Scientific Publications

Studies have demonstrated the role of ulcerative and non-ulcerative sexually transmitted infections (STI) in HIV transmission/acquisition risk; less is understood about the role of non-specific inflammatory genital abnormalities.

Protection against acquiring HIV infection may not require a vaccine in the conventional sense, because broadly neutralizing antibodies (bNAbs) alone prevent HIV infection in relevant animal challenge models. Additionally, bNAbs as therapeutics can effectively suppress HIV replication in infected humans and in animal models. Combinations of bNAbs are generally even more effective and bNAb-derived multivalent antibody-like molecules also inhibit HIV replication both in vitro and in vivo. To expand the available array of multi-specific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAbs that recognize HIV Env exclusively, a bispecific CrossMab targeting two epitopes on the major HIV co-receptor, CCR5, and bi- and trispecifics that cross-target both Env and CCR5. These newly designed molecules displayed exceptional breadth, neutralizing 98-100% of a 109-virus panel, as well as additivity and potency compared to the individual parental control IgGs. The bispecific molecules designed as tandem single chain variable fragments (scFvs; 10E8fv-N6fv and m36.4-PRO 140fv) displayed a median IC of 0.0685 and 0.0131 μg/ml, respectively. A trispecific containing 10E8-PGT121-PGDM1400 Env-specific binding sites was equally potent (median IC of 0.0135 μg/ml), while the trispecific molecule targeting Env and CCR5 simultaneously (10E8Fab-PGDM1400fv-PRO 140fv), demonstrated even greater potency with a median IC of 0.007 μg/ml. By design, some of these molecules lacked Fc-mediated effector function; therefore, we also constructed a trispecific prototype possessing reconstituted CH2-CH3 domains to restore Fc receptor binding capacity. The molecules developed here, along with those described previously, possess promise as prophylactic and therapeutic agents against HIV. Broadly neutralizing antibodies (bNAbs) prevent HIV infection in monkey challenge models and suppress HIV replication in infected humans. Combinations of bNAbs are more effective at suppression and antibody-like molecules engineered to have 2 or 3 bNAb combining sites also inhibit HIV replication in monkeys and other animal models. To expand the available array of multi-specific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAb binding sites that recognize the HIV envelope glycoprotein (Env), the HIV co-receptor (CCR5), or that cross-target both Env and CCR5. Several of the bi- and trispecific molecules neutralized most viruses in a diverse cross-clade panel with greater breadth and potency than the individual parental bNAbs. The molecules described here provide additional options to prevent or suppress HIV infection.

A critical property of a prophylactic HIV vaccine is likely to be its ability to elicit broadly neutralizing antibodies (bnAbs). BnAbs typically have multiple unusual features and are generated in a fraction of HIV-infected individuals through complex pathways. Current vaccine design approaches seek to trigger rare B cell precursors and then steer affinity maturation toward bnAbs in a multi-stage multi-component immunization approach. These vaccine design strategies have been facilitated by molecular descriptions of bnAb interactions with stabilized HIV trimers, the use of an array of sophisticated approaches for immunogen design, the development of novel animal models for immunogen evaluation and advanced technologies to interrogate antibody responses. In this review, we will discuss leading HIV bnAb vaccine immunogens, immunization strategies and future improvements.

Broadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex.

Symptoms of acute retroviral syndrome (ARS) may be used to identify patients with acute HIV-1 infection who seek care. ARS symptoms in African adults differ by region. We assessed whether reporting of ARS was associated with HIV-1 subtype in a multicentre African cohort study representing countries with predominant HIV-1 subtypes A, C, and D.

Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine.

HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates.

Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan 'hole' naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 , elicited low-to-no tier 1 virus neutralization , indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses .

A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.

Furin cleavage of the HIV envelope glycoprotein is an essential step for cell entry that enables formation of well-folded, native-like glycosylated trimers, releases constraints on the fusion peptide, and limits enzymatic processing of the N-glycan shield. Here, we show that a cleavage-independent, stabilized, soluble Env trimer mimic (BG505 NFL.664) exhibits a 'closed-form', native-like, prefusion conformation akin to furin-cleaved Env trimers. The crystal structure of BG505 NFL.664 at 3.39 Å resolution with two potent bNAbs also identifies the full epitopes of PGV19 and PGT122 that target the receptor binding site and N332 supersite, respectively. Quantitative site-specific analysis of the glycan shield reveals that native-like glycan processing is maintained despite furin-independent maturation in the secretory pathway. Thus, cleavage-independent NFL Env trimers exhibit quaternary protein and carbohydrate structures similar to the native viral spike that further validate their potential as vaccine immunogen candidates.