Bryan Briney

Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Significance Despite the high antigenic diversity of the HIV envelope trimer (Env), broadly neutralizing antibodies (bnAbs) have identified conserved regions that serve as targets for vaccine design. One of these regions is located at the apex of Env and is expressed fully only in the context of the correctly folded trimer. This work describes the isolation of bnAbs that target this region using a recombinant native-like Env trimer as an affinity reagent to sort specific antibody-producing cells. Characterization of these antibodies reveals a highly diverse antibody response against the trimer apex and provides molecular information that will be useful in the design of immunogens to elicit bnAbs to this region of Env. Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC50 of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.

Promiscuous Glycan Site Recognition by Antibodies to the High-Mannose Patch of gp120 Broadens Neutralization of HIV

HIV broadly neutralizing monoclonal antibodies targeting the high-mannose patch of Env can use alternate glycan sites for neutralization. Neutralizing Antibodies with a Sweet Tooth Sugar can be quite tempting—as anyone who’s seen a kid rip into birthday cake can attest. Yet, antibodies can also have a sweet tooth, targeting glycan modifications on the surface of proteins. Indeed, some antibodies that neutralize multiple HIV strains—broadly neutralizing monoclonal antibodies (bnmAbs)—target a high-mannose patch on the HIV protein Env. Although this high-mannose patch is centered around the glycan at position 332 (N332), it has remained unclear if the N332 glycan is absolutely required for neutralization and, if not, why not. Sok et al. found that these mannose patch–targeting antibodies can bind alternate glycans in the absence of N332, which helps to explain their ability to neutralize many strains of HIV. Specifically, some bnmAbs can bind to the N334 site when that replaces the N332 site and some can form more interactions with other glycans, particularly complex-type glycans on variable loops, if the N332 sugar is absent. These data also suggest that mannose patch–targeting bnmAbs can work in combination to neutralize a wider range of different strains than single bnmAbs. The promiscuity of glycan binding by these sugar-loving antibodies is important to consider for both vaccine and therapeutic antibody development. Broadly neutralizing monoclonal antibodies (bnmAbs) that target the high-mannose patch centered around the glycan at position 332 on HIV Env are promising vaccine leads and therapeutic candidates because they effectively protect against mucosal SHIV challenge and strongly suppress SHIV viremia in established infection in macaque models. However, these antibodies demonstrate varying degrees of dependency on the N332 glycan site, and the origins of their neutralization breadth are not always obvious. By measuring neutralization on an extended range of glycan site viral variants, we found that some bnmAbs can use alternate N-linked glycans in the absence of the N332 glycan site and therefore neutralize a substantial number of viruses lacking the site. Furthermore, many of the antibodies can neutralize viruses in which the N332 glycan site is shifted to the 334 position. Finally, we found that a combination of three antibody families that target the high-mannose patch can lead to 99% neutralization coverage of a large panel of viruses containing the N332/N334 glycan site and up to 66% coverage for viruses that lack the N332/N334 glycan site. The results indicate that a diverse response against the high-mannose patch may provide near-equivalent coverage as a combination of bnmAbs targeting multiple epitopes. Additionally, the ability of some bnmAbs to use other N-linked glycan sites can help counter neutralization escape mediated by shifting of glycosylation sites. Overall, this work highlights the importance of promiscuous glycan binding properties in bnmAbs to the high-mannose patch for optimal antiviral activity in either protective or therapeutic modalities.

HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

Baby steps toward bNAbs Some HIV-infected individuals develop heavily mutated, broadly neutralizing antibodies (bNAbs) that target HIV. Scientists aim to design vaccines that would elicit such antibodies. Jardine et al. report an important step toward this goal: They engineered an immunogen that could engage B cells from HIV-uninfected individuals that express the germline versions of the immunoglobulin genes harbored by a particular class of bNAbs. The frequencies of these B cells, their affinities for the immunogen, and structural analysis suggest that the immunogen is a promising candidate. Further shaping of the B cell response with subsequent immunogens may eventually elicit bNAbs in people. Science, this issue p. 1458 People that have not been infected with HIV can harbor HIV-1 broadly neutralizing antibody B cell precursors. Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.

Summary Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.

Human Peripheral Blood Antibodies with Long HCDR3s Are Established Primarily at Original Recombination Using a Limited Subset of Germline Genes

A number of antibodies that efficiently neutralize microbial targets contain long heavy chain complementarity determining region 3 (HCDR3) loops. For HIV, several of the most broad and potently neutralizing antibodies have exceptionally long HCDR3s. Two broad potently neutralizing HIV-specific antibodies, PG9 and PG16, exhibit secondary structure. Two other long HCDR3 antibodies, 2F5 and 4E10, protect against mucosal challenge with SHIV. Induction of such long HCDR3 antibodies may be critical to the design of an effective vaccine strategy for HIV and other pathogens, however it is unclear at present how to induce such antibodies. Here, we present genetic evidence that human peripheral blood antibodies containing long HCDR3s are not primarily generated by insertions introduced during the somatic hypermutation process. Instead, they are typically formed by processes occurring as part of the original recombination event. Thus, the response of B cells encoding antibodies with long HCDR3s results from selection of unusual clones from the naïve repertoire rather than through accumulation of insertions. These antibodies typically use a small subset of D and J gene segments that are particularly suited to encoding long HCDR3s, resulting in the incorporation of highly conserved genetic elements in the majority of antibody sequences encoding long HCDR3s.

Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

Summary Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.

Epitope-Specific Human Influenza Antibody Repertoires Diversify by B Cell Intraclonal Sequence Divergence and Interclonal Convergence

We generated from a single blood sample five independent human mAbs that recognized the Sa antigenic site on the head of influenza hemagglutinin and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the VH3-7 and JH6 gene segments, but at least four independent clones were identified by junctional analysis. High-throughput sequence analysis of circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple diverging lineages of these clones, including many containing very few mutations in the Ab genes, persisted in the circulation. Conversely, we noted frequent instances of amino acid sequence convergence in the Ag combining sites exhibited by members of independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly mutating virus Ags, such as influenza hemagglutinin. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human Abs.

Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.

High-throughput antibody sequencing reveals genetic evidence of global regulation of the naïve and memory repertoires that extends across individuals

Vast diversity in the antibody repertoire is a key component of the adaptive immune response. This diversity is generated centrally through the assembly of variable, diversity and joining gene segments, and peripherally by somatic hypermutation and class-switch recombination. The peripheral diversification process is thought to only occur in response to antigenic stimulus, producing antigen-selected memory B cells. Surprisingly, analyses of the variable, diversity and joining gene segments have revealed that the naïve and memory subsets are composed of similar proportions of these elements. Lacking, however, is a more detailed study, analyzing the repertoires of naïve and memory subsets at the level of the complete V(D)J recombinant. This report presents a thorough examination of V(D)J recombinants in the human peripheral blood repertoire, revealing surprisingly large repertoire differences between circulating B-cell subsets and providing genetic evidence for global control of repertoire diversity in naïve and memory circulating B-cell subsets.

Priming HIV-1 broadly neutralizing antibody precursors in human Ig loci transgenic mice

A major obstacle to a broadly neutralizing antibody (bnAb)–based HIV vaccine is the activation of appropriate B cell precursors. Germline-targeting immunogens must be capable of priming rare bnAb precursors in the physiological setting. We tested the ability of the VRC01-class bnAb germline-targeting immunogen eOD-GT8 60mer (60-subunit self-assembling nanoparticle) to activate appropriate precursors in mice transgenic for human immunoglobulin (Ig) loci. Despite an average frequency of, at most, about one VRC01-class precursor per mouse, we found that at least 29% of singly immunized mice produced a VRC01-class memory response, suggesting that priming generally succeeded when at least one precursor was present. The results demonstrate the feasibility of using germline targeting to prime specific and exceedingly rare bnAb-precursor B cells within a humanlike repertoire.