The Ontogeny of Vaccine-Induced HIV-1 Glycan-Reactive Antibodies

Abstract

The HIV-1 envelope (Env) glycoprotein trimer expressed on the virion surface is the target for both non-neutralizing and neutralizing antibodies (nAbs). In both infection and vaccination, the dominant neutralizing antibody responses are strain-specific, however, during chronic infection, antibodies that can potently neutralize genetically diverse HIV-1 isolates have been identified. These antibodies have been termed broadly neutralizing antibodies (bnAbs). BnAbs target conserved sites on the HIV-1 Env glycoprotein trimer including the membrane proximal external region, the gp120-gp41 interface, the CD4 binding site, V1V2-loop plus glycans, and the V3 loop plus glycans. Passive infusion studies with broadly neutralizing antibodies (bnAbs) of various specificities have been shown to be protective as well as control viral load. Thus, one component of a protective HIV-1 vaccine will likely require the induction of broad and potent neutralizing antibodies, however, it is not understood how such a response would be elicited through vaccination as these antibodies are rare and restricted by immune tolerance mechanisms due to their unusual features such as extensive somatic hypermutation, presence of a long HCDR3, glycan-reactivity, and poly- and or auto-reactivity. Due to the counter selection of B cells bearing antibodies with these characteristics by the host immune system, bnAb precursor B cells are rare in the B cell repertoire and would likely be difficult to engage through vaccination. As such, vaccinations using HIV-1 Env have induced dominant strain-specific antibody responses, but not broadly neutralizing antibody responses. To efficiently engage and expand bnAb-precursor B cells, production of stable homogeneous immunogens that selectively express bnAb epitopes may be necessary.Following the Introduction (Chapter 1), Methods (Chapter 2), Chapter 3 of this dissertation describes antibody reagents that are used to characterize recombinantly-produced HIV-1 Env glycoproteins. Chapters 4 and 5 present data relating to the ontogeny of vaccine-induced antibody responses to the first and second variable loops of the Env glycoprotein plus glycan (V1V2-glycan epitope) while Chapters 6 and 7 present data relating to the ontogeny of vaccine-induced antibody responses to the third variable loop plus glycan (V3-glycan epitope).Chapter 4 of this dissertation describes a study of the antibody responses in rhesus macaques following immunization with a synthetic glycopeptide mimic of the epitope bound by antibodies that target the first and second variable loops plus glycan (V1V2-glycan bnAb epitope). This V1V2 glycopeptide induced robust plasma antibody binding responses to HIV Env that contained the same V1V2 loop sequence as the immunogen and binding of plasma antibodies to HIV Env was dependent on a lysine at position 169 (K169). Dependency on K169 is common for antibodies that target the V1V2 loop, such as the strain-specific antibodies CH58 and CH59, that recognize only a peptidic epitope of the V1V2 loop, but also for V1V2-glycan bnAbs such as CH01 that recognize both the V1V2 loop peptide backbone in addition to glycans at positions N156 and N160). Even though binding of vaccine-induced antibodies was dependent on the presence of K169, no glycan-reactive responses were detected, thus suggesting the presence of strain-specific, peptide-reactive CH58 and CH59-like antibodies. Moreover, the heavy and light chain sequences of vaccine-induced antibodies were isolated and majority of Env-reactive antibodies paired with the rhesus Vλ3-17 lambda chain gene segment. It has been previously demonstrated that the rhesus Vλ3-17 lambda chain gene segment is the rhesus ortholog to the human Vλ3-10 gene as used by the strain-specific vaccine-induced human antibody, CH59. This is due to a germline-encoded glutamic acid and aspartic acid motif in the antibody HCDR2 loop (ED motif). The ED motif forms a salt bridge with the HIV-1 Env K169 and thus generates germline-encoded HIV-1 Env reactivity. In summary, immunization with the V1V2 glycopeptide minimal immunogen that was designed to target and expand rare V1V2-glycan reactive precursors elicited a dominant antibody response that solely recognized peptide, but not glycan. Thus, these results demonstrate that despite vaccinating with the V1V2-glycopeptide that preferentially expressed the V1V2-glycan epitope, V1V2 antibody responses that recognized only peptide were profoundly immunodominant. As V1V2-glycan bnAbs typically have long HCDR3 loops and long HCDR3-bearing B cells are rare in the peripheral B cell repertoire, the observed results could be due to lack of B cells that recognize the V1V2-glycan epitope. From this work, however, it is unclear if the observed results were due to a deficiency of these types of B cells in the periphery or due to instability of the vaccine immunogen in vivo.Chapter 5 describes the characterization and immunization of a humanized mouse model bearing the CH01 V1V2-glycan bnAb unmutated common ancestor antibody; i.e. the B cells in these mice bear the CH01 antibody heavy and light chain sequences that were predicted to be germline-encoded prior to somatic hypermutation (germline-reverted). With a long HCDR3 of 24 amino acids, B cells bearing the germline-reverted CH01 heavy and light chain were not deleted in the bone marrow, and peripheral B cell development was comparable to the background strain, C57BL/6, demonstrating that B cells bearing CH01-like antibodies could be responsive to an appropriately designed vaccine immunogen. Mice bearing only the germline reverted CH01 heavy chain were then immunized with HIV-1 Env. Immunization of these mice expanded B cell populations that were dependent on the presence of the V1V2-loop glycan N160. Furthermore, immunization of these mice elicited neutralization against difficult to neutralize (tier 2) HIV-1 isolates. Following immunization, B cells bearing antibodies whose binding to HIV-1 Env was N160 glycan-dependent were isolated, and heavy and light chain sequences were recovered. Analysis of the recovered heavy and light chains revealed that 18 different murine variable-kappa chain genes paired with the knocked in CH01 UCA and these pairings conferred N160 dependence. However, the knocked CH01 UCA heavy chain sequences were largely unmutated. These data suggest that long HCDR3-bearing B cells that are specific for the V1V2-glycan epitope (dependent on the presence of glycan at N160) are not deleted in the bone marrow and as such, vaccine-elicitation of antibodies targeting the V1V2-glycan epitope in wild-type animals should be feasible. Moreover, these data suggest that a critical first step for the elicitation of V1V2-glycan targeting antibody responses will be expansion of the B cells from which these responses derive, but additional antigenic diversity will likely be required to induce the full neutralization breadth and potency observed by V1V2-glycan bnAbs isolated from HIV-1 infected donors.Chapter 6 details the characterization of a stable synthetic glycopeptide that was designed to mimic the conformation of the peptide component of the HIV-1 V3-loop plus glycans, termed “Man9-V3.” Broadly neutralizing antibodies that recognize this epitope, termed “V3-glycan bnAbs” bound to Man9-V3 glycopeptide and bound with affinities comparable to those observed for native-like gp140 Env trimers. Moreover, both fluorophore-labeled Man9-V3 and native-like trimers similarly bound to bnAb memory B cells, and by flow sorting, members of a V3-glycan bnAb clonal lineage from an HIV-1-infected individual were isolated. Thus, these data suggest that Man9-V3 glycopeptide is a structural mimic of the HIV-1 Env epitope bound by V3-glycan bnAbs and is a candidate immunogen to initiate V3-glycan bnAb lineage maturation.In Chapter 7, the immunogenicity of Man9-V3 glycopeptide was tested in rhesus macaques. Using Man9-V3 as an immunogen, V3-glycan antibody responses were elicited. Combining flow sorting with next generation sequencing of immunoglobulin genes, the ontogeny of a vaccine-elicited V3-glycan antibody lineage, termed DH717 was studied. This lineage targeted the base of the HIV-1 V3-loop in addition to the V3-loop N301 and N332 glycans. Neutralization however, was limited to Env pseudoviruses bearing only high-mannose glycans as these antibodies could not neutralize viruses bearing native glycoforms. The structure of the most broad and potent member of the DH717 lineage, DH717.1 was determined using X-ray crystallography. This revealed that the rhesus DH717.1 V3-glycan antibody and 2G12, a human V3-glycan bnAb isolated from a HIV-1 infected donor, showed remarkable similarity in accommodation of high-mannose glycans. Specifically, DH717.1 and 2G12 antibodies accommodate terminal branches of high-mannose glycans through the formation of a binding pocket comprised of the HCDR1 and HCDR2 loops. Furthermore, DH717, like 2G12, bound the yeast Candida albicans in a glycan-dependent manner. With regards to the ontogeny of the V3-glycan DH717 lineage, next generation sequencing at pre-immunization time points revealed the DH717 lineage to be present prior to vaccination and to be mutated with regards to the computationally inferred DH717 germline sequence. While the already mutated pre-vaccination DH717 lineage member bound to both the yeast Candida albicans, and to Man9-V3 glycopeptide, the DH717 unmutated common ancestor bearing a computationally inferred germline sequence bound only to Candida albicans, and not Man9-V3. It was only after acquiring somatic mutations prior to immunization did the lineage acquire the ability to bind to Man9-V3, suggesting a role for high-mannose-bearing environmental antigens for priming such responses. After further acquisition of somatic mutations following vaccination did the lineage acquire the ability to bind the V3-glycan bnAb epitope as presented on a stabilized, native-like soluble recombinant HIV-1 Env trimer. This suggests that vaccination with a synthetic glycopeptide affinity matured a pre-existing, yeast-reactive B cell lineage to the HIV-1 V3-glycan bnAb epitope. Together, the studies described in Chapters 4-7 suggest that vaccine-induction of V1V2- and V3-glycan bnAbs may be feasible, and that to do so, stable glycopeptides that mimic bnAb epitopes will be needed to select for and expand the precursors for the desired glycan-bnAb response.