The transmitted HIV-1 subtype C: Characterization of the transmitted/founder full-length virus genome and the influence of early immune selective pressure on virus replication Melissa-Rose Hilda Abrahams Univeristy of Cape Town Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY in the Department of Clinical Laboratory Sciences UNIVERSITY OF CAPE TOWN October 2014 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Table of Contents Table of Contents ................................................................................................................... i Declaration ............................................................................................................................ iii Abstract................................................................................................................................. iv Acknowledgements ............................................................................................................... v List of Abbreviations .............................................................................................................. vi Chapter 1: Literature Review .............................................................................................. 1 1.1 Current perspective of the global HIV-1 pandemic and the progress of vaccine development ............................................................................................................... 2 1.2 Viral diversity ............................................................................................................... 5 1.3 The transmitted virus ................................................................................................... 7 1.4 High viral mutability ................................................................................................... 11 1.5 Immune responses to HIV-1 infection ........................................................................ 13 1.6 Immune selection and CTL escape............................................................................ 16 1.7 CTL responses and viral control ................................................................................ 19 1.8 Challenges to HIV-1 vaccine design .......................................................................... 21 1.9 Study rationale........................................................................................................... 24 Chapter 2: Identification of near full-length HIV-1 transmitted/founder subtype C virus genomes and their early diversification .......................................................................... 25 2.1 Abstract ..................................................................................................................... 26 2.2 Introduction ................................................................................................................ 27 2.3 Research Aim and Objectives .................................................................................... 29 2.4 Rationale ................................................................................................................... 30 2.5 Methods and Materials .............................................................................................. 30 2.6 Results ...................................................................................................................... 37 2.7 Discussion ................................................................................................................. 57 Chapter 3: Mapping immune selection in early subtype C infection ............................. 60 3.1 Abstract ..................................................................................................................... 61 3.2 Introduction ................................................................................................................ 62 i 3.3 Research Aim and Objectives .................................................................................... 65 3.4 Rationale ................................................................................................................... 65 3.5 Methods and Materials .............................................................................................. 65 3.6 Results ...................................................................................................................... 67 3.7 Discussion ................................................................................................................. 84 Chapter 4: The effect of CTL escape mutations on viral replication fitness ................. 89 4.1 Abstract ..................................................................................................................... 90 4.2 Introduction ................................................................................................................ 91 4.3 Research Aim and Objectives .................................................................................... 93 4.4 Rationale ................................................................................................................... 94 4.5 Methods and Materials .............................................................................................. 94 4.6 Results .................................................................................................................... 101 4.7 Discussion ............................................................................................................... 112 Chapter 5: General Discussion and Conclusion ........................................................... 116 5.1 Discussion ............................................................................................................... 116 5.2 Conclusion ............................................................................................................... 117 References ....................................................................................................................... 119 Appendix 1: Published manuscript Abrahams et al., 2009 J Virol ...................................... 145 Appendix 2: Published manuscript Abrahams et al., 2013 AIDS ....................................... 146 Appendix 3: Supplemental near full-length and sub-genomic sequences .......................... 147 Appendix 4: Limiting dilution amplification of gag, vif and nef ............................................ 148 Appendix 5: Construction of IMC pCAP200.t/f ................................................................... 151 ii Declaration I, Melissa-Rose Hilda Abrahams, hereby declare that the work on which this thesis/dissertation is based is my original work (except where acknowledgements indicate otherwise) and that neither the whole work nor any part of it has been, is being, or is to be submitted for another degree in this or any other university. I authorise the University to reproduce for the purpose of research either the whole or any portion of the contents in any manner whatsoever. Signature:……………………………………………….Date: ………………………………………. iii Abstract The identification of targets of early immune responses associated with control of HIV-1 infection will inform immunogen design for vaccine interventions. The early evolution of transmitted/founder subtype C virus sequences was investigated to determine the location and frequency of immune selection, and the impact of early immune escape mutations on viral replicative capacity. Single-genome amplified env sequences from 26 acutely-infected women were evaluated for conformance to a model of random evolution to elucidate multiplicity of infection. Near full- length genome sequences from the first six months of infection were generated for five women and sites evolving under immune selection were mapped. CD8+ cytotoxic T- lymphocyte escape mutations in HLA-B-restricted epitopes were introduced into infectious molecular clones of cognate transmitted/founder viruses by site-directed mutagenesis and their impact on viral replicative fitness was evaluated using parallel replication assays. In 77% of women (n=20) a single transmitted/founder variant established infection and two to five variants in the remaining 23% (n=6). Near full-length genome sequencing in five women confirmed single variant/low-diversity transmission and identified fifty-five genome regions evolving under immune selection, 40% of which was attributed to CD8+ cytotoxic T- lymphocyte pressure, 35% to antibody-mediated pressure, 16% to reversion and 9% could not be classified. The rate of sequence diversification and number of sites evolving under immune selection was highest in nef. The majority of evolving CD8+ cytotoxic T-lymphocyte epitopes (82%) contained shuffling/toggling mutations. A novel B*15:10-associated mutation, A164T, combined with a V85A Pol mutation reduced viral replication capacity in one individual. In a second individual, the attenuating HLA-B*58:01-associated mutation, T242N, enhanced viral replication capacity due to pre-existing compensatory polymorphisms in the transmitted/founder virus. A third individual, who had extremely rapid disease progression, was infected with the virus