Characterisation of Cellular Responses to Pandemic Influenza in Naturally Infected Individuals Shaima Begom Imperial College London National Heart and Lung Institute Submitted for the degree of PhD February 2015 I DECLARATION OF ORIGINALITY I certify that this thesis, and the research to which it refers, are the product of my own work, conducted during the years 2010 and 2014 of the PhD in the Section of Respiratory Medicine at Imperial College London. Any ideas or quotations from the work of other people published or otherwise, or from my own previous work are fully acknowledged in accordance with the standard referencing practices of the discipline. Furthermore, I would like to acknowledge Professor Ajit Lalvani and Dr Saranya Sridhar who designed the ImmunoFlu study cohort. Dr Sridhar recruited subjects for the ImmunoFlu study, and I participated in collection and bio-banking of biological samples during the PhD period. Dr Sridhar and I equally contributed to the laboratory work presented in Results sections 3.2 and 3.3 of this thesis. II COPYRIGHT DECLARATION “The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work.” Registry, Imperial College London. “Since 2003, ownership of copyright in original research articles remains with the Authors*, and provided that, when reproducing the Contribution or extracts from it, the Authors acknowledge first and reference publication in the Journal, the Authors retain the following non-exclusive rights: a. To reproduce the Contribution in whole or in part in any printed volume (book or thesis) of which they are the author(s). b. They and any academic institution where they work at the time may reproduce the Contribution for the purpose of course teaching. c. To reuse figures or tables created by them and contained in the Contribution in other works created by them. d. To post a copy of the Contribution as accepted for publication after peer review (in Word or Tex format) on the Author's own web site, or the Author's institutional repository, or the Author's funding body's archive, six months after publication of the printed or online edition of the Journal, provided that they also link to the Journal article on NPG's web site (eg through the DOI). NPG encourages the self-archiving of the accepted version of your manuscript in your funding agency's or institution's repository, six months after publication. This policy complements the recently announced policies of the US National Institutes of Health, Wellcome Trust and other research funding bodies around the world. NPG recognizes the efforts of funding bodies to increase access to the research they fund, and we strongly encourage authors to participate in such efforts. Authors wishing to use the published version of their article for promotional use or on a web site must request in the normal way.* Commissioned material is still subject to copyright transfer conditions.” Nature Publishing Group, Terms of re-use by Authors. III ACKNOWLEDGEMENTS Firstly, all praise is to Allah. A special thank you to my supervisors Prof Ajit Lalvani and Dr Saranya Sridhar, for giving me the opportunity and inspiration to undertake this project, for training me in various techniques and for support, advice and insightful discussions throughout. Thank you to all the volunteers who donated samples used in this study. Thanks to everyone in the department of Respiratory Medicine at St Mary’s hospital for making the last 4 years an enjoyable experience. The ‘Flubercoidosis Protocols’ will serve as a constant reminder to me of the wonderful friends and colleagues I have worked with. Thank you to Robert Sampson for help and advice on the flow cytometry staining panel. Thank you to Dr Nazneen Siddiqui for your encouragement during the final phase of writing this thesis. This thesis is dedicated to my family; my parents, husband and siblings, your constant support and patience made this dream a reality. IV AUTHORED PUBLICATIONS AND PRESENTATIONS Published papers Sridhar S, Begom S, Bermingham A, Ziegler T, Roberts KL, Barclay WS, Openshaw, P, Lalvani A. Predominance of heterosubtypic IFN-gamma-only-secreting effector memory T cells in pandemic H1N1 naive adults. European Journal of Immunology. 2012 Nov; 42(11):2913-24. Sridhar S, Begom S, Bermingham A, Hoschler K, Adamson W., Carman W., Lalvani A. Higher incidence of natural infection during the third pandemic wave compared to the second pandemic wave. Emerging Infectious Diseases, 2013. 19: 1866-1869. Sridhar S, Begom S, Bermingham A, Hoschler K, Adamson W., Carman W., Barclay WS, Lalvani A. Cellular Immune Correlates for protection against pandemic influenza. Nature Medicine, 2013 Oct, 19(10):1305-12. Sridhar S, Begom S, Hoschler K, Bermingham A, Adamson W, Carman W, Riley S, Lalvani A. Longevity and Determinants of Protective Humoral Immunity Following Pandemic Influenza Infection. AJRCCM, 2014 Dec. [Epub ahead of print]. Manuscripts in preparation Begom S, Sridhar S, et al., Evolution, longevity and immune determinants of protection-associated CD8+ effector T cells following natural pandemic influenza infection. Begom S, Sridhar S, Siddiqui N, Kwok J and Lalvani A. IFN-γ/IL-2 fluorescence- immunospot: a reproducible assay for quantifying clinically relevant functional T cell subsets in human studies. Conference presentations V 6th - 10th Dec 2010. British Society for Immunology Congress, Liverpool, UK. Poster presentation: Shaima Begom, Saranya Sridhar, Alison Bermingham, Samuel Bremang, Lisa Grass, Murphy Magtoto, Lee Potiphar, Peter Openshaw, Ajit Lalvani. ‘Cross-reactive T cells to pandemic influenza H1N1 are predominantly of an effector phenotype’. 29th May – 1st June, 2013. Mechanisms of Antigen Specific Immune Responses (MASIR), Dubrovnik, Croatia. Poster presentation: Shaima Begom, Saranya Sridhar, Chi Kwok, Nazneen Siddiqui, Prof Ajit Lalvani. ‘Assessing clinical performance parameters of a fluorescence-immunospot assay to quantify dual cytokine-secreting cells’. 5th -10th September 2013. Options for the Control of Influenza VIII. Cape Town, South Africa. Poster presentation: Saranya Sridhar, Shaima Begom, Alison Bermingham, Katja Hoschler, Walt Adamson, William Carman, Thomas Bean, Wendy Barclay, Jonathan Deeks, Ajit Lalvani. ‘CD8+ T cells correlate with protection against symptomatic illness following natural pandemic influenza infection in humans’. VI ABBREVIATIONS AF700 Alexa Fluor 700 APC Antigen presenting cell (or allophycocyanin antibody congugate) APC-Cy7 Allophycocyanin conjugated to a Cyanine-7 ASC Antibody secreting cell BSA Bovine serum albumin BV570 Brilliant Violet 570 CD Cluster of differentiation CI Confidence interval CMV Cytomegalovirus CTL Cytotoxic T lymphocyte CV Coefficient of variation DC Dendritic cell DMSO Dimethyl sulphoxide EBV Epstein bar virus EDTA Ethylenediaminetetraacetic acid ELISpot Enzyme-linked immunosorbent spot ESAT-6 6kDa early secreted antigenic target from Mycobacterium tuberculosis FACS Fluorescence activated cell sorting FBS Fetal bovine serum Fig. Figure FITC Fluorescin Isothiocyanate FMO Fluorescence minus one GALT Gut-associated lymphoid tissue Gamma-delta HA Haemagglutinin HAI Haemagglutination inhibition HIV-1 Human immunodeficiency virus-1 HLA Human leukocyte antigen HSI Heterosubtypic immunity IAV Influenza A virus ICS Intracellular cytokine staining IFN Interferon IFN- Interferon-gamma IgG Immunoglobulin G IgA Immunoglobulin A IgM Immunoglobulin M IL-1 Interleukin-1 IL-2 Interleukin-2 IL-4 Interleukin-4 IL-5 Interleukin-5 IL-6 Interleukin-6 IL-10 Interleukin-10 IL-12 Interleukin-12 IL-13 Interleukin-13 IL-17 Interleukin-17 ILI Influenza-like illness ILC Innate lymphoid cell iNKT Invariant Natural Killer T cell VII IQR Interquartile range L/D Live/Dead LAIV Live attenuated influenza vaccine LCMV Lymphocytic choriomeningitis virus LM Listeria monocytogenes LRT Lower respiratory tract M1 Matrix protein 1 mAb Monoclonal antibody MACS Magnetic activated cell sorting MAIT Mucosa-associated invariant T cell MALT Mucosa-associated lymphoid tissue MHC Major Histocompatibility Complex MOI Multiplicity of infection NA Neuraminidase NALT Nasopharynx-associated lymphoid tissue NK Natural Killer NLR Nod-like receptor NP Nucleoprotein OR Odds ratio PB1 Polymerase basic protein 1 PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PE Phycoerythrin PE-CF594 Phycoerithrin conjugated to Carmen Fluorochrome 594 PE-Cy5 Phycoerythrin conjugated to Cyanine-5 PE-Cy7 Phycoerythrin conjugated to Cyanine-7 PerCP-Cy5.5 Peridinin Chlorophyll Protein conjugated to Cyanine-5.5 pH1N1 2009 pandemic H1N1 PHE Public Health England PMA/I Phorbol myristate acetate/Ionomycin PPD Purified protein derivative QDot605 Quantum Dot 605 Qdot655 Quantum Dot 655 RT-PCR Reverse transcriptase polymerase chain reaction SA Strepavidin SD Standard deviation SEM Standard error of mean SFC Spot-forming cell Tcm Central memory T cell TCR T cell receptor Tem Effector memory T cell Temra Late effector memory T cell Th1 T helper 1 Th2 T helper 2 Th17 T helper 17 TIV Trivalent inactivated vaccine TLR Toll-like receptor TNF- Tumour necrosis factor alpha Trm Resident memory T cell URT Upper