Identification, isolation, and characterisation of HIV-1 neutralising antibodies

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. 8th August 2016A 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.MT201