) against radiologically confirmed pneumonia in <24 month olds and a 34% (95% CI, 4.8-54.3%) reduction in <12 month olds [206]. There was no efficacy demonstrated against clinical pneumonia defined as cough and fast breathing [206].

Comparing OM outcome between studies is more challenging as the definitions of a case of OM varies between studies. However a Cochrane review pooling results from 4 trials in disparate subjects ranging from healthy infants or toddlers, to toddlers with recurrent OM, found a small effect (RR 0.921; 95% CI, 0.894-0.95) on the prevention of acute OM [207]. A study in Finnish infants found that PCV had a small and non-significant effect (6% reduction; 95% CI, -4-16%) on all OM [161]. These children were followed up at 4 to 5 years of age and the vaccinees were found to have a 39% (95% CI, 4-61%) reduction in tympanostomy tube placement [208]. In US children, the PCV vaccine efficacy against any OM episode using per protocol analysis was 7% (95% CI, 4.1-9.7%) and for tympanostomy tube placement was 20.1% (95% CI, 1.5-35.2%) [103]. Children from this study were followed for up to 3.5 years and PCV was found to reduce OM visits by 7.8% (95%CI, 5.4-10.2%) and tympanostomy tube placements were reduced by 24% (95% CI, 12-35%) [209]. The vaccine efficacy of a 7-valent pneumococcal conjugate vaccine conjugated to meningococcal outer membrane protein complex was similar to PCV, and a booster with 23vPPS had similar efficacy to the conjugate booster despite higher antibodies generated following the PCV/23vPPS combination [188]. The vaccine efficacy against acute OM in infants who had received an 11-valent pneumococcal conjugate vaccine with each serotype conjugated to Haemophilus influenzae- derived protein D at 3, 4, 5, and 12-15 months was 33.6% (95% CI, 20.8-44.3%) [210]. This increased benefit over the other trialed conjugate vaccines may be due to the H.influenzae- derived protein D carrier protein which provided efficacy against non-typable H.influenzae, a common cause of acute OM but this remains to be proved [210].

1.6.2.2.4. Effectiveness Since the introduction of PCV into the national immunisation schedule in the US in 2000, there had been an impressive 69% reduction in IPD in children <2 year old [52]. Similarly in Australia, Germany, Portugal, Spain, Denmark, and Norway significant declines in IPD rates in young children have been reported [211-216]. The IPD rate in the high risk White Mountain Apache children has declined to its lowest rate ever since the introduction of PCV [217]. An observational study in the US using routine administrative data found that following PCV introduction the average annualised pneumococcal meningitis rate in children <2 years old decreased by 66%, and had declined in children 2 to 4 years old and those ≥65 years old compared with rates pre –vaccination [218]. For those children in

25 the US who developed IPD despite being vaccinated, IPD resulted primarily from NVT, or in children who were incompletely vaccinated or had co-morbid conditions [219].

Approximately 4 years following PCV introduction in the US replacement IPD, particularly due to serotype 19A, developed due to capsular switching and clonal expansion [220, 221]. Serotype 19A has become the predominant cause of IPD in children [222-224] and serogroups 15 and 33 have been reported as increasing causes of IPD in children in a multicentre study in the US [225]. The annual incidence of NVT IPD has significantly increased in the <5 year olds and ≥65 year olds in one study [222] and in children and adults in another [226]. Serotype replacement IPD is more common in the immunocompromised population but occurs in both the vaccinated group and older age groups [227]. Despite there being a significant reduction overall in pneumococcal meningitis in the US, a recent study has shown that the rate of NVT meningitis has significantly increased from 0.32 to 0.51, an increase of 60.5% (p<0.001) and the proportion of penicillin non-susceptible pneumococcal meningitis isolates has increased significantly compared with pre-vaccine levels [228]. The success of the introduction of PCV into the UK’s national schedule has been comprised to some extent by rapid NVT replacement [229].

The impact of PCV introduction on hospitalised pneumonia was investigated in an interrupted times series analysis that used all-cause pneumonia and pneumococcal pneumonia admission rates as the main outcome [230]. The advantage of this approach was that it could account for seasonal and secular trends that were present before the intervention [231]. Admission rates were compared before and after the introduction of PCV and rates of dehydration were used as a comparison. All-cause pneumonia rates had declined by 39% (95% CI, 22-52%) and pneumococcal pneumonia rates declined by 64% (95% CI, 47-77%) [230]. Dehydration rates remained steady throughout the study period. However biases including differences in physician diagnoses, coding, or admission practice following the introduction of PCV may have contributed to these findings. A study in the US assessing the impact of PCV on inpatient pneumonia rates found a suggestion of a decrease (IRR 0.6, p=0.07) in infants in the post- introduction period [232]. This same study found a significant decrease in the risk (IRR, 0.74, p=0.02) of confirmed outpatient pneumonia in the period after PCV introduction compared to the period prior to PCV introduction [232]. In contrast, another study found PCV to have no impact on the rate of outpatient pneumonia presentations in the US in children <2 years of age [233]. In Australia, a recent study using a national electronic database found that after adjustment for background and seasonal

26 trends, a reduction of 38% (95%CI, 36-40%) and 29% (95%CI, 26-31%) in all-cause pneumonia was found following PCV introduction (3 infant PCV doses with no booster) in children <2 years and 2 to 4 years respectively [234].

In contrast, recent studies have found an increase in empyema hospitalisations in US children [235, 236]. Using nationally representative dataset in the US, one observational study found that empyema hospitalisations of children ≤18 years old increased almost 70% and complicated pneumonia (including empyema, pleural effusion, or bacterial pneumonia requiring a chest tube or decortication) increased compared to the pre-vaccine era despite the rate of all hospitalised bacterial pneumonia and IPD significantly decreasing [235]. These findings, if real, may reflect increases in serotype replacement causing NVT pneumococcal pneumonia, reflecting that NVT may be more pathogenic than VTs, or an increase in complicated pneumonia due to other bacterial pathogens occupying pneumocci’s former niche and causing disease. Similarly an ecologic study using a different national dataset in the US found a doubling of pneumonia admissions complicated by empyema in the post- vaccine era with an increase in streptococcal and staphylococcal empyemas [236]. However this upward trend in empyema admissions was found prior to PCV introduction [236].

National outpatient OM visit rates were compared before and after the introduction of PCV in the US [233]. OM visit rates significantly declined by 20% (p=0.014) in children <2 years of age [233]. The risks of developing frequent OM or having tympanostomy tubes inserted were compared in children <2 years of age from 2 states in the US before and after the introduction of PCV [237]. Frequent OM visits declined by 17% and 28%, and tympanostomy tube insertion declined by 16% and 23% in Tennessee and New York children respectively [237]. In Australia there was a significant reduction in tympanostomy tube insertion in children up to 2 years of age following the introduction of PCV in the national immunisation schedule [238]. In a before and after PCV introduction study in a very high risk population of Australian Indigenous infants, it appeared that no benefit was found of PCV on OM rates althoughthe authors state bias and confounding may have impacted on the findings due to the nature of the design of the study [239]. A small prospective study in the US documenting the otopathogens in children 6 to 36 months of age with receurrent OM and children with infrequent acute OM found that 6 to 8 years post PCV introduction, NVT had replaced VT, and the frequency of isolation of S. pneumoniae was nearly equal to that of non-typeable Haemophilus influenzae, with other pathogens being less frequently isolated from middle ear fluid [240].

1.6.2.2.5. Reduced dose PCV schedules Although 3 PCV doses were originally considered to be required for an optimal immune response to PCV some studies have suggested that a single dose may be sufficient at least where stimulation with high pneumococcal carriage and earlier carrier priming has occurred [198, 241]. Because conjugate vaccines induce priming, it is possible that they will protect even in the absence of a circulating antibody response. This raises the possibility that fewer doses of conjugate vaccine than are presently recommended, perhaps even a single dose, may be sufficient to protect from serious invasive disease or death. When the introduction of PCV into the US national immunisation schedule was met with a global shortage of vaccine, many children received fewer than the recommended 4 doses of vaccine. A case control study documenting the impact of this on IPD due to vaccine serotypes found that one and 2 dose schedules given to infants <7 months of age had an effectiveness of 73% (95%CI, 43-87%) and 96% (95%CI, 88-99%) respectively [242]. Models however have predicted that a single PCV dose given between 5-10 months of age could prevent a significant amount of VT IPD [243]. A South African trial showed a significant and potentially protective antibody response to most serotypes following a single dose at 6 weeks of age with at least 70% of infants producing antibody concentrations >0.15μg/mL after a single PCV dose and at least 95% doing so after 2 doses [198]. A study in Filipino infants with an 11-valent pneumococcal vaccine conjugated to either tetanus protein or diphtheria-toxoid showed that a single dose at 18 weeks of age elicited similar antibody concentrations at 9 months of age compared with those that had received 3 doses [241]. A model estimating the herd effect on IPD incidence predicted that even in situations where PCV coverage is less than 3 or 4 doses, PCV may still induce herd effects [244].

The immunogenicity of 3 versus a 2 dose pneumococcal primary series with different co- administered vaccines, is different in different settings [157, 170, 182, 194, 245, 246] (Table 2). Following 3 doses of the 9-valent pneumococcal conjugate vaccine in Icelandic infants, 7 out of the 9 serotypes had significantly higher post primary antibody concentrations compared to the 2 dose group [194]. However the proportion of infants in the 2 dose group with antibody levels >0.35μg/mL (the estimated protective level) was only significantly lower compared to the 3 dose group for serotype 6B [194]. A randomised controlled trial from Israel in which infants received either PCV at 2, 4, 6, and 12 months of age, 4, 6, and 12 months of age, or at 2, 4, and 6 months of age found that the “2+1” group had lower post

28 primary IgG concentrations for serotypes 6B, 14, 18C, and 23F compared to the 3 PCV dose groups [247]. A cohort study from the Netherlands compared a “2+1” PCV schedule (given at 2, 4 and 11 months of age) with a “3+1” (given at 2, 3, 4 and 11 months of age) schedule found that pre-booster levels were comparable for 6 of 7 PCV serotypes (except serotypes 6B) and post-booster levels were comparable for 5 of 7 PCV serotypes (except 6B and 19F) [248]. A study using an 11-valent pneumococcal conjugate vaccine in Israeli infants showed a 2 dose schedule was less immunogenic than a 3 dose post primary series for serotypes 6B, 14, 18C, and 23F with a significantly lower proportion of infants with antibody levels ≥0.35μg/mL for serotypes 6B, 18C, and 23F. This study used an unlicensed 11-valent pneumococcal conjugate vaccine conjugated to diphtheria and tetanus carrier proteins [246]. PCV given to US infants showed 3 doses were needed to achieve an immunological response for serotype 6B but 2 doses were sufficient for the other 6 PCV serotypes [182]. PCV was less immunogenic for serotype 6B in Italian infants after 2 PCV doses than described in the US and Finland, but similar for the other PCV serotypes [161, 173, 245]. A randomised controlled trial in Denmark, Norway, Slovakia, and Sweden comparing a “2+1” 10-valent pneumococcal non-typeable H. influenzae protein D-conjugate vaccine schedule (given at 3, 5 and 11-12 months of age) with a “3+1” schedule (given at 3, 4, 5 and 11-12 months of age) found for most vaccine serotypes a trend towards lower post-primary and post-booster immune responses in children primed with the 2 dose primary series [249].

In contrast, other studies have found minimal immunological differences between a 3 or 2 PCV dose primary series [157, 245]. In a non-randomised study in UK infants no significant differences in GMC following a 3 or 2 dose schedule were found [157]. There were no significant differences in the proportion of infants achieving antibody concentrations >0.35μg/mL except for serotype 14 for which there was a higher proportions achieved in the 3 dose group [157]. Two doses may well be sufficient to protect against most PCV serotypes. In some European countries and Italy, routine immunisations are given in a 2 dose primary series with a booster at or before the end of the first year of life. Norway introduced a “2+1” PCV schedule (given at 3, 5 and 12 months of age) in 2006 [215]. Following this, the incidence of IPD in children less than 2 years of age rapidly declined and the incidence rate of NVT IPD remained stable [215]. The vaccine efficacy was estimated to be 74% (95% CI, 57- 85%) [215]. Liguria, in Italy, found a statistically significant reduction in hospitalisation rates for all-cause (15.2%; 95%CI 2.8-26.1) and pneumococcal pneumonia (70.5%; 95%CI, 9.7- 90.4%), and for acute OM (36.4%; 95%CI, 24.1-46.7%) in children born after the introduction of universal PCV [250]. Effectiveness data from the UK using the “2+1” schedule has shown a

29 reduction in all infant IPD [251]. There has been a marked reduction in VT disease [251]. However the incidence of NVT IPD has been increasing and remains a concern [251]. Three doses may be required to protect against serotype 6B as breakthrough cases of 6B have occurred [252]. Ongoing surveillance will determine whether these breakthrough cases will be a significant and consistent finding.

Table 2: Summary of serotype-specific GMC data from trials of reduced dose pneumococcal conjugate vaccines Co- GMC (μg/mL) Ref. Country PCV Schedule n administered 1 4 5 6B 9V 14 18C 19F 23F Reduced dose GMC data compared with 3 doses (if performed),vaccines one month post primary series [194] Iceland 9v-MCC 3,5m DTaP-IPV/Hib 108 3.62 2.34 1.2 0.69 1.73 4.69 1.52 3.2 0.91

9v-MCC 3,4,5m DTaP-IPV/Hib 110 3.34 2.97 1.52 1.94 1.99 6.95 1.83 4.19 1.77

[247] Israel 7v 4,6m DTaP-IPV-PRPT 157 2.22 0.57 1.38 3.57 1.22 2.13 0.63

7v 2,4,6m1 DTaP-IPV-PRPT 145 1.94 2.32 1.46 5.22 1.54 1.79 1.26

7v 2,4,6m1 DTaP-IPV-PRPT 157 1.97 1.86 1.43 5.03 1.70 2.10 1.04

DTaP-HBV- [245] Italy 7v 3,5m 46 4.19 1.08 2.83 6.02 2.17 5.84 1.23 IPV/Hib [170] Sweden 7v 3,5m DTaP-IPV/Hib 75 4.43 0.30 3.17 3.37 2.47 5.03 0.88

[157] UK 9v 2,4m DTaP-Hib, MCC 82 3.41 2.05 1.42 1.01 1.57 3.2 1.54 4.11 1.15

9v 2,3,4m DTaP-Hib, MCC 73 2.77 1.82 1.49 1.12 1.60 4.78 1.68 3.00 1.52

DTaP-IPV-Hib 154- [253] Mexico 13v 2,4m 3.42 3.54 1.89 0.83 1.82 5.59 1.81 4.16 0.85 HBV, rotavirus 197 [201] The Gambia 5v 2,4m DTwP,HBV2 30 2.96 3.87 4.08 1.89 3.03

5v 2,3,4m DTwP,HBV2 30 3.61 7.59 3.76 2.49 4.29

Co- GMC (μg/mL) Ref. Country PCV Schedule n administered 1 4 5 6B 9V 14 18C 19F 23F Reduced dose GMC data compared with 3 doses (if performed),vaccines one month post primary series 231- [198] South Africa 9v 6,(10,14w) 3 DTwP-Hib,HBV 2.98 1.01 2.03 0.46 0.65 1.04 0.81 1.09 0.27 234

227- 9v 6,10,(14w) 4 DTwP-Hib,HBV 5.68 5.28 5.4 1.77 2.98 2.28 3.21 3.2 1.14 233

199- 9v 6,10,14w DTwP-Hib,HBV 5.30 4.02 6.18 5.87 3.24 3.61 4.78 2.99 2.73 206 GMC following primary series dose one or 2 compared with post-dose 3 levels

[177] Bangladesh 7v 18,(24,28w)5 DTwP-Hib6 106 4.92 0.56 0.94 1.02 0.73 1.00 0.68

7v 18,24,28w DTwP-Hib6 106 8.61 13.34 3.68 10.23 5.14 5.75 6.65

[182] US 7v 2,4,(6m)7 DTwP/Hib 90 1.48 0.26 0.44 1.97 0.74 2.2 0.32

7v 2,4,6m DTwP/Hib 90 1.36 1.37 0.98 3.48 1.24 3.45 1.8

US Navajo/ White DTaP, Hib, IPV, [197] 7v 6w,(4,6m)8 215 1.0 0.25 0.41 0.7 0.53 0.63 0.2 Mountain HBV9 Apache

DTaP, Hib, IPV, 7v 6w,4,(6m)10 202 2.97 1.6 1.73 4.58 1.59 1.68 1.02 HBV9

Co- GMC (μg/mL) Ref. Country PCV Schedule n administered 1 4 5 6B 9V 14 18C 19F 23F Reduced dose GMC data compared with 3 doses (if performed),vaccines one month post primary series DTaP, Hib, IPV, 7v 6w,4,6m 223 3.21 8.25 2.47 6.81 2.61 2.74 2.59 HBV9

1 GMC from 2 3 PCV dose primary series groups shown. At 12 months, each 3 dose group either received a PCV booster or no booster. 2HBV co-administered with 1st and 3rd dose of PCV only 3 GMC following one dose 4 GMC following 2 doses 5Blood drawn at 24 weeks of age (~6 weeks post-dose 1) 6DTwP-Hib and OPV co-administered with 1st dose of PCV only 7Blood drawn at 6 months pre-dose 3 8 Blood drawn 2 months post-dose 1 9IPV and Hib co-administered with 1st and 2nd dose of PCV only; HBV co-administered with 1st dose of PCV only 10Bood drawn 2 months post-dose 2

1.6.2.3 23vPPS The relatively inexpensive 23vPPS covered approximately 85-90% of IPD isolates in adults in the US pre-PCV era [254]. The 23vPPS is licensed for use in adults and children ≥ 2 years old who have certain underlying conditions which are risk factors for IPD [254]. The characteristics of the immune response to 23vPPS compared with that after polysaccharide protein conjugate vaccines include: a poorer immune response, especially in infants and young children; absent booster responses on repeated administration of the vaccine; minimal maturation of the immune response as indicated by a predominance of low affinity antibody; and no priming. Immunological priming allows a rapid antibody response when an organism is encountered later and may indicate long-term immunity.

A 0.5mL dose of 23vPPS contains 25μg of purified capsular polysaccharide from each of the 23 serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 20, 22F, 23F, and 33F). The 23vPPS is administered as a single intramuscular or subcutaneous injection [254].

1.6.2.3.1. Safety Local reactions are the most common adverse event (AE) following administration of 23vPPS. These reactions usually resolve within 2 days of vaccination and are usually mild [255, 256]. Some studies assessing AEs following re-vaccination reported large local reactions and limb swelling in healthy children and adults vaccinated within 3 or 4 years of first vaccination [257-259]. Re-vaccination at an interval of at least 5 years since first vaccination has subsequently been shown to be well tolerated in adults [260-262].

1.6.2.3.2. Immunogenicity Antibody responses to many of the pathogenic pneumococcal serotypes in the 23vPPS are poor in infants, as polysaccharide antigens induce a T-cell independent immune response [263]. Therefore they are poorly immunogenic in those aged < 2 years and fail to induce immunologic memory as a single dose of PPS followed by a booster dose of PPS in children <5 years old does not result in higher antibody levels than a single dose of PPS [264]. Higher antibody concentrations would be expected following re-vaccination if priming had occurred with the first PPS dose. Serotype-specific IgG responses following a single dose of 23vPPS are age and serotype dependent [146, 263-268] with poor responses demonstrated in most infant studies for serogroups 6 [146, 263-268], 19 [146, 263, 264, 267, 268], and 23 [263-268], and inconsistent responses in other studies for serotypes/groups 1 [263, 264], 12 [146, 266], 14 [264, 266], and 18 [268]. Serotype 3 can induce antibodies in infants as young as 3 months of age [269]. In the Gambia, antibody concentrations were measured to 6 pneumococcal

34 polysaccharides in children before and one month following 23vPPS at the ages of 2, 4, 6, or 9 months or at 5 to 10 years of age [263]. Antibody responses to serotypes 1, 3, and 5 were seen in all age groups but responses to serotypes 19F and 23F were only seen in the older children [263]. Hence responses are particularly poor to those “paediatric serotypes” that are carried for longer periods, but are better to the more pathogenic serotypes.

1.6.2.3.3. Immunological hyporesponsivesness A particular concern relating to the administration of PPS to young children is the theoretical risk that hyporesponsiveness may occur following re-challenge or subsequent pneumococcal exposure following PPS [189]. These concerns are largely based on 2 observations. First, it has been demonstrated that repeated vaccination with meningococcal C polysaccharide vaccine results in diminished immune response to subsequent doses of the vaccine, even in adults [270, 271]. A study documenting immunologic memory 5 years after meningococcal A/C conjugate vaccination in infancy showed that challenge with the meningococcal polysaccharide or conjugate at 2 years of age displayed evidence of immunologic memory [271]. However subsequent challenge with the polysaccharide vaccine at 5 years of age failed to induce a similar memory response in the polysaccharide vaccinated group. The authors concluded that the initial polysaccharide immunisation at 2 years of age interfered with the immune response to subsequent polysaccharide vaccination [271]. No adverse clinical effects have ever been documented from repeated exposure to the meningococcal polysaccharide vaccine. The clinical significance of this finding is unknown, and meningococcal C polysaccharide vaccine continues to be used throughout the world, especially in countries of the African “meningitis belt” [272]. In some communities, children are vaccinated yearly. There is no epidemiological evidence of increased susceptibility to meningococcal disease in communities or individuals that have been exposed to repeated vaccination [272]. Secondly, a small pneumococcal vaccine trial undertaken in the Netherlands in children up to 6 years of age with a history of OM randomised to receive a combined PCV and 23vPPS regimen, or a control vaccine found that whilst the primary endpoint (the recurrence rate of OM) was identical in the 2 groups, there was an increase in the total number of OM episodes suffered by the pneumococcal vaccinated group, which just achieved statistical significance at the p=0.05 level [273].

For Australian Indigenous children, the national immunisation schedule consisted of 3 infant doses of PCV followed by a 23vPPS booster at 18 months of age. In a recent retrospective cohort study of Indigenous children in the Northern Territory, Australia, the findings

35 suggested an increased risk of hospitalised acute LRTI after pneumococcal vaccination particularly after receipt of the 23vPPS (adjusted hazard ratio after vs no dose, 1.39;95% CI, 1.12-1.17; p=0.002) [274]. In contrast, results from Western Australia using a population based data linkage system, found significant reductions in acute LRTI in Indigenous children [275]. However it is unclear what proportion of children had received the 23vPPS. Similarly national data from a national electronic database found a significant decrease in Australian Indigenous childrens’ hospitalisations for pneumonia [276]. In addition, there is suggestive evidence that the incidence of 19A IPD is less in Indigenous children living in regions where the 23vPPS is given in the second year of life [277].

Immunogenicity studies following re-vaccination with PPS in young children using different valencies and formulations ranging from 5 to 100μg/serotype of PPS have shown inconsistent results including reduced responses to some serotypes following re-vaccination [257, 265]. Conversely, one infant study showed no evidence of hyporesponsiveness on revaccination with PPS [266]. The assays used in these studies were less specific than techniques currently in use. A more recent study in children over 12 months of age who were randomised to receive one dose of the 7-valent pneumococcal polysaccharide- meningococcal outer membrane protein complex conjugate vaccine or 23vPPS at 12, 15, or 18 months of age were further randomised to receive a booster of the conjugate vaccine or 23vPPS 12 months later [186]. Those that had received the conjugate as a primary dose followed by a 23vPPS booster achieved higher antibody concentrations post booster than those that had received the 23vPPS as a primary and booster dose. In the same study, in a non-randomised comparison comparing the immunogenicity of a single primary dose of 23vPPS at 24, 27, or 30 months of age to a booster dose of 23vPPS at 24, 27, or 30 months of age following a priming dose of 23vPPS at 12, 15, or 18 months, lower antibodies were potentially achieved for serotypes 4, 6B, 9V, and 23F. The authors concluded that 23vPPS given at 12-18 months may induce tolerance to an additional 23vPPS vaccination 12 months later [186]. A small study in which 11 children aged between 2-8 years with recurrent infections who received a second 23vPPS due to an inadequate response to the same vaccine administered 6 months previously, found that the GMC following the second dose compared to those following the first dose appeared to be similar for most serotypes but lower for serotypes 6B and 23F, although these differences were not statistically different [278].

In vitro studies have suggested that polysaccharides antigens may be able to down regulate B cells [279], and that newly formed antibody via IgG, IgM, or immune complexes can bind to inhibitory Fc receptors and prevent antibody production [280]. Both plasma and memory B cells are stimulated following exposure to PPS. In contrast to T-independent immune responses, priming by either PCV, previous encounter with S. pneumoniae or a cross-reacting antigen prior to 23vPPS vaccination, could stimulate immunologic memory by presentation of polysaccharide-protein conjugate antigens to the immune system (T-dependent) [281]. Given the T-independent nature of PPS antigens, 23vPPS may stimulate the existing pool of memory B cells to differentiate into plasma cells and secrete antibody without replenishment of the memory B cell pool. This has been proposed as one mechanism for the hyporesponsiveness observed following meningococcal polysaccharide vaccine administration [282]. Upon subsequent booster with 23vPPS or a natural infection, immune hyporesponsiveness could be induced as a result of a decreased memory B cell population.

The development of immune hyporesponsiveness may also be the result of immune regulation via the establishment of pneumococcal-specific tolerogenic immune responses. Increased expression of the immunosuppressive cytokine IL-10 [264, 283] and suppressor T cell activity may suppress the response to PPS [284]. Recent evidence also suggests a role for CD4+ T-lymphocytes in the immune response to pneumococcal antigens [285]. Studies have demonstrated the importance of co-stimulatory signals (CD40-CD40L) for a robust immune response to pneumococcal antigens and that CD4+ T-lymphocytes can protect mice against pneumococcal colonisation independent of specific antibody. These findings strongly suggest a role for cellular immunity in protection against pneumococcal infection [98, 99, 286-288]. Furthermore it is possible that regulatory T-lymphocytes (Treg) may suppress antibody production and other immune responses in the context of chronic antigen exposure. Hyporesponsiveness induced by Treg has been described during bacterial, viral and parasitic infections with up-regulation of CD4+CD25+ Treg and IL-10 and TGF-β secretion [284, 289]. Limited data are available on the role of Treg in the attenuation of the immune response to pneumococcal antigens. However a high level of exposure to pneumococci, particularly in early life, could induce Treg activity that suppresses serotype-specific IgG, thereby increasing IPD risk following 23vPPS immunization.

The clinical relevance of these immunological findings is not known. There is one case report documenting immunological “paralysis” for four years to the causative pneumococcal

37 serotype in a 9 month old infant who had pneumococcal meningitis, despite demonstrating normal immune responses to other protein and polysaccharide antigens [290].

1.6.2.3.4. Efficacy Several studies in the 1980s assessed the efficacy of the 8- or 14- valent PPS vaccine in preventing OM in children under 6 years of age [207, 267, 291-297]. Most studies evaluating the impact of PPS immunisation in the absence of additional PCV in infants or children have not shown any impact on pneumococcal disease or carriage [267, 292, 298]. In Australia, a randomised, controlled trial showed no difference (RR 1.01; 95% CI, 0.81-1.25) in the risk of acute OM in children <2 years of age and a possible trend towards reduction (RR 0.83; 95% CI, 0.69-1.01) in those children >2 years of age [292]. In contrast, a study in Papua New Guinea, where more than 7,000 children aged 6 months to 5 years of age were given either the 14- or 23vPPS in one or 2 doses according to age, there was a non-significant 19% reduction in all-cause mortality, and a 50% reduction in pneumonia mortality (95% CI, 1-75%) [299]. Natural exposure in a population with a high incidence of pneumococcal infections, resulting in regular antigenic stimulation may explain this finding [189]. This study has not been widely accepted, largely due to lack of supportive serological data and questions regarding the study design [300]. The study has not been replicated anywhere, and the vaccine is not used currently for infants anywhere in the developing world, not even Papua New Guinea despite the vaccine showing a significant reduction in mortality (hazard ratio 0.42; 95%CI, 0.19-0.93) for children aged between 12 and 24 months [301]. A Finnish study using the 14-valent PPS in infants aged 3 months to 6 years showed significant efficacy of 52% against VT recurrent OM for children <2 years of age if serogroup 6 was excluded [146]. A meta-analysis found no evidence for an overall effect in risk on OM in children <2 years of age who received PPS, but in the pooled analysis of children >24 months of age there was evidence of a vaccine effect (RR 0.78; 95% CI, 0.63- 0.97) [207].

1.6.2.4 Combined PCV/23vPPS schedules Pneumococcal conjugate vaccines with broader serotype coverage were, until recently, not available. As such some health authorities had decided on or were considering a combination of an infant PCV primary series with a booster of the 23vPPS in the second year of life to address the limited serotype coverage offered by PCV. The theoretical advantage of this approach is to prime with PCV then boost with 23vPPS which is cheaper and may offer some protection against disease due to the serotypes not included in the PCV. These non-PCV serotypes have become more important in IPD now that PCV use is widespread, as the impact of serotype replacement

38 leads to a larger proportion of circulating serotypes coming from those serotypes not included in the PCV [19]. Until recently, Australian Indigenous infants routinely receive 3 PCV doses in infancy followed by a booster of the 23vPPS at 18 months of age. This schedule has been found to be immunogenic for most serotypes [302]. For these reasons, and because of its lower cost, the 23vPPS may be the most suitable booster for use in developing countries.

There have been several studies involving children in a number of countries using different pneumococcal conjugate formulations and schedules, comparing the immunogenicity of a 23vPPS or PCV booster following a pneumococcal conjugate vaccine primary series. The majority of studies have shown that serotype-specific antibody concentrations are generally higher following 23vPPS than PCV booster [157, 172, 185-188]. The higher response may be due to the higher dose of pneumococcal polysaccharide in the 23vPPS, compared to PCV, enhancing the stimulation of memory B cells or by stimulating a greater number of B cells overall [189]. A randomised controlled trial of infants in Papua New Guinea primed with PCV given at 0-1-2 months, 1-2-3 months, or no PCV followed by 23vPPS at 9 months of age found a an excellent booster response for the PCV VT, and a 2.8 to 12.4 fold rise in the 3 NVT tested (serotypes 2, 5, and 7F) [303].

A study of vaccine efficacy against acute OM found that a PCV/23vPPS compared to a PCV/PCV schedule had similar results despite higher antibodies generated post PCV/23vPPS [188]. Previous studies have found that the quality of antibody, measured by avidity or OPA, can differ in those that have received 23vPPS or PCV as a booster, however results have been conflicting and therefore inconclusive [154, 157, 186, 304, 305]. Finnish studies have shown the concentration of antibodies required for 50% killing was higher [304] and that the avidity of such antibodies was lower after PCV/23vPPS compared with PCV/PCV [147, 154, 186]. In contrast, another study in Finland using the 11-valent pneumococcal conjugate vaccine showed that OPA was better in the group that received a 23vPPS booster at 12-15 months than those that had the conjugate booster [305]. A study in Israeli children who received a single dose of the 7-valent pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine followed by either a conjugate or 23vPPS booster, achieved similar opsonic antibody titers in each group for the one serotype tested (6B) [186]. The avidity of antibodies in a limited number of studies on a limited number of serotypes increased following a conjugate booster but not a PPS booster [147, 154].

1.6.2.5 Impact of pneumococcal vaccination on nasopharyngeal carriage Acquisition and carriage of pneumococci is associated with the occurrence of acute OM [11],

39 bacteraemia [9], and pneumonia [10]. In developing countries, colonisation rates can be >60% by 2 months of age [15, 16]. An intervention that reduces or delays carriage could result in a decrease in IPD and possibly a decrease in mucosal disease. PPS vaccines do not significantly impact on NP carriage [306]. In contrast, clinical trials using the 5, 7, or 9-valent pneumococcal conjugate vaccines have shown a reduction in VT carriage compared with unvaccinated infants [306-308] or toddlers [309-311]. However the overall rate of pneumococcal NP carriage has remained essentially unchanged due to serotype replacement from NVT [306, 307, 311-313]. Since the routine introduction of PCV into infant national immunisation schedules, there have been a number of carriage surveys documenting the effect of PCV on pneumococcal NP carriage. Similar to the clinical trials, all studies have found that there has been a reduction in VT carriage [314-321]. For NVT, colonisation has increased following vaccination, with serogroups 11 and 15 being commonly reported in many studies [314-316, 318, 320, 321], and more recently the newly identified serotype 6C has been reported [322]. Moreover, the widespread use of infant PCV in the US has resulted in significant protection of unimmunised individuals [17, 323] presumably mediated by reduced NP carriage interrupting the transmission of pneumococci [17, 18].

In the UK and some Scandinavian countries, a 2 PCV dose schedule in infancy followed by a PCV booster towards the end of the first year of life is routinely given. Little is known about the effect of reduced dose PCV schedules in terms of their impact on carriage and subsequent effect on herd immunity. There is one published randomised controlled trial reporting the effect of reduced dose pneumococcal conjugate vaccine schedules on NP carriage [324]. This study from the Netherlands compared carriage rates following 2 PCV doses at 2 and 4 months of age with a “2+1” schedule at 2, 4, and 11 months of age, and an unvaccinated control group. Both vaccinated groups had significant reductions in VT carriage in the second year of life compared with controls [324]. The booster dose resulted in an earlier further reduction in VT carriage at 18 months compared with no booster dose (24% vs 16%). However by 2 years of age both vaccinated groups had similar VT carriage rates (15% each) [324]. Similarly, in a case-control study Gambian infants, vaccinated with either 3 or 2 doses of a 5-valent pneumococcal conjugate vaccine in infancy followed by 23vPPS at 18 months of age showed a significant reduction in VT carriage compared with unvaccinated matched controls at 2 years of age [306]. An observational study of Portuguese children aged between 12 and 24 months of age in day care centres showed a single PCV dose led to serotype replacement between VT and NVT isolates, both in single and multiple serotype

40 carriers, in contrast to the unvaccinated control group where no replacement phenomenon was detected [325]. This was evident at both the population and individual level [325]. In addition, a single dose of PCV decreases VT colonisation as it prevents de novo acquisition and increases NVT colonisation by enhancing NVT unmasking [325].

PCV has had larger than anticipated herd immunity effects on IPD in the unvaccinated elderly and other age groups [19, 52]. However replacement disease, particularly due to serotype 19A, has developed due to capsular switching and clonal expansion [220]. Despite being a significant reduction overall in pneumococcal meningitis in the US, a recent study has shown that the rate of non-PCV serotype meningitis has significantly increased by 60.5% and the proportion of penicillin non-susceptible pneumococcal meningitis isolates increased significantly to pre-vaccine levels [228]. However, serotype replacement may be more of a concern for the control of OM and pneumonia although there is no evidence of this so far. Finnish infants given PCV in a clinical trial showed an increase in NVT OM incidence by 33% although the overall effect was a reduction in disease [161].

In contrast to PCV, studies using PPS have shown no effect on pneumococcal carriage [326- 330]. One study in the Gambia where 5-valent pneumococcal conjugate vaccinees or Hib vaccinated infants were given 23vPPS at 18 months of age found that the matched controls had signifcantly higher rates of VT carriage than the pneumococcal vaccinated groups [306]. NVT were found more frequently in the pneumococcal vaccinated groups than the control group indicating 23vPPS had no beneficial effect on NVT carriage [306]. In the Netherlands PCV followed by a 23vPPS booster given to children aged between one and 7 years with recurrent acute OM found no beneficial effect from the booster vaccine [273]. A Finnish study of vaccine efficacy against acute OM in infants found that infants given a 3 dose PCV followed by a 23vPPS booster had no further beneficial effects to a PCV booster following a 3 PCV dose primary series a despite higher antibodies generated post the PCV/23vPPS schedule [188].

It is important, therefore, to assess NP carriage and serotype distribution with the introduction of PCV, as the impact of serotype replacement is likely to be dependent on the pattern of serotypes circulating in the community. Moreover, as NP carriage of VT may be an indicator of vaccine efficacy, the combination of this effect with immunogenicity data may help to identify which immunisation schedules are likely to be more effective.

1.7 Rationale

1.7.1 Access to Vaccines

Approximately 73% and 69% of infants in sub-Saharan Africa and south Asia, respectively, have received 3 doses of diphtheria-tetanus-pertussis vaccine [331]. Deaths from ARI occur mainly in communities with poor health care access [332]. Vaccine provision to children in these countries depends on health care access and financial resources. Children in remote regions often receive incomplete immunisation courses, and doses are usually given later than recommended. At the commencement of this study the price of PCV was approximately US$50 per dose, and as such the recommended schedule of 4 doses, at the time, was unlikely to reach those children at greatest risk. At the commencement of this study there was only one licensed vaccine (Prevenar , formerly Wyeth Vaccines now Pfizer Inc.) in the US and Australia. In 2010 the 10- and 13-valent pneumococcal conjugate vaccines have now reached licensure. Therefore, the present PCV was the only one of its type available at the time this project commenced and its price was likely to remain unaffordable for developing countries for quite some time. Recently novel financing mechanisms for low income countries have been developed called the Advanced Market Commitment and the International Finance Facility for Immunisation. However for low- middle income countries (including Fiji), who will not benefit from these financing mechanisms, it is important to investigate new strategies to deliver this vaccine that are safe and effective, but also affordable and that provide flexibility in terms of the number and timing of doses.

1.7.2 Evaluation of Alternative Pneumococcal Vaccine Schedules is a Research Priority

In 2001, a meeting was held at the National Institutes of Health (NIH), Bethesda, Maryland, to determine research priorities to facilitate the use of pneumococcal vaccines in developing countries. The discussions spanned epidemiological, clinical, technical, policy and logistical impediments to the introduction of these vaccines to the world’s poorest countries. The recommendations from the meeting included a list of priority research activities, including the need to evaluate alternative regimens of PCV, to provide regimens that are more affordable, provide more flexibility for countries where repeated and timely immunisation visits are atypical, and provide protection in early infancy. The following strategies, amongst others, were identified for evaluation: regimens that include only one or 2 doses of PCV

(rather than 3 or 4), those that combine PCV and the 23vPPS, and those that include a neonatal dose. The vaccine trial in this thesis addresses the first 2 strategies.

1.7.3 Immunological Basis to the Vaccine Trial Design

Because PCV induces priming, it is possible that it will protect even in the absence of a detectable antibody response. This raises the possibility that fewer doses of PCV than are presently recommended, perhaps even a single dose, may be sufficient to protect from serious IPD. Antibody levels achieved after 3 doses are usually sustained for only a few months, and then decline close to pre-immunisation levels. However a dose of pneumococcal vaccine, either 23vPPS or PCV, administered during the second year of life to children primed with 2 or 3 doses of any of the PCV, generally induces a rapid, 10-fold increase in antibody concentrations to most serotypes, consistent with priming [173, 175, 176, 178-184, 333].

23vPPS has been shown to elicit a booster response following 2 or 3 doses of PCV for the shared 7 PCV serotypes. The additional 16 non-PCV serotypes may offer broader serotype protection. However the immunological safety of 23vPPS booster in young children needs to be evaluated. Vaccine schedules that combine one to 3 doses of PCV with a dose of 23vPPS may lead to later hyporesponsiveness in vaccinated children similar to that found with the meningococcal C polysaccharide vaccine [271, 282]. It may be that this hyporesponsiveness is found in susceptible individuals only, or it may be that it is found with certain serotypes only, or both. Therefore, the Fiji study has been designed to address this question, as well as addressing the immunogenicity and impact on carriage of regimens combining PCV and 23vPPS.

For the vaccine study we aimed to find a vaccination strategy for resource poor countries in terms of serotype coverage, flexibility, and affordability. We undertook a Phase II vaccine trial to document the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination regimens combining one, 2, or 3 doses of PCV in infancy. In order to broaden the serotype coverage, the additional benefit of a booster of 23vPPS at 12 months of age was also assessed. To address the theoretical concerns of hyporesponsiveness to 23vPPS following re-challenge, the immunological responses at 17 months of age to a small challenge dose of 20% of 23vPPS (mPPS) in infants who had or had not received the 23vPPS at 12 months of age was undertaken.

1.7.4 Knowledge to be Gained

Pneumococcal infections are the most common cause of morbidity and mortality in children globally. This study has profound implications for the early introduction of pneumococcal vaccines, in particular, to the world’s poorest countries. As the current schedule recommends 3 doses in infancy, it is unlikely to be affordable for most developing countries. Evidence from the US suggests that fewer doses are required for protection against IPD, and even modest immunity may be sufficient to prevent children from dying from pneumococcal disease. In addition, many infants in developing countries have episodic access to health care and immunisations. It is important, therefore, to investigate affordable, safe, flexible and effective ways to deliver this vaccine. The need to evaluate alternative regimens of PCV was identified as an important research priority by a recent WHO/ GAVI joint meeting to address impediments to the introduction of these vaccines in developing countries. This study has been deliberately formulated with that need in mind. For the development of the study, we imagined a possible future set of recommendations for the use of pneumococcal vaccines in remote areas with minimal access to health care. Under such circumstances, children presenting during the first 6 months of life could receive one to 3 doses of conjugate, while those presenting later with any experience of conjugate vaccination could receive a dose of 23vPPS.

1.7.5 Evaluation of Alternative Pneumococcal Schedules in Fiji

Fiji is a developing country with a strong immunisation system and an established track record in the introduction of new vaccines. It was likely that Fiji would have been one of the first developing countries to introduce PCV, provided the disease burden is established and an appropriate regimen defined. Hepatitis B vaccine was introduced in Fiji over 10 years ago. Fiji was the first country in the Pacific to have introduced Hib vaccine. Negotiations with Wyeth-Lederle in 1995 resulted in 150,000 free doses of Hib vaccine being donated to the government. The initial Hib campaign commenced in mid- 1994 and ceased in mid-1995. A cluster survey in 1996 found that 79% of children less than 12 months old had completed the primary series. Routine immunisation began again in 1997. In early 2009, a coverage survey found the third dose coverage of DTP-Hib/HepB to be >98%.

1.8 Objectives

My thesis comprises a series of studies documenting the pneumococcal disease burden and a Phase II pneumococcal vaccine trial in the resource poor setting, Fiji. The overall objective

44 was to gather sufficient evidence for the Fiji MoH to decide whether to introduce the pneumococcal vaccination into its national schedule and define an appropriate and affordable vaccination strategy.

The objectives were to: 1) Document the prevalence of NP carriage of pneumococci, risk factors for carriage, antimicrobial susceptibility patterns of carried pneumococci, and the serotypes of carried pneumococci in healthy young children in Fiji; 2) Document the burden of IPD and serotype distribution in all ages in Fiji.

The primary objective of the vaccine trial was to:

1) Demonstrate non-inferiority in GMC for 11 or more of the 23 23vPPS serotypes, one month post-mPPS, in the groups that have or have not received the 12 month 23vPPS.

The secondary objectives of the vaccine trial were to:

1) Demonstrate non-inferiority one month post-mPPS, between those groups receiving 23vPPS at 12 months and those who do not with respect to the proportion of children:

a) in each group with antibody levels ≥0.35 g/mL to each of the 23 serotypes included in the 23vPPS, by ELISA. b) with OI ≥8 to each of the 11 serotypes for which OI data were available.

2) Compare the proportion of infants at 18 weeks of age with antibody levels by ELISA, to 7 PCV serotypes ≥0.35 and 1 g/mL following a primary series of 3 doses of PCV in infancy or an alternative schedule of 0, 1 or 2 doses.

3) Compare the GMC of ELISA antibody at 18 weeks of age following a primary series of 3 doses of PCV in infancy or an alternative schedule of 0, 1 or 2 doses.

4) Compare the GMC for 23 23vPPS serotypes, at 12½ months of age following a booster of 23vPPS at 12 months of age, following a 0, 1, 2, or 3 dose primary series of PCV in infancy.

5) Compare the proportions of children who prior to mPPS carry VT and NVT pneumococci in the nasopharynx among children who have received one, 2, or 3 doses of PCV in infancy with or without a booster dose of 23vPPS at 12 months of age.

Tertiary objectives were to compare:

1) Serotype-specific GMC for 3, 2, 1 or 0 dose PCV groups with or without the 12 month 23vPPS one month following mPPS.

2) The median avid antibody levels in those who did or did not receive the 12 month 23vPPS booster for the 23 23vPPS serotypes and had 3, 2, 1 or 0 dose PCV groups for the 7 PCV serotypes.

3) The proportions of children carrying a VT and a NVT for the 7 PCV serotypes for NP swabs taken at 6, 9, 12, and 17 months of age for the different groups.

1.8.1 Change to the Original Objectives

The FiPP vaccine trial commenced recruitment in October 2004 with support from NIAID and the National Health and Medical Research Council (NHMRC). However, recruitment was suspended by NIAID in February 2005 after 228 infants had been recruited. Due to an administrative oversight within NIAID the trial had not passed through the required internal review within NIH, so recruitment was suspended pending that review. According to the original protocol, infants were randomised to receive one, 2 or 3 doses of PCV in early infancy and a booster dose of 23vPPS at 6 or 9 months. Two control groups were also recruited to receive either 23vPPS at 9 months or no pneumococcal vaccine prior to 15 months of age. Outcomes to be measured were antibody levels (ELISA, OPA, and avidity), pneumococcal NP carriage and response to mPPS at 15 months of age. Following the review by NIAID this design was rejected on the grounds that they believe there was insufficient safety data to support the use of 23vPPS vaccine in infants under 12 months of age. Specifically fears had been raised that administration of one or more of the serotypes included in the 23vPPS vaccine may lead to diminished response to some pneumococcal antigens on subsequent re-exposure, potentially leading to an increased risk of disease. This had not been specifically addressed in earlier studies.

This concern, raised by the Technical Review Group convened by NIAID, was based on recent studies of meningococcal C polysaccharide vaccine, in both children and adults, which indicate that this widely used vaccine leads to lower responses to the same vaccine on revaccination [271]. The NIAID advisors expressed concerns that this phenomenon may also occur with one or several of the serotypes included in the 23vPPS vaccine, either when this vaccine is given alone or following earlier PCV. The protective efficacy of this vaccine against pneumonia mortality when delivered to infants in Papua New Guinea provides reassurance

46 in this regard [299, 301]. On the other hand, in a recent study from the Netherlands in which older children with recurrent OM were vaccinated with a combined PCV and 23vPPS regimen, while the proportion of vaccinated children suffering subsequent OM was identical to controls, the total number of episodes suffered by vaccinees appeared to be greater [273].

Following the NIAID review, we were instructed not to give 23vPPS to infants at either 6 or 9 months of age and instead to redesign the study to evaluate the immunological safety of 23vPPS at 12 months. We were also advised not to proceed with the original sample size of 1,060 but redesign a smaller study. As a result of these concerns, the design had changed to evaluate the immunological safety of 23vPPS at the same time as evaluating alternative pneumococcal vaccination regimens. Therefore the redesigned FiPP had the primary objective of testing the following hypothesis:

1) To demonstrate non-inferiority one month post-mPPS, between those groups receiving 23vPPS at 12 months and those who do not with respect to the proportion of children with OI ≥8 to each of the 11 serotypes for which OI data are available.

Furthermore, during the course of the trial and following discussion with the Data Safety and Monitoring Board (DSMB) in November 2007, the definition of “hyporeponsiveness” changed from what was originally defined in the protocol. The original definition of “hyporesponsiveness” has been changed to “non-responsiveness”. As such the primary objective was as stated in section Objectives 1.8.

1.8.2 Potential Risks

There was a small risk of local reactions following 23vPPS administration to children who have been previously immunised with PCV. In addition, there was a risk that some children may have reduced responses to some pneumococcal antigens following receipt of 23vPPS. As was discussed previously, this was based on experience with meningococcal C vaccine, which represents the only model of hyporesponsiveness following polysaccharide administration in humans [271]. There is no evidence that this phenomenon has led to any adverse effects in the millions of individuals who receive meningococcal C polysaccharide each year [272]. While it was possible that this study may uncover hyporesponsiveness to one or more pneumococcal polysaccharide serotypes, it was unlikely that this would be of clinical significance. None of the studies that have been conducted up to now have documented any adverse effects in children as young as 3 months of age receiving 23vPPS

[146, 292, 299, 334, 335]. The combination of PCV and 23vPPS being employed in this study was similar to that used as a regimen for high risk Indigenous infants in Australia until the 10-valant vaccine was introduced in the third quarter of 2009 with a 4 dose schedule.

Pain was likely to be felt by the infants with each venipuncture. There was a small risk of bruising and redness at the venipuncture site. Rarely infections may occur as a result of venipuncture.

1.8.3 Known Potential Benefits

The potential benefits of vaccinating individual infants for protection against pneumococcal disease outweighs the potential risks. There is extensive experience with PCV in trials in developed and developing countries, and in all cases it has been shown to be safe and efficacious [52, 336]. All children in this trial will receive pneumococcal immunisation that is likely to be effective. Those groups that receive 0 or 1 dose of PCV in infancy will receive a dose of PCV at the end of the trial, at 2 years of age, when a single dose is known to be effective.

1.9 Hypothesis

The original primary hypothesis for analysis was that 23vPPS at 12 months will not lead to a greater proportion of non-responsiveness to mPPS at 18 months for any of the 11 serotypes for which functional analyses are available. This was to be tested by determining whether, for each of the 11 serotypes, if the proportion of children showing non-responsiveness was 15% greater (in absolute terms) in the 23vPPS group than in the group that had not received the 12 month 23vPPS. Differences in proportions and their confidence intervals were to be calculated using the standard normal approximation, in view of the large sample size. The test was to be the standard test of non-inferiority of proportions, single sided (as recommended by ICH guidelines for non-inferiority trials), with a significance level of 5%. To determine non-inferiority we were to calculate single-sided 90% confidence intervals for the difference. The proportion of child who had or had not received the 12 month 23vPPS with serotype-specific OI ≥8 will be declared non-significant if the lower bound of these confidence intervals excludes -10%. It was anticipated that within a child, serotype responses would not be independent of each other. In other words, it was likely that a child who was non-responsive to one serotype would be non-responsive to a number of serotypes. It was assumed that for any given serotype, 30% of children were expected to be

48 non-responsive, and 30% of children will be non-responsive to at least half of the serotypes (4 or more).

2 MATERIALS AND METHODS

2.1 Setting

The Republic of Fiji islands lies within the South Pacific Ocean. It comprises two large islands, Viti Levu and Vanua Levu and more than 300 smaller islands (Figure 2). Fiji’s population of 827,900 is primarily comprised of Indigenous Fijians (57%), who are predominantly Melanesian, and Indo-Fijians (38%) who are of Indian ethnicity (2007 Census). Over 75% of the population lives on Viti Levu which has two medical divisions, the Western and Central divisions. The population in the Central Medical division is 371,850. The total population of Suva, the capital, is 75,225 and the population of Nausori, its neighbouring satellite, town is 24,630. Together they contain approximately one third of the Central Division’s population and from here the participants of this study were recruited.

The literacy rate is approximately 90% with English as the official language, but Fijian and English are taught in schools. Hindi is the third language. Fiji is classified as a low middle income country. The poverty rate is 35% and the GDP per head of population in 2006 was approximately FJ $5474. (Source: Fiji Islands Bureau of Statistics, 2008).

The infant mortality rate is 18.4 per 1,000 live births (Fiji Ministry of Health Annual Report, 2007). The <5 child mortality rate is 22.4 per 1,000 live births. In 2007 there were 19,298 live births (Fiji Ministry of Health Annual Report, 2007) of which approximately one third were in the Suva and Nausori area.

Figure 2: Map of the Republic of the Fiji islands showing the 4 medical divisions

Source: Fiji Islands Bureau of Statistics, 2008

2.1.1 Health Infrastructure

All health care infrastructure in Fiji is co-ordinated by the Ministry of Health (MoH). Health care is largely provided free of charge, although for some care (eg, hospitalisation) a small co-payment is sometimes collected from patients. The Suva urban region has 6 health centres, and one private hospital, which does not provide inpatient paediatric services.

2.1.2 Drug Licensing Procedure

The MoH’s Fiji National Drug and Therapeutics Committee approves the use of all drugs and vaccines for use in Fiji. The study vaccines PCV, 23vPPS, and DTwP/Hib-HepB were approved for use by this committee prior to the commencement of the study.

2.1.3 Study Sites

2.1.3.1 Colonial War Memorial Hospital (CWMH) CWMH is the principal tertiary referral hospital for Fiji, and also provides primary and secondary level care for the residents of Suva and surrounds. CWMH is the only hospital within the proposed study area with a dedicated children’s hospital, and is therefore the main hospital to which any study children would be admitted. The children’s hospital is separate but adjacent to the main hospital and treats children less than 12 years old. There are dedicated neonatal and paediatric intensive care units, both with mechanical ventilatory capacity.

Hand-written patient records are filed by hospital number. Discharge data are entered into a computerised database (PATIS) including: hospital number, name, address, date of birth (DOB), and discharge diagnosis. Patients can be recalled by name or hospital number. PATIS combines information from all divisional, and many sub-divisional, hospitals.

Most children with acute illness in Suva attend the paediatric outpatient department (OPD) and approximately 100 children are assessed daily. CWMH provides over 90% of all antenatal care for Suva, and there are approximately 6000 births at CWMH per year.

2.1.3.2 Nausori Health Centre Nausori Health Centre is located approximately 30-minutes by car from CWMH and serves a semi-urban population of approximately 68,000. Nausori Maternity Hospital provides obstetric care for up to 1000 births per year. All Maternal and Child Health (MCH) care including routine infant immunisations, growth surveillance, well baby checks, and developmental assessments are performed in the MCH clinic. All information is recorded on the MoH parent held record. For this study, a room was made available specifically for the project and contained a dedicated ice-lined refrigerator, lockable cupboards, and clinic furniture.

2.1.3.3 Valelevu Health Centre Valelevu Health Centre is located within 15-minutes drive from CWMH and serves a population of approximately 75,000 to 80,000. It provides health services to a poor, rapidly growing, densely populated urban area. Similar to Nausori Health Centre, all MCH visits are provided for this catchment population. A dedicated area was reserved for the study staff and participants.

2.1.3.4 Makoi Health Centre Makoi Health Centre is a small health centre located within 5- minutes drive from Valelevu Health Centre. The health centre provides similar services as the Valelevu Health Centre to the nearby population.

2.1.4 Suva Study Team

The study team was headed by the onsite principal investigator (PI) with the field work monitoring conducted by an on-site study co-ordinator. The study doctor oversaw all clinical evaluations. The study co-ordinator and doctor had daily contact with the PI. The study nurses undertook recruitment, informed consent, study visits, and collected study samples. The research assistants transported specimens, monitored the storage of the specimens, and performed data entry. The laboratory technician processed the samples, and oversaw the storage and overseas shipment of study samples.

2.1.5 Ethical Procedures

The Fiji National Research Council (FNRC) is convened by the MoH, and reviews and approves any large-scale health research projects in Fiji, before any project can proceed. The study was submitted to this committee and was approved. Upon receipt of approval from the FNRC formal ethical review was undertaken by the Fiji School of Medicine’s Fiji National Research Ethics Review Committee, which approved the project protocol, consent materials, and protocol amendments before the project proceeded (reference number #2002-001). In addition, the project was separately reviewed and approved by the University of Melbourne Human Research Ethics Committee (reference number #010570). The ethics procedures of both institutions follow the accepted international standards and the principles of the World Medical Association’s Declaration of Helsinki. The PI ensured that this study was conducted in full conformity with the International Conference on Harmonisation Good Clinical Practice (ICH-GCP) regulations and guidelines.

2.1.5.1 Payment to parents/legal guardians The only payments to parents were as reimbursement for bus fares at each study visit. This was approved by both ethics committees.

2.1.5.2 Subject confidentiality Subject confidentiality was strictly held in trust by the investigators, the study staff, and the sponsor(s) and their agents. This confidentiality was extended to cover testing of biological samples in addition to the clinical information relating to participants.

All infants were allocated a study file, which had their unique study number, study workbook, the signed informed consent form, the signed plain language statement, eligibility form, case reporting forms (CRF), reactogenicity forms, and serious adverse event (SAE) and withdrawal form, if appropriate. This file was stored in a locked cupboard in the clinics or in the study office. The study protocol, documentation, data and all other information generated were held in strict confidence. No information concerning the study or the data were released to any unauthorized third party.

The study monitor and other authorised representatives of the sponsor (such as PPD, the company commissioned to provide external independent monitoring) were permitted to inspect all documents and records required to be maintained by the PI, including the medical records (office, clinic and/or hospital) for the participants in this study. The clinical study site permitted access to such records.

2.1.5.3 Study discontinuation Recommendations for study discontinuation were to be discussed between the DSMB, the investigators, and all the funding and participating parties. The final decision to discontinue the study was to be made by the Department of Microbiology and Infectious Diseases (DMID), National Institutes of Health (NIH), USA. Should a decision for discontinuation be made, the investigators would be asked to propose a safety follow-up strategy and/or rescue vaccination plan.

2.2 Study Design

The study was a single blind, open-label, randomised controlled trial of 550 healthy infants in Suva, Fiji.

2.2.1 Selection of Study Participants

Parents/legal guardians of young infants were shown a video in English explaining the study, whilst waiting in well baby clinics at Nausori, Valelevu, and Makoi Health Centres. Antenatal mothers in the last trimester of pregnancy were approached as they waited for their outpatient appointments at CWMH. In addition, postnatal mothers, who were well and had delivered healthy infants, were contacted in the wards at CWMH and Nausori Health Centres. The initial contact was to introduce the trial, discuss the rationale for the project, and answer any questions the parents/legal guardians may have had (see Appendix 1, SOP 2: Process of informed consent). Brochures (written in English, Fijian, and Hindi) were given to the women to take home (see Appendix 2: Informed consent materials: brochure). This enabled parents/legal guardians to discuss with their family members the

possibility of participating in the trial before being formally asked for consent. All subjects screened were entered into a Screening Log. At the second point of contact, the study nurse discussed the trial again and the parents/legal guardians were asked whether they would like their infant to participate in the trial. For women at CWMH a follow-up phone call one to 2 weeks following delivery was made by the study nurse to see whether they may be interested in their infant participating in the trial.

2.2.2 Informed Consent

Written informed consent was freely obtained from the parent/legal guardian in their first language (i.e. Fijian, Hindi or English), prior to enrolment of their infant into the study (see Appendix 1, SOP 2, Process of informed consent). In the case of underage mothers, we ensured that the consent process also involved their parent/legal guardian. The consent was witnessed by a study nurse confirming that she/he had explained the trial fully, the parent understood the rationale for the study and the study process, and that they were free to withdraw their infant from the study at any time without interference to their normal health care.

2.2.3 Eligibility Criteria

If informed consent was granted, the infant was examined by a study nurse (see Appendix 1, SOP 4: General history and medical examination) who would ensure the infant fitted the inclusion criteria and that no exclusion criteria were present (see Appendix 1, SOP 3: Checking eligibility criteria). If the study nurse had any concerns or queries regarding the eligibility criteria they contacted the study doctor for further clarification or assistance.

2.2.3.1 Subject inclusion criteria Infants were eligible for the study if they meet the following criteria: Healthy infant aged between 6 and 8 weeks;

No significant maternal or perinatal history;

Written and signed parental/legal guardian consent;

Lived within 30 minutes of the health clinic;

Family anticipated living in the study area for 2 years.

2.2.3.2 Subject exclusion criteria Infants were excluded if they had any of the following:

Known allergy to any component of the vaccine;

Allergic reaction or anaphylactoid reaction to previous vaccines;

Known immunodeficiency disorder;

HIV positive mother (most women were tested for HIV antenatally; HIV status was based on clinic records or self report);

Known thrombocytopenia or coagulation disorder;

On immunosuppressive medication;

Received any blood product since birth;

Severe congenital anomaly;

Chronic or progressive disease;

Seizure disorder;

History of invasive pneumococcal, meningococcal, or Haemophilus influenzae diseases;

Moderate or severe acute infection (temporary exclusion) (see Appendix 1, SOP 3: Checking eligibility criteria). Minor illnesses such as an uncomplicated upper respiratory tract infection, localised skin infections, or mild diarrhoea were not exclusion criteria.

If the infant met the eligibility criteria, the infant was formally enrolled in the study (see Appendix 1, SOP 5: Enrollment and randomisation procedure) and documented in the Enrollment Log. Infants with a moderate or severe illness were asked to return in one week and were reassessed at that time.

2.2.4 Enrollment, Randomisation and Masking Procedures

As pneumococcal disease burden is considerably higher in the indigenous Fijian population compared with the Indo-Fijian population, infants were stratified by ethnicity then randomised using a computer-generated list of random numbers in blocks of variable size to one of 8 groups to receive 0, 1, 2, or 3 doses of PCV, with half of each group receiving a booster of the 23vPPS at 12 months of age. The randomisation list was prepared by the Clinical Epidemiology and Biostatistics Unit, Royal Children’s Hospital, Melbourne (see Appendix 1, SOP 5: Enrollment and Randomisation procedure). Group allocation was concealed in opaque envelopes which study nurses removed sequentially from a box. The control group (Group H) received no infant doses of PCV nor the 23vPPS at 12 months. At 17 months of age, all infants received a small re-challenge dose (mPPS) equivalent to 20% of the full

dose. At 2 years of age, any child who had received a single or no PCV doses (Groups E, F, G and H) received a single dose of PCV to ensure all infants were immunised by the completion of the study.

Vaccine administrators and the assessors for reactogenicity were not blinded to the study group allocation of each infant. Most of the reactogenicity variables that were measured were objective (respiratory rate, temperature). Those measures that were not objective, for example “irritability,” were graded by the parent rather than the assessor. Laboratory staff members were blinded to group allocation.

Each infant was assigned a unique study identification number that was attached to all clinic records and the parent held child health record that was brought to all health clinic attendances. This sticker had the contact number of study personel and their unique identification number. Non-study staff in the clinics and CWMH were requested to contact the study personnel if the infant presented to them with any illness.

2.2.5 Study Vaccines, Vaccine Storage and Administration

2.2.5.1 Formulation and labeling PCV was manufactured by Wyeth Vaccines (now Pfizer Inc.) and contains saccharides of the capsular antigen of S. pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F,

23F individually conjugated to diphtheria CRM197 protein. CRM197 is a nontoxic variant of diphtheria toxin isolated from cultures of Corynebacterium diphtheriae strain C7 ( 197) and/ or C. diphtheriae strain C7 ( 197) pPx 350. Each 0.5 mL contains 2 micrograms of polysaccharide for serotypes 4, 9V, 14, 19F, and 23F; 2 micrograms of oligosaccharide 18C, and 4 micrograms of type 6B.

The 23vPPS (PneumovaxTM, Merck & Co., Inc.) consists of a purified mixture of capsular polysaccharide from 23 pneumococcal serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F). 25 µg of each serotype was contained in this standard 0.5-mL dose. The 23vPPS has been in use since 1983 and has a good safety profile.

The GlaxoSmithKline pentavalent vaccine (HiberixTM/TritanrixTM-HepBTM) was used as the routine co- administered vaccines given at 6, 10, and 14 weeks of age. It contains a lyophilized Hib vaccine comprising of 10 µg of purified capsular polysaccharide covalently bound to approximately 30 µg tetanus toxoid. A 0.5mL dose of DTwP-HepB vaccine acts as the diluent for the Hib vaccine and contains not less than 30 IU of adsorbed D toxoid, not less than 60 IU of adsorbed T toxoid, not less than 4 IU of wP, and 10 µg of recombinant Hepatitis B surface antigen protein.

All vaccines were shipped according to the manufacturer’s specifications and arrived with a Certificate of Analysis. Cold chain monitoring in transit was documented and adequate temperatures were maintained throughout their journey.

As this was an open label study and the vaccines were supplied from Australia, labelling of the PCV and 23vPPS were as per the requirements of the Therapeutic Goods Administration, Canberra, Australia.

2.2.5.2 Product storage and stability The vaccines were stored in dedicated ice-line refrigerators in the study office and distributed to the dedicated clinic ice-lined refrigerators in pre- chilled UNICEF cool boxes each Monday morning. The number of vials kept at the central site and distributed to the clinics was documented. Vaccines were returned to the central store from the clinics at the end of each week.

The vaccines were stored at +2 to +8 C and had appropriate cold chain monitoring (see Appendix 1, SOP 7: Cold chain maintenance and vaccine logistics). Twenty-four hour temperature monitoring occurred. Loggers were kept with the vaccines at all times and the temperature records were electronically downloaded monthly and filed. Any breach in the cold chain was reported to the PI and the vaccines were not used until a decision was made on the outcome of the breach in cold chain.

2.2.5.3 Vaccine administration The PCV and 23vPPS were not mixed with any other vaccine or product. Before use, the vaccine was shaken to obtain a homogenous suspension. The vaccine was not used if it could not be uniformly suspended. The study vaccine was administered immediately after being drawn up into the syringe. A new, disposable syringe and needle was used for each injection. These were disposed of in a puncture-proof container (see Appendix 1, SOP 8: Vaccine administration).

TritanrixTM-HepBTM vaccine was drawn up using a sterile needle and a 2 mL syringe. The vaccine was then added to the vial containing powdered HiberixTM vaccine to allow reconstitution. The vial was shaken until no particulate matter remained. The mixed vaccines were then drawn up into the same syringe ensuring an aseptic technique was adhered to at all times. The 0.5 mL of PCV was given in the left anterolateral thigh except for those children receiving the 2-year-old PCV dose, whereby the vaccine was administered in the deltoid muscle (see Appendix 1, SOP 8: Vaccine administration). Co-administered vaccines were given in the right anterolateral thigh, except the measles-rubella vaccine, which was given in the right deltoid muscle. All 23vPPS was administered in the left deltoid. The 12-month dose of 23vPPS was 0.5 mL and the mPPS at 17 months of age was 0.1 mL of the 23vPPS. If a child had a moderate or

severe illness at the time of vaccination the visit was deferred and they were referred, treated appropriately, and followed up the following week (see Appendix 1, SOP 6: Deferred visits). Minor illnesses such as uncomplicated upper respiratory tract infections, localized skin infections, and mild diarrhoea were not contraindications to vaccination.

2.2.5.4 Accountability procedures for the study vaccine The study nurse recorded immunisation details in the infant’s workbook, the parent health record, the clinic study diary, and on the appropriate “Vaccine Accountability Form.” In addition, the co-administered vaccines were recorded in the MoH’s clinic records. The study number and study visit was recorded on each vial. The study coordinator was responsible for checking each vial with the Vaccine Accountability forms to ensure that infants received their vaccines according to the protocol. The used vials were stored in the study office.

2.2.5.5 Assessment of compliance with study vaccines Window periods for vaccination are shown in Appendix 1; SOP 9: Study visit windows. Study visit dates were calculated according to a Julian calendar in an excel spreadsheet, which were developed for each study group to ensure that visits were correctly scheduled. Calendars of study visits were stored in each clinic. The study coordinator maintained an Excel monitoring database, which was updated following each study visit. Missed visits were checked weekly with the clinic diary and participants were offered another appointment time to ensure that infants were followed up within the window period.

2.2.6 Study Visits

2.2.6.1 Vaccination schedule The Study visit outline (see Appendix 1, SOP 10) provides an overview of the study groups and vaccination schedule for the study. The schedule is shown in Table 3.

Table 3: Outline of study visits

Birth 6 wks 10 wks 14 wks 18w 6m 9m 12m 2w 17m 18m 2y Gp post BCG PCV1 PCV2 PCV3 B1 B2 12mb B4 B5 A OPV DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 MR mPPS HBV OPV2 OPV3 OPV4 NP1 NP2 NP3 NP4 BCG PCV1 PCV2 PCV3 B1 12mb B3 B4 B5 B OPV DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 MR mPPS HBV OPV2 OPV3 OPV4 NP1 NP2 23vPPS NP3 NP4 BCG PCV1 PCV2 B1 B2 12mb B4 B5 C OPV DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 MR mPPS HBV OPV2 OPV3 OPV4 NP1 NP2 NP3 NP4 BCG PCV1 PCV2 B1 12mb B3 B4 B5 D OPV DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 MR mPPS HBV OPV2 OPV3 OPV4 NP1 NP2 23vPPS NP4 NP3 BCG PCV1 B1 B2 12mb B4 B5 PCV2 E OPV DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 MR mPPS HBV OPV2 OPV3 OPV4 NP1 NP2 NP3 NP4 BCG PCV1 B1 12mb B3 B4 B5 PCV2 F OPV DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 MR mPPS HBV OPV2 OPV3 OPV4 23vPPS NP1 NP2 NP3 NP4 BCG DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 B1 B2 12mb B3 B4 B5 PCV1 G OPV OPV2 OPV3 OPV4 MR mPPS HBV 23vPPS NP1 NP2 NP3 NP4 BCG DTPw-Hib-HBV1 DTPw-Hib-HBV2 DTPw-Hib-HBV3 B1 12mb B4 B5 PCV1 H OPV OPV2 OPV3 OPV4 MR mPPS HBV NP1 NP2 NP3 NP4 BCG: Bacille Calmette-Guerin vaccine OPV: Oral polio vaccine HBV: Hepatitis B vaccine PCV: 7-valent pneumococcal conjugate vaccine DTPw-Hib-HBV: combined Diphtheria-Tetanus- whole cell Pertussis-H. influenzae type b-HBV B: Blood draw NP: Nasopharyngeal swab 12mb: 12 month blood test 23vPPS: 23-valent pneumococcal polysaccharide vaccine MR: Measles-rubella vaccine mPPS: Micro dose (20%) of 23vPPS

During the course of the trial there was a measles epidemic in Fiji so all children received a second dose of measles-rubella vaccine one month following their first dose of measles-rubella vaccine as per MoH policy.

2.2.7 Follow-up

It is routine child health policy for the Fiji MoH that all infants are seen at a MCH clinic on a monthly basis for well baby checks until they are 2 years old. These checks were performed by the study nurses in addition to the study visits.

The study visits occured as outlined in Appendix 1, SOP 10: Study visit outline. Appointment times were made for each study visit and families were asked to present on a certain day. If they failed to attend, a phone call was made to remind the family and have their study visit rebooked within the designated window (see Apendix 1, SOP 9: Study windows). If the phone contact was unsuccessful the study nurse visited the infant in their home and either performed the study visit or made another appointment time.

Prior to each vaccination the identity of the infant was confirmed by asking the name of the child, the father’s name, and checking the parent held record and unique identification number. Administration of the vaccine was recorded on the vaccine administration form, the parent held record, and the clinic register.

Each infant’s workbook outlined the study visits, the medical assessment, the vaccines due, questions on the occurrence of any SAE, pneumococcal NP carriage risk factors (the number of children in the household who were ≤ 5 years of age, family income, exposure to household cigarette smoking, breastfeeding status, whether the child had symptoms of an upper respiratory tract infection at the time of the visit, and whether the child had received antimicrobials in the preceding 2 weeks), the procedures to take place, and the reactogenicity visits. Prior to each 23vPPS or PCV vaccine visit, the parents were asked whether the infant had been in the hospital since the last study visit and whether a significant nonserious AE had occurred that may contraindicate the study vaccine, the infant’s axillary temperature was taken, any elimination criteria were documented, a general medical history and examination was performed, and withdrawal was documented if an exclusion criterion had developed. Study nurses contacted the study doctor for advice if any abnormality was detected. Standard operating procedures were developed for all of these topics (see Appendix 1) and worksheet developed to record information (Appendix 3). Each visit was completed with a checklist, so that study nurses were reminded to complete all study tasks

including the completion of all study documentation. The parents were informed at each study visit when the next study visit was due. This was documented on the parent held record and the clinic appointment book.

2.2.7.1 Final study visit The final study visit was at 18 months for groups A, B, C, and D, and at 2 years of age for groups E, F, G, and H. For groups A-D, parents were instructed to contact study staff if the infant was hospitalised or developed a serious illness up until the child turned 2 years old. However, SAEs were monitored until all children reached 2 years of age through the computerised hospital discharge records and the postmortem registers.

2.2.8 Withdrawal of a Participant From the Study

Any parent/legal guardian could withdraw their infant from the study at any time (see Appendix 1, SOP 11: Withdrawal of an infant from the study). Parents were reassured that withdrawal from the trial would not affect any health care provided for the infant and infants were referred to the MoH for ongoing routine MCH care.

The study doctor in consultation with the PI was always contacted prior to an infant being formally withdrawn from the study.

The reasons a participant may have been withdrawal from the study included:

Voluntary withdrawal. Any parent/legal guardian could withdraw their infant from the study without reason. They were reassured that withdrawing their child from the study had no adverse effect on their usual medical care. They were informed that their infant was unlikely to be protected from the 7 serotypes of pneumococci covered by PCV, if they had not completed all vaccinations.

They may have developed a newly diagnosed elimination criteria. A child was withdrawn from the study if they develop in the course of the study one or more of the elimination criteria. These criteria were checked at PCV and 23vPPS visits.

Failure to return for follow-up;

Failure to return for study visits within the specified window periods;

Protocol violation;

Parent/caregiver refused future study procedures;

Death;

Reaction to vaccine (either non-serious or serious) that was determined by the study doctor to be possibly related to vaccine and/or was a contraindication to receiving further study vaccine.

Depending on the reason for withdrawal, the study doctor in consultation with the PI decided if any off-study vaccine or study investigations were recommended (see Appendix 1, SOP 11: Withdrawal of an infant from the study, and SOP 12: Flowchart for off-study PCV)

2.2.9 Assessment of Safety

2.2.9.1 Adverse events At each study visit the study nurse asked the parent/caregiver whether their child had experienced any significant signs/symptoms since their last visit. AEs were not monitored post PCV as sufficient safety information has already been analysed following the clinical trials and post marketing surveillance of this vaccine. However symptoms/signs of reactions that would contra-indicate further administration of PCV were asked about at subsequent study visits.

All AEs occurring within 7 days of 23vPPS at 12 months and mPPS at 17 months, including local and systemic reactions not meeting the criteria for an SAE were captured on the appropriate CRF. Information collected included event description, time of onset, investigator assessment of severity, relationship to study vaccine, and time of resolution/stabilisation of the event. All AEs occurring within 7 days of 23vPPS were documented appropriately regardless of relationship. AEs were followed to adequate resolution. If the AE was considered longstanding or permanent, medical care was taken over by the CWMH paediatric staff. All SAEs were reported from enrollment, regardless of temporal relationship and causality.

Any medical conditions that were present at the time that the patient was screened or just prior to the receipt of 23vPPS were recorded and considered as baseline and not reported as an AE. However, if the condition deteriorated within 7 days of 23vPPS administration it was recorded as an AE.

Appropriate medical treatment and supervision was available in case of an anaphylactic event following administration of the vaccine. All infants were supervised for at least 30 minutes immediately following administration of the vaccine. All staff were trained in the treatment of anaphylaxis (see Appendix 1, SOP 13: Adverse events reporting). Adrenaline (1:1000, 0.01 mg/kg) was available in all participating centres. This was to be given by deep intramuscular injection if required, followed by immediate medical attention and referral to hospital.

2.2.9.2 Reactogenicity Reactogenicity post-vaccination was documented only for those children receiving the 23vPPS at 12 months and again at 17 months (see Appendix 1, SOP 14: Follow-up for reactogenicity). This was documented in reactogenicity forms and filed in the infant’s folder. Reactogenicity was assessed on days one, 2, and 7 post-booster vaccination, recording local reactions, systemic symptoms, and concomitant medications by interviews at each visit. Evidence of local induration, tenderness, and redness were measured in millimeters at the widest margin. Fever, irritability, and feeding difficulties were recorded. The study nurse recorded the axillary temperature and respiratory rate. Any infant with marked swelling or any significant systemic symptom was referred to the clinic for further assessment and followed by the medical and nursing staff there until resolution. The study doctor was notified of AEs and causality was assigned. Parents were encouraged to report other symptoms outside this surveillance period.

Reactogenicity was recorded on worksheets by the study nurses on days 1, 2, and 7 post-23vPPS as outlined previously. This information was transcribed onto the CRFs, and collated and presented to the DSMB.

The following were classified as severe AEs of interest:

Local redness and/or swelling > 30 mm at the widest margin (approximately one quarter the circumference of the thigh) within 72 hours of their last vaccination;

Ulceration or abscess at the injection site within 10 days of vaccination;

Convulsion within 72 hours of vaccination;

Severe irritability, fever, poor feeding, vomiting, or drowsiness (grade 3 –prevented normal activities eg breastfeeding) within 72 hours of vaccination.

Fever >38o C per axilla

Any severe AE of interest but not technically an SAE were reported to the DMID Medical Monitor and the DMID Protocol Champion on a monthly basis and the DSMB at each meeting.

2.2.9.3 Serious adverse events

A SAE was defined as an AE meeting one of the following conditions:

Death during the period of protocol defined surveillance

Life threatening event (defined as an infant at immediate risk of death at the time of the event)

An event requiring inpatient hospitalisation or prolongation of existing hospitalisation during the period of protocol defined surveillance

Results in congenital anomaly or birth defect

Results in a persistent or significant disability/incapacity

Any other important medical event that may not result in death, be life threatening, or require hospitalisation, may be considered a SAE when, based upon appropriate medical judgment, the event may jeopardise the infant and may require medical or surgical intervention to prevent one of the outcomes listed above.

Staff in the hospital were requested to contact the study staff if the infant presented to them with any illness. A study doctor regularly checked the hospital computerised discharge records and postmortem records to find any missed notifications of illness or deaths.

2.2.9.4 Intensity of event

All AEs were assessed by the investigator as:

Mild: events that required minimal or no treatment and did not interfere with the infant’s daily activities.

Moderate: events that resulted in a low level of inconvenience or concern with the therapeutic measures. Moderate events may have caused some interference with functioning.

Severe: events that interrupted an infant’s usual daily activity and may have required systemic drug therapy or other treatment. Severe events were usually incapacitating.

Life threatening: Any adverse vaccine experience that placed the infant, in the view of the investigator, at immediate risk of death from the reaction as it occurred.

Changes in the severity of an AE were documented to allow an assessment of the duration of the event at each level of intensity.

2.2.9.5 Reporting procedures If there was any doubt as to whether a clinical observation was an AE, the event was reported. All AEs had their relationship to study product assessed using the following terms: associated or not associated.

Associated – The event was temporarily related to the administration of the study vaccine and no other aetiology explained this event.

Not Associated –The event was temporarily independent of the study vaccine and/or the event appeared to be explained by another aetiology.

All SAEs were:

Recorded on the SAE case worksheet by the study doctor

Followed until satisfactory resolution or until the PI deemed the event to be chronic or the patient to be stable

Reviewed by the study doctor

Any AE considered serious by the PI or which met the aforementioned criteria was submitted on the DMID SAE form to the Independent Safety Monitor (Prof Keith Grimwood), the PI, GlaxoSmithKline, and the DMID pharmacovigilance contractor PPD. Provisions were made to serotype any invasive pneumococcal specimen isolated from a study infant. The Study Doctor completed a SAE Report Form within the following timelines:

All deaths and immediately life threatening events, whether related or unrelated to the study, were recorded on the SAE Form and sent by fax within 24 hours of site awareness.

SAEs other than death and immediately life threatening events, regardless of relationship, were reported via fax by the site within 72 hours of becoming aware of the event.

Other supporting documentation of the event was provided as soon as possible. The independent safety monitor reviewed all SAEs and independently assigned causality. The study had a designated independent DSMB to assess all reported SAEs. The DSMB was an independent group of experts that advised DMID and the study investigators. The primary responsibilities of the DSMB were to 1) periodically review and evaluate the accumulated study data for participant safety, study conduct and progress, and 2) make recommendations to DMID concerning the continuation, modification, or termination of the trial.

The University of Melbourne Human Research Ethics Committee and the Fiji National Research Ethics Review Committee received an annual report from the PI of all AEs or more frequently as determined by the DSMB.

2.2.9.6 Procedures to be followed in the event of abnormal laboratory test or clinical finding In the event of a laboratory test or finding that necessitated medical treatment, the PI ensured that such treatment was instituted without delay. Where a laboratory finding emerged that suggested that a child may have diminished immunity to pneumococcus as a result of vaccination undertaken

in this project, the child was to be recalled by the PI for review. The case would be referred to the DSMB for guidance. In some cases an extra dose of PCV could be required.

2.2.9.7 AE follow up All SAEs were followed until satisfactory resolution or until the PI deemed the event to be chronic or the infant stable. The nonserious AEs were followed until resolution.

2.2.9.8 Halting rules It was determined that in the case of the following events, an extraordinary meeting of the DSMB would be called to discuss the potential need to stop the study on the grounds of safety:

1. There was a life threatening or fatal SAE which, in the view of the PI or the Independent Safety Monitor, was attributable to one of the study vaccines.

2. There was an unusual cluster of SAEs that, in the view of the PI or the Independent Safety Monitor, could be due to one of the study vaccines.

3. Evidence emerges from another study that indicates that one or more of the regimens being investigated in this study were unsafe.

2.2.9.9 Procedure in the case of a child, or group of children, being found to be hyporesponsive If a child was found to be non-responsive, or poorly responsive to serotypes included in the PCV, a further dose of PCV would be given at an age determined by the DSMB, and the child bled again for antibodies after one month, provided consent was given by the parents.

In the case of hyporesponsiveness to other serotypes in the 23vPPS, the parents were to be counselled regarding the finding and given access to paediatric staff and advised to report without delay should the child develop a severe febrile illness. In all cases the course of action would be discussed with the DSMB.

2.2.10 Sample Collection

2.2.10.1 Blood tests Each infant had 5 blood tests, except those in Group H, who had 2 blood tests (Table 3). A 5mL venous blood sample was drawn 4 weeks following the completion of the primary series of PCV (at 18 weeks of age) for all children except those in Group H who received no PCV at this time. For those who were randomised to receive 23vPPS at 12 months, 5mL blood samples were drawn immediately pre- and 2 weeks post- this booster vaccination to assess immunological memory following one to 3 doses of PCV for the 7 serotypes in PCV and assess the primary response to the 16 non-PCV serotypes. For those not randomised to receive the 12-month dose of 23vPPS, 5mL blood samples were taken at 9 and 12 months of age, (except for Group H) to

assess long term protection, avidity maturation, and functional activity following a 1, 2, or 3 dose primary series. For all infants in the study, blood samples were taken at 17 months and 18 months (i.e. pre- and one month post the mPPS dose at 17 months of age) to assess long term protection and hyporesponsiveness to a re-challenge dose (see Appendix 1, SOP 15: Collection and management of blood samples). For infants aged at least 12 months the parents were asked if they preferred their infant to have local anaesthetic cream applied to the skin to reduce discomfort. For all infants distraction techniques were utilised to minimise discomfort.

If participants did not attend on the designated day, all attempts were made to bleed the child as close as possible to Day 14 post vaccination to ensure comparability between groups. Blood was separated in the health centre by centrifugation and the serum collected. These samples were kept chilled and delivered to the CWMH laboratory in a chilled UNICEF box (see Appendix 1, SOP 16: Laboratory processing of blood samples). Each sample of serum was divided into 4 aliquots. Aliquots 1 and 4 were stored in the -70 C freezer (avidity and OPA). Aliquots 2 and 3 were stored in 2 separate -20 C freezers (for ELISA analysis).

2.2.10.2 Nasopharyngeal samples Each infant had 4 NP swabs taken at 6, 9, 12, and 17 months of age. Two trained study staff performed the procedure to ensure no injury was caused by an infant‘s sudden movement (see Appendix 1, SOP 19: Collection and management of NP swabs). Buffered cotton NP swabs (Sarstedt, Australia) were taken by horizontal insertion into the nares. The swab was left in situ for five seconds and rotated, then immediately placed into a sterile cryovial tube (Simport, Canada) containing 1 mL of skim-milk-tryptone-glucose-glycerol (STGG) transport medium [337]. The swabs were transported to CWMH in chilled UNICEF boxes.

The swabs were processed and stored according to the consensus guidelines from a World Health Organization working group [338] (see Appendix 1, SOP 20: Laboratory processing of NP swab samples). The cryovials containing the swabs were vigorously shaken (using a vortex) and the supernatant and swab were either stored at -70 C until culture or immediately plated on receipt in the laboratory.

To culture S.pneumoniae isolates from the NP supernatant, 50 μL was inoculated onto a 2.5 mg/L gentamicin 5% citrated sheep blood Columbia agar (Oxoid) plate [4]. Plates were incubated at 37 C in 5% CO2 for 18 to 24 hours. S.pneumoniae colonies were initially identified by α-hemolysis, colony morphology, and optochin (BD Difco, Franklin Lakes, NJ) sensitivity (see Appendix 1, SOP 20: Laboratory processing of NP swabs). Isolates with intermediate Optochin sensitivity were confirmed as pneumococci by bile solubility testing. Single colonies were subcultured and pure colonies were

sent to the Pneumococcal Reference Laboratory, Centre for Infectious Diseases & Microbiology, ICPMR, Westmead, NSW, Australia, where they were serotyped by multiplex-PCR and reverse-line blot assay [339, 340]. In addition, all isolates were also serotyped by Quellung reaction using specific antisera (Statens Serum Institute, Copenhagen, Denmark). When certain serotypes could not be distinguished by the reverse-line blot method (eg 10A/10B result), the final serotype was determined by Quellung. Laboratory staff members were blinded to group allocation during the processing of each isolate.

2.2.11 Sample Transportation - International

Transportation of aliquots of both sera and isolates was via consignments approximately every 4 months to minimise potential loss. Sera samples were sent to the Pneumococcal Laboratory, Murdoch Children’s Research Institute, Melbourne and the Bacterial Respiratory Pathogen Reference Laboratory, University of Alabama, USA. NP samples and isolates were sent to the Murdoch Children’s Research Institute Pneumococcal laboratory and the Pneumococcal Reference Laboratory, Centre for Infectious Diseases & Microbiology, Westmead Hospital, Sydney, Australia. Specimens were packed according to IATA 650 specifications and shipped on dry ice (see Appendix 1, SOP 21: Transport of specimens overseas). Shipment lists of individual specimens sent were maintained by the FiPP office and the Murdoch Children’s Research Institute Pneumococcal laboratory. On receipt of the samples at their destination a brief report documented their integrity on arrival.

For most participants consent was obtained for the future use of samples and these samples were stored at the reference laboratories. Where this level of consent was not obtained the samples were discarded at the end of analysis described below.

2.2.12 Antibody Assays

In the first 12 months anticapsular pneumococcal antibody levels were assayed for all 7 PCV serotypes (4, 6B, 9V, 14, 18C, 19F, 23F). From 12 months onwards, sera were assayed for the presence of antibodies to the remaining 16 serotypes in 23vPPS (1, 2, 3, , 5, , 7F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 19A, 20, 22F, 33F) (including pre/post the 12 month 23vPPS), using a modified 3rd generation enzyme-linked immunosorbent assay (ELISA) based on current WHO recommendations [341]. In brief, microtitre wells were coated with pneumococcal polysaccharide diluted in phosphate buffered saline by incubating at room temperature overnight. To neutralise non-specific antibodies to cell wall polysaccharide (C-PS) , serum samples for all serotypes (except serotype 22F) were diluted 1/100 in pre-absorption buffer containing C-PS (10µg/mL) and serotype 22F (30µg/mL) and

incubated overnight at 4oC. Absorption with 30 µg/ml serotype 22F overnight has been reported previously [123, 342] and unpublished data from our laboratory have shown this to further improve the specificity of the pneumococcal ELISA. The reference serum standard 89-SF (Food and Drug Administration, Bethesda MD) and samples for measurement of specific IgG to serotype 22F were pre-absorbed with C-PS at 10µg/mL and incubated overnight at 4oC. Horseradish peroxidase conjugated anti-human IgG and a TMB (3.3’, 5.5’-tetramethylbenzidine) substrate solution was used for detection. A high, medium, and low control serum were used on each plate to assess assay performance and inter-assay variation. Results from an inter-laboratory comparison between Wyeth Vaccines and the KTL Finland laboratory demonstrated a good correlation in measurement of serotype-specific antibody concentrations [343].

Avidity of IgG antibodies to capsular polysaccharide was determined by enzyme immunoassay (EIA) for all 7 PCV serotypes in the first 12 months and for the remaining 16 pneumococcal serotypes in 23vPPS fro 12 months onwards [147, 154, 344]. After washing, 0.5 M sodium thiocyanate (NaSCN) in F-PBS was added to the serum samples to dissociate weak antibody-antigen complexes. The sample was incubated for 15 minutes at room temperature, after which the plates were washed and antibody binding was detected by the addition of horse radish peroxidase -conjugated anti-human IgG. The colour was developed by the TMB Peroxidase Substrate system. Absorbance at 405 nm, reference filter 620 nm was read on an EIA reader. Control serum was added to each plate to assess reproducibility.

OPA were performed for 8 serotypes (serotypes 1, 4, 5, 6B, 9V, 14, 18C, and 23F) at each blood draw and on 6 additional serotypes (3, 6A, 6C, 7F, 19A, 19F) for the pre- and post-mPPS blood draws using a fourfold multiplexed method [139]. In brief, all serum samples were incubated at 56°C for 30 min before serial dilutions were prepared. Frozen aliquots of target pneumococci were thawed, washed twice (unless otherwise indicated) with opsonization buffer B (Hanks’ balanced salt solution *HBSS+ with Mg and Ca, 0.1% gelatin, and 10% FBS) by centrifugation (12,000 X g, 2 min), and diluted to the proper bacterial density (~105 CFU/ml for single-serotype assays and ~2 X 105 CFU/ml of each serotype for multiplexed assays). Equal volumes of four bacterial suspensions in one assay group were pooled. Ten microliters of bacterial suspension was added to each well. After 30 min of incubation at room temperature, 10μl of complement and 40μl of HL60 cells were added to each well. HL60 cells were washed twice before use with HBSS by centrifugation (350 X g, 5 min), and 4 X 105 cells were added to each well (unless otherwise indicated). Plates were incubated in a tissue culture incubator (37°C, 5% CO2) with shaking (mini orbital shaker; Bellco Biotechnology, Vineland, NJ) at 700 rpm. After a 45-minincubation, plates were placed on ice for 10 to 15 min and an aliquot

of the final reaction mixture (10μl) was spotted onto four different Todd-Hewitt broth–yeast extract (0.5%) agar plates [345]. After application of an overlay agar containing one of the four antibiotics to each Todd-Hewitt broth–yeast extract (0.5%) agar plate and overnight incubation at 37°C, the number of bacterial colonies in the agar plates was enumerated [345].

2.3 Data Management

Study nurses, the study doctor, and the laboratory technician were responsible for transcribing data from the source documents to the CRFs. The fieldwork source documents were initially filed in a lockable cupboard in Nausori and Valelevu Health Centres (including Makoi’s files), then transferred to the study office at CWMH for long-term storage.

The study coordinator reviewed all source documents on a weekly basis and was responsible to ensure the completeness, accuracy, legibility and timeliness of the data reported. Once monitored, the CRFs were transported to the study office where double data entry was performed into a password-protected EpiData version 3.1 database (see Appendix 1, SOP 23: Data management). A copy of the fieldwork CRF was filed in the infant’s workbook and the original was stored in the study office. The original laboratory CRFs were stored securely in the study office and copies stored in the PI’s office.

AE reporting was completed by the study nurses and monitored by the study co-ordinator and the study doctor. The on-site PI monitored all AE source documents and CRFs and was responsible for the complete and accurate documentation of the study. Causality of AEs was assigned by the PI. SAE reports were collated by the study doctor into reports. These AE data were presented quarterly to the FiPP Steering Committee, DMID, and to the DSMB.

Double data entry accuracy was attained by running a validation program in EpiData version 3.1. Any discrepancies were corrected immediately by checking with the original CRF stored in the study office. The validation program reports were monitored by the PI. On a monthly basis, the research assistants exported the validated database into Stata (version 9.0, Stata Corporation, College Station, Texas) to run a series of commands to further check for any errors.

2.3.1 Data capture methods

Paper CRFs were used. Once monitored, they were transported to the study office on a daily basis. The data were entered in an ongoing way. The number of CRFs entered into the database was documented in the weekly and quarterly monitoring report.

2.3.1.1 Timing of Reports Data validation and cleaning occurred on a monthly basis as outlined above (see Appendix 1, SOP 23: Data management). AEs and SAEs were reported as described previously. Data were not analysed until the end of the study unless indicated by the DSMB.

Once the data were cleaned, the database was locked so that further changes could not be made. A copy of the cleaned database was stored on an external hard drive and sent to CEBU. The on-site principal investigator was responsible for sending a copy of the completed datasets to DMID at the end of the study.

2.3.1.2 Study Records Retention The data will be stored indefinitely at the study office and the University of Melbourne. Approval from the ethics committees and DMID will be required to destroy any records.

2.4 Clinical Monitoring Plan

Site monitoring was conducted to ensure the human subject protection, study procedures, laboratory, and data collection processes were of high quality and met DMID, GCP/ICH guidelines.

Quality assurance was maintained by quality site visits performed by an external auditor as directed by DMID (see Appendix 1 SOP 24: Quality monitoring plan). Any recommendations forthcoming were enacted by the on-site PI.

2.4.1 Source Documents

Each participating site maintained appropriate medical and research records for the duration of the study, in compliance with ICH E6 GCP, Section 4.9 and regulatory and institutional requirements for the protection of confidentiality of subjects. Study staff and investigators had access to the records. The site permitted authorised representatives of DMID and its representatives to examine clinical records for the purposes of audits and evaluation of the study safety and progress.

2.4.2 Protocol Deviations

A protocol deviation was any noncompliance with the clinical trial protocol, GCP, or Manual of Procedures requirements. The noncompliance could be either on the part of the subject, the investigator, or the study site staff. As a result of deviations, corrective actions were developed by the site and implemented promptly. See Appendix 1, SOP 25: Protocol compliance.

It was the collective responsibility of all study staff to use continuous vigilance to identify and report deviations within 5 working days of identification of the protocol deviation, or within 5 working days

of the scheduled Protocol-required activity. All deviations were promptly reported to DMID, via the PPD web- or fax-based system.

All deviations from the Protocol were addressed in study subject source documents. A completed copy of the DMID Protocol Deviation Form was maintained in the Regulatory File, as well as in the subject’s source document. Protocol deviations were sent to the ethics committees as per their guidelines. The site PI/study staff were responsible for knowing and adhering to the ethics committees requirements.

2.4.3 Quality Control and Quality Assurance

The day to day monitoring of the study was the responsibility of the study coordinator. Following written standard operating procedures, the monitor verified that the clinical trial was conducted and data were generated, documented (recorded), and reported in compliance with the protocol, GCP, and the applicable regulatory requirements. Reports were regularly submitted to DMID on monitoring activities.

Each week the internal monitor compiled a weekly report. Each quarter, the FiPP steering committee and DMID were forwarded the report. The report compiled study visit status including outstanding visits, withdrawals, SAEs; monitoring status, database management, cold chain issues, vaccine wastage protocol deviations, laboratory issues, and GCP issues. The final quarterly report can be found in Appendix 4.

DMID provided external monitoring through its contract to PPD Development to independently assess protocol and GCP compliance. A monitoring plan was developed and visits started in the second quarter of 2005 through study-close-out in August 2008.

2.5 Statistical Methods

2.5.1 Background to Original Study Protocol

This study began as a Phase II vaccine trial in which, using a factorial design, regimens containing one, 2 and 3 doses of PCV, supplemented by a dose of 23vPPS at 6 or 9 months of age were compared with respect to immunogenicity and impact on NP carriage. Analyses were based on the demonstration of noninferiority, initially between the 2-dose and 3-dose PCV regimens, then between the 1-dose and 3-dose regimens, and finally between the 6-month and 9-month 23vPPS dose regimens. Immunogenicity was described using standard parameters for each serotype and carriage was described at specified timepoints as carriage of VT and NVT pneumococci. Following the revision of the study design (outlined in section 1.8.1) similar comparisons were still available,

except that instead of comparing 6-month and 9-month 23vPPS dose regimens, comparisons were made between those who receive 23vPPS at 12 months and those who did not. As the focus of the study had shifted to investigate the potential for later hyporesponsiveness following 23vPPS administration, the sample size, and primary and secondary objectives were adjusted as outlined in the Objectives of the study, section 1.8.

The original primary hypothesis of the revised design on which the sample size was based was that 23vPPS vaccination at 12 months would not lead to a greater proportion of hyporesponsiveness to mPPS at 17 months for any of the 11 serotypes for which functional analyses were available. Formally this was tested by attempting to reject the null hypothesis that, for at least one of the 11 serotypes, the proportion of children showing hyporesponsiveness was 15% greater (in absolute terms) in the 23vPPS group than the group receiving no vaccine at 12 months. The test was the standard single sided test of non-inferiority of proportions (as recommended by ICH guidelines for noninferiority trials), with a significance level of 5%.

Following discussion with the DSMB in November 2007, the definition of “hyporeponsiveness” was changed from what was originally defined in the protocol. The original definition of “hyporesponsiveness” was changed to “non-responsiveness”. The new (and final) primary endpoint was to demonstrate non-inferiority in GMC one month post mPPS for 8 or more of the 23 23vPPS serotypes, one month post mPPS, in the groups that had or had not received the 12 month 23vPPS.. However given that the sample size was calculated based on the original proposal of the revised design, the original primary endpoint was analysed as a secondary endpoint.

2.5.2 Sample Size Calculation

Sample size calculations were based on the original primary hypothesis of the revised design, as mentioned previously, that the proportion of infants showing hyporesponsiveness was not greater in the 23vPPS group for each of the 11 serotypes for which functional assays were expected to be available. In other words, it was sought to simultaneously reject, at the =0.05 level, 11 null hypotheses of inferiority: that the proportions of infants with hyporesponsiveness were greater by 15% in the 23vPPS group vs the non 23vPPS group. It was assumed that the true proportion showing hyporesponsiveness to each serotype was 30% regardless of group allocation as children under 2 years of age do not respond as well as older children and adults.

Hyporesponsiveness may be infant specific or serotype-specific. In other words, there may be children who, following receipt of 23vPPS, respond poorly to a number of pneumococcal serotypes. Alternatively, there may be individual serotypes, possibly amongst those that are not usually

assayed, in which 23vPPS administration leads to subsequent hyporesponsiveness in a significant proportion of individuals.

In ELISA terms, a child was defined as a poor responder to a specific serotype if, following mPPS at 17 months, they have an antibody level of <0.35 ug/mL. In OPA terms, a child was defined as a poor responder to a specific serotype if, following mPPS at 17 months, they have an OPS titre <8. Noninferiority of the 23vPPS group would be established with respect to both infant specific and serotype-specific hyporesponsiveness. This was done by comparing children who did or did not receive 23vPPS at 12 months of age:

1) For each serotype assayed (23 for ELISA and 11 for OPA) the proportion of children responding to the mPPS dose at 17 months.

2) The proportion of children who were poor responders, defined as poor response by the above definition, to more than 5 of the 11 serotypes in the 23vPPS for which there was an OPA assay.

For each serotype it was assumed that 30% of children were poor responders to mPPS at 17 months. To achieve 80% power to show that the 23vPPS recipients have a non-inferior response at level =0.05, inferiority margin =0.15 in each of 11 serotypes would require a power of 98% in each individual serotype tested if these tests were independent. However there was likely to be significant intra-child correlation of hyporesponsiveness. To account for this, hyporesponsiveness was modelled via beta-binomial distribution. Firstly, each child was assumed to have a propensity p for hyporesponsiveness drawn from the beta distribution. Then for each of the 11 serotypes subject to OPA testing, that child was made hyporesponsive with probability p.

The alpha and beta parameters of the beta distribution were chosen so that:

a) for any given serotype, 30% of children were expected to be hyporesponsive; and

b) it was expected that the proportion of children hyporesponsive to more than half of the serotypes (6 or more) was also 30%.

These conditions implied the beta-distribution parameters alpha=0.628 and beta=1.465. Running 10,000 simulations of the trial with 250 children in each arm estimated the power to be 83% to reject inferiority for overall hyporesponsiveness. Decreasing the inferiority margin to 10% reduced the power to 27% with 250 children in each group. Having 500 in each group increased the power to 77%.

The majority of children were assumed to have a very low probability of hyporesponsiveness to any given serotype. The cumulative distribution showed that approximately 20% of children were assumed to have a per-serotype probability of hyporesponsiveness of 5% or less. For 30% of children it is 10% or less. Only 23% of children were assumed to have a >50% probability of hyporesponsiveness to any given serotype. Hence a total recruitment of 550 infants was required to allow for 10% loss of follow-up while maintaining acceptable power.

The secondary analysis did not require any assumptions other than the proportion of overall hyporesponsiveness in each group. This was fixed at 30%, consistent with the model used for the per-serotype analysis. For this noninferiority test, power was calculated using the Farrington & Manning maximum-likelihood standard-error approach to noninferiority tests [346]. With an inferiority margin of 10%, 250 children in each group were sufficient to achieve 80% power to reject the secondary null hypothesis.

The secondary hypothesis was that the proportion of children showing hyporesponsiveness at 18 months to more than half of all 23 serotypes, as defined by an antibody level of <0.35 ug/mL by ELISA, will be no greater in those children who received 23vPPS at 12 months of age than those who did not.

Whilst OPA may be more sensitive at detecting hyporesponsiveness, they were not available for all 23 serotypes in the 23vPPS and they are time-consuming and expensive to perform. Therefore it was proposed to address this by ELISA assays. In ELISA terms, a child was defined as a poor responder to a specific serotype if, following mPPS at 17 months, he/she had an antibody level of <0.35 ug/mL. The proportion of children who were poor responders by ELISA to 12 or more serotypes were compared between children who received 23vPPS at 12 months and those who did not. An inferiority margin of 10% was used.

For this secondary analysis, it was anticipate that 30% of 18-month old children in each group will be poor responders. The proposed sample size would provide 79% power to reject the null hypothesis that the proportion of poor responders will be 10% greater (in absolute terms) in the 23vPPS groups. As a tertiary analysis, the primary and secondary analyses were repeated, stratified according to the receipt of none, one, two or three doses of PCV in the first year. This will proceed in 2 steps: initial stratification will be into 2 groups, defined by the receipt of >2 doses of PCV versus one or none.

We will further explore the inter-relationship of total serotype-specific antibody (measured by ELISA), antibody avidity (measured by thiocyanate-eluted ELISA) and OPA. It is currently believed that the physiological significance of the serotype-specific antibody response is likely to be best

quantified with functional antibody measurement, followed in turn by antibody avidity and total antibody. Yet the field to date has focused more on total antibody levels and less on avidity or OPA. ELISA and avidity assays for all 23 serotypes present in the 23vPPS were performed, and OPA for the serotypes for which they were available. At the time of analysis, OPA assays were available for 13 serotypes.

2.5.3 Definition of Outcome Measures

Serotype-specific antibody concentrations by ELISA were log (base e) transformed to calculate the geometric mean concentration (GMC). An antibody level ≥0.35 g/mL, determined from pooled analysis of data from efficacy trials [16, 17], and theoretically implying an immunological correlate of protection, was used as a cut off for comparing groups.

Avidity results were expressed as an avidity index (AI), defined as the percentage of antibodies that remain bound to the antigens after NaSCN elution. The AI was calculated for each serotype by dividing the end point titre of the serum sample with NaSCN treatment by the end-point titre of the sample without NaSCN treatment and multiplying by 100. The results were expressed at each time point and by group allocation as a serotype-specific median AI.

The OPA results were expressed as an opsonization index (OI) defined as the interpolated dilution of serum that killed 50% of bacteria exposed to the sample under analysis. The lower limit of detection in the assay is 4. The OIs of samples that did not kill 50% of bacteria were reported as “2” for analysis purposes. The threshold of the assay was set at 8 [127], a level that has been shown to confer protection against IPD in a mouse model and correlates with an IgG concentration of 0.2 to 0.35μg/mL [123]. In addition, serotype-specific OIs were log (base e) transformed to calculate geometric mean titers (GMT).

Serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were classified as VT, all other viable isolates including those that were non-typable were classified as NVT. At the time of writing the original protocol, significant cross protection between serotypes (for example 6B with 6A) was not identified and hence a “vaccine related serotype” category was not included. Rates of NP carriage were calculated using the number of total pneumococcal, VT, or NVT isolates in each group at each time point divided by the total number of children who had a NP swab taken for each group at each time point.

2.5.4 Statistical Analysis of Primary Objective

In addition to the changes to the study design and primary endpoint as outlined above, during the course of the trial and following discussion with the DSMB in November 2007, the definition of

“hyporeponsiveness” changed from what was originally defined in the protocol. The original definition of “hyporesponsiveness” had been changed to “non-responsiveness”. As such the the primary objective was:

1. To demonstrate non-inferiority in GMC one month post mPPS for 8 or more of the 23 23vPPS serotypes, one month post mPPS, in the groups that had or had not received the 12 month 23vPPS. A group was defined as hyporesponsive if a significant difference was found in serotype-specific GMC for 8 or more of the 23 serotypes one month post mPPS between those groups that had (groups B, D, F and G) or had not (groups A, C, E and H) received the 12 month 23vPPS. GMC and 95% confidence intervals were calculated. Comparisons of serotype-specific GMC in those groups who have or have not received the 12 month 23vPPS were performed using a 2 sample t test for 23 23vPPS serotypes, at 18 months of age following mPPS. A significance level of ≤0.01 was considered statistically significant for all analyses due to the multiple comparisons.

2.5.5 Statistical Analysis of Secondary Objectives

The secondary objectives were:

1. To demonstrate non-inferiority one month post mPPS, between those groups receiving 23vPPS at 12 months and those who did not with respect to:

a. The proportion of children in each group with antibody levels ≥0.35 g/mL to each of the 23 serotypes included in the 23vPPS, by ELISA.

b. The proportion of children with OI ≥8 to each of the 8 serotypes for which OI data were available.

The proportion of children with antibody levels ≥0.35 g/mL to each of the 23 serotypes included in the 23vPPS, by ELISA were calculated then compared by using Fisher’s exact test in the groups that received or did not receive the 12 month 23vPPS.

The proportions of children with OIs ≥ 8 in those who did or did not receive the 12 month 23vPPS booster were calculated. A comparison of the proportion with OIs ≥ 8 in those who did or did not receive the 12 month 23vPPS booster using Fisher’s exact test was made.

2. To compare the proportion of infants at 18 weeks of age with antibody levels by ELISA, to 7 PCV serotypes ≥0.35 and ≥1 g/mL following a primary series of 3 doses of PCV in infancy or an alternative schedule of 0, 1 or 2 doses.

The proportion of children with antibody levels ≥0.35 g/mL and ≥1 g/mL to the 7 PCV serotypes from those that received 3 doses of PCV was calculated then compared with the proportions of children in the groups that received 0, 1, or 2 doses of PCV in infancy using Fisher’s exact test.

3. To compare the GMC of ELISA antibody at 18 weeks of age following a primary series of 3 doses of PCV in infancy or an alternative schedule of 0, 1 or 2 doses.

GMC and 95% confidence intervals were calculated. Comparisons of serotype-specific GMC for 7 PCV serotypes in the 3 dose PCV group were calculated and compared with the GMC for the 0, 1, or 2 dose PCV groups using the 2 sample t test.

4. To compare the GMC for 23 23vPPS serotypes, at 12 ½ months of age in those who did and did not receive a booster of 23vPPS at 12 months of age, following a 0, 1, 2, or 3 dose primary series of PCV in infancy.

GMC and 95% confidence intervals were calculated. Comparisons of serotype-specific GMC in those groups who did or did not receive the 12 month 23vPPS and by 0, 1, 2, or 3 doses of PCV in infancy were performed using the 2 sample t test for 23 23vPPS serotypes.

5.To compare the proportions of children who prior to mPPS (NP4) carried VT and NVT pneumococci in the nasopharynx among children who had received one, 2, or 3 PCV doses in infancy with or without a booster dose of 23vPPS at 12 months of age.

Rates of NP carriage were calculated using the number of total pneumococcal, VT, or NVT isolates in each group at each time point divided by the total number of children who had a NP swab taken for each group at each time point. Analyses comparing the proportions of infants with NP carriage of any pneumococcus, VT and NVT carriage were performed using Fisher’s exact test.

2.5.6 Statistical Analysis of Tertiary Objectives

Hyporesponsiveness was further analysed by comparing serotype-specific GMC for 3, 2, 1 or 0 dose PCV groups with or without the 12 month 23vPPS using the 2 sample t test for 23 23vPPS serotypes, one month following mPPS. This was adjusted for pre-existing antibody level using multivariable regression analysis. To prevent an inflated type 1 error due to multiple comparisons, and obtain a single p-value for the null hypothesis of mPPS having no impact on the antibody response to any of the 23 serotypes, a joint test of all the regression coefficients from the aforementioned multivariable regression analysis was performed [347].

A comparison of median avidity of antibody levels in those who did or did not receive the 12 month 23vPPS booster for the 23 23vPPS serotypes and had 3, 2, 1 or 0 dose PCV groups for the 7 PCV serotypes was performed. To test for differences in distributions, between group comparisons of the median serotype-specific AI were performed using the Rank sum test, and paired comparisons of the median serotype-specific AI within groups were performed using the Sign rank test.

At each point where a NP swab was collected for ascertainment of carriage, comparisons were made between the groups with respect to the proportion of children carrying a VT and a NVT for the 7 PCV serotypes for NP swabs 1, 2, and 3 using a Fisher’s exact test, for 0-3 dose PCV groups.

3 PNEUMOCOCCAL NASOPHARYNGEAL CARRIAGE AND PATTERNS OF PENICILLIN RESISTANCE IN YOUNG CHILDREN IN FIJI

3.1 Abstract

Background: Little is known about nasopharyngeal carriage and the patterns of antibiotic resistance of pneumococci in Pacific nations. We set out to document pneumococcal nasopharyngeal carriage and associated risk factors, antimicrobial resistance and serotypes in healthy children in Fiji.

Methods: A cross-sectional survey of healthy children aged 3–13 months was conducted. NP swabs were collected from each child and processed according to standard methods. Antimicrobial resistance was determined by disk diffusion and confirmed by E-testing. Serotyping was performed by the Quellung reaction.

Results: Of 440 consecutive NP swabs taken, 195 were S. pneumoniae-positive (carriage rate 44.3%). Higher rates were found in the indigenous Fijian population. Penicillin non-susceptibility was found in 11.4% of isolates, with one isolate demonstrating high-level resistance. Cotrimoxazole resistance was common (20.3%) and no isolates were chloramphenicol-resistant. Multi-drug resistance was uncommon. The commonest serotypes were 6A (13.2%), 23F (8.3%), 19F (7.4%), and 6B (6.2%). Thirty per cent were included in the 7-valent pneumococcal conjugate vaccine (PCV), 53.7% if cross- reacting strains were included. Being indigenous Fijian or having symptoms of acute respiratory infection were independent risk factors for carriage.

Conclusions: Pneumococcal NP carriage is common in Fijian children. Penicillin resistance has been documented for the first time in Fiji and, as a result, first-line treatment for meningitis has been altered. Being indigenous Fijian is a risk factor for disease, although the reasons for this are unclear. A low proportion of carriage serotypes are covered by the existing PCV. 3.2 Introduction

S. pneumoniae is a significant cause of morbidity and mortality in children worldwide. The nasopharynx is the main reservoir for pneumococci and plays an important role in the spread of the organism. NP acquisition and carriage of pneumococci is associated with the occurrence of AOM[11], bacteraemia [9] and pneumonia [10]. In developing countries, NP colonisation rates can be greater than 60% by 2 months of age [15, 16]. Studies of nasopharyngeal carriage can offer insights into the pneumococcal disease burden in a community and are a convenient way of determining a population’s level of antibiotic resistance.

Increasing rates of penicillin resistance in pneumococci have been reported globally over the past decade [348]. This is creating increasing problems in the treatment of meningitis [349] and OM [350]. Moreover, isolates with high-level penicillin resistance are frequently multi-drug-resistant. The

81 prevalence of carriage of penicillin-resistant pneumococci varies according to geographical location and has been reported to be as high as 100% in Romania [351]. Antimicrobial use by the individual and at community level has been shown to be strongly associated with NP carriage of penicillin- resistant pneumococci [352].

Pneumococcal antimicrobial resistance is serotype-specific with most resistant strains belonging to serotypes that are included in PCV [307]. Community-wide administration of PCV has led to a reduction in the carriage of these serotypes [129, 353]. In the USA, there has been a 50% reduction in antimicrobial resistance in invasive disease isolates in PCV-vaccinated children under 2 years of age [353]. Immunisation of South African infants with the 9-valent pneumococcal conjugate vaccine reduced by 67% (95% CI 19–88) the incidence of first episodes of invasive pneumococcal disease caused by penicillin-resistant pneumococci [129].

In the Pacific, little is known of the prevalence of NP carriage of S. pneumoniae or antimicrobial resistance patterns. No antimicrobial resistance of S. pneumoniae has been reported previously in Fiji, where PCV is not routinely administered. This study was undertaken to document the prevalence of NP carriage of pneumococci, risk factors for carriage, antimicrobial susceptibility patterns of carried pneumococci, and the serotypes of carried pneumococci in healthy young children in Fiji.

3.3 Methods

3.3.1 Study Site

The Republic of Fiji Islands lies in the Pacific Ocean. It comprises two large islands, Viti Levu and Vanua Levu, and many smaller islands. The population of Fiji is approximately 775,000 (based on the 1996 census) with approximately 51% of the total population being indigenous Fijian and 44% Indo- Fijian. About 70% of the population lives on Viti Levu which has two medical divisions, the Western and Central divisions. The population of under-5s in the Central Medical division is 35,759.

3.3.2 Study Design

We conducted a cross-sectional survey of healthy children aged 3–13 months attending maternal and child health centres for routine immunisations from a selection of eight urban and 11 rural villages in the Central Division of the island of Viti Levu between October 2003 and April 2004. Approximately 10% of children in this age cohort and living in this division were enrolled in the study.

3.3.3 Risk Factor Evaluation

Parents/guardians were interviewed regarding the following risk factors associated with NP pneumococcal carriage: gender, age, ethnicity, number of siblings in the household, breastfeeding status, socio-economic status, exposure to tobacco or indoor cooking smoke, symptoms of ARI (cough, fever, or coryza), hay fever symptoms, prior antibiotic use, and any hospitalisations. Birthweight and current weight were confirmed by examining the child’s health card.

3.3.4 Laboratory Methods

Buffered cotton NP swabs (Sarstedt, aluminium shaft-buffered, Australia) were inserted horizontally into the nare, left in situ for 5 seconds and rotated, then immediately placed into a sterile cryovial tube (Simport, Canada) containing 2 ml of skim milk-tryptone-glucose-glycerol (STGG) transport medium [337]. This was transported to the laboratory on the same day at a temperature of <5°C. The swabs were processed according to the consensus guidelines from a World Health Organization working group [338]. The swabs were vortexed and either stored at -70 C until plated or plated upon arrival at the laboratory. For frozen samples, the swab was thawed at room temperature. Each sample was vortexed and 50 μl was inoculated onto a 2.5 mg/L gentamicin 5% sheep blood

Columbia (Oxoid) agar plate with an optochin disc (Diffco). Plates were incubated at 37 C in 5% CO2 for 18–24 hours. Pneumococcal isolates were initially identified by α-haemolysis, colony morphology and optochin sensitivity. Isolates with intermediate optochin were confirmed as pneumococci by bile solubility testing. One colony per plate was harvested for subculture on 5% sheep blood–agar plates and plates were re-incubated for a further 24 hours. All isolates were stored at -70 C.

Resistance was initially determined by the Bauer and Kirby disk diffusion method on Mueller Hinton media (Oxoid) with 5% sheep blood. The isolates were tested for susceptibility using 1 μg oxacillin discs, 15 μg erythromycin discs, 30 μg chloramphenicol discs and 1.25/23.75 μg trimethoprim/sulfamethoxazole discs (Diffco). The level of resistance was confirmed by minimum inhibitory concentrations (MIC) using E tests (Ab Biodisk, Dalvagen, Sweden). National Committee for Clinical Laboratory Standards (NCCLS) criteria were used to classify whether the isolates were sensitive, intermediate or resistant [354]. Isolates with an oxacillin zone size of ≥20 mm were considered penicillin-susceptible. Isolates non-susceptible to oxacillin had E tests performed to penicillin. In those that were penicillin-resistant or intermediate on E test, E tests were performed for erythromycin, chloramphenicol, cotrimoxazole and ceftriaxone, regardless of the disk sensitivity result. Strains found to be non-susceptible to three or more antibiotics were classified as multi-drug- resistant. Serogrouping/serotyping was performed by the Quellung reaction using specific antisera (Statens Seruminstitut, Copenhagen, Denmark). Those that were non-typeable or OMNI negative had lytPCR performed [355] to confirm that they were S. pneumoniae.

3.3.5 Statistical Analysis

Data were analysed using Stata version 9 (Stata Corporation, College Station, Texas). Weight-for-age Z-scores were calculated using USA reference standards. Underweight was defined as a weight-for- age Z-score ≤-2. Unadjusted odds ratios were calculated for risk factors for NP pneumococcal carriage. Variables with a p-value of <0.25 in the univariate analysis were evaluated in a multiple logistic regression model. Statistical significance was defined as p<0.05.

3.3.6 Ethics Approval

This study was approved by the University of Melbourne Human Research Ethics Committee and the Fiji National Research Ethics Review Committee.

3.4 Results

A total of 774 children were enrolled in the study. Seven did not have NP swabs taken. Owing to technical difficulties, only 440 consecutive NP swabs were taken with culture results available to determine a carriage rate (Figure 3). Altogether, 246 and 239 isolates were available, respectively, for susceptibility testing and serotyping. Five non-typeable isolates were lytPCR-negative and were therefore not S. pneumoniae. These PCR-negative isolates were not included in the analyses.

Figure 3 : Flowchart of nasopharyngeal swabs and sensitivity and serotype of pneumococcal isolates

774 eligible children

7 had no NP swab taken 767 NP swabs taken 327 NP swabs inappropriately stored 440 consecutive NP swabs available to determine NP carriage rate

195 positive 51 isolates pneumococcal survived storage isolates 246 isolates available for susceptibility testing

7 isolates not viable 239 isolates serotyped

223 with 7 OMNI- 9 non-typable serotype negative result

The characteristics of the 440 study children are shown in Table 4. Over three-quarters of participants were from an urban area. Over half the children were exposed to cigarette smoke in the home and over one-quarter were exposed to indoor cooking smoke.

Of the 440 swabs, 195 were positive for S. pneumoniae, an NP carriage rate of 44.3%. Higher carriage rates were found in the indigenous Fijian population (55.8%) than in other ethnic groups (21.3% and 30.3% for Indo-Fijian and others, respectively). The relative risk of having pneumococcal NP carriage in indigenous Fijians (158/283) compared with other ethnic groups (36/155) was 2.4 (95% CI 1.77–3.26).

Eleven per cent of isolates were penicillin-resistant (Table 5). Resistance to cotrimoxazole was common (20.3%). No isolates were resistant to chloramphenicol and resistance to erythromycin was uncommon (1.6%). Both isolates resistant to ceftriaxone were also resistant to penicillin. Three (1.2%) isolates were multi-drug-resistant. There was no clear association between antibiotic- resistant pneumococcal carriage and being an indigenous Fijian NP carrier (OR 2.56, 95% CI 0.36– 19.79).

One isolate had a high level of penicillin resistance (Table 6) and was susceptible to ceftriaxone. Over half the isolates resistant to cotrimoxazole had a high level of resistance.

Table 4: Characteristics of the study children (n=440) Characteristics n=440 Median age (range), mths 9.4 (2.5–13.3) Ethnicity,1 n (%): Indigenous Fijian 283 (64.6) Indo-Fijian 122 (27.9) Other 33 (7.5) Sex, n (%):2 Male 232 (52.9) Female 206 (47.0) Residence, n (%): Rural 97 (22.1) Urban 343 (77.9) Median family income per annum in FJ$ (range) 7020 (0–91,000) Low socio-economic status3, n (%) 158 (35.9) Median birthweight (range), g 3250 (500–4850) Low birthweight4 50 (11.3%) Malnourished5 34 (7.7%) Median number of children living in the same household (range) 2 (1–11) Median number <5 years old (range) 1 (1–5) Cigarette smokers at home, n (%) 239 (54.6) Cooking fuel used, n (%): Gas 263 (60.1) Electricity 212 (48.4) Wood 224 (51.1) Kerosene 343 (78.3) If wood or kerosene used, proportion of cooking done indoors, n (%) 224 (51.1) Hayfever symptoms, n (%) 55 (12.5) Symptoms of ARI, n (%) 64 (30.2) Hospitalisation within the last 3 mths, n (%) 13 (2.9) Antibiotics taken in last month, n (%) 60 (13.7) Currently breastfeeding, n (%) 334 (76.3) 1Ethnicity unknown for two children 2Sex was not recorded for two children 3Family income

Table 5: Antimicrobial resistance patterns of Streptococcus pneumoniae isolates (n=246) Antimicrobial Resistant isolates n (%) Penicillin1 28 (11.4) Erythromycin 4 (1.6) Chloramphenicol 0 Cotrimoxazole 50 (20.3) Ceftriaxone 2 (0.8) 1Of the 28 penicillin non-susceptible isolates, three were also resistant to erythromycin, 20 were also resistant to cotrimoxazole, two were also resistant to ceftriaxone and three were also resistant to both erythromycin and cotrimoxazole.

Table 6: Level of antimicrobial resistance of Streptococcus pneumoniae isolates (n=246) Number (%) of isolates resistant at: Antimicrobial Intermediate level1 High level2 MIC range (μg/ml) Penicillin 27 (10.9) 1 (0.4) 0.064–3.0 Erythromycin 1 (0.4) 3 (1.2) 0.5–16.0 Chloramphenicol NA3 0 Cotrimoxazole 23 (9.3) 27 (10.9) 0.75–16.0 Ceftriaxone 1 (0.4) 1 (0.4) 1.5–4.0 1Intermediate: penicillin 0.12–1.0 μg/ml, erythromycin 0.5–1.0 μg/ml, trimethoprim component of cotrimoxazole 1–2 μg/ml 2High level: penicillin ≥2 μg/ml, erythromycin ≥1 μg/ml, chloramphenicol ≥8 μg/ml, trimethoprim component of cotrimoxazole ≥8 μg/ml 3Not applicable. There is no intermediate level of resistance to chloramphenicol.

The commonest pneumococcal serotype was 6A (13.2%), followed by 23F (8.3%), 19F (7.4%) and 6B (6.2%) (Table 7). Thirty per cent of S. pneumoniae isolates were represented in PCV, 54.3% if the potentially cross-reactive strains are included (Table 7). Forty-six per cent of isolates were represented in the 23-valent pneumococcal polysaccharide vaccine, and 68% of isolates were represented in either PCV, including the cross-reactive serotypes, or 23vPPS. Of the 28 penicillin- resistant isolates, 65.5% were represented in PCV. The most common penicillin-resistant isolates were serotype 23F (34.5%), followed by 19F (10.3%) and 14 (10.3%).

Being indigenous Fijian, living in a rural area, being of low birthweight, having two or more children under 5 years of age in the household, exposure to indoor cooking smoke and having symptoms of ARI were all identified in the univariate analysis as statistically significant risk factors for NP pneumococcal carriage (Table 8). Breastfeeding was found to be protective. The results of multiple logistic regression showed that being indigenous Fijian or having symptoms of acute ARI were independent risk factors for NP pneumococcal carriage (Table 8). Having two or more children aged

88 under 5 years in the household or having a rural residence showed a trend towards statistical significance (Table 8). As the number of isolates was small, risk factors for penicillin-resistant NP carriage were not analysed.

Table 7: Serogroups/types of Streptococcus pneumoniae isolates from study children (n=239) Serogroups/types Pneumococci n (%) Serotypes included in PCV: 1 4 4 6B 15 9V 4 14 10 18C 3 19F 18 23F 20 Total 74 (30.9) Serotypes potentially cross reactive with those included in PCV: 1 6A 32 9N 3 18A 3 19A 8 19B 1 23A 7 23B 2 Total 56 (54.3% coverage by 7-valent PCV & cross reactive serotypes) Other serotypes included in the 23vPPS: 2 3 3 7F 2 8 4 12F 2 15B 12 17F 7 20 2 22F 1 33F 1 Total 110 (46% coverage by 23vPPS†) Other serotypes/groups 59 (24.7) OMNI-negative 7 (2.9) Non-typeable 9 (3.8) 17-valent pneumococcal conjugate vaccine 223vPPS: 23-valent pneumococcal polysaccharide vaccine

Table 8: Risk factors for pneumococcal nasopharyngeal carriage n Unadjusted odds ratio p-value Adjusted odds ratio p-value (95% CI) (95% CI) Indigenous Fijian1 283 4.09 (2.65–6.35) <0.001 2.81 (1.76–4.49) <0.001

Male 232 1.12 (0.77–1.64) 0.541 Low family income2 158 0.92 (0.51–1.64) 0.777 Rural residence1 97 2.36 (1.49–3.75) <0.001 1.64 (0.96–2.80) 0.073 Low birth weight1,3 50 0.40 (0.21–0.78) 0.007 0.61 (0.29–1.24) 0.172 Weight-for-age ≤-2 SD 34 1.16 (0.48–2.77) 0.741 ≥2 children living in the same house & 184 1.55 (1.06–2.27) 0.024 1.49 (0.98–2.25) 0.061 aged <5 y1 Exposure to cigarette smoke 239 1.21 (0.83–1.76) 0.328 Indoor cooking with wood1 224 2.17 (1.48–3.18) <0.001 1.43 (0.91–2.24) 0.123 Any hospitalisation in last 3 months 13 0.78 (0.25–2.42) 0.667 Antibiotics taken within last month 60 0.75 (0.43–1.31) 0.316 Hayfever symptoms1 55 1.58 (0.99–2.54) 0.057 1.15 (0.68–1.92) 0.605 Current ARI symptoms1 64 2.28 (1.55–3.35) <0.001 1.93 (1.27–2.91) 0.002 Breastfeeding1 334 0.56 (0.35–0.88) 0.013 0.74 (0.45–1.23) 0.244 1 Variables included in the multivariate analysis 2Annual family income

3.5 Discussion

Pneumococcal NP carriage is common (44.3%) in young children in Fiji. This is similar to findings in a nearby Pacific island country, New Caledonia, where 52% of children aged <2 years had pneumococcal NP carriage[356] but lower than the 100% of young infants in Papua New Guinea [357]. The prevalence of NP carriage of pneumococci varies according to geographical location and has been reported to be as low as 5.7% in France [358]. Pneumococcal carriage rates parallel rates of invasive pneumococcal disease, [9, 10] so these data suggest that Fijian children are likely to suffer a high burden of pneumococcal disease.

Indigenous Fijian children were at greater risk of pneumococcal NP carriage than Indo-Fijian children (RR 2.4). This was an independent risk factor for NP pneumococcal carriage and might be related to overcrowding, genetic susceptibility or other unknown factors. This might partly explain the findings of a recent study in Fiji which showed that indigenous Fijian children were 29 times more likely than Indo-Fijian children to have chest X-ray-confirmed pneumonia [83]. Acquisition and NP carriage of pneumococci has been shown to be associated with the occurrence of pneumonia [10]. Differences in carriage rates in children of different ethnicity living in the same country have been reported previously in neighbouring New Caledonia, where being an indigenous child (Melanesian) was a risk factor for pneumococcal NP carriage [356]. Pneumococcal NP carriage rates in Chinese and Vietnamese children living in Hong Kong demonstrated a pneumococcal NP carriage rate of 10.8% and 55.7%, respectively [359]. Healthy, young Jewish and Bedouin children in Israel had carriage rates of 35% and 67%, respectively [360]. This higher NP carriage rate in certain ethnicities might be owing to children living in lower socio-economic circumstances being more at risk of pneumococcal NP carriage. In this study, 41.7% (n=118) and 19.7% (n=24) of the indigenous Fijian and Indo-Fijian populations, respectively, were of low socio-economic status as defined by the Fiji Department of Statistics. In this study, low family income was not found to be a risk factor for carriage. Perhaps the sample is too small or genetic factors play a more important role in this population, or the definition of low socio-economic status is invalid.

This study found that having symptoms of ARI was also an independent risk factor for pneumococcal NP carriage. Having two or more children under 5 years in the household or residing in a rural area showed a trend towards statistical significance. Other studies have shown that risk factors for NP pneumococcal carriage include attendance at a day care centre [361-364], young age, current AOM [365] or ARI [363, 364], recent use of antibiotics [365, 366] and living with more than one sibling [364]. It is very uncommon for children in Fiji to attend day care.

Penicillin resistance has been documented for the first time in Fiji. High-level resistance is currently uncommon but ongoing surveillance is required, particularly of invasive pneumococcal isolates. Penicillin resistance has increased worldwide over the past 2 decades, but geographical patterns vary considerably. The level of resistance in Fiji is currently relatively low (11%) and most likely reflects the regulated prescribing practices in the country as antimicrobial use, both by individuals and at community level, has been shown to be strongly associated with NP carriage of penicillin- resistant pneumococci [352].

Resistance to cotrimoxazole is high (20.3%) and this antibiotic is no longer recommended for the treatment of pneumonia in Fiji. Macrolide resistance was found to be low (1.6%), probably because of the low level of prescribing this antimicrobial in Fiji. Most countries have relatively low macrolide resistance rates, although the geographical prevalence is variable [367-369]. In Hungary and South Africa, where rates of macrolide use are high, the resistance rate is approximately 50% [369, 370]. We found no chloramphenicol resistance and a low rate of multidrug resistance.

As carriage with pneumococci has been correlated with clinical disease [9-11, 371, 372], the first-line treatment for paediatric meningitis in Fiji has now changed from penicillin and chloramphenicol to 3rd-generation cephalosporins. Because clinical failures have been documented despite chloramphenicol susceptibility in vitro [349], chloramphenicol is not recommended as an alternative for treatment of meningitis where there are penicillin-resistant pneumococci. High-dose penicillin will continue to be recommended for treating pneumonia [373].

There is low serotype coverage (30%) among carriage isolates for the PCV. This is lower than that found in New Caledonia where there was 46% coverage for those isolates included in the PCV [356]. In Vietnam, the rate of NP carriage coverage of the PCV was 80% [374]. The rate of NP carriage coverage of the PCV was approximately 64.6% in Asian children [374], 47.5 % in young Australian Aboriginal children with ARI [375]and approximately 76% in young French children [376]. We found that the additive coverage of the 11-valent PCV (covering the additional serotypes 1, 3, 5 and 7F) was negligible. NP carriage studies tend to over-represent ‘paediatric’ serotypes in the PCV, and more virulent serotypes, such as serotype 1, are carried less frequently [63]. In this study, NP carriage isolates have a wider serotype distribution and therefore coverage of invasive pneumococcal serotypes by PCV might also be low.

About two-thirds of the resistant pneumococcal isolates were covered by the PCV which is lower than has been reported in most other settings, except South India where 42% of carriage isolates were penicillin-resistant [377]. The rates of NP carriage of resistant pneumococcal isolates covered by the PCV in Greece, Uganda, South Africa, Brazil, and China were 72%, 80% , 42%, 86%, 91%

93 respectively [378-382]. In this study, serotype 23F was the commonest penicillin-resistant serotype. Penicillin resistance has been predominantly associated with serotypes 6B, 14, 19F and 23F.

In conclusion, pneumococcal NP carriage is common in Fijian children and might correlate with a high burden of pneumococcal disease. Penicillin resistance has been documented for the first time in Fiji and, consequently, first-line treatment for meningitis has been altered. Being indigenous Fijian is a risk factor for carriage, the cause of which is unclear. We found low serotype coverage among carriage isolates for the PCV, which might translate to low coverage among invasive isolates. These results highlight the value of carriage studies in providing a profile of pneumococcal strains and antimicrobial resistance patterns in a population.

4 EPIDEMIOLOGY OF INVASIVE PNEUMOCOCCAL DISEASE IN FIJI: THE POTENTIAL IMPACT OF PNEUMOCOCCAL CONJUGATE VACCINE

4.1 Abstract

IPD epidemiology and the potential impact of the pneumococcal conjugate vaccine in Fiji was documented. The annual incidence was 26.5 and 10.9 in those aged <5 and >55 years per 100,000 respectively. The CFR was 9.4% and 67% in <5 and >65 year olds respectively. One pneumococcal death and case would be prevented in <5 years olds for every 1,930 and 128 infants vaccinated with PCV respectively.

4.2 Introduction

In 2000, over 14 million episodes of serious pneumococcal disease were estimated to have occurred worldwide, with over 800,000 deaths in children <5 years [41]. PCV in the USA has led to an impressive reduction in childhood IPD [52] and has had unanticipated herd immunity effects [52]. The importance of including all ages in the epidemiological estimates assists in calculating the true economic burden and the likely direct and indirect effects of PCV introduction. No IPD burden studies involving all ages have previously been performed in low or middle income countries.

This study aims to document age-specific IPD in terms of burden and mortality, clinical syndromes, serotype distribution, and the potential coverage of IPD by pneumococcal conjugate vaccines in all ages in the Central Medical Division, Fiji, prior to PCV introduction. In addition, the potential impact of PCV on IPD and chest radiograph confirmed pneumonia was estimated.

4.3 Methods Fiji is a low-middle income country and consists of Indigenous Fijians (57%) and Indo-Fijians (38%) who are of Indian ethnicity. Over 75% of the population lives on one island, Viti Levu, which has two medical divisions, the Western and Central divisions. The IMR is 18.4 per 1,000 live births (Fiji Ministry of Health Annual Report, 2007). HIV prevalence is <1%.

This was a prospective laboratory based surveillance study. All invasive S. pneumoniae isolates from the CWMH microbiology laboratory, Suva, from 1st July 2004 to 31st October, 2007 were collected. CWMH is the only tertiary referral hospital in Fiji, the referral hospital for serious illness, and the only public hospital that provides microbiology services in the Central division. There are 4 sub-divisional hospitals

within the catchment, however all children with severe illness are referred to CWMH for medical care. PCV and the 23vPPS are available privately but their current use is negligible.

As a routine, all hospitalized febrile pediatric patients have blood cultures drawn, hospitalized adult patients with suspected pneumonia may have blood cultures drawn and all patients with suspected sepsis or pyrexia of unknown origin have blood cultures drawn. All suspected meningitis cases have blood cultures and lumbar punctures performed unless contra-indicated. Identification of pneumococcal isolates in the CWMH laboratory is based on routine methods. All isolates were stored at -70ºC prior to shipment to Westmead Hospital, Centre for Infectious Diseases and Microbiology, Australia for serotyping by Quellung reaction. In addition, any serotype 6A and 6B isolate identified was also serotyped by multiplex-PCR and reverse-line blot assay which is able to detect serotype 6C and 6D [58, 340].

Clinical syndromes were assigned based on presenting symptoms, clinical findings, and laboratory results. Meningitis was defined as S. pneumoniae CSF culture positive or a clinical diagnosis of meningitis with positive blood culture. Bactaeremic pneumonia was defined as S. pneumoniae blood culture positive and physician diagnosis of pneumonia. Pneumococcal sepsis was defined as S. pneumonia blood culture positive, no clinical focus of infection, and severe enough to warrant ICU admission, and/or newly diagnosed organ failure or cardiovascular instability. Pneumococcal bacteremia without a focus was defined as S. pneumoniae blood culture positive without a clinical focus of infection, and no documented evidence of sepsis.

Data were exported to Stata version 9.0 (Stata Corporation, College Station, Texas) for analysis. The numerator for the incidence rates was calculated using the number of annual IPD cases and the denominator was the catchment population of the Central Medical division from the 2007 Census. The data from this study based in the Central Medical division was extrapolated to make national estimates for IPD in children aged <5 years assuming the distribution of IPD cases was equal in all divisions. This data was used to calculate the number of PCV preventable episodes of IPD in <5 year olds using the following formula extrapolated from the Hib Rapid Assessment Tool [383]: the estimated annual number of IPD cases/deaths in Fiji was multiplied by the vaccine efficacy (VE) of 97.4% for PCV from published IPD data [103], multiplied by the local serotype coverage for IPD in <5 year olds (ascertained from our study), multiplied by Fiji’s DTP3 immunisation coverage of 98% (Fiji Immunisation coverage survey, 2009). For chest radiograph confirmed pneumonia in <5 year olds, the number of PCV preventable

episodes was calculated using the following formula: the number of cases/deaths were ascertained from previously published Fiji data [83], and multiplied by the published VE for chest radiograph confirmed pneumonia of 25.5% [205], multiplied by an DTP3 coverage rate of 98%.

This study was approved by the University of Melbourne Human Research Ethics Committee and the Fiji National Research Ethics Review Committee.

4.4 Results

There were 83 episodes of IPD identified. The annual incidence of IPD was 26.5 and 10.9 in <5 and ≥55 year old population per 100,000 respectively. The annual incidence by age shows a peak at both extremes of age (Figure 4). Compared with Indo-Fijians, indigenous Fijians were over 4 times more likely to develop IPD (IRR 4.3, 95% CI 2.1-10.3).

Figure 4: Annual incidence of IPD by age group in the Central Medical Division, Fiji from 1st July 2004 to 31st October, 2007

35 Indo-Fijian Fijian 30 Total

10 Annual incidence per 100,000 per Annualincidence

0-4 5-9y 75+ 15-19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74 10-14y Age (years)

Meningitis and bactaeremic pneumonia were the commonest clinical manifestations in <5 year olds (Table 9). Bactaeremic pneumonia accounted for 56.9% of IPD cases in those aged ≥5 years. Meningitis

was more common in the <5s than those aged ≥5 years. The length of hospital stay was longer in those <5 years compared with the older age group, and a higher proportion (31.3% versus 3.9%) were admitted to ICU largely because meningitis was a more common clinical manifestation in young children.

Table 9: Clinical manifestations of IPD cases by age (n=83) Clinical Manifestations <5 years (n=32) ≥5 years (n=51) n (%) n (%) Meningitis 11 (34.4) 5 (9.8) Pneumonia 11 (34.4) 29 (56.9) Sepsis 4 (12.5) 11 (21.6) Bacteremia without focus 5 (15.6) 4 (7.8) Pericarditis 1 (3.1) 0 Septic arthritis 0 1 (2) Unknown 0 1 (2) Underlying conditions Immunodeficiency/suppression 0 2 (3.9) Cancer 0 1 (2) HIV 0 1 (2) Diabetes 0 7 (13.7) Congenital heart disease 2 (6.3) 1 (2) Other cardiovascular disease1 2 (6.3) 11 (21.6) Chronic lung disease 0 3 (5.9) Chronic renal disease 0 2 (3.9) Malnutrition 2 (6.3) 3 (5.9) Pre-term infant 1 (3.1) NA Management Length of hospital stay in days 11 (5-15) 2 6 (3-9) 2 For meningitis 14 (10-15) 2 33 For bacteraemic pneumonia 12 (5-21) 2 6 (4-8)2 Admitted to ICU 10 (31.3) 2 (3.9) ICU length of stay in days 4 (3-8) 2 6.5 (2-10)2 Deaths (n=17) 3 (9.4) 14 (27.5) 1Includes ischemic heart disease, hypertension, arrhythmias, cor pulmonale, rheumatic heart disease 2Median (interquartile range) c3One case only HIV Human immunodeficiency virus ICU Intensive care unit NA Not applicable

There were 17 deaths (CFR 20.5%, 9.4% in those aged <5 years and 53.3% in those aged ≥55 years). The CFR by age group is shown in Figure 5. Nearly half of the deaths (47.1%) were in those aged ≥55 years. Of those that died, 47.1% had sepsis. Underlying conditions were present in 64.7% of those that died.

Figure 5: IPD case fatality rates in the Central Medical Division, Fiji, by age group

20 Case Fatality Rate, % Rate, CaseFatality 10

0 <1 1-4y 5-14y 15-54 55-64 65+ Age group (years)

The commonest serotypes were 14 (33.3%) and 6B (13.3%) in children <5 years old and serotypes 7F (18.8%) and 1 (16.7%) in those ≥5 years (Figure 6). Six serotypes (14, 6B, 1, 3, 6A, 7F) accounted for 68.8% of all serotypes in children <5 years. The potential coverage of IPD cases by pneumococcal conjugate vaccine improved as the valency increased (Figure 7). The 23vPPS would have no potential additional benefit to the 13-valent PCV in the <5s due to the high prevalence of serotype 6A which is included in the 13-valent PCV but not included in the 23vPPS.

100

Figure 6: Serotype distribution amongst IPD cases (n=78)

35 100

90 30 80

25 70

60 % 20 All ages 50 <5y

15 Cumulative % of isolatesof% Cumulative % all ages 40

10 30

20 5 10

0 0

1 4 3 8 14 7F 6B 9V 6C 20 12F 19F 6A 10F 19A 28A 10A 11A 15A 18F Serotypes

Figure 7: Proportion of IPD isolates, by age, potentially covered by the 7, 10, and 13-valent pneumococcal conjugate vaccine, and the 23-valent pneumococcal polysaccharide vaccine

100 93.8% <5y ≥5y 90 83.3% 83.3% 80 72.9% 66.7% 66.7% 70

60 53.3% 50

40 31.3%

30 Percent of isolates, % 20

0 7-valent conjugate 10-valent 13-valent 23-valent conjugate conjugate polysaccharide Pneumococcal vaccine

101

The estimated annual numbers of IPD and chest radiograph confirmed pneumonia cases and deaths in <5 year olds and the number of potentially averted cases and deaths if PCV were introduced are shown in Table 10. For every 1,930 infants vaccinated, one death would be prevented. For every 128 infants vaccinated one case would be averted.

Table 10: Estimated annual number of IPD and hospitalized chest X-ray confirmed pneumonia cases and deaths in <5 year olds in Fiji, and the estimated number of cases and deaths averted if PCV were introduced Cases Deaths Cases Deaths averted averted IPD 22 2 11 1 CXR-confirmed pneumonia 562 39 140 9 TOTAL 584 41 151 10

4.5 Discussion

To our knowledge this is the first report of IPD which includes all ages from a low-middle income country. In this study IPD is common at both age spectra. The higher rate of IPD between different ethnic groups living within the same geographical region has been described elsewhere and may be related to overcrowding, genetic susceptibility, poorer living conditions, or other unknown factors.

Our data differ substantially from the global burden of disease estimates of 1,367 cases per 100,000 <5 year olds and a CFR of 1.9% [41]. Our estimates have fewer cases but higher mortality compared to these estimates. These differences are likely related to the methodologies employed and the reliability of routinely reported data to make global estimates compared to data collected in special studies.

Comparing disease burden rates between geographical sites is difficult due to many differing clinical and laboratory practices. Recently WHO and the PneumoADIP standardized case definitions for the surveillance of pneumococcal disease [48]. Where IPD incidence rates were calculated and countries had similar IMR rates to Fiji, Vietnam, a low-income country, had a higher IPD rate (48.7 per 100,000 <5s) [49], and rural Thailand, a middle-income country, had a similar IPD rate (10.6-28.9 per 100,000 <5s) to our study [50]. There are no published IPD data for all age groups from other Pacific island countries but our data is likely to be similar and relevant for these regional countries.

102

In this study the CFR of IPD in children <5 years was approximately double the reported rate in industrialized countries but similar to that reported in Chile (10%) [46] and Mozambique [384]. For the elderly, the presence of underlying conditions often contributes to the high CFR. In those aged >65 years, the CFR was <30% in industrialized countries. The rate in Fiji was much higher at 67% in those aged >65 years. This may be due to our study being hospital based and therefore only included persons with more severe disease who were sick enough to be hospitalized. This may indicate that the true burden of disease is higher and overall CFR is lower than measured in this study. There is no data to compare to other low-middle income countries. Over half who died in this age group had underlying conditions.

The commonest serotypes causing IPD in <5 year olds in Fiji shows a similar distribution to other countries [61]. The potential coverage of IPD in Fiji by the PCV is low and substantially lower than that reported in the USA prior to vaccine introduction [51, 54]. Only hospitalised IPD cases were included in our study which may partially explain this disparity. The 13-valent PCV would provide good universal coverage for all age groups in Fiji due to the addition of serotypes 1 and 7F.

In conclusion, this study provides a reliable baseline of IPD in Fiji in the pre-vaccination era. The importance of including all ages in the estimate assists in calculating the true economic burden and the likely direct and indirect effects of PCV. The major challenge for Fiji in PCV introduction is the issue of vaccine financing. At US$53 per dose, the cost to fully vaccinate the birth cohort with 3 doses would require the total vaccine procurement budget to increase over 6-fold.

103

5 IMMUNOGENICITY FOLLOWING ONE, TWO, OR THREE DOSES OF THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE

5.1 Abstract

The aim was to identify an appropriate infant pneumococcal vaccination strategy for resource poor countries. Fijian infants received 0, 1, 2, or 3 doses of 7-valent pneumococcal conjugate vaccine (PCV) in early infancy. Following 3 PCV doses, geometric mean concentration (GMC) to all 7 serotypes were ≥1.0µg/mL, and >85% of children achieved antibody levels ≥0.35µg/mL at 18 weeks. Following 2 doses, GMC were lower for 6B, 14, and 23F, but higher for 19F compared with 3 doses. Following a single dose, significant responses were seen for all serotypes post primary series compared with the unvaccinated. By 12 months, differences between 2 and 3 doses persisted for serotype 14 only. Although GMC following 3 doses are higher than after 2 doses, the differences were small. A single dose may offer some protection for most serotypes.

5.2 Introduction

S. pneumoniae is the most common cause of bacterial pneumonia in children worldwide. It is the leading vaccine preventable cause of serious infection in infants [3]. An estimated 1.6 million deaths are attributable to pneumococcal disease each year with the majority of these deaths occurring in low income countries primarily in children and the elderly [4]. The case fatality rate is particularly high in infants less than 6 months old [5]. Over 40 serogroups comprising of over ninety serotypes of pneumococcus have been identified [57]. Within serogroups, serotypes cross-react immunologically, and in some cases this translates into cross-protection. The association of particular serotypes with disease varies according to age, geography, and clinical site. Serotypes 6B, 14, and 19F are important worldwide, while serotype 5 is mainly found in low income countries and 18C is more common in affluent countries [59]. In general, the range of serotypes causing disease in affluent countries like the United States and in Europe are relatively narrow and largely confined to the serotypes found in PCV. PCV includes only serotypes 4, 6A, 9V, 14, 18C, 19F, 23F. In contrast, the range of serotypes causing disease in low income countries is wider. Therefore PCV covers a smaller proportion of the pneumococcal serotypes causing disease in children in low income countries compared to more affluent countries.

In the USA, PCV has been shown to be safe and efficacious in a 3 dose primary series with a booster in the second year of life [103]. Following the introduction of PCV into the US national immunisation

104

schedule in 2000 there has been a significant decline in vaccine VT IPD in all ages [52]. Replacement disease, particularly due to serotype 19A has developed due to capsular switching and clonal expansion [220]. A study from the UK trialled alternative schedules with the aim of reducing the number of PCV doses in the primary series and administering an earlier booster at 12 months of age (“2+1” schedule) [157]. The UK subsequently introduced a “2+1” schedule in 2007, in which children receive PCV at 2, 4 and 13 months. In some Scandinavian countries and Italy, routine immunisations are given in a 2 dose primary series with a booster at or before the end of the first year of life. When the introduction of PCV into the USA national immunisation schedule was met with a global shortage of vaccine, many children received fewer than the recommended 4 doses of vaccine. A case control study documenting the impact of this on IPD due to vaccine serotypes showed that one and 2 dose schedules given to infants less than 7 months of age had an effectiveness of 73% (95%CI 43-87%) and 96% (95%CI 88-99%) respectively [242].

From seroprevalence data collected in Fiji, the 7 serotypes included in PCV would cover 55% of episodes of IPD in children aged under 5 [385]. This would potentially increase to 83% if 23vPPS was used, and due to the high prevalence of serotype 6A which is not included in 23vPPS, this would increase to 87% if the new 13-valent pneumococcal conjugate vaccine produced by Wyeth Vaccines (which includes serotypes 1, 3, 5, 6A, 7F and 19A) was used [385]. The aim of this study was to find a vaccination strategy for resource poor countries in terms of serotype coverage, flexibility, and affordability. To address these issues, we undertook a Phase II vaccine trial in Fiji to document the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination regimens combining 1, 2, or 3 doses of PCV in infancy. In order to broaden the serotype coverage, the additional benefit of a booster of 23vPPS at 12 months of age was also assessed. This paper compares the geometric mean serotype specific IgG antibody concentrations following the different PCV primary series, and up to 12 months of age.

105

5.3 Methods

5.3.1 Study Participants

The study was a single blind, open-label randomised Phase II vaccine trial in Suva, Fiji. Healthy infants aged between 6 and 8 weeks were eligible for enrolment if they had no significant maternal or perinatal disease history; they resided within 30 minutes of one of the three participating health centres; and the family anticipated living in the study area for 2 years. Infants were excluded if they had: a known allergy to any component of the vaccine; an allergic reaction or anaphylactoid reaction with previous vaccines; a known immunodeficiency disorder; a HIV positive mother; known thrombocytopenia or coagulation disorder; were on immunosuppressive medication; received any blood product since birth; a severe congenital anomaly; a chronic or progressive disease; a seizure disorder; or a history of invasive pneumococcal, meningococcal, or H. influenzae diseases prior to study entry.

The study was conducted and monitored according to Good Clinical Practice. It was jointly approved by the Fiji National Research Ethics Review Committee and the University of Melbourne Human Research Ethics Committee. Written, informed consent was sought from families of children eligible to join the study according to methods approved by the overseeing ethics committees.

5.3.2 Study Procedures and Vaccines

Infants were enrolled at the time they presented to any one of 3 participating health centres to receive their first dose of the combined Diphtheria-Tetanus- whole cell Pertussis-H. influenzae type b- Hepatitis B vaccine (Hiberix containing 10μg of purified Hib capsular polysaccharide covalently bound to approximately 30μg tetanus toxoid mixed with Tritanrix -HepB containing not less than 30 IU of adsorbed D toxoid, not less than 60 IU of adsorbed T toxoid, not less than 4 IU of wP, and 10µg of recombinant HBsAg protein, GlaxoSmithKline) at 6 weeks of age. Randomisation lists were produced by the study statistician and group allocation was concealed in opaque envelopes which study nurses removed sequentially from a box. Eligible infants were stratified by ethnicity and randomised using a computer-generated list of random numbers in blocks of variable size to one of 8 groups to receive 0,

1, 2, or 3 doses of PCV. The 7-valent CRM197 protein-polysaccharide conjugate vaccine containing polysaccharide antigen from pneumococcal serotypes 4, 6B, 9V, 14, 18C, 19F, 23F (Prevnar , Wyeth Vaccines) was used. The vaccine contains 2 g/serotype, except serotype 6B which is 4 g. The 3 dose group received PCV at 6 weeks (window 6-8 weeks of age), 10 weeks (window 8-12 weeks of age) and

106

14 weeks of age (window 12-16 weeks of age). The 2 dose group received PCV at 6 and 14 weeks of age and the one dose group received PCV at 14 weeks of age. Vaccines were given a minimum of 25 days apart. Routine vaccines (Hiberix mixed with Tritanrix-HepB ) and oral polio were given at 6, 10, and 14 weeks of age. Hiberix /Tritanrix-HepB and PCV were given in the right and left anterolateral thigh respectively. The children in all primary series groups were further randomized to receive 23vPPS (Pneumovax , Merck Sharpe Dohme, which consists of a purified mixture of 25µg of capsular polysaccharide from each 23 pneumococcal serotypes) or no booster at 12 months of age. All children received Measles-Rubella vaccine at 12 months of age. Responses to the 12 month 23vPPS vaccination will be presented elsewhere.

5.3.3 Laboratory Procedures

All children had blood taken at 18 weeks and 12 months of age. Those children that were not randomized to receive the 12 month 23vPPS additionally had blood taken at 9 months of age. The 12 month blood sample was taken prior to the administration of the 23vPPS, so that the results presented in this manuscript are from the 7 groups that had blood taken in the first year of life. Blood was separated in the health centre, kept chilled and transported to the Colonial War Memorial Hospital laboratory where it was divided into aliquots and stored at –70 C or -20 C on the same day, until transported to the Pneumococcal Laboratory, Murdoch Childrens Research Institute, Melbourne on dry ice for analysis.

Anticapsular pneumococcal antibody levels were assayed for all serotypes in PCV at 18 weeks, 9 and 12 months of age, using a modified WHO ELISA method [343]. In brief, microtitre wells were coated with pneumococcal polysaccharide diluted in phosphate buffered saline by incubating at room temperature overnight. To neutralise non-specific cell wall polysaccharide (CPS) antibodies, serum samples were diluted 1/100 in pre-absorption buffer containing CPS (10µg/mL) and serotype 22F (30µg/mL) and incubated overnight at 4oC. The reference serum standard 89-SF (Food and Drug Administration, Bethesda MD) was pre-absorbed with CPS at 10µg/mL and incubated overnight at 4oC. Horseradish peroxidase conjugated anti-human IgG and a TMB (3.3’, 5.5’-tetramethylbenzidine) substrate solution were used for detection. A high, medium, and low control serum were used on each plate to assess assay performance and inter-assay variation. Results from an inter-laboratory correlation between Wyeth Vaccines and the KTL Finland laboratory demonstrated a good correlation

107

in serotype specific antibody concentrations [343]. Laboratory staff were blinded to the group allocation of each serum sample.

5.3.4 Statistical Analysis

This manuscript reports analytic results concerning the secondary purpose of the trial. All case reporting forms were monitored prior to double data entry. Cleaned data were exported to Stata version 9.0 (Stata Corporation, College Station, Texas) for analysis. Serotype specific antibody concentrations by ELISA were log (base e) transformed to calculate GMC. Pair-wise comparisons of serotype specific GMC between 0-3 dose PCV groups were performed using a two sample t-test. Comparisons of the proportion of infants between groups with serotype specific antibody concentrations ≥0.35 and ≥1µg/mL for 0-3 dose PCV groups were performed using Fisher’s exact test. A p-value of <0.01 was considered statistically significant due to the multiple comparisons.

5.4 Results

There were 552 children enrolled in the study (Figure 8). Characteristics and the number of children randomized each PCV group are shown in Table 11. The consent rate was 30.5%. At 12 months of age the withdrawal rate among children who had consented to join the study was 10%. No participant was withdrawn due to a reaction to any of the vaccines.

At 18 weeks of age, GMC in the 2 dose group were significantly lower (each p<0.001) for 3 of 7 serotypes (6B, 14, 23F), while the GMC for serotype 19F was significantly higher (p=0.003) than in the 3 dose group (Table 12). Similarly, the proportions of infants with antibody concentrations ≥0.35μg/mL were significantly lower for serotypes 14 (p=0.001) and 23F (p=0.003) when comparing the 2 and 3 dose groups. The proportions of infants with antibody concentrations ≥1μg/mL were significantly lower for serotypes 6B (p<0.001), 14 (p=0.004), and 23F (p<0.001) in the 2 dose group when compared to the 3 dose group (Table 13).

By 9 months of age there were minimal differences between antibody levels in the 2 and 3 dose groups. At 12 months of age antibody levels against serotype 14 were still significantly lower (p=0.001) in the 2 dose group, with serotype 18C also being lower (p=0.011) (Table 12). The proportions of children with antibody concentrations ≥0.35μg/mL and ≥1μg/mL were not significantly different in the 2 dose group compared to the 3 dose group for any serotype (Table 13).

Figure 8: CONSORT chart of the screened and enrolled children to 12 months of age 108

1811 screened 1259 did not consent

552 randomised

A=71 B=65 C=76 D=80 E=62 F=66 G=63 H=692

Withdrawn=4 Withdrawn=3 Withdrawn=3 Withdrawn=2 Withdrawn=3 Withdrawn=3 Withdrawn=1 ●Parent refused study ●Parent refused ●Failure to ●Development ●Parent refused ●Protocol ●Parent refused procedure=1 study procedure=1 return for follow of exclusion study violation=2 study procedure ●Voluntary ●Moved out of up=2 criteria=1 procedure=1 ●Development withdrawal=2 study area=2 ●Moved out of ●Voluntary ●Voluntary of an exclusion ●Moved out of study study area=1 withdrawal=1 withdrawal=2 criteria=1 area=1 3PCV=67 3PCV=62 2PCV=73 2PCV=78 1PCV=59 1PCV=63

Withdrawn=1 Withdrawn=1 Withdrawn=3 ●Parent refused ●Protocol ●Voluntary Unsuccessful study procedure violation withdrawal=2 blood draw=1 ●Unsuccessful ●Moved out of blood draw=1 study area=1

18w 18w 18w 18w 18w 18w 18w blood=62 18w blood=NA blood=67 blood=61 blood=71 blood=75 blood=59 blood=62

Withdrawn=5 Withdrawn=8 Withdrawn=1 Withdrawn=3 Withdrawn=4 Withdrawn=2 Withdrawn=3 ●Failure to return for ●Protocol ●Development ●Protocol ●Development ●Moved out of ●Failure to follow up=2 amendment=6 of an exclusion amendment=2 of an exclusion study area=1 return for follow ●Parent refused study ●Moved out of criteria=1 ●Moved out of criteria=1 ●Protocol up=1 procedure=1 study area=2 study area=1 ●Moved out of amendment=1 ●Moved out of ●Development of an study area=3 study area=1 exclusion criteria=1 ●Parent

9m blood=49 9m blood=43 refused study 1 NA =22 NA1=12 procedures= 9m blood=37 1 NA1=24 Refused blood=1 Withdrawn=1 ●Moved out of Not taken=1 study area

12m blood=60 12m blood=53 12m blood=70 12m blood=72 12m blood=54 12m blood=61 12m blood=59 12m blood=NA

A=3PCV; B=3PCV+ 12 month 23vPPS; C=2PCV; D=2PCV+ 12 month 23vPPS; E=1PCV; F=1PCV+ 12 month 23vPPS;G=12 month 23vPPS; H=no vaccines in the first 12 months 1NA=Not applicable due to a protocol amendment in which the timing of the 23vPPS was changed to 12 months from 6 or 9 months of age after 228 children had been recruited. 2 Group H did not contribute data to this manuscript as no blood samples were taken in the first 12 months.

1NA: Not applicable due to protocol amendments following enrollment 2Group H did not contribute data for this manuscript as no blood samples were taken in the first 12 months 109

Table 11: Baseline characteristics of infants at enrolment and randomised to the different PCV groups Characteristic 3 PCV 2 PCV 1 PCV 0 PCV (n=136) (n=156) (n=128) (n=63) Gender Male 71 (52%) 70 (45%) 59 (46%) 32 (51%) Female 65 (48%) 86 (55%) 69 (54%) 31 (49%) Median age in weeks 6.7 6.4 6.5 6.5 Ethnicity Indigenous Fijian 82 (60%) 106 (68%) 83 (65%) 35 (55%) Indo-Fijian 46 (34%) 45 (29%) 39 (31%) 20 (32%) Other 8 (6%) 5 (3%) 6 (4%) 8 (13%) Median weight in grams 4900 4800 4825 4750

110

Table 12: Geometric mean concentrations (GMC) of serotype-specific IgG titres taken 4 weeks following the PCV primary series, and at 9 and 12 months of age, by number of PCV doses administered in the primary series Serotypes 3 PCV (n=1251) 2 PCV (n=146) 1 PCV (n=121) 0 PCV 3,4 (n=62) GMC GMC 3 vs 2 GMC 3 vs 1 GMC (95%CI) (95%CI) p value2 (95%CI) p value2 (95%CI) 4 weeks post primary series

4 5.47 (4.84-6.19) 5.23 (4.46-6.13) 0.661 2.20 (1.80-2.70) <0.001 0.04 (0.03-0.04) 6B 1.66 (1.33-2.07) 0.86 (0.70-1.07) <0.001 0.19 (0.16-0.22) <0.001 0.11 (0.10-0.13) 9V 4.76 (4.19-5.40) 4.71 (3.88-5.71) 0.933 0.90 (0.74-1.09) <0.001 0.07 (0.06-0.08) 14 5.51 (4.50-6.76) 3.12 (2.42-4.03) <0.001 1.07 (0.89-1.27) <0.001 0.34 (0.27-0.43) 18C 3.20 (2.66-3.86) 2.67 (2.16-3.31) 0.22 0.58 (0.45-0.74) <0.001 0.06 (0.05-0.07) 19F 5.52 (4.79-6.36) 7.99 (6.62-9.64) 0.003 0.84 (0.70-1.00) <0.001 0.25 (0.21-0.30) 23F 2.93 (2.39-3.59) 1.65 (1.29-2.11) <0.001 0.23 (0.20-0.27) <0.001 0.11 (0.10-0.14) 9 months (n=37) (n=49) (n=43)

4 0.79 (0.55-1.14) 0.86 (0.67-1.12) 0.682 0.60 (0.42-0.85) 0.277 NA 6B 0.82 (0.58-1.17) 0.81 (0.59-1.12) 0.949 0.39 (0.29-0.52) 0.001 NA 9V 0.91 (0.71-1.16) 1.00 (0.72-1.38) 0.661 0.56 (0.40-0.77) 0.021 NA 14 3.99 (2.86-5.57) 1.93 (1.20-3.09) 0.02 1.11 (0.79-1.57) <0.001 NA 18C 0.49 (0.37-0.65) 0.41 (0.33-0.53) 0.353 0.18 (0.14-0.24) <0.001 NA 19F 1.04 (0.70-1.54) 1.40 (1.05-1.86) 0.207 0.89 (0.61-1.29) 0.568 NA

111

23F 0.65 (0.46-0.94) 0.44 (0.33-0.60) 0.094 0.24 (0.18-0.32) <0.001 NA 12 months (n=113) (n=142) (n=1145) (n=59)

4 0.48 (0.41-0.57) 0.47 (0.40-0.54) 0.73 0.63 (0.50-0.81) 0.066 0.07 (0.06-0.09) 6B 0.86 (0.72-1.03) 0.76 (0.63-0.92) 0.356 0.57 (0.46-0.71) 0.005 0.14 (0.12-0.17) 9V 0.59 (0.51-0.67) 0.62 (0.54-0.71) 0.522 0.50 (0.41-0.62) 0.221 0.09 (0.07-0.11) 14 2.38 (1.98-2.86) 1.52 (1.26-1.84) 0.001 1.16 (0.94-1.44) <0.001 0.19 (0.16-0.24) 18C 0.32 (0.27-0.38) 0.24 (0.21-0.28) 0.011 0.17 (0.15-0.20) <0.001 0.06 (0.05-0.08) 19F 1.05 (0.83-1.34) 1.14 (0.95-1.36) 0.592 0.93 (0.76-1.15) 0.462 0.46 (0.35-0.59) 23F 0.54 (0.44-0.66) 0.42 (0.35-0.50) 0.07 0.26 (0.21-0.31) <0.001 0.07 (0.06-0.09) 1Three bloods not available for testing 2Two sample t test was applied to compare GMC following 2 doses or a single dose of PCV with the GMC following 3 doses of PCV 3Two sample t test was applied to compare GMC following a single dose of PCV with the GMC following 0 doses of PCV. For all serotypes the p-value was <0.001 except for serotype 23F at 12 months of age whereby the p-value was 0.042. 4 Two sample t test was applied to compare GMC following 0 doses of PCV with the GMC following 3 doses of PCV. For all serotypes and all time points the p-value was <0.001. 5 One blood not available for testing NA Not applicable

112

Table 13: Proportion of infants with antibody concentrations ≥0.35 and ≥1μg/mL at 4 weeks post primary series, and at 9 and 12 months of age, by number of PCV doses administered in the primary series ≥0.35μg/mL ≥1μg/mL Serotypes 3 PCV 2 PCV 3 vs 2 1 PCV 3 vs 1 0 PCV 1 vs 0 3 PCV 2 PCV 3 vs 2 1 PCV 3 vs 1 0 PCV1 1 vs 0 p value p value p value p p value p value value 4 weeks post primary series (n=1252) (n=146) (n=121) (n=62) (n=1252) (n=146) (n=121) (n=62) 4 100 98.6 0.501 95.9 0.028 0 <0.001 97.6 93.8 0.152 76.9 <0.001 0 <0.001 6B 87.2 77.4 0.04 15.7 <0.001 6.5 0.099 66.4 39.0 <0.001 3.3 <0.001 0 0.301 9V 100 95.2 0.016 84.3 <0.001 3.2 <0.001 96.8 90.4 0.049 43.8 <0.001 1.6 <0.001 14 99.2 90.4 0.001 86.8 <0.001 53.2 <0.001 87.2 72.6 0.004 52.9 <0.001 12.9 <0.001 18C 93.6 90.4 0.379 61.2 <0.001 3.2 <0.001 86.4 79.5 0.149 42.1 <0.001 0 <0.001 19F 99.2 98.6 1.000 81.8 <0.001 33.9 <0.001 97.6 93.2 0.152 33.1 <0.001 4.8 <0.001 23F 94.4 82.2 0.003 29.8 <0.001 9.7 0.003 80.8 60.9 <0.001 4.1 <0.001 0 0.169 9 months (n=37) (n=49) (n=43) (n=37) (n=49) (n=43) 4 89.2 85.7 0.751 67.4 0.031 NA NA 35.1 42.9 0.511 30.2 0.811 NA NA 6B 75.7 71.4 0.806 53.5 0.061 NA NA 43.2 44.9 1.000 13.9 0.005 NA NA 9V 91.9 85.7 0.504 69.8 0.023 NA NA 48.6 57.1 0.514 32.6 0.173 NA NA 14 100 79.6 0.004 86.0 0.028 NA NA 89.2 67.3 0.021 53.5 0.001 NA NA 18C 70.3 61.2 0.494 25.6 <0.001 NA NA 18.9 10.2 0.348 4.7 0.073 NA NA 19F 89.2 95.9 0.395 76.7 0.237 NA NA 40.5 59.2 0.127 34.9 0.648 NA NA

113

23F 73.0 55.1 0.116 25.6 <0.001 NA NA 35.1 20.4 0.146 6.9 0.002 NA NA 12 months (n=113) (n=142) (n=1143) (n=59) (n=113) (n=142) (n=1143) (n=59) 4 64.6 66.2 0.793 63.2 0.89 6.8 <0.001 16.8 18.3 0.869 32.5 0.009 0 <0.001 6B 82.3 73.2 0.099 64.9 0.004 11.9 <0.001 48.7 34.5 0.029 27.2 0.001 3.4 <0.001 9V 72.6 81.0 0.133 67.5 0.469 8.5 <0.001 25.7 28.9 0.672 18.4 0.203 1.7 0.001 14 95.6 88.0 0.042 86.0 0.02 11.9 <0.001 83.2 69.0 0.013 57.9 <0.001 8.5 <0.001 18C 41.6 31.7 0.116 19.3 <0.001 5.1 0.012 10.6 4.9 0.097 1.8 0.006 0 0.548 19F 90.3 88.7 0.838 80.7 0.059 54.2 <0.001 41.6 57.0 0.017 44.7 0.688 23.7 0.008 23F 61.9 57.0 0.444 30.7 <0.001 20.3 0.155 29.2 17.6 0.035 9.6 <0.001 5.1 0.386 1Fisher’s exact test was applied to compare 3 PCV doses with no dose. For all serotypes the p-value was <0.001 except serotypes 18C (p=0.009) and 19F (p=0.029) for the ≥1μg/mL comparison at 12 months of age. 2Three bloods not available for testing 3One blood not available for testing NA Not applicable

114

5.5 Discussion

Our study has shown that a 3 dose PCV schedule is more immunogenic than alternative schedules with one or 2 PCV doses. Despite a difference in post primary GMC between the 3 and 2 dose group, the proportions of infants with antibody concentrations above the estimated protective level of 0.35μg/mL 4 weeks after the primary series was >90% for all serotypes in the 2 dose group except 6B (77.4%) and 23F (82.2%). However the differences between the 3 and 2 PCV dose groups were minimal by 12 months of age. There was a natural decline in levels of circulating serotype-specific antibodies from 18 weeks to 12 months of age in the 3 and 2 dose groups with no significant difference between the 2 groups in the proportion of children with antibodies ≥0.35μg/mL by 12 months of age.

The immunogenicity of 3 versus a 2 dose pneumococcal primary series with different coadministered vaccines, has shown different results in different settings [157, 170, 182, 194, 245, 246]. Following 3 doses of the 9-valent pneumococcal conjugate vaccine in Icelandic infants, 7 out of the 9 serotypes had significantly higher post primary antibody levels compared to the 2 dose group [194]. However the proportion of infants in the 2 dose group with antibody levels >0.35μg/mL was only significantly lower compared to the 3 dose group for serotype 6B [194]. A study using an 11-valent pneumococcal conjugate vaccine in Israeli infants showed a 2 dose schedule was less immunogenic than a 3 dose post primary series for serotypes 6B, 14, 18C, and 23F with a significantly lower proportion of infants with antibody levels ≥0.35μg/mL for serotypes 6B, 18C, and 23F. This study used an unlicensed 11- valent pneumococcal conjugate vaccine conjugated to diphtheria and tetanus carrier proteins[246]. PCV given to US infants showed 3 doses were needed to achieve an immunological response for serotype 6B but 2 doses were sufficient for the other 6 PCV serotypes [182]. PCV was less immunogenic for serotype 6B in Italian infants after 2 PCV doses than described in the US and Finland, but similar for the other PCV serotypes [161, 173, 245]. In contrast, other studies have found minimal immunological differences between a 3 or 2 PCV dose primary series [157, 245]. A study in UK infants showed no significant difference in GMC following a 3 or 2 dose schedule [157]. There was no significant difference in the proportion of infants achieving antibody concentrations >0.35μg/mL except for serotype 14 for which there was a higher proportion of infants in the 3 dose group [157]. Nevertheless, 2 doses may well be sufficient to protect against most PCV serotypes as early effectiveness data from the UK using the “2+1” schedule has shown a reduction in all infant IPD [252].

115

However 3 doses may be required to protect against serotype 6B as breakthrough cases of 6B have occurred [252]. Ongoing surveillance will determine whether these breakthrough cases will be a significant and consistent finding.

Whilst it is difficult to directly compare our immunogenicity results with other studies using different conjugate and co-administered vaccines in different ethnic groups, our data show at least comparable GMC results post primary series for all serotypes except serotype 6B and 23F following a 2 dose primary series compared with immunogenicity data from a USA trial [173]. In addition, our results for both the 3 and 2 dose groups were similar to those of Gambian infants who received 3 doses of an unlicensed 9-valent pneumococcal conjugate vaccine also produced by Wyeth Vaccines [202]. The only exception was that a lower proportion of Fijian children who received 2 doses had antibody concentrations ≥0.35μg/mL for serotype 23F compared with the Gambian infants [202]. Our GMC were, in general, similar following 2 or 3 PCV doses compared with the GMC from South African infants, except serotype 6B was less immunogenic in the Fijian 2 dose group [199]. Immunogenicity results for a 2 dose primary series from this study suggests similar protection may be obtained for IPD in the first 12 months of life as achieved in vaccine effectiveness and clinical trials [52, 68, 129], except perhaps for serotype 6B.

Comparisons of immunogenicity results between studies is also difficult as laboratory methods may differ. Our laboratory’s results showed excellent inter-laboratory correlation between the Finnish KTL laboratory and Wyeth laboratories [343]. Dissimilar results between studies may also be influenced by the common circulating serotypes at different study sites, which provide natural exposure and boosting [174]. However in this study, the poorer immunogenicity of serotypes 6B and 23F is most likely due to these serotypes being inherently less immunogenic. It is noteworthy that the GMC for serotype 14 in this study is better maintained over time for all schedules. This serotype is the commonest cause (33%) of IPD in children under 5 years old in Fiji and as such may have provided natural boosting [385].

Our study has shown that a single PCV dose administered at 14 weeks of age induces a significant immunological response and is likely to offer some protection for most serotypes. This is consistent with findings from a South African trial which showed a significant and potentially protective antibody response to most serotypes following a single dose at 6 weeks of age with at least 70% of infants producing antibody concentrations >0.15μg/mL after a single PCV dose and at least 95% doing so after

116

2 doses [198]. A case control study in the USA similarly documented that a single dose schedule administered to infants less than 7 months of age had an effectiveness of 73% (95%CI 43-87%) on VT IPD [242]. Models however have predicted that a single PCV dose given between 5-10 months of age could prevent a significant amount of VT IPD [243]. However, it is unlikely that significant protection, from a single PCV dose administered early in infancy, would persist for children throughout the highest risk period for IPD and pneumonia and an early booster at 6 or 9 months of age (“1+1” schedule) is worthy of further investigation for use in developing countries. Indeed our study has shown that booster responses tend to be stronger after a single dose of PCV rather than 2 or 3 doses [386]. This may be particularly relevant for low income countries where access to immunisation services is irregular, and middle income countries who have a significant burden of disease and are unable to pay affluent country vaccine prices but who do not benefit from immunisation financing mechanisms such as the Advanced Market Commitment or the International Finance Facility for Immunisation.

When interpreting our study it is important to note that two factors may have increased the immunogenicity of the one and 2 dose groups. Firstly, the co-administration of Diphtheria-Tetanus- whole cell Pertussis (DTwP) may have enhanced the immune response due to its potential adjuvant effect [387]. Although there are no studies in the literature directly comparing immunogenicity of PCV with Diphtheria-Tetanus-acellular Pertussis (DTaP) and DTwP, studies with co-administered DTwP tend to have stronger immune responses than those in which PCV is co-administered with DTaP. Furthermore, studies from Israel of an unlicenced 11-valent pneumococcal conjugate vaccine showed that co-administration with DTaP resulted in substantially lower immune responses compared to co- administration with DTwP [388]. Thus, in the present study, co-administration with DTwP may have augmented the immunogenicity of PCV.

Secondly, the carrier protein in PCV is CRM197, a modified diphtheria toxin. Experience with Hib vaccines has shown that prior administration of DTwP may augment responses to the CRM197 based conjugate vaccines [144, 388]. Thus, the groups that received one or two doses of PCV may have had augmented responses to their 14 week dose of PCV as a result of extra doses of DTwP. Therefore, care should be taken before using these data to support a two dose schedule in which only 2 doses of either DTwP or DTaP are given.

Based on re-analysis of existing phase 3 efficacy trials, it has been estimated that 0.35μg/mL represents a protective level above which an individual is protected against IPD, particularly meningitis

117

[102]. While the level required to protect children against pneumonia may be higher, it is unlikely that such an absolute level exists, and more likely that higher levels may be more helpful where other risk factors are present. Our results show that the proportion of infants with antibody concentrations ≥1μg/mL is significantly higher at 18 weeks of age in the 3 dose group compared with the 2 dose group for 3 of 7 PCV serotypes. However by 9 and 12 months of age the proportion of infants achieving this level is higher in the 2 dose group as compared to the 3 dose group for 4 of 7 and 3 of 7 serotypes, respectively. Hence infants may be protected from most VT IPD and severe pneumococcal disease but be less protected from non-bacteraemic pneumonia and OM in the first 9 months of life with a 2 dose schedule. For countries with high mortality, a one or 2 dose primary series may be sufficient to reduce mortality and the burden of severe pneumococcal disease.

In summary, the immunogenicity of 3 PCV doses is better than 2 doses. However it is likely that a 2 dose PCV primary series would offer similar protection as provided by 3 doses for most serotypes except possibly 6B. One PCV dose is likely to offer some protection for many serotypes. Further results documenting the avidity and OPA post primary series, the responses post booster, and the impact on NP carriage will follow.

118

6 SAFETY AND IMMUNOGENICITY OF THE 23-VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE, FOLLOWING ONE, TWO, OR THREES DOSES OF THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE IN INFANCY

6.1 Abstract

Fijian infants aged 6 weeks were stratified by ethnicity and randomised to receive 0, 1, 2, or 3 PCV doses with or without the 23vPPS at 12 months. Strong booster effects for all 7 PCV serotypes were elicited, and for 4/7 serotypes these responses were highest in the single PCV group. There were fourfold rises in GMC for all non-PCV serotypes. By 17 months the 23vPPS group still had significantly higher GMC (each p<0.001) for all serotypes. The 23vPPS was well tolerated and induced excellent responses for all serotypes which were greatest in the single PCV group.

6.2 Introduction

S.pneumoniae is the most common cause of bacterial pneumonia in children worldwide. It is the leading vaccine preventable cause of serious infection in infants [3]. A recent review estimated that over 14 million episodes of serious pneumococcal disease occurred worldwide in the year 2000, with over 800,000 deaths in children under 5 years [41]. The CFR is particularly high in infants less than 6 months old [5]. At least 48 serogroups comprising over 90 serotypes of pneumococcus have been identified [57]. Within serogroups, some serotypes cross-react immunologically, and in some cases this translates into cross-protection such as antibodies against 6B which provide cross-protection against 6A [52]. The association of particular serotypes with disease varies according to age, geography, and clinical presentation [61]. In general, the range of serotypes causing IPD in affluent countries like the US and in Europe is relatively narrow and largely confined to the serotypes found in PCV. In contrast, the range of serotypes causing disease in low-income countries is wider.

The 10-valent pneumococcal conjugate vaccine has recently been licensed in some countries, and a 13-valent vaccine is likely to be licensed by 2010. Some health authorities have decided or are considering a combination of an infant PCV primary series with a booster of the 23vPPS in the second year of life to address the limited serotype coverage offered by PCV. There have been several studies involving children in a number of countries using different pneumococcal conjugate formulations and schedules, comparing the immunogenicity of a 23vPPS or PCV

119

booster following a pneumococcal conjugate vaccine primary series. The majority of studies have shown that serotype-specific antibody concentrations are generally higher following 23vPPS than PCV booster [157, 172, 185-188]. The higher response may be due to the higher dose of pneumococcal polysaccharide in the 23vPPS, compared to PCV, enhancing the stimulation of memory B cells or by stimulating a greater number of B cells overall [189]. Despite this, only pneumococcal conjugate vaccines provide mucosal immunity and have shown a reduction in NP carriage (NP carriage being an antecedent event for all pneumococcal disease) for conjugate serotypes, compared with unvaccinated infants [306-308] or toddlers [309-311]. In contrast, pneumococcal polysaccharide vaccines have shown no effect on pneumococcal carriage [326-330]. Most studies evaluating the impact of pneumococcal polysaccharide immunisation in the absence of additional PCV in infants or children have not shown any impact on pneumococcal disease or carriage [267, 292, 298].

Data from Fiji shows that the 7 serotypes included in PCV, plus the cross reactive serotype 6A, would potentially cover 63.3% of IPD cases in children under 5 years [385]. This coverage would potentially increase to 83% if the 23vPPS was used, and would increase to 87% if the new 13- valent pneumococcal conjugate vaccine produced by Wyeth Vaccines (which includes serotypes 1, 3, 5, 6A, 7F and 19A) was used, largely due to the inclusion of 6A which is not included in the 23vPPS [385]. The aim of this study was to find an optimal vaccination strategy suitable for resource poor countries in terms of serotype coverage, flexibility, and affordability. To address these issues, we undertook a Phase II vaccine trial in Fiji to document the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination regimens combining 1, 2, or 3 doses of PCV in infancy. In order to broaden the serotype coverage, the additional benefit of a 23vPPS booster at 12 months of age was also assessed. Presented are the geometric mean serotype-specific IgG antibody concentrations (GMC) prior to and 2 weeks following the 12 month 23vPPS, and at 17 months of age.

120

6.3 Methods

6.3.1 Study Participants

The study was a single blind, open-label randomised Phase II vaccine trial undertaken in Suva, the capital of Fiji. Healthy infants aged between six and eight weeks were eligible for enrolment. Details of the selection criteria and the randomisation procedure have been reported elsewhere [389].

The study was conducted and monitored according to Good Clinical Practice. It was approved by the Fiji National Research Ethics Review Committee and the University of Melbourne Human Research Ethics Committee.

6.3.2 Study Procedures and Vaccines

Infants were stratified by ethnicity and randomized into one of eight groups. The seven-valent

CRM197 protein-polysaccharide conjugate vaccine containing polysaccharide antigen from pneumococcal serotypes 4, 6B, 9V, 14, 18C, 19F, 23F (PrevenarTM, Wyeth Vaccines) was used. The vaccine contains 2 g of each serotype, except serotype 6B which contains 4 g. The three dose group received PCV at 6, 10, and 14 weeks of age, the 2 dose group received PCV at 6 and 14 weeks of age and the single dose group received PCV at 14 weeks of age. Routine vaccines (HiberixTM mixed with TritanrixTM-HepBTM, GlaxoSmithKline) and oral polio were given with the primary series. HiberixTM contains 10μg of purified Hib capsular polysaccharide covalently bound to approximately 30μg tetanus toxoid mixed with TritanrixTM-HepBTM which contains not less than 30 IU of adsorbed D toxoid, not less than 60 IU of adsorbed T toxoid, not less than 4 IU of whole cell Pertussis, and 10µg of recombinant HBsAg protein. The children in all primary series groups were further randomized to receive a dose of 23vPPS (PneumovaxTM, Merck & Co., Inc., which consists of a purified mixture of 25µg of capsular polysaccharide from 23 pneumococcal serotypes) or no vaccine at 12 months of age (window: 12 months plus 4 weeks). In addition, all children received Measles-Rubella vaccine at 12 months of age co-administered with 23vPPS. The children randomized to receive 0 or 1 PCV dose in infancy had a single dose of PCV administered at 2 years of age.

121

Children were reviewed on day 1, 2 and 7 following 23vPPS and assessed for any AE. An AE was defined as any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of 23vPPS, whether or not related to 23vPPS. A severe non-serious AE was defined as an event which prevented normal activities but did not meet the criteria of a SAE. A SAE was defined as an AE meeting one of the following conditions: death in the 2 year follow up period; a life threatening event; hospitalisation or prolongation of existing hospitalisation during the 2 year period; or resulting in a persistent or significant disability/incapacity.

SAEs were sourced from parent interview at each study visit and via a search of computerized hospital discharge data. Causality of any non-serious AE were assigned by the study doctor and reviewed by a paediatrician (FR). Causality of SAEs were assigned by the study doctor and assessed by an independent external safety monitor and regularly reviewed by the study’s DSMB.

6.3.3 Laboratory Procedures

Children who received the 12 month 23vPPS had blood drawn immediately prior to and 14 days following the 23vPPS (window: 10-21 days post 23vPPS). All children had blood drawn at 17 months of age. Blood was separated by centrifugation in the health centre, kept chilled and transported to the CWMH laboratory, Suva, where it was divided into aliquots and stored at - 20 C on the same day, until transported to the Pneumococcal Laboratory, Murdoch Childrens Research Institute, Melbourne, on dry ice for analysis.

122

previously [123, 342] and unpublished data from our laboratory have shown this to further improve the specificity of the pneumococcal ELISA. The reference serum standard 89-SF (Food and Drug Administration, Bethesda MD) and samples for measurement of specific IgG to serotype 22F were pre-absorbed with C-PS at 10µg/mL and incubated overnight at 4oC. Horseradish peroxidase conjugated anti-human IgG and a TMB (3.3’, 5.5’-tetramethylbenzidine) substrate solution was used for detection. A high, medium, and low control serum were used on each plate to assess assay performance and inter-assay variation. Results from an inter- laboratory comparison between the Pneumococcal Laboratory, Murdoch Childrens Research Institute, (Melbourne, Australia), Wyeth Vaccine Research Laboratory (USA) and the KTL laboratory (Finland) demonstrated a good correlation of serotype-specific antibody concentrations [343]. Laboratory staff members were blinded to the group allocation of each serum sample.

6.3.4 Statistical Analysis

This manuscript reports analytic results concerning the secondary purpose of the trial. Cleaned data were exported to Stata version 9.0 (Stata Corporation, College Station, Texas) for analysis. Serotype-specific antibody concentrations by ELISA were log (base e) transformed to calculate GMC. Comparisons of serotype-specific GMC between 0-3 dose PCV groups were performed using a two sample t-test. Comparisons of serotype-specific GMC before and after the 23vPPS were performed using the paired t test. Comparisons of the proportion of infants between groups with serotype-specific antibody concentrations ≥0.35 and ≥1µg/mL were performed using Fisher’s exact test. Comparisons of serotype-specific antibody concentrations ≥0.35 and ≥1µg/mL before and after the 23vPPS were performed using exact McNemar’s test. A p-value of <0.01 was considered statistically significant due to the multiple comparisons.

6.4 Results

There were 552 infants enrolled in the study (Figure 9) and the characteristics of the randomised infants have been described elsewhere [390]. The 552 participants represent a consent rate of 30.5%, of which 10% had withdrawn by 12 months and 15% by 17 months of age. The commonest reason for withdrawal was relocation outside the study area. No participant was withdrawn due to a reaction to any of the vaccines. The 12 month 23vPPS was administered to

123

245 children with all groups having blood drawn a median of 14 days (IQR 14-15 days) post booster.

6.4.1 Immunogenicity to PCV Serotypes

Two weeks following the 23vPPS, GMC were significantly higher (each p<0.001) for all PCV serotypes for children that had received either 1, 2, or 3 PCV doses in the primary series compared to levels prior to receiving 23vPPS (Table 14). For 4 of 7 serotypes (4, 9V, 18C, 19F) this response was most profound in the single PCV dose group. There were no significant differences in GMC 2 weeks following the 23vPPS for any PCV serotype between the 3 and 2 PCV dose groups. GMC were significantly higher (each p<0.001) 2 weeks following the 23vPPS compared with the pre-23vPPS levels, for all PCV serotypes in the group that had not received PCV in infancy (Table 14).

Two weeks following the 12 month 23vPPS, there was no significant difference between the 3 and 2 dose PCV groups or between the 3 and single dose groups in the proportion of children with antibody concentrations ≥0.35 and ≥1µg/mL for the PCV serotypes (Table 15).

At 17 months of age the groups that had received the 12 month 23vPPS continued to have significantly higher GMC (each p<0.001) for all PCV serotypes compared to those that had not received the 12 month 23vPPS but the same number of PCV doses (Table 16). The single PCV dose group which received the 23vPPS continued to have higher GMC compared to the 2 or 3 dose PCV groups which did or did not receive the 23vPPS. There were significantly higher proportions with antibody concentrations ≥1µg/mL for the PCV serotypes in those groups that had received the 12 month 23vPPS compared with those that had not received the 23vPPS (Table 16).

124 Figure 9: CONSORT chart of the screened and enrolled children to 17 months of age

1811 screened

1259 did not enroll

552 Randomised

A=71 B=65 C=76 D=80 E=62 F=66 G=63 H=69

Withdrawn=9 Withdrawn=12 Withdrawn=5 Withdrawn=8 Withdrawn=8 Withdrawn=5 Withdrawn=4

Refused=1 Refused=1 Not taken=1

12m blood=60 12m blood=53 12m blood=70 12m blood=72 12m blood=54 12m blood=61 12m blood=59

Not taken=1 Withdrawn=2 Unsuccessful=1 Not taken=2 Not taken=2

Not taken=1

Blood test 2 weeks later=52 Blood test 2 weeks later =69 Blood test 2 weeks later Blood test 2 weeks later =58 =57 Withdrawn=3 Withdrawn=4 Withdrawn=3 Withdrawn=3 Withdrawn=5 Withdrawn=2 Withdrawn=2 Withdrawn=6 Unsuccessful=1 Unsuccessful =1

17m blood=59 17m 17m blood=68 17m blood=67 17m blood=49 17m 17m 17m blood=62 blood=48 blood=59 blood=57

A=3PCV-7; B=3PCV-7+ 12 month PPV-23; C=2PCV-7; D=2PCV-7+ 12 month PPV-23; E=1PCV-7; F=1PCV-7+ 12 month PPV-23; G=12 month PPV-23; H=no vaccines in the first 12 months

125

Table 14: Serotype-specific IgG geometric mean concentrations (GMC and 95% confidence intervals) to PCV serotypes before and 14 days following the 12 month 23vPPS and by number of PCV doses administered in the primary series Pre-23vPPS at 12 months of age 14 days post-12 month 23vPPS 3 PCV 2 PCV 1 PCV 0 PCV 3 PCV1,2 2 PCV1,2 1 PCV1-3 0 PCV1,3 (n=52) (n=69) (n=58) (n=57) (n=52) (n=69) (n=58) (n=57) 4 0.46 0.42 0.76 0.08 14.68 14.70 46.47 2.36 (0.37-0.57) (0.34-0.52) (0.53-1.08) (0.06-0.09) (11.10-19.42) (11.72-18.43) (36.65-58.92) (1.76-3.16) 6B 0.85 0.79 0.69 0.14 29.58 23.83 18.07 0.31 (0.64-1.12) (0.59-1.07) (0.49-0.98) (0.12-0.17) (21.58-40.54) (17.99-31.57) (13.55-24.11) (0.23-0.42) 9V 0.59 0.62 0.67 0.09 14.10 15.20 34.84 1.20 (0.49-0.73) (0.53-0.71) (0.49-0.89) (0.07-0.11) (10.60-18.75) (12.03-19.21) (27.03-44.90) (0.90-1.61) 14 2.00 1.55 1.36 0.19 15.73 13.91 20.08 0.41 (1.48-2.72) (1.21-1.99) (1.01-1.83) (0.16-0.24) (10.48-23.60) (9.87-19.60) (12.19-33.05) (0.29-0.58) 18C 0.37 0.23 0.22 0.06 8.71 10.51 16.03 1.22 (0.27-0.49) (0.19-0.27) (0.17-0.29) (0.05-0.08) (6.70-11.34) (8.21-13.46) (11.33-22.67) (0.88-1.69) 19F 0.86 1.20 1.05 0.47 27.91 24.77 84.47 1.13 (0.64-1.16) (0.93-1.55) (0.79-1.39) (0.36-0.61) (19.98-38.99) (18.21-33.68) (55.80-127.87) (0.80-1.59) 23F 0.52 0.41 0.31 0.19 10.87 10.29 8.33 0.42 (0.39-0.69) (0.32-0.53) (0.23-0.42) (0.14-0.25) (7.41-15.96) (7.19-14.73) (5.25-13.20) (0.31-0.57) 1 P-values were <0.001 for all serotypes comparing GMC pre/post 12 month 23vPPS for all PCV dosage groups. 2For GMC comparisons between a single or 2 doses of PCV with 3 PCV doses, the p-values were not significant for all serotypes except for the 3 versus single dose comparison for serotypes 4, 9V, 18C, and 19F (each p<0.01). 3 P-values were <0.001 for all serotypes comparing GMC between 0 or a single dose of PCV with 3 PCV doses.

126

Table 15: Proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to PCV serotypes before and 14 days post-12 month 23vPPS and by number of PCV doses administered in the primary series Pre-23vPPS at 12 months of age 14 days post-12 month 23vPPS 3 PCV 2 PCV 1 PCV 0 PCV 3 PCV 2 PCV 1 PCV 0 PCV (n=52) (n=69) (n=58) (n=57) (n=52) (n=69) (n=58) (n=57) ≥0.35µg/mL 4 63.5 60.9 64.9 7.0 98.1 1002 1002 94.72,3 6B 78.8 72.5 70.2 12.3 100 1002 1002 38.6 9V 73.1 81.2 75.4 8.8 100 1002 1002 87.72 14 92.3 92.8 89.5 12.3 94.21 95.71, 2 93.11, 2 43.9 18C 44.2 27.5 31.6 5.3 100 1002 98.32 82.5 19F 86.5 91.3 84.2 54.4 1001 1001,2 1002 84.2 23F 59.6 53.6 42.1 21.1 98.1 98.62 93.12 50.9 ≥1µg/mL 4 13.5 8.7 36.8 0 98.1 1002 1002 84.2 6B 46.2 36.2 33.3 3.5 98.1 1002 1002 14.01 9V 26.9 27.5 21.1 1.8 98.1 1002 1002 54.4 14 76.9 72.5 63.2 8.8 92.3 95.72 89.72 21.11 18C 13.5 2.9 3.5 0 98.1 95.72 96.62 57.9 19F 34.6 57.9 52.6 24.6 98.1 98.62 96.62 50.9 23F 30.8 15.9 15.8 5.3 94.2 88.42 86.22 17.51 A significant difference was observed for all comparisons except those marked: 1Comparison of proportions pre/post 12 month 23vPPS. 2Comparison of proportions following 0, 1, or 2 doses with 3 doses of PCV. 3Comparison of proportions following a single and no dose of PCV.

127

Table 16: Serotype-specific IgG GMC (and 95% confidence intervals) and proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to PCV serotypes at 17 months in those who did or did not receive the 12 month 23vPPS and by number of PCV doses in the primary series

3 PCV, 3 PCV, 2 PCV, 2 PCV, 1 PCV, 1 PCV, 0 PCV, 0 PCV, no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS (n=59) (n=48) (n=68) (n=67) (n=49) (n=59) (n=62) (n=57) GMC (95%CI) 4 0.35 2.19 0.43 2.03 0.56 6.36 0.11 0.70 (0.29-0.43) (1.79-2.69) (0.33-0.56) (1.70-2.42) (0.39-0.80) (4.94-8.19) (0.09-0.13) (0.53-0.91) 6B 0.91 4.35 0.78 3.85 0.62 3.88 0.20 (0.16- 0.21 (0.69-1.21) (3.22-5.89) (0.58-1.04) (3.10-4.79) (0.47-0.83) (2.87-5.25) 0.24) (0.17-0.26) 1 9V 0.41 2.25 0.49 2.50 0.51 5.49 0.14 0.38 (0.34-0.49) (1.74-2.91) (0.39-0.62) (2.02-3.08) (0.36-0.71) (4.23-7.12) (0.11-0.17) (0.31-0.47) 14 1.78 4.31 1.12 3.58 0.93 4.85 0.31 0.67 (1.42-2.24) (3.00-6.19) (0.86-1.46) (2.72-4.71) (0.66-1.32) (3.27-7.19) (0.25-0.38) (0.47-0.96) 18C 0.21 1.28 0.20 1.13 0.15 1.79 0.10 0.50 (0.18-0.26) (1.07-1.53) (0.16-0.25) (0.92-1.38) (0.12-0.19) (1.37-2.34) (0.08-0.12) (0.39-0.66) 19F 1.19 5.55 1.06 4.53 0.92 13.47 0.59 0.79 (0.84-1.67) (4.24-7.26) (0.82-1.38) (3.54-5.79) (0.66-1.26) (9.89-18.33) (0.47-0.75) (0.61-1.04) 1 23F 0.57 1.68 0.43 1.41 0.32 1.78 0.19 0.27 (0.43-0.75) (1.29-2.20) (0.32-0.58) (1.06-1.86) (0.22-0.48) (1.28-2.49) (0.16-0.23) (0.21-0.34) 1

128

3 PCV, 3 PCV, 2 PCV, 2 PCV, 1 PCV, 1 PCV, 0 PCV, 0 PCV, no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS (n=59) (n=48) (n=68) (n=67) (n=49) (n=59) (n=62) (n=57) Proportion≥0.35µg/mL 4 52.5 100 51.5 100 63.3 98.3 6.5 73.7 6B 83.1 97.92 72.1 100 73.5 98.3 21.0 24.62 9V 61.0 97.9 60.3 98.5 61.2 100 12.9 56.1 14 98.3 95.82 91.2 97.02 83.7 94.92 41.9 73.7 18C 22.0 97.9 22.1 94.0 10.2 93.2 9.7 66.7 19F 89.8 1002 91.2 1002 87.8 98.32 72.6 82.52 23F 64.4 93.8 54.4 88.1 34.7 89.3 17.7 43.9 Proportion ≥1µg/mL 4 5.1 85.4 23.5 88.1 30.6 96.6 1.6 38.6 6B 39.0 89.6 41.2 95.5 34.7 84.7 3.2 5.32 9V 10.2 85.4 16.2 82.1 22.4 91.5 1.6 5.32 14 69.5 89.6 47.1 85.1 42.9 81.4 9.7 28.12 18C 3.4 66.7 5.9 59.7 2.0 69.5 3.2 31.6 19F 40.7 95.8 47.1 94.0 42.9 94.9 29.0 29.82 23F 22.0 75.0 22.1 65.7 10.2 72.9 1.6 5.32 All comparisons were significant except: 1 Comparison of GMC for 3, 2, 1 or 0 PCV doses with or without the 12 month 23vPPS. 2 Comparison of the proportions with antibody concentrations ≥0.35 and ≥1µg/mL for 3, 2, 1 or 0 PCV doses with or without the 12 month 23vPPS.

129

6.4.2 Immunogenicity to Non-PCV Serotypes

Two weeks following the 12 month 23vPPS, GMC and the proportions with antibody concentrations ≥0.35 and ≥1µg/mL for all non-PCV serotypes in the 23vPPS were significantly higher (each p<0.001) than pre-23vPPS levels (Table 17). To assess for non-specific effects, the proportion of children with antibody concentrations ≥0.35µg/mL were compared between the 3, 2, and single PCV dose groups with the group that had received no prior PCV. There were no significant differences in responses to the non-PCV serotypes following the 12 month 23vPPS between the 3 and 0 PCV dose groups (data not shown). However for serotypes 15B and 19A, the proportion of children with antibody concentrations ≥0.35µg/mL were significantly higher in the 2 and single dose groups compared with the 0 PCV dose group (data not shown).

By 17 months of age, GMC and the proportion with antibody concentrations ≥0.35µg/mL were still significantly higher (each p<0.001) for all non-PCV serotypes in the groups that had received the 23vPPS vaccine at 12 months compared to the groups that had not (Table 18).

130

Table 17: Serotype-specific IgG GMC (and 95%CI) and proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to non-PCV serotypes before and 14 days post-12 month 23vPPS Pre-23vPPS at 12 months of age (n=235) 1 14 days post-12 month 23vPPS (n=235) 1 Serotype GMC2 (95%CI) % ≥0.35µg/mL3 % ≥1µg/mL3 GMC2 (95%CI) % ≥0.35µg/mL3 % ≥1µg/mL3 1 0.17 (0.15-0.19) 17.9 6.4 1.59 (1.38-1.82) 91.9 65.9 2 0.41(0.36-0.46) 54.5 13.6 10.73 (9.50-12.11) 100 98.7 3 0.27 (0.23-0.32) 34.9 10.6 8.28 (7.26-9.44) 99.1 96.6 5 0.26 (0.23-29) 35.7 11.5 2.26 (2.01-2.55) 97.9 78.7 7F 0.09 (0.08-0.10) 11.5 2.9 1.73 (1.51-1.99) 92.3 73.6 8 0.24 (0.21-0.28) 30.6 7.7 8.88 (7.82-10.09) 98.7 97.4 9N 0.23 (0.19-0.26) 27.7 7.7 8.31 (7.04-9.82) 98.3 93.6 10A 0.21 (0.19-0.24) 22.9 6.4 0.76 (0.66-0.89) 73.6 38.7 11A 0.09 (0.09-0.11) 12.8 7.2 1.51 (1.28-1.77) 87.2 68.9 12F 0.07 (0.07-0.08) 7.2 1.3 0.37 (0.31-0.43) 50.6 23.4 15B 0.29 (0.27-0.34) 37.9 9.4 2.15 (1.84-2.51) 91.1 76.2 17F 0.10 (0.09-0.11) 5.1 1.3 0.81 (0.68-0.96) 73.2 43.2 19A 0.46 (0.40-0.51) 56.6 17.9 1.93 (1.60-2.32) 86.8 63.8 20 0.09 (0.09-0.10) 4.3 1.3 0.68 (0.57-0.83) 66.1 39.1 22F 0.40 (0.36-0.46) 54.5 15.3 4.73 (3.81-5.87) 94.9 81.3 33F 0.13 (0.12-0.14) 7.7 2.9 1.66 (1.40-1.97) 84.3 69.4 1 235 pairs available for comparison. One pre-23vPPS sample was not available for testing. 2P-values were <0.001 for all pre/post 23vPPS GMC comparisons for all serotypes. 3P-values were <0.001 for comparisons of proportions with antibody concentrations ≥0.35µg/mL and ≥1µg/mL for all serotypes.

131

Table 18: Serotype-specific IgG GMC (and 95% confidence intervals) and proportions of children with antibody concentrations ≥0.35 and ≥1µg/mL to non- PCV serotypes at 17 months of age in those that did or did not receive the 12 month 23vPPS No 23vPPS at 12 months of age (n=238) 23vPPS at 12 months of age (n=231)

1 3 1 2 3 GMC (95%CI) % ≥0.35µg/mL2 % ≥1µg/mL GMC (95%CI) % ≥0.35µg/mL % ≥1µg/mL 1 0.23 (0.21-0.25) 25.6 3.4 0.63 (0.56-0.71) 77.5 27.3 2 0.51 (0.45-0.57) 65.5 22.3 2.88 (2.60-3.18) 99.1 92.2 3 0.30 (0.26-0.34) 34.9 10.1 1.46 (1.30-1.63) 96.1 69.3 5 0.31 (0.28-0.34) 41.6 7.1 0.77 (0.69-0.86) 81.4 39.4 7F 0.12 (0.10-0.13) 11.3 3.8 0.51 (0.45-0.57) 63.6 22.9 8 0.29 (0.26-0.33) 34.9 8.4 2.31 (2.08-2.56) 99.1 86.6 9N 0.23 (0.20-0.26) 24.4 4.6 1.90 (1.64-2.19) 92.6 74.0 10A 0.19 (0.17-0.21) 16.4 2.5 0.27 (0.25-0.31) 36.8 6.5 11A 0.14 (0.12-0.16) 19.7 11.3 0.35 (0.31-0.41) 51.5 15.6 12F 0.09 (0.08-0.10) 5.0 0.8 0.18 (0.16-0.21) 23.8 6.1 15B 0.30 (0.27-0.34) 37.4 9.7 0.74 (0.66-0.84) 79.2 39.8 17F 0.12 (0.11-0.13) 6.3 0.4 0.36 (0.31-0.41) 48.5 16.9 19A 0.59 (0.53-0.67) 71.4 24.4 1.14 (1.00-1.31) 87.0 57.1 20 0.13 (0.12-0.15) 12.6 4.2 0.26 (0.23-0.30) 36.4 10.0 22F 0.46 (0.41-0.51) 59.7 18.9 1.43 (1.22-1.68) 91.3 59.7 33F 0.18 (0.17-0.20) 15.1 3.4 0.62 (0.54-0.71) 70.6 36.4 1 P-values were <0.001 for all serotypes. 2P-values were <0.001 comparing proportions ≥0.35µg/mL for all serotypes. 3 P-values were <0.01 comparing proportions ≥1µg/mL for all serotypes except serotypes 10A and 11A.

132

6.4.3 Adverse Events

Following 23vPPS at 12 months of age, low grade fever was common (28.2%) while high grade fever occurred in 6.1%. The description of other general reactions are shown in Table 19. Local injection site reactions occurred in a minority of recipients. All events resolved within 48 hours. There were 101 SAEs throughout the 2 year follow up period, with none attributable to receipt of any of the study vaccines. One child who had received 2 doses of PCV at 6 and 14 weeks of age died at 9 months of age from dehydration secondary to acute gastroenteritis. For children over 12 months of age, there were 14 SAEs in children who had received the 12 month 23vPPS and 22 SAEs in children who had not received 23vPPS, during the follow up period up to 2 years of age. For children over 12 months of age, there were 4 cases of inpatient pneumonia in children who had received the 12 month 23vPPS compared with 7 cases in those that had not during the same follow up period. There were no cases of IPD throughout the study period.

Table 19: Non-serious adverse events1 in those children who received 23vPPS at 12 months of age (n=245) Systemic adverse events Number (%) High fever (≥38ºC per axilla) 15 (6.1) Low grade fever (>37ºC & <38ºC per axilla) 69 (28.2) Diarrhoea 2 (0.8) Vomiting 4 (1.6) Severe vomiting2 1 (0.4) Anorexia 11 (4.5) Severe anorexia2 2 (0.8) Drowsiness 13 (5.3) Severe drowsiness2 2 (0.8) Irritability 23 (9.4) Severe irritability3 2 (0.8) Local reactions Erythema 15 (6.1) >30mm2 3 (1.2) Tenderness 13 (5.3) Induration 13 (5.3) >30mm2 3 (1.2) 1All cases were classified as mild or moderate unless otherwise stated 2Resolved within 2 days 3Resolved within 1 day

133

6.5 Discussion

This study has shown that 1, 2, or 3 doses of PCV in infancy primed infants sufficiently elicit an excellent booster response to the 23vPPS at 12 months of age for all PCV serotypes. Furthermore, there were good antibody responses to the 16 non-PCV serotypes following 23vPPS at 12 months. The antibody concentrations for all 23 serotypes remained significantly higher at 17 months of age in the 23vPPS group compared to the group that had not received 23vPPS.

In addition, this study has shown that priming with a single PCV dose in infancy produced the greatest booster (memory) response for most serotypes following 23vPPS at 12 months compared with 2 or 3 PCV doses. Responses following the 23vPPS were similar for those children that had received either 2 or 3 PCV doses in infancy and lower than that in children who received a single PCV dose. The immunological explanation for the single PCV dose having a better booster response is not clear. Post booster antibody concentrations are usually higher in those that have had a stronger primary response [145]. One study found that a stronger primary response was more likely following higher doses of antigen and/or a higher concentration of carrier protein, possibly through the enhanced induction of antibody producing plasma cells [178]. However this would not explain the findings in our study of a better booster response in the single dose group as our previously published data has shown that a single PCV dose (lower antigen dose) administered at 14 weeks of age induced a weaker primary response [389]. In that previous study, a significant immunological response was found in the single dose group compared with an unvaccinated control group, but significantly lower GMC for all PCV serotypes compared to 2 or 3 PCV doses [389].

Another possible explanation for the better booster response in the single PCV dose group may be that a single antigen challenge rather than multiple antigen exposures, may preferentially drive the induction of memory B cells (which are required for a booster response), rather than plasma cells [391]. Having a greater pool of memory B cells would subsequently elicit a greater booster response. A fewer dose (single PCV dose) primary series may preferentially induce B cell differentiation away from plasma cells, towards memory B cells compared to repeated antigen exposure associated with 2 or 3 PCV dose primary series [186, 187]. In summary, our findings are consistent with the suggestion that 2 or 3 PCV doses in infancy are more effective at producing plasma cells [389] and less effective at producing

134

memory B cells than a single PCV dose. Alternatively, it is speculated that our findings may be explained by some form of immunological tolerance following 2 or 3 PCV doses.

Our findings indicate that PCV/23vPPS compared to the PCV primary series without a booster should offer superior protection from pneumococcal disease lasting at least 5 months following the 12 month 23vPPS. A recent study of asthmatic children aged 2-5 years underwent sequential immunisation of PCV followed by 23vPPS either 2 or 10 months post PCV [392]. Antibody concentrations for PCV and 2 non-PCV serotypes (5 and 7F) were higher following the 23vPPS booster than after PCV alone [392]. Despite superior antibody concentrations being demonstrated for PCV/23vPPS compared with PCV/PCV, we would not advise PCV/23vPPS for 3 reasons. Firstly, superior vaccine efficacy using PCV/23vPPS against clinical disease has not been demonstrated. A study of vaccine efficacy against AOM found that a PCV/23vPPS compared to a PCV/PCV schedule had similar results despite higher antibodies generated post PCV/23vPPS [188]. This may be due to inferior quality of antibodies being produced following 23vPPS. However previous studies have found that the quality of antibody, measured by avidity or OPA, can differ in those that have received 23vPPS or PCV as a booster, however results have been conflicting and therefore inconclusive [154, 157, 186, 304, 305]. Finnish studies have shown the concentration of antibodies required for 50% killing was higher [304] and that the avidity of such antibodies was lower after PCV/23vPPS compared with PCV/PCV [147, 154, 186]. In contrast, another study in Finland using the 11- valent pneumococcal conjugate vaccine showed that OPA was better in the group that received a 23vPPS booster at 12-15 months than those that had the conjugate booster [305]. A study in Israeli children who received 1 dose of the 7-valent pneumococcal polysaccharide- meningococcal outer membrane protein complex conjugate vaccine followed by either a conjugate or 23vPPS booster, achieved similar opsonic antibody titres in each group for the 1 serotype tested (6B) [186]. Data from the assessment of functional antibody responses in our study documenting the avidity to 23 serotypes and OPA to 8 serotypes will be forthcoming.

Secondly, conjugate vaccines are the only vaccines that provide mucosal immunity. As NP carriage is an antecedent event in IPD, the reduction or prevention of NP carriage reduces the transmission of pneumococci and prevents IPD in the vaccinated individual and provides herd immunity [17, 18, 323]. In contrast, pneumococcal polysaccharide vaccines have shown no effect on pneumococcal carriage [326-330]. Most studies evaluating the impact of pneumococcal polysaccharide immunisation in the absence of additional PCV in infants or children have not shown any impact on pneumococcal disease or carriage [267, 292, 298]. This

135

finding may be at least partially explained by the lack of effect that pneumococcal polysaccharide vaccine has on NP carriage. In contrast, one study in Papua New Guinea, where children aged six months to five years of age were given either the 14-valent or 23vPPS in one or two doses according to age, there was a (non-significant) 19% reduction in mortality from any cause, and a 50% reduction in pneumonia mortality (95%CI, 1-75%) [299]. Natural exposure in a population with a high incidence of pneumococcal infections, resulting in regular antigenic stimulation may explain this finding [189].

Thirdly, immunological hyporesponsiveness following 23vPPS at 12 months of age has been demonstrated by reduced responses to a small re-challenge dose of 23vPPS administered at 17 months of age [390]. This attenuated response to the re-challenge dose may be due to depletion of the memory B cell pool [271]. A study documenting immunologic memory 5 years after meningococcal A/C conjugate vaccination in infancy showed that challenge with the meningococcal polysaccharide or conjugate at 2 years of age demonstrated immunologic memory. However subsequent challenge with polysaccharide at 5 years of age resulted in an inability to demonstrate memory in the polysaccharide group. The authors concluded that polysaccharide immunisation at 2 years of age interfered with the immune response to subsequent polysaccharide vaccination [271]. One explanation for this is that polysaccharide immunisation induces memory B cells to differentiate into plasma cells and secrete antibody but does not replenish the memory B cell pool [282]. Subsequent challenge with 23vPPS may then result in immune hyporesponsiveness. No adverse clinical effects have ever been documented due to repeated exposure to the meningococcal polysaccharide vaccine. In this study we demonstrated no adverse clinical consequences, although the study was not designed to evaluate this effect.

In summary, 23vPPS at 12 months induces an excellent booster response following 1, 2, or 3 doses of PCV in infancy for all PCV and significant responses for non-PCV serotypes up to 5 months following vaccination. Booster responses were greatest for a single PCV dose compared to 2 or 3 doses of PCV.

136

7 HYPORESPONSIVENESS TO RE-CHALLENGE DOSE FOLLOWING PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE, A RANDOMIZED CONTROLLED TRIAL

7.1 Abstract

Aim: To evaluate the immunological impact of 23vPPS at 12 months, for children who have received zero to three infant doses of PCV, on responses to a subsequent exposure to a small dose of 23vPPS (mPPS).

Methods: Five hundred and fifty-two Fijian infants were stratified by ethnicity and randomized into eight groups to receive zero, one, two, or three PCV doses at 14 weeks, six and 14 weeks, or six, ten, and 14 weeks. Within each group, half received 23vPPS at 12 months and all received mPPS at 17 months. Sera were taken prior and one month post-mPPS.

Results: By 17 months, GMC to all 23 serotypes in 23vPPS were significantly higher in children who had received 23vPPS at 12 months compared to those who had not. Post-mPPS, children who had not received the 12 month 23vPPS had a significantly higher GMC for all PCV serotypes compared with those who had (each p<0.02). For the non-PCV serotypes, children who had not received the 12 month 23vPPS had significantly higher GMC for six of 16 non-PCV serotypes (7F, 9N, 12F, 19A, 22F, 33F) than those who did (each p<0.02). After adjusting for the pre-mPPS level, exposure to 23vPPS was associated with a lower response to mPPS for all serotypes (each p<0.001).

Conclusion: Despite higher antibody concentrations at 17 months in children who had received 23vPPS at 12 months, the response to a re-challenge was poor for all 23 serotypes compared to children who had not received the 12 month 23vPPS.

7.2 Introduction

Pneumococcal disease is estimated to cause 1.6 million deaths each year, primarily in children and the elderly. The majority of these deaths occur in low income countries [4]. Over 90 serotypes in 48 serogroups of pneumococcus have been identified [57]. Most serious pneumococcal disease is caused by a relatively small number of serotypes. However these vary by age, geography, and clinical presentation [393]. The range of serotypes causing disease in affluent societies is largely confined to the serotypes found in PCV. In contrast, the range of serotypes causing disease in low income countries is wider [59]. The 10-valent pneumococcal conjugate vaccine has recently been licensed in some countries, and a 13-valent vaccine is likely to be licensed by 2010.

137

The use of 23vPPS as a booster following PCV in infancy (PCV/23vPPS) has the theoretical advantage of boosting the seven serotypes shared between PCV and 23vPPS, while broadening the serotype coverage with the addition of 16 non-PCV serotypes. For this reason it has been routinely given to Australian Indigenous children as a booster at 18 months of age following three doses of PCV in infancy. The majority of immunological studies have shown PCV/23vPPS to produce at least similar or higher antibody levels for all shared serotypes compared with a PCV boost [154, 157, 185, 187, 188, 273, 394, 395]. Studies describing qualitative function such as OPA and avidity are limited and have shown inconsistent results [154, 157]. A T-cell independent response, which is immature in infancy, is required for an immunological response to the non-PCV serotypes using the combined PCV/23vPPS approach. However the immunogenicity of 23vPPS varies by age and serotype [146, 263-268] with poor responses demonstrated in most infant studies for serogroups 6 [146, 263-268], 19 [146, 263, 264, 267, 268], and 23 [263-268], and inconsistent responses in other studies for serotypes/groups 1 [263, 264], 12 [146, 266], 14 [264, 266], and 18 [268].

A particular concern relating to the administration of pneumococcal polysaccharide vaccine (PPS) to unprimed young children is the theoretical risk that hyporesponsiveness may occur following re- challenge or subsequent pneumococcal exposure following PPS [189]. This phenomenon has been demonstrated in studies with Group A and C meningococcal polysaccharide vaccine [271]. Studies in young children using different valencies and formulations ranging from five to100 μg/serotype of PPS have shown inconsistent results including reduced responses to some serotypes following revaccination [257, 265]. Conversely, one infant study showed no evidence of hyporesponsiveness on revaccination with PPS [266]. The assays used in these studies were less specific than techniques currently in use, and the clinical relevance of these immunological findings remains unknown.

The seven serotypes included in PCV are responsible for 55% of IPD episodes in children aged under 5 in Fiji [385]. This potential serotype coverage would increase to 83% if the 23vPPS, which does not contain serotype 6A, was used, and 87%, if the new 13-valent pneumococcal conjugate vaccine produced by Wyeth Vaccines (which includes serotypes 1, 3, 5, 6A, 7F and 19A) was used [385]. The aim of this study was to find an optimal vaccination strategy for resource poor countries in terms of serotype coverage, flexibility, and affordability. We undertook a Phase II vaccine trial in Fiji to document the safety and immunogenicity of various pneumococcal vaccination regimens combining one, two, or three doses of PCV in infancy. To broaden serotype coverage, the additional benefit of a booster of 23vPPS at 12 months of age was also assessed. To address the concerns of hyporesponsiveness to PPS following re-challenge, this paper presents the

138

immunological response at 17 months of age to a small challenge dose of 20% of the 23vPPS (mPPS) in infants who had or had not received the 23vPPS at 12 months of age.

7.3 Methods

7.3.1 Study Participants

7.3.2 Study Procedures and Vaccines

Infants were stratified by ethnicity and randomised into one of eight groups The seven-valent

CRM197 protein-polysaccharide conjugate vaccine containing polysaccharide antigen from pneumococcal serotypes 4, 6B, 9V, 14, 18C, 19F, 23F (Prevenar , Wyeth Vaccines) was used. The vaccine contains 2 g/serotype, except serotype 6B which is 4 g. The three dose group received PCV at six, ten, and 14 weeks of age, the two dose group received PCV at six and 14 weeks of age and the one dose group received PCV at 14 weeks of age. Routine vaccines (HiberixTM mixed with TritanrixTM-HepBTM, GlaxoSmithKline) and oral polio were given with the primary series. HiberixTM contains 10μg of purified Hib capsular polysaccharide covalently bound to approximately 30μg tetanus toxoid mixed with TritanrixTM-HepBTM which contains not less than 30 IU of adsorbed D toxoid, not less than 60 IU of adsorbed T toxoid, not less than 4 IU of wP, and 10µg of recombinant HBsAg protein. The children in all primary series groups were further randomized to receive a dose of 23vPPS (PneumovaxTM, Merck & Co., Inc., which consists of a purified mixture of 25µg of capsular polysaccharide from 23 pneumococcal serotypes) or no vaccine at 12 months of age (window: 12 months plus four weeks). In addition, all children received Measles-Rubella vaccine at 12 months of age co-administered with 23vPPS. All children received 20% of the 23vPPS (mPPS) at 17 months of age (window: 17 months plus eight weeks). The children randomised to receive 0 or 1 PCV dose in infancy, had a single dose of PCV administered at 2 years of age.

Children were followed up for serious adverse events (SAE’s) to any of the study vaccines throughout the two year study period. The occurrence of SAE’s were sourced from parent interviews at each visit and by searching the national computerised hospital discharge records every quarter. Causality of any SAE was assigned by the study doctor and assessed by an independent safety monitor. All SAE’s were periodically reviewed by an independent DSMB.

139

7.3.3 Laboratory Procedures

Children who received the 12 month 23vPPS had bloods drawn prior to and 14 days post 23vPPS. All children had blood taken before and four weeks following the 17 month mPPS. Blood was separated by centrifugation at the health centre, kept chilled and transported to the CWMH laboratory, Suva, where it was divided into aliquots and stored at -20 C on the same day, until transported to the Pneumococcal Laboratory, Murdoch Childrens Research Institute, Melbourne on dry ice for analysis.

Anticapsular pneumococcal antibody levels were assayed for all 23vPPS serotypes (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F), using a modified 3rd generation ELISA based on current WHO recommendations[341]. Briefly 96-well medium binding polystyrene plates (Greiner microlon, Germany) were coated with pneumococcal polysaccharides (ATCC, USA) and incubated overnight at room temperature. Non-specific, non-opsonic antibodies were absorbed from sera by incubation overnight at 4oC with PBS containing 10% foetal bovine serum (PBS/FCS), cell wall polysaccharide (C-PS 10 g/ml) and serotype 22F (30 g/ml). The reference serum 89SF [125, 396] (Dr Milan Blake, FDA, USA) and samples for anti serotype 22F IgG quantitation were absorbed with PBS/FCS and C-PS. Plates were blocked with PBS/FCS and patient samples and standards added. The reaction was detected with a secondary antibody HRP conjugated anti-human IgG (Chemicon, Australia) and enzyme substrate solution, TMB (3,3’,5,5’- tetramethylbenzidine, KPL, USA) followed by a 1M H3PO4 stop solution. The absorbance (OD) was measured at 450 nm (reference filter 630 nm) on a Bio-Tek Elx808 (Bio-Tek Instruments, USA). OD was converted to antibody concentrations ( g/ml) using KCJunior software (Bio-Tek Instruments, USA). Sample dilutions were analyzed in duplicate and three controls (low, medium and high) were included on each plate to assess assay performance and inter-assay variation. Results from an inter-laboratory comparison between Wyeth Vaccines and the KTL Finland laboratory demonstrated a good correlation in measurement of serotype-specific antibody concentrations [397]. Laboratory staff members were blinded to the group allocation of each serum sample.

7.3.4 Data Management and Statistical Analysis

Cleaned data were exported to Stata version 9.0 (Stata Corporation, College Station, Texas) for analysis. Serotype-specific antibody concentrations by ELISA were log transformed (to base e) to calculate GMC. Comparisons of pre- and post-mPPS GMC and between group comparisons were performed using a paired t-test and two sample t-test respectively. Simple and multi-variable regression analyses were undertaken to adjust for both the pre-mPPS log antibody concentration

140

for all 23 serotypes, and the number of PCV doses administered for all seven PCV serotypes. A p- value of <0.05 was considered statistically significant.

The primary endpoint was serotype-specific GMC response to mPPS at 18 months of age in children who had received the 12 months 23vPPS compared to children who had not received the 23vPPS. We defined hyporesponsiveness to a particular serotype as a significantly lower GMC observed post-mPPS, in the 12 month 23vPPS group compared to the no 12 month 23vPPS group, controlling for pre-mPPS antibody levels, using multivariable regression analysis. To prevent an inflated type 1 error due to multiple comparisons, and obtain a single p-value for the null hypothesis of mPPS having no impact on the antibody response to any of the 23 serotypes, a joint test of all the regression coefficients from the aforementioned multivariable regression analysis was performed [347].

7.3.5 Ethical Approval

The study was approved by the Fiji National Research Ethics Review Committee and the University of Melbourne Human Research Ethics Committee.

7.4 Results

There were 552 children enrolled in the study (Figure 10) which represents a consent rate of 30.5%. There were 90 (16.3%) withdrawals and no child was withdrawn due to an adverse event resulting from administration of any of the vaccines. Characteristics and the number of children randomised to the eight groups are shown in Table 20.

Following the 12 month 23vPPS, there were significantly higher GMC (each p<0.001) for all PCV serotypes. For four of seven PCV serotypes (4, 9V, 18C, 19F) this response was most profound in the group that had received only a single dose of PCV (each p<0.001). Children who received the 23vPPS at 12 months showed significant higher GMC (each p<0.001) for all non-PCV serotypes in the 23vPPS.

Five months following the 12 month 23vPPS and prior to the administration of the re-challenge dose of mPPS at 17 months of age, the group that had received 23vPPS at 12 months had significantly higher GMC for all the PCV and non-PCV serotypes compared with the groups that had not received the 12 month 23vPPS (Figure 11 and Figure 13 respectively; each p<0.001).

GMC to the PCV serotypes following the re-challenge dose of mPPS at 17 months are shown in

141

Figure 12. The groups that did not receive the 12 month 23vPPS had better responses and significantly higher GMC for all PCV serotypes than those groups that had received the 12 month 23vPPS (Figure 12). Response to mPPS for the non-PCV serotypes are shown in Figure 14. The groups that did not receive the 12 month 23vPPS had significantly higher GMC for six of 16 non- PCV serotypes (7F, 9N, 12F, 19A, 22F, 33F) compared with those groups that did have the 12 month 23vPPS (Figure 14).

To examine the effect of 23vPPS at 12 months and the number of PCV doses in early infancy, we performed graphical examination to assess whether the poor response to mPPS in the 12 month 23vPPS recipients was due to the higher pre-mPPS antibody concentrations. Figure 15 shows the post-mPPS log antibody concentration (y-axis) against the pre-mPPS log antibody concentration (x- axis) for the non-PCV serotypes 1, 5, 7F, and 19A. For any given log antibody concentration pre- mPPS, children who had not received the 23vPPS at 12 months had higher log antibody concentrations one month post-mPPS. A similar pattern is seen for all other non-PCV serotypes (data not shown). For PCV serotypes, a similar pattern was demonstrated. Figure 16 and Figure 17 show the post-mPPS log antibody concentration for serotypes 4 and 6B respectively, against the pre-mPPS concentration.

For the PCV serotypes further adjustment for prior receipt of one, two or three PCV doses in addition to 23vPPS exposure and pre-mPPS antibody concentration was undertaken. Adjustment for the number of PCV dosages had limited impact on the overall effect of prior receipt of 23vPPS on the response to mPPS. For each of the PCV dosage groups and any given pre-mPPS antibody concentration, those who did not receive 23vPPS at 12 months of age had a higher log antibody concentration post-mPPS, shown in Figure 18 and Figure 19 for serotypes 4 and 6B respectively.

1811 screened 142 1259 did not consent 552 enrolled

3 PCV=71 3PCV+12m 23vPPS=65 2PCV=76 2PCV+12m 23vPPS=80 1PCV=62 1PCV+12m 23v PPS=66 12m23vPPS=63 0PCV no12m 23v PPS=69

Withdrawn =11 Withdrawn=16 Withdrawn=8 Withdrawn=13 Withdrawn=13 Withdrawn=7 Withdrawn=6 Withdrawn=6

Refused=1 Unsuccessful blood Unsuccessful draw=1 blood draw=1

Pre-mPPS1=59 Pre-mPPS1=48 Pre-mPPS1=68 Pre-mPPS1=67 Pre-mPPS1=49 Pre-mPPS1=59 Pre-mPPS1=57 Pre-mPPS1=63

No mPPS=1 No mPPS=2 No mPPS=1 No mPPS=2 =given=2

mPPS2=58 mPPS2=49 mPPS2=68 mPPS2=65 mPPS2=48 mPPS2=59 mPPS2=57 mPPS2=61

Withdrawn =1 Withdrawn=1 Withdrawn=3 Withdrawn=1 Withdrawn=1 Withdrawn=1 Withdrawn=3

3 3 3 3 3 3 3 3 Post-mPPS =59 Post-mPPS =49 Post-mPPS =67 Post-mPPS =64 Post-mPPS =48 Post-mPPS =58 Post-mPPS =56 Post-mPPS =60

Figure 10: CONSORT chart of the screened and enrolled children to 18 months of age, showing the number having the pre-mPPS blood test1, mPPS at 17 2 3 months of age , and blood test one month post-mPPS

Withdrawals: 34 moved out of the study area, 16 failed to return for follow up, 13 refused a study procedure, nine developed an exclusion criterion, nine were withdrawn due to a protocol amendment, for six no reason was given, two had protocol violations, and one died from causes unrelated to receipt of the study vaccine.

143

Table 20: Baseline characteristics of infants at enrolment and on randomisation to one of eight groups Characteristics 3 PCV 2 PCV 1 PCV 0 PCV No 12m 12m No 12m 12m No 12m 12m 12m No 12m 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS (n=71) (n=65) (n=76) (n=80) (n=62) (n=66) (n=63) (n=69) Gender Male 38 (53.5%) 33 (50.8%) 31 (40.8%) 39 (48.8%) 31 (50%) 28 (42.4%) 32 (50.8%) 32 (46.4%) Female 33 (46.5%) 32 (49.2%) 45 (59.2%) 41 (51.2%) 31 (50%) 38 (57.6%) 31 (49.2%) 37 (53.6%) Median age in 6.7 6.5 6.5 6.4 6.5 6.5 6.5 6.4 weeks Ethnicity Indigenous 39 (54.9%) 43 (66.1%) 48 (63.1%) 58 (72.5%) 41 (66.1%) 42 (63.7%) 35 (55.6%) 45 (65.2%) Fijian Indo-Fijian 29 (40.9%) 17 (26.2%) 24 (31.6%) 21 (26.3%) 17 (27.4%) 22 (33.3%) 20 (31.7%) 20 (29.0%) Other 3 (4.2%) 5 (7.7%) 4 (5.3%) 1 (1.2%) 4 (6.5%) 2 (3.0%) 8 (12.7%) 4 (5.8%) Median weight 4850 5000 4775 4800 4900 4750 4750 4800 in grams

Figure 11: Serotype-specific IgG GMC (μg/mL) to PCV serotypes at 17 months of age pre-mPPS in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs.

3 GMC (mcg/mL) GMC

0 4 6B 9V 14 18C 19F 23F no 12m 23vPPS 12m 23vPPS

144

Figure 12: Serotype-specific IgG GMC (μg/mL) to PCV serotypes at 17 months of age post-mPPS in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs.

3 GMC (mcg/mL) GMC 2

0 4 6B 9V 14 18C 19F 23F no 12m 23vPPS 12m 23vPPS

Figure 13: Serotype-specific IgG GMC (μg/mL) to non-PCV serotypes at 17 months of age pre-mPPS in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs.

3.5

2.5

1.5

1 GMC GMC (mcg/mL)

0.5

0 1 2 3 5 7F 8 9N 10A 11A 12F 15B 17F 19A 20 22F 33F no 12m 23vPPS 12m 23vPPS

145

Figure 14: Serotype-specific IgG GMC (μg/mL) to non-PCV serotypes one month post-mPPS in children who did or did not receive the 12 month 23vPPS. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs.

3.5

2.5

1.5

1 GMC (mcg/mL)

0.5

0 1 2 3 5 7F 8 9N 10A 11A 12F 15B 17F 19A 20 22F 33F no 12m 23vPPS 12m 23vPPS

146

Serotype 1 Serotype 5 3

3 2

2 1 1 0 0

-1 -1

-2 -2

Post mPPS, Log antibody concentration antibody Log mPPS, Post Post mPPS, Log antibody concentration antibody Log mPPS, Post

-3 -3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 Pre mPPS, Log Ab concentration Pre mPPS, Log Ab concentration

Serotype 7F Serotype 19A 3

2 3

1 2

0 1

0 -1

-1 -2

-2

Post mPPS, Log antibody concentration antibody Log mPPS, Post Post mPPS, Log antibody concentration antibody Log mPPS, Post

-3 -3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 Pre mPPS, Log Ab concentration Pre mPPS, Log Ab concentration Footnote: A 45-degree line, which indicates no change before and after the re-challenge, is super-imposed. The pre- and post-mPPS log antibody concentrations of those who received 23vPPS at 12 months mostly fell along the 45-degree line, indicating no response to mPPS. In contrast, most children who did not receive 23vPPS had an increase in antibody concentration, as indicated by the data points falling above the 45- degree line. The distribution of pre-mPPS log antibody concentrations of those that did and did not receive the 12 month 23vPPS mainly overlapped in the range between -2 to 0 for serotype 1. Within this range, it is clear that at any given log antibody concentration pre-mPPS, children who had not received the 23vPPS at 12 months had higher log antibody concentrations one month post-mPPS.

147

Figure 16: Pre- and one month post-mPPS log antibody concentrations for serotype 4 for children who did (+) or did not (o) receive 23vPPS at 12 months of age

Serotype 4

-1

Post mPPS,Log Ab concentration Ab mPPS,Log Post -2

-3

-3 -2 -1 0 1 2 3 Pre mPPS, Log Ab concentration

Figure 17: Pre- and one month post-mPPS log antibody concentrations for serotype 6B for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series

Serotype 6B

-1 Post mPPS, Log Ab concentration Ab Log mPPS, Post -2

-3 -3 -2 -1 0 1 2 3 Pre mPPS, Log Ab concentration

148

Figure 18: Pre- and one month post-mPPS log antibody concentrations for serotype 4 for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series

Serotype 4

PCV==0 PCV==1

3 2 1 0 -1 -2 -3

PCV==2 PCV==3

3 2 1 0

Post mPPS, Log Ab concentration Ab Log mPPS, Post -1 -2 -3

-3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 Pre mPPS, Log Ab concentration Graphs by PCV

Figure 19: Pre- and one month post-mPPS log antibody concentrations for serotype 6B for children who did (+) or did not (o) receive 23vPPS at 12 months of age and by the number of PCV doses in the primary series

Serotype 6B

PCV==0 PCV==1

3 2 1 0 -1 -2 -3

PCV==2 PCV==3

3 2 1 0 Post mPPS, Log Ab concentration Ab Log mPPS, Post -1 -2 -3 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3 Pre mPPS, Log Ab concentration Graphs by PCV

149

To quantify the above graphical examination, simple and multi-variable regression analyses were undertaken to adjust for the pre-mPPS log antibody concentration for each serotype, and then by number of PCV doses administered for the PCV serotypes. Without adjusting for the pre-mPPS log antibody concentration, the log antibody concentrations for the non-PCV serotypes one month post-mPPS were not significantly different between those who had or had not received 23vPPS at 12 months of age for ten of 16 non-PCV serotypes (1, 2, 3, 5, 8, 10F, 11A, 15B, 17F and 20) but were significantly lower for the remaining six of 16 serotypes in those who had received the 12 month 23vPPS. The log antibody concentrations one month post-mPPS are significantly associated with the pre-mPPS antibody concentration for all 16 non-PCV serotypes (each p<0.001). Having adjusted for the pre-mPPS log antibody concentration, exposure to 23vPPS was associated with a lower response to mPPS for all 16 non-PCV serotypes (each p<0.001). For PCV serotypes, a similar response was demonstrated. The response one month post-mPPS was significantly associated with the pre-mPPS antibody concentration for all seven PCV serotypes (p<0.001) and having adjusted for the pre-mPPS concentration, prior exposure to 23vPPS was associated with a lower response to mPPS (each p<0.001). In contrast, most children who had not received 23vPPS had an increase in antibody concentration. A joint test rejected the null hypothesis of mPPS having no impact on the antibody response to any of the 23 serotypes, having adjusted for the pre-mPPS antibody concentrations (p<0.001).

There were 101 SAE’s throughout the study period with none attributable to receipt of any of the study vaccines. In children over 12 months of age, there were 14 SAE’s in the 12 month 23vPPS group and 22 SAE’s in the group that did not receive the 23vPPS. There were four cases of inpatient pneumonia in children who had received the 12 month 23vPPS compared to seven cases in those that had not, in infants aged over 12 months of age. There were no cases of IPD throughout the study period.

7.5 Discussion

This is the first study in children, using the third generation WHO ELISA assay to measure antibody responses to all 23vPPS serotypes following receipt of that vaccine. The results show that prior receipt of 23vPPS causes immune hyporesponsiveness to a subsequent 23vPPS challenge. Despite those children who received the 12 month 23vPPS having higher circulating antibody concentrations at 17 months of age, their responses to a re-challenge with a small dose of 23vPPS demonstrated a profound lack of response to all 23 serotypes after adjusting for the pre-existing

150

antibody concentration. In contrast, those children who had not received the 12 month 23vPPS could clearly mount a satisfactory response to mPPS.

There are a number of potential immunological mechanisms that may explain these findings. In vitro studies have suggested that polysaccharides antigens may be able to down regulate B cells [279], and that newly formed antibody via IgG, IgM, or immune complexes can bind to inhibitory Fc receptors and prevent antibody production [280]. The critical role of pneumococcal-specific memory B cells in first line of defense against pneumococcal infection has recently become an important area of research. IgM+CD27+ and ‘switched’ IgG+CD27+ memory B cells are considered to play a role in the immune response to 23vPPS since these cell populations are deficient in patients with primary immunodeficiency syndromes who are susceptible to recurrent infections with encapsulated bacteria [108, 109]. Both plasma and memory B cells are stimulated following exposure to PPS. In contrast to T-independent immune responses, priming by either PCV, previous encounter with S. pneumoniae or a cross-reacting antigen prior to 23vPPS vaccination, could stimulate immunological memory by presentation of polysaccharide-protein conjugate antigens to the immune system (T-dependent) [281]. Given the T-independent nature of PPS antigens, 23vPPS may stimulate the existing pool of memory B cells to differentiate into plasma cells and secrete antibody without replenishment of the memory B cell pool. This has been proposed as one mechanism for the hyporesponsiveness observed following polysaccharide vaccine administration [282]. Upon subsequent booster with 23vPPS or a natural infection, immune hyporesponsiveness could be induced as a result of a decreased memory B cell population and result in the reduced antibody concentrations observed in this study.

In addition, the development of immune hyporesponsiveness may also be the result of immune regulation via the establishment of pneumococcal-specific tolerogenic immune responses. Increased expression of the immunosuppressive cytokine interleukin 10 [264, 283] and suppressor T cell activity may suppress the response to PPS [284]. Recent evidence also suggests a role for CD4+ T-lymphocytes in the immune response to pneumococcal antigens [285]. Studies have demonstrated the importance of co-stimulatory signals (CD40-CD40L) for a robust immune response to pneumococcal antigens and that CD4+ T-lymphocytes can protect mice against pneumococcal colonization independent of specific antibody. These findings strongly suggest a role for cellular immunity in protection against pneumococcal infection [98, 99, 286-288]. Furthermore it is possible that regulatory T-lymphocytes (Treg) may suppress antibody production and other immune responses in the context of chronic antigen exposure. Hyporesponsiveness induced by Treg has been described during bacterial, viral and parasitic infections with up-

151

regulation of CD4+CD25+ Treg and IL-10 and TGF-β secretion [284, 289]. Limited data is available on the role of Treg in the attenuation immune response to pneumococcal antigens. However a high level of exposure to pneumococci, particularly in early life, could induce Treg activity that suppresses serotype-specific IgG, thereby increasing IPD risk following 23vPPS immunization.

The clinical relevance of this immunological finding in this study are not known. There is one case report documenting immunological paralysis for four years to the causative pneumococcal serotype in a nine month old infant who had pneumococcal meningitis, despite demonstrating normal immune responses to other protein and polysaccharide antigens [290]. Most studies evaluating the impact of PPS immunization in the absence of additional PCV in infants or children have not shown any impact on pneumococcal disease or carriage [267, 292, 298]. In contrast, a study in Papua New Guinea, where children aged six months to five years of age were given either the 14 or 23vPPS in one or two doses according to age, there was a (non-significant) 19% reduction in mortality from any cause, and a 50% reduction in pneumonia mortality (95%CI 1-75%) [299]. Natural exposure in a population with a high incidence of pneumococcal infections, resulting in regular antigenic stimulation may explain this finding [189]. However, a Finnish study of the 14- valent PPS in infants aged three months to six years showed significant efficacy against vaccine type recurrent otitis media was 52% for children less than two years of age if serogroup 6 was excluded [146].

A study documenting immunological memory five years after meningococcal A/C conjugate vaccination in infancy showed that challenge with the meningococcal polysaccharide or conjugate at two years of age induced immunological memory [271]. However subsequent challenge with polysaccharide at five years of age failed to induce a similar memory response in the polysaccharide group. The authors concluded that the initial polysaccharide immunization at two years of age interfered with the immune response to subsequent polysaccharide vaccination, a finding similar to our current results with 23vPPS [271]. No adverse clinical effects have ever been documented from repeated exposure to the meningococcal polysaccharide vaccine and in this study we demonstrated no increase in clinical adverse effects to the 23vPPS, although the numbers were small and the study was not designed to study this. There was no increase in nasopharyngeal carriage of non-PCV serotypes five months after receipt of the 12 month 23vPPS (FM Russell et al. in press). We intend to follow the children from this study to assess NP carriage as an increase in carriage of non-PCV types in the 12 month 23vPPS group would indicate that this immunological finding may have a biological effect. This would provide the first indication that these children may have increased susceptibility to pneumococcal disease. Further results

152

documenting the avidity and OPA post 23vPPS and mPPS, and the impact on NP carriage will follow. In addition, immunological assays to assess B cell subsets will enable a more comprehensive assessment of the impact of 23vPPS on immunological functioning. However, our findings suggest that additional immunization with the 23vPPS following a primary series of PCV does not provide added benefit for antibody production and instead results in impaired immune responses following a subsequent PPS antigen challenge. Whether this observation is associated with adverse clinical effects remains to be determined. Nevertheless, our findings raise some doubt over the value of the combined PCV/23vPPS schedule.

153

8 OPSONOPHAGOCYTIC ACTIVITY FOLLOWING A REDUCED DOSE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE INFANT PRIMARY SERIES AND 23-VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE AT 12 MONTHS OF AGE

8.1 Abstract

Opsonophagocytic activity (OPA) was measured following reduced infant doses of 7-valent pneumococcal conjugate vaccine (PCV) with or without 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months, and subsequent re-exposure to a small dose of pneumococcal polysaccharide antigens (mPPS) at 17 months. Fijian infants were randomized to receive 0, 1, 2, or 3 PCV doses. Half received 23vPPS at 12 months and all received mPPS at 17 months. OPA was performed on up to 14 serotypes. Three and 2 PCV doses resulted in similar OPA for most PCV serotypes up to 9 months and for half of the serotypes at 12 months. A single dose improved OPA compared with the unvaccinated group. 23vPPS significantly improved OPA for all serotypes tested but in general, was associated with diminished responses following re-challenge.

8.2 Introduction

Streptococcus pneumoniae is the leading vaccine preventable cause of serious infection in infants [3]. A recent review estimated that over 14 million episodes of serious pneumococcal disease occurred worldwide in the year 2000, with over 800,000 deaths in children under 5 years or age [41]. Pneumococcal capsular polysaccharide is a major virulence factor of S. pneumoniae and this capsule resists phagocytosis by the host immune cells [398, 399]. The primary mechanism of protection in the host against invasive pneumococcal disease (IPD) is mediated by the presence of opsonophagocytic antibodies [131]. Antibodies to pneumococcal polysaccharide offer protection by opsonizing pneumococci and activating complement thereby promoting phagocytosis of pneumococci. The ability of anti-pneumococcal antibodies to induce phagocytosis and killing is believed to be a surrogate for protection. As both antibody concentration and avidity contribute to opsonophagocytic activity (OPA) and confer protection in a mice challenge models [102, 132-135], OPA is considered to be the best marker of vaccine efficacy. While it is recommended that antibody concentration, as measured by a standardized enzyme immunoassay (ELISA), should be the primary measure when licensing new pneumococcal conjugate vaccines [102], OPA has been accepted as a necessary additional measure and the reference method for measuring the protective capacity of pneumococcal antibodies [126]. A recent study has shown that only OPA can be a surrogate for immune protection for the vaccine related serotype 19A [400]. Moreover, OPA levels have been found to be more meaningful than ELISA titers with respect to the prediction of protective efficacy against acute otitis media (AOM) [401].

154

The aim of this study in Fiji was to find an optimal pneumococcal vaccination strategy for resource poor countries in terms of serotype coverage, flexibility, and affordability. We undertook a Phase II vaccine trial in Fiji to document the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination regimens combining one, 2, or 3 doses of PCV in infancy. To broaden serotype coverage, the additional benefit of a booster of 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months of age was also assessed. To address the concerns of immunological hyporesponsiveness to 23VPPS in young children following re-challenge, the immunological response at 17 months of age to a small challenge dose of 20% of the 23VPPS (mPPS) was evaluated.

Our findings to date include evidence of similar immunogenicity following either a 2 or 3 dose PCV primary series, as measured by IgG antibody concentrations to capsular polysaccharides by ELISA [389]. We also found that a single PCV dose would offer protection in the first 12 months of life for many serotypes [389]. The one or 2 dose PCV schedules induced immunologic memory [386]. There were significant responses for the non-PCV serotypes included in 23vPPS booster which persisted for at least 5 months following vaccination [386]. Memory responses following 23VPPS were most profound for children who had received only a single dose of PCV previously, compared with the 2 or 3 dose groups [386]. However despite higher antibody concentrations at 17 months in children who had received 23vPPS at 12 months, the response to a re-challenge was poor to all 23 serotypes compared to children who did not receive the 12 month 23vPPS indicating immunological hyporesponsiveness [390]. In this manuscript we present the functional antibody responses to these different pneumococcal vaccination schedules, as measured by OPA.

8.3 Materials and Methods

8.3.1 Study Participants

The study was a single blind, open-label randomized Phase II vaccine trial undertaken in Suva, Fiji. Healthy infants aged between 6 and 8 weeks were eligible for enrolment. Details of the selection criteria and the randomization procedure have been reported elsewhere [386, 389, 390].

The study was conducted and monitored according to Good Clinical Practice. Monitoring was undertaken by Pharmaceutical Product Development Inc. It was approved by the Fiji National Research Ethics Review Committee and the University of Melbourne Human Research Ethics Committee.

155

8.3.2 Study Procedures and Vaccines

Infants were stratified by ethnicity and randomized into one of 8 groups to receive 0, 1, 2 or 3 doses of PCV in infancy with or without a 23VPPS booster at 12 months of age. Infants received the

7-valent CRM197 protein-polysaccharide conjugate vaccine containing polysaccharide antigen from pneumococcal serotypes 4, 6B, 9V, 14, 18C, 19F, 23F (PrevenarTM, Wyeth Vaccines). The vaccine contains 2 g per serotype, except serotype 6B which contains 4 g. The 3 dose group received PCV at 6 weeks, 10 weeks, and 14 weeks of age. The 2 dose group received PCV at 6 and 14 weeks of age and the single dose group received PCV at 14 weeks of age. Routine vaccines, with combined diphtheria-tetanus-pertussis-Hepatitis B and Hemophilus influenzae type b (HiberixTM mixed with TritanrixTM-HepBTM, GlaxoSmithKline) and oral polio were given at 6, 10, and 14 weeks of age. Half the children in all primary series groups were randomized to receive a full dose (0.5mL) of 23vPPS (PneumovaxTM, Merck & Co., Inc., which consists of a purified mixture of 25µg of capsular polysaccharide from 23 pneumococcal serotypes: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F) at 12 months of age in the left upper deltoid muscle. All children received Measles-Rubella vaccine at 12 months of age in the right upper deltoid muscle. All children received 0.1mL of the 23VPPS (mPPS) at 17 months of age. The children randomised to receive 0 or one PCV dose in infancy, had a single dose of PCV administered at 2 years of age.

8.3.3 Laboratory Assays

All children except the group randomized to receive no PCV or 23vPPS in infancy had blood drawn at 18 weeks and 12 months of age. Those children who were randomized to not receive the 12 month 23vPPS additionally had blood drawn at 9 months of age. Children who received the 12 month 23vPPS additionally had blood drawn 14 days post-23VPPS. All children had blood drawn before and 4 weeks following mPPS. Serum was separated from blood by centrifugation at the health centre, kept chilled, and transported to the Colonial War Memorial Hospital laboratory, Suva, where it was divided into aliquots and stored at -70 C on the same day, until transported to the Pneumococcal Laboratory, Murdoch Childrens Research Institute, Melbourne on dry ice for analysis, then to the Bacterial Respiratory Pathogen Reference Laboratory, University of Alabama at Birmingham, USA for OPA analysis.

Opsonophagocytic assays were performed for 8 serotypes (serotypes 1, 4, 5, 6B, 9V, 14, 18C, and 23F) at each blood draw and on 6 additional serotypes (3, 6A, 6C, 7F, 19A, 19F) for the pre- and post-mPPS blood draws using a fourfold multiplexed OPA (MOPA) [139]. In brief, all serum samples were incubated at 56°C for 30 min before serial dilutions. Frozen aliquots of target pneumococci

156

were thawed, washed twice with opsonization buffer B (Hanks’ balanced salt solution *HBSS+ with Mg and Ca, 0.1% gelatin, and 10% FBS) by centrifugation (12,000 X g, 2 min), and diluted to the proper bacterial density (~5 X 104 CFU/ml for each of the 4 serotypes). Ten microliters of bacterial suspension was added to each well. After 30 min of incubation at room temperature, 10 μl of complement and 40μl of HL60 cells were added to each well. HL60 cells were washed twice before use with HBSS by centrifugation (350 X g, 5 min), and 4 X 105 cells were added to each well. Plates were incubated in a tissue culture incubator (37°C, 5% CO2) with shaking (mini orbital shaker; Bellco Biotechnology, Vineland, NJ) at 700 rpm. After a 45-min incubation, plates were placed on ice for 10 to 15 min and an aliquot of the final reaction mixture (10 μl) was spotted onto four different Todd-Hewitt broth–yeast extract (0.5%) agar plates [345]. After application of an overlay agar containing one of the four antibiotics to each Todd-Hewitt broth–yeast extract (0.5%) agar plate and overnight incubation at 37°C, the number of bacterial colonies in the agar plates was enumerated [345]. The MOPA results are expressed as opsonization indices (OIs). An OI is defined as the interpolated dilution of serum that killed 50% of bacteria.

Serum anti-pneumococcal antibody concentrations to each of the serotypes were measured by the third generation ELISA which included a pre-absorption step with 22F polysaccharide to increase the specificity of the assay. The details have been described elsewhere [386, 389, 390].

8.3.4 Statistical Analysis

The results presented are based on secondary analyses nested within the main trial. All data collection forms were monitored prior to double data entry. Cleaned data were exported to Stata version 9.0 (Stata Corporation, College Station, Texas) for analysis. In the MOPA, the lowest dilution of serum tested in the assay is 4. The OIs of samples that did not kill 50% of bacteria were reported as “2” for analysis purposes. The threshold of the assay was set at 8 [401]. Pair-wise comparisons of proportions with OIs ≥ 8 were performed using exact McNemar’s test. Comparisons of the proportion of infants with OIs ≥ 8 between groups were performed using Fisher’s exact test. Serotype-specific OIs were natural log transformed to calculate geometric mean titers (GMT). Simple and multi-variable regression analyses were undertaken to adjust for both the pre-mPPS OPA titer for all 23 serotypes, and the number of PCV doses administered for all 7 PCV serotypes. We defined hyporesponsiveness to a particular serotype as a significantly lower OPA titer observed post-mPPS, in the 12 month 23vPPS group compared to the no 12 month 23vPPS group, controlling for pre-mPPS OPA titer levels, using multivariable regression analysis. Serotype- specific antibody concentrations by ELISA were log (base e) transformed to calculate geometric mean concentrations (GMC). Serotype-specific IgG GMC are presented but analyses have been

157

reported in detail elsewhere [386, 389, 390]. PCV serotypes were classified as serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. All other serotypes were classified as non-PCV serotypes. Due to the multiple comparisons, a p-value of <0.01 was considered statistically significant.

8.4 Results

There were 552 children enrolled in the study. Characteristics and the number of children randomized to the 8 groups have been reported elsewhere [390, 402]. There were 90 (16.3%) withdrawals. No participants were withdrawn due to an adverse event attributable to any of the vaccines.

8.4.1 PCV Serotypes

One month following the PCV primary series there were no significant differences between the 3 and 2 PCV dose groups in the proportion of infants with OI ≥8 for 4 of 6 PCV serotypes (Table 21). OPA GMT following 2 PCV doses were 3 to10-fold lower for serotypes 23F and 6B respectively compared with a 3 dose schedule, with serotype 14 showing a similar trend. A single PCV dose was inferior to 3 doses but significantly increased the proportion of infants with OI ≥8 compared with the unvaccinated group for all serotypes except 6B.

At 9 months of age there were no significant differences between the 3 and 2 PCV dose groups in the proportion of infants with OI ≥8 for any PCV serotype except serotype 23F (Table 21). However there was a trend for higher OPA GMT in the 3 dose group compared to the 2 dose group. The proportion of infants with OI ≥8 was significantly higher in the 3 dose group compared with the single dose group for all serotypes except serotype 4.

At 12 months of age the proportion of infants with OI ≥8 were significantly higher in the 3 dose group compared to the 2 dose group for 3 of 6 PCV serotypes (6B, 18C, 23F) with approximately 24% fewer children in the 2 dose group having an OI ≥8 for serotype 6B compared to the 3 dose group (Table 21). There was a trend for higher OPA GMT for serotype 14 in the 3 PCV dose group compared to the 2 PCV dose group. The proportion of infants with OI ≥8 was significantly higher for all serotypes in the 3 PCV dose group compared with the single dose (except serotype 4) and the unvaccinated groups. A single PCV dose significantly increased the proportion of infants with OI ≥8 compared to the unvaccinated group for all serotypes except 6B.

158

Table 21: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of infants with OI ≥8 to 6 PCV serotypes 4 weeks post primary series, and at 9 and 12 months of age, by number of PCV administered in the primary series Serotype 3 PCV 2 PCV 1 PCV 0 PCV GMC OPA GMT % GMC OPA GMT % GMC OPA GMT % GMC OPA GMT % (95%CI) (95%CI) OI ≥8 (95%CI) (95%CI) OI ≥8 (95%CI) (95%CI) OI (95%CI) (95%CI) OI ≥8 n=125 n=146 n=121 ≥8 n=62 n=119 n=141 n=118 n=61 4 weeks post primary series 4 5.47 872.3 100 5.23 935.6 99 2.20 219.0 942 0.04 2.3 22,3 (4.84-6.19) (709.6-1072.2) (4.46-6.13) (745.0-1175.0) (1.80-2.70) (153.0-313.5) (0.03-0.04) (1.9-2.7) 6B 1.66 660.6 95 0.86 63.6 682 0.19 3.7 142 0.11 2.2 32 (1.33-2.07) (423.6-1030.2) (0.70-1.07) (40.0-101.2) (0.16-0.22) (2.8-4.9) (0.10-0.13) (1.9-2.4) 9V 4.76 801.7 100 4.71 794.8 97 0.90 84.3 762 0.07 2.1 22,3 (4.19-5.40) (609.9-1053.8) (3.88-5.71) (581.8-1085.9) (0.74-1.09) (53.6-132.6) (0.06-0.08) (1.9-2.4) 14 5.51 984.4 94 3.12 430.4 87 1.07 137.6 752 0.34 3.3 232,3 (4.50-6.76) (633.4-1529.9) (2.42-4.03) (267.4-692.6) (0.89-1.27) (78.8-240.5) (0.27-0.43) (2.6-4.2) 18C 3.20 696.2 98 2.67 576.1 98 0.58 35.6 632 0.06 2.2 22,3 (2.66-3.86) (508.0-954.1) (2.16-3.31) (435.5-762.0) (0.45-0.74) (23.1-54.8) (0.05-0.07) (2.0-2.3) 23F 2.93 750.4 97 1.65 278.8 872 0.23 18.2 462 0.11 2.2 22,3 (2.39-3.59) (515.2-1093) (1.29-2.11) (182.3-426.5) (0.20-0.27) (11.1-29.5) (0.10-0.14) (2.0-2.4) 9 months n=37 n=36 n=49 n=48 n=43 n=43 4 0.79 101.0 97 0.86 70.3 90 0.60 49.9 81 NA NA NA (0.55-1.14) (60.0-170.1) (0.67-1.12) (43.1-114.6) (0.42-0.85) (26.0-95.8) 6B 0.82 65.8 78 0.81 48.2 75 0.39 5.8 262 NA NA NA (0.58-1.17) (29.6-146.5) (0.59-1.12) (25.5-91.2) (0.29-0.52) (3.0-11.2) 9V 0.91 104.1 97 1.00 62.0 85 0.56 25.1 562 NA NA NA (0.71-1.16) (56.2-192.9) (0.72-1.38) (35.1-109.7) (0.40-0.77) (12.4-50.8) 14 3.99 401.4 94 1.93 124.2 81 1.11 55.4 672 NA NA NA (2.86-5.57) (207-778.5) (1.20-3.09) (62.3-247.4) (0.79-1.57) (24.0-127.6) 18C 0.49 134.4 94 0.41 51.8 88 0.18 13.7 652 NA NA NA (0.37-0.65) (80.4-224.2) (0.33-0.53) (33.2-80.7) (0.14-0.24) (8.7-21.7) 23F 0.65 117.2 94 0.44 29.2 632 0.24 10.5 472 NA NA NA

159

Serotype 3 PCV 2 PCV 1 PCV 0 PCV GMC OPA GMT % GMC OPA GMT % GMC OPA GMT % GMC OPA GMT % (95%CI) (95%CI) OI ≥8 (95%CI) (95%CI) OI ≥8 (95%CI) (95%CI) OI (95%CI) (95%CI) OI ≥8 n=125 n=146 n=121 ≥8 n=62 n=119 n=141 n=118 n=61 (0.46-0.94) (59.6-230.3) (0.33-0.60) (14.4-59.2) (0.18-0.32) (5.6-19.7) 12 months n=113 n=112 n=142 n=141 n=114 n=111 n=59 n=56 4 0.48 32.5 71 0.47 27.4 74 0.63 63.2 812 0.07 6.1 232,3 (0.41-0.57) (21.6-48.9) (0.40-0.54) (20.1-37.5) (0.50-0.81) (41.6-96.1) (0.06-0.09) (3.5-10.6) 6B 0.86 71.0 77 0.76 27.0 592 0.57 4.2 202 0.14 3.0 72 (0.72-1.03) (45.6-110.6) (0.63-0.92) (17.5-41.6) (0.46-0.71) (3.1-5.8) (0.12-0.17) (2.0-4.5) 9V 0.59 48.4 78 0.62 56.9 79 0.50 37.7 662 0.09 4.9 192,3 (0.51-0.67) (31.7-73.7) (0.54-0.71) (38.2-84.7) (0.41-0.62) (23.9-59.3) (0.07-0.11) (2.9-8.5) 14 2.38 204.6 87 1.52 79.4 79 1.16 48.6 732 0.19 3.8 132,3 (1.98-2.86) (134.5-311.2) (1.26-1.84) (52.5-120.0) (0.94-1.44) (30.3-77.9) (0.16-0.24) (2.3-6.1) 18C 0.32 46.8 84 0.24 ( 22.7 712 0.17 12.4 592 0.06 2.6 52,3 (0.27-0.38) (33.0-66.4) 0.21-0.28) (16.9-30.3) (0.15-0.20) (9.0-17.2) (0.05-0.08) (1.9-3.5) 23F 0.54 64.2 79 0.42 35.4 652 0.26 12.7 432 0.07 4.5 162,3 (0.44-0.66) (42.0-98.3) (0.35-0.50) (23.8-52.5) (0.21-0.31) (8.2-19.7) (0.06-0.09) (2.7-7.7) 1Dilution able to kill 50% of bacteria All 3 vs 2, 3 vs 1, 3 vs 0, 1 vs 0 PCV dose comparisons of proportions with OI≥8 were not significant except those marked: 2Significant difference (p<0.01) comparing proportions with OI≥8 following 0, 1, or 2 doses PCV to the proportions following 3 PCV doses 3Significant difference (p<0.01) comparing proportions with OI≥8 following a single PCV dose to the proportions following no PCVdose

160

Two weeks following the 23vPPS at 12 months of age there were significant increases in the proportion of children with OI ≥8 for all PCV serotypes for all PCV dosage groups. There were no significant differences between the 3 and 2 PCV dose groups, and the 3 versus single PCV dose groups in the proportion of children with OI ≥8 for any PCV serotype (Table 22). However there was a trend for higher OPA GMT in the 3 dose groups compared with the 2 dose group for serotypes 6B, 14, and 23F. The proportion of children with OI ≥8 was significantly higher for all PCV serotypes in the 3 dose group compared with those children who had not received PCV. A single PCV dose significantly increased the proportion of children with OI ≥8 compared to the unvaccinated group for all PCV serotypes.

At 17 months of age, the proportion of children with OI ≥8 for the 3 and 2 PCV dose groups who had received the 23vPPS at 12 months continued to be significantly higher for all PCV serotypes compared with the corresponding PCV group who had not received the 12 month 23vPPS, except for serotype 14 and 23F (Table 23). For the single dose group who had received the 12 month 23vPPS, there were significantly higher proportions of children with OI≥8 for 5 of 7 PCV serotypes compared with the single dose group who had not received the 12 month 23vPPS. For the groups that had received no PCV there were no significant differences in the proportion of children with OI ≥8 for any PCV serotype except serotype 4 which was significantly higher in the group that had received the 12 month 23VPPS.

At 18 months of age, one month post-mPPS, there were no significant differences in the proportion of children with OI ≥8 for any PCV serotype comparing the 3, 2, or single PCV dose groups who had also received the 12 month 23vPPS compared with the corresponding PCV group who had not received the 12 month 23vPPS. For example, in the 3 PCV groups, 100% of children who did not receive 23vPPS at 12 months had an OI ≥8 for serotype 4, compared with 98% who did receive 23vPPS. The proportion of children with OI ≥8 was significantly higher for 3 of the PCV serotypes in the group that had received no PCV in infancy or 23vPPS at 12 months compared with the group that had received no PCV and the 12 month 23vPPS.

To examine the effect of 23vPPS at 12 months and the number of PCV doses in early infancy, multiple variable regression was undertaken. Prior to adjusting for the pre-existing OPA titer, the post-mPPS OPA titers were significantly lower for all serotypes (except 7F) in the 23vPPS group compared with the group that had not received the 12 month 23vPPS.

161

Table 22: OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI ≥8 to 6 PCV serotypes pre- and 14 days post-23vPPS at 12 months of age and by number of PCV administered in the primary series Pre-23vPPS at 12 months of age 14 days post-12 month 23vPPS 3 PCV 2 PCV 1 PCV 0 PCV 3 PCV 2 PCV 1 PCV 0 PCV (n=52) (n=67) (n=58) (n=55) (n=52) (n=67) (n=58) (n=55) OPA GMT % OPA GMT % OPA GMT % OPA GMT % OPA GMT % OPA GMT % OPA GMT % OPA GMT % (95%CI) OI (95%CI) OI (95%CI) OI (95%CI) OI (95%CI) OI (95%CI) OI (95%CI) OI (95%CI) OI ≥8 ≥8 ≥8 ≥8 ≥8 ≥8 ≥8 ≥8 4 28.7 69 18.2 70 65.0 81 6.2 24 4571.8 100 4808.0 100 23392.4 100 249.9 852,3 (15.9-51.8) (11.8-28.0) (36.0-117.4) (3.5-10.9) (3104-6733.6) (3729.5- (16960.1- (126.8-492.5) 6198.4) 32264.1) 6B 95.5 83 19.8 51 4.6 22 3.0 7 6446.7 94 3560.0 96 1361.4 91 4.8 152,3 (48.7-187.2) (10.6-36.8) (2.9-7.1) (2.0-4.6) (3419.9-12152.5) (2149.1- (718.6-2579.2) (2.6-8.6) 5897.3) 9V 48.9 81 48.2 82 61.0 76 5.0 19 5093.3 100 4391.1 100 13747.9 100 81.4 672,3 (26.7-89.6) (28.0-83.0) (32.5-114.7) (2.9-8.7) (3730.5-6954.0) (3417.2- (9792.4-19301.3) (37.0-179.0) 5642.6) 14 156.8 81 86.4 82 60.6 78 3.8 13 3023.5 92 2261.3 97 2618.5 90 9.0 262,3 (79.0-311.2) (48.1-155.0) (32.2-114.3) (2.3-6.2) (1531.6-5968.8) (1352.7- (1221.4-5613.6) (4.3-19.0) 3780.0) 18C 59.8 88 18.5 64 18.5 66 2.6 5 3975.9 100 3262.8 100 5302.7 100 66.7 752,3 (35.3-101.2) (12.0-28.5) (11.4-30.2) (1.9-3.6) (3018.5-5237.1) (2513.6- (3433.1-8190.4) (33.3-133.8) 4235.4) 23F 52.4 75 26.9 61 17.0 48 4.6 16 4109.5 98 2295.0 97 2119.6 93 18.1 422,3 (27.5-99.7) (15.3-47.5) (8.8-33.0) (2.7-7.9) (2422.8-6970.6) (1420.2- (1161-3869.8) (8.5-38.5) 3708.5) 1Dilution able to kill 50% of bacteria. All post 23vPPS comparisons comparing 3 vs 2, 3 vs 1, 3 vs 0, and 1 vs 0 PCV doses were not significant except those marked: 2Significant difference (p<0.01) comparing proportions with OI≥8 following no PCV dose to the proportions following 3 doses of PCV 3Significant difference (p<0.01) comparing proportions with OI≥8 following a single PCV dose to the proportions following no PCV dose

162

Table 23: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI)1, OPA Geometric Mean Titers2 (GMT and 95%CI) and proportions of infants with OI ≥8 pre-mPPS at 17 months of age and one month post-mPPS in those that have or have not received the 12 month 23vPPS and by number of PCV doses in the primary series

GMC 0.35 4.69 2.19 2.01 0.43 3.99 2.03 2.00 0.56 10.39 6.36 6.37 0.11 0.79 0.70 0.81 (0.29- (3.62- (1.79- (1.66- (0.33- (3.10- (1.70-2.42) (1.67- (0.39- (7.38- (4.94- (4.93- (0.09- (0.59- (0.53- (0.63- 0.43) 6.07) 2.69) 2.43) 0.56) 5.12) 2.40) 0.80) 14.63) 8.19) 8.23) 0.13) 1.07) 0.91) 1.04) GMT 18.6 1627.8 403.3 444.1 37.1 1286.3 297.5 340.5 56.3 3478.8 1598.7 1829.7 4.9 204.9 18.9 19.1 (11.3- (1209.1- (282.2- (307.2- (20.1- (922.1- (217.0- (254.7- (30.3- (1957.6- (1089.9- (1273.5- (3.1- (116.0- (11.0- (10.7- 30.7) 2191.6) 576.3) 642.1) 68.7) 1794.5) 407.7) 455.1) 104.7) 6182.1) 2344.9) 2628.7) 7.6) 362.0) 32.6) 34.2) OI ≥8 64 100 98 983, 5 72 100 97 1003, 5 84 953 984 983, 5 24 88 65 61 6B GMC 0.91 12.77 4.35 4.08 0.78 10.57 3.85 4.08 0.62 5.82 3.88 3.83 0.20 0.30 0.21 0.22 (0.69- (9.52- (3.22- (3.17- (0.58- (7.73- (3.10-4.79) (3.18- (0.47- (3.90- (2.87- (2.80- (0.16- (0.24- (0.17- (0.17- 1.21) 17.12) 5.89) 5.26) 1.04) 14.44) 5.25) 0.83) 8.68) 5.25 5.26) 0.24) 0.37) 0.26) 0.29) GMT 50.6 2774.9 539.8 639.7 39.9 1898.8 223.9 239.4 6.2 302.9 81.4 89.9 3.3 5.4 4.3 3.7 (27.5- (1941.0- (325.8- (390.7- (20.9- (1134.4 (134.1- (144.6- (3.4- (131.2- (40.3- (45.2- (2.2- (3.1-9.4) (2.6- (2.3- 92.9) 3967.1) 894.3) 1047.4) 76.1) - 373.7) 396.3) 11.2) 698.9) 164.1) 178.9) 5.0) 7.1) 5.9) 3178.1) OI ≥8 74 100 96 963, 5 65 97 92 923, 5 30 81 76 803, 5 10 213 174 13 5 9V GMC 0.41 3.92 2.25 2.10 0.49 4.34 2.50 2.46 0.51 9.26 5.49 5.21 0.14 0.51 0.38 0.50 (0.34- (3.08- (1.74- (1.67- (0.39- (3.33- (2.02- (1.98- (0.36- (6.45- (4.23- (4.05- (0.11- (0.39- (0.31- (0.40- 0.49) 4.99) 2.91) 2.64) 0.62) 5.66) 3.08) 3.05) 0.71) 13.28) 7.12) 6.70) 0.17) 0.68) 0.47) 0.62) GMT 29.8 1317.7 383.7 431.9 48.7 1284.8 300.9 306.6 40.0 1786.5 1127.6 902.3 8.8 60.8 9.6 9.6

163

3 PCV no 23vPPS 3 PCV + 23vPPS 2 PCV no 23vPPS 2 PCV + 23vPPS 1 PCV no 23vPPS 1 PCV + 23vPPS 0 PCV no 23vPPS 0 PCV + 23vPPS Pre- Post- Pre- Post- Pre- Post - Pre- Post- Pre- Post- Pre- Post - Pre- Post- Pre- Post- mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS (16.7- (982.5- (245.2- (300.7- (27.4- (894.4- (212.0- (210.2- (19.6- (1074.3- (852.7- (619.4- (4.9- (28.7- (5.4- (5.3- 53.1) 1767.3) 600.5) 620.4) 86.4) 1845.8) 427.1) 447.0) 81.8) 2971.1) 1491.1) 1314..3) 15.7) 128.9) 17.0) 17.4) OI ≥8 67 100 100 1003, 5 75 98 97 973, 5 74 100 100 983, 5 34 62 394 37 14 GMC 1.78 10.25 4.31 4.26 1.12 6.84 3.58 3.83 0.93 11.09 4.85 4.94 0.31 0.51 0.67 0.97 (1.42- (7.79- (3.00- (3.08- (0.86- (4.80- (2.72- (2.91- (0.66- (6.95- (3.27- (3.39- (0.25- (0.37- (0.47- (0.68- 2.24) 13.49) 6.19) 5.90) 1.46) 9.77) 4.71) 5.03) 1.32) 17.69) 7.19) 7.19) 0.38) 0.71) 0.96) 1.38) GMT 117.7 2123.9 405.6 507.0 50.0 930.0 276.0 316.7 34.8 1005.6 248.1 328.1 8.7 18.4 7.6 24.3 (62.4- (1547.4- (222.6- (290.7- (24.2- (497.8- (157.2- (182.1- (15.7- (432.7- (114.0- (156.0- (4.4- (8.0-42.2) (3.8- (10.6- 222.1) 2915.2) 739.1) 884.1) 103.6) 1737.3) 484.4) 550.6) 77.3) 2336.8) 540.1) 689.8) 17.2) 15.2) 55.4) OI ≥8 79 100 914 943, 5 63 90 87 893, 5 65 88 814 833, 5 26 343 284 46 5 18C GMC 0.21 1.94 1.28 1.25 0.20 2.55 1.13 1.23 0.15 2.80 1.79 1.87 0.10 0.48 0.50 0.62 (0.18- (1.56- (1.07- (1.04- (0.16- (1.97- (0.92- (1.00- (0.12- (2.02- (1.37- (1.46- (0.08- (0.35- (0.39- (0.46- 0.26) 2.42) 1.53) 1.49) 0.25) 3.29) 1.38) 1.51) 0.19) 3.87) 2.34) 2.39) 0.12) 0.65) 0.66) 0.83) GMT 18.7 937.6 260.8 249.9 15.6 1025.8 151.0 156.0 8.4 1033.0 322.4 309.7 4.2 32.4 6.7 7.1 (12.1- (705.6- (193.9- (184.6- (9.9- (768.9- (100.4- (105.0- (5.1- (647.9- (201.7- (192.1- (2.8- (17.1- (4.3- (4.5- 28.9) 1246.0) 350.9) 338.3) 24.8) 1368.5) 227.3 231.8) 13.8) 1647.1) 515.2) 499.1) 6.4) 61.5) 10.7) 11.3) OI ≥8 69 100 100 1003, 5 67 100 95 953, 5 53 98 94 943, 5 22 64 394 38 5 19F GMC 1.19 15.33 5.55 5.42 1.06 15.95 4.53 5.09 0.92 34.74 13.47 14.34 0.59 0.99 0.79 0.88 (0.84- (11.27- (4.24- (4.17- (0.81- (11.59- (3.54- (3.93- (0.66- (23.17- (9.90- (10.72- (0.47- (0.77- (0.61- (0.67- 1.67) 20.86) 7.26) 7.06) 1.38) 21.94) 5.79) 6.61) 1.26) 52.11) 18.33) 19.18) 0.75) 1.26) 1.04) 1.15) GMT 10.4 1917.5 391.2 479.2 11.5 1621.3 257.2 285.0 9.3 3657.4 1086.4 1470.4 3.2 13.0 5.9 9.7 (5.7- (1222.9- (243.5- (318.9- (6.4- (952.3- (160.2- (177.0- (5.0- (1793.1- (626.7- (929.2- (2.2- (7.4-22.7) (3.5- (5.6- 19.0) 3006.6) 628.5) 719.9) 20.5) 2760.3) 412.9) 458.9) 17.2) 7459.8) 1883.3) 2326.7) 4.6) 9.8) 16.8)

164

3 PCV no 23vPPS 3 PCV + 23vPPS 2 PCV no 23vPPS 2 PCV + 23vPPS 1 PCV no 23vPPS 1 PCV + 23vPPS 0 PCV no 23vPPS 0 PCV + 23vPPS Pre- Post- Pre- Post- Pre- Post - Pre- Post- Pre- Post- Pre- Post - Pre- Post- Pre- Post- mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS OI ≥8 40 97 98 983, 5 45 97 95 953, 5 44 93 93 963, 5 12 50 314 44 5 23F GMC 0.57 4.10 1.68 1.54 0.43 3.74 1.41 1.52 0.32 3.34 1.78 1.54 0.19 0.37 0.27 0.27 (0.43- (3.26- (1.29- (1.21- (0.32- (2.74- (1.06-1.86) (1.17- (0.22- (2.22- (1.28- (1.10- (0.16- (0.29- (0.21- (0.21- 0.75) 5.17) 2.20) 1.96) 0.58) 5.12) 1.97) 0.48) 5.03) 2.49) 2.16) 0.23) 0.47) 0.34) 0.35) GMT 71.7 1716.2 312.5 268.2 43.7 971.9 141.7 156.0 10.4 507.2 175.0 139.0 6.7 27.0 5.4 4.3 (37.9- (1357.8- (189.8- (151.7- (22.0- (579.4- (81.5- (90.7- (4.8- (245.3- (97.5- (74.6- (3.7- (12.3- (3.1- (2.6- 135.6) 2169.3) 514.6) 474.2) 86.8) 1630.2) 246.6) 268.3) 22.6) 1048.5) 314.2) 258.9) 12.2) 59.4) 9.4) 7.5) OI ≥8 78 100 96 943, 5 63 95 844 873, 5 35 88 87 833, 5 24 47 224 17 1GMC sample size by group: 3 PCV no 23vPPS n=59, 3 PCV + 23vPPS n=48, 2 PCV no 23vPPS n=68, 2 PCV + 23vPPS n=67, 1 PCV no 23vPPS n=49, 1 PCV + 23VPPS n=59, 0 PCV no 23vPPS n=62, 0 PCV + 23VPPS n=57 2 Dilution able to kill 50% of bacteria. GMT and OI sample size by group: 3 PCV no 23vPPS n=58, 3 PCV + 23vPPS n=47, 2 PCV no 23vPPS n=60, 2 PCV + 23vPPS n=62, 1 PCV no 23vPPS n=43, 1 PCV + 23VPPS n=54, 0 PCV no 23VPPS n=58, 0 PCV + 23VPPS n=54 3All paired group comparisons of the proportion of children with OI≥8 pre/post mPPS were statistically significant (p<0.01) except those marked3. 4All comparisons of the proportion of children with OI≥8 at 17 months of age (pre-mPPS) between the 3 PCV/12 month 23vPPS group and the 3 PCV group, the 2 PCV/12 month 23vPPS group and the 2 PCV group, the one PCV/12 month 23vPPS group and the single PCV group, and the 12 month 23vPPS group and the unvaccinated group, were statistically significant (p<0.01) except those marked4. 5All comparisons of the proportion of children with OI≥8 at 18 months of age (post-mPPS) between the 3 PCV/12 month 23vPPS group and the 3 PCV group, the 2 PCV/12 month 23vPPS group and the 2 PCV group, the one PCV/12 month 23vPPS group and the single PCV group, and the 12 month 23vPPS group and the unvaccinated group, were statistically significant (p<0.01) except those marked5.

165

To assess whether the poor response to mPPS in the 12 month 23vPPS recipients was due to higher pre-mPPS OPA titers, adjustment was made for the pre-existing OPA titer. At any given pre-OPA titer, 23vPPS negatively affected the response to mPPS for all serotypes (examples using serotypes 4 and 6B are shown in Figure 20) except serotypes 1 and 5. At any given pre- mPPS OPA titer against serotype 4, most children who had received 23vPPS did not change titer level after re-exposure, as indicated by falling along the 45 degree line, whilst most children who did not receive 23vPPS at 12 months had an increase in OPA titer post-re- exposure. For serotypes 1 and 5, exposure to 23vPPS was not associated with a lower response to mPPS after adjusting for pre-existing OPA titer. For the PCV serotypes, the impact of 23vPPS on the mPPS response was not affected by the number of PCV doses received in the primary series (details not shown).

166

Figure 20: Pre- and one month post-mPPS OPA titer for serotypes 4 and 6B, in those that did (+) and did not (o) receive 23vPPS at 12 months of age. Data from infants receiving 0, 1, 2, or 3 PCV doses have been combined in these graphs.

Serotype 4

1000

100 Post mPPS,OPA titer PostmPPS,OPA

1 1 8 100 1000 Pre mPPS, OPA titer

Serotype 6B

1000

100 Post mPPS,OPA titer PostmPPS,OPA

1 1 8 100 1000 Pre mPPS, OPA titer

Footnote: A 45-degree line, which indicates no change before and after the re-challenge (mPPS), is super-imposed. The pre- and post-mPPS OPA titer of those who received 23vPPS at 12 months mostly fell along the 45-degree line, indicating no response to mPPS, except for serotypes 1 and 5. In contrast, most children who did not receive 23vPPS had an increase in OPA titer, as indicated by the data points falling above the 45-degree line.

167

8.4.2 Non-PCV and PCV related serotypes

Following the 12 month 23vPPS, there were significant increases in the proportion of children with OI ≥8 for the 2 serotypes (1 and 5) tested (Table 24). At 17 months of age (prior to mPPS) there were significantly higher proportions of children with OI ≥8 for 3 of 7 non-PCV serotypes (3, 5, 7F) in those that had received the 12 month 23vPPS compared with those that had not (Table 25). One month following mPPS, there were significantly higher proportions of children with OI ≥8 for 4 of 7 non-PCV serotypes (1, 5, 6A, 6C) in those that had not received the 23vPPS compared with those that had.

168

Table 24: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI ≥8 to the non-PCV serotypes 1 and 5 pre- and 14 days post-23vPPS at 12 months of age

Pre-23vPPS at 12 months 14 days post-12 month 23vPPS GMC OPA GMT % OI ≥8 GMC OPA GMT % OI ≥82 (95%CI) (95%CI) (95%CI) (95%CI) n=235 n=228 n=235 n=228 1 0.17 2.3 4 1.59 4.7 27 (0.15-0.19) (2.1-2.5) (1.38-1.82) (3.9-5.6) 5 0.26 2.1 2 2.26 22.2 77 (0.23-0.29) (2.0-2.3) (2.01-2.55) (18.2-27.1) 1Dilution able to kill 50% of bacteria 2P-value <0.001 for proportions with OI≥8 pre/post 23vPPS for both serotypes.

169

Table 25: Serotype-specific IgG Geometric Mean Concentrations (GMC and 95%CI), OPA Geometric Mean Titers1 (GMT and 95%CI) and proportions of children with OI ≥8 to 7 non-PCV serotypes at 17 months and one month post-mPPS No 12m 23vPPS 12m 23vPPS p-value2 GMC OPA GMT % OI ≥8 GMC OPA GMT % OI ≥8 (95%CI) (95%CI) (95%CI) (95%CI) n=238 n=224 n=231 n=220 Pre-mPPS at 17 months of age 1 0.23 (0.21-0.25) 2.2 (2.1-2.3) 4 0.63 (0.56-0.71) 2.4 (2.2-2.6) 6 0.167 3 0.30 (0.26-0.34) 7.4 (6.0-9.1) 45 1.46 (1.30-1.63) 41.2 (35.1-48.2) 92 <0.001 5 0.31 (0.28-0.34) 2.3 (2.1-2.5) 5 0.77 (0.69-0.86) 5.0 (4.3-5.8) 39 <0.001 6A NA 5.1 (3.9-6.6) 20 NA 8.1 (6.1-10.7) 35 0.028 6C NA 5.7 (4.2-7.9) 17 NA 5.2 (3.8-7.0) 16 0.317 7F 0.12 (0.10-0.13) 471.0 (321.2-690.8) 80 0.51 (0.45-0.57) 1593.1 (1240.7 -2045.6) 95 <0.001 19A 0.65 (0.57-0.74) 7.0 (5.4-9.1) 32 1.14 (1.00-1.31) 10.1 (7.8-13.1) 45 0.017 One month post-mPPS (18 months of age) 1 0.82 (0.73-0.93) 4.1 (3.4-4.9) 22 0.73 (0.65-0.81) 2.4 (2.2-2.5) 7 <0.001 3 2.11 (1.86-2.39) 111.2 (94.1-131.4) 96 2.32 (2.06-2.63) 77.2 (66.0-90.3) 95 0.512 5 0.86 (0.77-0.96) 12.3 (10.0-15.3) 60 0.85 (0.76-0.96) 5.7 (4.9-6.6) 41 <0.001 6A NA 33.3 (22.6-49.2) 54 NA 8.5 (6.3-11.4) 32 <0.001 6C NA 19.9 (13.0-30.3) 37 NA 6.3 (4.6-8.8) 20 <0.001 7F 0.83 (0.72-0.96) 3320.5 (2783.6-3960.9) 98 0.65 (0.57-0.74) 1949.2 (1538.7-2469.3) 96 0.172 19A 2.19 (1.83-2.62) 194.1 (138.4-272.2) 82 1.46 (1.28-1.68) 45.3 (33.8-60.7) 74 0.049 1Dilution able to kill 50% of bacteria 2 P-value for proportions with OI≥8 for each serotype mPPS comparing those that did not did not receive the 23vPPS

170

8.5 Discussion

This is the first randomised controlled trial of reduced pneumococcal dose schedules evaluating the OPA of all PCV and many non-PCV serotypes post 23VPPS booster. This study has shown that despite no significant difference found between many of the PCV serotypes comparing the 3 PCV dose group to the 2 dose group, a trend for higher OPA GMT were found for many of the serotypes. A significant difference may not have been found do to the small sample size. These findings are similar to the results of serotype-specific antibody concentrations measured by ELISA [389, 403]. Despite the OPA being significantly lower for all PCV serotypes following a single PCV dose compared with the 3 doses, a single dose clearly produces substantial functional antibodies compared with the unvaccinated control group. The response to a single PCV response may have been enhanced by the co-administration of Diphtheria-Tetanus-whole cell Pertussis (DTwP) due to its potential adjuvant effect [387, 389] and the groups that received one or 2 doses of PCV may have had augmented responses to their 14 week dose of PCV as a result of extra doses of DTwP [389]. Whilst there are no other single PCV dose studies for comparison our OPA findings largely replicate to the results of ELISA antibody concentrations post primary series [389].

The 23vPPS given at 12 months significantly improves the OPA for all PCV serotypes tested even for the group who had not previously been primed with PCV. Moreover, 2 weeks following the 12 month 23vPPS there were no significant differences in OPA for all serotypes between the single PCV dose group compared with the 3 dose group. In contrast, OPA was significantly higher for all serotypes comparing the single dose group and the group that had received no prior PCV indicating that priming even with a single PCV dose can produce better OPA responses than 23vPPS alone. These findings are similar to the results of serotype-specific antibody concentrations measured by ELISA whereby memory responses following 23vPPS were most profound for the single PCV dose group compared with the 2 or 3 dose groups [386].

The OPA continued to be significantly higher at 17 months of age for all PCV serotypes in those groups that had received the 23vPPS at 12 months compared to the groups that had not. Other combined pneumococcal conjugate vaccine/23vPPS studies have found inconsistent OPA results post booster. A 3 dose infant 11-valent pneumococcal conjugate vaccine followed by 23vPPS booster in Finnish children resulted in antibodies of greater OPA for vaccine serotypes than those children given the homologous conjugate booster [404]. However no difference was observed between groups of Israeli children vaccinated with the pneumococcal polysaccharide- meningococcal outer membrane protein complex conjugate vaccine (PncOMPC) and either the

171

homologous or polysaccharide booster [186]. In a randomised controlled trial of 4 doses of the 11- valent pneumococcal protein D conjugate vaccine (PD conjugate vaccine) followed by a 23vPPS dose in the fourth year of life in Czech children, vigorous OPA responses were found for all of the 11 vaccine serotypes tested post 23VPPS for both primed and unprimed children [401]. However, similar to our findings, the magnitude of the response was significantly higher in primed children for most of the serotypes [401]. These differences may be due to differences in vaccines (GSK versus Merck Inc.) and/or populations.

For the non-PCV serotypes, the 23VPPS at 12 months significantly improved the OPA for the 2 serotypes tested (serotypes 1 and 5). In the aforementioned study in Czech children, the strongest response post 23vPPS was found for serotype 1 (which is contained in both the PD conjugate vaccine and 23vPPS) and the weakest responses were for those serotypes that maintained the higher antibody levels prior to 23vPPS [401].

Serotype 6A is a vaccine related serotype as it differs only slightly in capsular structure and can cross react with antibodies to 6B. Despite a significant reduction in 6A IPD in vaccinated children in the US, herd immunity has not been evident amongst adults [222, 244]. However this lack of effect despite cross reactivity with 6B may be explained by the discovery of 6C which was originally typed as “6A” [57]. Vaccination with 6B provides some level of cross immunogenicity against 6C although the level of cross immunogenicity is much less than that of 6A. In the pre-mPPS samples, both groups showed some cross immunogenicity against both 6A and 6C. However, this data is difficult to interpret as the absolute responses are low, presumably due to the extended interval between the last vaccination and sample collection. In the group that did not receive the 12 month 23VPPS, there is a significant difference in the percentage of samples with OIs ≥8, again suggesting hyporesponsiveness.

For serotype 19A, the OPA titers were similar in both groups at 17 months of age, suggesting cross- opsonization from PCV serotype 19F. There are conflicting data regarding whether 19F provides cross-opsonization with 19A. In the Czech study, a fourth dose of PD conjugate vaccine (which does not contain serotype 19A) increased the OPA response to 19A (but 19A is in 23vPPS), again suggesting cross-opsonization with serotype 19F [401]. In contrast, another study found PCV to elicit antibodies binding to 19A although the majority of these were non-opsonic and thereby suggested that PCV would provide little immunoprotection against 19A if it were estimated by OPA and not ELISA [400, 405]. However a recent epidemiological study pooling data from a number of studies found modest protection against 19A IPD and AOM [406]. There is data suggesting another possible explanation for the apparent cross-opsonization of 19A, being the “Dob1” epitope, which

172

is common to both 6B and 19A but not 19F [407]. This cross- opsonisation may be population, vaccination schedule, and/or vaccine-dependent.

The lack of response to the re-challenge dose shown by the groups that had received prior 23vPPS at 12 months indicates hyporesponsiveness. We have shown previously that hyporesponsiveness, as demonstrated by a poor response to a small 23vPPS re-challenge dose (mPPS) by ELISA, occurs irrespective of pre-existing antibody level for all 23 serotypes [390]. This finding has been confirmed in this study by the attenuated OPA responses to re-challenge in the 23vPPS group which were not related to the pre-existing OPA level (except for serotypes 1 and 5). This attenuated response may be due to the depletion of the memory B cell pool [25, 26], with subsequent challenge with 23vPPS resulting in immune hyporesponsiveness. In this study we did not find any adverse clinical consequences from this immunological finding, although the study was not designed to evaluate this effect. For Australian Indigenous children, until recently the routine immunisation schedule consisted of 3 infant doses of PCV followed by a 23VPPS booster at 18 months of age. In a recent retrospective cohort study of Indigenous children in the Northern Territory, Australia, the findings suggested an increased risk of hospitalized acute lower respiratory infections after pneumococcal vaccination particularly after receipt of the 23vPPS [274]. In contrast, results from Western Australia using a population based data linkage system, found significant reductions in acute lower respiratory tract infections in Indigenous children [275].

The clinical relevance of immunological findings from vaccine trials is obviously best demonstrated in efficacy studies. In our study there are no pneumococcal disease endpoints to evaluate the clinical relevance of our immunological findings. However, separately we have evaluated the effect on nasopharyngeal (NP) carriage. We found a trend for a PCV dose effect on VT NP carriage with less VT carriage in the 3 PCV group (Russell FM et al. submitted for publication). A single PCV dose resulted in some initial reduction in PCV serotype carriage which provides supportive evidence that one dose may offer some early protection from IPD. The addition of 23vPPS at 12 months of age had no significant impact on carriage (but a trend for higher VT NP carriage), despite the substantial boosts in antibody levels observed [386] and despite better OPA (as demonstrated in this study) and better avidity (Russell FM et al. submitted for publication).

Vaccine responses are known to be serotype-specific. A recent study in the United Kingdom evaluating the immunogenicity of reduced dose PCV schedules found a 2 dose schedule given at 2 and 4 months of age had poor antibody responses to 6B [408]. The OPA for serotype 6B suggested that a lower antibody concentration of ~0.2 g/mL rather than 0.35 g/mL, correlated better with 6B OPA [408]. In our study, 68% of children following 2 PCV doses had functional antibodies post

173

primary series which is similar to that found in a study following 3 doses of 11-valent mixed carrier pneumococcal conjugate vaccine given at 2, 4, and 6 months to Israeli and Finnish children [155] and is higher than that found in Filipino infants using the same mixed carrier vaccine (43%) [409]. In contrast to the UK study, the Czech study, using PD conjugate vaccine, found 6B to have one of the highest OPA responses post primary series [401].

Another difficulty with the assay is the conflicting results found between the correlation of ELISA and OPA. The addition of pre-absorption not only with cell wall polysaccharide but also with pneumococcal 22F capsular polysaccharide, has not only improved ELISA specificity [123, 125] but has increased the correlation between anti-capsular polysaccharide antibody concentration and OPA [123, 126]. There is evidence of a low correlation between antibody concentration measured by ELISA and the measurement of functional antibodies especially in the sera of non-vaccinated individuals. Several studies have shown the presence of naturally acquired cross-reactive antibodies which are prevalent in sera of infants and adults suggesting that pneumococcal polysaccharides contain some non-protective cross-reactive epitopes or impurities, other than cell wall polysaccharide, that are common to many types of pneumococcal polysaccharide [410]. In general, the correlation between antibody concentration and OPA seems to be good in infants who have been immunized with conjugate vaccines [141, 142]. Correlates have been high for vaccine serotypes, but lower for cross-reacting serotypes like 6A and 19A [143, 405]. Our findings seem to indicate that there appears to be a reasonable correlation between the OPA and ELISA findings, but this will be further explored in a separate analysis.

In summary, this study has shown that 3 PCV doses in infancy have similar OPA compared with the 2 PCV dose group up to 9 months for most of the PCV serotypes except serotypes 6B and 23F. A single dose elicits OPA for all serotypes compared to the unvaccinated group. The 23VPPS significantly increases OPA for all serotypes tested. However, the poor response to re-challenge in those that had received prior 23VPPS suggests hyporesponsiveness.

174

9 SEROTYPE-SPECIFIC AVIDITY IS ACHIEVED FOLLOWING A SINGLE DOSE OF THE 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE, AND IS ENHANCED BY 23- VALENT PNEUMOCOCCAL POLYSACCHARIDE BOOSTER AT 12 MONTHS

9.1 Abstract

Aim: To evaluate whether avidity of serotype-specific IgG responses to pneumococcal serotypes is enhanced by an increased number of doses of the 7-valent pneumococcal conjugate vaccine (PCV) in infancy or by a 12 month 23-valent pneumococcal polysaccharide vaccine (23vPPS) booster, and /or subsequent re-exposure to a small dose of pneumococcal polysaccharide antigens (mPPS) at 17 months.

Methods: Fijian infants aged 6 weeks were recruited, stratified by ethnicity and randomised to 8 groups to receive 0, 1, 2, or 3 doses of PCV, with or without booster 23vPPS at 12 months. All children received mPPS at 17 months of age. Avidity of serotype-specific IgG for PCV serotypes in the first 12 months and for all 23vPPS serotypes thereafter was assessed by EIA after sodium thiocyanate elution.

Results: At 1 month post primary series, the 2 and 3 PCV dose groups demonstrated similar avidity, with the single dose group tending to have poorer avidity. However, by age 9 months, the single dose group had similar serotype-specific IgG avidity to the 2 and 3 PCV groups for most serotypes. The 23vPPS booster enhanced affinity maturation for most serotypes and this was most marked in those groups that received a single PCV dose. There was little further increase following the mPPS.

Conclusions: By 9 months of age, similar levels of high avidity serotype-specific IgG can be induced following one, 2 or 3 doses of PCV. A 23vPPS booster at 12 months enhanced affinity maturation with an increase in the amount of high avidity serotype-specific IgG for most serotypes. Subsequent re-challenge with mPPS at 17 months did not further enhance the avidity response in the 12 month 23vPPS groups.

175

9.2 Introduction

Invasive pneumococcal disease (IPD) is an important cause of morbidity and mortality, particularly in the very young and the elderly [4]. A recent review estimated that over 14 million episodes of serious pneumococcal disease occurred worldwide in the year 2000, with over 800,000 deaths in children under 5 years [41]. At least 40 serogroups comprising of over 90 serotypes of pneumococcus have been identified [57, 58]. In all regions of the world, serotypes 1, 5, and 14 account for 28-43% of IPD in under 5 year olds [61]. The 7-valent pneumococcal conjugate vaccine (PCV, PrevnarTM, Wyeth Vaccines) includes only serotypes 4, 6B, 9V, 14, 18C, 19F, 23F and covers a smaller proportion of the pneumococcal serotypes causing disease in children in low income countries compared to more affluent countries.

Serological criteria for the evaluation and licensure of new pneumococcal conjugate vaccines recommend evaluation of serotype-specific IgG antibody concentrations as other immunological measures have not been validated in the clinical setting [102]. Nevertheless, it has been argued that the qualitative characteristics of these antibodies may be just as important as their quantity in the assessment of PCV immunogenicity [147]. The efficiency of antibody-antigen binding is dependent on antibody affinity. The antigen binding capacity of polyclonal antibodies with different affinities can be measured as antibody avidity. Antibody avidity is an expression of the functional antibody affinity and is considered to be indicative of the protective efficacy of antibodies. In vitro, higher avidity antibody is associated with greater opsonophagocytic capacity [146-148]. Furthermore, findings from a study assessing the contribution of avidity, antibody concentration, and IgG subclass to opsonophagocytic activity demonstrated that lower amounts of high avidity antibody were sufficient for killing of bacteria whereas higher amounts of low avidity antibody were required for effective killing activity [147]. While the importance of avidity in determining protection from disease is unclear, studies of Haemophilus influenzae type b (Hib) conjugate vaccine have demonstrated that antibody avidity is strongly associated with functional activity of anti-Hib polysaccharide antibodies [149, 150], and anti-Hib polysaccharide antibodies of high avidity have been found to have protective efficacy in experimental Hib infection [151].

Immunological memory should be demonstrated for the licensure of new pneumococcal conjugate vaccine formulations [102]. Immunological memory can be demonstrated by increased serotype-specific IgG titres following administration of a booster dose of pneumococcal polysaccharide (PPS) or conjugate vaccine, or by enhanced antibody avidity [102]. An important feature of a memory response is the maturation of antibody avidity. Measurement of antibody

176

avidity following PCV immunisation provides information on both the development of B cell memory [152] and the functional activity of antibodies [132, 153]. One method of demonstrating immunological priming is to assess the ability of PPS to boost the immune response and elicit antibodies with increased affinity. Evaluation of affinity maturation has been used to assess priming capacity of PCV [147, 154-156] and efficacy of reduced dose schedules [157].

Due to the cost and the limited serotype coverage of PCV, some countries use reduced dose primary PCV series schedules with a 23-valent pneumococcal polysaccharide booster (23vPPS). In Fiji the 7 serotypes included in PCV would only cover 55% of IPD episodes in children under 5 years [385], so that the 23vPPS would potentially provide additional coverage against common disease associated serotypes. The aim of this study was to determine if a reduced number of doses of PCV would provide equivalent functional antibody activity (as assessed by antibody avidity) compared to a 3 dose primary series, and whether a booster with 23vPPS at 12 months would further enhance antibody avidity. To address the concerns of hyporesponsiveness to PPS following re- challenge, the immunological response at 17 months of age was assessed to a small challenge dose of 20% of the 23vPPS (mPPS) in infants who had or had not received the 23vPPS at 12 months of age.

9.3 Methods

9.3.1 Study Participants

A single blind, open-label randomized Phase II vaccine trial was completed in Suva, Fiji documenting the safety, immunogenicity and impact on pneumococcal carriage of various pneumococcal vaccination schedules combining one, 2, or 3 doses of PCV in infancy followed by a booster of 23vPPS. A small 23vPPS re-challenge dose was given to all children at 17 months of age. Healthy infants aged between 6 and 8 weeks of age were eligible for enrolment. Details of the selection criteria, randomisation procedure and immunogenicity results (serum levels of serotype- specific IgG) have been reported elsewhere [386, 389, 390]. This manuscript reports findings for avidity of serotype-specific IgG.

The study was approved by the Fiji National Research Ethics Review Committee and the University of Melbourne Human Research Ethics Committee and was conducted according to Good Clinical Practice.

177

9.3.2 Study Procedures and Vaccines

Infants were stratified by ethnicity and randomised into one of 8 groups. Infants received 1, 2, or 3 doses of the 7-valent CRM197 protein-polysaccharide conjugate vaccine (PCV) containing polysaccharide antigen from pneumococcal serotypes 4, 6B, 9V, 14, 18C, 19F, 23F (Prevnar TM, Wyeth Vaccines). The vaccine contains 2 g/serotype, except serotype 6B at 4 g. The 3 dose group received PCV at 6 weeks, 10 weeks, and 14 weeks of age. The 2 dose group received PCV at 6 and 14 weeks of age and the one dose group received PCV at 14 weeks of age. Vaccines were given a minimum of 25 days apart. Routine vaccines (HiberixTM mixed with TritanrixTM-HepBTM, GlaxoSmithKline) and oral polio were given at 6, 10, and 14 weeks of age. The children in all primary series groups were further randomised to receive a 23vPPS (23vPPS: PneumovaxTM, Merck & Co., Inc., which consists of a purified mixture of 25µg of capsular polysaccharide from 23 pneumococcal serotypes) or no 23vPPS at 12 months of age in addition to Measles-Rubella vaccine. A small re-challenge dose of 20% of the 23vPPS (mPPS) was administered to all groups at 17 months of age.

9.3.3 Laboratory Procedures

Table 26 presents the design of the study and the timing of blood draws. All children except the group randomised to receive no PCV or 23vPPS had blood drawn at 18 weeks and 12 months of age. In addition, those children who were not randomized to receive the 12 month 23vPPS had blood drawn at 9 months of age. Children who received the 12 month 23vPPS had additional bloods drawn 14 days post-23vPPS. All children had blood drawn at 17 months of age (prior to mPPS) and at 18 months (one month post-mPPS). Sera was separated by centrifugation in the health centre, kept chilled and transported to the Colonial War Memorial Hospital laboratory, Suva where it was divided into aliquots and stored at –70 C on the same day, until transported on dry ice to the Pneumococcal Laboratory, Murdoch Childrens Research Institute, Melbourne, for analysis.

178

Table 26: Timing of vaccination and blood draws for each of the 8 groups A B C D E F G H 6 weeks PCV PCV PCV PCV 10 weeks PCV PCV 14 weeks PCV PCV PCV PCV PCV PCV 18 weeks B B B B B B B 9 months B B B 12 months B B B B B B B 23vPPS 23vPPS 23vPPS 23vPPS 12 1/2 months B B B B 17 months B B B B B B B B mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS 18 months B B B B B B B B PCV: 7-valent pneumococcal conjugate vaccine B: Blood test 23vPPS: 23-valent pneumococcal polysaccharide vaccine mPPS: Micro-dose (20%) of the 23vPPS

Details of the ELISA laboratory methods have been reported elsewhere [397, 402]. Specific IgG was measured using a modified 3rd generation ELISA based on current WHO recommendations [341]. Briefly 96-well medium binding polystyrene plates (Greiner Bio-One International AG) were coated with pneumococcal polysaccharides 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F (ATCC, Rockville, MD) at 5 to 10 g/ml and incubated overnight at room temperature. Non-specific, non-opsonic antibodies were absorbed from patient and control sera prior to assay by incubation overnight at 4oC with PBS containing 10% foetal bovine serum (Invitrogen Corporation, USA), cell wall polysaccharide (C-PS 10 µg/ml, Statens Serum Institute, Denmark) and serotype 22F (30 µg/ml). Standard 89SF and samples to be processed for measurement of anti serotype 22F IgG quantitation were absorbed with PBS containing 10% foetal bovine serum and C-PS (10 µg/ml) without 22F. Plates were blocked with 10% foetal bovine serum/PBS for 1 hr at 37oC. Patient samples and standards were then added to plates and incubated for 2 hrs at 37oC before washing with PBS/0.05%Tween (PBS/T). Affinity isolated HRP conjugated anti human IgG (Chemicon, Australia Pty. Ltd.) was added for 2 hrs at 37oC before washing. An enzyme substrate TMB solution (3,3’, 5, 5’–tetramethylbenzidine, Kirkegaard Perry Laboratories, Washington DC) was added and allowed to develop for 9 minutes before stopping with 1M H3PO4. The absorbance was measured at 450 nm (reference filter 620

179

nm) on a Bio-Tek Elx808 (Bio- Tek Instruments, Vermont). The reference serum 89SF (Dr Milan Blake, FDA, Rockville, MD) was used as the reference serum [125, 396]. Optical density data was converted to antibody concentrations (µg/ml) using KCJunior software (Bio- Tek Instruments, Vermont). Each sample dilution was analyzed in duplicate and three controls (low, medium and high IgG) were included in each assay.

Avidity of serotype-specific IgG antibodies to all 23 pneumococcal serotypes in 23vPPS was determined by EIA [147, 154, 344]. Initial steps were as for the ELISA method described above. After incubation of serum samples and washing, 0.5 M sodium thiocyanate (NaSCN) in F-PBS was added to dissociate weak antibody-antigen complexes. NaSCN or F-PBS were incubated for 15 minutes at room temperature, after which the plates were washed and antibody binding was detected by the addition of horseradish peroxidase-conjugated anti-human IgG. The colour was developed by the TMB Peroxidase Substrate system. Absorbance at 450 nm, reference filter 620 nm was read on an EIA reader. Control serum was added to each plate to assess reproducibility. Laboratory staff members were blinded to the group allocation of each serum sample.

9.3.4 Statistical Analysis

Cleaned data were exported to Stata version 9.0 (Stata Corporation, College Station, Texas) for analysis. PCV serotypes were serotypes contained within PCV (4, 6B, 9V, 14, 18C, 19F, 23F). Non- PCV serotypes were those not contained in PCV but contained in 23vPPS (1, 2, 3, 5, 7F, 8, 9N, 10A, 11A, 12F, 15B, 17F, 19A, 20, 22F, 33F). For analyses of serotype-specific avidity responses to non- PCV serotypes following the 12 month 23vPPS booster, comparison of avidity responses was performed by pooling data from the 4 groups that received the12 month 23vPPS and comparing with pooled data from the 4 groups that did not receive the 12 month 23vPPS. Results are expressed as an avidity index (AI), assigned as the percentage of antibodies that remained bound to the antigens after NaSCN elution. The AI was calculated for each serotype assayed for each sample by dividing the end point titre of the serum sample with NaSCN treatment by the endpoint titre of the sample without NaSCN treatment and multiplying by 100. The results are expressed as a serotype-specific median AI (the median percentage of serotype-specific IgG that is avid) at each time point for each treatment group. To test for differences in distributions, between group comparisons of the median serotype-specific AI were performed using the Rank sum test, and paired comparisons of the median serotype-specific AI within groups were performed using the Sign rank test. A p-value of <0.01 was considered statistically significant.

180

9.4 Results

Table 27 shows the median AI for each PCV group 4 weeks post primary series (18 weeks of age), and at 9 and 12 months of age. The median AI were significantly higher (each p<0.01) in the 2 PCV dose group as compared to the 3 PCV dose group at 18 weeks for 4 of 7 PCV serotypes (4, 6B, 18C, 19F) and significantly lower for serotype 23F (p<0.001). By 9 months of age these differences were no longer evident. At 12 months of age the median AI were significantly higher in the 3 PCV dose group as compared to the 2 dose group for 3 of 7 serotypes - serotypes 6B (p<0.001), 14 (p=0.002), and 23F (p<0.001). When comparing the 3 PCV dose group with the single dose group, the median AI in the 3 dose group were significantly higher for all PCV serotypes post primary series (each p<0.001). Nevertheless, by 9 and 12 months of age these differences were no longer present, with similar median AI in the 2 groups for the majority of serotypes, except that the 3 PCV dose group had a higher median AI for serotype 23F (p≤0.001) at 9 months, and serotypes 14 (p<0.001) and 23F (p<0.001) at 12 months, as compared to the single PCV dose group. Figure 21 shows the kinetics of the serotype-specific avidity response for each PCV serotype during the first 12 months of life. Minimal differences can be seen between the one, 2, or 3 dose groups by 12 months of age for each serotype, except for 23F where the single dose group remained significantly lower than the 2 or 3 dose groups. These findings show that while 2 or 3 doses of PCV provide greater antibody avidity responses as compared with a single PCV dose in the early stages post primary series, avidity responses in the single PCV dose group increase over time and are similar to those in the 2 and 3 PCV dose groups by 9 months of age.

181

Table 27: Geometric mean (GM) concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of serotype-specific that is avid) for the PCV serotypes, taken 4 weeks following the PCV primary series, and at 9 and 12 months of age Serotype 3 PCV 2 PCV 1 PCV 0 PCV 18 weeks n=1252 (n=124)3 n=1462 (n=145)3 n=1212 (n=119)3 n=622,3 4 GM IgG 5.47 5.23 2.20 0.04 MAI (95%CI) 53 (49-56) 60 (55-64)4 29 (26-33)4 0 (-)4,5 6B GM IgG 1.66 0.86 0.19 0.11 MAI (95%CI) 35 (33-37) 46 (44-49)4 0 (-)4 0 (-)4,5 9V GM IgG 4.76 4.71 0.90 0.07 MAI (95%CI) 67 (64-69) 68 (65-72) 41 (35-45)4 0 (-)4,5 14 GM IgG 5.51 3.12 1.07 0.34 MAI (95%CI) 53 (47-57) 50 (44-55) 44 (36-49)4 0 (0-36)4,5 18C GM IgG 3.20 2.67 0.58 0.06 MAI (95%CI)) 64 (62-66) 69 (66-73)4 37 (29-42)4 0 (-)4,5 19F GM IgG 5.52 7.99 0.84 0.25 MAI (95%CI) 48 (45-51) 56 (54-58)4 29 (26-31)4 0 (-)4,5 23F GM IgG 2.93 1.65 0.23 0.11 MAI (95%CI) 44 (40-47) 34 (31-40)4 0 (-)4 0 (-)4,5 9 months n=372,3 n=492,3 n=432,3 4 GM IgG 0.79 0.86 0.60 NA MAI (95%CI) 65 (55-74) 64 (59-68) 62 (50-70) 6B GM IgG 0.82 0.81 0.39 NA MAI (95%CI) 54 (49-62) 45 (39-56) 48 (36-55) 9V GM IgG 0.91 1.00 0.56 NA MAI (95%CI) 72 (68-79) 69 (66-75) 63 (58-72) 14 GM IgG 3.99 1.93 1.11 NA MAI (95%CI) 65 (54-70) 58 (49-64) 51 (47-61) 18C GM IgG 0.49 0.41 0.18 NA MAI (95%CI)) 62 (53-69) 66 (59-76) 61 (50-69) 19F GM IgG 1.04 1.40 0.89 NA MAI (95%CI) 43 (37-49) 49 (45-51) 36 (28-44) 23F GM IgG 0.65 0.44 0.24 NA MAI (95%CI) 53 (45-58) 48 (36-52) 19 (0-38)4

182

Serotype 3 PCV 2 PCV 1 PCV 0 PCV 12 months n=1132 (n=111)3 n=1422 (n=138)3 n=1142 (n=110)3 n=592 (n=55)3 4 GM IgG 0.48 0.47 0.63 0.07 MAI (95%CI) 66 (62-69) 62 (59-66) 73 (69-77) 0 (-)4,5 6B GM IgG 0.86 0.76 0.57 0.14 MAI (95%CI) 63 (60-67) 51 (46-55)4 61 (53-66) 0 (-)4,5 9V GM IgG 0.59 0.62 0.50 0.09 MAI (95%CI) 75 (70-78) 71 (67-73) 72 (66-77) 0 (-)4,5 14 GM IgG 2.38 1.52 1.16 0.19 MAI (95%CI) 66 (63-69) 61 (56-64)4 53 (46-58)4 0 (-)4,5 18C GM IgG 0.32 0.24 0.17 0.06 MAI (95%CI)) 70 (65-74) 63 (60-69) 55 (33-67) 0 (-)4,5 19F GM IgG 1.05 1.14 0.93 0.46 MAI (95%CI) 47 (39-51) 42 (38-46) 43 (40-48) 0 (0-30)4,5 23F GM IgG 0.54 0.42 0.26 0.07 MAI (95%CI) 61 (52-64) 46 (35-54)4 0 (-)4 0 (-)4 1 Median AI – median percentage of serotype-specific IgG that is avid 2Sample size for GM IgG 3Sample size for AI 4P-values <0.01 for the 0, 1, or 2 PCV dose comparison of median AI to the 3 PCV dose group 5P-values <0.01 for the single PCV dose comparison with the 0 PCV dose group

183

Figure 21: Median AI (MAI, percentage of serotype-specific-IgG that is avid) for the PCV serotypes taken 4 weeks following the PCV primary series, and at 9 and 12 months of age

4 80 9V 80 70 70 60 60 50 50

40 40 30

Median AI, % Median 30 Median AI, % Median 20 20

10 10

0 0 18 weeks 9 months 12 months 18 weeks 9 months 12 months

6B 14 80 80

70 70

60 60

50 50

40 40

30 30

Median AI, % Median Median AI, % Median

20 20

10 10

0 0 18 weeks 9 months 12 months 18 weeks 9 months 12 months

184

18C 23F 80 80

70 70 60 60 50 50 40 40 30 30 AI, % Median Median AI, % Median 20 20 10 10 0 0 18 weeks 9 months 12 months 18 weeks 9 months 12 months

19F 80 -♦- 3 PCV 70 .-■-. 2 PCV 60 --▲-- 1 PCV 50 --■-- no PCV

30 Median AI, % Median 20

10 Footnote: 12 month blood taken prior to 12 month 23vPPS

0 18 weeks 9 months 12 months

185

To assess the effect of 23vPPS booster on avidity responses, the serotype-specific AI immediately prior to and 2 weeks following 23vPPS booster were compared. Table 28 shows the median AI results immediately prior to and 2 weeks following the 12 month 23vPPS. In all PCV dose groups, the 23vPPS booster resulted in a significant increase in median AI for the majority of PCV serotypes. A better response to 23vPPS was observed in the single PCV group as compared to the 2 or 3 PCV groups for several of the PCV serotypes. There were no significant differences in the median AI post-23vPPS for all PCV serotypes when comparing the 3 and 2 PCV dose groups. There were no significant differences in the median AI 2 weeks following the 23vPPS between the 3 and single PCV dose groups for 5 of 7 PCV serotypes with serotypes 4 (p<0.001) and 9V (p=0.004) being significantly higher in the single dose group. This suggests that a 23vPPS booster administered at 12 months results in similar post immunisation avidity responses for the majority of serotypes irrespective of whether infants received one, 2 or 3 PCV doses in their primary series. Furthermore, a single PCV dose followed by 23vPPS appeared to provide greater avidity responses when compared to the 3 dose PCV/23vPPS group for at least some serotypes. It has been reported previously that a single PCV dose resulted in higher serotype-specific IgG post- 23vPPS for 5 of 7 PCV serotypes [386], and in this study 4 of these 5 serotypes also demonstrated higher avidity when compared to the 2 or 3 PCV dose groups.

186

Table 28: Geometric mean concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of serotype-specific IgG that is avid (1) to PCV serotypes before and 14 days post 12 month 23vPPS, by number of PCV doses administered in the primary series in children randomized to receive 12 month 23vPPS 3 PCV 2 PCV 1 PCV 0 PCV (n=47) (n=63) (n=55) (n=55) Pre- Post- Pre- Post- Pre- Post- Pre- Post- 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS 23vPPS 4 GM IgG 0.46 14.68 0.42 14.7 0.76 46.47 0.08 2.36 MAI (95%CI) 61 (57-66) 68 (64-71) 62 (56-67) 66 (62-71) 74 (70-79) 82 (79-84) 2,3 0 (-) 23 (18-27) 2,4,5 6B GM IgG 0.85 29.58 0.79 23.83 0.69 18.07 0.14 0.31 MAI (95%CI) 63 (58-69) 64 (58-69) 51 (41-55) 60 (52-64) 2 64 (57-66) 69 (63-77) 2 0 (-) 0 (0-21) 4,5 9V GM IgG 0.59 14.10 0.62 15.2 0.67 34.84 0.09 1.2 MAI (95%CI) 72 (65-79) 74 (67-79) 69 (63-74) 72 (68-75) 2 74 (66-78) 82 (78-86) 2,3 0 (-) 45 (38-48)2,4,5 14 GM IgG 2.00 15.73 1.55 13.91 1.36 20.08 0.19 0.41 MAI (95%CI) 69 (64-76) 68 (60-72) 61 (55-65) 59 (50-66) 55 (44-65) 62 (56-70) 0 (-) 0 (0-12)2,4,5 18C GM IgG 0.37 8.71 0.23 10.51 0.22 16.03 0.06 1.22 MAI (95%CI) 69 (61-74) 76 (71-81) 2 62 (51-70) 74 (70-81) 2 61 (45-74) 82 (78-86) 2 0 (-) 43 (38-52) 2,4,5 19F GM IgG 0.86 27.91 1.2 24.77 1.05 84.47 0.47 1.13 MAI (95%CI) 44 (34-52) 65 (57-68) 2 40 (34-45) 64 (59-67) 2 43 (34-51) 69 (61-72) 2 0 (0-30) 25 (11-33)5 23F GM IgG 0.52 10.87 0.41 10.29 0.31 8.33 0.19 0.42 MAI (95%CI) 61 (48-67) 61 (53-63) 39 (30-51) 54 (48-59) 2 0 (0-28) 53 (46-60) 2 0 (-) 0 (0-20) 5 1Median AI – median percentage of serotype-specific IgG that is avid 2P-values <0.01 for comparing the difference in distribution of median AI pre- and post-23vPPS 3P-values <0.01 for comparing the difference between the 3 and 1 PCV dose groups post-23vPPS 4P-values <0.01 for comparing the difference between the 3 and 0 PCV dose groups post-23vPPS 5P-values <0.01 for comparing the difference between the 1 and 0 PCV dose groups post-23vPPS There were no significant differences between the 3 and 2 PCV dose groups post-23vPPS

187

Table 29 shows the median AI results immediately prior to mPPS at 17 months of age and one month post-mPPS for the PCV serotypes in all groups. By 17 months of age, for those who had received 3 PCV doses, there were no significant differences in the median AIs between those who had or had not received the 23vPPS at 12 months of age for any serotypes except 19F (p=0.002). Similar results were found for the 2 PCV dose group. In the single PCV dose group, the median AI at 17 months were significantly higher in those that had received the 12 month 23vPPS compared with those that had not received the 12 month 23vPPS for serotypes 6B (p=0.004), 18C (p<0.001), 19F (p<0.001), and 23F (p<0.001). For those groups that had received the 23vPPS at 12 months of age, there were no significant increases in avidity following the re-challenge with mPPS at 17 months of age (comparing the pre and post-mPPS sera within each PCV group) for all PCV serotypes except serotype 9V (p=0.001) in the 2 PCV/23vPPS group. For those groups that had not received the 23vPPS at 12 months, there were significant increases in avidity following the re-challenge dose (comparing pre and post-mPPS sera within each PCV group) for all PCV groups except serotypes 4 (p=0.731) in the 3 PCV dose group, and serotype 19F (p=0.914) in the no PCV dose group. Figure 22 shows the kinetics of the avidity response following the 23vPPS at 12 months of age for each PCV serotype. These findings suggest that although a 23vPPS booster at 12 months induces a marked increase in serum levels of high avidity serotype-specific IgG, a similar response is achieved with mPPS administered at 18 months. Indeed, the amounts of high avidity serotype-specific IgG were significantly higher at 18 months for many serotypes in the children who did not receive a prior 23vPPS at 12 months as compared to those who did.

188

Table 29: Geometric mean concentrations of serotype-specific IgG titres and median AI (MAI1, percentage of antibody that is avid) to PCV serotypes pre- mPPS at 17 months and one month post-mPPS in those who did or did not receive the 12 month 23vPPS shown by number of PCV doses in the primary series 3 PCV, 3 PCV, 2 PCV, 2 PCV, 1 PCV, 1 PCV, 0 PCV, 0 PCV, no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS Pre-mPPS at 17 months of age n=592 n=482 n=682 n=672 (n=65) n=492 (n=47) n=592 n=622 n=5722,3 (n=45)3 (n=67) 3 3 3 (n=57) 3 (n=60) 3 4 GM IgG 0.35 2.19 0.43 2.03 0.56 6.36 0.11 0.70 MAI (95%CI) 71 (66-77) 67 (63-72) 67 (62-71) 67 (60-72) 75 (71-80) 74 (71-80) 0 (-) 17 (5-26) 4 6B GM IgG 0.91 4.35 0.78 3.85 0.62 3.88 0.20 0.21 MAI (95%CI) 61 (54-68) 58 (48-63) 55 (51-62) 53 (47-58) 53 (46-61) 64 (60-72) 4 0 (-) 0 (-) 9V GM IgG 0.41 2.25 0.49 2.5 0.51 5.49 0.14 0.38 MAI (95%CI) 76 (69-79) 74 (69-80) 71 (68-75) 75 (70-78) 73 (62-81) 76 (74-81) 0 (-) 37 (20-48) 4 14 GM IgG 1.78 4.31 1.12 3.58 0.93 4.85 0.31 0.67 MAI (95%CI) 71 (64-75) 67 (55-74) 62 (54-68) 61 (52-67) 54 (30-60) 65 (55-69) 0 (-) 0 (0-23) 4 18C GM IgG 0.21 1.28 0.20 1.13 0.15 1.79 0.10 0.50 MAI (95%CI) 66 (54-71) 70 (64-75) 70 (56-72) 71 (68-77) 40 (0-67) 77 (74-82) 4 0 (-) 30 (20-42) 4 19F GM IgG 1.19 5.55 1.06 4.53 0.92 13.47 0.59 0.79 MAI (95%CI) 47 (33-55) 57 (51-63) 4 44 (35-53) 57 (52-62) 4 32 (16-41) 63 (57-67) 4 0 (0-28) 18 (0-28) 23F GM IgG 0.57 1.68 0.43 1.41 0.32 1.78 0.19 0.27 MAI (95%CI) 64 (54-71) 52 (45-61) 50 (30-61) 54 (50-60) 0 (0-4) 45 (43-55) 4 0 (-) 0 (-)

189

3 PCV, 3 PCV, 2 PCV, 2 PCV, 1 PCV, 1 PCV, 0 PCV, 0 PCV, no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS no 23vPPS 23vPPS One month post-mPPS n=59 (n=58) n=49 (n=48) n=67 (n=65) n=64 n=48 n=57 n=60 (n=57) n=56 4 GM IgG 4.69 2.01 3.99 2.00 10.39 6.37 0.79 0.81 MAI (95%CI) 72 (66-78) 66 (61-72) 69 (66-74) 64 (60-67) 83 (79-88) 76 (71-79) 29 (23-37) 21 (16-27) 6B GM IgG 12.77 4.08 10.57 4.08 5.82 3.83 0.30 0.22 MAI (95%CI) 68 (61-73) 56 (46-61) 57 (54-63) 53 (47-58) 62 (47-67) 63 (59-68) 0 (-) 0 (-) 9V GM IgG 3.92 2.10 4.34 2.46 9.26 5.21 0.51 0.50 MAI (95%CI) 80 (76-83) 72 (69-76) 4 77 (75-83) 69 (66-75) 4 84 (76-89) 80 (75-84) 36 (24-48) 41 (24-48) 14 GM IgG 10.25 4.26 6.84 3.83 11.09 4.94 0.51 0.97 MAI (95%CI) 69 (63-75) 66 (54-70) 62 (55-67) 60 (54-64) 62 (50-68) 60 (53-64) 0 (-) 21 (0-29) 18C GM IgG 1.94 1.25 2.55 1.23 2.80 1.87 0.48 0.62 MAI (95%CI) 77 (71-81) 74 (67-78) 75 (71-80) 75 (69-77) 82 (78-88) 79 (73-81) 49 (32-56) 36 (23-47) 19F GM IgG 15.33 5.42 15.95 5.09 34.74 14.34 0.99 0.88 MAI (95%CI) 66 (63-72) 55 (50-59) 4 69 (64-73) 54 (48-62) 4 71 (66-78) 63 (58-67) 19 (0-33) 24 (11-37) 23F GM IgG 4.10 1.54 3.74 1.52 3.34 1.54 0.37 0.27 MAI (95%CI) 68 (63-75) 50 (46-60) 4 68 (60-73) 55 (51-60) 4 57 (49-62) 46 (40-51) 0 (0-30) 0 (-) 1Median AI – median percentage of serotype-specific IgG that is avid 2Sample size for GM IgG 3Sample size for AI 4P-values <0.01 for comparing the difference in distribution of median AI between the 3 PCV dose group with or without 23vPPS, the 2 PCV dose group with or without 23vPPS, the single PCV dose group with or without 23vPPS, and the 0 PCV dose group with or without 23vPPS , at 17 and 18 months of age.

190

Figure 22: Median percentage of serotype-specific IgG that is avid for the PCV serotypes pre and post 12 month 23vPPS and pre and post 18 month mPPS in those that did or did not receive the 12 month 23vPPS

9V 4 90 90

80 80

70 70

60 60

50 50

40 40

Median AI, % AI, Median Median Median AI, % 30 30

20 20

10 10

0 0 12 months 121/2 months 17 months 18 months 12 months 121/2 months 17 months 18 months

6B 14 90 90

80 80

70 70 60 60 50 50

40 40 Median AI, % AI, Median

Median AI, % 30 30

20 20

10 10

0 0 12 months 121/2 months 17 months 18 months 12 months 121/2 months 17 months 18 months

191

18C 23F 90 90

80 80

70 70

60 60

50 50 40

40 Median AI,Median %

Median Median AI, % 30 30 20 20 10 10 0 0 12 months 121/2 months 17 months 18 months 12 months 121/2 months 17 months 18 months

19F 90

70 --▲-- 3PCV + 23vPPS __▲__3PCV 60 -- ■-- 2PCV + 23vPPS __■__ 2PCV 50 --♦-- 1PCV + 23vPPS __♦__ 1PCV 40

Median Median AI, % -- -- 23vPPS __ __ 0 PCV/23vPPS 30 ● ●

20 10

0 12 months 121/2 months 17 months 18 months

192

For the non-PCV serotypes, the median AI at 2 weeks following the 12 month 23vPPS were significantly higher in the groups that received the 12 month 23vPPS when compared with those that had not received the 12 month 23vPPS (each p<0.001) (Table 30). These differences between the groups that received the 12 month 23vPPS and those that did not receive the 23vPPS persisted to 17 months of age for all non-PCV serotypes (each p<0.01) (Table 31). At 18 months, following the mPPS booster, the median AI for all non-PCV serotypes were no longer significantly different between those groups that received the 23vPPS and those that did not for 9 of 16 non-PCV serotypes. Furthermore, there were no significant increases in median AI following the re- challenge mPPS dose in the groups that had received the 12 month 23vPPS for the majority of serotypes. The kinetics of the response to 8 of the non-PCV serotypes are shown in Figure 23.

193

Table 30: Geometric mean concentrations of serotype-specific IgG and median AI (MAI1, percentage of antibody that is avid) to non-PCV serotypes before and 14 days post 12 month 23vPPS (n=218) in infants randomized to receive 12 month 23vPPS Pre-23vPPS at 12 months of age2 14 days post 12 month 23vPPS2 Serotype 1 GM IgG 0.17 1.59 MAI (95%CI) 0 (-) 16 (14-19) 2 GM IgG 0.41 10.73 MAI (95%CI) 0 (0-27) 44 (39-47) 3 GM IgG 0.27 8.28 MAI (95%CI) 0 (-) 14 (13-15) 5 GM IgG 0.26 2.26 MAI (95%CI) 0 (-) 26 (23-30) 7F GM IgG 0.09 1.73 MAI (95%CI) 0 (-) 71 (67-73) 8 GM IgG 0.24 8.88 MAI (95%CI) 0 (-) 41 (39-43) 9N GM IgG 0.23 8.31 MAI (95%CI) 0 (0-29) 55 (51-57) 10A GM IgG 0.21 0.76 MAI (95%CI) 0 (-) 32 (25-38) 11A GM IgG 0.09 1.51 MAI (95%CI) 0 (-) 52 (48-57) 12F GM IgG 0.07 0.37 MAI (95%CI) 0 (-) 28 (20-34) 15B GM IgG 0.29 2.15 MAI (95%CI) 47 (40-55) 64 (60-67) 17F GM IgG 0.10 0.81 MAI (95%CI) 0 (-) 34 (31-37) 19A GM IgG 0.46 1.93 MAI (95%CI) 38 (30-44) 59 (53-63) 20 GM IgG 0.09 0.68 MAI (95%CI) 0 (-) 43 (33-51) 22F GM IgG 0.40 4.73 MAI (95%CI) 0 (0-30) 59 (55-66) 33F GM IgG 0.13 1.66 MAI (95%CI) 0 (-) 51 (48-57) 1Median AI – percentage of serotype-specific IgG that is avid 2 The p-value comparing the difference in the distribution of the median AI pre/post 23vPPS was <0.001 for all serotypes.

194

Table 31: Geometric mean concentrations of serotype-specific IgG and median AI (MAI1, percentage of serotype specific IgG that is avid) to non-PCV serotypes at 17 months of age and one month post mPPS, in those that did or did not receive the 12 month 23vPPS No 23vPPS at 12 months 23vPPS at 12 months Pre-mPPS at 17 months of age (n=233) (n=224) 1 GM IgG 0. 23 0.63 MAI (95%CI) 0 (-) 13 (0-17) 2 2 GM IgG 0.51 2.88 MAI (95%CI) 33 (3-42) 45 (40-49) 2 3 GM IgG 0.30 1.46 MAI (95%CI) 0 (-) 17 (15-18) 2 5 GM IgG 0.31 0.77 MAI (95%CI) 0 (-) 24 (19-30) 2 7F GM IgG 0.12 0.51 MAI (95%CI) 0 (-) 65 (61-68) 2 8 GM IgG 0.29 2.31 MAI (95%CI) 0 (0-20) 41 (39-43) 2 9N GM IgG 0.23 1.90 MAI (95%CI) 0 (-) 53 (50-57) 2 10A GM IgG 0.19 0.27 MAI (95%CI) 0 (-) 0 (-)2 11A GM IgG 0.14 0.35 MAI (95%CI) 0 (-) 33 (21-38) 2 12F GM IgG 0.09 0.18 MAI (95%CI) 0 (-) 0 (0-28) 2 15B GM IgG 0.30 0.74 MAI (95%CI) 35 (0-48) 58 (54-63) 2 17F GM IgG 0.12 0.36 MAI (95%CI) 0 (-) 0 (0-23) 2 19A GM IgG 0.59 1.14 MAI (95%CI) 42 (32-50) 59 (54-62) 2 20 GM IgG 0.13 0.26 MAI (95%CI) 0 (-) 0 (-)2 22F GM IgG 0.46 1.43 MAI (95%CI) 0 (0-22) 53 (47-55) 2 33F GM IgG 0.18 0.62 MAI (95%CI) 0 (-) 38 (25-43) 2 One month post-mPPS (n=228) (n=225) 1 GM IgG 0.82 0.73 MAI (95%CI) 15 (11-20) 14 (6-17) 2 GM IgG 3.61 3.14

195

No 23vPPS at 12 months 23vPPS at 12 months MAI (95%CI) 52 (48-55) 43 (39-47) 2 3 GM IgG 2. 11 2.32 MAI (95%CI) 18 (16-20) 15 (13-16) 2 5 GM IgG 0.86 0.85 MAI1(95%CI) 28 (21-31) 25 (21-29) 7F GM IgG 0.83 0.65 MAI (95%CI) 72 (68-75) 66 (64-68) 2 8 GM IgG 3.39 2.93 MAI (95%CI) 49 (47-52) 40 (37-42) 2 9N GM IgG 2.85 1.93 MAI (95%CI) 58 (55-61) 53 (49-55) 2 10A GM IgG 0.42 0.35 MAI (95%CI) 0 (0-24) 0 (0-25) 11A GM IgG 0.74 0.70 MAI (95%CI) 46 (43-50) 47 (41-52) 12F GM IgG 0.26 0.20 MAI (95%CI) 33 (25-39) 15 (0-30) 15B GM IgG 1.04 1.03 MAI (95%CI) 62 (58-65) 60 (56-63) 17F GM IgG 0.49 0.50 MAI (95%CI) 26 (12-36) 27 (23-32) 19A GM IgG 2.19 1.46 MAI (95%CI) 61 (56-66) 58 (53-60) 20 GM IgG 0.47 0.40 MAI (95%CI) 42 (26-49) 32 (0-44) 22F GM IgG 2.87 1.49 MAI (95%CI) 64 (59-67) 51 (47-55) 2 33F GM IgG 1.07 0.78 MAI (95%CI) 51 (47-54) 40 (34-47) 2 1Median AI – median percentage of serotype-specific IgG that is avid 2The p-values were <0.01 for testing differences in distribution of median AI at 17 and 18 months of age between those that had received 23vPPS at 12 months of age and those that had not.

196

Figure 23: Median AI (percentage of serotype-specific IgG that is avid) for selected non-PCV serotypes in those that did or did not receive the 12 month 23vPPS

80 1 80 10A 70 70 60 60 50 50 40 40

AI Medain 30 AIMedian 30

20 20

10 10 0 0 12 months 12 1/2 months 17 months 18 months 12 months 12 1/2 months 17 months 18 months

80 80 3 12F 70 70 60 60 50 50

40 40 Median AIMedian AI Median 30 30 20 20

10 10

0 0 12 months 12 1/2 months 17 months 18 months 12 months 12 1/2 months 17 months 18 months

80 80 5 15B 70 70

60 60 50 50

40 40 Median AIMedian

AI Median 30 30

20 20 10 10 0 0 12 months 12 1/2 months 17 months 18 months 12 months 12 1/2 months 17 months 18 months

80 80 7F 19A 70 70 60 60 50 50

40 40 Median AI Median AI Median 30 30 20 20

10 10

0 0 12 months 12 1/2 months 17 months 18 months 12 months 12 1/2 months 17 months 18 months

♦ no 12m 23vPPS --■-- 12m 23vPPS

197

9.5 Discussion

This is the first comprehensive study of avidity responses against all 23vPPS serotypes following different pneumococcal vaccination schedules. This study has shown that in the first 12 months of life, a 2 or 3 PCV dose primary series in infancy increased the amount of high avidity serotype- specific IgG for PCV serotypes. Surprisingly however, although the single PCV dose group induced minimal amounts of avid antibody immediately post primary series, levels of high avidity serotype- specific IgG increased progressively thereafter so that levels were similar to those observed for the 2 and 3 dose groups by 9 months, with the exception of serotype 23F. Consistent with our findings, a study from the United Kingdom trialing reduced PCV dose schedules together with a booster at 12 months of age [157] found that induction of high avidity serotype-specific IgG against the 3 serotypes tested (6B, 14, 23F) were similar for those receiving 2 or 3 PCV doses and increased significantly for both groups in the period following priming and post boosting [157].

From 12 to 17 months of age, the 12 month 23vPPS booster increased the median AI for most PCV serotypes and all non-PCV serotypes. At 17 months these differences persisted as there were significantly higher median AI for all non-PCV serotypes in the 23vPPS group compared to the group that had not received the 23vPPS at 12 months of age. Avidity may be seen as a surrogate measure of the induction of immunological memory for conjugate vaccines [152], so these findings indicate that one, 2, or 3 PCV doses prime infants to a similar degree. Furthermore, the single PCV dose group had at least similar or higher avidity for all PCV serotypes post the 12 month 23vPPS except for serotypes 14 and 23F as compared to 2 or 3 PCV dose groups, suggesting better priming following a single PCV dose. We have previously demonstrated similar trends for serum titres of serotype-specific IgG [386]. Others have found that a conjugate vaccine booster is better than a PPS booster [147, 154, 157, 411]. Infants primed with PCV and who received a PCV booster, but not PPS booster, had an increase in the amount of high avidity serotype-specific IgG [154] which suggested that the response to PCV was T cell dependent, while the T cell-independent PPS only activated existing memory B cells. A study in infants immunized at 2, 4, and 6 months of age with one of 4 different conjugate vaccines, and boosted at 14 months with the homologous conjugate or PPS found that while the induction of high avidity serotype-specific IgG against serotypes 6B, 14, 19F and 23F increased with age, only a booster dose of conjugate further increased avidity whereas a PPS booster did not [147]. Similarly, a reduced dose UK study found that a booster with PCV induced significantly higher avidity responses against the 3 serotypes tested (6B, 14, 23F) compared to a 23vPPS booster , and induction of high avidity serotype- specific IgG against 3

198

serotypes tested in Ghanaian children was higher in those who had received PCV as compared to 23vPPS [411].

In this study, following a re-challenge with mPPS, there were no further increases in the amount of high avidity serotype-specific IgG induced across the groups that received the 12 month 23vPPS. In contrast, there was a significant increase in high avidity serotype-specific IgG induced by mPPS in the groups that had not received the 23vPPS at 12 months. Nevertheless, at the end of the study there was little difference in avidity responses between any of the groups that were primed with PCV that had or had not received the 12 month 23vPPS. We have demonstrated previously that prior receipt of 23vPPS causes immunological hyporesponsiveness to a 23vPPS re-challenge [390]. Although the children who had received the 12 month 23vPPS had higher circulating antibody concentrations at 17 months of age, the response to a re-challenge dose demonstrated a lack of secondary response to all 23 serotypes irrespective of the pre-existing antibody level. In contrast, those who had not received the 12 month 23vPPS could clearly mount a response to mPPS [390]. The clinical relevance of this finding is unknown [389], as is the clinical relevance of the finding in this current study of no increase in antibody concentration or avidity response in the group that had received the 12 month 23vPPS prior to mPPS. Moreover, all vaccinated groups had similar avidity at the end of the study.

Different serotypes demonstrated differences in the kinetics of avidity responses following vaccination. The amount of high avidity serotype-specific IgG against serotype 6B increased post primary series following 2 or 3 PCV doses but not following a single PCV dose. However by 9 months of age, amounts of high avidity serotype-specific IgG were similar for serotype 6B across all PCV dose groups. In contrast to the avidity response for serotype 6B, the amount of high avidity serotype-specific IgG against serotype 23F did not increase by 9 months. Nevertheless, following the 12 month 23vPPS the amount of high avidity serotype-specific IgG against serotypes 6B and 23F increased with similar kinetics as observed for all other PCV groups. As expected, there was no significant increase in the avidity response for serotype-specific IgG against the majority of non- PCV serotypes (except 15B and 19A) from 9 months to 12 months of age, prior to 23vPPS, and 12 month 23vPPS resulted in an increased amount of high avidity serotype-specific IgG against all non-PCV serotypes compared to those that did not receive the vaccine. As this is the first study to document avidity responses for non-PCV serotypes there are no other studies to compare results with.

Antibody avidity is an expression of the functional antibody affinity and is considered to affect the protective efficacy of antibodies. In vitro, higher avidity antibody is associated with greater

199

opsonophagocytic capacity [146-148]. Avidity is therefore expected to correlate with specific IgG and OPA results [133], however this has not been consistently demonstrated. Results from this study and presented elsewhere assessing the correlation between OPA titre, specific IgG, and avid IgG demonstrated a good correlation between OPA titre and specific IgG and OPA titre and avid IgG for 5 of 6 PCV serotypes (4, 6B, 9V, 18C, 23F) both post-PCV and 23vPPS [412]. However no relationship was found between the OPA titre and avid IgG for the non-PCV serotypes 1 and 5 [412]. Another study showed no association between antibody avidity and opsonophagocytic capacity however there was a tendency to negative correlation between the antibody concentration needed for opsonophagocytosis and relative avidity, suggesting that lesser amounts of high avidity antibody than of low avidity antibody were needed for bacterial killing [133]. One caveat of our study is that avidity assays have not been standardized. This may account for the slight differences found in the first 12 months of life between our previously published serotype- specific IgG antibody concentrations [389] and the avidity results from this study. Previously we found that 3 or 2 PCV doses elicited higher serotype-specific IgG antibody concentrations for most serotypes than a single dose [389]. Cross reactivity between heterologous serotypes has been demonstrated [413]. It has been found that 9V immunity is associated with heterologous immunity to serotype 15 and 19A [413]. In this study, high avidity antibody against 2 non-PCV serotypes 15B and 19A were detected at 12 months of age prior to any vaccine induced immunity. Significant cross-reactivity between serotypes 9V, 15B and 19A has been identified from our study [413].

In summary, this is the first large study to evaluate and compare functional antibody responses following various immunisation schedules with PCV and 23vPPS. An important finding was that the functional antibody (avidity) response following a single PCV dose was similar to that observed following 2 or 3 PCV doses in the primary series. Furthermore, we have shown that booster responses tend to be stronger when preceded by a single dose of PCV as compared to 2 or 3 PCV doses, which is consistent with our previous findings in this cohort [386]. In this study, we have also shown that the serotype- specific antibody avidity response following a 23vPPS at 12 months of age was similar to that following a 17 month mPPS alone. Taken together, these findings support the use of abbreviated schedules in developing countries, either “2+1” or perhaps even a “1+1” schedule. However, there are several caveats to this approach: firstly, the value of a single PCV/23vPPS schedule is undermined by our current finding of immunological hyporesponsiveness post re-challenge (as also reported previously for this cohort [390]). Secondly, it is uncertain whether significant protection from a single PCV dose administered early in infancy would persist throughout the “at risk” period. Nevertheless, based on our current findings, the early

200

administration of a booster at 6 or 9 months of age (“1+1” schedule) would be worthy of further investigation for use in developing countries.

201

10 PNEUMOCOCCAL NASOPHARYNGEAL CARRIAGE FOLLOWING REDUCED DOSES OF 7-VALENT PNEUMOCOCCAL CONJUGATE VACCINE AND A 23-VALENT PNEUMOCOCCAL POLYSACCHARIDE VACCINE BOOSTER

10.1 Abstract

Background: To evaluate the effect of a reduced dose 7-valent pneumococcal conjugate vaccine (PCV) primary series followed by a 23-valent pneumococcal polysaccharide vaccine (23vPPS) booster on nasopharyngeal (NP) pneumococcal carriage.

Methods: Fijian infants aged 6 weeks were randomized to receive 0, 1, 2, or 3 PCV doses. Within each group, half received 23vPPS at 12 months. NP swabs were taken at 6, 9, 12, and 17 months and cultured for Streptococcus pneumoniae. Isolates were serotyped by multiplex-PCR and reverse-line blot assay.

Results: For PCV vaccine type (VT) carriage, there were no significant differences in carriage between the 3 and 2 dose groups at 12 months. VT NP carriage was significantly higher (p<0.01) in the unvaccinated group compared to the 3 dose group at 9 months of age. There appeared to be a PCV dose effect in the cumulative proportion of infants carrying VTs with less VT carriage occurring with more doses of PCV. Non-PCV serotype (NVT) carriage rates were similar for all PCV groups. When groups were pooled by receipt or no receipt of the 12 month 23vPPS, there were no differences in pneumococcal, VT, or NVT carriage rates between the 2 groups at 17 months of age.

Conclusions: There appeared to be a PCV dose effect on VT carriage with less VT carriage occurring with more doses of PCV. By 17 months of age NVT carriage rates were similar for all groups. 23vPPS had no impact on carriage, despite the substantial boosts in antibody levels. 202

10.2 Introduction

A recent review estimated that in the year 2000 over 14 million episodes of serious pneumococcal disease occurred worldwide, with over 800,000 deaths in children under 5 years [41]. Since the introduction of the 7-valent pneumococcal conjugate vaccine (PCV) in the USA there has been a dramatic reduction in vaccine type (VT) invasive pneumococcal disease (IPD) and a modest increase in non-vaccine type (NVT) IPD [52], particularly due to serotype 19A [323, 414]. As nasopharyngeal (NP) carriage is an antecedent event in IPD, the reduction or prevention of NP carriage may reduce the transmission of pneumococci. This has occurred in the USA whereby the widespread use of infant PCV has resulted in significant protection of unimmunized individuals [17, 323] presumably mediated by reduced NP carriage interrupting the transmission of pneumococci [17, 18].

Clinical trials using 5, 7, or 9-valent pneumococcal conjugate vaccines have shown a reduction in VT carriage compared with unvaccinated infants [306-308] or toddlers [309- 311]. However the overall rate of pneumococcal NP carriage remained essentially unchanged due to serotype replacement from NVT [306, 307, 311-313]. Since the routine introduction of PCV into infant national immunisation schedules, there have been a number of carriage surveys documenting the effect of PCV on pneumococcal NP carriage. Similar to the clinical trials, all studies have found that there has been a reduction in VT carriage (4, 8, 11, 15, 17, 18, 26, 34). NVT colonization has increased following vaccination, with serogroups 11 and 15 being commonly reported in many studies (4, 8, 11, 15, 18, 34), and more recently the newly identified serotype 6C [57].

203

In Fiji a vaccine trial has been completed with the aim of finding an optimal pneumococcal vaccination strategy for resource poor countries. A Phase II study was undertaken documenting the safety, immunogenicity and impact on carriage of various schedules combining 1, 2, or 3 doses of PCV in infancy followed by a booster of the 23-valent pneumococcal polysaccharide vaccine (23vPPS). Presented in this manuscript are the effects of the different vaccination schedules on overall NP carriage rates of pneumococci along with rates of VT and NVT carriage at 6, 9, 12 and 17 months of age.

10.3 Methods

10.3.1 Study Design

Details of the study design and randomization procedures have been reported elsewhere [58] [390]. In brief, following informed consent, healthy infants in Suva, Fiji were stratified by ethnicity, and within each stratum, randomized to receive 0, 1, 2, or 3 doses of PCV (PrevenarTM, Wyeth Vaccines, containing serotypes 4, 6B, 9V, 14, 18C, 19F, 23F; 2 g/serotype except serotype 6B which is 4 g)) at 6 weeks, 6 and 14 weeks, or 6, 10, and 14 weeks of age. The co-administered vaccines at 6, 10, and 14 weeks of age were TritanrixTM- HepBTM and HiberixTM, and oral polio vaccine. At 12 months of age, half the infants were randomized to receive 23vPPS (PneumovaxTM, Merck & Co., Inc., 25 g/serotype) and all infants received a microdose ((5 g/serotype) of 23vPPS (mPPS) at 17 months of age. All children received Measles-Rubella vaccine at 12 months of age. The children randomized to receive 0 or 1 PCV dose in infancy had a single dose of PCV administered at 2 years of age.

The study was approved by the Fiji National Research Ethics Review Committee and the University of Melbourne’s Human Research Ethics Committee. Parents or legal guardians signed a written informed consent document for their child to participate in the study. The study was conducted and monitored according to Good Clinical Practice.

10.3.2 Nasopharyngeal Swabs

Buffered cotton NP swabs (Sarstedt, aluminium shaft-buffered, Australia) were taken at 6, 9, 12, and 17 months of age by horizontal insertion into the nares by trained personnel. The swab was left in situ for five seconds and rotated, then immediately placed into a sterile cryovial tube (Simport, Canada) containing 1 mL of skim-milk-tryptone-glucose-glycerol (STGG) transport medium [337]. This was transported in a chilled carrier to the Colonial War Memorial Hospital laboratory, Suva, on the same day. The swabs were processed according to the consensus guidelines from a World Health Organization working group [338]. The

204

swabs were vortexed and either stored at -70 C until plated or plated on receipt in the laboratory. Fifty μL was inoculated onto a 2.5 mg/L gentamicin 5% citrated sheep blood [24]

Columbia agar (Oxoid) plate. Plates were incubated at 37 C in 5% CO2 for 18 to 24 hours. Pneumococcal isolates were initially identified by α-hemolysis, colony morphology, and optochin (Difco) sensitivity. Isolates with intermediate Optochin sensitivity were confirmed as pneumococci by bile solubility testing. Single colonies were subcultured and pure colonies were sent to the Pneumococcal Reference Laboratory, Centre for Infectious Diseases & Microbiology, ICPMR, Westmead, NSW, Australia, where they were serotyped by multiplex- PCR and reverse-line blot assay [339, 340]. Ten percent were also serotyped by Quellung reaction using specific antisera (Statens Serum Institute, Copenhagen, Denmark). Any discrepancy in serotype identified by the 2 methods were resolved by Quellung reaction. Laboratory staff members were blinded to group allocation for each isolate.

10.3.3 Questionnaires

At each of the 4 carriage study visits the following information was collected by the study nurse from a parent/guardian interview: the number of children in the household who were ≤ 5 years of age, family income, exposure to household cigarette smoking, breastfeeding status, whether the child had symptoms of an upper respiratory tract infection (coryza or cough) at the time of the visit, and whether the child had received antimicrobials in the preceding 2 weeks. Attendance at child care was not asked as this is uncommon in Fiji.

10.3.4 Statistical Analysis

All case reporting forms were monitored prior to data entry. Double data entry was performed on all case report forms. Cleaned data were exported to Stata version 9.0 (Stata Corporation, College Station, Texas) for analysis. Serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were classified as VT; all other viable isolates including those that were non-typeable were classified as NVT. Rates of NP carriage were calculated using the number of total pneumococcal, VT, or NVT isolates in each group at each time point divided by the total number of children who had a NP swab taken for each group at each time point. Analyses comparing the proportions of infants with NP carriage of any pneumococcus, NP VT and NP NVT carriage were performed using Fisher’s exact test. For each group, the cumulative proportion of children carrying a VT was calculated. Due to multiple comparisons a p-value <0.01 was considered statistically significant.

205

10.4 Results

There were 552 children enrolled in the study (Table 32). Characteristics of the study children are shown in Table 33. Age at enrollment was very comparable across the groups, but there were minor differences in some of the characteristics such as higher exposure to cigarette smoking in the 2 PCV dose group. Of the 2,208 NP swabs planned, 90.8% were taken, of which 47.8% were positive for Streptococcus pneumonaie. There were 959 pneumococcal isolates of which 33 (3.4%) were non-typeable and 13 (1.4%) were no longer viable for serotyping.

Table 32: Timing of vaccination and blood draws for each of the 8 groups A B C D E F G H 6 weeks PCV PCV PCV PCV 10 weeks PCV PCV 14 weeks PCV PCV PCV PCV PCV PCV 18 weeks B B B B B B B 9 months B B B 12 months B B B B B B B 23vPPS 23vPPS 23vPPS 23vPPS 12 1/2 months B B B B 17 months B B B B B B B B mPPS mPPS mPPS mPPS mPPS mPPS mPPS mPPS 18 months B B B B B B B B PCV: 7-valent pneumococcal conjugate vaccine B: Blood test 23vPPS: 23-valent pneumococcal polysaccharide vaccine mPPS: Micro-dose (20%) of the 23vPPS

206

Table 33: Characteristics of infants by group allocation at enrolment and at each of the 4 nasopharyngeal swab visits (%, unless otherwise stated) Time point, parameter 3 PCV 2 PCV 1 PCV 0 PCV No 12m 12m 23vPPS No 12m 12m 23vPPS No 12m 12m 23vPPS 12m 23vPPS No 12m 23vPPS (n=65) 23vPPS (n=80) 23vPPS (n=66) (n=63) 23vPPS (n=71) (n=76) (n=62) (n=69) At enrolment Male 38 (54) 33 (51) 31 (41) 39 (49) 31 (50) 28 (42) 32 (51) 32 (46) Median age in weeks 6.7 6.5 6.5 6.4 6.5 6.5 6.5 6.4 Ethnicity Indigenous 39 (55) 43 (66) 48 (63) 58 (73) 41 (66) 42 (64) 35 (55) 45 (65) Fijian Indo-Fijian 29 (41) 17 (26) 24 (32) 21 (26) 17 (27) 22 (33) 20 (32) 20 (29) Other 3 (4) 5 (8) 4 (5) 1 (1) 4 (7) 2 (3) 8 (13) 4 (6) Median weight in grams 4850 5000 4775 4800 4900 4750 4750 4800 Children ≤5 years old in 1.5 (1-4) 2 (1-5) 1 (1-3) 1 (0-4) 2 (0-4) 2 (1-4) 2 (1-5) 2 (1-4) household, median (range) Annual income1, 74 74 74 72 64 62 54 61 median (range) (15-270) (24-490) (20-392) (10-613) (10-245) (20-343) (20-294) (0-294) Exposure to cigarette 32 (49) 22 (36) 39 (54) 41 (54) 26 (44) 31 (49) 30 (50) 36 (54) smoking (%) At 6 months of age n=66 n=61 n=72 n=76 n=59 n=63 n=60 n=67 Breastfeeding (%) 55 (83) 50 (82) 57 (79) 66 (87) 50 (85) 50 (79) 55 (92) 56 (84) Current URTI (%) 18 (27) 15 (25) 20 (28) 30 (39) 16 (27) 14 (22) 20 (33) 20 (30) Antimicrobials in prior 9 (14) 5 (8) 11 (15) 10 (13) 6 (10) 7 (11) 5 (8) 13 (19) 2 weeks (%) At 9 months of age n=62 n=60 n=71 n=75 n=55 n=63 n=60 n=66 207

Time point, parameter 3 PCV 2 PCV 1 PCV 0 PCV No 12m 12m 23vPPS No 12m 12m 23vPPS No 12m 12m 23vPPS 12m 23vPPS No 12m 23vPPS (n=65) 23vPPS (n=80) 23vPPS (n=66) (n=63) 23vPPS (n=71) (n=76) (n=62) (n=69) Breastfeeding (%) 44 (71) 39 (65) 46 (65) 57 (76) 42 (76) 45 (71) 46 (77) 51 (77) Current URTI (%) 18 (29) 18 (30) 23 (32) 27 (36) 20 (36) 20 (32) 19 (32) 26 (39) Antimicrobials in prior 3 (5) 5 (8) 7 (10) 8 (11) 5 (9) 3 (5) 3 (5) 3 (5) 2 weeks (%) At 12 months of age n=61 n=53 n=71 n=72 n=54 n=61 n=59 n=66 Breastfeeding (%) 32 (52) 26 (49) 37 (52) 45 (63) 19 (35) 38 (62) 45 (76) 44 (67) Current URTI (%) 13 (21) 15 (28) 15 (21) 13 (18) 14 (26) 10 (16) 10 (17) 19 (29) Antimicrobials in prior 4 (7) 9 (17) 6 (8) 9 (13) 8 (15) 5 (8) 5 (8) 6 (9) 2 weeks (%) At 17 months of age n=60 n=49 n=68 n=67 n=49 n=59 n=57 n=63 Breastfeeding (%) 23 (38) 16 (33) 27 (40) 29 (43) 19 (39) 26 (44) 29 (51) 27 (43) Current URTI (%) 13 (22) 19 (39) 22 (32) 14 (21) 16 (33) 13 (22) 18 (32) 18 (29) Antimicrobials in prior 1 (2) 7 (14) 7 (10) 4 (6) 2 (4) 3 (5) 6 (11) 10 (16) 2 weeks (%) 1In USD: exchange rate USD0.49=1FJD URTI: Upper respiratory tract infection

208

Table 34 shows the impact of the different vaccination schedules on overall NP carriage in the first 12 months of life. There were no significant differences in NP pneumococcal carriage between any of the groups at any time point. There was a trend towards higher NP pneumococcal carriage rates in the single PCV dose group compared with unvaccinated group at 6 months of age (55% vs 42%, p=0.04). For VT NP carriage, the rates were significantly higher in the unvaccinated group at 9 months (16% vs 3%, p<0.01) compared with the 3 dose group. There was a trend towards higher VT NP carriage rates (10% vs 3%, p=0.03) in the 2 PCV dose group compared with the 3 PCV dose group and the unvaccinated group compared with the single dose group (16% vs 7%, p=0.03) at 9 months of age, and the unvaccinated and the 3 dose group at 12 months (16% vs 7%, p=0.03). Figure 24 shows little difference in the cumulative proportion of infants carrying a VT at 6 months of age. However there were substantial differences between the PCV groups and the unvaccinated group in the cumulative proportion of children carrying a VT at 9 and 12 months of age. Moreover there appears to be a PCV dose effect in the cumulative proportion of infants carrying a VT at 9 and 12 months of age with the 3 PCV group having the lowest cumulative proportion compared with the 2 and single dose groups. 209

Table 34: Nasopharyngeal (NP) carriage of all pneumococcal, 7-valent pneumococcal conjugate vaccine (PCV) serotypes (VT), and non-PCV serotypes (NVT) at 6, 9, and 12 months (m) of age following administration of 0, 1, 2, or 3 doses of PCV as a primary series 3 PCV 2 PCV p2 OR3 1 PCV p2 OR3 O PCV p2 OR3 p4 OR5 n=136 (%) n=156 (%) (95%CI) n=128 (%) (95%CI) n=132 (%) (95%CI) (95%CI) Pneumococcal NP carriage 6m1 64/127 71/148 0.72 1.1 67/122 0.53 0.83 53/127 0.17 1.44 0.04 1.72 (50) (48) (0.68-1.77) (55) (0.51-1.37) (42) (0.87-2.36) (1.04-2.86) 9m 58/122 81/146 0.15 0.7 59/118 0.7 0.9 60/126 0.9 0.95 0.8 1.06 (48) (56) (0.43-1.12) (50) (0.54-1.48) (48) (0.58-1.56) (0.64-1.75) 12m 48/114 64/143 0.39 0.8 59/115 0.1 0.65 56/125 0.37 0.78 0.53 1.2 (42) (45) (0.49-1.3) (51) (0.39-1.08) (45) (0.47-1.29) (0.73-1.98) VT NP carriage 6m1 13/127 16/148 0.84 0.88 14/122 1.0 0.96 16/127 0.29 0.61 0.3 0.63 (10) (11) (0.38-2.0) (11) (0.41-2.26) (13) (0.26-1.42) (0.27-1.45) 9m 4/122 15/146 0.03 0.30 8/118 0.25 0.46 20/126 <0.01 0.18 0.03 0.38 (3) (10) (0.09-0.9) (7) (0.13-1.59) (16) (0.06-0.54) (0.16-0.91) 12m 8/114 9/143 1.0 1.04 10/115 0.63 0.75 20/125 0.03 0.36 0.08 0.48 (7) (6) (0.39-2.78) (9) (0.29-1.98) (16) (0.15-0.87) (0.21-1.08) NVT NP carriage 6m1 51/127 53/148 0.54 1.17 52/121 0.7 0.9 31/127 0.03 1.78 0.01 1.97 (40) (36) (0.72-1.9) (43) (0.54-1.5) (24) (1.05-3.04) (1.15-3.38) 9m 52/122 64/146 1.0 1.02 49/118 0.9 1.06 34/126 0.01 1.92 0.03 1.81 (43) (44) (0.63-1.64) (42) (0.64-1.75) (27) (1.14-3.23) (1.07-3.06) 12m 40/114 55/143 0.9 1.05 49/115 0.53 0.84 36/125 0.07 1.63 0.01 1.94 (35) (38) (0.65-1.7) (43) (0.51-1.4) (29) (0.97-2.75) (1.14-3.28) 1NP swab taken a median of 11.6 weeks (IQR 11-12 weeks) after the completion of the PCV primary series 2Comparison of 0-2 PCV dose groups with the 3 PCV dose group 3Unadjusted odds ratio comparing NP carriage in children following 0, 1, or 2PCV doses with 3 PCV doses 4Comparison of the single PCV dose group with the unvaccinated group 5Unadjusted odds ratio comparing NP carriage in children following a single PCV dose with the unvaccinated group 210

Figure 24: Cumulative proportion of infants carrying a 7-valent pneumococcal conjugate vaccine (PCV) type at 6, 9, 12, and 17 months of age by PCV and 23-valent pneumococcal polysaccharide vaccine (23vPPS) group allocation

50 3PCV no 12m 23vPPS 3PCV 12m 23vPPS 40 2PCV no 12m 23vPPS 2PCV 12m 23vPPS 1PCV no 12m 23vPPS 30 1PCV 12m 23vPPS 0PCV no 12m 23vPPS Cumulative % Cumulative 0PCV 12m 23vPPS 20

0 6m 9m 12m 17m

For NVT NP carriage, there were no significant differences between any groups at any time point. Although there was a trend for higher NVT NP carriage in the 3 dose group compared with the unvaccinated group at 6 (40% vs 24%, p=0.03) and 9 months of age (43% vs 27%, p=0.01), and the single dose group compared with the unvaccinated groups at 6 (43% vs 24%, p=0.01), 9 (42% vs 27%, p=0.03) and 12 months of age (43% vs 29%, p=0.01).

At 17 months of age, there were no significant differences in pneumococcal, and VT carriage rates between those groups that had or had not received 23vPPS and by number of PCV doses (Figure 25). There was a trend for higher VT carriage among those that had received 0 or 1 PCV dose compared to the 2 or 3 PCV dose groups (Figure 25). Figure 24 shows the cumulative proportion of VT carriage at 17 months by PCV and 23vPPS group allocation. Minimal differences can be seen in VT carriage between each PCV group and whether they had or had not received 23vPPS at 12 months of age. However those that had received 23vPPS alone had the highest cumulative proportion of VT carriage at 17 months of age (p<0.001) compared to the 3 PCV only group. There were no significant differences in NVT carriage rates between those groups that had or had not received the 23vPPS at 12 months 211

of age (Table 35). When further analysed by PCV group allocation, there were no significant differences in NVT carriage at 17 months of age between any PCV group.

Figure 25: Nasopharyngeal (NP) carriage rates of all pneumococcal and 7-valent pneumococcal conjugate vaccine (PCV) serotypes (VT) at 17 months of age following 0, 1, 2, or 3 PCV doses of PCV in infancy with or without 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months

Frequency of NP carriage, % 10

12m 12m 12m 12m

No 12m 23vPPS 23vPPS No 12m 23vPPS 23vPPS No 12m 23vPPS 23vPPS No 12m 23vPPS 23vPPS 3 PCV 2 PCV 1 PCV 0 PCV

Pneumococcal carriage VT carriage

The p-values were not significant (all >0.45) for the following comparisons: 3 PCV with or without the 12 month 23vPPS, 2 PCV with or without the 12 month 23vPPS, a single PCV dose with or without the 12 month 23vPPS, and no PCV with or without the 12 month 23vPPS.

212

Table 35: Nasopharyngeal (NP) carriage of all pneumococcal and non-PCV serotypes (NVT) at 17 months of age in those who did or did not receive the 23-valent pneumococcal polysaccharide vaccine (23vPPS) at 12 months of age 12 month 23vPPS No 12 month 23vPPS p1 OR2 n=232 n=240 (95%CI) Pneumococcal NP 109 (47) 111 (46) 1.000 0.99 carriage n (%) (0.71-1.4) NVT NP carriage 89 (38) 93 (39) 0.733 0.87 n (%) (0.44-1.72) 1P-value comparing NP carriage in children who had or had not received the 12 month 23vPPS 2Unadjusted odds ratio comparing NP carriage in children who had or had not received the 12 month 23vPPS

Serotypes 19F and 23F were the most frequently carried PCV serotypes in all groups in the first 12 months, with 6B being the most frequently carried serotype by 17 months of age. For the non-PCV serotypes, 6A and 19A were the most frequently carried serotypes throughout the study.

10.5 Discussion

Our findings suggested a PCV dose effect on VT carriage with 3 PCV doses appearing to have a greater effect on VT carriage than fewer PCV doses. Unfortunately the sample size was too small to evaluate the impact of the different vaccination groups on the carriage of individual serotypes. We found that a single PCV dose results in some initial reduction in VT carriage (although not statistically significant) which along with immunogenicity data [389] provides supportive evidence that one dose may offer some early protection from IPD. Early differences (although not statistically significant) in NVT carriage rates between groups were no longer evident by 17 months of age. Furthermore, the addition of 23vPPS at 12 months of age had no impact on carriage, despite the substantial boosts in antibody levels observed [386].

One of the key questions is the duration of the effect of reduced dose schedules on NP carriage. In this study we found that the reduction in VT carriage was sustained to 17 months of age following a 3 or 2 PCV dose schedule. There is only one other randomized and published study reporting the effect of reduced dose PCV schedules on the effect of NP carriage. This study from the Netherlands compared carriage rates following 2 PCV doses at 2 and 4 months of age with a 2+1 schedule at 2, 4, and 11 months of age, with an unvaccinated control group. Both vaccinated groups had significant reductions in VT carriage

213

in the second year of life compared with controls [324]. The booster dose resulted in an earlier further reduction in VT carriage at 18 months compared with no booster dose (24% vs 16%). However by 2 years of age both vaccinated groups had similar VT carriage rates (15% each) [324]. Similarly, in a case-control study Gambian infants, vaccinated with either 3 or 2 doses of a 5-valent pneumococcal conjugate vaccine in infancy followed by 23vPPS at 18 months of age showed a significant reduction in VT carriage compared with unvaccinated matched controls at 2 years of age [306].

There are a number of studies evaluating the duration of effect of a 3 PCV dose schedule on VT carriage. In one study the effects on VT colonization were no longer evident by 2 to 5 years of age following 3 PCV doses in infancy and the 23vPPS at 13 months of age [308]. In contrast, a study in South Africa found in HIV non-infected children, VT colonization was significantly lower in the 3 PCV dose group compared to the placebo group 5 years after vaccination in infancy [415]. In the Gambia, the effects on carriage persisted at least 16 months post vaccination following 3 doses of infant 9-valent pneumococcal conjugate vaccine [416]. In another study following vaccination of toddlers under 2 years of age, the reduction in VT carriage continued for at least one year [309] and in 2 other studies to 4 years of age but not beyond [310, 311]. Since the routine introduction of PCV into infant national immunization schedules, there have been a number of carriage surveys documenting the effect of PCV on pneumococcal NP carriage. Similar to the clinical trials, all studies have reported a reduction in VT carriage (4, 8, 11, 15, 17, 18, 26, 34) particularly in those children who are up to date with their immunizations (4, 17, 26), have had a PCV booster in the second year of life (4, 11), or have not had prolonged intervals between PCV doses [319].

One of the disadvantages of the currently available PCV is serotype replacement by NVT filling the ecological vacant niche following vaccination [417]. In our study higher (although not statistically significant) NVT carriage rates in the PCV groups were found in the first 12 months of life compared to unvaccinated controls. However by 17 months of age this trend was no longer evident suggesting that this initial effect was not sustained. Our NVT rate (approximately 40%) is higher than reported in the reduced dose Netherlands study whereby NVT carriage rates of 15-17% were reported in the vaccinated groups and 8% in the unvaccinated control group [324]. The NVT rates reported in observational studies of children from industrialized countries following PCV tend to be lower (18% in the US [315], 15.5% in France [314]). However NVT carriage rates following vaccination in children from

214

non-industrialized countries (77% in the Gambia [306], 36% in South Africa [307]) and disadvantaged high risk communities (39.2% Navajo and White Mountain Apache children [312]) tend to be higher. This suggests that the impact of PCV on NVT may be greater in geographical settings with high burdens of pneumococcal disease as the direct and indirect effects of PCV vary with age and the presence of underlying conditions such as HIV [227]. Ongoing surveillance is needed to detect changes in rates of NVT IPD in additional to VT IPD following the introduction of PCV.

To our knowledge this is the first published randomized study to show the impact of a single PCV dose on NP carriage. There appeared to be a PCV dose effect on VT carriage with a single PCV dose having less effect on VT carriage than a 2 or 3 dose schedule. A single PCV dose initially reduced VT carriage (although this was not statistically significant) but this effect was not sustained and by 12 months of age there was no difference in rates compared to the unvaccinated controls. The single dose group had a higher (although not statistically significant) NVT carriage rate compared to unvaccinated controls up to 12 months of age but there were no significant differences in NVT carriage rates between any of the groups by 17 months of age. In contrast, results from a series of prevalence surveys in Canada showed that children who had received 0, 1, or 2 PCV doses were less likely to be colonized with NVT than those who had received 3 PCV doses [320]. In a small observational study coinciding with a shortfall of PCV supply in the US, NVT carriage was similar for those children that had received one or 2 doses (22 vs 27%) but higher in the 3 dose group (47%) [319]. Data from our study combined with our previously reported immunogenicity data, in which a single PCV dose elicited significant responses for all serotypes post primary series compared with the unvaccinated [389], provides additional evidence that a single PCV dose in infancy would offer some protection in the first 12 months of life. Moreover, memory responses were most profound for children who had received only a single dose of PCV previously, compared with the 2 or 3 dose groups [386]. The results from this study and previously published immunogenicity data [386, 389] raises the intriguing possibility that a schedule based on a single dose of vaccine early in infancy with an early booster might provide adequate protection while avoiding the problems of serotype replacement.

The 12 month 23vPPS booster had no additional effect on pneumococcal carriage rates nor any statistically significant effect on VT or NVT carriage rates despite significant boosts in antibody levels [386]. Similarly, studies using other pneumococcal polysaccharide vaccines have shown no effect on pneumococcal carriage [294, 326, 327, 329, 330]. PCV followed by

215

23vPPS given to 1 to 7 year old children with recurrent acute otitis media in the Netherlands found no beneficial effect from the booster vaccine [273]. In addition to having no effect of carriage, the 23vPPS may have deleterious immunological effects. In this study there was a suggestion that VT carriage was higher at 17 months of age in the group that received 23vPPS alone compared with all other groups. Despite our study previously demonstrating the potential value of a PCV/23vPPS schedule [386] immunological hyporesponsiveness to a small re-challenge dose of 23vPPS has been demonstrated [390]. These findings suggested that additional immunization with the 23vPPS following a primary series of PCV does not provide added benefit for antibody production and instead results in impaired immune responses following a subsequent pneumococcal polysaccharide antigen challenge.

Despite 2 or 3 doses having an impact on VT carriage the overall pneumococcal carriage rates did not change due to the increase in NVT. This has been consistently found in many other studies in different settings [307, 310-313, 315-318, 320, 321]. Considering this effect and the potential loss of natural boosting after widespread implementation of reduced doses, ongoing surveillance on the impact on carriage and IPD is important as a booster dose may be necessary for long term protection [418, 419].

In conclusion, although the sample size was small there appeared to be a PCV dose effect on VT carriage with less VT carriage occurring with more doses of PCV. A single PCV dose resulted in some initial reduction in VT carriage, and along with supportive immunogenicity data, provides further evidence that a single dose would offer some protection from IPD. There was no significant difference in NVT carriage for all groups. The addition of 23vPPS at 12 months of age had no impact on carriage.

216

11 CONCLUSIONS

The overall objective of these studies was to gather sufficient evidence for the Fiji MoH to decide whether to introduce the pneumococcal vaccination into its national schedule and define an appropriate and affordable vaccination strategy. These studies have found the morbidity and mortality of pneumococcal disease in Fiji to be significant. Potential PCV coverage for IPD is low in all ages. Hence there is sufficient burden of disease to warrant the introduction of a national pneumococcal vaccination strategy in Fiji. However use of PCV alone would not adequately cover the circulating serotypes and the newer generation of conjugate vaccines would be a preferable choice.

The results of the vaccine trial can be thought of in terms of defining both the optimal pneumococcal primary series and booster vaccination strategy. Regarding the primary series schedule, data from this study would support the introduction of a 2 dose primary series schedule. This is supported by similar levels of both binding antibody and functional antibody for many serotypes post-primary series compared to the 3 dose group. In addition, similar memory responses were seen post 23vPPS booster in the 2 PCV dose group compared with the 3 dose group. A single PCV dose would offer protection in the first 12 months of life for many serotypes and, indeed, memory responses following 23vPPS were most profound for children who had received only a single dose of PCV previously, compared with the 2 or 3 dose groups. This provides reassurance that children who receive only a single dose in the primary series are still likely to benefit.

The optimal booster regimen is yet to be defined. Although we found that a dose of 23vPPS at 12 months of age resulted in good booster responses to serotypes in the 7-valent conjugate vaccine, and also led to potentially protective responses to the other 16 non- primed serotypes which persisted for at least 5 months, we also found evidence of subsequent hyporesponsiveness following re-challenge. This hyporesponsiveness was independent of prior antibody levels, and occurred to all 23 serotypes. Any further use of the 23vPPS in children aged <2 years would need to consider the relative merits of its potential to provide protective antibody levels (whether following primary immunisation at age 12 months or as a booster to a previous conjugate vaccine primary course) compared to its potential to lead to hyporesponsiveness following re-challenge.

The clinical relevance of immunological findings from vaccine trials is obviously best demonstrated in efficacy studies. In our studies there were no pneumococcal disease

217

endpoints to evaluate the clinical relevance of our immunological findings. However, separately we evaluated the effect on NP carriage. We found that 2 or 3 PCV doses in infancy had a similar impact on NP pneumococcal carriage rates. A single PCV dose resulted in some initial reduction in PCV serotype carriage which provides supportive evidence that one dose may offer some early protection from IPD. The addition of 23vPPS at 12 months of age had no impact on carriage, despite the substantial boosts in antibody levels observed and despite better OPA and better antibody avidity. This challenges the understanding on the relationship between antibody levels, OPA, and NP carriage, particularly following 23vPPS. It has been advocated that effects on carriage may help provide evidence of protection against disease, and also that high levels of serum antibody (including OPA) should lead to reduced carriage and thus, at the population level, herd immunity. This has been previously demonstrated in other pneumococcal conjugate vaccine studies and post PCV introduction. However our findings following the 23vPPS do not support this indicating other immunological mechanisms, not induced by 23vPPS but induced by PCV, are important to result in this biological effect. Moreover, further analyses of the carriage data using more sensitive molecular techniques using both quantitative and qualitative methods may provide further insight on the impact on carriage.

Thus, while 23vPPS at 12 months was well tolerated and provided antibody responses that may be protective and last for at least 5 months, this occurred at the cost of significantly reduced responses to all 23 serotypes on subsequent exposure. The immunological basis for this is unknown, and it is unknown whether this hyporesponsiveness is transient or a lasting effect. It is also unknown whether or not this immunological observation is associated with any clinical risk. However no impact on NP carriage was observed, nor was there any increase in disease in the 23vPPS groups compared with those that had not received the 23vPPS at 12 months of age. Further research will be undertaken to further look at the B cell responses and quantify pneumococcal NP load.

11.1 Implications for Pneumococcal Vaccine Policy in Fiji and Other Countries

PCV would not adequately cover the prevailing pneumococcal serotypes in Fiji. However the results of the vaccine study casts considerable doubt over the value of the PCV/23vPPS schedule and is not recommended due to hyporesponsiveness demonstrated on re-exposure to pneumococcal polysaccharide antigens. The results of this study confirm that a 2 dose PCV primary series is likely to be just as adequate as a 3 dose primary series. Indeed our

218

study has shown that booster responses tend to be stronger after a single dose of PCV rather than 2 or 3 doses, suggesting that children who receive only one dose in infancy may still benefit, although this would not be a recommended regimen. However, it is unlikely that significant protection, from a single PCV dose administered early in infancy, would persist for children throughout the highest risk period for IPD and pneumonia and an early booster at 6 or 9 months of age (“1+1” schedule) is worthy of further investigation for use in developing countries. This may be particularly relevant for low income countries where access to immunisation services is irregular, and middle income countries, such as Fiji, which have a significant burden of disease but are unable to pay affluent country vaccine prices and do not benefit from immunisation financing mechanisms.

219

REFERENCES

[1] Sternberg G. A fatal form of septicaemia in the rabbit, produced by the subcutaneous injection of human saliva. An experimental research. Nat Bd Health Bull 1881;2:781-3. [2] Pasteur. Note sur la maladie nouvelle provoquee par la salive d'un enfant mort de la rage Bull Acad Med (Paris) 1881;10(2):94-163. [3] Conclusions from the WHO multicenter study of serious infections in young infants. The WHO Young Infants Study Group. Pediatr Infect Dis J 1999 Oct;18(10 Suppl):S32- 4. [4] Pneumococcal conjugate vaccine for childhood immunization--WHO position paper. Wkly Epidemiol Rec 2007 Mar 23;82(12):93-104. [5] Denny FW, Loda FA. Acute respiratory infections are the leading cause of death in children in developing countries. Am J Trop Med Hyg 1986 Jan;35(1):1-2. [6] Austrian R. Of gold and pneumococci. A history of pneumococcal vaccines in South Africa. Trans Am Clin Climatol Assoc 1977;89:141-61. [7] Austrian R. Pneumococcal otitis media and pneumococcal vaccines, a historical perspective. Vaccine 2000 Dec 8;19 Suppl 1:S71-7. [8] Breiman RF, Butler JC, Tenover FC, Elliott JA, Facklam RR. Emergence of drug- resistant pneumococcal infections in the United States. JAMA 1994 Jun 15;271(23):1831-5. [9] Gray BM, Converse GM, 3rd, Dillon HC, Jr. Epidemiologic studies of Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life. J Infect Dis 1980 Dec;142(6):923-33. [10] Hodges RG, Macleod CM, Bernhard WG. Epidemic pneumococcal pneumonia: III. Pneumococcal carrier studies. Am J Hyg 1946;44:207-30. [11] Faden H, Duffy L, Wasielewski R, Wolf J, Krystofik D, Tung Y. Relationship between nasopharyngeal colonization and the development of otitis media in children. Tonawanda/Williamsville Pediatrics. J Infect Dis 1997 Jun;175(6):1440-5. [12] Alanee SR, McGee L, Jackson D, Chiou CC, Feldman C, Morris AJ, et al. Association of serotypes of Streptococcus pneumoniae with disease severity and outcome in adults: an international study. Clin Infect Dis 2007 Jul 1;45(1):46-51. [13] Hjuler T, Wohlfahrt J, Staum Kaltoft M, Koch A, Biggar RJ, Melbye M. Risks of invasive pneumococcal disease in children with underlying chronic diseases. Pediatrics 2008 Jul;122(1):e26-32. [14] Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008 Apr;6(4):288-301. [15] Montgomery JM, Lehmann D, Smith T, Michael A, Joseph B, Lupiwa T, et al. Bacterial colonization of the upper respiratory tract and its association with acute lower respiratory tract infections in Highland children of Papua New Guinea. Rev Infect Dis 1990 Nov-Dec;12 Suppl 8:S1006-16. [16] Mastro TD, Nomani NK, Ishaq Z, Ghafoor A, Shaukat NF, Esko E, et al. Use of nasopharyngeal isolates of Streptococcus pneumoniae and Haemophilus influenzae from children in Pakistan for surveillance for antimicrobial resistance. Pediatr Infect Dis J 1993 Oct;12(10):824-30. [17] Hammitt LL, Bruden DL, Butler JC, Baggett HC, Hurlburt DA, Reasonover A, et al. Indirect effect of conjugate vaccine on adult carriage of Streptococcus pneumoniae: an explanation of trends in invasive pneumococcal disease. J Infect Dis 2006 Jun 1;193(11):1487-94.

220

[18] Millar EV, Watt JP, Bronsdon MA, Dallas J, Reid R, Santosham M, et al. Indirect effect of 7-valent pneumococcal conjugate vaccine on pneumococcal colonization among unvaccinated household members. Clin Infect Dis 2008 Oct 15;47(8):989-96. [19] Lexau CA, Lynfield R, Danila R, Pilishvili T, Facklam R, Farley MM, et al. Changing epidemiology of invasive pneumococcal disease among older adults in the era of pediatric pneumococcal conjugate vaccine. JAMA 2005 Oct 26;294(16):2043-51. [20] Pikis A, Campos JM, Rodriguez WJ, Keith JM. Optochin resistance in Streptococcus pneumoniae: mechanism, significance, and clinical implications. J Infect Dis 2001 Sep 1;184(5):582-90. [21] Anand C, Gordon R, Shaw H, Fonseca K, Olsen M. Pig and goat blood as substitutes for sheep blood in blood-supplemented agar media. J Clin Microbiol 2000 Feb;38(2):591-4. [22] Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Atlanta: Centers for Disease Control and Prevention 1998. [23] Gratten M, Battistutta D, Torzillo P, Dixon J, Manning K. Comparison of goat and horse blood as culture medium supplements for isolation and identification of Haemophilus influenzae and Streptococcus pneumoniae from upper respiratory tract secretions. J Clin Microbiol 1994 Nov;32(11):2871-2. [24] Russell FM, Biribo SS, Selvaraj G, Oppedisano F, Warren S, Seduadua A, et al. As a bacterial culture medium, citrated sheep blood agar is a practical alternative to citrated human blood agar in laboratories of developing countries. J Clin Microbiol 2006 Sep;44(9):3346-51. [25] Yeh E, Pinsky BA, Banaei N, Baron EJ. Hair sheep blood, citrated or defibrinated, fulfills all requirements of blood agar for diagnostic microbiology laboratory tests. PLoS One 2009;4(7):e6141. [26] Watson DA, Musher DM, Verhoef J. Pneumococcal virulence factors and host immune responses to them. Eur J Clin Microbiol Infect Dis 1995 Jun;14(6):479-90. [27] Nelson AL, Roche AM, Gould JM, Chim K, Ratner AJ, Weiser JN. Capsule enhances pneumococcal colonization by limiting mucus-mediated clearance. Infect Immun 2007 Jan;75(1):83-90. [28] MacLeod CM, Krauss MR. Relation of virulence of pneumococcal strains for mice to the quantity of capsular polysaccharide formed in vitro. J Exp Med 1950 Jul 1;92(1):1-9. [29] Brueggemann AB, Griffiths DT, Meats E, Peto T, Crook DW, Spratt BG. Clonal relationships between invasive and carriage Streptococcus pneumoniae and serotype- and clone-specific differences in invasive disease potential. J Infect Dis 2003 May 1;187(9):1424- 32. [30] Sleeman KL, Griffiths D, Shackley F, Diggle L, Gupta S, Maiden MC, et al. Capsular serotype-specific attack rates and duration of carriage of Streptococcus pneumoniae in a population of children. J Infect Dis 2006 Sep 1;194(5):682-8. [31] Sandgren A, Sjostrom K, Olsson-Liljequist B, Christensson B, Samuelsson A, Kronvall G, et al. Effect of clonal and serotype-specific properties on the invasive capacity of Streptococcus pneumoniae. J Infect Dis 2004 Mar 1;189(5):785-96. [32] Martens P, Worm SW, Lundgren B, Konradsen HB, Benfield T. Serotype-specific mortality from invasive Streptococcus pneumoniae disease revisited. BMC Infect Dis 2004 Jun 30;4:21. [33] Jansen AG, Rodenburg GD, de Greeff SC, Hak E, Veenhoven RH, Spanjaard L, et al. Invasive pneumococcal disease in the Netherlands: Syndromes, outcome and potential vaccine benefits. Vaccine 2009 Apr 14;27(17):2394-401.

221

[34] Harboe ZB, Thomsen RW, Riis A, Valentiner-Branth P, Christensen JJ, Lambertsen L, et al. Pneumococcal serotypes and mortality following invasive pneumococcal disease: a population-based cohort study. PLoS Med 2009 May 26;6(5):e1000081. [35] Ruckinger S, von Kries R, Siedler A, van der Linden M. Association of serotype of Streptococcus pneumoniae with risk of severe and fatal outcome. Pediatr Infect Dis J 2009 Feb;28(2):118-22. [36] Bender MH, Weiser JN. The atypical amino-terminal LPNTG-containing domain of the pneumococcal human IgA1-specific protease is required for proper enzyme localization and function. Mol Microbiol 2006 Jul;61(2): 526-43. [37] Sjostrom K, Spindler C, Ortqvist A, Kalin M, Sandgren A, Kuhlmann-Berenzon S, et al. Clonal and capsular types decide whether pneumococci will act as a primary or opportunistic pathogen. Clin Infect Dis 2006 Feb 15;42(4):451-9. [38] Jansen AG, Rodenburg GD, van der Ende A, van Alphen L, Veenhoven RH, Spanjaard L, et al. Invasive pneumococcal disease among adults: associations among serotypes, disease characteristics, and outcome. Clin Infect Dis 2009 Jul 15;49(2):e23-9. [39] Kelly T, Dillard JP, Yother J. Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae. Infect Immun 1994 May;62(5):1813-9. [40] Musher DM, Watson DA, Baughn RE. Does naturally acquired IgG antibody to cell wall polysaccharide protect human subjects against pneumococcal infection? J Infect Dis 1990 Apr;161(4):736-40. [41] O'Brien KL, Wolfson LJ, Watt JP, Henkle E, Deloria-Knoll M, McCall N, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet 2009 Sep 12;374(9693):893-902. [42] McIntosh ED, Fritzell B, Fletcher MA. Burden of paediatric invasive pneumococcal disease in Europe, 2005. Epidemiol Infect 2007 May;135(4):644-56. [43] Foster D, Knox K, Walker AS, Griffiths DT, Moore H, Haworth E, et al. Invasive pneumococcal disease: epidemiology in children and adults prior to implementation of the conjugate vaccine in the Oxfordshire region, England. J Med Microbiol 2008 Apr;57(Pt 4):480-7. [44] Hausdorff WP, Siber G, Paradiso PR. Geographical differences in invasive pneumococcal disease rates and serotype frequency in young children. Lancet 2001 Mar 24;357(9260):950-2. [45] Heffernan HM, Martin DR, Woodhouse RE, Morgan J, Blackmore TK. Invasive pneumococcal disease in New Zealand 1998-2005: capsular serotypes and antimicrobial resistance. Epidemiol Infect 2008 Mar;136(3):352-9. [46] Lagos R, Munoz A, Valenzuela MT, Heitmann I, Levine MM. Population-based surveillance for hospitalized and ambulatory pediatric invasive pneumococcal disease in Santiago, Chile. Pediatr Infect Dis J 2002 Dec;21(12):1115-23. [47] Scott JA. The preventable burden of pneumococcal disease in the developing world. Vaccine 2007 Mar 22;25(13):2398-405. [48] Levine OS, Cherian T, Hajjeh R, Knoll MD. Progress and future challenges in coordinated surveillance and detection of pneumococcal and Hib disease in developing countries. Clin Infect Dis 2009 Mar 1;48 Suppl 2:S33-6. [49] Anh DD, Kilgore PE, Slack MP, Nyambat B, Tho le H, Yoshida LM, et al. Surveillance of pneumococcal-associated disease among hospitalized children in Khanh Hoa Province, Vietnam. Clin Infect Dis 2009 Mar 1;48 Suppl 2:S57-64. [50] Baggett HC, Peruski LF, Olsen SJ, Thamthitiwat S, Rhodes J, Dejsirilert S, et al. Incidence of pneumococcal bacteremia requiring hospitalization in rural Thailand. Clin Infect Dis 2009 Mar 1;48 Suppl 2:S65-74. [51] Robinson KA, Baughman W, Rothrock G, Barrett NL, Pass M, Lexau C, et al. Epidemiology of invasive Streptococcus pneumoniae infections in the United States, 1995-

222

1998: Opportunities for prevention in the conjugate vaccine era. JAMA 2001 Apr 4;285(13):1729-35. [52] Whitney CG, Farley MM, Hadler J, Harrison LH, Bennett NM, Lynfield R, et al. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine. N Engl J Med 2003 May 1;348(18):1737-46. [53] Voss L, Lennon D, Okesene-Gafa K, Ameratunga S, Martin D. Invasive pneumococcal disease in a pediatric population, Auckland, New Zealand. Pediatr Infect Dis J 1994 Oct;13(10):873-8. [54] Roche PW, Krause VL, Bartlett M, Coleman D, Cook H, Davis C, et al. Invasive pneumococcal disease in Australia, 2004. Commun Dis Intell 2006;30(1):80-92. [55] Shouval DS, Greenberg D, Givon-Lavi N, Porat N, Dagan R. Serotype coverage of invasive and mucosal pneumococcal disease in Israeli children younger than 3 years by various pneumococcal conjugate vaccines. Pediatr Infect Dis J 2009 Apr;28(4):277-82. [56] Cortese MM, Wolff M, Almeido-Hill J, Reid R, Ketcham J, Santosham M. High incidence rates of invasive pneumococcal disease in the White Mountain Apache population. Arch Intern Med 1992 Nov;152(11):2277-82. [57] Park IH, Pritchard DG, Cartee R, Brandao A, Brandileone MC, Nahm MH. Discovery of a new capsular serotype (6C) within serogroup 6 of Streptococcus pneumoniae. J Clin Microbiol 2007 Apr;45(4):1225-33. [58] Jin P, Kong F, Xiao M, Oftadeh S, Zhou F, Liu C, et al. First Report of Putative Streptococcus pneumoniae Serotype 6D among Nasopharyngeal Isolates from Fijian Children. J Infect Dis 2009 Nov 1;200(9):1375-80. [59] Hausdorff WP, Bryant J, Kloek C, Paradiso PR, Siber GR. The contribution of specific pneumococcal serogroups to different disease manifestations: implications for conjugate vaccine formulation and use, part II. Clin Infect Dis 2000 Jan;30(1):122-40. [60] Fenoll A, Jado I, Vicioso D, Berron S, Yuste JE, Casal J. Streptococcus pneumoniae in children in Spain: 1990-1999. Acta Paediatr Suppl 2000 Dec;89(435):44-50. [61] GAVI's PneumoADIP, Department of International Health, Health. JHBSoP. Pneumococcal Regional Serotype Distribution for Pneumococcal AMC TPP; 2008. [62] Alexander MDHE, Craig MDHR, Shirley MDRG, Ellis C. Validity of etiological diagnosis of pneumonia in children by rapid typing from nasopharyngeal mucus The Journal of Pediatrics 1941 January;18(1):31-5. [63] Hausdorff WP, Feikin DR, Klugman KP. Epidemiological differences among pneumococcal serotypes. Lancet Infect Dis 2005 Feb;5(2):83-93. [64] Obaro SK, Monteil MA, Henderson DC. The pneumococcal problem. BMJ 1996;312:1521-5. [65] Soriano-Gabarró M, Schuchat A, Levine OS, Mulholland K, Feikin DR, Wenger J. Generic protocol to measure the burden of pneumococcal disease in children 0 to 23 months of age. [66] Tomashefski JF, Jr., Butler T, Islam M. Histopathology and etiology of childhood pneumonia: an autopsy study of 93 patients in Bangladesh. Pathology 1989;21(2):71-8. [67] Mulholland K. Magnitude of the problem of childhood pneumonia. Lancet 1999;354:590-2. [68] Cutts FT, Zaman SM, Enwere G, Jaffar S, Levine OS, Okoko JB, et al. Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial. Lancet 2005 Mar 26-Apr 1;365(9465):1139-46. [69] Bellamy C. The state of the world's children 2003. Geneva: UNICEF; 2002. [70] Levine MM, Lagos R, Levine OS, Heitmann I, Enriquez N, Pinto ME, et al. Epidemiology of invasive pneumococcal infections in infants and young children in

223

Metropolitan Santiago, Chile, a newly industrializing country. Pediatric Infectious Disease Journal 1998;17:287-93. [71] Vuori E, Peltola H, Kallio MJ, Leinonen M, Hedman K, Group S-TS. Etiology of pneumonia and other common childhood infections requiring hospitalisation and parenteral antimicrobial therapy. Clinical Infectious Diseases 1998;27:566-72. [72] Juven T, Mertsola J, Waris M, Leinonen M, Meurman O, Roivainen M, et al. Etiology of community-acquired pneumonia in 254 hospitalized children. Pediatric Infectious Disease Journal 2000;19(4):293-8. [73] Michaels RH, Poziviak CS. Countercurrent immunoelectrophoresis for the diagnosis of pneumococcal pneumonia in children. Journal of Pediatrics 1976;88(1):72-4. [74] Bonadio WA. Bacteremia in febrile children with lobar pneumonia and leukocytosis. Pediatric Emergency Care 1988;4(4):241-2. [75] Rusconi F, Rancilio L, Assael BM, Bonora G, Cerri M, Pietrogrande MC, et al. Counterimmunoelectrophoresis and latex particle agglutination in the etiologic diagnosis of presumed bacterial pneumonia in pediatric patients. Pediatric Infectious Disease Journal 1988;7(11):781-5. [76] Torzillo P, Dixon J, Manning K, Hutton S, Hueston L, Leinonen M, et al. Etiology of acute lower respiratory tract infections in Central Australian Aboriginal children. The Pediatric Infectious Disease Journal 1999;18(8):714-21. [77] Muhe L, Tilahun M, Lulseged S, Kebede S, Enaro D, Ringertz S, et al. Etiology of pneumonia, sepsis and meningitis in infants younger than three months of age in Ethiopia. Pediatric Infectious Disease Journal 1999;18(10 Suppl):S56-61. [78] Forgie IM, Campbell H, Lloyd-Evans N, Leinonen M, O'Neill KP, Saikku P, et al. Etiology of acute lower respiratory tract infections in children in a rural community in The Gambia. Pediatric Infectious Disease Journal 1992;11(6):466-73. [79] Ghafoor A, Nomani NK, Ishaq Z, Zaidi SZ, Anwar F, Burney MI, et al. Diagnosis of acute lower respiratory tract infections in children in Rawalpindi and Islamabad, Pakistan. Reviews of Infectious Diseases 1990;12(Supplement 8):S907-14. [80] Barker J, Gratten M, Riley I, Lehmann D, Montgomery J, Kajoi M, et al. Pneumonia in children in the eastern highlands of Papua New Guinea: a bacteriologic study of patients selected by standard clinical criteria. The Journal of Infectious Diseases 1989;159(2):348-52. [81] Sunakorn P, Chunchit L, Niltawat S, Wangweerawong M, Jacobs RF. Epidemiology of acute respiratory infections in young children from Thailand. Pediatric Infectious Disease Journal 1990;9(12):873-7. [82] Dagan R, Englehard D, Piccard E. Epidemiology of invasive childhood pneumococcal infections in Israel. Jama 1992;268(23):3328-32. [83] Magree HC, Russell FM, Sa'aga R, Greenwood P, Tikoduadua L, Pryor J, et al. Chest X-ray-confirmed pneumonia in children in Fiji. Bull World Health Organ 2005 Jun;83(6):427-33. [84] Cherian T, Mulholland EK, Carlin JB, Ostensen H, Amin R, de Campo M, et al. Standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies. Bull World Health Organ 2005 May;83(5):353-9. [85] Colquhoun S, Russell F, Carapetis J, Tikoduadua L, Pryor J, Waqatakirewa L, et al. Ethnic disparity in the burden of invasive pneumococcal disease in children aged less than 5 years in Fiji. 5th International Symposium on Pneumococci and Pneumococcal Disease; 2006 2-6 April, 2006; Alice Springs; 2006. [86] Colquhoun SM, Russell FM, Carapetis JR, Tikoduadua L, Pryor J, Mulholland E. A Cohort Study To Assess Quality Of Life In Young Fijian Children Who Have A History Of Bacterial Meningitis. 5th International Symposium on Pneumococci and Pneumococal Diseases; 2006 2-6 April, 2006; Alice Springs; 2006.

224

[87] Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001 Oct;2(10):947-50. [88] Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001 Aug;2(8):675-80. [89] Henderson B, Wilson M. Cytokine induction by bacteria: beyond lipopolysaccharide. Cytokine 1996 Apr;8(4):269-82. [90] Mond JJ, Lees A, Snapper CM. T cell-independent antigens type 2 Annual Review of Immunology 1995;13(1):655–92. [91] Mosier DE, J. J. Mond JJ, Goldings EA. The ontogeny of thymic independent antibody responses in vitro in normal mice and mice with an X-linked B cell defect. The Journal of Immunology 1977;119(6):1874–8. [92] Mond JJ, Vos Q, Lees A, Snapper CM. T cell independent antigens. Current Opinion in Immunology 1995;7(3):349–54. [93] Pabst R. The spleen in lymphocyte migration. Immunol Today 1988 Feb;9(2):43- 5. [94] Timens W, Boes A, Rozeboom-Uiterwijk T, Poppema S. Immaturity of the human splenic marginal zone in infancy. Possible contribution to the deficient infant immune response. The Journal of Immunology 1989;143(10):3200–6. [95] Zegers BJM, Van Der Giessen M, Reerink-Brongers EE, Stoop JW. The serum IgG subclass levels in healthy infants of 13–62 weeks of age. Clinica Chimica Acta 1980;101(2- 3):265–9. [96] Barrett DJ, Ayoub EM. IgG2 subclass restriction of antibody to pneumococcal polysaccharides. Clinical and Experimental Immunology 1986;63(1):127–34. [97] AlonsoDeVelasco E, Verheul AF, Verhoef J, Snippe H. Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines. Microbiol Rev 1995 Dec;59(4):591-603. [98] Malley R, Trzcinski K, Srivastava A, Thompson CM, Anderson PW, Lipsitch M. CD4+ T cells mediate antibody-independent acquired immunity to pneumococcal colonization. Proc Natl Acad Sci U S A 2005 Mar 29;102(13):4848-53. [99] McCool TL, Harding CV, Greenspan NS, Schreiber JR. B- and T-cell immune responses to pneumococcal conjugate vaccines: divergence between carrier- and polysaccharide-specific immunogenicity. Infect Immun 1999 Sep;67(9):4862-9. [100] Bogaert D, Thompson CM, Trzcinski K, Malley R, Lipsitch M. The role of complement in innate and adaptive immunity to pneumococcal colonization and sepsis in a murine model. Vaccine 2010 Jan 8;28(3):681-5. [101] Malley R. Antibody and cell-mediated immunity to Streptococcus pneumoniae: implications for vaccine development. J Mol Med 2010 Feb;88(2):135-42. [102] Jodar L, Butler J, Carlone G, Dagan R, Goldblatt D, Kayhty H, et al. Serological criteria for evaluation and licensure of new pneumococcal conjugate vaccine formulations for use in infants. Vaccine 2003 Jul 4;21(23):3265-72. [103] Black S, Shinefield H, Fireman B, Lewis E, Ray P, Hansen JR, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group. Pediatr Infect Dis J 2000 Mar;19(3):187-95. [104] Weinberger DM, Dagan R, Givon-Lavi N, Regev-Yochay G, Malley R, Lipsitch M. Epidemiologic evidence for serotype-specific acquired immunity to pneumococcal carriage. J Infect Dis 2008 Jun 1;197(11):1511-8. [105] Granat SM, Ollgren J, Herva E, Mia Z, Auranen K, Mäkelä PH. Epidemiological evidence for serotype-independent acquired immunity to pneumococcal carriage. J Infect Dis 2009 Jul 1;200(1):99-106.

225

[106] Malley R, Srivastava A, Lipsitch M, Thompson CM, Watkins C, Tzianabos A, et al. Antibody-independent, interleukin-17A-mediated, cross-serotype immunity to pneumococci in mice immunized intranasally with the cell wall polysaccharide. Infect Immun 2006 Apr;74(4):2187-95. [107] Basset A, Thompson CM, Hollingshead SK, Briles DE, Ades EW, Lipsitch M, et al. Antibody-independent, CD4+ T-cell-dependent protection against pneumococcal colonization elicited by intranasal immunization with purified pneumococcal proteins. Infect Immun 2007 Nov;75(11):5460-4. [108] Tangye SG, Good KL. Human IgM+CD27+ B cells: memory B cells or "memory" B cells? J Immunol 2007 Jul 1;179(1):13-9. [109] Moens L, Wuyts M, Meyts I, De Boeck K, Bossuyt X. Human memory B lymphocyte subsets fulfill distinct roles in the anti-polysaccharide and anti-protein immune response. J Immunol 2008 Oct 15;181(8):5306-12. [110] Harding CV, Roof RW, Allen PM, Unanue ER. Effects of pH and polysaccharides on peptide binding to class II major histocompatibility complex molecules. Proc Natl Acad Sci U S A 1991 Apr 1;88(7):2740-4. [111] Kadioglu A, Gingles NA, Grattan K, Kerr A, Mitchell TJ, Andrew PW. Host cellular immune response to pneumococcal lung infection in mice. Infect Immun 2000 Feb;68(2):492-501. [112] Zhang Q, Bagrade L, Bernatoniene J, Clarke E, Paton JC, Mitchell TJ, et al. Low CD4 T cell immunity to pneumolysin is associated with nasopharyngeal carriage of pneumococci in children. J Infect Dis 2007 Apr 15;195(8):1194-202. [113] Wright AE, Morgan W, Colbrook L, Dodgson R. Observations on prophylactic inoculation against pneumococcal infection and on the resultss which have been achieved by it. Lancet 1914;1:1-10,87-95. [114] Lister FS. Prophylactic inoculation of man against pneumococcal infections, and more particularly against lobar pneumonia. Publ S Afr Inst Med Res 1917;10:304-22. [115] Ekwurzel GM, Simmons JS, Dublin H, Felton LD. Studies on immunizing substances in pneumococci. VIII. Report on field test to determine the prophylactic value of a pneumocococcus antigen. Public Health Rep 1938;53:1877-93. [116] Felton LD. Studies on immunizing substances in pneumococci VII. Responses in human beings to antigenc pneumococcus polysaccharides, type I and II. Public Health Rep 1938;53:2855-77. [117] Austrian R, Gold J. Pneumococcal Bacteremia with Especial Reference to Bacteremic Pneumococcal Pneumonia. Ann Intern Med 1964 May;60:759-76. [118] Austrian R, Douglas RM, Schiffman G, Coetzee AM, Koornhof HJ, Hayden-Smith S, et al. Prevention of pneumococcal pneumonia by vaccination. Trans Assoc Am Physicians 1976;89:184-94. [119] Smit P, Oberholzer D, Hayden-Smith S, Koornhof HJ, Hilleman MR. Protective efficacy of pneumococcal polysaccharide vaccines. JAMA 1977 Dec 12;238(24):2613-6. [120] Feavers I, Knezevic I, Powell M, Griffiths E. Challenges in the evaluation and licensing of new pneumococcal vaccines, 7-8 July 2008, Ottawa, Canada. Vaccine 2009 Jun 8;27(28):3681-8. [121] WHO Pneumococcal Serology Reference Laboratories at the Institute of Child Health University College London England, USA TDoPatoAaBBA. Training manual for Enzyme linked immunosorbent assay for the quantitation of Streptococcus pneumoniae serotype specific IgG (Pn PS ELISA). [cited; Available from: [122] Coughlin RT, White AC, Anderson CA, Carlone GM, Klein DL, Treanor J. Characterization of pneumococcal specific antibodies in healthy unvaccinated adults. Vaccine 1998 Nov;16(18):1761-7.

226

[123] Concepcion NF, Frasch CE. Pneumococcal type 22f polysaccharide absorption improves the specificity of a pneumococcal-polysaccharide enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 2001 Mar;8(2):266-72. [124] Yu X, Sun Y, Frasch C, Concepcion N, Nahm MH. Pneumococcal capsular polysaccharide preparations may contain non-C-polysaccharide contaminants that are immunogenic. Clin Diagn Lab Immunol 1999 Jul;6(4):519-24. [125] Concepcion N, Frasch CE. Evaluation of previously assigned antibody concentrations in pneumococcal polysaccharide reference serum 89SF by the method of cross-standardization. Clin Diagn Lab Immunol 1998 Mar;5(2):199-204. [126] Romero-Steiner S, Frasch CE, Carlone G, Fleck RA, Goldblatt D, Nahm MH. Use of opsonophagocytosis for serological evaluation of pneumococcal vaccines. Clin Vaccine Immunol 2006 Feb;13(2):165-9. [127] Henckaerts I, Durant N, De Grave D, Schuerman L, Poolman J. Validation of a routine opsonophagocytosis assay to predict invasive pneumococcal disease efficacy of conjugate vaccine in children. Vaccine 2007 Mar 22;25(13):2518-27. [128] O'Brien KL, Moulton LH, Reid R, Weatherholtz R, Oski J, Brown L, et al. Efficacy and safety of seven-valent conjugate pneumococcal vaccine in American Indian children: group randomised trial. Lancet 2003 Aug 2;362(9381):355-61. [129] Klugman KP, Madhi SA, Huebner RE, Kohberger R, Mbelle N, Pierce N. A trial of a 9-valent pneumococcal conjugate vaccine in children with and those without HIV infection. N Engl J Med 2003 Oct 2;349(14):1341-8. [130] Siber GR, Chang I, Baker S, Fernsten P, O'Brien KL, Santosham M, et al. Estimating the protective concentration of anti-pneumococcal capsular polysaccharide antibodies. Vaccine 2007 May 10;25(19):3816-26. [131] Tuomanen EI, Austrian R, Masure HR. Pathogenesis of pneumococcal infection. N Engl J Med 1995 May 11;332(19):1280-4. [132] Usinger WR, Lucas AH. Avidity as a determinant of the protective efficacy of human antibodies to pneumococcal capsular polysaccharides. Infect Immun 1999 May;67(5):2366-70. [133] Anttila M, Voutilainen M, Jantti V, Eskola J, Kayhty H. Contribution of serotype- specific IgG concentration, IgG subclasses and relative antibody avidity to opsonophagocytic activity against Streptococcus pneumoniae. Clin Exp Immunol 1999 Dec;118(3):402-7. [134] Sun Y, Hwang Y, Nahm MH. Avidity, potency, and cross-reactivity of monoclonal antibodies to pneumococcal capsular polysaccharide serotype 6B. Infect Immun 2001 Jan;69(1):336-44. [135] Romero-Steiner S, Musher DM, Cetron MS, Pais LB, Groover JE, Fiore AE, et al. Reduction in functional antibody activity against Streptococcus pneumoniae in vaccinated elderly individuals highly correlates with decreased IgG antibody avidity. Clin Infect Dis 1999 Aug;29(2):281-8. [136] Romero-Steiner S, Frasch C, Concepcion N, Goldblatt D, Kayhty H, Vakevainen M, et al. Multilaboratory evaluation of a viability assay for measurement of opsonophagocytic antibodies specific to the capsular polysaccharides of Streptococcus pneumoniae. Clin Diagn Lab Immunol 2003 Nov;10(6):1019-24. [137] Bieging KT, Rajam G, Holder P, Udoff R, Carlone GM, Romero-Steiner S. Fluorescent multivalent opsonophagocytic assay for measurement of functional antibodies to Streptococcus pneumoniae. Clin Diagn Lab Immunol 2005 Oct;12(10):1238-42. [138] Martinez JE, Romero-Steiner S, Pilishvili T, Barnard S, Schinsky J, Goldblatt D, et al. A flow cytometric opsonophagocytic assay for measurement of functional antibodies elicited after vaccination with the 23-valent pneumococcal polysaccharide vaccine. Clin Diagn Lab Immunol 1999 Jul;6(4):581-6.

227

[139] Burton RL, Nahm MH. Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies. Clin Vaccine Immunol 2006 Sep;13(9):1004-9. [140] Bogaert D, Sluijter M, De Groot R, Hermans PW. Multiplex opsonophagocytosis assay (MOPA): a useful tool for the monitoring of the 7-valent pneumococcal conjugate vaccine. Vaccine 2004 Sep 28;22(29-30):4014-20. [141] Jokinen JT, Ahman H, Kilpi TM, Makela PH, Kayhty MH. Concentration of antipneumococcal antibodies as a serological correlate of protection: an application to acute otitis media. J Infect Dis 2004 Aug 1;190(3):545-50. [142] Vidarsson G, Sigurdardottir ST, Gudnason T, Kjartansson S, Kristinsson KG, Ingolfsdottir G, et al. Isotypes and opsonophagocytosis of pneumococcus type 6B antibodies elicited in infants and adults by an experimental pneumococcus type 6B-tetanus toxoid vaccine. Infect Immun 1998 Jun;66(6):2866-70. [143] Yu X, Gray B, Chang S, Ward JI, Edwards KM, Nahm MH. Immunity to cross- reactive serotypes induced by pneumococcal conjugate vaccines in infants. J Infect Dis 1999 Nov;180(5):1569-76. [144] Eskola J, Ward J, Dagan R, Goldblatt D, Zepp F, Siegrist CA. Combined vaccination of Haemophilus influenzae type b conjugate and diphtheria-tetanus-pertussis containing acellular pertussis. Lancet 1999 Dec 11;354(9195):2063-8. [145] Black S, Eskola J, Whitney CG, Shinefield H. Pneumococcal conjugate vaccine and pneumococcal common protein vaccines. In: Plotkin S, Orenstein W, Offit P, editors. Vaccines 5th Edition: Elsevier Inc., 2008: 531-67. [146] Makela PH, Sibakov M, Herva E, Henrichsen J, Luotonen J, Timonen M, et al. Pneumococcal vaccine and otitis media. Lancet 1980 Sep 13;2(8194):547-51. [147] Anttila M, Eskola J, Ahman H, Kayhty H. Differences in the avidity of antibodies evoked by four different pneumococcal conjugate vaccines in early childhood. Vaccine 1999 Apr 9;17(15-16):1970-7. [148] Lee LH, Frasch CE, Falk LA, Klein DL, Deal CD. Correlates of immunity for pneumococcal conjugate vaccines. Vaccine 2003 May 16;21(17-18):2190-6. [149] Schlesinger Y, Granoff DM. Avidity and bactericidal activity of antibody elicited by different Haemophilus influenzae type b conjugate vaccines. The Vaccine Study Group. JAMA 1992 Mar 18;267(11):1489-94. [150] Granoff DM, Lucas AH. Laboratory correlates of protection against Haemophilus influenzae type b disease. Importance of assessment of antibody avidity and immunologic memory. Ann N Y Acad Sci 1995 May 31;754:278-88. [151] Lucas AH, Granoff DM. Functional differences in idiotypically defined IgG1 anti- polysaccharide antibodies elicited by vaccination with Haemophilus influenzae type B polysaccharide-protein conjugates. J Immunol 1995 Apr 15;154(8):4195-202. [152] Goldblatt D, Vaz AR, Miller E. Antibody avidity as a surrogate marker of successful priming by Haemophilus influenzae type b conjugate vaccines following infant immunization. J Infect Dis 1998 Apr;177(4):1112-5. [153] Granoff DM, Anderson EL, Osterholm MT, Holmes SJ, McHugh JE, Belshe RB, et al. Differences in the immunogenicity of three Haemophilus influenzae type b conjugate vaccines in infants. J Pediatr 1992 Aug;121(2):187-94. [154] Anttila M, Eskola J, Ahman H, Kayhty H. Avidity of IgG for Streptococcus pneumoniae type 6B and 23F polysaccharides in infants primed with pneumococcal conjugates and boosted with polysaccharide or conjugate vaccines. J Infect Dis 1998 Jun;177(6):1614-21. [155] Wuorimaa TK, Dagan R, Bailleux F, Haikala R, Ekstrom N, Eskola J, et al. Functional activity of antibodies after immunization of Finnish and Israeli infants with an 11- valent pneumococcal conjugate vaccine. Vaccine 2005 Nov 16;23(46-47):5328-32.

228

[156] Ekstrom N, Ahman H, Verho J, Jokinen J, Vakevainen M, Kilpi T, et al. Kinetics and avidity of antibodies evoked by heptavalent pneumococcal conjugate vaccines PncCRM and PncOMPC in the Finnish Otitis Media Vaccine Trial. Infect Immun 2005 Jan;73(1):369-77. [157] Goldblatt D, Southern J, Ashton L, Richmond P, Burbidge P, Tasevska J, et al. Immunogenicity and boosting after a reduced number of doses of a pneumococcal conjugate vaccine in infants and toddlers. Pediatr Infect Dis J 2006 Apr;25(4):312-9. [158] Siber GR. Pneumococcal disease: prospects for a new generation of vaccines. Science 1994 Sep 2;265(5177):1385-7. [159] Falade AG, Lagunju IA, Bakare RA, Odekanmi AA, Adegbola RA. Invasive pneumococcal disease in children aged <5 years admitted to 3 urban hospitals in Ibadan, Nigeria. Clin Infect Dis 2009 Mar 1;48 Suppl 2:S190-6. [160] Center KJ, Strauss A. Safety experience with heptavalent pneumococcal CRM197-conjugate vaccine (Prevenar) since vaccine introduction. Vaccine 2009 May 26;27(25-26):3281-4. [161] Eskola J, Kilpi T, Palmu A, Jokinen J, Haapakoski J, Herva E, et al. Efficacy of a pneumococcal conjugate vaccine against acute otitis media. N Engl J Med 2001 Feb 8;344(6):403-9. [162] Knuf M, Habermehl P, Cimino C, Petersen G, Schmitt HJ. Immunogenicity, reactogenicity and safety of a 7-valent pneumococcal conjugate vaccine (PCV7) concurrently administered with a DTPa-HBV-IPV/Hib combination vaccine in healthy infants. Vaccine 2006 May 29;24(22):4727-36. [163] Scheifele DW, Halperin SA, Smith B, Ochnio J, Meloff K, Duarte-Monteiro D. Assessment of the compatibility of co-administered 7-valent pneumococcal conjugate, DTaP.IPV/PRP-T Hib and hepatitis B vaccines in infants 2-7 months of age. Vaccine 2006 Mar 15;24(12):2057-64. [164] Schmitt HJ, Faber J, Lorenz I, Schmole-Thoma B, Ahlers N. The safety, reactogenicity and immunogenicity of a 7-valent pneumococcal conjugate vaccine (7VPnC) concurrently administered with a combination DTaP-IPV-Hib vaccine. Vaccine 2003 Sep 8;21(25-26):3653-62. [165] Tichmann-Schumann I, Soemantri P, Behre U, Disselhoff J, Mahler H, Maechler G, et al. Immunogenicity and reactogenicity of four doses of diphtheria-tetanus-three- component acellular pertussis-hepatitis B-inactivated polio virus-Haemophilus influenzae type b vaccine coadministered with 7-valent pneumococcal conjugate Vaccine. Pediatr Infect Dis J 2005 Jan;24(1):70-7. [166] Wise RP, Iskander J, Pratt RD, Campbell S, Ball R, Pless RP, et al. Postlicensure safety surveillance for 7-valent pneumococcal conjugate vaccine. JAMA 2004 Oct 13;292(14):1702-10. [167] Black S, France E, Center K, Hansen J, E. L, Graepel J, et al. Postmarketing assessment of uncommon events following Prevnar, 7-valent pneumococcal conjugate vaccine. IDSA 44th annual meeting October, 2006; Toronto, Canada. [168] Black S, France E, Center K, Hansen J, E. L, Graepel J, et al. Kawasaki Disease (KD) following immunization: Case series from a large safety surveillance study of Prevnar, 7- valent pneumococcal conjugate vaccine. IDAS 44th annual meeting October, 2006; Toronto, Canada. [169] Center KJ, Hansen JR, Lewis E, Fireman BH, Hilton B. Lack of association of Kawasaki disease after immunization in a cohort of infants followed for multiple autoimmune diagnoses in a large, phase-4 observational database safety study of 7-valent pneumococcal conjugate vaccine: lack of association between Kawasaki disease and seven- valent pneumococcal conjugate vaccine. Pediatr Infect Dis J 2009 May;28(5):438-40.

229

[170] Kayhty H, Ahman H, Eriksson K, Sorberg M, Nilsson L. Immunogenicity and tolerability of a heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 12 months of age. Pediatr Infect Dis J 2005 Feb;24(2):108-14. [171] Sigurdardottir ST, Ingolfsdottir G, Davidsdottir K, Gudnason T, Kjartansson S, Kristinsson KG, et al. Immune response to octavalent diphtheria- and tetanus-conjugated pneumococcal vaccines is serotype- and carrier-specific: the choice for a mixed carrier vaccine. Pediatr Infect Dis J 2002 Jun;21(6):548-54. [172] Choo S, Seymour L, Morris R, Quataert S, Lockhart S, Cartwright K, et al. Immunogenicity and reactogenicity of a pneumococcal conjugate vaccine administered combined with a haemophilus influenzae type B conjugate vaccine in United Kingdom infants. Pediatr Infect Dis J 2000 Sep;19(9):854-62. [173] Shinefield HR, Black S, Ray P, Chang I, Lewis N, Fireman B, et al. Safety and immunogenicity of heptavalent pneumococcal CRM197 conjugate vaccine in infants and toddlers. Pediatr Infect Dis J 1999 Sep;18(9):757-63. [174] Puumalainen T, Dagan R, Wuorimaa T, Zeta-Capeding R, Lucero M, Ollgren J, et al. Greater antibody responses to an eleven valent mixed carrier diphtheria- or tetanus- conjugated pneumococcal vaccine in Filipino than in Finnish or Israeli infants. Pediatr Infect Dis J 2003 Feb;22(2):141-9. [175] Stein KE. Thymus-independent and thymus-dependent responses to polysaccharide antigens. J Infect Dis 1992 Jun;165 Suppl 1:S49-52. [176] Steinhoff MC, Edwards K, Keyserling H, Thoms ML, Johnson C, Madore D, et al. A randomized comparison of three bivalent Streptococcus pneumoniae glycoprotein conjugate vaccines in young children: effect of polysaccharide size and linkage characteristics. Pediatr Infect Dis J 1994 May;13(5):368-72. [177] Osendarp SJ, Prabhakar H, Fuchs GJ, van Raaij JM, Mahmud H, Tofail F, et al. Immunization with the heptavalent pneumococcal conjugate vaccine in Bangladeshi infants and effects of zinc supplementation. Vaccine 2007 Apr 30;25(17):3347-54. [178] Ahman H, Kayhty H, Vuorela A, Leroy O, Eskola J. Dose dependency of antibody response in infants and children to pneumococcal polysaccharides conjugated to tetanus toxoid. Vaccine 1999 Jun 4;17(20-21):2726-32. [179] Obaro SK, Adegbola RA, Chang I, Banya WA, Jaffar S, McAdam KW, et al. Safety and immunogenicity of a nonavalent pneumococcal vaccine conjugated to CRM197 administered simultaneously but in a separate syringe with diphtheria, tetanus and pertussis vaccines in Gambian infants. Pediatr Infect Dis J 2000 May;19(5):463-9. [180] Kayhty H, Ahman H, Ronnberg PR, Tillikainen R, Eskola J. Pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine is immunogenic in infants and children. J Infect Dis 1995 Nov;172(5):1273-8. [181] Dagan R, Igbaria K, Piglansky L, Melamed R, Willems P, Grossi A, et al. Safety and immunogenicity of a combined pentavalent diphtheria, tetanus, acellular pertussis, inactivated poliovirus and Haemophilus influenzae type b-tetanus conjugate vaccine in infants, compared with a whole cell pertussis pentavalent vaccine. Pediatr Infect Dis J 1997 Dec;16(12):1113-21. [182] Rennels MB, Edwards KM, Keyserling HL, Reisinger KS, Hogerman DA, Madore DV, et al. Safety and immunogenicity of heptavalent pneumococcal vaccine conjugated to CRM197 in United States infants. Pediatrics 1998 Apr;101(4 Pt 1):604-11. [183] Anderson EL, Kennedy DJ, Geldmacher KM, Donnelly J, Mendelman PM. Immunogenicity of heptavalent pneumococcal conjugate vaccine in infants. J Pediatr 1996 May;128(5 Pt 1):649-53. [184] Miernyk KM, Parkinson AJ, Rudolph KM, Petersen KM, Bulkow LR, Greenberg DP, et al. Immunogenicity of a heptavalent pneumococcal conjugate vaccine in Apache and

230

Navajo Indian, Alaska native, and non-native American children aged <2 years. Clin Infect Dis 2000 Jul;31(1):34-41. [185] Ahman H, Kayhty H, Lehtonen H, Leroy O, Froeschle J, Eskola J. Streptococcus pneumoniae capsular polysaccharide-diphtheria toxoid conjugate vaccine is immunogenic in early infancy and able to induce immunologic memory. Pediatr Infect Dis J 1998 Mar;17(3):211-6. [186] Blum MD, Dagan R, Mendelman PM, Pinsk V, Giordani M, Li S, et al. A comparison of multiple regimens of pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine and pneumococcal polysaccharide vaccine in toddlers. Vaccine 2000 May 8;18(22):2359-67. [187] Huebner RE, Mbelle N, Forrest B, Madore DV, Klugman KP. Long-term antibody levels and booster responses in South African children immunized with nonavalent pneumococcal conjugate vaccine. Vaccine 2004 Jul 29;22(21-22):2696-700. [188] Kilpi T, Ahman H, Jokinen J, Lankinen KS, Palmu A, Savolainen H, et al. Protective efficacy of a second pneumococcal conjugate vaccine against pneumococcal acute otitis media in infants and children: randomized, controlled trial of a 7-valent pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine in 1666 children. Clin Infect Dis 2003 Nov 1;37(9):1155-64. [189] O'Brien KL, Hochman M, Goldblatt D. Combined schedules of pneumococcal conjugate and polysaccharide vaccines: is hyporesponsiveness an issue? Lancet Infect Dis 2007 Sep;7(9):597-606. [190] Goldblatt D, Akoto A, Ashton L, et a. Immunogenicity and the generation of immune memory following 9-valent pneumococcal conjugate vaccination in Ghanaian infants with sickle cell disease. 40th Interscience Conference on Antimicrobial Agents and Chemotherapy; 2007 Sept 24–27; Toronto, Ontario, Canada; 2007. [191] Eskola J. Immunogenicity of pneumococcal conjugate vaccines. Pediatr Infect Dis J 2000 Apr;19(4):388-93. [192] Nurkka A, Ahman H, Korkeila M, Jantti V, Kayhty H, Eskola J. Serum and salivary anti-capsular antibodies in infants and children immunized with the heptavalent pneumococcal conjugate vaccine. Pediatr Infect Dis J 2001 Jan;20(1):25-33. [193] Reinert P, Guy M, Girier B, Szelechowski B, Baudoin B, Deberdt P, et al. [The safety and immunogenicity of an heptavalent pneumococcal polysaccharide conjugate vaccine (Prevenar) administered in association with a whole-cell pertussis-based pediatric combination vaccine (DTP-IPV/PRP-T) to French infants with a two-, three-, and four-month schedule]. Arch Pediatr 2003 Dec;10(12):1048-55. [194] Sigurdardottir ST, Davidsdottir K, Arason VA, Jonsdottir O, Laudat F, Gruber WC, et al. Safety and immunogenicity of CRM197-conjugated pneumococcal-meningococcal C combination vaccine (9vPnC-MnCC) whether given in two or three primary doses. Vaccine 2008 Aug 5;26(33):4178-86. [195] Buttery JP, Riddell A, McVernon J, Chantler T, Lane L, Bowen-Morris J, et al. Immunogenicity and safety of a combination pneumococcal-meningococcal vaccine in infants: a randomized controlled trial. JAMA 2005 Apr 13;293(14):1751-8. [196] Pichichero ME, Bernstein H, Blatter MM, Schuerman L, Cheuvart B, Holmes SJ. Immunogenicity and safety of a combination diphtheria, tetanus toxoid, acellular pertussis, hepatitis B, and inactivated poliovirus vaccine coadministered with a 7-valent pneumococcal conjugate vaccine and a Haemophilus influenzae type b conjugate vaccine. J Pediatr 2007 Jul;151(1):43-9, 9 e1-2. [197] O'Brien KL, Moisi J, Moulton LH, Madore D, Eick A, Reid R, et al. Predictors of pneumococcal conjugate vaccine immunogenicity among infants and toddlers in an American Indian PnCRM7 efficacy trial. J Infect Dis 2007 Jul 1;196(1):104-14.

231

[198] Huebner RE, Mbelle N, Forrest B, Madore DV, Klugman KP. Immunogenicity after one, two or three doses and impact on the antibody response to coadministered antigens of a nonavalent pneumococcal conjugate vaccine in infants of Soweto, South Africa. Pediatr Infect Dis J 2002 Nov;21(11):1004-7. [199] Madhi SA, Kuwanda L, Cutland C, Holm A, Kayhty H, Klugman KP. Quantitative and qualitative antibody response to pneumococcal conjugate vaccine among African human immunodeficiency virus-infected and uninfected children. Pediatr Infect Dis J 2005 May;24(5):410-6. [200] Obaro SK, Enwere GC, Deloria M, Jaffar S, Goldblatt D, Brainsby K, et al. Safety and immunogenicity of pneumococcal conjugate vaccine in combination with diphtheria, tetanus toxoid, pertussis and Haemophilus influenzae type b conjugate vaccine. Pediatr Infect Dis J 2002 Oct;21(10):940-7. [201] Leach A, Ceesay SJ, Banya WA, Greenwood BM. Pilot trial of a pentavalent pneumococcal polysaccharide/protein conjugate vaccine in Gambian infants. Pediatr Infect Dis J 1996 Apr;15(4):333-9. [202] Saaka M, Okoko BJ, Kohberger RC, Jaffar S, Enwere G, Biney EE, et al. Immunogenicity and serotype-specific efficacy of a 9-valent pneumococcal conjugate vaccine (PCV-9) determined during an efficacy trial in The Gambia. Vaccine 2008 Jul 4;26(29- 30):3719-26. [203] Madhi SA, Klugman KP. World Health Organisation definition of "radiologically- confirmed pneumonia" may under-estimate the true public health value of conjugate pneumococcal vaccines. Vaccine 2007 Mar 22;25(13):2413-9. [204] Madhi SA, Whitney CG, Nohynek H. Lessons learned from clinical trials evaluating pneumococcal conjugate vaccine efficacy against pneumonia and invasive disease. Vaccine 2008 Jun 16;26 Suppl 2:B9-B15. [205] Hansen J, Black S, Shinefield H, Cherian T, Benson J, Fireman B, et al. Effectiveness of heptavalent pneumococcal conjugate vaccine in children younger than 5 years of age for prevention of pneumonia: updated analysis using World Health Organization standardized interpretation of chest radiographs. Pediatr Infect Dis J 2006 Sep;25(9):779-81. [206] Lucero MG, Nohynek H, Williams G, Tallo V, Simoes EA, Lupisan S, et al. Efficacy of an 11-valent pneumococcal conjugate vaccine against radiologically confirmed pneumonia among children less than 2 years of age in the Philippines: a randomized, double- blind, placebo-controlled trial. Pediatr Infect Dis J 2009 Jun;28(6):455-62. [207] Straetemans M, Sanders EA, Veenhoven RH, Schilder AG, Damoiseaux RA, Zielhuis GA. Pneumococcal vaccines for preventing otitis media. Cochrane Database Syst Rev 2004(1):CD001480. [208] Palmu AA, Verho J, Jokinen J, Karma P, Kilpi TM. The seven-valent pneumococcal conjugate vaccine reduces tympanostomy tube placement in children. Pediatr Infect Dis J 2004 Aug;23(8):732-8. [209] Fireman B, Black SB, Shinefield HR, Lee J, Lewis E, Ray P. Impact of the pneumococcal conjugate vaccine on otitis media. Pediatr Infect Dis J 2003 Jan;22(1):10-6. [210] Prymula R, Peeters P, Chrobok V, Kriz P, Novakova E, Kaliskova E, et al. Pneumococcal capsular polysaccharides conjugated to protein D for prevention of acute otitis media caused by both Streptococcus pneumoniae and non-typable Haemophilus influenzae: a randomised double-blind efficacy study. Lancet 2006 Mar 4;367(9512):740-8. [211] Aristegui J, Bernaola E, Pocheville I, Garcia C, Arranz L, Duran G, et al. Reduction in pediatric invasive pneumococcal disease in the Basque Country and Navarre, Spain, after introduction of the heptavalent pneumococcal conjugate vaccine. Eur J Clin Microbiol Infect Dis 2007 May;26(5):303-10. [212] Roche PW, Krause V, Cook H, Barralet J, Coleman D, Sweeny A, et al. Invasive pneumococcal disease in Australia, 2006. Commun Dis Intell 2008 Mar;32(1):18-30.

232

[213] Ruckinger S, van der Linden M, Reinert RR, von Kries R, Burckhardt F, Siedler A. Reduction in the incidence of invasive pneumococcal disease after general vaccination with 7-valent pneumococcal conjugate vaccine in Germany. Vaccine 2009 Jun 24;27(31):4136-41. [214] Dias R, Canica M. Invasive pneumococcal disease in Portugal prior to and after the introduction of pneumococcal heptavalent conjugate vaccine. FEMS Immunol Med Microbiol 2007 Oct;51(1):35-42. [215] Vestrheim DF, Lovoll O, Aaberge IS, Caugant DA, Hoiby EA, Bakke H, et al. Effectiveness of a 2+1 dose schedule pneumococcal conjugate vaccination programme on invasive pneumococcal disease among children in Norway. Vaccine 2008 Jun 19;26(26):3277-81. [216] Harboe Z, Valentiner-Branth P, Benfield T, Christensen J, Andersen P, Howitz M, et al. Early effectiveness of heptavalent conjugate pneumococcal vaccination on invasive pneumococcal disease after the introduction in the Danish Childhood Immunization Programme. Vaccine 2010 Mar 19;28(14):2642-7. [217] Lacapa R, Bliss SJ, Larzelere-Hinton F, Eagle KJ, McGinty DJ, Parkinson AJ, et al. Changing epidemiology of invasive pneumococcal disease among White Mountain Apache persons in the era of the pneumococcal conjugate vaccine. Clin Infect Dis 2008 Aug 15;47(4):476-84. [218] Tsai CJ, Griffin MR, Nuorti JP, Grijalva CG. Changing epidemiology of pneumococcal meningitis after the introduction of pneumococcal conjugate vaccine in the United States. Clin Infect Dis 2008 Jun 1;46(11):1664-72. [219] Park S, Van Beneden C, Pilishvili T, Martin M, Facklam R, Whitney C, et al. Invasive pneumococcal infections among vaccinated children in the United States. J Pediatr 2010 Mar;156(3):478-83. [220] Moore MR, Gertz RE, Jr., Woodbury RL, Barkocy-Gallagher GA, Schaffner W, Lexau C, et al. Population snapshot of emergent Streptococcus pneumoniae serotype 19A in the United States, 2005. J Infect Dis 2008 Apr 1;197(7):1016-27. [221] Pilishvili T, Lexau C, Farley M, Hadler J, Harrison L, Bennett N, et al. Sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine. J Infect Dis 2010 Jan 1;201(1):32-41. [222] Hicks LA, Harrison LH, Flannery B, Hadler JL, Schaffner W, Craig AS, et al. Incidence of pneumococcal disease due to non-pneumococcal conjugate vaccine (PCV7) serotypes in the United States during the era of widespread PCV7 vaccination, 1998-2004. J Infect Dis 2007 Nov 1;196(9):1346-54. [223] Hsu K, Shea K, Stevenson A, Pelton S, Health. MDoP. Changing serotypes causing childhood invasive pneumococcal disease: Massachusetts, 2001-2007. Pediatr Infect Dis J 2010 Apr;29(4):289-93. [224] Techasaensiri C, Messina A, Katz K, Ahmad N, Huang R, McCracken GJ. Epidemiology and evolution of invasive pneumococcal disease caused by multidrug resistant serotypes of 19A in the 8 years after implementation of pneumococcal conjugate vaccine immunization in Dallas, Texas. Pediatr Infect Dis J 2010 Apr;29(4):294-300. [225] Gonzalez BE, Hulten KG, Lamberth L, Kaplan SL, Mason EO, Jr. Streptococcus pneumoniae serogroups 15 and 33: an increasing cause of pneumococcal infections in children in the United States after the introduction of the pneumococcal 7-valent conjugate vaccine. Pediatr Infect Dis J 2006 Apr;25(4):301-5. [226] Albrich WC, Baughman W, Schmotzer B, Farley MM. Changing characteristics of invasive pneumococcal disease in Metropolitan Atlanta, Georgia, after introduction of a 7- valent pneumococcal conjugate vaccine. Clin Infect Dis 2007 Jun 15;44(12):1569-76. [227] Flannery B, Heffernan RT, Harrison LH, Ray SM, Reingold AL, Hadler J, et al. Changes in invasive Pneumococcal disease among HIV-infected adults living in the era of childhood pneumococcal immunization. Ann Intern Med 2006 Jan 3;144(1):1-9.

233

[228] Hsu HE, Shutt KA, Moore MR, Beall BW, Bennett NM, Craig AS, et al. Effect of pneumococcal conjugate vaccine on pneumococcal meningitis. N Engl J Med 2009 Jan 15;360(3):244-56. [229] Pichon B, Moyce L, Efstratiou A, Slack M, George R. Serotype replacement following the introduction of the PCV7 in the UK - Impact on the characteristics of pneumococci causing meningitis. Meningitis and Septicaemia in Children and Adults, Meningitis Research Foundation. London 2009. [230] Grijalva CG, Nuorti JP, Arbogast PG, Martin SW, Edwards KM, Griffin MR. Decline in pneumonia admissions after routine childhood immunisation with pneumococcal conjugate vaccine in the USA: a time-series analysis. Lancet 2007 Apr 7;369(9568):1179-86. [231] Grijalva CG. Recognising pneumonia burden through prevention. Vaccine 2009 Aug 21;27 Suppl 3:C6-8. [232] Nelson JC, Jackson M, Yu O, Whitney CG, Bounds L, Bittner R, et al. Impact of the introduction of pneumococcal conjugate vaccine on rates of community acquired pneumonia in children and adults. Vaccine 2008 Sep 8;26(38):4947-54. [233] Grijalva CG, Poehling KA, Nuorti JP, Zhu Y, Martin SW, Edwards KM, et al. National impact of universal childhood immunization with pneumococcal conjugate vaccine on outpatient medical care visits in the United States. Pediatrics 2006 Sep;118(3):865-73. [234] Jardine A, Menzies RI, McIntyre PB. Reduction in hospitalizations for pneumonia associated with the introduction of a pneumococcal conjugate vaccination schedule without a booster dose in Australia. Pediatr Infect Dis J 2010 Jul;29(7). [235] Li S, Tancredi D. Empyema hospitalizations increased in US children despite pneumococcal conjugate vaccine. Pediatrics 2010 Jan;125(1):26-33. [236] Grijalva C, Nuorti J, Zhu Y, Griffin M. Increasing incidence of empyema complicating childhood community-acquired pneumonia in the United States. Clin Infect Dis 2010 Mar 15;50(6):805-13. [237] Poehling KA, Szilagyi PG, Grijalva CG, Martin SW, LaFleur B, Mitchel E, et al. Reduction of frequent otitis media and pressure-equalizing tube insertions in children after introduction of pneumococcal conjugate vaccine. Pediatrics 2007 Apr;119(4):707-15. [238] Jardine A, Menzies RI, Deeks SL, Patel MS, McIntyre PB. The Impact of Pneumococcal Conjugate Vaccine on Rates of Myringotomy With Ventilation Tube Insertion in Australia. Pediatr Infect Dis J 2009 Sep;28(9):761-5. [239] Mackenzie GA, Carapetis JR, Leach AJ, Morris PS. Pneumococcal vaccination and otitis media in Australian Aboriginal infants: comparison of two birth cohorts before and after introduction of vaccination. BMC Pediatr 2009;9:14. [240] Casey J, Adlowitz D, Pichichero M. New patterns in the otopathogens causing acute otitis media six to eight years after introduction of pneumococcal conjugate vaccine. Pediatr Infect Dis J 2010 Apr;29(4):304-9. [241] Lucero MG, Puumalainen T, Ugpo JM, Williams G, Kayhty H, Nohynek H. Similar antibody concentrations in Filipino infants at age 9 months, after 1 or 3 doses of an adjuvanted, 11-valent pneumococcal diphtheria/tetanus-conjugated vaccine: a randomized controlled trial. J Infect Dis 2004 Jun 1;189(11):2077-84. [242] Whitney CG, Pilishvili T, Farley MM, Schaffner W, Craig AS, Lynfield R, et al. Effectiveness of seven-valent pneumococcal conjugate vaccine against invasive pneumococcal disease: a matched case-control study. Lancet 2006 Oct 28;368(9546):1495- 502. [243] Barzilay EJ, O'Brien KL, Kwok YS, Hoekstra RM, Zell ER, Reid R, et al. Could a single dose of pneumococcal conjugate vaccine in children be effective? Modeling the optimal age of vaccination. Vaccine 2006 Feb 13;24(7):904-13.

234

[244] Haber M, Barskey A, Baughman W, Barker L, Whitney CG, Shaw KM, et al. Herd immunity and pneumococcal conjugate vaccine: a quantitative model. Vaccine 2007 Jul 20;25(29):5390-8. [245] Esposito S, Pugni L, Bosis S, Proto A, Cesati L, Bianchi C, et al. Immunogenicity, safety and tolerability of heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 11 months post-natally to pre- and full-term infants. Vaccine 2005 Feb 25;23(14):1703- 8. [246] Dagan R, Givon-Lavi N, Chimney S, Jancu J, Greenberg D. Immunogenicity of CRM-Conjugated 7-Valent Pneumococcal Vaccine (PCV7) Administered at a Licensed 3-dose primary Schedule (3+1) Compared to a reduced 2-Dose Primary Schedule (2+1)- A Randomized Study. I 45th meeting of IDSA; 2007 October 4-7; San Diego 2007. [247] Givon-Lavi N, Greenberg D, Dagan R. Immunogenicity of alternative regimens of the conjugated 7-valent pneumococcal vaccine a randomized controlled trial. Pediatr Infect Dis J 2010 Aug;29(8). [248] Rodenburg G, van Gils E, Veenhoven R, Jones N, Tcherniaeva I, Hak E, et al. Comparability of antibody response to a booster dose of 7-valent pneumococcal conjugate vaccine in infants primed with either 2 or 3 doses. Vaccine 2010 Feb 3;28(5):1391-6. [249] Silfverdal S, Hogh B, Bergsaker M, Skerlikova H, Lommel P, Borys D, et al. Immunogenicity of a 2-dose priming and booster vaccination with the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine. Pediatr Infect Dis J 2009 Oct;28(10):e276-82. [250] Durando P, Crovari P, Ansaldi F, Sticchi L, Sticchi C, Turello V, et al. Universal childhood immunisation against Streptococcus pneumoniae: the five-year experience of Liguria Region, Italy. Vaccine 2009 May 26;27(25-26):3459-62. [251] Health Protection Agency. http://wwwhpaorguk/Topics/InfectiousDiseases/InfectionsAZ/Pneumococcal/Epidemiologic alDataPneumococcal/ 2010 [cited 21 Apr]; Available from: [252] Goldblatt D. Immunology and Impact of Reduced and Alternative Schedules of Pneumococcal Conjugate Vaccination. 6th International Symposium of Pneumococci and Pneumococal Diseases; 2008 8-12 June 2008; Reykjavik, Iceland.; 2008. [253] Gutierrez-Brito M, Girgenti D, Giardina P, Sarkozy D, Gruber WC, Emini E, et al. Immunogenicity, safety, and tolerabililty, of 13-valent pneumococcal conjugate vaccine given to infants with routine pediatric vaccinations in Mexico: interim report 7th International Symposium on Pneumococci and Pneumococcal Diseases. Tel Aviv, Israel, 2010. [254] 23-valent pneumococcal polysaccharide vaccine. WHO position paper. Wkly Epidemiol Rec 2008 Oct 17;83(42):373-84. [255] Lee HJ, Kang JH, Henrichsen J, Konradsen HB, Jang SH, Shin HY, et al. Immunogenicity and safety of a 23-valent pneumococcal polysaccharide vaccine in healthy children and in children at increased risk of pneumococcal infection. Vaccine 1995 Nov;13(16):1533-8. [256] King JC, Jr., Vink PE, Farley JJ, Parks M, Smilie M, Madore D, et al. Comparison of the safety and immunogenicity of a pneumococcal conjugate with a licensed polysaccharide vaccine in human immunodeficiency virus and non-human immunodeficiency virus-infected children. Pediatr Infect Dis J 1996 Mar;15(3):192-6. [257] Lawrence EM, Edwards KM, Schiffman G, Thompson JM, Vaughn WK, Wright PF. Pneumococcal vaccine in normal children. Primary and secondary vaccination. Am J Dis Child 1983 Sep;137(9):846-50. [258] Nelson K, Goldman JA, Perlino CA. Severe local reactions to pneumococcal vaccine. South Med J 1980 Feb;73(2):264-5.

235

[259] Kaplan J, Sarnaik S, Schiffman G. Revaccination with polyvalent pneumococcal vaccine in children with sickle cell anemia. Am J Pediatr Hematol Oncol 1986 Spring;8(1):80- 2. [260] Torling J, Hedlund J, Konradsen HB, Ortqvist A. Revaccination with the 23-valent pneumococcal polysaccharide vaccine in middle-aged and elderly persons previously treated for pneumonia. Vaccine 2003 Dec 8;22(1):96-103. [261] Davidson M, Bulkow LR, Grabman J, Parkinson AJ, Chamblee C, Williams WW, et al. Immunogenicity of pneumococcal revaccination in patients with chronic disease. Arch Intern Med 1994 Oct 10;154(19):2209-14. [262] Mufson MA, Hughey DF, Turner CE, Schiffman G. Revaccination with pneumococcal vaccine of elderly persons 6 years after primary vaccination. Vaccine 1991 Jun;9(6):403-7. [263] Temple K, Greenwood B, Inskip H, Hall A, Koskela M, Leinonen M. Antibody response to pneumococcal capsular polysaccharide vaccine in African children. Pediatr Infect Dis J 1991 May;10(5):386-90. [264] Douglas RM, Paton JC, Duncan SJ, Hansman DJ. Antibody response to pneumococcal vaccination in children younger than five years of age. J Infect Dis 1983 Jul;148(1):131-7. [265] Koskela M, Leinonen M, Haiva VM, Timonen M, Makela PH. First and second dose antibody responses to pneumococcal polysaccharide vaccine in infants. Pediatr Infect Dis 1986 Jan-Feb;5(1):45-50. [266] Borgono JM, McLean AA, Vella PP, Woodhour AF, Canepa I, Davidson WL, et al. Vaccination and revaccination with polyvalent pneumococcal polysaccharide vaccines in adults and infants. Proc Soc Exp Biol Med 1978 Jan;157(1):148-54. [267] Sloyer JL, Jr., Ploussard JH, Howie VM. Efficacy of pneumococcal polysaccharide vaccine in preventing acute otitis media in infants in Huntsville, Alabama. Rev Infect Dis 1981 Mar-Apr;3 Suppl:S119-23. [268] Leinonen M, Sakkinen A, Kalliokoski R, Luotonen J, Timonen M, Makela PH. Antibody response to 14-valent pneumococcal capsular polysaccharide vaccine in pre-school age children. Pediatr Infect Dis 1986 Jan-Feb;5(1):39-44. [269] Jackson L, Neuzil K. Pneumococcal Polysaccharide Vaccines. In: Plotkin S, Orenstein W, Offit P, editors. Vaccines 5th edition: Elsevier, 2008. [270] Borrow R, Joseph H, Andrews N, Acuna M, Longworth E, Martin S, et al. Reduced antibody response to revaccination with meningococcal serogroup A polysaccharide vaccine in adults. Vaccine 2000 Dec 8;19(9-10):1129-32. [271] MacLennan J, Obaro S, Deeks J, Lake D, Elie C, Carlone G, et al. Immunologic memory 5 years after meningococcal A/C conjugate vaccination in infancy. J Infect Dis 2001 Jan 1;183(1):97-104. [272] Greenwood B. The changing face of meningococcal disease in West Africa. Epidemiol Infect 2007 Jul;135(5):703-5. [273] Veenhoven R, Bogaert D, Uiterwaal C, Brouwer C, Kiezebrink H, Bruin J, et al. Effect of conjugate pneumococcal vaccine followed by polysaccharide pneumococcal vaccine on recurrent acute otitis media: a randomised study. Lancet 2003 Jun 28;361(9376):2189-95. [274] O'Grady K, Lee K, Carlin J, Torzillo P, Chang A, Mulholland E, et al. Increased risk of hospitalization for acute lower respiratory tract infection among Australian indigenous infants 5-23 months of age following pneumococcal vaccination: a cohort study. Clin Infect Dis 2010 Apr 1;50(7):970-8. [275] Moore H, Lehmann D, de Klerk N, Jacoby P, Richmond P. Reduction in disparity in pneumonia hospitilisations between Aboriginal and Non-Aboriginal children in Western Australia 7th International Symposium on Pneumococci and Pneumococcal Diseases. Tel Aviv, Israel, 2010: Poster 16.

236

[276] Jardine A, Menzies R, McIntyre P. The impact of pneumococcal conjugate vaccine hospitalisation for respiratory infections in the Australian Indigenous population. 7th Symposium of Pneumococci and Pneumococcal Diseases. Tel Aviv, Israel, 2010: Poster 30. [277] McIntyre P, Chiu C, Jayasinghe S, Menzies R, Krause V, Cook H, et al. Reduction in 19A invasive pneumococcal disease in Indigenous Australian children receiving conjugate pneumococcal vaccine with polysaccharide booster. 7th International Symposium on Pneumococci and Pneumococcal Diseases. Tel Aviv, 2010: Poster 118. [278] Sorensen RU, Leiva LE, Giangrosso PA, Butler B, Javier FC, 3rd, Sacerdote DM, et al. Response to a heptavalent conjugate Streptococcus pneumoniae vaccine in children with recurrent infections who are unresponsive to the polysaccharide vaccine. Pediatr Infect Dis J 1998 Aug;17(8):685-91. [279] Yokochi T, Kato Y, Sugiyama T, Koide N, Morikawa A, Jiang GZ, et al. Lipopolysaccharide induces apoptotic cell death of B memory cells and regulates B cell memory in antigen-nonspecific manner. FEMS Immunol Med Microbiol 1996 Aug;15(1):1-8. [280] Heyman B. Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu Rev Immunol 2000;18:709-37. [281] Baxendale HE, Davis Z, White HN, Spellerberg MB, Stevenson FK, Goldblatt D. Immunogenetic analysis of the immune response to pneumococcal polysaccharide. Eur J Immunol 2000 Apr;30(4):1214-23. [282] Granoff DM, Pollard AJ. Reconsideration of the use of meningococcal polysaccharide vaccine. Pediatr Infect Dis J 2007 Aug;26(8):716-22. [283] Balazs M, Martin F, Zhou T, Kearney J. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 2002 Sep;17(3):341-52. [284] Noel C, Florquin S, Goldman M, Braun MY. Chronic exposure to superantigen induces regulatory CD4(+) T cells with IL-10-mediated suppressive activity. Int Immunol 2001 Apr;13(4):431-9. [285] Wuorimaa T, Kayhty H, Eskola J, Bloigu A, Leroy O, Surcel HM. Activation of cell- mediated immunity following immunization with pneumococcal conjugate or polysaccharide vaccine. Scand J Immunol 2001 Apr;53(4):422-8. [286] Khan AQ, Lees A, Snapper CM. Differential regulation of IgG anti-capsular polysaccharide and antiprotein responses to intact Streptococcus pneumoniae in the presence of cognate CD4+ T cell help. J Immunol 2004 Jan 1;172(1):532-9. [287] Guttormsen HK, Sharpe AH, Chandraker AK, Brigtsen AK, Sayegh MH, Kasper DL. Cognate stimulatory B-cell-T-cell interactions are critical for T-cell help recruited by glycoconjugate vaccines. Infect Immun 1999 Dec;67(12):6375-84. [288] Dullforce P, Sutton DC, Heath AW. Enhancement of T cell-independent immune responses in vivo by CD40 antibodies. Nat Med 1998 Jan;4(1):88-91. [289] Snapper CM. Differential regulation of protein- and polysaccharide-specific Ig isotype production in vivo in response to intact Streptococcus pneumoniae. Curr Protein Pept Sci 2006 Aug;7(4):295-305. [290] Pichichero ME. Immunological paralysis to pneumococcal polysaccharide in man. Lancet 1985 Aug 31;2(8453):468-71. [291] Makela PH, Leinonen M, Pukander J, Karma P. A study of the pneumococcal vaccine in prevention of clinically acute atttacks of recurrent otitis media. Rev Infect Dis 1981 Mar-Apr;3 Suppl:S124-32. [292] Douglas RM, Miles HB. Vaccination against Streptococcus pneumoniae in childhood: lack of demonstrable benefit in young Australian children. J Infect Dis 1984 Jun;149(6):861-9.

237

[293] Karma P, Pukander J, Sipila M, Timonen M, Pontynen S, Herva E, et al. Prevention of otitis media in children by pneumococcal vaccination. Am J Otolaryngol 1985 May-Jun;6(3):173-84. [294] Rosen C, Christensen P, Henrichsen J, Hovelius B, Prellner K. Beneficial effect of pneumococcal vaccination on otitis media in children over two years old. Int J Pediatr Otorhinolaryngol 1984 Jul;7(3):239-46. [295] Douglas RM, Hansman D, McDonald B, Paton J, Kirke K. Pneumococcal vaccine in aboriginal children--a randomized controlled trial involving 60 children. Community Health Stud 1986;10(2):189-96. [296] Teele DW, Klein JO, Bratton L, Fisch GR, Mathieu OR, Porter PJ, et al. Use of pneumococcal vaccine for prevention of recurrent acute otitis media in infants in Boston. The Greater Boston Collaborative Otitis Media Study Group. Rev Infect Dis 1981 Mar-Apr;3 Suppl:S113-8. [297] Howie VM, Ploussard J, Sloyer JL, Hill JC. Use of pneumococcal polysaccharide vaccine in preventing otitis media in infants: different results between racial groups. Pediatrics 1984 Jan;73(1):79-81. [298] Rosen C, Christensen P, Hovelius B, Prellner K. Effect of pneumococcal vaccination on upper respiratory tract infections in children. Design of a follow-up study. Scand J Infect Dis Suppl 1983;39:39-44. [299] Riley ID, Lehmann D, Alpers MP, Marshall TF, Gratten H, Smith D. Pneumococcal vaccine prevents death from acute lower-respiratory-tract infections in Papua New Guinean children. Lancet 1986 Oct 18;2(8512):877-81. [300] Obaro S, Leach A, McAdam KW. Use of pneumococcal polysaccharide vaccine in children. Lancet 1998 Aug 15;352(9127):575. [301] Lehmann D, Vail J, Firth MJ, de Klerk NH, Alpers MP. Benefits of routine immunizations on childhood survival in Tari, Southern Highlands Province, Papua New Guinea. Int J Epidemiol 2005 Feb;34(1):138-48. [302] Leach AJ, Morris PS, Mackenzie G, McDonnell J, Balloch A, Carapetis J, et al. Immunogenicity for 16 serotypes of a unique schedule of pneumococcal vaccines in a high- risk population. Vaccine 2008 Jul 23;26(31):3885-91. [303] Pomat W, Phuanukoonnon S, van den Biggelaar A, Francis J, Orami T. Alternative pneumococcal vaccination schedules for high risk populations: immunogenicity of neonatal pneumococcal conjugate vaccine and pneumococcal polysachharide vaccine in PNG children. 7th International Symposium of Pneumococci and Pneumococcal Diseases. Tel Aviv, 2010: Poster 34. [304] Ekstrom N, Vakevainen M, Verho J, Kilpi T, Kayhty H. Functional antibodies elicited by two heptavalent pneumococcal conjugate vaccines in the Finnish Otitis Media Vaccine Trial. Infect Immun 2007 Apr;75(4):1794-800. [305] Nurkka A, Poolman J, Henckaerts Iea. Opsonophagocytic activity of antibodies induced by 11-valent pneumococcal conjugate vaccine. 4th International Symposium on Pneumococci and Pneumococcal Disease; 2004 May 9–13, 2004; Helsinki, Finland; 2004. [306] Obaro SK, Adegbola RA, Banya WA, Greenwood BM. Carriage of pneumococci after pneumococcal vaccination. Lancet 1996 Jul 27;348(9022):271-2. [307] Mbelle N, Huebner RE, Wasas AD, Kimura A, Chang I, Klugman KP. Immunogenicity and impact on nasopharyngeal carriage of a nonavalent pneumococcal conjugate vaccine. J Infect Dis 1999 Oct;180(4):1171-6. [308] Lakshman R, Murdoch C, Race G, Burkinshaw R, Shaw L, Finn A. Pneumococcal nasopharyngeal carriage in children following heptavalent pneumococcal conjugate vaccination in infancy. Arch Dis Child 2003 Mar;88(3):211-4.

238

[309] Dagan R, Melamed R, Muallem M, Piglansky L, Greenberg D, Abramson O, et al. Reduction of nasopharyngeal carriage of pneumococci during the second year of life by a heptavalent conjugate pneumococcal vaccine. J Infect Dis 1996 Dec;174(6):1271-8. [310] Veenhoven RH, Bogaert D, Schilder AG, Rijkers GT, Uiterwaal CS, Kiezebrink HH, et al. Nasopharyngeal pneumococcal carriage after combined pneumococcal conjugate and polysaccharide vaccination in children with a history of recurrent acute otitis media. Clin Infect Dis 2004 Oct 1;39(7):911-9. [311] Dagan R, Givon-Lavi N, Zamir O, Sikuler-Cohen M, Guy L, Janco J, et al. Reduction of nasopharyngeal carriage of Streptococcus pneumoniae after administration of a 9-valent pneumococcal conjugate vaccine to toddlers attending day care centers. J Infect Dis 2002 Apr 1;185(7):927-36. [312] Millar EV, O'Brien KL, Watt JP, Bronsdon MA, Dallas J, Whitney CG, et al. Effect of community-wide conjugate pneumococcal vaccine use in infancy on nasopharyngeal carriage through 3 years of age: a cross-sectional study in a high-risk population. Clin Infect Dis 2006 Jul 1;43(1):8-15. [313] Frazao N, Brito-Avo A, Simas C, Saldanha J, Mato R, Nunes S, et al. Effect of the seven-valent conjugate pneumococcal vaccine on carriage and drug resistance of Streptococcus pneumoniae in healthy children attending day-care centers in Lisbon. Pediatr Infect Dis J 2005 Mar;24(3):243-52. [314] Cohen R, Levy C, de La Rocque F, Gelbert N, Wollner A, Fritzell B, et al. Impact of pneumococcal conjugate vaccine and of reduction of antibiotic use on nasopharyngeal carriage of nonsusceptible pneumococci in children with acute otitis media. Pediatr Infect Dis J 2006 Nov;25(11):1001-7. [315] Ghaffar F, Barton T, Lozano J, Muniz LS, Hicks P, Gan V, et al. Effect of the 7- valent pneumococcal conjugate vaccine on nasopharyngeal colonization by Streptococcus pneumoniae in the first 2 years of life. Clin Infect Dis 2004 Oct 1;39(7):930-8. [316] Pelton SI, Loughlin AM, Marchant CD. Seven valent pneumococcal conjugate vaccine immunization in two Boston communities: changes in serotypes and antimicrobial susceptibility among Streptococcus pneumoniae isolates. Pediatr Infect Dis J 2004 Nov;23(11):1015-22. [317] Moore MR, Hyde TB, Hennessy TW, Parks DJ, Reasonover AL, Harker-Jones M, et al. Impact of a conjugate vaccine on community-wide carriage of nonsusceptible Streptococcus pneumoniae in Alaska. J Infect Dis 2004 Dec 1;190(11):2031-8. [318] Huang SS, Platt R, Rifas-Shiman SL, Pelton SI, Goldmann D, Finkelstein JA. Post- PCV7 changes in colonizing pneumococcal serotypes in 16 Massachusetts communities, 2001 and 2004. Pediatrics 2005 Sep;116(3):e408-13. [319] Jones VF, Harrison C, Stout GG, Hopkins J. Nasopharyngeal colonization with heptavalent pneumococcal conjugate vaccine serotypes of Streptococcus pneumoniae with prolonged vaccine dosing intervals. Pediatr Infect Dis J 2005 Nov;24(11):969-73. [320] Kellner JD, Scheifele D, Vanderkooi OG, Macdonald J, Church DL, Tyrrell GJ. Effects of routine infant vaccination with the 7-valent pneumococcal conjugate vaccine on nasopharyngeal colonization with streptococcus pneumoniae in children in Calgary, Canada. Pediatr Infect Dis J 2008 Jun;27(6):526-32. [321] Dunais B, Bruno P, Carsenti-Dellamonica H, Touboul P, Dellamonica P, Pradier C. Trends in nasopharyngeal carriage of Streptococcus pneumoniae among children attending daycare centers in southeastern France from 1999 to 2006. Pediatr Infect Dis J 2008 Nov;27(11):1033-5. [322] Nahm MH, Lin J, Finkelstein JA, Pelton SI. Increase in the prevalence of the newly discovered pneumococcal serotype 6C in the nasopharynx after introduction of pneumococcal conjugate vaccine. J Infect Dis 2009 Feb 1;199(3):320-5.

239

[323] Pelton SI, Huot H, Finkelstein JA, Bishop CJ, Hsu KK, Kellenberg J, et al. Emergence of 19A as virulent and multidrug resistant Pneumococcus in Massachusetts following universal immunization of infants with pneumococcal conjugate vaccine. Pediatr Infect Dis J 2007 Jun;26(6):468-72. [324] van Gils EJ, Veenhoven RH, Hak E, Rodenburg GD, Bogaert D, Ijzerman EP, et al. Effect of reduced-dose schedules with 7-valent pneumococcal conjugate vaccine on nasopharyngeal pneumococcal carriage in children: a randomized controlled trial. JAMA 2009 Jul 8;302(2):159-67. [325] Frazão N, Sá-Leão R, de Lencastre H. Impact of a single dose of the 7-valent pneumococcal conjugate vaccine on colonization. Vaccine 2010 Apr 26;28(19):3445-52. [326] Herva E, Luotonen J, Timonen M, Sibakov M, Karma P, Makela PH. The effect of polyvalent pneumococcal polysaccharide vaccine on nasopharyngeal and nasal carriage of Streptococcus pneumoniae. Scand J Infect Dis 1980;12(2):97-100. [327] Wright PF, Sell SH, Vaughn WK, Andrews C, McConnell KB, Schiffman G. Clinical studies of pneumococcal vaccines in infants. II. Efficacy and effect on nasopharyngeal carriage. Rev Infect Dis 1981 Mar-Apr;3 Suppl:S108-12. [328] Rosen C, Christensen P, Hovelius B, Prellner K. A longitudinal study of the nasopharyngeal carriage of pneumococci as related to pneumococcal vaccination in children attending day-care centres. Acta Otolaryngol 1984 Nov-Dec;98(5-6):524-32. [329] Douglas RM, Hansman D, Miles HB, Paton JC. Pneumococcal carriage and type- specific antibody. Failure of a 14-valent vaccine to reduce carriage in healthy children. Am J Dis Child 1986 Nov;140(11):1183-5. [330] Christensen P, Hovelius B, Prellner K, Rosen C, Christensen KK, Kurl DN, et al. Effects of pneumococcal vaccination on tonsillo-pharyngitis and upper respiratory tract flora. Int Arch Allergy Appl Immunol 1985;78(2):161-6. [331] (UNICEF) UNsCF. The state of the world’s children 2009. New York; 2008. [332] Mulholland K. Magnitude of the problem of childhood pneumonia. Lancet 1999 Aug 14;354(9178):590-2. [333] O'Brien KL, Steinhoff MC, Edwards K, Keyserling H, Thoms ML, Madore D. Immunologic priming of young children by pneumococcal glycoprotein conjugate, but not polysaccharide, vaccines. Pediatr Infect Dis J 1996 May;15(5):425-30. [334] Karma P, Luotonen J, Timonen M, Pontynen S, Pukander J, Herva E, et al. Efficacy of pneumococcal vaccination against recurrent otitis media. Preliminary results of a field trial in Finland. Ann Otol Rhinol Laryngol Suppl 1980 May-Jun;89(3 Pt 2):357-62. [335] Riley ID, Everingham FA, Smith DE, Douglas RM. Immunisation with a polyvalent pneumococcal vaccine. Effect of respiratory mortality in children living in the New Guinea highlands. Arch Dis Child 1981 May;56(5):354-7. [336] Eskola J. Polysaccharide-based pneumococcal vaccines in the prevention of acute otitis media. Vaccine 2000 Dec 8;19 Suppl 1:S78-82. [337] O'Brien KL, Bronsdon MA, Dagan R, Yagupsky P, Janco J, Elliott J, et al. Evaluation of a medium (STGG) for transport and optimal recovery of Streptococcus pneumoniae from nasopharyngeal secretions collected during field studies. J Clin Microbiol 2001 Mar;39(3):1021-4. [338] O'Brien KL, Nohynek H. Report from a WHO working group: standard method for detecting upper respiratory carriage of Streptococcus pneumoniae. Pediatr Infect Dis J 2003 Feb;22(2):133-40. [339] Kong F, Brown M, Sabananthan A, Zeng X, Gilbert GL. Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes. J Clin Microbiol 2006 May;44(5):1887-91.

240

[340] Zhou F, Kong F, Tong Z, Gilbert GL. Identification of less-common Streptococcus pneumoniae serotypes by a multiplex PCR-based reverse line blot hybridization assay. J Clin Microbiol 2007 Oct;45(10):3411-5. [341] Nahm MH, Goldblatt D. Training manual for enzyme linked immunosorbent assay for the quantitation of Streptococcus pneumoniae serotype specific IgG (Pn PS ELISA); http://www.vaccine.uab.edu/ELISA%20Protocol.pdf., 2006. [342] Henckaerts I, Goldblatt D, Ashton L, Poolman J. Critical differences between pneumococcal polysaccharide enzyme-linked immunosorbent assays with and without 22F inhibition at low antibody concentrations in pediatric sera. Clin Vaccine Immunol 2006 Mar;13(3):356-60. [343] Balloch A, Licciardi PV, Leach A, Nurkka A, Tang ML. Results from an inter- laboratory comparison of pneumococcal serotype-specific IgG measurement and critical parameters that affect assay performance. Vaccine 2010 Feb 3;28(5):1333-40. [344] Wuorimaa T, Dagan R, Vakevainen M, Bailleux F, Haikala R, Yaich M, et al. Avidity and subclasses of IgG after immunization of infants with an 11-valent pneumococcal conjugate vaccine with or without aluminum adjuvant. J Infect Dis 2001 Nov 1;184(9):1211- 5. [345] Kim KH, Yu J, Nahm MH. Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies. Clin Diagn Lab Immunol 2003 Jul;10(4):616-21. [346] Farrington CP, Manning G. Test statistics and sample size formulae for comparative binomial trials with null hypothesis of non-zero risk difference or non-unity relative risk. Stat Med 1990 Dec;9(12):1447-54. [347] Zellner A. Estimators for seemingly unrelated regression equations. J American Statistical Association 1963;58:977-92. [348] Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol Rev 1990 Apr;3(2):171-96. [349] Friedland IR, Klugman KP. Failure of chloramphenicol therapy in penicillin- resistant pneumococcal meningitis. Lancet 1992 Feb 15;339(8790):405-8. [350] Dagan R, Abramson O, Leibovitz E, Lang R, Goshen S, Greenberg D, et al. Impaired bacteriologic response to oral cephalosporins in acute otitis media caused by pneumococci with intermediate resistance to penicillin. Pediatr Infect Dis J 1996 Nov;15(11):980-5. [351] Leibovitz E, Dragomir C, Sfartz S, Porat N, Yagupsky P, Jica S, et al. Nasopharyngeal carriage of multidrug-resistant Streptococcus pneumoniae in institutionalized HIV-infected and HIV-negative children in northeastern Romania. Int J Infect Dis 1999 Summer;3(4):211-5. [352] Arason VA, Kristinsson KG, Sigurdsson JA, Stefansdottir G, Molstad S, Gudmundsson S. Do antimicrobials increase the carriage rate of penicillin resistant pneumococci in children? Cross sectional prevalence study. BMJ 1996 Aug 17;313(7054):387-91. [353] Kyaw MH, Lynfield R, Schaffner W, Craig AS, Hadler J, Reingold A, et al. Effect of introduction of the pneumococcal conjugate vaccine on drug-resistant Streptococcus pneumoniae. N Engl J Med 2006 Apr 6;354(14):1455-63. [354] Standards NCfCL. Performance standards for antimicrobial susceptibility testing. NCCLS document M100-S10. Villanova, Pennsylvania, 2002. [355] Messmer TO, Sampson JS, Stinson A, Wong B, Carlone GM, Facklam RR. Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniae. Diagn Microbiol Infect Dis 2004 Aug;49(4):249-54.

241

[356] Charveriat MA, Chomarat M, Watson M, Garin B. [Nasopharyngeal carriage of Streptococcus pneumoniae in healthy children, 2 to 24 months of age, in New-Caledonia]. Med Mal Infect 2005 Oct;35(10):500-6. [357] Gratten M, Gratten H, Poli A, Carrad E, Raymer M, Koki G. Colonisation of Haemophilus influenzae and Streptococcus pneumoniae in the upper respiratory tract of neonates in Papua New Guinea: primary acquisition, duration of carriage, and relationship to carriage in mothers. Biol Neonate 1986;50(2):114-20. [358] Guillemot D, Carbon C, Balkau B, Geslin P, Lecoeur H, Vauzelle-Kervroedan F, et al. Low dosage and long treatment duration of beta-lactam: risk factors for carriage of penicillin-resistant Streptococcus pneumoniae. JAMA 1998 Feb 4;279(5):365-70. [359] Sung RY, Ling JM, Fung SM, Oppenheimer SJ, Crook DW, Lau JT, et al. Carriage of Haemophilus influenzae and Streptococcus pneumoniae in healthy Chinese and Vietnamese children in Hong Kong. Acta Paediatr 1995 Nov;84(11):1262-7. [360] Aran A, Fraser D, Dagan R. [Characteristics of nasopharyngeal carriage of Streptococcus pneumoniae in children during acute respiratory disease]. Harefuah 2001 Apr;140(4):300-5, 68. [361] Borres MP, Alestig K, Krantz I, Larsson P, Norvenius G, Stenqvist K. Carriage of penicillin-susceptible and non-susceptible pneumococci in healthy young children in Goteborg, Sweden. J Infect 2000 Mar;40(2):141-4. [362] Nilsson P, Laurell MH. Impact of socioeconomic factors and antibiotic prescribing on penicillin- non-susceptible Streptococcus pneumoniae in the city of Malmo. Scand J Infect Dis 2005;37(6-7):436-41. [363] Ciftci E, Dogru U, Aysev D, Ince E, Guriz H. Nasopharyngeal colonization with penicillin-resistant Streptococcus pneumoniae in Turkish children. Pediatr Int 2000 Oct;42(5):552-6. [364] Finkelstein JA, Huang SS, Daniel J, Rifas-Shiman SL, Kleinman K, Goldmann D, et al. Antibiotic-resistant Streptococcus pneumoniae in the heptavalent pneumococcal conjugate vaccine era: predictors of carriage in a multicommunity sample. Pediatrics 2003 Oct;112(4):862-9. [365] Duchin JS, Breiman RF, Diamond A, Lipman HB, Block SL, Hedrick JA, et al. High prevalence of multidrug-resistant Streptococcus pneumoniae among children in a rural Kentucky community. Pediatr Infect Dis J 1995 Sep;14(9):745-50. [366] Fairchok MP, Ashton WS, Fischer GW. Carriage of penicillin-resistant pneumococci in a military population in Washington, DC: risk factors and correlation with clinical isolates. Clin Infect Dis 1996 Jun;22(6):966-72. [367] Dagan R, Yagupsky P, Goldbart A, Wasas A, Klugman K. Increasing prevalence of penicillin-resistant pneumococcal infections in children in southern Israel: implications for future immunization policies. Pediatr Infect Dis J 1994 Sep;13(9):782-6. [368] Fenoll A, Martin Bourgon C, Munoz R, Vicioso D, Casal J. Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing systemic infections in Spain, 1979-1989. Rev Infect Dis 1991 Jan-Feb;13(1):56-60. [369] Marton A, Gulyas M, Munoz R, Tomasz A. Extremely high incidence of antibiotic resistance in clinical isolates of Streptococcus pneumoniae in Hungary. J Infect Dis 1991 Mar;163(3):542-8. [370] Leach AJ, Boswell JB, Asche V, Nienhuys TG, Mathews JD. Bacterial colonization of the nasopharynx predicts very early onset and persistence of otitis media in Australian aboriginal infants. Pediatr Infect Dis J 1994 Nov;13(11):983-9. [371] Musher DM, Groover JE, Reichler MR, Riedo FX, Schwartz B, Watson DA, et al. Emergence of antibody to capsular polysaccharides of Streptococcus pneumoniae during outbreaks of pneumonia: association with nasopharyngeal colonization. Clin Infect Dis 1997 Mar;24(3):441-6.

242

[372] Friedland IR, Klugman KP. Antibiotic-resistant pneumococcal disease in South African children. Am J Dis Child 1992 Aug;146(8):920-3. [373] Bogaert D, Ha NT, Sluijter M, Lemmens N, De Groot R, Hermans PW. Molecular epidemiology of pneumococcal carriage among children with upper respiratory tract infections in Hanoi, Vietnam. J Clin Microbiol 2002 Nov;40(11):3903-8. [374] Lee NY, Song JH, Kim S, Peck KR, Ahn KM, Lee SI, et al. Carriage of antibiotic- resistant pneumococci among Asian children: a multinational surveillance by the Asian Network for Surveillance of Resistant Pathogens (ANSORP). Clin Infect Dis 2001 May 15;32(10):1463-9. [375] Gratten M, Manning K, Dixon J, Morey F, Torzillo P, Hanna J, et al. Upper airway carriage by Haemophilus influenzae and Streptococcus pneumoniae in Australian aboriginal children hospitalised with acute lower respiratory infection. Southeast Asian J Trop Med Public Health 1994 Mar;25(1):123-31. [376] Dellamonica P, Pradier C, Dunais B, Carsenti H. New perspectives offered by a French study of antibiotic resistance in day-care centers. Chemotherapy 1998 Sep;44 Suppl 1:10-4. [377] Coles CL, Kanungo R, Rahmathullah L, Thulasiraj RD, Katz J, Santosham M, et al. Pneumococcal nasopharyngeal colonization in young South Indian infants. Pediatr Infect Dis J 2001 Mar;20(3):289-95. [378] Syrogiannopoulos GA, Grivea IN, Katopodis GD, Geslin P, Jacobs MR, Beratis NG. Carriage of antibiotic-resistant Streptococcus pneumoniae in Greek infants and toddlers. Eur J Clin Microbiol Infect Dis 2000 Apr;19(4):288-93. [379] Joloba ML, Bajaksouzian S, Palavecino E, Whalen C, Jacobs MR. High prevalence of carriage of antibiotic-resistant Streptococcus pneumoniae in children in Kampala Uganda. Int J Antimicrob Agents 2001 May;17(5):395-400. [380] Klugman KP, Koornhof HJ, Wasas A, Storey K, Gilbertson I. Carriage of penicillin resistant pneumococci. Arch Dis Child 1986 Apr;61(4):377-81. [381] Rey LC, Wolf B, Moreira JL, Milatovic D, Verhoef J, Farhat CK. Antimicrobial susceptibility and serotypes of nasopharyngeal Streptococcus pneumoniae in children with pneumonia and in children attending day-care centres in Fortaleza, Brazil. Int J Antimicrob Agents 2002 Aug;20(2):86-92. [382] McGee L, Wang H, Wasas A, Huebner R, Chen M, Klugman KP. Prevalence of serotypes and molecular epidemiology of Streptococcus pneumoniae strains isolated from children in Beijing, China: identification of two novel multiply-resistant clones. Microb Drug Resist 2001 Spring;7(1):55-63. [383] World Health Organization. Estimating the local burden of Haemophilus influenzae type b (Hib) disease preventable by vaccination: a rapid assessment tool, 2001. WHO document no. WHO/V&B/01.27. Geneva: WHO, 2001. [384] Madhi SA, Kohler M, Kuwanda L, Cutland C, Klugman KP. Usefulness of C- reactive protein to define pneumococcal conjugate vaccine efficacy in the prevention of pneumonia. Pediatr Infect Dis J 2006 Jan;25(1):30-6. [385] Russell F, Chandra R, Carapetis J, Seduadua A, Tikoduadua L, Buadromo E, et al. Epidemiology and Serotypes of Invasive Pneumococcal Disease in all ages in Fiji. 6th International Symposium of Pneumococci and Pneumococal Diseases; 2008 June 8-12, 2008.; Reykjavik, Iceland; 2008. [386] Russell FM, Licciardi PV, Balloch A, Biaukula V, Tikoduadua L, Carapetis JR, et al. Safety and immunogenicity of the 23-valent pneumococcal polysaccharide vaccine at 12 months of age, following one, two, or threes doses of the 7-valent pneumococcal conjugate vaccine in infancy. Vaccine 2010 Apr 19;28(18):3086-94.

243

[387] Dagan R, Goldblatt D, Maleckar JR, Yaich M, Eskola J. Reduction of antibody response to an 11-valent pneumococcal vaccine coadministered with a vaccine containing acellular pertussis components. Infect Immun 2004 Sep;72(9):5383-91. [388] Bell F, Heath P, Shackley F, MacLennan J, Shearstone N, Diggle L, et al. Effect of combination with an acellular pertussis, diphtheria, tetanus vaccine on antibody response to Hib vaccine (PRP-T). Vaccine 1998 Apr;16(6):637-42. [389] Russell FM, Balloch A, Tang ML, Carapetis JR, Licciardi P, Nelson J, et al. Immunogenicity following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine. Vaccine 2009 Sep 18;27(41):5685-91. [390] Russell FM, Carapetis JR, Balloch A, Licciardi PV, Jenney AW, Tikoduadua L, et al. Hyporesponsiveness to re-challenge dose following pneumococcal polysaccharide vaccine at 12 months of age, a randomized controlled trial. Vaccine 2010 Apr 26;28(19):3341-9. [391] Siegrist C. Vaccine Immunology. In: Plotkin S, Orenstein W, Offit P, editors. Vaccines 5th Edition: Elsevier, 2008: 17-36. [392] Wenger JD, Zulz T, Bruden D, Singleton R, Bruce MG, Bulkow L, et al. Invasive Pneumococcal Disease in Alaskan Children: Impact of the Seven-Valent Pneumococcal Conjugate Vaccine and the Role of Water Supply. Pediatr Infect Dis J 2009 Nov 30. [393] Cherian T. WHO expert consultation on serotype composition of pneumococcal conjugate vaccines for use in resource-poor developing countries, 26-27 October 2006, Geneva. Vaccine 2007 Sep 4;25(36):6557-64. [394] Zangwill KM, Greenberg DP, Chiu CY, Mendelman P, Wong VK, Chang SJ, et al. Safety and immunogenicity of a heptavalent pneumococcal conjugate vaccine in infants. Vaccine 2003 May 16;21(17-18):1894-900. [395] Obaro SK, Huo Z, Banya WA, Henderson DC, Monteil MA, Leach A, et al. A glycoprotein pneumococcal conjugate vaccine primes for antibody responses to a pneumococcal polysaccharide vaccine in Gambian children. Pediatr Infect Dis J 1997 Dec;16(12):1135-40. [396] Quataert SA, Kirch CS, Wiedl LJ, Phipps DC, Strohmeyer S, Cimino CO, et al. Assignment of weight-based antibody units to a human antipneumococcal standard reference serum, lot 89-S. Clin Diagn Lab Immunol 1995 Sep;2(5):590-7. [397] Balloch A, Mininni T, Nurkka A, Mackenzie G, Leach A, Kayhty H. Interlaboratory comparison of the specific IgG response to serotypes in Prevenar. 5th International Symposium on Pneumococci and Pneumococcal Diseases; 2006 April 2-6, 2006; Alice Springs, Australia; 2006. [398] Bruyn GA, Zegers BJ, van Furth R. Mechanisms of host defense against infection with Streptococcus pneumoniae. Clin Infect Dis 1992 Jan;14(1):251-62. [399] Austrian R. Some observations on the pneumococcus and on the current status of pneumococcal disease and its prevention. Rev Infect Dis 1981 Mar-Apr;3 Suppl:S1-17. [400] Lee H, Nahm MH, Burton R, Kim KH. Immune response in infants to the heptavalent pneumococcal conjugate vaccine against vaccine-related serotypes 6A and 19A. Clin Vaccine Immunol 2009 Mar;16(3):376-81. [401] Schuerman L, Prymula R, Henckaerts I, Poolman J. ELISA IgG concentrations and opsonophagocytic activity following pneumococcal protein D conjugate vaccination and relationship to efficacy against acute otitis media. Vaccine 2007 Mar 1;25(11):1962-8. [402] Russell FM, Balloch A, Tang ML, Carapetis JR, Licciardi P, Nelson J, et al. Immunogenicity following one, two, or three doses of the 7-valent pneumococcal conjugate vaccine. Vaccine 2009 Jul 17. [403] Givon-Lavi N, Greenberg D, Dagan R. Immunogenicity of alternative regimens of the conjugated 7-valent pneumococcal vaccine: a randomized controlled trial. Pediatr Infect Dis J 2010 Aug;29(8):756-62.

244

[404] Nurkka A, Poolman J, Henckaerts I, Kilpi TM, Käyhty H. Opsonophagocytic activity of antibodies induced by 11-valent pneumococcal conjugate vaccine. 4th International Symposium on Pneumococci and Pneumococcal Disease Helsinki, Finland; May 9-13, 2004. Abstract PSV-41. [405] Fernsten P, Hu B, Yu X. Specificty of the opsonic response to serotype 19A and 19F conjugate vaccines. 6th International Symposium on Pneumococci and Pneumococcal Diseases. Reykjavik, Iceland, 2008. [406] Hausdorff W, Hoet B, Schuerman L. Do pneumococcal conjugate vaccines provide any cross-protection against serotype 19A? BMC Pediatr 2010 Feb 2;10(4). [407] Park S, Parameswar A, Demchenko A, Nahm M. Identification of a simple chemical structure associated with protective human antibodies against multiple pneumococcal serogroups. Infect Immun 2009;77(8):3374-9. [408] Goldblatt D, Southern J, Ashton L, Andrews N, Woodgate S, Burbidge P, et al. Immunogenicity of a Reduced Schedule of Pneumococcal Conjugate Vaccine in Healthy Infants and Correlates of Protection for Serotype 6B in the United Kingdom. Pediatr Infect Dis J 2009 Dec 11. [409] Puumalainen T, Ekstrom N, Zeta-Capeding R, Ollgren J, Jousimies K, Lucero M, et al. Functional antibodies elicited by an 11-valent diphtheria-tetanus toxoid-conjugated pneumococcal vaccine. J Infect Dis 2003 Jun 1;187(11):1704-8. [410] Soininen A, van den Dobbelsteen G, Oomen L, Kayhty H. Are the enzyme immunoassays for antibodies to pneumococcal capsular polysaccharides serotype specific? Clin Diagn Lab Immunol 2000 May;7(3):468-76. [411] Goldblatt D, Akoto A, Ashton L, al. e. Immunogenicity and the generation of immune memory following 9-valent pneumococcal conjugate vaccination in Ghanaian infants with sickle cell disease. 40th Interscience Conference on Antimicrobial Agents and Chemotherapy; 2000 Sept 24–27; Toronto, Ontario, Canada; 2000. [412] Balloch A, Licciardi P, Russell F, Burton R, Nahm M, Mulholland K, et al. Does serotype-specific IgG and avidity to Streptococcus pneumoniae following infant immunisation correlate with functional opsonophagocytic activity? 6th International Symposium on Pneumococci and Pneumococcal Diseases. Reykjavik, Iceland, 2008. [413] Licciardi PV, Balloch A, Russell FM, Mulholland EK, Tang MLK. Antibodies to serotype 9V exhibit novel serogroup cross-reactivity following infant pneumococcal immunization. Vaccine 2010 May 14;28(22):3793-800. [414] Singleton RJ, Hennessy TW, Bulkow LR, Hammitt LL, Zulz T, Hurlburt DA, et al. Invasive pneumococcal disease caused by nonvaccine serotypes among alaska native children with high levels of 7-valent pneumococcal conjugate vaccine coverage. JAMA 2007 Apr 25;297(16):1784-92. [415] Adrian P, van Niekerk N, Jones S, Cutland C, von Gottberg A, de Gouveia L, et al. Long term effects of a 9-valent pneumococcal conjugate vaccine (PCV) on nasopharyngeal colonization with pneumococcal serotypes included in the vaccine. th International Symposium on Pneumococci and Pneumococcal Diseases; 2006 April 2-6, 2006.; Alice Springs, Australia; 2006. [416] Cheung YB, Zaman SM, Nsekpong ED, Van Beneden CA, Adegbola RA, Greenwood B, et al. Nasopharyngeal Carriage of Streptococcus pneumoniae in Gambian Children who Participated in a 9-valent Pneumococcal Conjugate Vaccine Trial and in Their Younger Siblings. Pediatr Infect Dis J 2009 Jun 16. [417] Lipsitch M, O'Neill K, Cordy D, Bugalter B, Trzcinski K, Thompson CM, et al. Strain characteristics of Streptococcus pneumoniae carriage and invasive disease isolates during a cluster-randomized clinical trial of the 7-valent pneumococcal conjugate vaccine. J Infect Dis 2007 Oct 15;196(8):1221-7.

245

[418] Heath PT, McVernon J. The UK Hib vaccine experience. Arch Dis Child 2002 Jun;86(6):396-9. [419] Kelly DF, Moxon ER, Pollard AJ. Haemophilus influenzae type b conjugate vaccines. Immunology 2004 Oct;113(2):163-74.

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: RUSSELL, FIONA MARY

Title: Alternative pneumococcal vaccination schedules for infants in Fiji and pneumococcal epidemiology

Date: 2010

Citation: Russell, F. M. (2010). Alternative pneumococcal vaccination schedules for infants in Fiji and pneumococcal epidemiology. PhD thesis, Department of Paediatrics, Faculty of Medicine, The University of Melbourne.

Publication Status: Unpublished

Persistent Link: http://hdl.handle.net/11343/35820

File Description: Alternative pneumococcal vaccination schedules for infants in Fiji and pneumococcal epidemiology

Terms and Conditions: Terms and Conditions: Copyright in works deposited in Minerva Access is retained by the copyright owner. The work may not be altered without permission from the copyright owner. Readers may only download, print and save electronic copies of whole works for their own personal non-commercial use. Any use that exceeds these limits requires permission from the copyright owner. Attribution is essential when quoting or paraphrasing from these works.