BMS-818251, a fostemsavir analog, is a next-generation HIV-1 attachment inhibitor with enhanced potency and a similar resistance profile. By using ex vivo viral outgrowth assays with HIV+ donor samples, we demonstrate here that BMS-818251 exhibits superior viral suppression compared to temsavir, the active form of fostemsavir. To map potential resistance pathways, we employed deep mutational scanning and pseudotyped virus neutralization assays to identify escape mutations within the HIV-1 envelope glycoprotein (Env). These mutations were largely clustered around the BMS-818251 binding site, with key resistance mutations reducing drug-binding affinity. Several of the enriched mutations, such as S375I/N, M426L, and M475I, have been previously observed in fostemsavir-treated patients, highlighting their clinical relevance. Isothermal titration calorimetry revealed reduced binding affinity as the primary mechanism of resistance, though with notable exceptions, such as R429G, suggesting additional factors to influence viral escape. Ex vivo Env sequencing confirmed selection of resistance mutations under BMS-818251 pressure, reinforcing the predictive value of deep mutational scanning for in vivo resistance monitoring. Compared to fostemsavir, BMS-818251 achieved more effective viral suppression at lower concentrations, even in donor samples harboring preexisting resistance mutations. These findings support the continued development of BMS-818251 as a promising alternative to fostemsavir, with potential benefits for patients with multidrug-resistant HIV-1.

The elicitation of cross-neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) by vaccination remains a major challenge. Here, we immunized previously Env-immunized nonhuman primates with a series of near-native trimers that possessed N-glycan deletions proximal to the conserved CD4 binding site (CD4bs) to focus B cells to this region. Following heterologous boosting with fully glycosylated trimers, we detected tier 2 cross-neutralizing activity in the serum of several animals. Isolation of 185 matched heavy- and light-chain sequences from Env-binding memory B cells from an early responder identified a broadly neutralizing antibody lineage, LJF-0034, which neutralized nearly 70% of an 84-member HIV-1 global panel. High-resolution cryoelectron microscopy (cryo-EM) structures revealed a bifurcated binding mode that bridged the CD4bs to V3 across the gp120:120 interface on two adjacent protomers, evading the proximal N276 glycan impediment to the CD4bs, allowing neutralization breadth. This quaternary epitope defines a potential target for future HIV-1 vaccine development.

Motivation:Broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) of human immunodeficiency virus-1 (HIV-1) have been utilized in clinical trials aimed at preventing and treating HIV-1 infections. However, the emergence of neutralization resistance to bNAbs occurs rapidly due to the high mutation rate of HIV-1. Previous studies have suggested the use of in silico methods to effectively predict the resistance of HIV-1 isolates to bNAbs. In this study, we present a novel machine learning approach called BRAVE (Bnab Resistance Analysis Via Evolutionary scale modeling 2) designed to predict HIV-1 resistance against 33 known bNAbs. This innovative tool employs a Random Forests classifier that uses a protein language model to reliably capture protein features. Results:BRAVE outperformed leading resistance prediction tools on various performance metrics, attaining the highest performance in established classification measures including accuracy, area under the curve, logarithmic loss, and F1-score. Importantly, rigorous statistical comparisons (p<0.001) show that BRAVE is significantly more accurate than state-of-the-art neutralization prediction tools. BRAVE will facilitate informed decisions of antibody usage and sequence-based monitoring of viral escape in clinical settings.

Enveloped viruses responsible for global health pandemics often display a glycan shield on their surface envelope glycoproteins. In HIV, the glycan shield is formed by clusters of high-mannose glycans and plays essential roles in viral fitness and immune evasion. A few mannose-binding lectins potently inactivate HIV but have not been fully exploited due to poor pharmacokinetics and short serum half-lives. To address this, we engineered an antibody-lectin conjugate comprising the anti-HIV lectin griffithsin (GRFT) to the Fc region of human IgG1, with the aim of extending its serum half-life and augmenting anti-HIV activity by inducing immune effector responses. Engineered mGRFT-Fc produced in bacteria exhibited picomolar anti-HIV activity and an extended serum half-life, and mGRFT-Fc produced in mammalian cells (mGRFT-Fcglyc) elicited immune effector responses. In HIV-infected CD34+-humanized mice, both GRFT and mGRFT-Fcglyc effectively suppressed viral loads for up to 8 weeks after a single dose. Significantly, mGRFT-Fcglyc prevented HIV infection by neutralizing HIV and provided sustained protection from break-through infections via Fc-mediated immune effector responses, exhibiting a dual mode of protection. This study demonstrates the successful engineering of a lectin-based biologic and provides early evidence that a glycan-targeting agent alone can confer protection from viral infection in vivo.

Soluble 'SOSIP'-stabilized HIV-1 envelope glycoprotein (Env) trimers elicit dominant antibody responses targeting their glycan-free base regions, potentially diminishing neutralizing responses. Previously, using a nonhuman primate model, we demonstrated that priming with fusion peptide (FP)-carrier conjugate immunogens followed by boosting with Env trimers reduced the anti-base response. Further, we demonstrated that longer immunization intervals further reduced anti-base responses and increased neutralization breadth. Here, we demonstrate that long trimer-boosting intervals, but not long FP immunization intervals, reduce the anti-base response. Additionally, we identify that FP priming before trimer immunization enhances antibody avidity to the Env trimer. We also establish that adjuvants Matrix M and Adjuplex further reduce anti-base responses and increase neutralizing titers. FP priming, long trimer-immunization interval, and an appropriate adjuvant can thus reduce anti-base antibody responses and improve Env-directed vaccine outcomes.

An antibody-based HIV-1 vaccine will require the induction of potent cross-reactive HIV-1-neutralizing responses. To demonstrate feasibility toward this goal, we combined vaccination targeting the fusion-peptide site of vulnerability with infection by simian-human immunodeficiency virus (SHIV). In four macaques with vaccine-induced neutralizing responses, SHIV infection boosted plasma neutralization to 45%-77% breadth (geometric mean 50% inhibitory dilution [ID50] similar to 100) on a 208-strain panel. Molecular dissection of these responses by antibody isolation and cryo-electron microscopy (cryo-EM) structure determination revealed 15 of 16 antibody lineages with cross-clade neutralization to be directed toward the fusion-peptide site of vulnerability. In each macaque, isolated antibodies from memory B cells recapitulated the plasma-neutralizing response, with fusion-peptide-binding antibodies reaching breadths of 40%-60% (50% inhibitory concentration [IC50] < 50 mu g/mL) and total lineage-concentrations estimates of 50-200 mu g/mL. Longitudinal mapping indicated that these responses arose prior to SHIV infection. Collectively, these results provide in vivo molecular examples for one to a few B cell lineages affording potent, broadly neutralizing plasma responses.

Broadly neutralizing antibodies (bNAbs) are promising candidates for the treatment and prevention of HIV-1 infections. Despite their critical importance, automatic detection of HIV-1 bNAbs from immune repertoires is still lacking. Here, we develop a straightforward computational method for the Rapid Automatic Identification of bNAbs (RAIN) based on machine learning methods. In contrast to other approaches, which use one-hot encoding amino acid sequences or structural alignment for prediction, RAIN uses a combination of selected sequence-based features for the accurate prediction of HIV-1 bNAbs. We demonstrate the performance of our approach on non-biased, experimentally obtained and sequenced BCR repertoires from HIV-1 immune donors. RAIN processing leads to the successful identification of distinct HIV-1 bNAbs targeting the CD4-binding site of the envelope glycoprotein. In addition, we validate the identified bNAbs using an in vitro neutralization assay and we solve the structure of one of them in complex with the soluble native-like heterotrimeric envelope glycoprotein by single-particle cryo-electron microscopy (cryo-EM). Overall, we propose a method to facilitate and accelerate HIV-1 bNAbs discovery from non-selected immune repertoires. Artificial intelligence holds great promise to improve diagnosis of numerous immune-related or infectious diseases. Here, the authors show that machine learning can be used to identify HIV-1 specific broad neutralising antibody.

A subset of people living with HIV (PLWH) can produce broadly neutralizing antibodies (bNAbs) against HIV, but the lymph node (LN) dynamics that promote the generation of these antibodies are poorly understood. Here, we explored LN-associated histological, immunological, and virological mechanisms of bNAb generation in a cohort of anti-retroviral therapy (ART)-naïve PLWH. We found that participants who produce bNAbs, termed neutralizers, have a superior LN-associated B cell follicle architecture compared with PLWH who do not. The latter was associated with a significantly higher in situ prevalence of Bcl-6hi follicular helper CD4 T cells (TFH), expressing a molecular program that favors their differentiation and stemness, and significantly reduced IL-10 follicular suppressor CD4 T cells. Furthermore, our data reveal possible molecular targets mediating TFH- B cell interactions in neutralizers. Together, we identify cellular and molecular mechanisms that contribute to the development of bNAbs in PLWH.

A mucosal route of vaccination could prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication at the site of infection and limit transmission. We compared protection against heterologous XBB.1.16 challenge in nonhuman primates (NHPs) ~5 months following intramuscular boosting with bivalent mRNA encoding WA1 and BA.5 spike proteins or mucosal boosting with a WA1-BA.5 bivalent chimpanzee adenoviral-vectored vaccine delivered by intranasal or aerosol device. NHPs boosted by either mucosal route had minimal virus replication in the nose and lungs, respectively. By contrast, protection by intramuscular mRNA was limited to the lower airways. The mucosally delivered vaccine elicited durable airway IgG and IgA responses and, unlike the intramuscular mRNA vaccine, induced spike-specific B cells in the lungs. IgG, IgA and T cell responses correlated with protection in the lungs, whereas mucosal IgA alone correlated with upper airway protection. This study highlights differential mucosal and serum correlates of protection and how mucosal vaccines can durably prevent infection against SARS-CoV-2.

AbstractBroadly neutralizing antibodies are proposed as therapeutic and prophylactic agents against HIV‐1, but their potency and breadth are less than optimal. This study describes the immunization of a llama with the prefusion‐stabilized HIV‐1 envelope (Env) trimer, BG505 DS‐SOSIP, and the identification and improvement of potent neutralizing nanobodies recognizing the CD4‐binding site (CD4bs) of vulnerability. Two of the vaccine‐elicited CD4bs‐targeting nanobodies, G36 and R27, when engineered into a triple tandem format with llama IgG2a‐hinge region and human IgG1‐constant region (G36×3‐IgG2a and R27×3‐IgG2a), neutralized 96% of a multiclade 208‐strain panel at geometric mean IC80s of 0.314 and 0.033 µg mL−1, respectively. Cryo‐EM structures of these nanobodies in complex with Env trimer revealed the two nanobodies to neutralize HIV‐1 by mimicking the recognition of the CD4 receptor. To enhance their neutralizing potency and breadth, nanobodies are linked to the light chain of the V2‐apex‐targeting broadly neutralizing antibody, CAP256V2LS. The resultant human‐llama bispecific antibody CAP256L‐R27×3LS exhibited ultrapotent neutralization and breadth exceeding other published HIV‐1 broadly neutralizing antibodies, with pharmacokinetics determined in FcRn‐Fc mice similar to the parent CAP256V2LS. Vaccine‐elicited llama nanobodies, when combined with V2‐apex broadly neutralizing antibodies, may therefore be able to fulfill anti‐HIV‐1 therapeutic and prophylactic clinical goals.

Elicitation of antibodies that neutralize the tier-2 neutralization-resistant isolates that typify HIV-1 transmission has been a long-sought goal. Success with prefusion-stabilized envelope trimers eliciting autologous neutralizing antibodies has been reported in multiple vaccine-test species, though not in humans. To investigate elicitation of HIV-1 neutralizing antibodies in humans, here, we analyze B cells from a phase I clinical trial of the "DS-SOSIP"-stabilized envelope trimer from strain BG505, identifying two antibodies, N751 2C06.01 and N751-2C09.01 (named for donor-lineage.clone), that neutralize the autologous tier-2 strain, BG505. Though derived from distinct lineages, these antibodies form a reproducible antibody class that targets the HIV-1 fusion peptide. Both antibodies are highly strain specific, which we attribute to their partial recognition of a BG505-specific glycan hole and to their binding requirements for a few BG505-specific residues. Prefusion-stabilized envelope trimers can thus elicit autologous tier-2 neutralizing antibodies in humans, with initially identified neutralizing antibodies recognizing the fusion-peptide site of vulnerability.

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence.

Soluble HIV-1-envelope (Env) trimers elicit immune responses that target their solvent-exposed protein bases, the result of removing these trimers from their native membrane-bound context. To assess whether glycosylation could limit these base responses, we introduced sequons encoding potential N-linked glycosylation sites (PNGSs) into base-proximal regions. Expression and antigenic analyses indicated trimers bearing six-introduced PNGSs to have reduced base recognition. Cryo-EM analysis revealed trimers with introduced PNGSs to be prone to disassembly and introduced PNGS to be disordered. Protein-base and glycan-base trimers induced reciprocally symmetric ELISA responses, in which only a small fraction of the antibody response to glycan-base trimers recognized protein-base trimers and vice versa. EM polyclonal epitope mapping revealed glycan-base trimers -even those that were stable biochemically- to elicit antibodies that recognized disassembled trimers. Introduced glycans can thus mask the protein base but their introduction may yield neo-epitopes that dominate the immune response.

Summary: Broadly neutralizing antibodies (bNAbs) against HIV can reduce viral transmission in humans, but an effective therapeutic will require unusually high breadth and potency of neutralization. We employ the OSPREY computational protein design software to engineer variants of two apex-directed bNAbs, PGT145 and PG9RSH, resulting in increases in potency of over 100-fold against some viruses. The top designed variants improve neutralization breadth from 39% to 54% at clinically relevant concentrations (IC80 < 1 μg/mL) and improve median potency (IC80) by up to 4-fold over a cross-clade panel of 208 strains. To investigate the mechanisms of improvement, we determine cryoelectron microscopy structures of each variant in complex with the HIV envelope trimer. Surprisingly, we find the largest increases in breadth to be a result of optimizing side-chain interactions with highly variable epitope residues. These results provide insight into mechanisms of neutralization breadth and inform strategies for antibody design and improvement.

Waning immunity and continued virus evolution have limited the durability of protection from symptomatic infection mediated by intramuscularly (IM)-delivered mRNA vaccines against COVID-19 although protection from severe disease remains high. Mucosal vaccination has been proposed as a strategy to increase protection at the site of SARS-CoV-2 infection by enhancing airway immunity, potentially reducing rates of infection and transmission. Here, we compared protection against XBB.1.16 virus challenge 5 months following IM or mucosal boosting in non-human primates (NHP) that had previously received a two-dose mRNA-1273 primary vaccine regimen. The mucosal boost was composed of a bivalent chimpanzee adenoviral-vectored vaccine encoding for both SARS-CoV-2 WA1 and BA.5 spike proteins (ChAd-SARS-CoV-2-S) and delivered either by an intranasal mist or an inhaled aerosol. An additional group of animals was boosted by the IM route with bivalent WA1/BA.5 spike-matched mRNA (mRNA-1273.222) as a benchmark control. NHP were challenged in the upper and lower airways 18 weeks after boosting with XBB.1.16, a heterologous Omicron lineage strain. Cohorts boosted with ChAd-SARS-CoV-2-S by an aerosolized or intranasal route had low to undetectable virus replication as assessed by levels of subgenomic SARS-CoV-2 RNA in the lungs and nose, respectively. In contrast, animals that received the mRNA-1273.222 boost by the IM route showed minimal protection against virus replication in the upper airway but substantial reduction of virus RNA levels in the lower airway. Immune analysis showed that the mucosal vaccines elicited more durable antibody and T cell responses than the IM vaccine. Protection elicited by the aerosolized vaccine was associated with mucosal IgG and IgA responses, whereas protection elicited by intranasal delivery was mediated primarily by mucosal IgA. Thus, durable immunity and effective protection against a highly transmissible heterologous variant in both the upper and lower airways can be achieved by mucosal delivery of a virus-vectored vaccine. Our study provides a template for the development of mucosal vaccines that limit infection and transmission against respiratory pathogens.Graphical abstract:

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures. The SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme provides a real-time risk assessment of SARS-CoV-2 variants with the potential to affect transmission, virulence and resistance to infection- and vaccine-induced immunity.