Characterisation of Immune Responses to Varicella Vaccination in Relation to Clinical Outcome Suzanna Leonie Rose McDonald Centre for Infectious Disease, Institute of Cell and Molecular Science, Bart‟s and the London School of Medicine and Dentistry, Queen Mary University of London 2010 Submitted to the University of London for the degree of Doctor of Philosophy 1 DECLARATION: I declare that the work presented in this thesis is my own. Signed: Suzanna Leonie Rose McDonald ABSTRACT: This thesis examines both humoral and cellular adaptive immune responses to varicella vaccination (up to 18 months post immunisation), in an ethnically diverse population of healthcare workers. Using two parameters of humoral immunity at six weeks post first vaccination; (avidity readings 60%, and a TRFIA reading 400mIU/mL) a cut-off of 130mIU/mL was defined for a more sensitive in house immuno assay (TRFIA), in this vaccinated adult population. Using these cut-offs, three patterns of antibody responses were identified; primary responders who seroconverted following vaccination, secondary responders who had pre- existing immunity and subjects who responded poorly to vaccination. Demographic and immunological characteristics of each subset were examined. An association between black ethnicity and lower antibody titre to vaccination in primary responders was identified, whilst Caucasians were more likely to have a history and pre-existing immunity, in keeping with the epidemiology of chickenpox in temperate climates. The follow-up study revealed that affinity maturation to VZV can take longer than 18 months in response to vaccination. At follow-up, 25% of subjects recruited at this time point were seronegative by TRFIA. Seroconversion after two doses of vaccine and a TRFIA titre of <500mIU/ml after two doses were significantly associated with waning antibody titre over time. Positive IFN- ELISPOT responses at 18 months did not necessarily correspond with TRFIA seropositive status. 2 ACKNOWLEDGEMENTS AND DEDICATION: First, I would like to thank my supervisor, Prof. Judy Breuer, for giving me the opportunity to undertake this PhD and for the immeasurable amount of time and energy she has invested in this adventure; for her continued support, encouragement and guidance throughout. I would also like to thank my second supervisor, Prof. Ping Wang. Thanks to Dr. Dan Pennington and Dr. Sourenna Kiani for their helpful comments and advice with this thesis, and for their support and belief in me over the last few years. Thanks also to Dr. Jon Dodd (Imperial College) for comments and advice with the introduction chapter of this thesis. I would also like to thank Dr. Rav Kanda, Mrs. Fiona Scott, Miss Christine Tomlins and Dr. Alison Steele for proof reading parts of this thesis. I would like to thank all the ROVE study participants; without their samples this study would not have been possible. I would also like to say a special thanks to Mrs. Fiona Scott for coordinating the ROVE study and carrying out sample collection; and Mr. Mahmoud Al-Bassam, Miss Ingrid Green and Mr. Gavin Wall for technical assistance with processing ROVE samples. I would like to thank collaborators who have assisted with the ROVE study; Dr. Chris Maple (HPA; London) for his assistance with serology; Dr. Paul Sinnott and Mr. Sharham Hemmatpour (Bart‟s and the London NHS Trust) for assistance with Luminex HLA typing; Dr. Graham Ogg and Dr. Louise Jones (University of Oxford) for their assistance with tetramer assays and Sharon Steinberg in the lab of Prof. Anne Gershon (Columbia University, USA) for carrying out FAMA assays. For general statistical assistance, I would like to thank Mrs. Mary Leedham Green, as well as Dr. Nick Andrews (HPA; London) and Dr. Karen Ayres (University of Reading) for their invaluable advice with mixture modelling and probability density curves and ROC analysis respectively. I would like to pay a special tribute to the members of the Breuer Laboratory, past and present; for the vast amount of support along the way; Dr. Mark Quinlivan; Dr. Inga Dry; Dr. Nitu Sengupta, Dr. Karin 3 Averbeck, Dr. Manuraj Singh, Dr. Meleri Jones and Mr. Forrest Ran along with members of the Wang laboratory past and present; Dr. Vassia Sofra and Dr. Salah Mansour, Major Dr. Bo Zhu, Dr. Alistair Symonds and Dr. Martine Barel. I would also like to thank Dr. Gary Warnes for his assistance with FACS and imaging techniques over the last few years. I would like to say a special thank you to colleagues (too numerous to mention by name) at Bart‟s and the London School of Medicine and Dentistry, in the Centre of Infectious Diseases, the Centre of Adult and Paediatric Gastroenterology (and the many other departments I have infiltrated), for immeasurable amounts of advice and support over the years. Thank you all for creating such a wonderful and happy environment to carry out my PhD; many of the friendships I have made at the Institute of Cell and Molecular Science will last a lifetime, and I was blessed to share the journey with you. I would like to say a special thank you for the continued love and support I have received from my friends and family over the last four years; many of whom didn‟t realise what completing a PhD entailed, but have been supportive, patient and forgiving nonetheless. I would also like to take this opportunity to apologise to all of them, for having seen a lot less of me over the last few years, and for having to endure a grumpy monster most of the time when they have seen me. I would like to extend that apology to the ladies of Jamaica St. over the years, and say thank you for helping to make a lovely nest to come home too. Lastly, I would like to thank my Mum and my Nan for their financial support over the last few months and my concluding thanks are to them, my Sister and my Brother-in-law, for their never ending emotional support. This thesis is dedicated to an autodidactic biologist; my Granddad. 4 CONTENTS: DECLARATION 2 ABSTRACT 2 ACKNOWLEDGEMENTS AND DEDICATION 3 TABLE OF CONTENTS 5 LIST OF FIGURES 13 LIST OF TABLES 18 LIST OF ABBREVIATIONS 20 CHAPTER 1: INTRODUCTION 1.1 Varicella Zoster Virus in Context 29 1.2 Classification and Virus Structure 29 1.2.1 Structure and Morphology of the Varicella Zoster Virus 32 1.3 Genomic Structure and Organisation 33 1.4 Functions and Properties of Key VZV Proteins 39 1.4.1 Glycoproteins and Structural Proteins 39 1.4.2 Proteins Involved in Replication 44 1.5 VZV Infection at a Cellular Level 47 1.5.1 Cell Attachment and Entry 47 1.5.2 Initial Events in Viral Replication 52 1.5.2.1 Viral DNA Replication 52 1.5.3 Protein Synthesis 54 1.5.4 Virion Assembly 56 1.5.4.1 The Nucleocapsid 56 1.5.4.2 Acquisition of Tegument Proteins 59 1.5.4.3 Glycoprotein Synthesis and Trafficking 60 1.5.4.4 Envelopment and Virion Morphogenesis 62 1.5.5 Egress and Cell-to-Cell Spread 67 1.6 The Natural History of VZV Infection 70 1.6.1 Pathogenesis of Varicella 70 1.6.1.1 Rash Formation and the Host Immune Response 76 1.6.1.2 The Role of Individual VZV Proteins in Varicella Pathogenesis 78 1.6.2 Pathogenesis of Latency and Reactivation 86 1.6.2.1 The Role of Individual VZV Proteins in the Pathogenesis of Latency 90 5 1.7 Host Immune Responses to VZV, and Immune Modulation and Evasion by the Virus 92 1.7.1 NK Cell Mediated Immunity and VZV Induced Immune Modulation 93 1.7.2 Immune Modulation of Dendritic Cells 94 1.7.3 Modulation of Apoptosis 95 1.7.4 Immune Modulation of Adhesion Molecules 96 1.7.5 VZV Modulation of the Major Histocompatibility Complexes (MHC-I and MHC-II) 96 1.7.6 Cytokines Induced by VZV Infection, and the Immune Modulation of Cytokine Pathways by VZV 100 1.7.6.1 Type I (IFN-) and Type II (IFN- and IFN-) Interferons 103 1.7.7 Modulation of the NFB Transactivation Pathway 105 1.8 Prevention and Treatment 106 1.8.1 Antiviral Therapy 106 1.8.2 Varicella Vaccination 107 1.8.2.1 Development of the Varicella Oka Vaccine 107 1.8.2.2 Comparison of POka and VOka Viruses 107 1.8.2.3 Vaccine Virus Attention 108 1.8.2.4 Uses of the Varicella Vaccine 110 1.8.2.5 Vaccine Efficacy 111 1.8.2.6 The Aim of Universal Vaccination and Achievements Ten Years Post Introduction 111 1.8.2.7 The Effect of Varicella Vaccination on Rates of Zoster 112 1.8.3 Development of the Zoster Vaccine 113 CHAPTER 2: MATERIALS AND METHODS 2.1 List of Buffers and Reagents 115 2.2 Patient Recruitment and Sample Collection 115 2.2.1 ROVE (Response to Oka Vaccine Evaluation) Study 115 2.2.2 Sample Collection in the Case of ROVE Rash Development, Post Vaccination 118 2.3 Sample Processing and Storage 118 2.3.1 ROVE Blood Samples 118 6 2.3.2 Whole Blood 118 2.3.3 Plasma Isolation 118 2.3.4 Serum Isolation 118 2.3.5 Peripheral Blood Mononuclear Cells (PBMC) Separation 119 2.3.6 DNA Extraction 119 2.3.7 Cell Freezing 120 2.3.8 Thawing Cell Aliquots 120 2.3.9 Viable Cell Counts 120 2.3.10 Sample Storage 121 2.4 Serology Based Assays 121 2.4.1 ELISA Based Assays 121 2.4.1.1 VZV IgG TRFIA 121 2.4.1.2 VZV IgG Avidity (EUROIMMUN) Assay 122 2.4.2 SDS-PAGE and Western Blotting 123 2.4.2.1 Coomassie Staining 125 2.4.2.2 Bradford Assay 125 2.4.3 Fluorescent Antibody to Membrane Antigen (FAMA) Technique 125 2.5 Cell Based Assays 126 2.5.1 IFN- ELISPOT 126 2.5.1.1 Ex Vivo Overnight Stimulation on ROVE HCW PBMCs Using Whole VZV Lysate as Antigen 126 2.5.1.2 Assessing PBMC Aggregation at Various Incubation Temperatures for ELISPOT Optimisation 127 2.5.2 DRB1*1501 Specific Peptide Stimulation of PBMCs T Cell Lines 128 2.5.3 Flow Cytometry Analysis 128 2.5.3.1 Flow Cytometry Analysis