i IMPACT OF IMMUNE-DRIVEN SEQUENCE VARIATION IN HIV-1 SUBTYPE C GAG- PROTEASE ON VIRAL FITNESS AND DISEASE PROGRESSION SUBMITTED BY: JACLYN WRIGHT SUPERVISED BY: PROFESSOR THUMBI NDUNG’U Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in Virology in the Nelson R. Mandela School of Medicine, College of Health Sciences, University of KwaZulu-Natal, Durban. 2011 i This project represents original work done by the author and where others have made contributions it has been acknowledged in the text. The experimental work described in this thesis was performed in the HIV Pathogenesis Programme Laboratory, Hasso Plattner Research Laboratory, and Africa Centre Laboratory, in the Doris Duke Medical Research Institute, Nelson R. Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa, under the supervision of Professor T. Ndung’u. J. Wright: Date: Professor T. Ndung’u: Date: As the candidate’s supervisor I agree to the submission of this thesis. Professor T. Ndung’u: Date: ii DECLARATION I, Jaclyn Wright, declare that (i) The research reported in this thesis, except where otherwise indicated, is my original work. (ii) This thesis has not been submitted for any degree or examination at any other university. (iii) This thesis does not contain other person’s data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. (iv) This thesis does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: (a) Their words have been re-written but the general information attributed to them has been referenced; (b) where their exact words have been used, their writing has been placed inside quotation marks, and referenced. (v) Where I have reproduced a publication of which I am author, co-author or editor, I have indicated in detail which part of the publication was actually written by myself alone and have fully referenced such publications. (vi) This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the References sections, Signed: Date: iii PUBLICATIONS AND PRESENTATIONS Peer-reviewed publications: 1. Wright, J.K., Brumme, Z.L., Carlson, J.M., Heckerman, D., Kadie, C.M., Brumme, C.J., Wang, B., Losina, E., Miura, T., Chonco, F., van der Stok, M., Mncube, Z., Bishop, K., Goulder, P.J.R., Walker, B.D., Brockman, M.A., Ndung’u T. 2010. Gag- Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters. Journal of Virology 84(20): 10820-10831. 2. Wright, J.K., Novitsky, V., Brockman, M.A., Brumme, Z.L., Brumme, C.J., Carlson, J.M., Heckerman, D., Wang, B., Losina, E., Leshwedi, M., van der Stok, M., Maphumulo, L., Mkhwanazi, N., Chonco, F., Goulder, P.J.R., Essex, M., Walker, B.D., Ndung’u, T. 2011. Influence of Gag-Protease-Mediated Replication Capacity on Disease Progression in Individuals Recently Infected with HIV-1 Subtype C. Journal of Virology 85(8): 3996-4006. 3. Radebe, M., Nair, K., Chonco, F., Bishop, K., Wright, J.K., van der Stok, M., Bassett, I.V., Mncube, Z., Altfeld, M., Walker, B.D., Ndung’u, T. 2011. Limited immunogenicity of HIV CD8+ T-cell epitopes in acute clade C virus infection. Journal of Infectious Diseases 204(5): 768-76. 4. Wright, J.K., Brumme, Z.L., Julg, B., van der Stok, M., Mncube, Z., Gao, X., Carlson, J.M., Goulder, P.J., Walker, B.D., Brockman, M.A., Ndung'u, T. 2011. Lack of Association between HLA Class II Alleles and In Vitro Replication Capacities of Recombinant Viruses Encoding HIV-1 Subtype C Gag-Protease from Chronically Infected Individuals. Journal of Virology 86(2): 1273-1276. 5. Wright, J.K., Naidoo, V.L., Brumme, Z.L., Prince, J.L., Claiborne, D.T., Goulder, P.J.R., Brockman, M.A., Hunter, E., Ndung’u, T. 2012. The impact of HLA-B*81- associated mutations in HIV-1 Gag on viral replication capacity. Journal of Virology, in press. Conference presentations: 1. Wright, J.K., Brockman, M.A., Brumme, Z.L., Goulder, P.J.R., Walker, B.D., Ndung’u, T. Impact of Immune-Driven Sequence Variation in HIV-1 Subtype C Gag-Protease on Viral Fitness and Disease Progression. AIDS Vaccine Conference 2009, 19-22 October, 2009, Paris, France, Abstract 164471. iv 2. Wright, J.K., Brockman, M.A., Brumme, Z.L., Reddy, S., Maphumulo, L., Bishop, K., Goulder, P.J.R., Walker, B.D., Ndung’u, T. Evidence of Higher HIV-1 Subtype B Gag-Protease-Mediated Viral Replicative Fitness Compared to HIV-1 Subtype C. South African Immunology Society meeting, 9-11 December, 2009, Cape Town, South Africa, Abstract LB-61. 3. Wright, J.K., Brockman, M.A., Brumme, Z.L., Nair, K., Chonco, F., van der Stok, M., Mncube, Z., Bishop, K., Walker, B.D., Ndung’u, T. Gag-Protease-mediated fitness is associated with viral set point in individuals acutely infected with HIV-1 subtype C. Seventeenth Conference on Retroviruses and Opportunistic Infections, 16-19 February, 2010, San Francisco, USA, Abstract F-112. 4. Wright, J.K., Brockman, M.A., Brumme, Z.L., Carlson, J.M., Heckerman, D., Goulder, P.J.R., Walker, B.D., Ndung’u, T. Mutations in Gag-Protease associated with changes in replication capacity in chronic HIV-1 subtype C infection. XVIII International AIDS Conference, 18-23 July, 2010, Vienna, Austria, Abstract TUPE0035. 5. Wright, J.K., Novitsky, V., Brockman, M.A., Brumme, Z.L., Brumme, C.J., Carlson, J.M., Leshwedi, M., Ndung’u, T. Influence of Gag-Protease-Mediated Replication Capacity on Disease Progression in Individuals Recently Infected with HIV-1 Subtype C. 5th South African AIDS conference, 7-10 June, 2011, Durban, South Africa, Abstract 77. 6. Wright, J.K., Brockman, M.A., Brumme, Z.L., Carlson, J.M., Heckerman, D., Naidoo, V.L., Jaggernath, M., Goulder, P.J.R., Walker, B.D., Ndung’u, T. The impact of Gag and specific HLA-B*81-associated Gag mutations on viral replication capacity. 6th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 17-20 July, 2011, Rome, Italy, Abstract MOPE061. v STATEMENT The following publications, fully referenced here, have been reproduced in part non- continuously throughout the thesis: 1. Wright, J.K., Brumme, Z.L., Carlson, J.M., Heckerman, D., Kadie, C.M., Brumme, C.J., Wang, B., Losina, E., Miura, T., Chonco, F., van der Stok, M., Mncube, Z., Bishop, K., Goulder, P.J.R., Walker, B.D., Brockman, M.A., Ndung’u T. 2010. Gag- Protease-Mediated Replication Capacity in HIV-1 Subtype C Chronic Infection: Associations with HLA Type and Clinical Parameters. Journal of Virology 84(20): 10820-10831. 2. Wright, J.K., Novitsky, V., Brockman, M.A., Brumme, Z.L., Brumme, C.J., Carlson, J.M., Heckerman, D., Wang, B., Losina, E., Leshwedi, M., van der Stok, M., Maphumulo, L., Mkhwanazi, N., Chonco, F., Goulder, P.J.R., Essex, M., Walker, B.D., Ndung’u, T. 2011. Influence of Gag-Protease-Mediated Replication Capacity on Disease Progression in Individuals Recently Infected with HIV-1 Subtype C. Journal of Virology 85(8): 3996-4006. The candidate performed the experiments described in these papers, and where others made contributions it has been duly acknowledged in the text. The candidate drafted these publications in full and they were reviewed by co-authors. J. Wright: Date: Professor T. Ndung’u: Date: vi ACKNOWLEDGEMENTS I would like to thank: - Professor Ndung’u for his supervision, encouragement, and support throughout the project - Dr. Mark Brockman and Dr. Zabrina Brumme for their hospitality and mentorship - Jennifer Sela and Pamela Rosato for demonstrating laboratory assays - Staff and students at the HIV Pathogenesis Programme, Hasso Plattner Research Laboratory and Africa Centre for assistance, in particular Keshni Hiramen, Taryn Green, and Vanessa Naidoo - Collaborators as acknowledged in the text - Dr. Johannes Viljoen and Africa Centre laboratory for providing access to tissue culture and sequencing facilities - Sinikithemba Study, HIV Pathogenesis Programme Acute Infection Study, and Tshedimoso Study teams - Clinic/hospital staff as well as the study participants - The National Research Foundation, Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, and the HIV Pathogenesis Programme for financial assistance vii ABSTRACT Introduction Understanding of the viral and host factors that determine time for progression to acquired immunodeficiency syndrome (AIDS) in individuals infected with human immunodeficiency virus type 1 (HIV-1) could aid in the design of an effective HIV-1 vaccine. Human leukocyte antigen (HLA) class I profile is strongly and consistently associated with differential rates of HIV-1 disease progression, however the mechanisms explaining this are not well understood. It has been hypothesised that “protective” HLA alleles select escape mutations in functionally important epitopes in the conserved group specific antigen (Gag) protein resulting in HIV-1 attenuation, which may result in slower disease progression. Many of the studies investigating the fitness cost of Gag escape mutations have concentrated on a few pre-selected mutations and have not assessed fitness consequences in the natural sequence background. Furthermore, the majority of studies have focussed on HIV-1 subtype B, while HIV-1 subtype C is the most prevalent subtype worldwide. Therefore, in the present study, a large population-based approach and clinically-derived Gag-protease sequences were used to comprehensively investigate the relationship between immune- driven sequence variation in Gag, viral replication capacity and markers of disease progression in HIV-1 subtype C chronic infection. The influence of Gag function on HIV-1 disease progression was further investigated in early HIV-1 subtype C infection.