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Optimmunize: Improving the beneficial effects of vaccines 19 - 21 February 2020 ABSTRACT BOOK ABSTRACT Name:

Optimmunize: Improving the beneficial effects of vaccines

Wellcome Genome Campus Conference Centre, Hinxton, Cambridge, UK 19-21 February 2020

Scientific Programme Committee:

Christine Stabell Benn University of Southern Denmark, Denmark

Rebecca Chandler Uppsala Monitoring Centre, Sweden

Nigel Curtis The University of Melbourne, Australia

Eleanor Fish University of Toronto, Canada

Sabra Klein Johns Hopkins University, USA

Tweet about it: #optimmunize20

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Scientific Programme Committee

Christine Stabell Benn Rebecca Chandler University of Southern Denmark, Denmark Uppsala Monitoring Centre, Sweden

Nigel Curtis Eleanor Fish The University of Melbourne, Australia University of Toronto, Canada

Sabra Klein Johns Hopkins University, USA

Wellcome Genome Campus Scientific Conferences Team:

Jemma Beard Nicole Schatlowski Conference and Events Scientific Programme Organiser Officer

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Dear colleague,

I would like to offer you a warm welcome to the Wellcome Genome Campus Advanced Courses and Scientific Conferences: Optimmunize: Improving the beneficial effects of vaccines. I hope you will find the talks interesting and stimulating, and find opportunities for networking throughout the schedule.

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General Information

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Even number poster assignments will be presenting in poster session 2, which takes place on Thursday, 20 February, at 18:00 – 19:00

The page number of your abstract in the abstract book indicates your assigned poster board number. An index of poster numbers appears in the back of this book.

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 13:15 to Stansted and Heathrow airports.  13:30 to Cambridge train station and city centre (Downing Street).

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Contact numbers Wellcome Genome Campus Conference Centre – 01223 495000 (or Ext. 5000) Wellcome Genome Campus Conference Organiser (Jemma) – 07473 934631

If you have any queries or comments, please do not hesitate to contact a member of staff who will be pleased to help you.

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Conference Summary

Wednesday, 19 February 11:30 – 12:50 Registration with buffet lunch 12:50 – 13:00 Welcome and introduction 13:00 – 14:30 Session1: Why the no-specific effects of vaccines are important 14:30 – 15:30 Keynote lecture: Mihai Netea, Radboud University, The Netherlands 15:30 – 16:00 Afternoon tea 16:00 – 17:30 Session 2: Potential non-specific effects of vaccines – evidence from clinical trials 17:30 – 18:00 Lightning talks 18:00 – 19:00 Poster session 1 (odd numbers) and drinks reception 19:00 – 21:00 Dinner

Thursday, 20 February 09:00 – 10:45 Session 3: Effects of vaccines on immunity and the epigenome 10:45 – 11:15 Morning coffee 11:15 – 12:30 Session 4: Sex-differnces in immunity and response to vaccines 12:30 – 14:00 Lunch 14:00 – 15:30 Session 5: Potential off-target use of vaccines 15:30 – 16:00 Afternoon tea 16:00 – 17:30 Session 6: Conference Q&A 17:30 – 18:00 Lightning talks 18:00 – 19:00 Poster session 2 (even numbers) and drinks reception 19:00 – 21:00 Dinner

Friday, 21 February 09:00 – 10:00 Session 7: Vaccination of the elderly 10:00 – 10:30 Session 8: Non-specific effects of vaccines: A complex and controversial topic 10:30 – 10:45 Morning coffee 10:45 – 12:45 Session 9: Looking toward the future: New vaccines/testing products in the pipeline 12:45 – 12:55 Closing remarks 12:55 – 13:15 Lunch 13:15 Coach departs to London Heathrow airport via London Stansted airport 12:45 Coach departs to Cambridge train station and city centre (Downing Street)

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https://www.mdpi.com/journal/vaccines

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Optimmunize: Improving the beneficial effects of vaccines

Wellcome Genome Campus Conference Centre, Hinxton, Cambridge

19 – 21 February 2020

Lectures to be held in the Francis Crick Auditorium Lunch and dinner to be held in the Hall Restaurant Poster sessions to be held in the Conference Centre

Spoken presentations - If you are an invited speaker, or your abstract has been selected for a spoken presentation, please give an electronic version of your talk to the AV technician.

Poster presentations – If your abstract has been selected for a poster, please display this in the Conference Centre on arrival.

Conference programme

Wednesday, 19 February

11:30-12:50 Registration with lunch

12:50-13:00 Welcome and Introductions Programme Committee

13:00-14:30 Session 1: Why the non-specific effects of vaccines are important Chair: Nigel Curtis

13:00 Opening note Nigel Curtis Melbourne University, Australia

13.10 Why the non-specific effects of vaccines are important Peter Aaby Bandim Health Project, Guinea-Bissau

13:30 Off target effects: the box is finally open but the contents are still confusing Adam Finn University of Bristol, UK

13:50 Q&A & discussion

14:30-15:30 Keynote Lecture: Trained immunity: a memory for innate host defense Mihai Netea Radboud University, The Netherlands

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15:30-16:00 Afternoon Tea

16:00-17:30 Session 2: Potential non-specific effects of vaccines – evidence from clinical trials Chair: Sabra Klein

16:00 The Melbourne Infant Study: BCG for Allergy and Infection Reduction (MIS BAIR) Nigel Curtis Melbourne University, Australia 16:30 Repurposing of Vaccines: What is in a name? Tobias Kollmann Telethon Kids Institute, Australia 17:00 Immune system development varies according to age, location and anemia in African children Danika Hill Babraham Institute, UK

17:15 Evidence for BCG-induced protection against heterologous infectious disease in Ugandan neonates: an investigator-blind randomised controlled trial. Sarah Prentice East and North Hertfordshire NHS Trust, UK

17:30-18:00 Lightning talks

18:00-19:00 Poster Session 1 (odd numbers) with drinks reception

19:00 Dinner

Thursday, 20 February

09:00-10:45 Session 3: Effects of vaccines on immunity and the epigenome Chair: Mihai Netea

09:00 What have we learnt about BCG vaccination in the last 20 years? (and can we improve its efficacy?) Hazel Dockrell LSHTM, UK

09.30 Non-specific effects of vaccines and nanoparticles: potential use in cancer patients Magda Plebanski RMIT, Australia

10.00 BCG and DTPw vaccination induce altered programmes of “trained” innate immunity in mice David Lynn South Australian Health and Medical Research Institute, Australia

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10.15 Exploring the role of RORa on trained immunity in human monocytes Gizem Kilic Radboud University Medical Center, The Netherlands

10.30 Systemic bacterial burden of sepsis-challenged neonatal mice significantly associated with survival Byron Brook University of British Columbia, Canada

10:45-11:15 Morning Coffee

11:15-12:30 Session 4: Sex-differences in immunity and response to vaccines Chair: Eleanor Fish

11:15 Age-associated sex differences in immunity to seasonal inactivated influenza vaccines Sabra Klein Johns Hopkins University, USA

11:45 Vertically transferred immunity in neonates: Mothers, mechanisms and mediators Petra Arck University Medical Center Hamburg-Eppendorf, Germany

12:15 Female-biased effects of aging on a chimeric hemagglutinin-based universal influenza virus vaccine in mice Santosh Dhakal Johns Hopkins University, USA

12:30-14:00 Lunch

14:00-15:30 Session 5: Potential off-target use of vaccines Chair: Christine Stabell Benn

14:00 BCG vaccines off target effects: so many good things with an emphasis on type 1 diabetes Denise Faustman Massachusetts General Hospital, USA

14:30 Sex-differential effects of rabies vaccine on survival in young free- roaming dogs, a novel animal model for the study of non-specific effects of vaccines in high-mortality populations Darryn Knobel Ross University School of Veterinary Medicine, West Indies

15:00 BCG vaccination is associated with reduced malaria prevalence in children under the age of five in sub-Saharan Africa Mike Berendsen Bandim Health Project, Guinea-Bissau

15:15 Discussion: How can we use off-target effects to optimize vaccination?

15:30-16:00 Afternoon Tea

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16:00-17:30 Session 6: Conference Q&A Chair: Christine Stabell Benn Young investigators(< 10 yrs post last degree (PhD, MD) will be asked to provide one critical question related to the conference subject when registering. The organizers will select the 2 best abstracts to be presented during this session. In addition, the organizers will select the 5 most thought provoking questions, and they will be discussed in a subsequent panel session that will include relevant speakers and the young researchers who posed the critical questions.

17:30-18:00 Lightning talks

18:00-19:00 Poster Session 2 (even numbers) with drinks reception

19:00 Dinner

Friday, 21 February

09:00-10:00 Session 7: Vaccination of the elderly Chair: Sarah Prentice

09:00 The impact of immunosenescence on immune responses after influenza vaccination in older individuals Inna Ovsyannikova, Mayo Clinic, USA

09:20 Evidence for immunomodulatory effects of vaccination in aging Australians Katie Flanagan Monash University, Australia

09:40 Panel discussion: Can vaccines be used to combat immunosenescence?

10:00-10:30 Session 8: Non-specific effects of vaccines: a complex and controversial topic Chair: Eleanor Fish

10:00 Non-specific effects of vaccines: Complex and controversial….and a challenge for current vaccine paradigms Rebecca Chandler Uppsala Monitoring Centre, Sweden

10:15 Discussion

10:30-10:45 Morning Coffee

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10:45-12:45 Session 9: Looking toward the future: New vaccines/testing products in pipeline Chair: Rebecca Chandler

10:45 Specific and off-target effects of novel live attenuated pertussis vaccine Camille Locht Institute Pasteur, France

11:15 Impact of adjuvants, sex, and the microbiome on SIV-vaccine-induced immunity and protective efficacy in rhesus macaques Marjorie Robert-Guroff National Cancer Institute, USA

11:45 Precision adjuvants for next generation vaccines tailored to vulnerable populations Ofer Levy Harvard University, USA

12:15 New live attenuated tuberculosis vaccine MTBVAC induces trained immunity and confers protection against experimental lethal pneumonia Jorge Dominguez Andres Radboud University Medical Center, The Netherlands

12:30 MV130 as a Trained Immunity-based Vaccine, clinical implications and mechanism of action Laura Conejero Immunotek, Spain

12.45-12:55 Closing remarks

12:55 -13:15 Lunch

13:15 Coach depart to Heathrow Airport via Stansted Airport 13:30 Coach departs to Cambridge City Centre and Train Station

The following abstracts should not be cited in bibliographies. Materials contained herein should be treated as personal communication and should be cited as such only with consent of the author.

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2020 CONFERENCES COURSES Evolutionary Systems Biology LABORATORY COURSES LECTURE/DISCUSSION COURSES 12-14 February Genomics and Clinical Microbiology Clinical Genomics: Fundamentals of Optimmunize: Improving the beneficial 19-24 January Variant Interpretation in Clinical Practice effects of vaccines NEW Genomics and Clinical Virology 29-31 January 19-21 February 23–28 February Genomic Practice for Genetic Counsellors Single Cell Biology Genetic Engineering of Mammalian 3-5 February 11-13 March Stem Cells Practical Aspects of Small Molecule Genomics of Brain Disorders 15–27 March Drug Discovery 18-20 March Next Generation Sequencing 21-26 June Genomics of Rare Diseases 20–27 April Evolutionary Biology and Ecology 25-27 March Low Input Epigenomics NEW of Cancer Proteomics in Cell Biology and 12-20 May 29 June-3 July Disease Mechanisms RNA Transcriptomics Science Policy: Improving the 30 March-1 April 17-26 June Uptake of Research into UK Policy Longitudinal Studies Single Cell Technologies and Analysis 19-21 August 20-22 April 24-31 July Genomics for Dermatology Nursing, Genomics and Healthcare NEW Molecular Pathology and 25-27 November 27-29 April Diagnosis of Cancer OVERSEAS COURSES Antimicrobial Resistance - Genome 22-27 November Next Generation Big Data and Emerging Technologies Derivation and Culture of Human 4-6 May Sequencing Bioinformatics Induced Pluripotent Stem Cells (hiPSCs) 19-24 January (Chile) Curating the Clinical Genome 14-18 December 20-22 May 9-14 February (Malaysia) Healthy Ageing COMPUTATIONAL COURSES Molecular Approaches to Clinical 27-29 May Mathematical Models for Infectious Microbiology in Africa 21-27 March (The Gambia) Genomic Epidemiology of Malaria Disease Dynamics 7-10 June 24 February-6 March Genomics and Epidemiological Surveillance of Bacterial Pathogens Virus Genomics and Evolution Fungal Pathogen Genomics 19-24 April (Paraguay) 15-17 June 11-16 May Reproducibility, Replicability and Trust Summer School in Bioinformatics Working with Pathogen Genomes in Science NEW 22-26 June 10-15 May (Vietnam) 9-11 September Systems Biology: From Large Viral Genomics and Bioinformatics Genome Informatics Datasets to Biological Insight 7-12 June (Uruguay) 14-17 September 6-10 July Antimicrobial Resistance of CRISPR and beyond: perturbations at Genetic Analysis of Mendelian Bacterial Pathogens scale to understand genomes and Complex Disorders 27 September-3 October (Kenya) 15-21 July 23-25 September Malaria Experimental Genetics Proteomics Bioinformatics Genomic Imprinting - from Biology 8-13 November (The Gambia) to Disease NEW 26-31 July 28-30 September Genetic Analysis of Practical Aspects of Drug Discovery Exploring Human Host-Microbiome Population-based Association Studies 29 November-4 December (Uruguay) Interactions in Health and Disease 21-25 September 21-23 October Working with Protozoan Parasite ONLINE COURSES Database Resources Bacterial Genomes - 4 courses 4-9 October Genetic Counselling - 1 course Next Generation Please see our website for more details Sequencing Bioinformatics and scheduling of online courses 18-24 October Computational Systems Biology for Complex Human Disease NEW 6-11 December

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WGC_Courses_and_Conferences_2020-Abstract-Books(Blue-FullColour)december2019.indd 1 03/12/2019 11:08:04 Spoken Presentations

Why the non-specific effects of vaccines are important

Peter Aaby Statens Serum Institut, Denmark For public health, a vaccine serves to prevent a specific disease. Thus, the effect of a vaccine on overall survival is assumed to be proportional to the number of specific infections prevented. However, from measuring the overall effects of vaccines on morbidity and mortality has come a growing number of contradictions of this assumption, and an increasing realisation that vaccines also have important non-specific effects (NSEs). With this knowledge, we can define new vaccination policies with more profound effects on survival. While there is now amble evidence that vaccines not only have specific effects, we are still in the process of understanding the NSEs, but some strong principles have emerged: First, so far it has been shown that four live vaccines enhance resistance to unrelated infections (beneficial NSEs) by reducing child mortality more than can be explained by prevention of the vaccine-targeted infection. Second, in contrast, six live vaccines have deleterious NSEs, increasing the risk of other infections, particularly for females. Third, combining live and non- live vaccines have variable effects. Fourth, females and males react differently to vaccines. Fifth, the sequence and combination of vaccines is important since the most recent vaccine has the strongest NSEs. Sixth, vaccinating with live vaccines in the presence of existing maternal immunity amplifies the beneficial NSEs. Seventh, boosting with live vaccines amplifies the beneficial NSEs. Eight, as immune-stimulants vaccines may interact beneficially or deleteriously with other interventions affecting the immune system. Systematic searches for the effect of various vaccines and combinations will undoubtedly produce more principles of NSEs in the future. As illustrated by a few examples, public health will benefit if the NSEs are taken into consideration. First, the live high-titre measles vaccine (HTMV) was effective in preventing measles infection, but had to be withdrawn by WHO because it was associated with two-fold increased female mortality. This negative effects was due to non-live vaccines being given after the live measles vaccine. Second, campaigns with measles vaccine and live oral polio vaccine (OPV) have played a major role in reducing child mortality in low-income countries in the last decades. Hence, stopping live vaccines after eradication may increase mortality as happened with smallpox vaccine and may soon happen with OPV. Third, several non-live vaccines including the malaria vaccine (RTS,S) currently being tested in Africa have been associated with increased female-to-male mortality. Co-administration of non-live vaccine with a live vaccine may reduce the negative effect for girls.

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Off target effects: the box is finally open but the contents are still confusing Adam Finn University of Bristol, UK Vaccines are developed, tested, licensed and, usually, implemented based on evidence showing their direct (on target) protective effects and safety in recipients. Evidence of their indirect effects (impact on transmission of the target infection in the wider population) usually follows universal implementation and often exceeds expectations. Recently models predicting such effects have begun to be incorporated into cost-benefit models which drive implementation and even targeting strategies (e.g. paediatric influenza and adolescent meningococcal programmes). However the extent to which immunisation, especially in early childhood, may have off target effects is much less clear. Epidemiological evidence of some important downstream off-target effects (when prevention of the target infection in turn prevents secondary infections that otherwise would have ensued e.g. varicella-group A streptococcus; influenza-pneumococcal pneumonia; measles-multiple infections) are beginning to emerge with supporting explanatory mechanisms in some cases. However the picture with respect to pure off-target effects (where administration of the vaccine itself alters the recipients’ susceptibility to unrelated infections) currently appears to be both complex and hard-to-call. Confronted with the apparent paradox of modest efficacy of the candidate sporozoite vaccine RTS,S against malaria alongside a numerically small but proportionally (between groups) large safety signal with an excess of cases of meningitis, diverse in both aetiology and timing, and even of cerebral malaria in the vaccine recipients, we developed an alternative hypothesis that the non-live rabies vaccine, given to controls in the older study group in whom the apparent central nervous system infection signal was observed, might actually have been protective in some non-specific off target way. We found several studies in the literature published over a 50 year period that support this theory as well as studies suggesting that components of the rabies virus present in the inactivated vaccine may activate multiple unrelated lymphocyte clones. We are currently investigating possible mechanisms for such an effect in more detail. If proven to exist, any added protective value of such non-specific effects of rabies vaccine might drive its introduction in communities in which deaths from paediatric rabies cases occur but not in sufficient numbers to justify the cost of universal prevention.

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Trained immunity: a memory for innate host defense

Mihai G. Netea, Department of Medicine, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands

The inability of innate immunity to build an immunological memory, considered one of the main characteristics differentiating it from adaptive immunity, has been recently challenged by studies in plants, invertebrates, and mammals. Long-term reprogramming of innate immunity, that induces adaptive traits and has been termed trained immunity characterizes prototypical innate immune cells such as natural killer cells and monocytes, and provides protection against reinfection in a T/B-cell-independent manner. In contrast, trained immunity has been shown to be able to induce protection against reinfection in a lymphocyte- independent manner. Non-specific protective effects dependent on trained immunity have also been shown to be induced after BCG vaccination in humans. Specific signaling mechanisms including the dectin-1/Raf1 and NOD2-mediated pathways induce trained immunity, through induction of histone modifications (methylation, acetylation) and epigenetic reprogramming of monocyte function. Complex immunological and metabolic circuits link cell stimulation to long-term epigenetic reprogramming of the function of myeloid cells and their bone marrow progenitors. The concept of trained immunity represents a paradigm change in immunity and its putative role in infection and inflammation may represent the next step in the design of future vaccines and immunotherapeutic approaches.

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The Melbourne Infant Study: BCG for Allergy and Infection Reduction (MIS BAIR)

Nigel Curtis University of Melbourne, Australia

The Melbourne Infant Study: BCG for Allergy and Infection Reduction (MIS BAIR) [misbair.org.au] is a randomised controlled trial of neonatal BCG vaccination in which over 1200 newborns in Melbourne have been recruited. The clinical outcomes, measured over the first five years of life, are respiratory infections, eczema, allergic sensitisation (measured by skin prick test) and asthma. Parent-completed online questionnaires have been used to collect extensive and detailed antenatal, perinatal and postnatal data. The trial includes an extensive biobank of blood and stool samples [lifecourse.melbournechildrens.com] for laboratory studies to investigate the immunological and molecular basis for the heterologous (non-specific) effects of BCG vaccine.

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Repurposing of Vaccines: What is in a name?

Tobias R. Kollmann, Nelly Amenyogbe

Telethon Kids Institute; Perth Children's Hospital; University of Western Australia

Vaccination induces changes far beyond the intended immune memory response targeting a specific pathogen. These effects have variously been labelled non-specific, off-target or heterologous. All of these terms suggest a vague, unintended impact. While that may have been true during the original discovery of effects that go beyond those targeting a specific pathogen, there now is ample data to suggest that the host modulatory functions of vaccines can specifically be harnessed to provide targeted benefit from e.g. preventing newborn mortality to reducing several non-communicable diseases. To reflect this confidence in the science associated with this stream of vaccine evaluation, we suggest use of the term 'repurposing of vaccines' to capture the active, intentional harnessing of this vast and largely untapped field of science. In analogy to the field of repurposing drugs, this name would also help demystify current research efforts on this topic and emphasize the benefits to be gained, ranging from accelerating vaccine discovery and design to increased safety to reduced R&D expenditure, thus market value.

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Immune system development varies according to age, location and anemia in African children

Danika L. Hill 1,2‡, Edward J. Carr1,3, Tobias Rutishauser4,5, Gemma Moncunill6, Joseph J. Campo6, Silvia Innocentin1, Maxmillian Mpina4,5,7, Augusto Nhabomba8, Anneth Tumbo4,5,7, Chenjerai Jairoce8, Henriëtte A. Moll9, Menno C. van Zelm2, Carlota Dobaño6,8*, Claudia Daubenberger4,5*‡, Michelle A. Linterman1*‡.

1. Lymphocyte Signalling & Development, Babraham Institute, Cambridge, UK. 2. Department of Immunology and Pathology, Central Clinical School, Monash University and Alfred Hospital, Melbourne, Victoria, Australia 3. Department of Medicine, University of Cambridge, Cambridge, UK 4. Swiss Tropical and Public Health Institute, Basel, Switzerland. 5. University of Basel, Switzerland. 6. ISGlobal, Barcelona Centre for International Health Research, Hospital Clínic–Universitat de Barcelona, Catalonia, Spain. 7. Ifakara Health Institute, Bagamoyo, Tanzania 8. Centro de Investigação em Saúde de Manhiça, Maputo, Mozambique. 9. Department of Pediatrics, Sophia Children’s Hospital, Erasmus MC, University Medical Center, Rotterdam, the Netherlands *Joint senior authors ‡ Corresponding authors

The high incidence of infectious disease in children from low- and middle-income countries creates a challenge for their immune systems in first few years of life. These children have the greatest need of the protection afforded by vaccination, however vaccines often show reduced efficacy in these populations. An improved understanding of how age, infection, nutrition, and genetics influence immune ontogeny and function is key to informing vaccine design for this at-risk population. We sought to identify factors that shape immune development in children under five years of age from Tanzania and Mozambique by detailed immunophenotyping of longitudinal blood samples collected during the RTS,S malaria vaccine phase III trial. In this cohort, the composition of the immune system is dynamically altered during the first years of life, and this was further influenced by geographical location, with some immune cell types showing an altered rate of development in Tanzanian children compared to Dutch children enrolled in the Generation R population-based cohort study. High titer antibody responses to the RTS,S/AS01E vaccine were associated with an activated immune profile at the time of vaccination, including an increased frequency of antibody secreting plasmablasts and follicular helper T cells. Anemic children had lower frequencies of recent thymic emigrant T cells, isotype-switched memory B cells and plasmablasts, and modulating iron bioavailability in vitro could recapitulate the B cell defects observed in anemic children. Our findings demonstrate that the composition of the immune system in children varies according to age, geographical location and anemia status.

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Evidence for BCG-induced protection against heterologous infectious disease in Ugandan neonates: an investigator-blind randomised controlled trial.

Prentice S (a,b), Webb EL (c) Akello F (b) Kiwudhu F (b) Akurut H (b) Elliott AM (a,b) Dockrell HM (d) Cose S (b,d) The Delayed BCG Study Team a: Clinical Research Department, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT b: MRC/UVRI and LSHTM Uganda Research Unit, 51-59 Nakiwogo Road, Entebbe, P.O. Box 49 c: Department of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT d: Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT

Trials conducted in low birth-weight infants in West Africa suggest that BCG vaccination produces heterologous protection against non-tuberculous infectious disease during the neonatal period. Uncertainty exists as to the generalisability of these findings, and thus their broader public health implications. We conducted an investigator-blind randomised controlled trial in 560 healthy neonates in East Africa, comparing BCG vaccination on the day of birth with BCG vaccination at 6-weeks of age. Illness incidence during the first 10- weeks of life was measured by active participant follow-up, comprising free access to physician review and investigations, regular routine physician reviews and weekly telephone interviews. Infants randomised to receive BCG at birth had a 29% reduction in physician-diagnosed non-tuberculous infectious disease incidence compared to BCG unvaccinated infants, during the 6-week period prior to BCG vaccination in the delayed group (Hazard Ratio (HR) 0.71, 95% CI (0.53 to 0.95)). In pre-specified sub-group analysis, this reduction was more pronounced in low birth-weight infants ≤2500g (HR 0.10 (0.01 to 0.75)) compared to normal birth-weight infants (HR 0.79 (0.59 to 1.07)) interaction p-value 0.04). After BCG vaccination in the delayed group at 6-weeks of age, there was no significant difference in physician- diagnosed, non-tuberculous infectious disease incidence between the groups. We conclude that BCG vaccination produces heterologous protection against non- tuberculous infectious disease during the neonatal period, in addition to having TB specific effects. This corroborates findings from other groups and suggests that the beneficial non- specific effects of BCG are applicable in disparate high-mortality settings. Prioritisation of BCG vaccination on the first day of life may have significant public health benefits through reductions in all-cause infectious morbidity and mortality.

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What have we learnt about BCG vaccination in the last 20 years? (and can we improve its efficacy?) H.M. Dockrell and S.G. Smith London School of Hygiene & Tropical Medicine Recent trials of new TB vaccines have used BCG vaccination as a control arm or are carried out in populations where BCG has been given previously. These and other cross-population observational studies that aimed to better understand the variable efficacy of BCG vaccination have shown that although we have immunological assays that read out the immunogenicity of BCG vaccination, we still lack proven correlates of protection. Induction of antigen-specific Th1 responses are part of the protective immune response but not sufficient. Antibodies and innate lymphoid cells have also been implicated. BCG vaccination has also been shown to induce beneficial non-specific effects. Epigenetic and metabolic changes in innate cells give rise to trained innate immunity which could explain such effects. Genetically modified BCG vaccines, or even repeat BCG vaccination may give enhanced protection. Exploiting the effects of particular metabolites, which by themselves appear to induce trained immunity, may give another means of improving BCG’s efficacy. What is clear is that this old vaccine still has much to teach us, and what we learn can inform the design of improved vaccines for TB as well as providing protection against other infections in young children as well as immunotherapies for use in autoimmune disease.

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Non-specific effects of vaccines and nanoparticles: potential use in cancer patients Magdalena Plebanski School of Health and Biomedical Sciences, RMIT University, Melbourne, Victoria, Australia The immune systems reactions to particulate material has been selected over evolution based on environmental challenges, including nano and micro-sized viruses, bacteria and parasites. However, such reactions have to be balanced against the need to maintain homeostasis in response to internal particulate materials such as exosomes, as well as non- lethal external particles, such as non-toxic fine and ultra-fine particulates in the air. Even as current vaccines elicit sophisticated non-specific effects, that re-frame immune system process, with significant impact on overall morbidity and mortality in high burden environments, new generation vaccines engaging synthetic nanotechnology, are also likely to trigger non-specific effects, based substantially on the rules of interaction established over evolution between particulates and immunity, particularly at the level of antigen presenting cells. Over the years we have mapped some of these rules, and show that nanoparticles and microparticles used as vaccine components can elicit large non-specific effects, capable of accelerating the clearance of infections such as malaria and influenza, as well as preventing the elicitation of allergic airways inflammation. These non-specific effects are in turn modulated by particle size, shape and charge. For example, dendritic cells exquisitely internalize nanoparticles 40-60 nm, whereas other phagocytes prefer to take up nanoparticles 500-2000 nm. After being taken up, synthetic nanoparticles also signal inside these cells, promoting epigenetic imprinting and altered antigen presenting cell reactivity. These studies are supporting the development of new generation synthetic nanovaccines, with a priori defined non-specific effects, to treat cancer. We will further finalize this presentation with considerations on the nature of immune systems alterations in cancer patients, specifically ovarian cancer patients, which can alter their response to vaccines, including those based on new generation synthetic nanoparticles.

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BCG and DTPw vaccination induce altered programmes of “trained” innate immunity in mice.

David J. Lynn, Marjolein van Wolfswinkel 1, Natalie Stevens 1, Georgina Eden 1, Miriam A. Lynn 1, Damon Tumes 2

1 Computational and Systems Biology Program, Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia.

2 Allergy and Cancer Immunology Laboratory, Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia.

3 College of Medicine and Public Health, Flinders University, Bedford Park, South Australia.

Vaccines prevent >2 million deaths annually by eliciting antigen-specific adaptive immunity, however increasing evidence suggests that vaccines also elicit nonspecific effects on morbidity and mortality unrelated to the targeted disease. Live vaccinations (e.g. BCG) are generally associated with beneficial nonspecific effects, while some inactivated vaccines (e.g. DTPw) have controversially been associated with increased all-cause mortality. We hypothesised that BCG and DTPw induce altered programmes of 'trained innate immunity' i.e. epigenetic reprogramming which alters innate responses to subsequent stimuli. To investigate this, we immunized 4-week-old mice with BCG or DTPw alone, or with both vaccines in two different schedules (BCG followed two weeks later by DTPw or DTPw followed by BCG). At 12 weeks post-immunisation, we assessed cytokine responses in monocytes and DCs by ICS, following stimulation with bacterial, fungal or viral antigens. BCG immunisation led to significantly increased TNF production by monocytes following stimulation with heat-killed Candida albicans (HKCA), as has been previously reported. In contrast, DTPw immunisation induced significantly increased IL6 production by DCs stimulated with either HKCA or LPS. Interestingly, immunisation with BCG prior to DTPw supressed IL6 production compared to DTPw alone or mock vaccination. Our findings indicate that immunisation of mice with BCG or DTPw induces altered programmes of trained innate immunity altering cytokine responses to subsequent microbial encounters. Furthermore, our data demonstrate that previously administered vaccines may modify the programme of trained innate immunity induced by other subsequently administered vaccines. Our ongoing studies are now investigating the transcriptomic and epigenetic basis for the observed phenotypes.

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Exploring the role of RORa on trained immunity in human monocytes

Gizem Kilic, Simone JCFM Moorlag, Rob ter Horst, Valerie ACM Koeken, L Charlotte J de Bree, Vera P Mourits, Leo AB Joosten, Jorge Domínguez-Andrés, Mihai G Netea

Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

Bacille Calmette-Guérin (BCG) is a live attenuated vaccine that has been traditionally used against tuberculosis. Besides the development of strong and fast memory B and T cell responses upon encounter with Mycobacterium tuberculosis, recent studies have shown that BCG vaccination is also able to improve protection against non-specific infections by mediating epigenetic and metabolic reprogramming of innate immune cells such as monocytes, neutrophils and NK cells, what has been termed "trained immunity". However, the strength and degree of BCG-induced trained immunity highly varies among people and depends on age, sex and genetic variants. Among them, the transcription factor RORα is known for its role as a regulator in the inflammatory response in monocytes and cholesterol metabolism. Preliminary analyses in two cohorts of healthy volunteers showed that several SNPs related to RORα might alter inflammatory responses and immune cell training after BCG vaccination. With this in mind, we investigated the potential role of RORα on BCG- induced trained immunity. To test the role of RORα on training, human monocytes isolated from buffy coat were stimulated with BCG (5 µg/ml) in the presence and absence of RORα inhibitor SR3335 for 24 hours, then cells were washed, and fresh media were added. Following 5 days of resting period, cells were re-stimulated with LPS for 24 hours, and cytokine levels were measured by ELISA. To study if IL-1 signaling has role on training promoted by RORα inhibition, IL-1 signaling was blocked by interleukin 1 receptor antagonist (IL-1RA), and monocytes were trained with BCG in presence of SR3335. Data revealed that inhibition of RORα by SR3335 significantly increased TNFα and IL-6 cytokine production in BCG-trained monocytes, in a dose dependent manner, upon re- challenge with LPS. Furthermore, even in the absence of training stimuli, pre-exposure of monocytes to SR3335 elevated pro-inflammatory cytokine production following re-stimulation of monocytes with LPS. Of note, RORα inhibition with or without TLR ligands and BCG stimulation for 24 hours did not lead to further increase in pro-inflammatory cytokine production, indicating that RORα inhibition does not cause acute inflammation. Lastly, impaired IL-1 signaling did not abolish increased production in TNFα and IL-6 mediated by RORα inhibition. Collectively, RORα was identified as a modulator of BCG-induced trained immunity in monocytes. To understand its role and regulatory function in detail, future studies will be focusing on whether RORα inhibition regulates monocyte metabolism and epigenome, similar to what is observed after BCG-induced trained immunity.

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Systemic bacterial burden of sepsis-challenged neonatal mice significantly associated with survival.

Byron Brook*, Danny Harbeson*, Nelly Amenyogbe, Rym Ben-Othman, Tobias R. Kollmann‡, Radhouane Aniba‡. *,‡ authors contributed equally

University of British Columbia, Department of Experimental Medicine.

Neonates suffer a disproportionately high rate of mortality due to infectious disease compared to adults, and neonatal interventions have had modest success in reducing mortality. This has largely been attributed to different neonatal specific immune responses, but the mechanism of how neonatal systems differ from adults has not been fully explored. Understanding what controls life and death can identify biologically important targets for future preventative treatments like vaccines, or future therapies, and death from bacterial sepsis can be driven by hyper-inflammation, or bacterial-induced organ damage. A neonatal mouse model of bacterial sepsis was employed to determine what controls survival. Observing neonatal mice was essential for age-dependent responses, but were limiting due to mice being too small for serial sampling. To obtain systemic samples mice were euthanized, but then final outcome could not be directly observed, therefore we required a method of predicting outcome so that systemic responses could be correlated with survival. Measures of health like righting reflex, degree of mobility, and weight were observed throughout the course of disease, and final outcome noted to determine whether the health features could be used to separate survivors from non-survivors. Each health feature was not accurate enough on its own to predict mortality, but their combination through algorithm and machine learning techniques led to the development of a predictor with repeated 85% accuracy and 93% sensitivity. This predictor of mortality allowed for the euthanasia of mice, collection of samples, and determination of whether systemic bacterial burden was correlated with survival. Mice predicted to survive had 10 - 1000 times reduced bacterial burden compared to non-survivors. This implicated bacterial clearance as an essential functional mediator of outcome, and that a 1 - 3 log reduction in systemic bacterial burden was physiologically relevant to survival. These results however do not rule out inflammation induced damage, which is an area of study for future experiments. It is important to measure the pathways that actually associate with life and death during the implementation of new neonatal vaccines and therapies to increase the chance of success for the application of new interventions to human cohorts in preventing death and saving lives. The ability to predict mortality can be used to further explore how neonates respond to sepsis, but could also be adapted and employed to other challenge or vaccination models.

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Age-associated sex differences in immunity to seasonal inactivated influenza vaccines Sabra L. Klein, Johns Hopkins Bloomberg School of Public Health Influenza is an ongoing threat to human health, despite the recommended annual vaccination. Among adults, females typically develop greater vaccine-induced immunity and protection than males, which is caused by sex differential epigenetic modifications in X- linked genes in B cells as well as sex steroid signaling. Furthermore, inactivated as well as universal influenza vaccine-induced immunity declines with age, but to a greater degree in females than males, which is associated with hormonal changes occurring during reproductive senescence. Taken together, both sex steroids and X-linked genes affect antibody production and results in sex-specific differences in the efficacy of vaccination against influenza.

This work was supported by the NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N272201400007C and the NIH/ORWH/NIA Specialized Center of Research Excellence in Sex Differences U54AG062333.

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Vertically transferred immunity in neonates: Mothers, mechanisms and mediators Petra Clara Arck1 1Laboratory for Experimental Feto- Maternal Medicine, Department of Gynecology and Obstetrics, University Clinic of Hamburg- Eppendorf, Germany ([email protected]) Early life immunity is fragile and neonates are prone to infections, which creates a major threat to offspring’s survival and future health. A sex-specific bias has often been reported, with boys being more susceptible to early life infections. Besides congentinal infections, vaccine-preventable infectious diseases pose a threat to neonatal immunity, as their immune system is still immature. However, the vulnerability during infancy can be mitigated by the transplacental transfer of pathogen-specific antibodies and possibly other mediators of immunity from mother to the fetus during pregnancy, followed postnatally by breast milk- mediated immunity. Since this largely antibody-mediated passive immunity can prevent the newborn from infections, neonatal immunity depends strongly on the maternal immune status, including the concentration of respective specific antibodies during pregnancy. Moreover, emerging concepts propose that mothers may transfer immunity to the newborns via vertical transfer of immune cells. Here, a preferential differentiation of hematopoietic stem and progenitor cells towards myelopoiesis induced by maternal immune cells could be detected in fetal mice, along with an improved resilience against cytomegalovirus infection in neonates. In humans, low number of maternal immune cells in cord blood have been associated with an increased number of infections in the first year of life, primarily in male newborns. Hence, neonatal immunity is crucially determined by the mother and adjustments of e.g. prenatal vaccination regimes may mitigate the risk for early life infections.

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Female-biased effects of aging on a chimeric hemagglutinin-based universal influenza virus vaccine in mice

Santosh Dhakal (1), Sharvari DeshPande (1), Meagan McMahon (2), Florian Krammer (2), Sabra L. Klein (1)

(1) Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA, 21205. (2) Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029.

Adult females, both in humans and mice, develop greater antibody responses to inactivated influenza vaccines (IIV) than males, which has implications for vaccine efficacy. The female- biased immunity to IIV diminishes with advanced age. Inactivated influenza vaccines are 10- 60% effective and, therefore, efforts to develop more efficacious influenza vaccines are underway. Targeting the conserved stalk region of the hemagglutinin (HA) protein of influenza viruses has shown promise as a universal influenza vaccine candidate. Whether biological sex and age affect immunity and protection provided by universal influenza vaccines has not been evaluated and formed the basis of this study. We used chimeric HA (cHA)-based universal influenza vaccine constructs expressing the same H1 stalk with an irrelevant head from H9, H11, or H12 influenza A viruses. Adult (8-10 weeks) or aged (68-70 weeks) C57BL/6 mice were immunized with influenza B virus expressing cH9/1, followed by cH11/1 and cH12/1 protein and subsequent challenge with mouse-adapted 2009 H1N1. Following vaccination, adult mice immunized with the chimeric HA-based universal influenza vaccine developed significantly greater serum IgG, and IgG2c but not IgG1 antibody titers against different group 1 influenza A viruses than vaccinated aged mice. The age-specific decline in vaccine-induced antibody titers was significantly more pronounced in female than male mice. After virus challenge, vaccinated aged females experiences greater morbidity than vaccinated adult females, with age-associated changes in protection being significantly less pronounced among males. The extent to which age-associated changes in sex steroid hormones, including estradiol, contribute to immunosenescence is currently under investigation. These data suggest female-biased effects of aging on cHA-based universal influenza vaccine-induced antibody responses and protection and highlight the importance of inclusion of sex and age in the design and dosage of diverse influenza vaccine platforms.

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BCG vaccines off target effects: so many good things with an emphasis on type 1 diabetes Denise Faustman Massachusetts General Hospital, USA

Abstract unavailable at time of printing

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Sex-differential effects of rabies vaccine on survival in young free-roaming dogs, a novel animal model for the study of non-specific effects of vaccines in high-mortality populations Darryn L. Knobel1,2 (presenter), Sintayehu M. Arega1, Anne Conan1 1 Center for Conservation Medicine and Ecosystem Health, Ross University School of Veterinary Medicine, St. Kitts ([email protected]) 2 Department of Veterinary Tropical Diseases, University of Pretoria, South Africa The preponderance of epidemiological evidence in support of non-specific effects (NSEs) of vaccines is derived from populations with high burdens of mortality due to infectious and parasitic diseases. There are considerable methodological and ethical challenges to the study of vaccine NSEs in these populations. We propose a novel animal model system for the study of vaccine NSEs: populations of owned, free-roaming domestic dogs. Free- roaming dogs are a useful model for the epidemiological study of NSEs, due to their abundance, accessibility, high mortality rates due to infectious and parasitic diseases, and low rates of routine vaccination. The ethical acceptability of (indeed, encouragement of) surgical sterilization (gonadectomy) in these populations also provides avenues to investigate the role of gonadal sex hormones in modification of vaccine NSEs by sex. In a recent randomized controlled trial comparing the effect of animal rabies vaccine (ARV, an inactivated, aluminum-adjuvanted vaccine) on all-cause mortality against a placebo injection in such a population of young dogs, we found that ARV substantially increased the hazard rate of death in female but not male dogs. We also observed that, in the placebo group, male mortality was substantially greater than female mortality, an effect which we presume is due to greater male susceptibility to prevalent infectious and parasitic diseases in the population. We contend that these two findings–relatively low female compared to male mortality in the placebo group and increased female but not male mortality in the vaccinated group compared to the placebo group–are related and point to a plausible mechanistic explanation. We hypothesize that the detrimental effect of ARV (and possibly other vaccines) on female survival is mediated through an effect on the immune system that alters susceptibility to concurrent or subsequent unrelated infections, to which females are normally less susceptible than males due to the influence of sex-related factors (hormonal and/or genetic). We present a causal structure of this hypothesis, and propose to test it in populations of free-roaming dogs through factorial randomized controlled trials, stratified by sex, that could feasibly investigate the joint effects of vaccination, treatment against concurrent or subsequent unrelated infections, and gonadectomy on mortality rates. Integrating measures of immunological changes following interventions will allow evaluation of the mediating effects of the immune system. We propose that establishment of free- roaming dogs in high-mortality populations as a novel animal model system will open up new avenues for causal inference in the study of NSEs of vaccines.

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BCG vaccination is associated with reduced malaria prevalence in children under the age of five in sub-Saharan Africa

Mike Berendsen MSc1,2,3,4, Sjors van Gijzel BSc1 Jeroen Smits PhD2 Quirijn de Mast MD1 Peter Aaby DMSc3,5 Christine Benn DMSc3,4 Mihai Netea MD1,6,7 Andre van der Ven MD1

1 Department of Internal Medicine, Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands 2 Global Data Lab, Institute for Management Research, Radboud University, Nijmegen, The Netherlands 3 Research Center for Vitamins and Vaccines (CVIVA), Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark. 4 Open, Odense Patient Data Explorative Network, University of Southern Denmark, Odense University Hospital, Odense, Denmark 5 Bandim Health Project, Indepth Network, Bissau, Guinea-Bissau. 6 Department for Genomics & Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, Germany 7 Human Genomics Laboratory, Craiova University of Medicine and Pharmacy, Craiova, Romania

Introduction Malaria continues to be a major cause of morbidity and mortality in sub-Saharan Africa (SSA) without effective interventions. Bacillus Calmette-Guérin (BCG) vaccine possesses protective non-specific effects (NSEs). This study explores whether BCG is associated with protection against malaria in children under the age of five in SSA. Methods We used data from the Demographic Health Survey (DHS) program, including 34,206 children from 13 SSA countries. BCG status was taken from vaccination cards when present; if not, mother's recall was used. Presence of malaria was defined as a positive rapid diagnostic test (RDT). Maternally reported presence or absence of fever in the previous two weeks defined symptomatic status. Multilevel logistic regression was used to account for the two-stage cluster sampling method. Results Of the 34,206 children, 12,325 (36.0%) children were malaria positive and 29,766 (87.0%) were BCG vaccinated. BCG vaccination was associated with a lower malaria prevalence (adjusted Odds Ratio (aOR)=0.94, [0.90 to 0.98], especially among children of whom BCG information was retrieved from a vaccination card (aORcard=0.88, [0.82 to 0.94]). Restricting the analysis to children from regions with suboptimal BCG coverage increased the association (aORcard=0.81, [0.73 to 0.89]). BCG associations were similar for asymptomatic (aORcard=0.86, [0.81 to 0.92]) and symptomatic malaria (aORcard=0.89, [0.78 to 1.01]). Conclusion BCG vaccination is associated with protection against malaria. These results indicate a possible role for timely BCG vaccination in the protection and elimination of malaria. If confirmed in further research, our findings have substantial implications for global efforts to reduce malaria burden.

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The Impact of Immunosenescnece on Immune Responses after Influenza Vaccination in Older Individuals

Inna G. Ovsyannikova, Richard B. Kennedy, Iana H. Haralambieva, Diane E. Grill, Gregory A. Poland

Mayo Clinic Vaccine Research Group, Mayo Clinic, Rochester, Minnesota, USA

Influenza is a major threat to human health worldwide and is responsible for up to 650,000 deaths globally each year. Age and immunosenescence, and their impact on immune responses after influenza vaccination, are of increasing importance for the development of better vaccines for the elderly. The aim of this study was to examine associations between age and immunosenescence markers and vaccine-induced immune responses and identify immunosenescence- associated differences in gene expression and gene regulation in an older study population receiving seasonal influenza A/H1N1 vaccination. A cohort of 159 healthy recipients who received seasonal TIV (Fluarix) was used for this study. Baseline median HAI/VNA antibody titers (1/80; IQR 1/40-1/320) demonstrated the presence of pre-existing antibodies. A positive correlation (r=0.49) was observed between the %CD4+CD28- and the %CD8+CD28- cells and a negative correlation (r=-0.33) was found between TREC and the %CD8+CD28- cells. Telomerase (TERT) activity was positively correlated with the observed increase in the influenza-specific memory B-cell Elispot response at Day 28 compared to baseline (p=0.025). T cell receptor excision circle (TREC) levels were positively correlated with the baseline and early (Day 3) influenza A/H1N1-specific memory B-cell Elispot response (p=0.04 and 0.035, respectively). We identified an interconnected set of gene expression pathways (antigen processing/presentation, MAPK, mTOR, TCR, BCR, calcium signaling, and other cellular activities) that were associated with immunosenescence markers. Using miRNA sequencing, several miRNAs (miR-320a, miR-320b, and miR-320d) associated with markers of immunosenescence, such as age, %CD4+CD28- cells and CD4+/CD8+ T cell ratio (p<0.003), were discovered. We found several CpG methylation sites (examples include KLF14 regulating TGFβ signaling and TSPAN33 having a role in cell differentiation) associated with age within the genes controlling immune function. In conclusion, our findings provide further evidence that immunosenescence is a complex phenomenon involving multiple aspects of immunity. These data suggest that influenza- specific immunity is significantly influenced by age, and that specific markers of immunosenescence are correlated with different humoral immune response outcomes observed after vaccination in older individuals.

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Evidence for immunomodulatory effects of vaccination in aging Australians Katie Flanagan Monash University, Australia Introduction: This talk will describe preliminary results from the VITAL Trial: Vaccine Immunomodulation Throughout the Aging Lifespan. The trial is taking a systems vaccinology approach to study how the diphtheria-tetanus-acellular pertussis (dTap) and seasonal influenza vaccines interact in aging Australians. It is further exploring the processes of immunosenescence and inflammaging that occur with advancing age, and how they impact vaccine immunogenicity. The rationale is that the elderly respond poorly to these vaccines and that non-targeted effects of vaccines have not been explored in the elderly. Methods: 300 adults (20-50 years) and 300 elderly (>65 years) individuals are being randomized into one of three vaccine groups. Group 1: dTap vaccination followed by seasonal influenza vaccination 4 weeks later; Group 2: influenza vaccination alone; Group3: dTap plus influenza vaccination. Blood is taken at multiple timepoints for transcriptome analysis, epigenetics, multicolour flow cytometry, cytokine multiplex and vaccine antibodies, FBC, CRP; and stool for microbiome sequencing at baseline. Reactogenicity / adverse event data are collected at each study visit. The trial is registered at www.clinicaltrials.gov identifier NCT02765126. Results: To date 264 people have been recruited into the study. Herein, we report reactogenicity, CRP, FBC and influenza antibody data. Preliminary flow cytometry, microbiome and transcriptome data will also be presented. Prior or concomitant dTap negatively affects influenza antibody levels in younger males and the elderly. CRP and FBC parameters were altered by dTap vaccination and the dual vaccination group experienced significantly greater reactogenicity. Conclusion: Prior or concomitant dTap vaccination impacts responses to seasonal influenza vaccination in this interim analysis of an ongoing trial. Disclosure of Interest Statement: KLF has recently served on the influenza vaccine advisory boards for Seqiris and Sanofi- Pasteur but they played no role in the design or funding for this study. She is a member of the Australian Technical Advisory Group on Immunisation but this abstract represents her personal views and not necessarily those of ATAGI.

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Specific and off-target effects of novel live attenuated pertussis vaccine Camille Locht Center for Infection and Immunity of Lille, Inserm U1019, CNRS UMR8204, Institut Pasteur de Lille Pertussis, mainly caused by Bordetella pertussis, is a severe respiratory disease affecting all age groups, but is most severe in less than 6-old months infants. While the introduction of vaccines in the mid-20th century has led to a substantial decrease in the incidence of the disease, it has not been controlled in any country, despite a > 85% global vaccine coverage. In recent years many countries have even experienced a dramatic resurgence of pertussis. Although several factors may contribute to this resurgence, the most parsimonious explanation appears to be asymptomatic transmission of B. pertussis. None of the currently available vaccines prevent infection by and transmission of B. pertussis, while they provide good protection against disease. Unlike current vaccines, infection by B. pertussis induces sterilizing immunity. We have thus developed a live attenuated pertussis vaccine to be delivered nasally. This vaccine, named BPZE1, was shown to be safe in pre-clinical models, including severely immunocompromised mice, and to provide strong protection against nasal infection in both mice and non-human primates. Clinical studies have shown that it is safe in humans, up to a dose of 109 CFU, able to transiently colonize the human respiratory tract and to induce B- and T-cell responses to B. pertussis antigens after a single nasal delivery. This vaccine is currently undergoing a clinical phase 2 trial involving 300 volunteers. In pre- clinical studies we found that BPZE1 protects also against other Bordetella species, such as Bordetella parapertussis and Bordetella bronchiseptica. Interestingly, protection against B. bronchiseptica was based on a dual mechanism: antigen-specific inhibition of colonization, due to cross-reactive antigens, and non-specific inhibition of B. bronchiseptica-induced inflammation. This latter off-target effect was further investigated in Bordetella-unrelated infectious models. BPZE1 was found to abolish lung inflammation, cytokine storm and death induced by influenza virus A, although no cross-reactive B- or T-cell epitope exist between the influenza virus and BPZE1. Similarly, in a murine model of RSV infection, the vaccine showed protection that lasted for at least 8 weeks and was found to depend on BPZE1- induced IL-17. Furthermore, in a model of non-infectious inflammation, such as allergic asthma, vaccination with BPZE1 prior to sensitization or between sensitization and challenge with house-dust mite prevented lung inflammation and restored the respiratory capacity of the mice. These observations are in line with important off-target effects of live attenuated vaccines, but, in contrast to other vaccines, BPZE1 has potent anti-inflammatory properties, without being immunosuppressive.

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Impact of adjuvants, sex, and the microbiome on SIV-vaccine-induced immunity and protective efficacy in rhesus macaques Thomas Musich1, Vishal Thovarai2, Slim Fourati3, David J. Venzon4, Venkatramanan Mohanram1, Iskra Tuero1, Leia K. Miller-Novak1, Sabrina Helmold Hait1, Mohammad Arif Rahman1, Ruth Hunegnaw1, Wuxing Yuan2, Colm O’hUigin2, Mark J. Cameron5, Luca Schifanella6, Monica Vaccari6, Yongjun Sui7, Celia Labranche8, David Montefiori8, Jay A. Berzofsky7, George N. Pavlakis9, Barbara K. Felber10, Genoveffa Franchini6, and Marjorie Robert-Guroff1 1Immune Biology of Retroviral Infection Section, 4Biostatistics and Data Management Section, 6Animal Models and Retroviral Vaccines Section, and 7Molecular Immunogenetics and Vaccine Research Section, Vaccine Branch, NCI, Bethesda, MD; 2Cancer and Inflammation Program, NCI, Bethesda, MD; 3Department of Pathology and 5Department of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH; 8Department of Surgery, Duke University Medical Center, Durham, NC; 9Human Retrovirus Section and 10Human Retrovirus Pathogenesis Section, Vaccine Branch, NCI, Frederick, MD.

We compared two vaccine strategies in the SIV rhesus macaque model that aimed to elicit mucosal as well as systemic immunity. Rhesus macaques were mucosally primed (first via intranasal and oral routes and next by the intratracheal route) with replicating Adenovirus SIV-recombinants encoding SIV Env and Gag. Subsequently they were boosted twice intramuscularly with ALVAC encoding SIV Gag, Pro, and Env plus Env protein in alum, or DNA encoding SIV Env, Gag, and rhesus IL-12 plus Env protein in alum. Both regimens induced strong viral-specific cellular and humoral immune responses in mucosal and systemic compartments. Following repeated low-dose intrarectal SIV challenges, significant delay of viral infection in vaccinated macaques compared to adjuvant controls that received empty Adenovirus vector plus alum only was not observed. However, delayed SIV acquisition in both the ALVAC/Env and DNA&Env vaccine arms and adjuvant controls was observed when compared to naïve controls. Transcriptomic analysis identified signatures associated with protective innate immunity. Vaccinated females that became infected, but not males, exhibited significantly lower acute viremia compared to same-sex controls, confirming our previously reported sex-difference in SIV vaccine outcomes. The rectal microbiome of females and males responded differently to the prime-boost regimen, especially following the recombinant adenovirus priming immunizations, and differentially associated with viremia control and systemic and mucosal humoral immunity. We conclude that the impact on protective efficacy of vaccine-induced microbiome alteration in males and females as well as trained innate immunity are factors of significant importance in vaccine outcome.

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Precision adjuvants for next generation vaccines tailored to vulnerable populations Ofer Levy MD, PhD Director, Precision Vaccines Program, Boston Children’s Hospital Professor, Harvard Medical School Associate Member, Broad Institute of MIT & Harvard Vaccines are the most important biomedical intervention over the past century, yet their path to discovery and development has been ad hoc, slow and frequently unsuccessful. Moreover, distinct immunity of the young and elderly renders these populations simultaneously more susceptible to infection as well as more difficult to successfully protect via immunization. Next generation approaches to de-risk and accelerate effective vaccine development include targeted clinical studies coupled with systems vaccinology to generate hypotheses re vaccine immunogenicity, population-specific human in vitro modeling for discovery of novel adjuvants and to characterize mechanism of action and benchmark to licensed vaccines, as well as select animal models that . While live vaccines have the potential to induce trained immunity and heterologous benefit, this approach is limited by divergence of vaccine strains propagated under diverse conditions, as is noted with Bacille Calmette-Guérin (BCG), as well as by the risk of inadvertent administration of a live vaccine to those with primary immunodeficiencies. In this context, identification of scalable small molecule adjuvants that are effective towards a target population is an attractive solution to enhance vaccine effectiveness in the most vulnerable. Supported by the U.S. National Institutes of Health/National Institute of Allergy & Infectious Diseases, the Precision Vaccines Program has undertaken several rounds of small molecule screening against human leukocytes that have identified several lead families targeting diverse pattern recognition receptors. Remarkably, there is a distinct ontogeny to innate immune responses to adjuvants and their combinations with distinct activity towards leukocytes that can be synergistic, additive or antagonistic depending on the age of a study participant. The horizon for progress towards next generation vaccines includes adjuvanted vaccines tailored to vulnerable populations that enhance both specific and heterologous (“non-specific”) immunity.

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New live attenuated tuberculosis vaccine MTBVAC induces trained immunity and confers protection against experimental lethal pneumonia

Jorge Domínguez-Andrés, Raquel Tarancón, Santiago Uranga, Anaísa V. Ferreira, Laszlo A. Groh, Mirian Domenech, Fernando González-Camacho, Niels P. Riksen, Nacho Aguilo, José Yuste, Carlos Martín, Mihai G. Netea

Department of Microbiology, Faculty of Medicine, University of Zaragoza, Spain CIBERES and Research Network on Respiratory Diseases, Spanish Ministry of Health and Instituto de Salud Carlos III, Madrid, Spain Department of Internal Medicine and Radboud Center for Infectious diseases (RCI), Radboud University Nijmegen Medical Centre, Geert Grooteplein 8, 6500HB Nijmegen, the Netherlands. Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, 4050- 313, Portugal. Centro Nacional de Microbiología. Instituto de Salud Carlos III, Madrid, Spain Servicio de Microbiología, Hospital Miguel Servet, ISS Aragón, Zaragoza, Spain Department for Genomics & Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, 53115 Bonn, Germany Human Genomics Laboratory, Craiova University of Medicine and Pharmacy, Craiova, Romania

Among the infectious diseases, tuberculosis is the leading cause of death worldwide, and represents a serious threat especially in developing countries. The protective effects of Bacillus Calmette-Guerin (BCG), the current vaccine against tuberculosis, have been related not only to specific induction of T-cell immunity, but also with the long-term epigenetic and metabolic reprogramming of the cells from the innate immune system through a process termed trained immunity. Here we show that MTBVAC, a live attenuated strain of Mycobacterium tuberculosis, that has been shown to be safe and immunogenic against tuberculosis antigens in adults and newborns, is also able to induce trained immunity through the induction of glycolysis and glutaminolysis, accumulation of histone methylation marks at the promoters of proinflammatory genes, facilitating an enhanced response after secondary challenge. Importantly, these findings in human primary myeloid cells are complemented by a strong MTBVAC-induced heterologous protection against a lethal challenge with Streptococcus pneumoniae in an experimental murine model of pneumonia. Collectively, our evidence underlines that MTBVAC is able to exert metabolic and epigenetic immunomodulatory effects through mechanisms similar to those of BCG. In line with this, vaccination with MTBVAC is likely to recapitulate the non-specific protective effects of BCG vaccination against heterologous infections, while at the same time improving the specific responses against Mtb infection. All together, these results underline the potential that MTBVAC has as an excellent candidate for mass vaccination, representing a possible improvement compared to the currently available BCG vaccine.

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S48

MV130 as a Trained Immunity-based Vaccine, clinical implications and mechanism of action

Antonio Nieto García 1, Paola Brandi 2, Laura Conejero 2,3, Ángel Mazón 1, María Nieto 1, Francisco J. Cueto 2, Sarai Martínez-Cano 2, Paula Saz-Leal 2,3, Michel Enamorado 2, Joaquín Amores-Iniesta 2, Miguel Casanovas 3, José Luis Subiza3, David Sancho2

1 Servicio de Alergia y Neumología Pediátrica. Hospital Universitario La Fe. Valencia, Spain 2 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) Madrid, Spain. 3 Inmunotek S.L., Madrid, Spain

Background Despite recurrent wheezing affects one third of children and is, therefore, a global health problem with considerable expenditure and impact on quality of life, specific vaccines licensed for its prevention are still lacking. Virtually all wheezing attacks (WA) in young children are of viral etiology. In clinical practice, bacterial immunostimulants have offered a strategy for the prevention of recurrent respiratory tract infections, including those of viral origin. However, the underlying immune mechanisms are poorly understood. Trained immunity-based vaccines (TIbV) have been recently described as those composed of trained immunity inducers that are intended to be effective not only against the specific pathogens targeted by the vaccine, but also against heterologous pathogens. Aim To evaluate the efficacy and safety of a sublingual bacterial preparation (MV130) based on heat‐inactivated whole‐cell bacteria in preventing WA in children, and to investigate in vitro and in experimental models, its underlying mechanism of protection. Methods A phase III randomized, double‐blind, placebo‐controlled, parallel‐group trial, including 120 children <3years old with ≥3 WA during the previous year was performed. They were randomly allocated to receive active treatment (MV130) or placebo for six months; the primary endpoint was the number of WA within one year after the first dose. The main secondary endpoints were the duration of WA (days) and symptoms and medication scores. Mechanisms of action of MV130 were addressed experimentally in mouse models of viral and fungal infection and in vitro assays in human monocytes. Results A reduction of 40% in the number of WA in the active vs the placebo group (p<0.001) was observed with an excellent safety profile. Secondary outcomes were also significantly improved in the active group vs placebo. Both outcomes were significant not only during the treatment (1st half year) but once it was discontinued (2nd half year). MV130 induces trained immunity hallmarks in human monocytes (enhanced glycolytic metabolism and cytokine production), and in vivo against C. albicans reinfection. In addition, administration of MV130 in mice confers protection to intranasal viral infection mediated by trained immunity. Discussion We provide evidence for the first time that a mucosal polybacterial preparation induces trained immunity, confers protection against unrelated pathogens, and shows a quite long lasting clinical efficacy against a virus‐related condition such as recurrent WA. Although the TIbV concept is emerging, we believe that several current anti-infectious vaccines and immunostimulants, including MV130, may already fall in this category.

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Poster Presentations

Non-live rabies vaccine at six weeks of age increases all-cause mortality rate in female but not male puppies: a randomized controlled trial

Sintayehu Arega, Anne Conan, Darryn Knobel

Center for Conservation Medicine and Ecosystem Health, Ross University School of Veterinary Medicine, St. Kitts.

Epidemiological evidence suggests that some non-live vaccines may have non-specific detrimental effects in females but not males in high-mortality populations. We investigated this proposition in a randomized controlled trial in a population of owned, free-roaming dogs. The objectives of the trial were to evaluate whether injection of non-live rabies vaccine (RV) in puppies at 6 six weeks of age affected survival (to all causes of mortality) through to 13 weeks of age, compared to injection of sterile water (SW), and to determine whether this effect was modified by sex. The study design was a single-site, owner-blinded, randomized, placebo-controlled trial in South Africa. At six weeks of age, puppies born to vaccinated mothers (dams), were randomly assigned within litters in a 1:1 ratio to receive a subcutaneous injection of either RV or SW. A total of 358 puppies were randomized (with approximately equal male-to-female ratio in each group). The primary outcome was death to all causes through seven weeks after injection. Effect size was estimated using a mixed- effects Cox proportional hazards model stratified by body weight and adjusting for sex, with a random effect for litter. Mortality rate was 2,664/1,000 dog-years in the RV group and 1,955/1,000 dog-years in the SW group (hazard ratio 1.35; 95% confidence intervals (CI) 0.83-2.18). The effect of RV on survival was modified by sex (p-value of likelihood ratio test for interaction = 0.02): In the RV group relative to the SW group, female survival was reduced (hazard ratio in females 3.09; 95% CI 1.24-7.69) while there was no evidence that survival in males was affected (hazard ratio in males 0.79; 95% CI 0.41-1.53). We conclude that non-live rabies vaccine at six weeks of age increased all-cause mortality in female puppies, with no evidence of an effect in male puppies. Further randomized controlled trials should be conducted in other high-mortality dog populations to confirm these findings. Free- roaming dog populations provide opportunities to test additional hypotheses that may otherwise be challenging in humans, including the effects of vaccination with live and non- live vaccines.

P1

Non-specific effects of rabies vaccine on mortality in a Danish pig herd

Kristoffer Jarlov Jensen, Lola Kathe Tolstrup, Darryn Knobel, Charlotte Sonne Kristensen, Gregers Jungersen, Christine Stabell Benn

The new malaria vaccine RTS,S was recently tested in phase-III randomized controlled trials in malaria-endemic African sites. Despite a modest specific protection against malaria, girls receiving the malaria vaccine had a worryingly 2-fold increased all-cause mortality compared to the comparator vaccine recipients. The inactivated rabies vaccine was used as a comparator vaccine in one of the study arms. The explanations for the adverse association could be a female specific detrimental non-specific effect of the new non-live malaria vaccine. Alternatively, it was hypothesised that the comparator rabies vaccine had beneficial protective effects on mortality, corroborated by reduced meningitis incidences among the rabies vaccine recipients. A few historical experimental murine studies and lately also an observational study in free roaming dogs have indicated that rabies vaccine may non- specifically reduce non-rabid mortality. Overall, whereas there is a growing body of evidence of non-specific effects of human vaccines, there is a paucity of systematic investigations of potential non-specific effects of veterinary routine vaccines. The present study was designed to investigate if the veterinary inactivated rabies vaccine may have non-specific effects of morbidity and mortality in young pigs.

In a Danish herd of commercial pigs free of wildtype rabies virus using a 2x2 factorial randomized design, 575 pregnant sows (2-3 weeks before scheduled farrowing) and 5747 of their subsequent offspring (median 6 days of age) were allocated (1:1) to rabies vaccine or no rabies vaccine. Except for the rabies vaccine, all animals were treated as customary. Death or antibiotics treatment were observed until departure from the observation stables (approximately 12 weeks of age / 20 kgs).

Until 21 days of life, overall mortality was 1.6% (90 pigs died, rabies vaccine: n=50; control: n=40), the mortality rate ratio being 1.27 (95% confidence interval: 0.85-1.90). Prior sow rabies vaccination did not modify the effect of neonatal vaccination. Nonetheless, for mortality as well as antibiotic treatments within 21 days of life, there was non-significant indication of a beneficial effect of rabies vaccine in female piglets, but a negative effect in male piglets from rabies-naïve sows, and a negative effect of rabies vaccine in female piglets from rabies-vaccinated sows. These effects had waned at 12 weeks of age.

With a slightly higher mortality in the piglets receiving inactivated rabies vaccine, the study did not lend support to the proposed beneficial effect of the rabies vaccine used in the human malaria vaccine trial in African children. Although the low mortality impaired the power of subgroup analyses, the study indicated an interacting effect modification by sex and maternal vaccination.

P2

BCG vaccination induces innate immune training in adults over 50 years of age.

Mike Berendsen1,2,3, Pauli Bles1,2, Charlotte de Bree1, Kristoffer Jensen2,4, Clara Jensen5, Delfim Mendes6, Mihai Netea1,7,8, Christian Wejse9,10, Christine Benn3,5

1 Department of Internal Medicine, Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands. 2 Bandim Health Project, Indepth Network, Bissau, Guinea-Bissau. 3 Open, Odense Patient data Explorative Network, University of Southern Denmark, Odense University Hospital, Odense, Denmark. 4 Experimental and Translational Immunology, Department of Health Technology, Technical University of Denmark, Kgs Lyngby, Denmark 5 Research Center for Vitamins and Vaccines (CVIVA), Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark 6 Hospital Raoul Follereau, Bissau, Guinea-Bissau 7 Department for Genomics & Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, Germany 8 Human Genomics Laboratory, Craiova University of Medicine and Pharmacy, Craiova, Romania 9 Department of Public Health, Aarhus University, Aarhus, Denmark 10 Department of Infectious Diseases, Skejby University Hospital, Aarhus, Denmark

Introduction Bacillus Calmette-Guérin (BCG) was developed as a vaccine against tuberculosis, but many studies have shown its ability to induce potent protection against other infectious diseases. These non-specific effects (NSEs) could be explained by an increased production of innate cytokines after BCG vaccination, the so called 'innate immune training', which has been seen in children and adolescents. However, it is not known if BCG induces similar responses in elderly. Methods Randomized placebo-controlled trial (RCT) among 40 HIV-negative, TB-negative, Bissau- Guinean adults above 50 years of age, randomized 1:1 to BCG-Japan or solvent. Levels of interleukin (IL)-1β, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α were measured from supernatants of peripheral blood mononuclear cells (PBMCs) stimulated with heat-killed M. Tuberculosis, Lipopolysaccharide (LPS)/Phytohaemagglutinin (PHA), S. Aureus, C. Albicans and S. Pneumoniae before immunization, 2 weeks and 2 months after immunization. Results Among the 40 participants, only 2 (5%) had visible signs of previous BCG vaccination, while 32 (80%) had a positive Quantiferon test indicating that a large part of our population had either latent or passed M. Tuberculosis infection. Two weeks after vaccination, cytokine responses in the BCG-vaccinated group were lower than the responses in the solvent group. This was, however, completely reversed at two months after vaccination, where cytokine responses were highest in the BCG-vaccinated group; an increase that was slightly higher when restricted to Quantiferon positive individuals. Conclusion This study indicates that BCG vaccination can induce innate immune training also in adults above 50 years and might be an interesting tool in counteracting immunosenescence in elderly. The fact that responses were higher in Quantiferon positive individuals suggests a boosting effect regarding the NSEs of BCG.

P3

Lifestyle Differences Between Western European and African Populations Contribute to Differential Immune Responses in the Elderly

Ozlem Bulut, Godfrey Temba, Valerie Koeken, Simone Moorlag, Charlotte de Bree, Vera Mourits, Martin Jaeger, Jorge Dominguez Andres, Andre van der Ven, Leo A.B. Joosten, Quirijn de Mast, Mihai Netea

Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands

Impairment of adaptive immune responses with old age leads to higher susceptibility to infections and increased mortality. Adaptive immunity defects are also believed to be the underlying reason of poor vaccination efficiency in the elderly. Trained immunity is the adaptation of innate host defense which leads to non-specific immunological memory in innate immune cells through epigenetic and metabolic reprogramming. It is the proposed mechanism for non-specific protective effects of vaccines. Since innate immunity is mostly intact in the elderly, induction of trained immunity could be utilized to improve vaccination. The aim of this study is to identify the factors affecting the innate response in the elderly with the hope of identifying new targets for improving vaccine efficacy. To this end, metabolomic profiles were measured by high-throughput mass spectrometry from the plasmas of a Western European cohort of 324 healthy individuals between the ages of 18-71 and a Tanzanian cohort of 323 healthy individuals between the ages of 18-62. Metabolome data was analyzed using MetaboAnalyst v4.0. Out of 1376 metabolites, 57 were significantly more abundant and 77 were diminished in the elderly (>50 years old) in both cohorts compared to younger individuals. Pathway analyses revealed that metabolites from histidine metabolism and primary bile acid biosynthesis pathways were significantly downregulated in the elderly in both cohorts. When the elderly of the two cohorts were compared, 403 metabolites were significantly more abundant in Europeans. These include metabolites contributing to oxidative stress, whereas the 398 metabolites more abundant in the Tanzanians include metabolites with anti-oxidant and anti-inflammatory properties. Although age-related defects are observed in the production of T-cell derived cytokines such as IFNγ in both cohorts, there is also a lower production of monocyte-derived cytokines IL-6 and TNFα in Tanzanians in contrast to Europeans. These differences of the innate immune response could partially be explained with the different metabolic repertoire. Most of the metabolites which are highly different between two cohorts are food-derived and have not yet been linked to immunological functions. These results indicate that differences in lifestyle, especially diet, reflected by the metabolomic signature might influence the aging of the immune system. Both unique and shared metabolites found in the European and African cohorts would provide valuable insights to the mechanism of immune aging. The next step will be investigating the effect of the most differentially regulated metabolites on trained immunity response in vitro and at a later stage potentially in vivo.

P4

The influence of β-1,3-1,6-glucans on rabies vaccination titers in cats

Byrne J., Butaye P.

Ross University School of Veterinary Medicine

The aim of this study was to evaluate the immune stimulation capacity of a fully formulated diet with β-1,3-1,6 glucans in cats by assessing the rabies antibody titer after vaccination. Therefore, we recruited thirty-five healthy rabies antibody negative cats. The cats were broken into two groups and fed standard diet of feed in accordance to body weight. One group had the β-glucans incorporated; the other group served as control group. After two weeks, of digestive adjustment and immunological stimulation, the rabies vaccine (Imrab3TF) was administered on days 14 and 35. Blood samples were taken on days 14, 35, and 56. Titers are determined with the Rapid Florescent Foci Inhibition Test (RFFIT). Titers will be compared with the student t-test to determine significant differences between the titers of the two groups.

P5

EXPLORATIVE STUDY TO THE EFFECT OF BCG BOOSTER METHODS ON THE

Priya A. Debisarun 1, Gizem Kiliç 1, Charlotte de Bree 1, Jorge Domínguez-Andrés 1, Mihai Netea 1,2,3

1. Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands 2. Department for Genomics & Immunoregulation, Life and Medical Sciences Institute (LIMES), University of Bonn, 53115 Bonn, Germany 3. Human Genomics Laboratory, Craiova University of Medicine and Pharmacy, Craiova, Romania.

Rationale: the live attenuated vaccine Bacillus Calmette-Guérin is able to induce non- specific beneficial effects and protect against non-related infectious diseases via epigenetic and metabolic rewiring of innate immune cells, which has been termed 'trained immunity' or innate immune memory. However, the effect of BCG on innate immune responses wanes after three months, and the response is highly variable between individuals. Therefore, development of methods to boost the non-specific effects is of great importance. There are indications that the beneficial, non- specific effects of BCG and other live vaccines may be enhanced by means of revaccination. Albeit BCG revaccination is not a general WHO policy, there are many countries where this is standard care. This study will investigate the effect of BCG revaccination as well as high-dose primary BCG vaccinations on the induction and course of trained immunity in healthy volunteers. We hypothesize that this may increase the duration and strength of its beneficial non-specific effects, and thereby contribute to the changing perspective on the general immunological effects of vaccines and optimize the use of already existing vaccines. Objective: determine the effect of BCG revaccination and high dose BCG vaccination on the induction of innate immune memory. Study design: a randomized controlled explorative pilot study. Study population: 50 healthy adult volunteers. Intervention: participants will be randomized 3:3:3:1 to four different intervention groups. Group 1 will receive standard dose BCG vaccination followed by a subsequent standard dose BCG vaccination three months later. Intervention group 2 will receive placebo vaccination followed by a high dose BCG vaccination three months later. Intervention group 3 will receive placebo vaccination followed by standard dose BCG vaccination three months later. Intervention group 4 will receive two times placebo vaccination. Throughout the study blood will be drawn from healthy volunteers at timepoints 0, 3 months and 6 months, to analyze changes in immune responses. Main study parameters/endpoints: comparison of cytokine responses after ex-vivo PBMC and NK-cell re-stimulation, epigenetic and RNA transcriptome profiles.

P6

Timeliness of DTaP-IPV-Hib vaccination and development of atopic dermatitis before one year of age - A register-based cohort study of the Danish birth cohorts 1997-2012

Lise Gehrt 1, 2, Andreas Rieckmann1, 7, Nicholas Kiraly3, Aksel Karl Georg Jensen1, 4, Peter Aaby1, 5, Christine Stabell Benn1, 2, Signe Sørup1, 6

1 Research Center for Vitamins and Vaccines (CVIVA), Bandim Health Project, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark 2 OPEN, Open Patient data Explorative Network, Odense University Hospital/Department of Clinical Research, University of Southern Denmark, J.B. Winsløws Vej 9 A, 3. Sal, DK-5000 Odense C, Denmark 3 Gastro and Food Allergy, Murdoch Children’s Research Institute, Royal Children's Hospital, 50 Flemington Rd Parkville, Victoria 3052 Australia 4 Section of Biostatistics, University of Copenhagen, Øster Farimagsgade 5, DK-1014 Copenhagen K, Denmark 5 Bandim Health Project, Indepth Network, Apartado 861, 1004 Bissau Codex, Guinea-Bissau 6 Department of Clinical Epidemiology, Aarhus University, Olof Palmes Allé 43-45, DK-8200 Aarhus N, Denmark 7 Section of Epidemiology, Department of Public Health, University of Copenhagen, Copenhagen, Denmark

Abstract Background Vaccines may have non-specific effects affecting the susceptibility towards conditions not targeted by the vaccine. A study from Australia including 4433 children found that delayed Diphtheria- Tetanus-acellular Pertussis (DTaP) containing vaccination was associated with a reduced risk of developing atopic dermatitis (AD) and reduced use of prescription medicines for AD before 1 year of age. We tested if delayed vaccination with DTaP-containing vaccines reduced the risk of developing AD before 1 year of age in Danish children. Methods We used nationwide registers to follow 883,160 children born in Denmark from 1997 to 2012, and to identify disease specific prescriptions and hospital contacts for AD to define onset of AD. Binary regression models adjusting for potential confounding factors were applied to estimate adjusted relative risks (aRR) of developing AD comparing children with delayed vaccination against diphtheria, tetanus, acellular pertussis, polio, and Haemophilus Influenzae type b (DTaPIPV-Hib) and children without delayed DTaP-IPV-Hib vaccination. Results Among 143,429 children with a delayed first dose of DTaP-IPV-Hib, 4847 (3.4%) developed AD before 1 year of age, compared with 27,628 (3.7%) among 739,731 children not having delayed DTaP-IPV-Hib (aRR 0.94 (95% confidence interval (CI): 0.91-0.97)). The aRR was 0.93 (95% CI: 0.90-0.98) for children with a delayed second dose, and aRR=0.91 (95% CI: 0.87-0.95) comparing children with delayed first and second dose with all timely vaccinated children. Conclusion The results support the hypothesis that delayed vaccination with DTaP-IPV-Hib is associated with reduced risk of developing AD. The dose-dependent relationship further strengthens the evidence of a causal relationship. Bias related to health care seeking behavior may explain some of the observed effect and further evidence from other settings is required.

P7

Innate immune priming by Influenza vaccines Helen R. Wagstaffe1, Anna Rodrigues Galan1, Ciara Lusa1, Jason P. Mooney1,2, Asia-Sophia Wolf1,4, Natalie Prevatt5, Ron H. Behrens5, Eleanor M. Riley1,2, Martin R. Goodier1 1 Department of Infection biology, London School of Hygiene and Tropical Medicine. 2 The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh. 3 Division of Infection and Immunity, University College London. 4 Department of Clinical Research, London School of Hygiene and Tropical Medicine.

Seasonal influenza vaccines are potent inducers of myeloid cell derived cytokines with the potential to promote the responses of innate and adaptive effectors cells. Natural killer cells are innate lymphoid effector cells which are activated by cytokines, by immune complexes and by signals transduced via activating ligands on other host cells. After vaccination adaptive immune responses can enhance both cytokine-driven and antibody-dependent NK cell responses. Our recent studies provide mechanistic insights into how influenza vaccination also primes human natural killer cells both directly, through cell-intrinsic mechanisms, and indirectly via the priming of NK cell activating cytokine production by myeloid cells. An initial study of 52 adult volunteers revealed that seasonal trivalent inactivated influenza intramuscular and intradermal and live attenuated intranasal vaccines primes NK cells for enhanced in-vitro responsiveness (CD25 expression and IFN- production) to activating cytokines (IL-12 and IL-18) with effects being observed up to 3 months post vaccination. Vaccine induced enhancement demonstrated several characteristics of cytokine-induced memory-like NK cells (CIML-NK), involves enhancement of responses within less differentiated subsets and is reflected in vaccine effects measured directly ex-vivo. We subsequently demonstrate in a further 51 volunteers that seasonal influenza vaccination promotes increased myeloid cell-derived cytokine production on in-vitro re-stimulation. IL-15 alone, in the absence of vaccine antigen reveals enhancement of GMCSF, IL-10, IL-1 , TNF- and IFN- secretion from post vaccination compared to baseline samples. Parallel enhancement of NK cell function involves the production of IL-12 from myeloid DC. We conclude that influenza vaccination-induced training of accessory cells combines with the generation of CIML NK cells to enhance the overall NK cell response post-vaccination. These studies raise several important questions surrounding vaccine induced innate immune priming, including the impact of vaccination route and the durability of these responses.

P8

Nasal challenge with a TLR7/8 agonist (R848) induces in vivo mucosal interferons and chemokines with increased responses observed in allergy and asthma

Akhilesh Jha, Ryan S Thwaites, Tanushree Tunstall, Onn Min Kon, Robin J Shattock, Trevor T Hansel, Peter J M Openshaw

National Heart and Lung Institute, Imperial College London, UK Department of Medicine, University of Cambridge, UK Department of Infectious Diseases, Imperial College London, UK

Background Respiratory viral infections causes significant global morbidity and mortality, especially in vulnerable populations such as those with respiratory disease. Host recognition of viruses is mediated in particular by Toll-like receptors (TLRs) to elicit innate immune responses. The development of respiratory mucosal vaccine adjuvants and methods to accurately assess respiratory mucosal immune responses are lacking.

Objectives A synthetic analogue of single-stranded RNA (ssRNA) and TLR7/8 agonist (R848) was administered in vivo to assess nasal mucosal innate immune responses and to study the effect of allergy and asthma.

Methods Nasal spray with saline and R848 was administered to healthy non-allergic (n=12), allergic rhinitis (n=12) and allergic asthma (n=11) participants. Serial non-invasive nasal mucosal samples and blood was collected to assess immune mediators and mucosal gene expression.

Results R848 was well tolerated with no evidence of systemic immune activation. R848 significantly induced nasal mucosal IFN-α2a, IFN-γ, pro-inflammatory cytokines (TNF-α, IL-2, IL-12p70) and chemokines (CXCL10, CCL2, CCL3, CCL4 and CCL13) compared to saline. Participants with allergic rhinitis and asthma had increased IFN-α2a, CCL3 and CCL13 relative to healthy participants, whilst those with asthma alone had increased gene expression of interferon stimulated genes DDX58, MX1 and IFIT3.

Conclusions These results confirm that nasal delivery of R848 is well tolerated and innate immune function can be assessed non-invasively and with precision. It highlights it as a tool to study innate immune responses in airway disease and other host disease states, and has potential for use as a vaccine adjuvant.

Future work In-vivo and ex-vivo experiments utilising R848 will be performed to assess the epigenetic mechanisms of innate immune memory in airway epithelial and immune cells using a single- cell transcriptomic approach.

P9

Sex differences in immune response to influenza vaccine in healthcare workers during the 2017-2018 influenza season

Helen Kuo, Rosemary Morgan, Kathryn Shaw-Saliba, Katherine Fenstermarcher, Santosh Dhakal, Richard Rothman, Andrew Pekosz, Sabra Klein

Johns Hopkins School of Public Health

Seasonal influenza epidemics affect five to fifteen percent of the world population with an estimated 290,000 to 650,000 annual respiratory deaths due to influenza (1). The Center for Disease Control recommends annual influenza vaccination for healthcare workers (HCWs) because not only does their occupation increase their risk of contracting influenza, but HCWs care for people at higher risk for developing influenza related complications (2). Seroconversion, a 4-fold difference between pre and post vaccination, is often used as an indirect measurement of seroprotection against influenza post vaccination. Studies have shown immune responses, such as seroconversion, differ between male and female vaccine recipients, but few have focused on HCWs and their influenza vaccine responses by sex. To understand the sex differences in immune response to the influenza vaccine in the HCWs population, 111 participants (38 male; 73 female) were recruited during the 2017-2018 influenza season as part of the Johns Hopkins Centers for Excellence in Influenza Research and Surveillance (JH-CEIRS) annual serological surveys among Johns Hopkins Hospital HCWs that take part in the annual hospital-wide vaccination campaigns. Participants were recruited just prior to receiving their annual influenza vaccine, which protects against influenza type A/Michigan H1N1, A/Hong Kong H3N2, and B/Brisbane viruses, and blood was collected to measure baseline antibody titer. After 28 days, blood was collected to assess post vaccination antibody titer levels. Neutralizing antibody assays were used to measure pre and post antibody titers against A/Michigan H1N1 and A/Hong Kong H3N2. Pre-vaccination antibody titers were higher among those who do not seroconvert compared to those who did seroconvert for both A strain influenza viruses. Male participants were 2.86 times more likely to seroconvert for the H1N1 antibody and 1.15 times more likely to seroconvert for the H3N2 antibody than female participants. However, female participants had much higher pre-vaccination antibody titer compared to male participants, suggesting female participants were maintaining their high antibody titers throughout the year. Factors, such as sex, BMI, age and direct patient contact were explored in a regression model as possible correlations for female participants maintaining their pre-vaccination antibody titers. Sex was the only statistically significant (p<0.01) factor that appears to show correlation to seroconversion. Additional studies are needed to understand sex differences in immune response to the influenza vaccine.

P10

A Brief Review of Adversomics: Understanding Genetic Drivers for Adverse Events after Vaccination

Inna G. Ovsyannikova, Richard B. Kennedy, Gregory A. Poland

Mayo Clinic Vaccine Research Group, Mayo Clinic, Rochester, Minnesota, USA

Despite the huge population benefits of routine immunization, the rare occurrence of vaccine severe adverse events (SAEs) inhibits vaccine acceptance and infectious disease prevention. The molecular mechanisms underlying the immune response during SAEs after vaccination in humans are not well understood, but may be predictable rather than random.

Introduced in 2009, the term “adversomics” refers to the application of immunogenetics, immunogenomics, and systems biology approaches to identifying genetic and molecular signatures for vaccine SAEs at the individual and population level.

The current state of the adversomics field includes limited studies exploring genetic and proteomic signatures/biomarkers of AEs following smallpox, 17D-derived yellow fever (YF), measles-mumps-rubella (MMR), influenza and hepatitis B (HBV) vaccines.

As described in published literature, genetic polymorphisms in the IL1, IL4, and IL18 genes/haplotypes and inflammatory cytokine response pathways (ICAM-1, IL-10, and CSF- 3) have been associated with systemic AEs after smallpox vaccination. YF vaccine- associated disease was characterized by viremia, robust T/B cell responses, and polymorphisms in the cytokine receptor CCR5 (delta32) and its ligand RANTES (403 G/A) genes. With respect to both MMR vaccine-related febrile seizures and measles neutralizing antibody responses, GWAS associations of CD46 (rs1318653) and IFI44L (rs273259) genetic variants with febrile seizures and decreased antibodies have been recently published. Furthermore, an increased incidence of narcolepsy after adjuvanted influenza pH1N1 vaccine has been associated with HLA-DQB1*06:02/DQA1*01:02 alleles, suggesting that antigen presentation by these alleles is crucial. HBV-associated major AEs have been linked to class II HLA-DRB1* (*01:01/*03:01/*04:01/*13:01/*15:01) alleles/haplotypes and class I HLA-A2 gene interactions.

Understanding and preventing vaccine SAEs is critical in order to improve public trust in vaccine safety and to develop new safe and effective vaccines that minimize or avoid SAEs. In this presentation I will review the current knowledge about the field of adversomics, as well as the known associations and mechanisms of vaccine SAEs.

P11

Unconventional protection to Mycobacterium tuberculosis infection conferred by molds

Marta ROMANO, Pauline PERCIER; Giresse TIMA; Fabienne JURION and Olivier DENIS

Service Immune Response, Directorate Infectious Diseases in Humans; SCIENSANO, Brussels, Belgium

Introduction: Molds are ubiquitous filamentous fungi and more than one hundred different indoor and outdoor mold species have been recognized as major sources of aeroallergens that can cause respiratory forms of allergy, ranging from allergic rhinitis to asthma. Conflicting results have been reported on the association between allergy and infection with M. tuberculosis and/or active Tuberculosis disease. In this study, we wanted to address if a previous allergic sensitization with the outdoor mold Alternaria alternata could have an effect on the susceptibility to infection with M. tuberculosis. Material & Methods: For that purpose, well defined murine models of sensitization with different compounds isolated from A. alternata or heat-killed spores were used. Intranasal instillations with these induce lung inflammation with different profiles (differences in type of cells recruited to the airway and in the local cytokine and chemokine expression profiles induced). Mice were treated with the mold antigens before or after pulmonary M. tuberculosis infection. Results: Interestingly, our results indicate that previous sensitization with an A. alternata fraction inducing a genuine eosinophilic / pro-Th2 response has no effect on susceptibility to infection with M. tuberculosis, while treatment with fractions of A. alternata inducing a mixed pro-Th2/pro-Th17 response results in an improved control of M. tuberculosis infection. This control of pulmonary growth of M. tuberculosis is observed as early as 6 days after infection, it is maintained for several weeks without need of continuous instillation of A. alternata and M. tuberculosis specific adaptive immune response is not influenced by the previous treatment with A. alternata. In addition, treatment of M. tuberculosis infected mice with heat-killed A. alternata spores results in a control of the bacterial burden. Discussion-Conclusion: Even if our results warrant further mechanistic analysis, our data collectively indicate and confirm that the pulmonary environment can significantly shape outcome of infection with M. tuberculosis infection.

P12

Immunological mechanisms of MV140, a polybacterial vaccine to prevent urinary tract infections

Paula Saz-Leal 1, 2, Caihong Wang 2, Marianne M. Ligon 2, Carmen M. Díez-Rivero 1, Laura Conejero 1 and Indira U. Mysorekar 2,3

1 Inmunotek, SL. Alcalá de Henares, Madrid, Spain. 2 Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO, USA. 3 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA.

Background Urinary tract infections (UTIs) are the most reported bacterial infections in women. High rates of recurrence lead to 5-10% of total women suffering from recurrent UTIs (rUTI). Because of the lack of effective alternatives, antibiotic prophylaxis is still the gold standard treatment for rUTI, contributing to the massive rise of antimicrobial resistance. MV140 is a sublingual vaccine based on a mixture of whole-cell heat-inactivated bacteria indicated for rUTI prophylaxis. Clinical studies in more than 1,000 subjects have demonstrated that MV140 significantly reduces UTI episodes compared to antibiotic prophylaxis, preventing recurrence in 80-90% of individuals during the year following vaccination. Moreover, MV140 is effective against the specific pathogens targeted by the vaccine, but also against heterologous infections. Mechanistically, sublingual immunization with MV140 showed to induce a systemic CD4+ T cell response in experimental models, but the underlying mechanisms in the context of infection were still unknown. Aim To evaluate the protection conferred by the polybacterial preparation MV140 in an experimental model of uropathogenic Escherichia coli (UPEC)-mediated UTI and to investigate its mechanism of action. Additionally, to compare MV140 effect to that provided by a monobacterial vaccine consisting of the specific UPEC infectious challenge. Methods Mice were vaccinated intranasally either with MV140 (E. coli, K. pneumoniae, E. faecalis, P. vulgaris) or a specific UPEC strain (UTI89)-based vaccine, both heat-inactivated, and subsequently subjected to a UTI89-induced acute UTI. Bacterial load, as well as the immune response in urine and bladder, were measured following infection. Otherwise, the systemic immune response to both MV140 and UPEC was evaluated in the spleens of vaccinated versus control uninfected mice. Results Both MV140 and the UPEC-based vaccine conferred significant protection to infection with an early reduction in bacterial load compared to controls. These results were accompanied with increased numbers of myeloid immune cells in the urine, and a heightened CD4+ T cell response in the bladder. Both types of vaccination induced similar systemic T helper (Th) 1, Th17 and IL-10-mediated responses against the MV140 and UTI89. Discussion We demonstrate for the first time that MV140 confers protection in an acute UTI experimental model. This is accompanied by the induction of local myeloid and adaptative T cell responses, the latter correlating with that observed systemically. The protection conferred by MV140 against a UTI89 challenge is comparable to that provided by its specific vaccine, and may further support a quite broad-spectrum anti- infectious profile of this polybacterial preparation.

P13

BCG skin reactions by 2 months of age are associated with better survival in infancy: A prospective observational study

Frederik Schaltz-Buchholzer1,2,3, Mike Berendsen1,2,3,4, Adam Roth5,6, Kristoffer Jarlov Jensen1,7, Morten Bjerregaard-Andersen2,3, Marcus Kjær Sørensen2,3, Ivan Monteiro2, Peter Aaby2, Christine Stabell Benn1,2,3,8

1 Bandim Health Project, Institute of Clinical Research, Uni. Southern Denmark and Odense University Hospital, Odense, Denmark: FSB (MD), MB (MSc), CSB (Professor, DMSc). 2 Bandim Health Project, INDEPTH Network, Bissau, Guinea-Bissau: FSB, MB, KJJ (PhD), MBA (MD, PhD), MKS (medical student), IM (trial supervisor), PA (Professor, DMSc), CSB. 3 Research Centre for Vitamins and Vaccines, Copenhagen, Denmark: FSB, MB, MBA, MKS, CSB. 4 Department of Internal Medicine, Radboud Centre for Infectious Diseases, Radboud University Medical Centre, Nijmegen, The Netherlands: MB. 5 Public Health Agency of Sweden, Nobels väg 18, 171 82 Solna, Sweden: AR (MD, PhD). 6 Institution for Translational Medicine, Lund University, J Waldenströms g 35, CRC, hus 92, plan 11, Malmö, Sweden: AR. 7 Experimental and Translational Immunology, Department of Health Technology, Technical University of Denmark, Kgs Lyngby, Denmark: KJJ. 8 Danish Institute of Advanced Science, Uni. Southern Denmark, Odense, Denmark: CSB.

Objectives: Receiving Bacillus Calmette-Guérin (BCG)-Denmark vaccine at birth has been associated with a ~40% reduction in all-cause neonatal mortality and among BCG- vaccinated infants, developing a BCG scar during infancy has been associated with a similar reduction in subsequent mortality. We evaluated determinants of early BCG reactions and the reaction size by 2 months of age and tested whether having developed a BCG reaction by 2 months and the reaction size were associated with subsequent all-cause mortality. Design: Prospective observational study within five trials providing BCG-at-birth. The BCG reaction status and reaction size was evaluated at home-visits by 2 months of age; mortality up to 12 months was assessed at subsequent home-visits. Setting: Bissau, Guinea-Bissau. Participants: 6,012 infants BCG-vaccinated within 1 week of birth. Main outcome measures: BCG reaction determinants were evaluated by binomial regression providing Risk Ratios (RRs). Cox-models providing adjusted Mortality Rate Ratios (aMRRs) were used to assess the association between 1) having a 2-month reaction (yes/no) and 2) the reaction size tertiles and the subsequent 2-12-month mortality risk. A subgroup of infants had their BCG reaction evaluated and were bled at age 4 weeks; samples underwent in vitro analysis for specific and non-specific cytokine responses. Results: 5,804 infants (97%) had developed a BCG reaction by 2 months of age; 208 (3%) had no reaction. The BCG strain was the main determinant for both developing a 2-month BCG reaction and the reaction size: the RR for large-reaction comparing BCG-Russia vs. BCG-Denmark was 0.38 (0.30-0.47) and 0.61 (0.51-0.72) for BCG-Russia vs. BCG-Japan. The 2-12-month mortality risk was 4.8% (10/208) for non-reactors, 2.9% (64/2,213) for small reactors, 1.8% (30/1,710) for medium reactors and 0.8% (15/1,881) for large reactors. The reactor/non-reactor aMRR was 0.49 (0.26-0.95) and there was a linear trend of decreasing mortality with increasing reaction size (p for trend<0.001). BCG reactors had higher 4-week specific and non-specific cytokine responses, responses that were highest among those with large reactions. Conclusion: Among BCG-vaccinated infants, having a BCG skin reaction by 2 months of age was associated with markedly better survival, as was the reaction size. Our findings support that BCG has substantial non-specific effects on all-cause mortality and indicate that emphasising at-birth vaccination with immunogenic BCG strains and revaccinating non- reactors could have major public health benefits.

P14

Re-vaccination with measles-mumps-rubella vaccine and hospitalization for infections lasting 2 days or longer in Denmark and Sweden – an interrupted time-series analysis

Signe Sørup, Hélène Englund, Ida Laake, Heta Nieminen, Adam Roth, Christine Benn

Research Center for Vitamins and Vaccines (CVIVA), Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark (Signe Sørup and Christine Benn); Department of Clinical Epidemiology, Aarhus University, Aarhus, Denmark (Signe Sørup); Public Health Agency of Sweden, Nobels väg 18, 171 82 Solna, Sweden (Hélène Englund and Adam Roth); Department of Infectious Disease Epidemiology and Modelling, The Norwegian Institute of Public Health, Oslo, Norway (Ida Laake); Department of Public Health Solutions, Finnish Institute for Health and Welfare, Tampere, Finland (Heta Nieminen); Department of Translational Medicine, Lund University, Malmö, Sweden (Adam Roth); OPEN, Open Patient data Explorative Network, Odense University Hospital/Department of Clinical Research, University of Southern Denmark, Odense, Denmark (Christine Benn)

Background: In low-income countries, re-vaccination with live vaccines has been associated with reduced morbidity and mortality not related to the targeted diseases. In a previous cohort study of 4-year-old Danish children, re-vaccination with the live measles-mumps- rubella (MMR) vaccine was associated with a 16% reduction in the rate of hospitalizations for infections lasting 2 days or longer. Aim: To examine if the introduction of a second MMR-dose (MMR-2) at 4 years of age in Denmark (spring 2008) and at 6-9 years of age in Sweden (Autumn 2009) was associated with a reduction in the rate of hospitalizations for infections lasting 2 days or longer on a population level. Methods: We performed an interrupted time series analysis using Poisson regression to estimate the change in the level of the rate of hospitalizations for infections lasting 2 days or longer following the introduction of MMR-2 adjusting for seasonality (dummy variables for season: spring, summer, autumn, and winter). We included the 4-year olds in Denmark and the 9-year olds in Sweden. We defined the pre-intervention, phase-in (excluded from the model), and post-intervention periods to be March 2005 to February 2008, March 2008 to February 2009, and March 2009 to February 2012 in Denmark and September 2005 to August 2009, September 2009 to August 2012 and September 2012 to August 2015 in Sweden. We estimated the observed rate of hospitalizations for infection lasting 2 days or longer for each season and year based on the number of hospitalizations from nationwide registers (infections identified by ICD-10 codes) and person-years at risk approximated from population statistics. Results: The crude rate of hospitalizations for infections lasting 2 days or longer among 4 year-olds in Denmark was 8.3 per 1000 person-years before and 7.9 per 1000 person-years after the introduction of MMR-2. The corresponding rates among 9 year-olds in Sweden were 2.7 and 2.5 per 1000 person-years, respectively. Although the rate decreased over the entire study period, the Poisson regression predicted that the rate was 0.5 per 1000 person- years higher after the phase-in of MMR-2 in Denmark (p value for level change=0.48) and 0.2 per 1000 person-years higher in Sweden (p value for level change=0.56) than what would have occurred without the introduction of MMR-2. Conclusion: On a population level, the introduction of MMR-2 was not associated with a reduction in the rate of hospitalizations for infection lasting 2 days or longer in Denmark nor in Sweden.

P15

Investigation of Mother to Child Transmission of HLA Antibodies DM Savulescu1, M Groome2, SCK Malfeld1, S Madhi2, A Koen2, S Jones2 and M Suchard1,3

1. Centre for Vaccines and Immunology (CVI), National Institute for Communicable Diseases (NICD), a division of the National Health Laboratory Service, South Africa 2. Respiratory and Meningeal Pathogens Research Unit (RMPRU), University of the Witwatersrand, South Africa 3. Chemical Pathology, Faculty of Health Sciences, University of the Witwatersrand, South Africa Vaccination during pregnancy is a growing field of interest, however many aspects of humoral immunity during pregnancy remain poorly understood. Human leukocyte antigens (HLA) are proteins responsible for presentation of peptide fragments to T lymphocytes. HLA- encoding genes are extremely polymorphic, and the foetus has a different HLA antigen repertoire from the mother. Late in pregnancy, women produce and transfer high amounts of antibodies, including antibodies agains HLA (HLA antibodies), to the fetus, where presumably the antibodies may act as autoantibodies. Little is known about specificity or transfer rate of HLA antibodies to the foetus. We describe the prevalence of HLA antibodies in serum of thirty mother-infant pairs six weeks post-partum. We used Luminex single angtigen technology that allows for highly sensitive antibody detection. We found that 72% of babies and 80% of mothers expressed HLA antibodies. In babies, 77% of HLA-I antibodies and 50% of HLA-II antibodies matched those of their mothers, suggesting that the remaining antibodies were self-made. In contrast, the proportion of maternal antibodies that matched those of their babies was only 16% for HLA-I and 24% for HLA-II, suggesting that not all HLA antibodies produced during pregnancy cross to the fetus. Production of self-made antibodies by six weeks of age indicates early capacity of the neonatal immune system to generate HLA antibodies; either natural antibodies or preceded by exposure to specific HLA antigens. Our findings further indicate that there is selective antibody transfer of particular antibody specificities from mother to infant. Because HLA antibodies may act against paternal antigens present in the neonate, the mechanism of selective antibody transfer or depletion may give insights into mechanisms of infant immune tolerance.

P16

Sex-specific effects of early life influenza vaccination on protection against diverse influenza viruses in mice

Rebecca L Ursin, Yishak Woldetstadik, Georgia Stavrakis, Kristyn E. Sylvia, Harrison R. Powell, Hsuan Liu, Andrew Pekosz, and Sabra L. Klein

Johns Hopkins University Bloomberg School of Public Health, Departments of Biochemistry and Molecular Biology, and the W. Harry Feinstone Department of Molecular Microbiology and Immunology, Baltimore, MD 21205

Influenza viruses kills tens of thousands of people annually worldwide, with seasonal influenza vaccines serving as the best mode for protection. Vaccine efficacy depends on a myriad of factors, including intrinsic characteristics of the virus, such as mutations that enable viruses to escape host immunity, as well as host factors, including sex, age, and immune history. To test the hypothesis that early life exposure to influenza vaccines may differentially affect responses to vaccination and infection later in life, we immunized and boosted male and female C57BL/6 mice beginning at postnatal day (PND) 11-14 with a 2009 H1N1 inactivated influenza vaccine (IIV) followed by re-vaccination in adulthood (PND 63- 75) and subsequent infection with a 2009 H1N1 drift variant virus. Prior to puberty, both males and females mounted low neutralizing antibody (nAb) titers, with no sex differences in vaccine-induced immune responses. Upon vaccination in adulthood, robust, sex- differentiated responses were observed in which females had greater nAb titers than males and were better protected following challenge with the drift variant virus. To determine the range of cross-neutralization and protection against mutating influenza viruses, we developed a panel of 2009 H1N1 viruses that contain the following mutations in the immunodominant hemagglutinin (HA) protein: a single amino acid mutant virus with a mutation at position K166Q; a double mutant at positions K166Q and G157E; a triple mutant with K166Q, G157E, and N211D; and a virus with the K166Q mutation plus an entire antigenic region substituted with a non-human HA. Using these viruses, we determined that regardless of the mutations in HA, unvaccinated adult females experience greater morbidity and mortality and have a lower lethal dose 50 (LD50) than their male counterparts. Whether immunization with IIV prior to puberty and/or in adulthood differentially affects cross- neutralization and protection against the panel of 2009 H1N1 viruses that contain HA mutations is currently under investigation. These studies are critical for understanding how pre-existing immunity affects protection against viral infection and will help to develop better influenza vaccines, including universal influenza vaccines, that provide equal protection against circulating strains of influenza viruses in both males and females.

P17

Towards a Vaccine Based on Enterococcal Membrane Vesicles

Theresa Wagner (1), Mona Johannessen (1), Kristin Hegstad (1,2)

(1) Research group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø – The Arctic University of Norway (2) Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North-Norway, Tromsø, Norway

Enterococcus faecium is a leading cause of nosocomial infections, especially in immunocompromised patients. Multi-resistant E. faecium, including Vancomycin Resistant Enterococci (VRE) impair therapy and limit treatment options. Thus, novel therapeutic approaches and alternatives for infection control are desperately needed to combat VRE. Vaccines are promising alternatives in times of increasing antimicrobial resistance, since the risk of resistance development is low and vaccines can be used prophylactically. For enterococci, there are no available vaccines yet, even though research has been ongoing to identify vaccine candidates. In a previous study, we isolated and characterized membrane vesicles (MVs) produced by E. faecium. We described their proteomic content in four clinically relevant strains, including VRE. The E. faecium MV contained known virulence factors, among them adhesins, collagen binding proteins, fibronectin binding protein, as well as biofilm promoting factors. Also, antimicrobial resistance related proteins were found to be MV-associated, including all proteins of the vanA-cluster of a VRE. Furthermore, we found six described vaccine candidates to be associated with enterococcal MVs. MVs would be auspicious vaccine candidates since they are highly stable, non-infectious and non-replicative. They contain major immunogenic proteins, and would be able to elicit protective immune responses and act as adjuvant. Since high amounts of pure MVs will be needed for vaccination studies, we optimized the production of MV, by investigating the influence of antibiotic exposure, as well as different isolation methods, on the outcome. The cytotoxicity of MVs was tested in various cell lines. At present we are immunizing rabbits with E. faecium MV and are planning to characterize their serum. In the future, we would like to explore the potential use of MVs to elicit protective immunity against E. faecium, including VRE, infection.

P18

Notes

P19

Notes

P20

Delegate List LAURA CONEJERO INMUNOTEK [email protected] Peter Aaby

Statens Serum Institut Maria Giulia Conti [email protected] Sapienza University of Rome

[email protected] Asimenia Angelidou

Boston Children's Hospital Nigel Curtis [email protected] University of Melbourne du [email protected]

Petra Arck Mia Damhus University Medical Center Hamburg Center for Nutrition and Therapy [email protected] [email protected]

Sintayehu Arega Joseph de Vrieze Ross University School of Veterinary Oost Indische Inkt Medicine [email protected] [email protected]

Priya Debisarun Christine Stabell Benn RadboudUMC University of Southern Denmark [email protected] [email protected]

Santosh Dhakal Mike Berendsen Johns Hopkins University Bandim Health Project [email protected] [email protected]

Hazel Dockrell Byron Brook London School of Hygiene & Tropical University of British Columbia Medicine [email protected] [email protected]

Ozlem Bulut Jorge Dominguez Andres Radboud University Medical Center Radboudumc [email protected] [email protected]

Egle Butkeviciute David Elliman LSHTM PHE/GOSH [email protected] [email protected]

Jake Byrne Jorge Esparza Gordillo Ross University School of Veterinary GlaxoSmithKline Medicine [email protected] [email protected]

Denise Faustman Rebecca Chandler Harvard Medical School Uppsala Monitoring Centre [email protected] [email protected]

Zoltan Fehervari Gizem Kilic Nature Research Radboudumc [email protected] [email protected]

Adam Finn Sabra Klein University of Bristol Johns Hopkins University [email protected] [email protected]

Eleanor Fish Henrik Kloverpris University of Toronto Africa Health Research Institute [email protected] [email protected]

Katie Flanagan Darryn Knobel UTAS/RMIT/Monash University RUSVM [email protected] [email protected]

Anna Fletcher Tobias Kollmann Micropathology LTD. Telethon Kids Institute [email protected] [email protected]

PILAR GARCA CORBEIRA Helen Kuo GlaxoSmithKline Johns Hopkins School of Public Health [email protected] [email protected]

Lise Gehrt Ofer Levy University of Southern Denmark Boston Children's Hospital [email protected] [email protected]

MARTIN R GOODIER Camille Locht LSHTM Inserm [email protected] [email protected]

Danika Hill David Lynn Babraham Institute SAHMRI [email protected] [email protected]

Akhilesh Jha Anne Marie Rosendahl Madsen University of Cambridge University of Southern Denmark [email protected] [email protected]

Kamilla Josefsdottir Siddhartha Mahanty Directorate of Health University of Melbourne [email protected] [email protected]

Konstantinos Karampatsas Hafza Zahira Manzoor St George's University of London University of Veterinary and Animal [email protected] Sciences [email protected] Christine Karanja Chege Kenyatta University [email protected] Francois Mayer Nature Microbiology Frederik Schaltz-Buchholzer [email protected] Bandim Health Project [email protected] Victoria McLelland ION Osteopaths Klaus Schwamborn [email protected] Valneva [email protected] Kirsty Minton Nature Reviews Immunology Frank Shann [email protected] University of Melbourne [email protected] Jessica Mwinyi Swedish Medical Products Agency Sven Arne Silfverdal [email protected] Umea University [email protected] Mihai Netea Radboud UMC Nijmegen Signe Soerup [email protected] Statens Serum Institut AND Aarhus UNiversity Inna Ovsyannikova [email protected] Mayo Clinic [email protected] Adrian Spillmann Valneva Magdalena Plebanski [email protected] RMIT University [email protected] Melinda Suchard National Institute for Communicable Katrina Pollock Diseases Imperial College London [email protected] [email protected] Rebecca Ursin Sarah Prentice Johns Hopkins University East and North Hertfordshire NHS Trust [email protected] [email protected] Theresa Wagner Marjorie Robert Guroff University of Tromsø National Institutes of Health [email protected] [email protected] Hilton Whittle Marta Romano LSHTM ISP-WIV Belgium [email protected] [email protected]

Christine Rueckert CureVac AG [email protected]

PAULA SAZ INMUNOTEK [email protected] Index

Aaby, P S1 Robert-Guroff, M S43 Arck, P S27 Romano, M P12 Arega, S P1 Saz, P P13 Benn, C. S P2 Schaltz-Buchholzer, F P14 Berendsen, M S35, P3 Soerup, S P15 Brook, B S23 Suchard, M P16 Bulut, O P4 Byrne, J P5 Ursin, R P17

Conejero, L S49 Wagner, T P18 Curtis, N S7

Debisarun, P P6 Dhakal, S S29 Dockrell, H S15 Dominguez Andres, J S47

Faustman, D S31 Finn, A S3 Flanagan, K S39

Gehrt, L P7 Goddier, M P8

Hill, D S11

Jha, A P9

Kilic, G S21 Klein, S S25 Knobel, D S33 Kollmann, T S9 Kuo, H P10

Levy, O S45 Locht, C S41 Lynn, D S19

Netea, M S5

Ovsyannikova, I S37, P11

Plebanski, M S17 Prentice, S S13 To Hinxton Village (Vehicle access via main exit to site) Willow Court (A&B) B = 230-243 330-343 Mulberry Court (C) A = 244-259 201-229 344-361 301-329 401-406 A B

C

Disabled Tennis Court Conference Centre Training Suite Reception Francis Crick Auditorium James Watson Pavilion Rosalind Franklin Pavilion Hinxton Hall Loft Room 1 and 2 Pompeiian Room Library Room Green Room Restaurant Lounges/Bar Bedrooms 362-367/407-410

Hinxton Hall

Conference Centre

EMBL - EBI The Sulston Laboratories

West Pavilion

The Cairns Pavilion The Data Centre RSF

Wet

Labs The Morgan Building Morgan The

Reception

Fire Assembly Point Designated Smoking Area EBI South @ACSCevents Wellcome Genome Campus Courses and Conferences wellcomegenomecampus.org /coursesandconferences