3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Defining virus-antibody interplay during the development of HIV-1 neutralization breadth to inform vaccine design(2016) Bhiman, Jinal NomathembaHuman Immunodeficiency Virus Type 1 (HIV-1) infects approximately two million people annually, highlighting the need for a preventative vaccine. An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs), which arise naturally in some infected individuals and recognize the envelopes (Env) of multiple HIV-1 strains. Understanding the molecular events that contribute to bNAb development during infection may provide a blueprint for vaccine strategies. Here we investigated the development of a V1V2-directed bNAb lineage in the context of viral co-evolution in an HIV-1 superinfected participant (CAP256). For this, clonally-related monoclonal antibodies (mAbs), with a range of neutralization breadth, were isolated. We determined their developmental pathway from strain-specificity towards neutralization breadth and identified viral variants responsible for initiating and maturing this bNAb lineage. MAbs were isolated by memory B cell culture or trimer-specific single B cell sorting and extensively characterized by Env-pseudotyped neutralization, cell surface-expressed Env binding, electron microscopy and epitope-predictive algorithms. Antibody next-generation sequencing (NGS) at multiple time-points enabled the inference of the unmutated common ancestor (UCA) of this lineage. Viral co-evolution was investigated using Env single genome amplification and V1V2 NGS. A family of 33 clonally-related mAbs, CAP256-VRC26.01-33, was isolated from samples spanning four years of infection. The UCA of this lineage possessed an unusually long heavy chain complementarity determining region 3 (CDRH3), which resulted from a unique recombination event. Surprisingly, this UCA potently neutralized later viral variants that had evolved from the superinfecting virus, which we termed bNAb-initiating Envs. Viral diversification, which peaked prior to the development of neutralization breadth, created multiple immunotypes at key residues in the V1V2 epitope. Exposure to these immunotypes allowed adaptation of some mAbs to tolerate this variation and thus mature towards neutralization breadth. Based on these data, we proposed a four-step immunization strategy which includes priming with bNAb-initiating Envs to engage rare B cells with a long CDRH3; followed by three sequential boosts (including select V1V2 immunotypes) to drive antibody maturation. In conclusion, this study has generated a testable HIV-1 vaccine immunization strategy through the delineation of mAb-virus co-evolution during the development of neutralization breadth.Item Autologous neutralising antibody specificities in HIV-1 subtype C: characterising the C3V4 region and defining the mechanisms of escape(2012) Bhiman, Jinal NomathembaIntroduction: Most new HIV-1 infections world-wide are caused by subtype C viruses. The C3V4 region, including the alpha2-helix and V4 loop, has been identified as a major target for autologous neutralising antibodies in subtype C infections. Factors associated with the immunogenicity of this region, and the mechanisms of escape from anti-C3V4 responses have not been described, although charge changes in the alpha2-helix have been proposed to mediate neutralisation escape. Methods: Seventeen HIV-1 subtype C infected individuals were classified as C3V4 responders or nonresponders using chimeric viruses in env-pseudotyped neutralisation assays. Longitudinal sequences obtained from C3V4 responders were used to identify putative neutralisation escape mutations. The role of these mutations in mediating escape was investigated using site-directed mutagenesis. Results: The C3V4 region was confirmed as a major target in HIV-1 subtype C infections. The development of an anti-C3V4 response was associated with shorter V4 loops and fewer potential N-linked glycans (PNGs) in the C3V4 region. Anti-C3V4 responses were associated with higher autologous neutralising titres. Neutralisation escape from an anti-C3V4 response was rarely mediated by charge changes in the alpha2-helix and generally occurred through mutations in other structurally proximal regions of the envelope. This study confirmed the use of glycan shuffling as a predominant escape pathway. In three individuals multiple mechanisms of escape were identified and in two other cases escape mutations within the C3V4 and structurally proximal regions clustered at opposite termini of the alpha2-helix, inconsistent with the surface area of a single epitope. Conclusion: A more exposed and accessible C3V4 region was more likely to elicit an anti-C3V4 response. The highly immunogenic nature of this region may contribute to the higher overall neutralisation titres in subtype C infections. Distinct clusters of mutations may suggest the existence of two “sub-epitopes” within the C3V4 domain that warrant further investigation. These findings emphasise the adaptability and plasticity of the C3V4 region in the context of viral evasion of host defences.