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Abstract The relative conservation of the influenza hemagglutinin (HA) stem compared to that of the immunodominant HA head makes the HA stem an attractive target for broadly protective influenza vaccines. Here we report the first-in-human, dose-escalation, open-label trial (NCT04579250) evaluating an unadjuvanted group 2 stabilized stem ferritin nanoparticle vaccine based on the H10 A/Jiangxi-Donghu/346/2013 influenza HA, H10ssF, in healthy adults. Participants received a single 20 mcg dose (n = 3) or two 60 mcg doses 16 weeks apart (n = 22). Vaccination with H10ssF was safe and well tolerated with only mild systemic and local reactogenicity reported. No serious adverse events occurred. Vaccination significantly increased homologous H10 HA stem binding and neutralizing antibodies at 2 weeks after both first and second vaccinations, and these responses remained above baseline at 40 weeks. Heterologous H3 and H7 binding antibodies also significantly increased after each vaccination and remained elevated throughout the study. These data indicate that the group 2 HA stem nanoparticle vaccine is safe and induces stem-directed binding and neutralizing antibodies.

Abstract Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

Abstract Ebola virus disease (EVD) is a filoviral infection caused by virus species of the Ebolavirus genus including Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV). We investigated the safety and immunogenicity of a heterologous prime-boost regimen involving a chimpanzee adenovirus 3 vectored Ebola vaccine [either monovalent (cAd3-EBOZ) or bivalent (cAd3-EBO)] prime followed by a recombinant modified vaccinia virus Ankara EBOV vaccine (MVA-EbolaZ) boost in two phase 1/1b randomized open-label clinical trials in healthy adults in the United States (US) and Uganda (UG). Trial US (NCT02408913) enrolled 140 participants, including 26 EVD vaccine-naïve and 114 cAd3-Ebola-experienced participants (April-November 2015). Trial UG (NCT02354404) enrolled 90 participants, including 60 EVD vaccine-naïve and 30 DNA Ebola vaccine-experienced participants (February-April 2015). All tested vaccines and regimens were safe and well tolerated with no serious adverse events reported related to study products. Solicited local and systemic reactogenicity was mostly mild to moderate in severity. The heterologous prime-boost regimen was immunogenic, including induction of durable antibody responses which peaked as early as two weeks and persisted up to one year after each vaccination. Different prime-boost intervals impacted the magnitude of humoral and cellular immune responses. The results from these studies demonstrate promising implications for use of these vaccines in both prophylactic and outbreak settings.

Abstract Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Summary: Identifying individual functional B cell receptors (BCRs) is common, but two-dimensional analysis of B cell frequency versus BCR potency would delineate both quantity and quality of antigen-specific memory B cells. We efficiently determine quantitative BCR neutralizing activities using a single-cell-derived antibody supernatant analysis (SCAN) workflow and develop a frequency-potency algorithm to estimate B cell frequencies at various neutralizing activity or binding affinity cutoffs. In an HIV-1 fusion peptide (FP) immunization study, frequency-potency curves elucidate the quantity and quality of FP-specific immunoglobulin G (IgG)+ memory B cells for different animals, time points, and antibody lineages at single-cell resolution. The BCR neutralizing activities are mainly determined by their affinities to soluble envelope trimer. Frequency analysis definitively demonstrates dominant neutralizing antibody lineages. These findings establish SCAN and frequency-potency analyses as promising approaches for general B cell analysis and monoclonal antibody (mAb) discovery. They also provide specific rationales for HIV-1 FP-directed vaccine optimization.

Abstract 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.

Abstract While several COVID-19 vaccines have been in use, more effective and durable vaccines are needed to combat the ongoing COVID-19 pandemic. Here, we report highly immunogenic self-assembling SARS-CoV-2 spike-HBsAg nanoparticles displaying a six-proline-stabilized WA1 (wild type, WT) spike S6P on a HBsAg core. These S6P-HBsAgs bound diverse domain-specific SARS-CoV-2 monoclonal antibodies. In mice with and without a HBV pre-vaccination, DNA immunization with S6P-HBsAgs elicited significantly more potent and durable neutralizing antibody (nAb) responses against diverse SARS-CoV-2 strains than that of soluble S2P or S6P, or full-length S2P with its coding sequence matching mRNA-1273. The nAb responses elicited by S6P-HBsAgs persisted substantially longer than by soluble S2P or S6P and appeared to be enhanced by HBsAg pre-exposure. These data show that genetic delivery of SARS-CoV-2 S6P-HBsAg nanoparticles can elicit greater and more durable nAb responses than non-nanoparticle forms of stabilized spike. Our findings highlight the potential of S6P-HBsAgs as next generation genetic vaccine candidates against SARS-CoV-2.

Abstract Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Summary: 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.

ABSTRACTThe amino-acid composition of the immunoglobulin variable region has been observed to impact antibody pharmacokinetics (PK). Here, we sought to improve the PK of the broad HIV−1-neutralizing VRC01-class antibodies, VRC07-523LS and N6LS, by reducing the net positive charge in their variable domains. We used a structure-guided approach to generate a panel of antibody variants incorporating select Arg or Lys substituted to Asp, Gln, Glu, or Ser. The engineered variants exhibited reduced affinity to heparin, reduced polyreactivity, and improved PK in human FcRn-transgenic mice. One variant, VRC07-523LS.v34, with three charge substitutions, had an observed in vivo half-life and an estimated human half-life of 10.8 and 60 days, respectively (versus 5.4 and 38 days for VRC07-523LS) and retained functionality, neutralizing 92% of a 208-strain panel at a geometric mean IC80

ABSTRACTAntibody CAP256-VRC26.25 targets the second hypervariable region (V2) at the apex of the HIV envelope (Env) trimer with extraordinary neutralization potency, although less than optimal breadth. To improve breadth, we linked the light chain of CAP256V2LS, an optimized version of CAP256-VRC26.25 currently under clinical evaluation, to the llama nanobody J3, which has broad CD4-binding site-directed neutralization. The J3-linked bispecific antibody exhibited improved breadth and potency over both J3 and CAP256V2LS, indicative of synergistic neutralization. The cryo-EM structure of the bispecific antibody in complex with a prefusion-closed Env trimer revealed simultaneous binding of J3 and CAP256V2LS. We further optimized the pharmacokinetics of the bispecific antibody by reducing the net positive charge of J3. The optimized bispecific antibody, which we named CAP256.J3LS, had a half-life similar to CAP256V2LS in human FcRn knock-in mice and exhibited suitable auto-reactivity, manufacturability, and biophysical risk. CAP256.J3LS neutralized over 97% of a multiclade 208-strain panel (geometric mean concentration for 80% inhibition (IC80) 0.079 μg/ml) and 100% of a 100-virus clade C panel (geometric mean IC80 of 0.05 μg/ml), suggesting its anti-HIV utility especially in regions where clade C dominates.

Vaccines against the WA1 SARS-CoV2 strain confer protection against other variants. However, the mechanisms underlying cross-protection are not fully understood. Here, the authors develop a method for rapid analysis of single B cells from patient samples and show that infection with a variant elicits convergent, public B cell responses to other variants.

Abstract The broadly neutralizing antibody (bNAb) CAP256-VRC26.25 has exceptional potency against HIV-1 and has been considered for clinical use. During the characterization and production of this bNAb, we observed several unusual features. First, the antibody appeared to adhere to pipette tips, requiring tips to be changed during serial dilution to accurately measure potency. Second, during production scale-up, proteolytic cleavage was discovered to target an extended heavy chain loop, which was attributed to a protease in spent medium from 2-week culture. To enable large scale production, we altered the site of cleavage via a single amino acid change, K100mA. The resultant antibody retained potency and breadth while avoiding protease cleavage. We also added the half-life extending mutation LS, which improved the in vivo persistence in animal models, but did not impact neutralization activity; we observed the same preservation of neutralization for bNAbs VRC01, N6, and PGDM1400 with LS on a 208-virus panel. The final engineered antibody, CAP256V2LS, retained the extraordinary neutralization potency of the parental antibody, had a favorable pharmacokinetic profile in animal models, and was negative in in vitro assessment of autoreactivity. CAP256V2LS has the requisite potency, developability and suitability for scale-up, allowing its advancement as a clinical candidate.

Summary: 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: Background: The phase 2b proof-of-concept Antibody Mediated Prevention (AMP) trials showed that VRC01, an anti-HIV-1 broadly neutralising antibody (bnAb), prevented acquisition of HIV-1 sensitive to VRC01. To inform future study design and dosing regimen selection of candidate bnAbs, we investigated the association of VRC01 serum concentration with HIV-1 acquisition using AMP trial data. Methods: The case–control sample included 107 VRC01 recipients who acquired HIV-1 and 82 VRC01 recipients who remained without HIV-1 during the study. We measured VRC01 serum concentrations with a qualified pharmacokinetic (PK) Binding Antibody Multiplex Assay. We employed nonlinear mixed effects PK modelling to estimate daily-grid VRC01 concentrations. Cox regression models were used to assess the association of VRC01 concentration at exposure and baseline body weight, with the hazard of HIV-1 acquisition and prevention efficacy as a function of VRC01 concentration. We also compared fixed dosing vs. body weight-based dosing via simulations. Findings: Estimated VRC01 concentrations in VRC01 recipients without HIV-1 were higher than those in VRC01 recipients who acquired HIV-1. Body weight was inversely associated with HIV-1 acquisition among both placebo and VRC01 recipients but did not modify the prevention efficacy of VRC01. VRC01 concentration was inversely correlated with HIV-1 acquisition, and positively correlated with prevention efficacy of VRC01. Simulation studies suggest that fixed dosing may be comparable to weight-based dosing in overall predicted prevention efficacy. Interpretation: These findings suggest that bnAb serum concentration may be a useful marker for dosing regimen selection, and operationally efficient fixed dosing regimens could be considered for future trials of HIV-1 bnAbs. Funding: Was provided by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID) (UM1 AI068614, to the HIV Vaccine Trials Network [HVTN]; UM1 AI068635, to the HVTN Statistical Data and Management Center [SDMC], Fred Hutchinson Cancer Center [FHCC]; 2R37 054165 to the FHCC; UM1 AI068618, to HVTN Laboratory Center, FHCC; UM1 AI068619, to the HPTN Leadership and Operations Center; UM1 AI068613, to the HIV Prevention Trials Network [HPTN] Laboratory Center; UM1 AI068617, to the HPTN SDMC; and P30 AI027757, to the Center for AIDS Research, Duke University (AI P30 AI064518) and University of Washington (P30 AI027757) Centers for AIDS Research; R37AI054165 from NIAID to the FHCC; and OPP1032144 CA-VIMC Bill & Melinda Gates Foundation.

Here, the authors apply positron emission tomography (PET) imaging to visualize HIV tissue-wide burden in infected individuals using a radiolabeled broadly neutralizing antibody, 89Zr-VRC01, and show that PET tracer lymph node uptake positively correlates with HIV protein levels measured directly from cells obtained from these tissues. This strategy may allow non-invasive characterization of residual HIV infection in the setting of therapeutic interventions.

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.

Summary: Broadly neutralizing antibodies (bNAbs) against HIV-1 are promising immunotherapeutic agents for treatment of HIV-1 infection. bNAbs can be administered to SHIV-infected rhesus macaques to assess their anti-viral efficacy; however, their delivery into macaques often leads to rapid formation of anti-drug antibody (ADA) responses limiting such assessment. Here, we depleted B cells in five SHIV-infected rhesus macaques by pretreatment with a depleting anti-CD20 antibody prior to bNAb infusions to reduce ADA. Peripheral B cells were depleted following anti-CD20 infusions and remained depleted for at least 9 weeks after the 1st anti-CD20 infusion. Plasma viremia dropped by more than 100-fold in viremic animals after the initial bNAb treatment. No significant humoral ADA responses were detected for as long as B cells remained depleted. Our results indicate that transient B cell depletion successfully inhibited emergence of ADA and improved the assessment of anti-viral efficacy of a bNAb in a SHIV-infected rhesus macaque model.

New vaccine delivery technologies, such as mRNA, have played a critical role in the rapid and efficient control of SARS-CoV-2, helping to end the COVID-19 pandemic. Enveloped virus-like particles (eVLPs) are often more immunogenic than protein subunit immunogens and could be an effective vaccine platform. Here, we investigated whether the genetic delivery of eVLPs could achieve strong immune responses in mice as previously reported with the immunization of in vitro purified eVLPs. We utilized Newcastle disease virus-like particles (NDVLPs) to display SARS-CoV-2 prefusion-stabilized spikes from the WA-1 or Beta variant (S-2P or S-2Pᵦ, respectively) and evaluated neutralizing murine immune responses achieved by a single-gene-transcript DNA construct for the WA-1 or Beta variant (which we named S-2P-NDVLP-1T and S-2Pᵦ-NDVLP-1T, respectively), by multiple-gene-transcript DNA constructs for the Beta variant (S-2Pᵦ-NDVLP-3T), and by a protein subunit–DNA construct for the WA-1 or Beta variant (S-2P-TM or S-2Pᵦ-TM, respectively). The genetic delivery of S-2P-NDVLP-1T or S-2Pᵦ-NDVLP-1T yielded modest neutralizing responses after a single immunization and high neutralizing responses after a second immunization, comparable to previously reported results in mice immunized with in vitro purified S-2P-NDVLPs. Notably, genetic delivery of S-2Pᵦ-NDVLP-3T yielded significantly higher neutralizing responses in mice after a second immunization than S-2Pᵦ-NDVLP-1T or S-2Pᵦ-TM. Genetic delivery also elicited high spike-specific T-cell responses. Collectively, these results indicate that genetic delivery can provide an effective means to immunize eVLPs and that a multiple-gene transcript eVLP platform may be especially efficacious and inform the design of improved vaccines.

Background: Advances in therapeutic drugs have increased life-expectancies for HIV-infected individuals, but the need for an effective vaccine remains. We assessed safety and immunogenicity of HIV-1 vaccine, Trimer 4571 (BG505 DS-SOSIP.664) adjuvanted with aluminum hydroxide (alum), in HIV-negative adults. Methods: We conducted a phase I, randomized, open-label, dose-escalation trial at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Eligible participants were HIV-negative, healthy adults between 18-50 years. Participants were randomized 1:1 to receive Trimer 4571 adjuvanted with 500 mcg alum by either the subcutaneous (SC) or intramuscular (IM) route at weeks 0, 8, and 20 in escalating doses of 100 mcg or 500 mcg. The primary objectives were to evaluate the safety and tolerability of Trimer 4571 with a secondary objective of evaluating vaccine-induced antibody responses. The primary and safety endpoints were evaluated in all participants who received at least one dose of Trimer 4571. Trial results were summarized using descriptive statistics. This trial is registered at ClinicalTrials.gov, NCT03783130. Findings: Between March 7 and September 11, 2019, 16 HIV-negative participants were enrolled, including six (38%) males and ten (62%) females. All participants received three doses of Trimer 4571. Solicited reactogenicity was mild to moderate in severity, with one isolated instance of severe injection site redness (6%) following a third 500 mcg SC administration. The most commonly reported solicited symptoms included mild injection site tenderness in 14 (88%) and mild myalgia in six (38%) participants. The most frequent unsolicited adverse event attributed to vaccination was mild injection site pruritus in six (38%) participants. Vaccine-induced seropositivity occurred in seven (44%) participants and resolved in all but one (6%). No serious adverse events occurred. Trimer 4571-specific binding antibodies were detected in all groups two weeks after regimen completion, primarily focused on the glycan-free trimer base. Neutralizing antibody activity was limited to the 500 mcg dose groups. Interpretation: Trimer 4571 was safe, well tolerated, and immunogenic in this first-in-human trial. While this phase 1 trial is limited in size, our results inform and support further evaluation of prefusion-stabilized HIV-1 envelope trimers as a component of vaccine design strategies to generate broadly neutralizing antibodies against HIV-1. Funding: Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Understanding the human antibody response to influenza A virus strains is important for vaccine development. Here, Creanga et al. generate a panel of 55 replication-deficient reporter viruses representing diversity of human H1N1 and H3N2, and pandemic subtypes and characterize the neutralization profile of 24 antibodies and polyclonal sera.

Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

Abstract Understanding maturation pathways of broadly neutralizing antibodies (bnAbs) against HIV‐1 can be highly informative for HIV‐1 vaccine development. A lineage of J038 bnAbs is now obtained from a long‐term SHIV‐infected macaque. J038 neutralizes 54% of global circulating HIV‐1 strains. Its binding induces a unique “up” conformation for one of the V2 loops in the trimeric envelope glycoprotein and is heavily dependent on glycan, which provides nearly half of the binding surface. Their unmutated common ancestor neutralizes the autologous virus. Continuous maturation enhances neutralization potency and breadth of J038 lineage antibodies via expanding antibody‐Env contact areas surrounding the core region contacted by germline‐encoded residues. Developmental details and recognition features of J038 lineage antibodies revealed here provide a new pathway for elicitation and maturation of V2‐targeting bnAbs.

Summary: Using the VRC01-class of anti-HIV-1 broadly neutralizing antibodies (bnAbs) elicited in sequentially immunized Ig-humanized mice as an example, we describe a protocol to identify key mutations for bnAb function by point mutagenesis and antibody binding and neutralization assays. We also describe steps to monitor how the key mutations arise in response to specific immunogens, which is critical for vaccine evaluation and design, via longitudinal antibody mutation profiling. This protocol can be customized for other V-gene-specific bnAbs and animal models.For complete details on the use and execution of this profile, please refer to Chen et al. (2021).

Influenza virus hemagglutinin (HA) stem between group 1 and 2 viruses has different glycosylation patterns, likely hampering cross-group protection. Here, Boyoglu-Barnum et al. show that introducing a group 2 glycan into a group 1 stem nanoparticle vaccine broadens antibody responses in mice to cross-react with group 2 HAs.

Elucidation of broadly neutralizing antibodies (bnAb) is a goal in HIV vaccine development. Here, Bradley et al. show that administration of CTLA-4 blocking antibody with vaccine antigens increases HIV-1 envelope antibody responses in macaques and a bnAb precursor mouse model.

Broadly neutralizing antibodies (bNAbs) are being evaluated for HIV post-exposure prophylaxis (PEP) in the setting of vertical transmission. Here, using a macaque model of perinatal SHIV infection, the authors show that PEP for infant macaques within 30–48 h of SHIV exposure is highly effective using either bNAbs or ART.

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccine-elicited immunity, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring of vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

Background: Respiratory syncytial virus (RSV) subtypes, A and B, co-circulate in annual epidemics and alternate in dominance. We have shown that a subtype A RSV fusion (F) glycoprotein, stabilized in its prefusion conformation by DS-Cav1 mutations, is a promising RSV-vaccine immunogen, capable of boosting RSV-neutralizing titers in healthy adults. In both humans and vaccine-tested animals, neutralizing titers elicited by this subtype A DS-Cav1 immunogen were ~ 2- to 3-fold higher against the homologous subtype A virus than against the heterologous subtype B virus. Methods: To understand the molecular basis for this subtype difference, we introduced DS-Cav1 mutations into RSV strain B18537 F, determined the trimeric crystal structure, and carried out immunogenicity studies. Results: The B18537 DS-Cav1 F structure at 2-Å resolution afforded a precise delineation of prefusion F characteristics, including those of antigenic site Ø, a key trimer-apex site. Structural comparison with the subtype A prefusion F indicated 11% of surface residues to be different, with an alpha-carbon root-mean-square deviation (RMSD) of 1.2 Å; antigenic site Ø, however, resulted in 23% of its surface residues and had an alpha-carbon RMSD of 2.2 Å. Immunization of vaccine-tested animals with DS-Cav1-stabilized B18537 F induced neutralizing responses ~100-fold higher than with postfusion B18537 F. Notably, elicited responses neutralized RSV subtypes A and B at similar levels and were directed towards both conserved equatorial and diverse apical regions. Conclusion: We propose that structural differences in apical and equatorial sites–coupled to differently focused immune responses–provide a molecular explanation for observed differences in elicited subtype A and B neutralizing responses. Keywords: antigenic site; crystal structure; epitope; fusion glycoprotein; immunogenicity; neutralization; RSV subtype; vaccine

Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.

Summary: Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.

Conjugate-vaccine immunogens require three components: a carrier protein, an antigen, and a crosslinker, capable of coupling antigen to carrier protein, while preserving both T-cell responses from carrier protein and B-cell responses from antigen. We previously showed that the N-terminal eight residues of the HIV-1 fusion peptide (FP8) as an antigen could prime for broad cross-clade neutralizing responses, that recombinant heavy chain of tetanus toxin (rTTHC) as a carrier protein provided optimal responses, and that choice of crosslinker could impact both antigenicity and immunogenicity. Here, we delve more deeply into the impact of varying the linker between FP8 and rTTHC. In specific, we assessed the physical properties, the antigenicity, and the immunogenicity of conjugates for crosslinkers ranging in spacer-arm length from 1.5 to 95.2 Å, with varying hydrophobicity and crosslinking-functional groups. Conjugates coupled with different degrees of multimerization and peptide-to-rTTHC stoichiometry, but all were well recognized by HIV-fusion-peptide-directed antibodies VRC34.01, VRC34.05, PGT151, and ACS202 except for the conjugate with the longest linker (24-PEGylated SMCC; SM(PEG)24), which had lower affinity for ACS202, as did the conjugate with the shortest linker (succinimidyl iodoacetate; SIA), which also had the lowest peptide-to-rTTHC stoichiometry. Murine immunizations testing seven FP8-rTTHC conjugates elicited fusion-peptide-directed antibody responses, with SIA- and SM(PEG)24-linked conjugates eliciting lower responses than the other five conjugates. After boosting with prefusion-closed envelope trimers from strains BG505 clade A and consensus clade C, trimer-directed antibody-binding responses were lower for the SIA-linked conjugate; elicited neutralizing responses were similar, however, though statistically lower for the SM(PEG)24-linked conjugate, when tested against a strain especially sensitive to fusion-peptide-directed responses. Overall, correlation analyses revealed the immunogenicity of FP8-rTTHC conjugates to be negatively impacted by hydrophilicity and extremes of length or low peptide-carrier stoichiometry, but robust to other linker parameters, with several commonly used crosslinkers yielding statistically indistinguishable serological results.

Summary: SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.

Here, the authors report that specific monoclonal antibodies isolated from vaccinated rhesus macaques can neutralize a subset of Tier 2 difficult-to-neutralize HIV-1 that express Env in an open conformation, suggesting that V3 loop-specific targeting can bias the estimation of vaccine-induced bnAbs.

Quaternary contacts mediated by an extended heavy-chain framework region 3 (FR3) have been shown to improve binding to HIV envelope and virus neutralization for a few antibodies. Here, Liu et al. engraft such an FR3 loop onto several potent broadly neutralizing antibodies, resulting in improved neutralization activity and pharmacokinetics.

Temsavir, a compound that inhibits HIV entry by binding envelope (Env), is currently in clinical development. Here, Lai et al. identify a more than 10-fold improved compound and, using lattice engineering, obtain crystal structures that give insights into improved inhibition between small molecules and Env.

Sequence signatures of multidonor broadly neutralizing influenza antibodies can be used to quantify the prevalence of B cells with virus-neutralizing potential to accelerate development of broadly protective vaccine strategies. Antibodies of the same class share similar recognition modes and developmental pathways, and several antibody classes have been identified that neutralize diverse group 1- and group 2-influenza A viruses and have been observed in multiple human donors. One such multidonor antibody class, the HV6-1-derived class, targets the stem region of hemagglutinin with extraordinary neutralization breadth. Here, we use an iterative process to combine informatics, biochemical, and structural analyses to delineate an improved sequence signature for HV6-1-class antibodies. Based on sequence and structure analyses of known HV6-1 class antibodies, we derived a more inclusive signature (version 1), which we used to search for matching B-cell transcripts from published next-generation sequencing datasets of influenza vaccination studies. We expressed selected antibodies, evaluated their function, and identified amino acid-level requirements from which to refine the sequence signature (version 2). The cryo-electron microscopy structure for one of the signature-identified antibodies in complex with hemagglutinin confirmed motif recognition to be similar to known HV6-1-class members, MEDI8852 and 56.a.09, despite differences in recognition-loop length. Threading indicated the refined signature to have increased accuracy, and signature-identified heavy chains, when paired with the light chain of MEDI8852, showed neutralization comparable to the most potent members of the class. Incorporating sequences of additional class members thus enables an improved sequence signature for HV6-1-class antibodies, which can identify class members with increased accuracy.

Summary: Soluble “SOSIP”-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high-titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the effect on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, here, we quantify the prevalence of trimer-base antibody responses in 49 non-human primates immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. Trimer-base responses account for ∼90% of the overall trimer response in animals immunized with trimer only, ∼70% in animals immunized with a cocktail of SOSIP trimer and FP conjugate, and ∼30% in animals primed with FP conjugates before trimer immunization. Notably, neutralization breadth in FP-conjugate-primed animals correlates inversely with trimer-base responses. Our data provide methods to quantify the prevalence of trimer-base responses and reveal that FP-conjugate priming, either alone or as part of a cocktail, can reduce the trimer-base response and improve the neutralization outcome.

The re-emergence of Zika virus (ZIKV) caused widespread infections that were linked to Guillain-Barré syndrome in adults and congenital malformation in fetuses, and epidemiological data suggest that ZIKV infection can induce protective antibody responses. A more detailed understanding of anti-ZIKV antibody responses may lead to enhanced antibody discovery and improved vaccine designs against ZIKV and related flaviviruses. Here, we applied recently-invented library-scale antibody screening technologies to determine comprehensive functional molecular and genetic profiles of naturally elicited human anti-ZIKV antibodies in three convalescent individuals. We leveraged natively paired antibody yeast display and NGS to predict antibody cross-reactivities and coarse-grain antibody affinities, to perform in-depth immune profiling of IgM, IgG, and IgA antibody repertoires in peripheral blood, and to reveal virus maturation state-dependent antibody interactions. Repertoire-scale comparison of ZIKV VLP-specific and non-specific antibodies in the same individuals also showed that mean antibody somatic hypermutation levels were substantially influenced by donor-intrinsic characteristics. These data provide insights into antiviral antibody responses to ZIKV disease and outline systems-level strategies to track human antibody immune responses to emergent viral infections.

Passive transfer of broadly neutralizing antibodies is showing promise in the treatment and prevention of HIV-1. One class of antibodies, the VRC01 class, appears especially promising. To improve VRC01-class antibodies, we combined structure-based design with a matrix-based approach to generate VRC01-class variants that filled an interfacial cavity, used diverse third-complementarity-determining regions, reduced potential steric clashes, or exploited extended contacts to a neighboring protomer within the envelope trimer. On a 208-strain panel, variant VRC01.23LS neutralized 90% of the panel at a geometric mean IC80 less than 1 μg/ml, and in transgenic mice with human neonatal-Fc receptor, the serum half-life of VRC01.23LS was indistinguishable from that of the parent VRC01LS, which has a half-life of 71 d in humans. A cryo-electron microscopy structure of VRC01.23 Fab in complex with BG505 DS-SOSIP.664 Env trimer determined at 3.4-Å resolution confirmed the structural basis for its ~10-fold improved potency relative to VRC01. Another variant, VRC07-523-F54-LS.v3, neutralized 95% of the 208-isolated panel at a geometric mean IC80 of less than 1 μg/ml, with a half-life comparable to that of the parental VRC07-523LS. Our matrix-based structural approach thus enables the engineering of VRC01 variants for HIV-1 therapy and prevention with improved potency, breadth, and pharmacokinetics.

Summary: The HIV fusion peptide (FP) is a promising vaccine target. FP-directed monoclonal antibodies from vaccinated macaques have been identified that neutralize up to ∼60% of HIV strains; these vaccinations, however, have involved ∼1 year with an extended neutralization-eclipse phase without measurable serum neutralization. Here, in 32 macaques, we test seven vaccination regimens, each comprising multiple immunizations of FP-carrier conjugates and HIV envelope (Env) trimers. Comparisons of vaccine regimens reveal FP-carrier conjugates to imprint cross-clade neutralizing responses and a cocktail of FP conjugate and Env trimer to elicit the earliest broad responses. We identify a signature, appearing as early as week 6 and involving the frequency of B cells recognizing both FP and Env trimer, predictive of vaccine-elicited breadth ∼1 year later. Immune monitoring of B cells in response to vaccination can thus enable vaccine insights even in the absence of serum neutralization, here identifying FP imprinting, cocktail approach, and early signature as means to improve FP-directed vaccine responses.

Licensed vaccines or therapeutics are rarely available for pathogens with epidemic or pandemic potential. Developing interventions for specific pathogens and defining generalizable approaches for related pathogens is a global priority and inherent to the UN Sustainable Development Goals. Nipah virus (NiV) poses a significant epidemic threat, and zoonotic transmission from bats-to-humans with high fatality rates occurs almost annually. Human-to-human transmission of NiV has been documented in recent outbreaks leading public health officials and government agencies to declare an urgent need for effective vaccines and therapeutics. Here, we evaluate NiV vaccine antigen design options including the fusion glycoprotein (F) and the major attachment glycoprotein (G). A stabilized prefusion F (pre-F), multimeric G constructs, and chimeric proteins containing both pre-F and G were developed as protein subunit candidate vaccines. The proteins were evaluated for antigenicity and structural integrity using kinetic binding assays, electron microscopy, and other biophysical properties. Immunogenicity of the vaccine antigens was evaluated in mice. The stabilized pre-F trimer and hexameric G immunogens both induced serum neutralizing activity in mice, while the post-F trimer immunogen did not elicit neutralizing activity. The pre-F trimer covalently linked to three G monomers (pre-F/G) induced potent neutralizing antibody activity, elicited responses to the greatest diversity of antigenic sites, and is the lead candidate for clinical development. The specific stabilizing mutations and immunogen designs utilized for NiV were successfully applied to other henipaviruses, supporting the concept of identifying generalizable solutions for prototype pathogens as an approach to pandemic preparedness.

Summary: Antibodies targeting the V1V2 apex of the HIV-1 envelope (Env) trimer comprise one of the most commonly elicited categories of broadly neutralizing antibodies. Structures of these antibodies indicate diverse modes of Env recognition typified by antibodies of the PG9 class and the PGT145 class. The mode of recognition, however, has been unclear for the most potent of the V1V2 apex-targeting antibodies, CAP256-VRC26.25 (named for donor-lineage.clone and referred to hereafter as VRC26.25). Here, we determine the cryoelectron microscopy structure at 3.7 Å resolution of the antigen-binding fragment of VRC26.25 in complex with the Env trimer thought to have initiated the lineage. The 36-residue protruding loop of VRC26.25 displays recognition incorporating both strand-C interactions similar to the PG9 class and V1V2 apex insertion similar to the PGT145 class. Structural elements of separate antibody classes can thus intermingle to form a “combined” class, which in this case yields an antibody of extraordinary potency. : Antibody CAP256-VRC26.25 is one of the most potent known HIV-1-neutralizing antibodies. Gorman et al. determine the cryo-EM structure of this antibody in complex with the Env trimer that initiated the antibody lineage. The structure reveals how elements of distinct antibody classes can intermingle to yield an antibody of extraordinary potency. Keywords: broadly neutralizing antibody, HIV-1 envelope trimer, multidonor antibody class, PCT64, PG9, PGDM1400, PGT145, tyrosine sulfation, V1V2 apex recognition

Broadly neutralizing antibodies are showing promise in the treatment and prevention of HIV-1, with several now being evaluated clinically. Some lead clinical candidates, including antibodies CAP256-VRC26.25, N6, PGT121, and VRC07-523, have one or more N-linked glycosylation sequons in their variable domains (Fvs) from somatic hypermutation, and these glycans increase chemical heterogeneity, complicating the manufacture of these antibodies as products. Here we propose a general method to remove Fv glycans and use this method to develop engineered versions of these four antibodies with Fv glycans removed. When germline residues were introduced to remove each glycan, antibody properties between wild type and mutant were not significantly altered for CAP256-VRC26.25 and PGT121; however, germline mutants for N6 and VRC07-523 showed increased polyreactivity, which is known to correlate with unfavorable in vivo pharmacokinetics. To reduce polyreactivity induced by removal of Fv glycan, we mutated aromatic residues and arginines structurally proximal to the removed glycan and identified Fv glycan-removed variants with low polyreactivity for N6 and VRC07-523. Two such variants, N6-N72LCQ-R18LCD and VRC07-523-N72LCQ-R24LCD, showed thermostability, neutralization potency and breadth, and half-life in humanized FcRn mice that were similar to their wild-type Fv-glycosylated counterparts. The removal of Fv glycan and reduction of chemical heterogeneity were confirmed by liquid chromatography-mass spectrometry. With reduced heterogeneity, the Fv-glycan-removed variants developed here may have utility as products for treating or preventing infection by HIV-1.

Knowledge on how antibody responses have evolved is critical for the induction of protective immunity. Here the authors analyse, using high-throughput sequencing of both exon and intron regions, the mutation and lineage development of an HIV-neutralizing antibody to find an unexpected early emergence of broadly neutralizing species.

Broadly neutralizing antibodies targeting HIV Env could potentially be utilized as therapeutics. Here, Steinhardt et al. engineer a trispecific antibody with specificity for the receptor-binding site, a conserved Env glycan patch and the Env membrane proximal region with nearly pan-isolate neutralization breadth and high potency.

Summary: Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ∼10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.

Summary: The elicitation of autologous neutralizing responses by immunization with HIV-1 envelope (Env) trimers conformationally stabilized in a prefusion closed state has generated considerable interest in the HIV-1 vaccine field. However, soluble prefusion closed Env trimers have been produced from only a handful of HIV-1 strains, limiting their utility as vaccine antigens and B cell probes. Here, we report the engineering from 81 HIV-1 strains of soluble, fully cleaved, prefusion Env trimers with appropriate antigenicity. We used a 96-well expression-screening format to assess the ability of artificial disulfides and Ile559Pro substitution (DS-SOSIP) to produce soluble cleaved-Env trimers; from 180 Env strains, 20 yielded prefusion closed trimers. We also created chimeras, by utilizing structure-based design to incorporate select regions from the well-behaved BG505 strain; from 180 Env strains, 78 DS-SOSIP-stabilized chimeras, including 61 additional strains, yielded prefusion closed trimers. Structure-based design thus enables the production of prefusion closed HIV-1-Env trimers from dozens of diverse strains. : Joyce et al. use a structure-based chimeric strategy to produce prefusion closed Env trimers from 81 strains of HIV-1 encompassing all major clades. These soluble Env trimers should have utility as B cell probes and HIV-1 immunogens, thereby enabling both antibody identification and vaccine development. Keywords: conformational stabilization, envelope trimer, HIV-1 vaccine, prefusion closed trimer, structure-based design

The events required for the induction of broad neutralizing antibodies (bnAbs) following HIV-1 envelope (Env) vaccination are unknown, and their induction in animal models as proof of concept would be critical. Here, we describe the induction of plasma antibodies capable of neutralizing heterologous primary (tier 2) HIV-1 strains in one macaque and two rabbits. Env immunogens were designed to induce CD4 binding site (CD4bs) bnAbs, but surprisingly, the macaque developed V1V2-glycan bnAbs. Env immunization of CD4bs bnAb heavy chain rearrangement (VHDJH) knockin mice similarly induced V1V2-glycan neutralizing antibodies (nAbs), wherein the human CD4bs VH chains were replaced with mouse rearrangements bearing diversity region (D)-D fusions, creating antibodies with long, tyrosine-rich HCDR3s. Our results show that Env vaccination can elicit broad neutralization of tier 2 HIV-1, demonstrate that V1V2-glycan bnAbs are more readily induced than CD4bs bnAbs, and define VH replacement and diversity region fusion as potential mechanisms for generating V1V2-glycan bnAb site antibodies.

An efficient HIV-1 vaccine will likely depend on eliciting broadly neutralizing antibodies (bnAb). Here the authors analyze the B cell repertoire in macaques and knock-in mice in response to sequential immunization with Env variants that induce a bnAb targeting the CD4-binding site of Env in a HIV-1 infected individual.