3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Identification, isolation, and characterisation of HIV-1 neutralising antibodies(2016) Wibmer, Constantinos KurtA preventative HIV-1 vaccine would contribute substantially to ending the AIDS epidemic. While the correlates of protection are unknown, broadly neutralizing HIV-1 antibodies (bNAbs) can prevent infection in animal models. However these antibodies are rare even in natural infection, their epitopes are still being characterized, and all the factors underlying their development have yet to be elucidated. Characterising bNAb targets, and defining how bNAbs develop in HIV-1 infected people, might therefore provide blueprints for the rational design of an HIV-1 vaccine. Here, we characterized the broadly neutralizing plasma specificities of two individuals in the CAPRISA cohort, CAP248 and CAP257. This paved the way for the isolation of a monoclonal antibody (mAb) from each donor, targeting two recently described sites of vulnerability on the HIV-1 envelope trimer. The CAP248-2B mAb targets a glycan-independent epitope in the gp120-gp41 interface, distinct from previously described epitopes. Using mutagenesis, protein crystallography, and electron microscopy, we identified key components of this epitope in the gp120 C terminus, and in gp41 upstream of the membrane proximal external region (MPER). Mutations that escaped CAP248-2B made heterologous strains up to 100-fold more sensitive to MPER bNAbs. For CAP257, we described the sequential development of three distinct bNAb specificities, and analogous to CAP248 we showed that escape from the earliest bNAbs exposed the epitopes for later bNAbs. Immunotype toggling during early CAP257 escape pathways resulted in viral diversification that immediately preceded the development of neutralization breadth. Lastly, we isolated a strain-specific CD4 binding site (CD4bs) antibody called CAP257-RH1, which recognises an N276 glycan-dependent epitope that overlapped with one of the CAP257 plasma bNAb specificities. CAP257-RH1 represents an early member of the CAP257 CD4bs response, and its strain-specificity could be attributed to a preference for unglycosylated V5 loops, found in an early minority population of autologous viral envelope sequences. This rare feature may be important for the induction of N276 glycan-dependent CD4bs antibodies. Thus, in this thesis we describe new targets for HIV-1 neutralizing antibodies, delineate the viral pathways that drove neutralization breadth, and identify unique viral variants that may potentially enhance the immunogenicity of the CD4bs and MPER. These data may serve as a roadmap for the induction of HIV-1 bNAbs by vaccination.Item Defining C3-V4 neutralisation epitopes on human immunodeficiency virus type-1 subtype c envelope glycoproteins(2012-01-17) Wibmer, Constantinos KurtThe rational design of an HIV-1 vaccine immunogen able to induce potent, cross-reactive, neutralising antibodies remains one of the single greatest challenges in the field of vaccine research today. Roughly a dozen broadly neutralising monoclonal antibodies have been isolated to date, and their epitopes represent important vaccination targets. Interestingly, apart from three that identify over-lapping epitopes in gp41, all of the broadly neutralising monoclonal antibodies target epitopes apparent on different conformations of gp120 (including the epitopes of PG9/PG16). Thus the gp120 monomer remains the most ideal template for immunogen design. Recently, epitopes in the C3-V4 region of gp120 have been shown to be major targets for early strain-specific neutralising antibodies in subtype C infected individuals. Autologous neutralising antibodies identify vulnerable sites on the envelope, and understanding the nature of antigenic “hotspots” on gp120 will help to guide rational vaccine design. This study sought to confirm in four individuals that the C3-V4 epitope was in fact apparent on monomeric gp120, and thereafter to better characterise the nature of viral escape from these antibodies. Using magnetic beads coated with one of 16 different recombinant gp120 proteins it was confirmed that the C3-V4 response was aimed at a monomer-specific epitope in all four cases. In two instances these antibodies were shown to contribute to autologous neutralisation, while in a third the existence of quaternary structure specific antibodies that could not be adsorbed with monomeric gp120 made this link impossible. In the forth instance transfer of the C3-V4 region was shown to expose a normally occluded epitope in the CD4 binding site. This research also provided evidence for other epitopes for autologous neutralising antibodies in C3, overlapping with the CD4 binding site and V5. Lastly, by introducing relevant escape mutations into the parental recombinant gp120s and then comparing the ability of these proteins to adsorb out anti-C3 antibodies, it was shown that while these mutations conferred complete resistance to neutralisation they did not prevent the antibodies from binding to their respective epitopes. The extensive characterisation of C3-related epitopes such as those described in this research should no doubt contribute to the rational design of a gp120 based vaccine immunogen aimed at eliciting broad and potent neutralising antibody responses.