Microbiology & Immunology - Theses
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ItemThe role of Major Histocompatibility Complex class I in Human Immunodeficiency Virus-1 and Simian Immunodeficiency Virus infections( 2016)The first official report of AIDS was in 1981. Since then, the HIV-1 infection has reached a pandemic state and high rates of infection are especially seen in poor developing countries. Intensive research has greatly prolonged the life expectancy of an HIV-1+ individual, but combined antiretroviral therapy (cART) is costly and the evolution of drug resistant HIV-1 strains are major obstacles to therapy. Thus, a prophylactic anti-HIV-1 vaccine will be ideal to address these challenges. The last thirty years have seen a few vaccine trials, with the most promising of these being the Thai RV144 trial with a vaccine efficacy of 31.2%. Subsequent research has demonstrated that the protective effect was likely conferred by anti-HIV-1 antibody-dependent cell-mediated cytotoxicity (ADCC) mediating IgG antibodies against Env, in the presence of low levels of IgA against Env. Cell types, such as, natural killer cells (NK cells), monocytes and neutrophils mediate ADCC. NK cell mediated ADCC is affected by NK cell education involving major histocompatibility complex class I (MHC I) alleles, as they are ligands for most NK cell receptors. A characteristic feature of HIV-1 infection is the peak viremia observed approximately two weeks after infection, which declines with the emergence of the cellular immune response such as CD8+ T cells. CD8+ T cells recognise virus infected cells through HIV-1 peptides bound to MHC I molecules on antigen presenting cells. Population studies have also demonstrated the role played by MHC I alleles in HIV-1 infection progression. Therefore, this thesis examined the role of MHC I in HIV-1 and SIV infections. SIV infected non-human primates are a valuable research tool to study immunity to HIV-1, trial candidate vaccines and drugs. The most well studied animal model is the SIV infected Indian rhesus macaque model. However, these macaques are difficult to obtain for research purposes due to export bans in India and the limited distribution of breeding colonies in the world. The macaque model we employ is the pig-tailed macaque sourced from breeding colonies in Australia and Indonesia. SIV infection in pig-tailed macaques closely mimics that in Indian rhesus macaques. Unlike in the SIV infected Indian rhesus macaque model, the MHC I alleles that control SIV infection progression in the pig-tailed macaque model are poorly defined. To date, only one allele, Mane-A1*084, has been linked with CTL escape in Gag and Tat proteins of SIV. In Chapter 2, we examined the role played by MHC I alleles in CTL escape in 44 SIVmac251 infected pig-tailed macaques. We extracted cellular RNA from 44 pig-tailed macaques and used 454 Roche pyrosequencing to determine the Mane haplotypes. Viral RNA was extracted from the plasma of these SIVmac251 infected pig-tailed macaques and we sequenced the whole genome of the virus using Illumina Nextera XT MiSeq sequencing. We also sequenced the challenge stock virus for the purposes of a reference genome during the bioinformatics analysis. Using a novel approach we first identified the well known CTL escape epitopes linked with Mane-A1*084, KP9 in Gag and KSA10 and KVA10 in Tat to validate our approach. We found over 70 potential CTL escape mutations. We validated one such result, a Nef CTL escape mutation associated with Mane-B028 using an intracellular cytokine staining (ICS) assay. Analysis of viral load data demonstrated that CTL escape driven by Mane-A1*084, as previously published by our group does not affect SIV infection control. Tetramer staining of expanded epitope specific CD8+ T cells, with tetramers synthesised for use in Indian rhesus macaques demonstrated that reagents used in rhesus macaques can also be used in pig-tailed macaques. Many studies have shown that cell-associated virus is more infectious than cell-free virus. We developed an assay to measure anti-HIV-1 NK cell activity against allogeneic T cells (Chapter 3) using a 29 cohort of HIV-1- individuals. We used gp120 coated T cells from donor 1, and combined whole blood from donor 2 in the presence of HIV-1+ plasma, brefeldin A, monensin and CD107a. NK cell activation was measured using intracellular IFNγ staining. NK cells were activated only in the presence of gp120 coated T cells and HIV-1+ plasma. We were able to modify this assay to investigate the contribution of different HLA/KIR interactions on anti-HIV-1 NK cell activation using an HIV-1- cohort. Epidemiological studies have demonstrated that the presence of KIR3DL1 and its ligand HLA-Bw4 confers protection and/or controls HIV-1 infection. We observed that educated KIR3DL1+ NK cells were able to overcome the inhibitory interaction between KIR3DL1 and HLA-Bw4, to show NK cell activation against HLA-Bw4+ gp120 coated allogeneic T cells. We also used a LDH cytotoxicity assay and showed cytolysis of gp120 coated CEM cells, in the presence of HIV-1+ antibodies. Another KIR/HLA combination of interest is the inhibitory KIR2DL1 and HLA-C2, which may protect from or control HIV-1. Studies on HIV-1+ African cohorts have shown that this KIR population expands following HIV-1 infection. In Chapter 4, we examined the role of this HLA/KIR combination using the above-mentioned modified assay. KIR2DL1+ NK cells from HLA-C2 donors showed higher levels of activation than KIR2DL1- NK cells, in the presence of gp120 coated CEM cells and HIV-1+ plasma. NK cell activation was not limited to antibody dependent stimulation, as a HLA devoid cell line called 721.221 also elicited NK cell activation in educated KIR2DL1+ NK cells. CD57 is a NK cell differentiation marker, and CD57+ NK cells showed higher NK cell activation than CD57- NK cells. CD57+ NK cells also had a higher frequency of KIR2DL1 expression as expected. Despite this, the enhanced activation observed in the educated KIR2DL1+ NK cell subset was not a result of differentiation, as CD57- KIR2DL1+ also showed high NK cell activation. An autologous ADCC assay showed that the functional advantage observed in educated KIR2DL1+ NK cells is a function of education. In conclusion, it is clear that host MHC I alleles play an important role in HIV-1 and SIV infections. We identified a number of MHC I alleles with the potential to drive CTL escape in SIV infection. Future studies to validate these findings will be useful for vaccine studies. This will enable the selection of macaques for vaccine trials and help determine the effect of the vaccine independent of host MHC I alleles. The Thai RV144 trial indicated that anti-HIV-1 ADCC, may be an important immune mechanism to target in anti-HIV-1 vaccine development. As detailed in Chapters 3 and 4, NK cell education plays an important role in the anti-HIV-1 NK cell activation in the presence of allogeneic T cells. Anti-HIV-1 ADCC was observed in the presence of low levels of surface Env on target T cells as discussed in Chapter 3, which is promising as HIV-1+ CD4 T cells are known to down-regulate the expression of CD4 and hence display low levels of surface HIV-1 Env. The studies in this thesis show that it is important to consider the influence of host genes in HIV-1 infection progression, in future anti-HIV-1 vaccine development studies.