Safety and immunogenicity of a polyvalent DNA-protein HIV vaccine with matched Env immunogens delivered as a prime-boost regimen or coadministered in HIV-uninfected adults in the USA (HVTN 124): a phase 1, placebo-controlled, double-blind randomised controlled trial.

BACKGROUND: An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. METHODS: HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. FINDINGS: Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. INTERPRETATION: Both vaccine regimens were safe, warranting evaluation in larger trials. FUNDING: US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells.

The envelope (Env) glycoproteins on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAbs) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes, including V2i, the gp120-g41 interface, and gp41-MPER, are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by the pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where the virus-mAb mix was pre-incubated/not pre-incubated for 1 hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein mediates viral entry and is the sole target of neutralizing antibodies. Our data suggest that antibody epitopes including V2q (e.g., PG9, PGT145), CD4bs (e.g., VRC01, 3BNC117), and V3 (2219, 2557) are masked on HIV-1 particles. The PG9 and 2219 epitopes became accessible for binding after conformational unmasking was induced by the pre-binding of select mAbs. Attempts to understand the masking mechanism led to the revelation that interaction between virus and host cells is needed to sensitize the virions for neutralization by broadly neutralizing antibodies (bNAbs). These data provide insight on how bNAbs may gain access to these occluded epitopes to exert their neutralization effects and block HIV-1 infection. These findings have important implications for the way we evaluate the neutralizing efficacy of antibodies and can potentially guide vaccine design.

Global Increases in Human Immunodeficiency Virus Neutralization Sensitivity Due to Alterations in the Membrane-Proximal External Region of the Envelope Glycoprotein Can Be Minimized by Distant State 1-Stabilizing Changes.

Binding to the receptor, CD4, drives the pretriggered, "closed" (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer ([gp120/gp41]3) into more "open" conformations. HIV-1 Env on the viral membrane is maintained in a State-1 conformation that resists binding and neutralization by commonly elicited antibodies. Premature triggering of Env before the virus engages a target cell typically leads to increased susceptibility to spontaneous inactivation or ligand-induced neutralization. Here, we showed that single amino acid substitutions in the gp41 membrane-proximal external region (MPER) of a primary HIV-1 strain resulted in viral phenotypes indicative of premature triggering of Env to downstream conformations. Specifically, the MPER changes reduced viral infectivity and globally increased virus sensitivity to poorly neutralizing antibodies, soluble CD4, a CD4-mimetic compound, and exposure to cold. In contrast, the MPER mutants exhibited decreased sensitivity to the State 1-preferring inhibitor, BMS-806, and to the PGT151 broadly neutralizing antibody. Depletion of cholesterol from virus particles did not produce the same State 1-destabilizing phenotypes as MPER alterations. Notably, State 1-stabilizing changes in Env distant from the MPER could minimize the phenotypic effects of MPER alteration but did not affect virus sensitivity to cholesterol depletion. Thus, membrane-proximal gp41 elements contribute to the maintenance of the pretriggered Env conformation. The conformationally disruptive effects of MPER changes can be minimized by distant State 1-stabilizing Env modifications, a strategy that may be useful in preserving the native pretriggered state of Env. IMPORTANCE The pretriggered shape of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) is a major target for antibodies that can neutralize many strains of the virus. An effective HIV-1 vaccine may need to raise these types of antibodies, but this goal has proven difficult. One reason is that the pretriggered shape of Env is unstable and dependent on interactions near the viral membrane. Here, we showed that the membrane-proximal external region (MPER) of Env plays an important role in maintaining Env in a pretriggered shape. Alterations in the MPER resulted in global changes in Env conformation that disrupted its pretriggered shape. We also found that these disruptive effects of MPER changes could be minimized by distant Env modifications that stabilized the pretriggered shape. These modifications may be useful for preserving the native shape of Env for structural and vaccine studies.

Differential V2-directed antibody responses in non-human primates infected with SHIVs or immunized with diverse HIV vaccines.

V2p and V2i antibodies (Abs) that are specific for epitopes in the V1V2 region of the HIV gp120 envelope (Env) do not effectively neutralize HIV but mediate Fc-dependent anti-viral activities that have been correlated with protection from, or control of HIV, SIV and SHIV infections. Here, we describe a novel molecular toolbox that allows the discrimination of antigenically and functionally distinct polyclonal V2 Ab responses. We identify different patterns of V2 Ab induction by SHIV infection and three separate vaccine regimens that aid in fine-tuning an optimized immunization protocol for inducing V2p and V2i Abs. We observe no, or weak and sporadic V2p and V2i Abs in non-vaccinated SHIV-infected NHPs, but strong V2p and/or V2i Ab responses after immunization with a V2-targeting vaccine protocol. The V2-focused vaccination is superior to both natural infection and to immunization with whole Env constructs for inducing functional V2p- and V2i-specific responses. Strikingly, levels of V2-directed Abs correlate inversely with Abs specific for peptides of V3 and C5. These data demonstrate that a V1V2-targeting vaccine has advantages over the imprecise targeting of SIV/SHIV infections and of whole Env-based immunization regimens for inducing a more focused functional V2p- and V2i-specific Ab response.

Chemoenzymatic Synthesis and Antibody Binding of HIV-1 V1/V2 Glycopeptide-Bacteriophage Qß Conjugates as a Vaccine Candidate.

The broadly neutralizing antibody PG9 recognizes a unique glycopeptide epitope in the V1V2 domain of HIV-1 gp120 envelope glycoprotein. The present study describes the design, synthesis, and antibody-binding analysis of HIV-1 V1V2 glycopeptide-Qß conjugates as a mimic of the proposed neutralizing epitope of PG9. The glycopeptides were synthesized using a highly efficient chemoenzymatic method. The alkyne-tagged glycopeptides were then conjugated to the recombinant bacteriophage (Qß), a virus-like nanoparticle, through a click reaction. Antibody-binding analysis indicated that the synthetic glycoconjugates showed significantly enhanced affinity for antibody PG9 compared with the monomeric glycopeptides. It was also shown that the affinity of the Qß-conjugates for antibody PG9 was dependent on the density of the glycopeptide antigen display. The glycopeptide-Qß conjugates synthesized represent a promising candidate of HIV-1 vaccine.

Safety, pharmacokinetics and antiviral activity of PGT121, a broadly neutralizing monoclonal antibody against HIV-1: a randomized, placebo-controlled, phase 1 clinical trial.

Human immunodeficiency virus (HIV)-1-specific broadly neutralizing monoclonal antibodies are currently under development to treat and prevent HIV-1 infection. We performed a single-center, randomized, double-blind, dose-escalation, placebo-controlled trial of a single administration of the HIV-1 V3-glycan-specific antibody PGT121 at 3, 10 and 30 mg kg-1 in HIV-uninfected adults and HIV-infected adults on antiretroviral therapy (ART), as well as a multicenter, open-label trial of one infusion of PGT121 at 30 mg kg-1 in viremic HIV-infected adults not on ART (no. NCT02960581). The primary endpoints were safety and tolerability, pharmacokinetics (PK) and antiviral activity in viremic HIV-infected adults not on ART. The secondary endpoints were changes in anti-PGT121 antibody titers and CD4+ T-cell count, and development of HIV-1 sequence variations associated with PGT121 resistance. Among 48 participants enrolled, no treatment-related serious adverse events, potential immune-mediated diseases or Grade 3 or higher adverse events were reported. The most common reactions among PGT121 recipients were intravenous/injection site tenderness, pain and headache. Absolute and relative CD4+ T-cell counts did not change following PGT121 infusion in HIV-infected participants. Neutralizing anti-drug antibodies were not elicited. PGT121 reduced plasma HIV RNA levels by a median of 1.77 log in viremic participants, with a viral load nadir at a median of 8.5 days. Two individuals with low baseline viral loads experienced ART-free viral suppression for ≥168 days following antibody infusion, and rebound viruses in these individuals demonstrated full or partial PGT121 sensitivity. The trial met the prespecified endpoints. These data suggest that further investigation of the potential of antibody-based therapeutic strategies for long-term suppression of HIV is warranted, including in individuals off ART and with low viral load.

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens.

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets-the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity-and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30-40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

HIV-1 Envelope Glycoprotein Cell Surface Localization Is Associated with Antibody-Induced Internalization.

To minimize immune responses against infected cells, HIV-1 has evolved different mechanisms to limit the surface expression of its envelope glycoproteins (Env). Recent observations suggest that the binding of certain broadly neutralizing antibodies (bNAbs) targeting the 'closed' conformation of Env induces its internalization. On the other hand, non-neutralizing antibodies (nNAbs) that preferentially target Env in its 'open' conformation, remain bound to Env on the cell surface for longer periods of time. In this study, we attempt to better understand the underlying mechanisms behind the differential rates of antibody-mediated Env internalization. We demonstrate that 'forcing' open Env using CD4 mimetics allows for nNAb binding and results in similar rates of Env internalization as those observed upon the bNAb binding. Moreover, we can identify distinct populations of Env that are differentially targeted by Abs that mediate faster rates of internalization, suggesting that the mechanism of antibody-induced Env internalization partially depends on the localization of Env on the cell surface.

Elimination of SHIV Infected Cells by Combinations of Bispecific HIVxCD3 DART® Molecules.

Bispecific HIVxCD3 DART molecules that co-engage the viral envelope glycoprotein (Env) on HIV-1-infected cells and the CD3 receptor on CD3+ T cells are designed to mediate the cytolysis of HIV-1-infected, Env-expressing cells. Using a novel ex vivo system with cells from rhesus macaques (RMs) infected with a chimeric Simian-Human Immunodeficiency Virus (SHIV) CH505 and maintained on ART, we tested the ability of HIVxCD3 DART molecules to mediate elimination of in vitro-reactivated CD4+ T cells in the absence or presence of autologous CD8+ T cells. HIVxCD3 DART molecules with the anti-HIV-1 Env specificities of A32 or 7B2 (non-neutralizing antibodies) or PGT145 (broadly neutralizing antibody) were evaluated individually or combined. DART molecule-mediated antiviral activity increased significantly in the presence of autologous CD8+ T cells. In this ex vivo system, the PGT145 DART molecule was more active than the 7B2 DART molecule, which was more active than the A32 DART molecule. A triple combination of the DART molecules exceeded the activity of the individual PGT145 DART molecule. Modified quantitative virus outgrowth assays confirmed the ability of the DART molecules to redirect RM CD3+ T cells to eliminate SHIV-infected RM CD4+ T cells as demonstrated by the decreased propagation of in vitro infection by the infected cells pre-incubated with DART molecules in presence of effector CD8+ T cells. While mediating cytotoxic activity, DART molecules did not increase proinflammatory cytokine production. In summary, combination of HIVxCD3 DART molecules that have broadly-neutralizing and non-neutralizing anti-HIV-1 Env specificities can leverage the host immune system for treatment of HIV-1 infection but will require appropriate reactivation of the latent reservoir.

Asymmetric Structures and Conformational Plasticity of the Uncleaved Full-Length Human Immunodeficiency Virus Envelope Glycoprotein Trimer.

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41)3] is produced by cleavage of a conformationally flexible gp160 precursor. gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pretriggered, "closed" (state 1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more "open" Env conformations (states 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and cross-linking decreased the exposure of pNAb epitopes on cell surface gp160; however, after detergent solubilization, cross-linked and BMS-806-treated gp160 sampled non-state-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume state-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a state-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage for a persistent virus like HIV-1.

Functional analysis of a monoclonal antibody reactive against the C1C2 of Env obtained from a patient infected with HIV-1 CRF02_AG.

BACKGROUND: Recent data suggest the importance of non-neutralizing antibodies (nnAbs) in the development of vaccines against HIV-1 because two types of nnAbs that recognize the coreceptor binding site (CoRBS) and the C1C2 region mediate antibody-dependent cellular-cytotoxicity (ADCC) against HIV-1-infected cells. However, many studies have been conducted with nnAbs obtained from subtype B-infected individuals, with few studies in patients with non-subtype B infections. RESULTS: We isolated a monoclonal antibody 1E5 from a CRF02_AG-infected individual and constructed two forms of antibody with constant regions of IgG1 or IgG3. The epitope of 1E5 belongs to the C1C2 of gp120, and 1E5 binds to 27 out of 35 strains (77 %) across the subtypes. The 1E5 showed strong ADCC activity, especially in the form of IgG3 in the presence of small CD4-mimetic compounds (CD4mc) and 4E9C (anti-CoRBS antibody), but did not show any neutralizing activity even against the isolates with strong binding activities. The enhancement in the binding of A32, anti-C1C2 antibody isolated from a patient with subtype B infection, was observed in the presence of 1E5 and the combination of 1E5, A32 and 4E9C mediated a strong ADCC activity. CONCLUSIONS: These results suggest that anti-C1C2 antibodies that are induced in patients with different HIV-1 subtype infections have common functional modality and may have unexpected interactions. These data may have implications for vaccine development against HIV-1.

Antibody Conjugates for Targeted Therapy Against HIV-1 as an Emerging Tool for HIV-1 Cure.

Although advances in antiretroviral therapy (ART) have significantly improved the life expectancy of people living with HIV-1 (PLWH) by suppressing HIV-1 replication, a cure for HIV/AIDS remains elusive. Recent findings of the emergence of drug resistance against various ART have resulted in an increased number of treatment failures, thus the development of novel strategies for HIV-1 cure is of immediate need. Antibody-based therapy is a well-established tool in the treatment of various diseases and the engineering of new antibody derivatives is expanding the realms of its application. An antibody-based carrier of anti-HIV-1 molecules, or antibody conjugates (ACs), could address the limitations of current HIV-1 ART by decreasing possible off-target effects, reduce toxicity, increasing the therapeutic index, and lowering production costs. Broadly neutralizing antibodies (bNAbs) with exceptional breadth and potency against HIV-1 are currently being explored to prevent or treat HIV-1 infection in the clinic. Moreover, bNAbs can be engineered to deliver cytotoxic or immune regulating molecules as ACs, further increasing its therapeutic potential for HIV-1 cure. ACs are currently an important component of anticancer treatment with several FDA-approved constructs, however, to date, no ACs are approved to treat viral infections. This review aims to outline the development of AC for HIV-1 cure, examine the variety of carriers and payloads used, and discuss the potential of ACs in the current HIV-1 cure landscape.

HIV envelope antigen valency on peptide nanofibers modulates antibody magnitude and binding breadth.

A major challenge in developing an effective vaccine against HIV-1 is the genetic diversity of its viral envelope. Because of the broad range of sequences exhibited by HIV-1 strains, protective antibodies must be able to bind and neutralize a widely mutated viral envelope protein. No vaccine has yet been designed which induces broadly neutralizing or protective immune responses against HIV in humans. Nanomaterial-based vaccines have shown the ability to generate antibody and cellular immune responses of increased breadth and neutralization potency. Thus, we have developed supramolecular nanofiber-based immunogens bearing the HIV gp120 envelope glycoprotein. These immunogens generated antibody responses that had increased magnitude and binding breadth compared to soluble gp120. By varying gp120 density on nanofibers, we determined that increased antigen valency was associated with increased antibody magnitude and germinal center responses. This study presents a proof-of-concept for a nanofiber vaccine platform generating broad, high binding antibody responses against the HIV-1 envelope glycoprotein.

Near-Pan-neutralizing, Plasma Deconvoluted Antibody N49P6 Mimics Host Receptor CD4 in Its Quaternary Interactions with the HIV-1 Envelope Trimer.

The first step in HIV-1 entry is the attachment of the envelope (Env) trimer to target cell CD4. As such, the CD4-binding site (CD4bs) remains one of the few universally accessible sites for antibodies (Abs). We recently described a method of isolating Abs directly from the circulating plasma and described a panel of broadly neutralizing Abs (bnAbs) from an HIV-1 "elite neutralizer" referred to as patient N49 (N49 Ab lineage [M. M. Sajadi, A. Dashti, Z. R. Tehrani, W. D. Tolbert, et al., Cell 173:1783-1795.e14, 2018, https://doi.org/10.1016/j.cell.2018.03.061]). Here, we describe the molecular details of antigen recognition by N49P6, an Ab of the N49 lineage that recapitulates most of the neutralization breadth and potency of the donor's plasma IgG. Our studies done in the context of monomeric and trimeric antigens indicate that N49P6 combines many characteristics of known CD4bs-specific bnAbs with features that are unique to the N49 Ab lineage to achieve its remarkable neutralization breadth. These include the omission of the CD4 Phe43 cavity and dependence instead on interactions with highly conserved gp120 inner domain layer 3. Interestingly, when bound to BG505 SOSIP, N49P6 closely mimics the initial contact of host receptor CD4 to the adjacent promoter of the HIV-1 Env trimer to lock the trimer in the closed conformation. Altogether, N49P6 defines a new class of near-pan-neutralizing, plasma deconvoluted CD4bs Abs that we refer to as the N49P series. The details of the mechanisms of action of this new Ab class pave the way for the next generation of HIV-1 bnAbs that can be used as vaccine components of therapeutics. IMPORTANCE Binding to target cell CD4 is the first crucial step required for HIV-1 infection. Thus, the CD4-binding site (CD4bs) is one of the most accessible sites for antibodies (Abs). However, due to steric constraints, only a few Abs are capable of targeting this site. Here, we show that the exceptional neutralization breadth and potency of N49P6, a near-pan-neutralizing Ab targeting the CD4bs isolated from the plasma of an HIV-1 "elite neutralizer," patient N49, are due to its signature combination of more typical CD4bs Ab-binding characteristics with unique interactions with the highly conserved gp120 inner domain. In addition, we also present a structural analysis of N49P6 in complex with the BG505 SOSIP trimer to show that N49P6 exhibits remarkable breadth in part by mimicking CD4's quaternary interaction with the neighboring gp120 protomer. In its mode of antigen interaction, N49P6 is unique and represents a new class of CD4bs-specific bnAbs.

Geospatial HIV-1 subtype C gp120 sequence diversity and its predicted impact on broadly neutralizing antibody sensitivity.

Evolving diversity in globally circulating HIV-1 subtypes presents a formidable challenge in defining and developing neutralizing antibodies for prevention and treatment. HIV-1 subtype C is responsible for majority of global HIV-1 infections. In the present study, we examined the diversity in genetic signatures and attributes that differentiate region-specific HIV-1 subtype C gp120 sequences associated with virus neutralization outcomes to key bnAbs having distinct epitope specificities. A total of 1814 full length HIV-1 subtype C gp120 sequence from 37 countries were retrieved from Los Alamos National Laboratory HIV database (www.hiv.lanl.gov). The amino acid sequences were assessed for their phylogenetic association, variable loop lengths and prevalence of potential N-linked glycosylation sites (pNLGS). Responses of these sequences to bnAbs were predicted with a machine learning algorithm 'bNAb-ReP' and compared with those reported in the CATNAP database. Subtype C sequences from Asian countries including India differed phylogenetically when compared with that from African countries. Variable loop lengths and charges within Indian and African clusters were also found to be distinct from each other, specifically for V1, V2 and V4 loops. Pairwise analyses at each of the 25 pNLG sites indicated distinct country specific profiles. Highly significant differences (p<0.001***) were observed in prevalence of four pNLGS (N130, N295, N392 and N448) between South Africa and India, having most disease burden associated with subtype C. Our findings highlight that distinctly evolving clusters within global intra-subtype C gp120 sequences are likely to influence the disparate region-specific sensitivity of circulating HIV-1 subtype C to bnAbs.

Neutralization diversity of HIV-1 Indian subtype C envelopes obtained from cross sectional and followed up individuals against broadly neutralizing monoclonal antibodies having distinct gp120 specificities.

BACKGROUND: The potential use of the broadly neutralizing monoclonal antibodies (bnAbs) towards prophylaxis and treatment to HIV-1 is currently being explored. While a number of promising bnAbs have been discovered and a few of them have progressed towards clinical development, their extent of neutralization coverage with respect to global HIV-1 variants given the existence of genetically distinct subtypes and recombinants circulating globally is not clearly known. In the present study, we examined the variation in the neutralization susceptibility of pseudoviruses expressing 71 full length primary HIV-1 subtype C envs obtained from limited cross-sectional individuals over different time points against four bnAbs that target gp120 with distinct specificities: VRC01, CAP256-VRC26.25, PGDM1400 and PGT121. RESULTS: We found significant variations in the susceptibility of Indian clade C to these four bnAbs. These variations were found to be distinct to that observed in African subtype C based on the existing datasets and concordant with their sequence diversity. Trend analysis indicated an increasing neutralization resistance observed over time with CAP25-VRC26.25, PGDM1400 and PGT121 when tested on pseudoviruses expressing envs obtained from 1999 to 2016. However, inconsistent trend in neutralization susceptibility was observed, when pseudoviruses expressing envs obtained from three followed up individuals were examined. Finally, through predictive analysis of the 98 Indian subtype C including those assessed in the present study by employing additive model implemented in CombiNAber ( http://www.hiv.lanl.gov ), we observed two possibilities where combinations of three bnAbs (VRC01/CAP56-VRC26.25/PGT121 and PGDM1400/CAP256-VRC26.25/PGT121) could achieve near 100% neutralization coverage. CONCLUSIONS: Our findings not only indicate disparate intra-clade C genetic vis-à-vis neutralization diversities but also warrant the need for more comprehensive study using additional isolates towards comparing inter and intra-clade neutralization diversities which will be necessary for selecting the bnAb combinations suitable for optimal coverage of the region-specific HIV-1 circulating subtypes. Expanding these efforts is imperative for designing efficacious bnAb based intervention strategies for India as well as subtype C in general.

Membrane Env Liposomes Facilitate Immunization with Multivalent Full-Length HIV Spikes.

A major goal of HIV vaccine design is to elicit broadly neutralizing antibodies (bNAbs). Such bNAbs target HIV's trimeric, membrane-embedded envelope glycoprotein spikes (mEnv). Soluble Env (sEnv) trimers have been used as vaccines, but engineering sEnvs for stability, multivalency, and desired antigenicity is problematic and deletes key neutralizing epitopes on glycoprotein 41 (gp41) while creating neoepitopes that elicit unwanted antibodies. Meanwhile, multivalent mEnv vaccines are challenging to develop due to trimer instability and low mEnv copy number amid other extraneous proteins on virus-like particles. Here, we describe a multivalent mEnv vaccine platform that does not require protein engineering or extraneous proteins. mEnv trimers were fixed, purified, and combined with naked liposomes in mild detergent. On removal of detergent, mEnv spikes were observed embedded in liposome particles (mean diameter, 133 nm) in correct orientation. These particles were recognized by HIV bNAbs and not non-NAbs and are designated mEnv liposomes (MELs). Following a sequential immunization scheme in rabbits, MELs elicited antibodies that neutralized tier 2 HIV isolates. Analysis of serum antibody specificities, including those to epitopes involving a missing conserved N-glycosylation site at position 197 near the CD4 binding site on two of the immunogens, provides clues on how NAb responses can be improved with modified immunogens. In sum, MELs are a biochemically defined platform that enables rational immunization strategies to elicit HIV bNAbs using multimerized mEnv. IMPORTANCE A vaccine that induced broadly neutralizing antibodies against HIV would likely end the AIDS pandemic. Such antibodies target membrane-embedded envelope glycoprotein spikes (mEnv) that HIV uses to enter cells. Due to HIV Env's low expression and instability, soluble stabilized Env trimers have been used as vaccine candidates, but these have an altered base that disrupts targets of HIV broadly neutralizing antibodies that bind near the membrane and are not available for all HIV isolates. Here, we describe membrane Env liposomes (MELs) that display a multivalent array of stable mEnvs on liposome particles. MELs showed the expected antibody recognition properties, including targeting parts of mEnv missing on soluble Envs. Immunization with MELs elicited antibodies that neutralized diverse HIV isolates. The MEL platform facilitates vaccine development with potentially any HIV Env at high valency, and a similar approach may be useful for eliciting antibodies to membrane-embedded targets of therapeutic interest.

DNA priming immunization is more effective than recombinant protein vaccine in eliciting antigen-specific B cell responses.

While DNA prime-protein boost vaccination approach has been widely used in preclinical and clinical studies especially in the field of HIV vaccine development, the exact role of DNA immunization has not been fully identified. Our previous work demonstrated that DNA immunization was able to elicit T follicular helper (Tfh) cell responses and germinal center (GC) B cell development in a mouse model. In the current report, a mouse immunogenicity study was conducted to further ask whether DNA immunization is able to elicit antigen-specific B cell responses. Using HIV-1 Env as model antigen delivered in the form of DNA prime-protein boost, our data demonstrated that DNA prime was able to enhance the antigen-specific B cell responses for both Env-specific antibody secreting cells (ASC) and memory B cells. Furthermore, the DNA priming can greatly reduce the need of including an adjuvant as part of the recombinant protein vaccine boost formulation. Our findings revealed one mechanism that supports the value of DNA priming in assisting the inductin of high affinity and long lasting antigen specific antibody responses.

Rational Engraftment of Quaternary-Interactive Acidic Loops for Anti-HIV-1 Antibody Improvement.

Broadly neutralizing antibodies (bNAbs) are the focus of increasing interest for human immunodeficiency virus type 1 (HIV-1) prevention and treatment. Although several bNAbs are already under clinical evaluation, the development of antibodies with even greater potency and breadth remains a priority. Recently, we reported a novel strategy for improving bNAbs against the CD4-binding site (CD4bs) of gp120 by engraftment of the elongated framework region 3 (FR3) from VRC03, which confers the ability to establish quaternary interactions with a second gp120 protomer. Here, we applied this strategy to a new series of anti-CD4bs bNAbs (N49 lineage) that already possess high potency and breadth. The resultant chimeric antibodies bound the HIV-1 envelope (Env) trimer with a higher affinity than their parental forms. Likewise, their neutralizing capacity against a global panel of HIV-1 Envs was also increased. The introduction of additional modifications further enhanced the neutralization potency. We also tried engrafting the elongated CDR1 of the heavy chain from bNAb 1-18, another highly potent quaternary-binding antibody, onto several VRC01-class bNAbs, but none of them was improved. These findings point to the highly selective requirements for the establishment of quaternary contact with the HIV-1 Env trimer. The improved anti-CD4bs antibodies reported here may provide a helpful complement to current antibody-based protocols for the therapy and prevention of HIV-1 infection.IMPORTANCE Monoclonal antibodies represent one of the most important recent innovations in the fight against infectious diseases. Although potent antibodies can be cloned from infected individuals, various strategies can be employed to improve their activity or pharmacological features. Here, we improved a lineage of very potent antibodies that target the receptor-binding site of HIV-1 by engineering chimeric molecules containing a fragment from a different monoclonal antibody. These engineered antibodies are promising candidates for development of therapeutic or preventive approaches against HIV/AIDS.