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

Defining C3-V4 neutralisation epitopes on human immunodeficiency virus type-1 subtype c envelope glycoproteins

The 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

HIV broadly neutralizing antibody targets.

CAPRISA, 2015.Abstract available in pdf

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing antibodies and identifies a set of mutations in the gp120 C terminus that exposes the membrane-proximal external region of gp41, with potential utility in HIV vaccine design

Structure of an N276-dependent HIV-1 neutralizing antibody targeting a rare V5 glycan hole adjacent to the CD4 binding site.

CAPRISA, 2016.Abstract available in pdf

Structure and recognition of a novel HIV-1 gp120-gp41 interface antibody that caused MPER exposure through viral escape.

CAPRISA, 2017.Abstract available in pdf

New member of the V1V2-directed CAP256-VRC26 lineage that shows increased breadth and exceptional potency.

CAPRISA, 2016.Abstract available in pdf

South African HIV-1 subtype C transmitted variants with a specific V2 motif show higher dependence on α4β7 for replication.

CAPRISA, 2015.Abstract available in pdf

Viral escape from HIV-1 neutralizing antibodies drives increased plasma neutralization breadth through sequential recognition of multiple epitopes and immunotypes.

Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient ppearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

CAPRISA, 2014.Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development