Final Report Summary - UNFITHIV (Understanding the importance of viral Fitness Impairment after Transmission of HIV: Implication on HIV vaccine design) There is an urgent need to develop effective vaccines to combat the human immunodeficiency virus (HIV)/AIDS pandemic, which currently causes ~2 million deaths per year worldwide. The challenges associated with induction of protective antibody responses to HIV, together with the wealth of evidence showing that virus-specific CD8 T cells play an important role in control of HIV replication, have prompted efforts to develop T cell-inducing vaccines to combat this infection; but vaccine design is hampered by the current lack of understanding of features of a protective HIV-specific T cell response. HIV’s ability to evade T cell control by mutating to escape T cell recognition suggests that it may be critical for vaccines to elicit T cell responses that limit viral escape. However the changes the virus undergoes to escape from T cell responses may reduce its ability to replicate, which could be beneficial. This project is designed to explore the importance of HIV’s replication capacity and its reduction by T cell escape mutations in determining the outcome of infection. A detailed analysis of viral interactions with the host CD8 T cell response in acute HIV infection has been performed in 9 infected individuals, 4 controlling infection well and 5 controlling infection poorly, to gain insight into the importance of i. the fitness of the transmitted virus ii. the extent and kinetics of viral escape from the earliest CD8 T cell responses and iii. the viral fitness cost incurred in escaping from these early responses, in determining the prognostically-important set-point persisting viral load. Three specific aims were proposed in the initial grant application to address these points. In Aim 1 we planned to evaluate the fitness of the transmitted founder (TF) virus and its relationship to the persisting viral load established in early HIV infection. In order to do so, viral isolates were generated from the earliest plasma sample available from 7 of the 9 HIV infected subjects (2 controllers and 5 non-controllers) and their replicative capacity in CD4 T cells was compared in vitro. In addition, HIV-1 infectious molecular clones (IMC) corresponding to the full length sequence of the TF virus from 2 controllers and 2 non-controllers were produced, infectious virus was generated from these constructs by 293FT transfection and the growth kinetics of these IMC-derived TF viruses in CD4 T cells were assessed in vitro. In experiments performed with both virus isolates and IMCs, there was a trend for viruses from HIV controllers to exhibit a lower replication capacity than those from non-controllers. Although the low number of subjects studied precluded detection of a statistically significant difference in the replicative fitness of viruses from controllers and non-controllers (or a correlation between the fitness of the transmitted founder virus and viral load at viral set point), these results lend support to the hypothesis that the fitness of the transmitted virus represents one of the factors that contributes to the level at which viral replication is controlled following acute HIV-1 infection.For Aim 2 we planned to analyse comprehensively the extent and kinetics of HIV-1 escape from the earliest virus-specific CD8 T cell responses and its relationship to the efficiency of control of early virus replication. This was achieved by mapping the epitopes recognised by the host T cell response during acute and early infection in all 9 study subjects, assessing viral evolution at these sites over the first year of infection by longitudinal sequence analysis, and assessing the effect of mutations identified within T cell epitope sequences on peptide recognition by epitope-specific T cells. It was apparent that viral escape from T cell recognition, resulting in a reduced or abolished T cell response, is a common phenomenon in early HIV infection as it was evident in all subjects regardless of infection outcome. There was a trend for subjects who failed to control viral replication efficiently to exhibit a higher percentage of escape from the initially dominant T cell responses by 3 months post-infection. However, this did not reach statistical significance and there were examples of subjects who controlled viremia efficiently but showed rapid escape from the initial T cell response and non-controllers that showed little evidence of early T cell escape. This raised the hypothesis (investigated in aim 3) that not just the extent of rapid escape from the earliest host T cell responses, but also the costs to viral fitness incurred in achieving this escape, may determine the setpoint viral load established following acute infection. Aim 2 was extended to incorporate cutting-edge research to address the hypothesis that CD8 T cells are able to recognise epitopes derived from the terminal regions of the HIV-1 genome, previously termed the untranslated regions (UTRs). This work was performed in collaboration Prof George Shaw’s group at the University of Pennsylvania, USA, who had identified several sites within the UTR of 5 acutely HIV-infected subjects where the transmitted founder sequence was rapidly replaced by mutations over time. We addressed whether these mutations could potentially have been driven by immune selection pressure exerted by T cell responses directed against UTR-derived epitopes. In order to evaluate whether CD8 T cell responses were directed against epitopes derived from the UTR, overlapping peptides corresponding to the putative translation products from all six reading frames of each subject’s autologous virus were synthesised and tested for T cell recognition in IFN ELISPOT assays. In two subjects, no responses were detected to any of the peptides tested. However in one subject we were able to find a T cell response towards the dimerization initiation sequence (DIS) region within the UTR. Further fine-mapping identified the optimal epitope peptide, and we confirmed that mutations selected for within this sequence over time in vivo reduced or abolished epitope recognition by the subject’s T cell response. We were also able to detect a UTR-specific T cell response in a second subject, which was directed towards an epitope within the U3 region of the UTR. Work on the fifth subject is still ongoing. These results show that the terminal regions of the HIV-1 genome constitute a previously-unrecognised source of novel targets for CD8 T cell responses that are able to exert biologically significant selection pressure on viral replication in vivo, the utility of which as HIV vaccine immunogens warrants future exploration.Aim 3 planned to address the fitness costs of mutations acquired to escape from the earliest host T cell responses in HIV controllers versus non-controllers. We engineered early T cell escape mutations into the TF IMCs generated in aim 1 by site-directed mutagenesis and compared the replication capacity of the TF and variant viruses in CD4 T cells in vitro. Two early T cell escape mutations selected for in the one TF controller virus studied to date each reduced the replicative capacity of the TF virus; but whereas an early T cell escape mutation was found to reduce autologous TF virus replication in one of the non-controllers studied, in the other non-controller early escape did not reduce the replication of the autologous TF virus. However the effects of early T cell escape mutations on the fitness of TF viruses from additional subjects need to be studied to determine whether there are significant differences in the fitness costs of escape mutations in controllers and non-controllers. In summary, the results from this in-depth study in a small number of infected individuals suggest the importance of infection with a transmitted founder virus that has a lower replication capacity and an associated induction of a broad primary HIV-specific T cell response, escape from which occurs with relatively delayed kinetics and may also be associated with high costs to viral fitness, to good viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that if they mutate incur a high fitness cost, emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes. Furthermore, we have identified the terminal regions of the HIV-1 genome as a novel source of CD8 T cell epitopes that could be targeted in vaccine design to increase response breadth and efficacy.