abstract / Microbiology / Doctoral / Doctor of Philosophy

An effective HIV-1 vaccine will likely elicit broadly neutralizing antibodies (bnAbs). However, development of vaccine immunogens that induce bnAbs remains a challenging goal. Understanding the somatic maturation pathways of known broadly neutralizing HIV-1 human monoclonal antibodies (bnmAbs) may help vaccine immunogen design. All known HIV-1 bnmAbs are highly somatically matured, and the putative germline antibodies of the known HIV-1 bnmAbs lack measurable binding activity to HIV-1 envelope glycoprotein (Env). Based on these observations, we hypothesize that somatic maturation of known HIV-1 bnmAbs may be initiated by primary immunogens which may not be related to HIV-1 Env; such primary immunogens trigger the somatic maturation of the germline antibodies and induce intermediate antibodies that bind to HIV-1 Env and further mature to bnAbs upon HIV-1 infection or Env vaccination. The main objective of my study is to identify intermediate antibodies of bnmAb b12 in uninfected and infected human individuals, as well as in uninfected rhesus macaques, the model animals for vaccine development. I constructed two nonimmune cDNA antibody VH1 scFv libraries using the mRNAs isolated from pooled PBMCs of 200 uninfected healthy human individuals and one uninfected rhesus macaque, respectively, and identified 5 and 10 possible b12 intermediate immunoglobulin heavy chain V-segments (IGHVs) from the human and macaque nonimmune libraries, respectively. 454 deep sequencing of the VHs and VLs in the nonimmune and two immune human cDNA Fab libraries confirmed the existence of b12 intermediate IGHVs, but we did not find further maturation of the b12 intermediate IGHVs in HIV-1-infected human individuals. Further sequence analysis revealed the extremely low frequency of the VHs with exactly the same V(D)J recombination as b12, which may explain the lack of further maturation of the intermediate IGHVs of b12 in HIV-1-infected humans. Characterization of HIV-1 Env trimer may aid in Env-based vaccine immunogen design. Therefore, I investigated the importance of Env variable loops in Env-mediated viral function. A panel of gp160JRFL loop deletion/replacement mutants were constructed and tested. The results indicate that, besides the CD4 binding loop and V3, V1V2 and loop D are also critical for virus entry into permissive cells. Deletion of variable V4 or V5 loop or replacement of V4 or V5 loop with a flexible linker of the same length abolish Env cell surface display, which may result from the conformational changes of the V4 or V5 loop deletion or replacement Env proteins. V4 or V5 deletion or replacement knocks out the CD4 binding site and CD4-induced site on Env, but enhances the exposure of the membrane-proximal external region (MPER) and N-trimer structure. In summary, my study demonstrated the existence of intermediate b12 IGHVs in uninfected and HIV-1-infected humans and rhesus macaques. These intermediate antibody fragments may be used to identify primary immunogens that initiate b12 somatic maturation. My study also showed the importance of Env variable loops for Env structural integrity and Env-mediated viral function. The enhanced exposure of the MPER in gp160JRFL ΔV4 or ΔV5 may be further explored for vaccine development to induce MPER-specific bnAbs. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

No abstract available. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2011.

Certain HLA-B-restricted CD8+ T cell responses are associated with control of viremia whereas HLA-Cw* restricted responses, including Gag epitopes are associated with high viremia. To better understand the role of HLA-Cw* restricted epitopes in viral control, HLA-Cw* restricted epitopes were optimally defined. Seventy eight study subjects from a cohort of 451 chronically infected participants had HLA-Cw* restricted CD8+ T cells responses as quantified by intracellular cytokine staining assessing IFN-γ secretion. Fine mapping and HLA restriction of the optimally defined HLA-Cw* restricted epitopes were performed using ELISPOT assay. Functional avidity of responses was assessed by peptide dilution in an ELISPOT assay. Two novel HLA-Cw* restricted epitopes Cw*04- TF10 (in reverse transcriptase) and Cw*08-RM9 (in gp120) were optimally defined. A previously described epitope, Cw*07- KY11 (Nef) was the most frequently targeted epitope in this cohort (30/78) and has high functional avidity compared to other HLA-Cw restricted CD8+ T cell responses. The polyfunctionality of HLA-B*57/5801-restricted Gag-specific HIV-1 CD8+ T cell responses and HLA-Cw*07-KY11 restricted CD8+ T cell responses within the same study subject was determined. Polyfunctionality of CD8+ T cell responses to HLAB* 57/5801 and HLA-Cw*07 restricted epitopes were determined in nine study subjects assessing IFN-γ, TNF-α, IL-2, MIP-1β, and CD107a by multicolour flow cytometry. Additionally gag and nef genes were sequenced from plasma. HLA-B*57/5801-restricted IFN-γ-producing CD8+ T cell responses were of lower magnitude than HLA-Cw*07 responses (p=0.0012) for the nine subjects. The majority of responses were monofunctional (75%), irrespective of HLA restriction. HLA-B*57/5801 and HLACw* 07 restricted CD8+ T cells did not differ significantly in polyfunctionality (p=0.84). Possession of ≥3 functions correlated positively with CD4+ T cell counts (r=0.85; p=0.006). The percentages of monofunctional CD8+ T cells inversely correlated with CD4+ T cell counts (r=-0.79; p=0.05). There was no correlation between polyfunctionality and viral load and sequence variation within targeted epitopes did not impact polyfunctionality. These results suggest that polyfunctionality of HIV-1-specific CD8+ T cells is associated with disease progression independent of restricting HLA alleles, and that loss of these polyfunctional cells correlates with increased in the frequency of monofunctional virus-specific CD8+ T cells. In addition, sequence variation does not appear to significantly impact CD8+ T cell polyfunctionality in chronic HIV infection. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2010.

Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (T_regs) and CD141⁺ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141⁺ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10^-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ T_regs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of T_regs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of T_regs with matched specificity for therapeutic intervention.

Despite the introduction of antiretroviral therapy, the HIV/HIV epidemic remains an unsolved global health problem. Amongst all the host defence mechanisms, HLA class I molecules have shown the strongest genetic association with delayed disease progression, in particular HLA-B alleles. Numerous studies have shown that the HLAmediated CD8+ T cell responses play a central role in the immune control of HIV. Yet our understanding of HLA-mediated immune control of HIV remains incomplete, even when considering the best-defined epitopes restricted by the protective HLA alleles at a population level. The studies I have conducted and describe herein focus on two well-charaterised protective HLA-B molecules, HLA-B*81:01 and HLA-B*27:05; a third protective molecule, HLA-B*52:01, that has not been well-studied hitherto; and finally the most prevalent HLAB allele in many Asian populations such as Taiwan, HLA-B*40:01, which has an apparently neutral effect on viral replication. This thesis is centred on the Gag-specific immune response, since previous studies have shown the benefits of CD8+ T-cell responses targeting this conserved and immunogenic region of the HIV proteome, in particular the p24 capsid protein. I have investigated here HLA footprints driven by CD8+ T-cell pressure on HIV that are evident in the viral sequences of individuals expressing these HLA molecules. These footprints include novel escape and putative compensatory mutations. The impact of these variants on viral replicative capacity (VRC) and on HIV disease outcome clinical outcomes was examined via fitness assays. These studies identified several escape mutations that effectively cripple HIV. The distinct compensatory pathways available to the virus to mitigate the fitness cost of particular escape mutations were evaluated. In the course of these analyses I have demonstrated the critical influence of the viral backbone, including HIV clade, in combination with particular viral variants, on VRC. Computational modelling analysis has been applied to facilitate understanding of the mechanism by which certain mutants affect the stability of interactions between HLA and viral capsid protein. This thesis offers novel insights into immune control of the key HIV subtypes – B- and C-clade – and of the most severely affected populations – in Africa (South Africa) and Asia (India and Taiwan) – within the global epidemic. This work helps to better define the viral mutation landscape that is essential both for future vaccines designed to corner the virus, and for successful HIV cure strategies.