Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody.

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies.

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico.

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.

SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies.

The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

Short CDRL1 in intermediate VRC01-like mAbs is not sufficient to overcome key glycan barriers on HIV-1 Env.

VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. IMPORTANCE: HIV-1 broadly neutralizing antibodies will be a key component of an effective HIV-1 vaccine, as they prevent viral acquisition. Over the past decade, numerous broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV. Despite an in-depth knowledge of their structures, epitopes, ontogenies, and, in a few rare cases, their maturation pathways during infection, bnAbs have, so far, not been elicited by vaccination. This necessitates the identification of key obstacles that prevent their elicitation by immunization and overcoming them. Here we examined whether CDRL1 shortening is a prerequisite for the broadly neutralizing potential of VRC01-class bnAbs, which bind within the CD4 receptor binding site of Env. Our findings indicate that CDRL1 shortening by itself is important but not sufficient for the acquisition of neutralization breadth, and suggest that particular combinations of amino acid mutations, not elicited so far by vaccination, are most likely required for the development of such a feature.

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.

Nanoparticles presenting clusters of CD4 expose a universal vulnerability of HIV-1 by mimicking target cells.

CD4-based decoy approaches against HIV-1 are attractive options for long-term viral control, but initial designs, including soluble CD4 (sCD4) and CD4-Ig, were ineffective. To evaluate a therapeutic that more accurately mimics HIV-1 target cells compared with monomeric sCD4 and dimeric CD4-Ig, we generated virus-like nanoparticles that present clusters of membrane-associated CD4 (CD4-VLPs) to permit high-avidity binding of trimeric HIV-1 envelope spikes. In neutralization assays, CD4-VLPs were >12,000-fold more potent than sCD4 and CD4-Ig and >100-fold more potent than the broadly neutralizing antibody (bNAb) 3BNC117, with >12,000-fold improvements against strains poorly neutralized by 3BNC117. CD4-VLPs also neutralized patient-derived viral isolates that were resistant to 3BNC117 and other bNAbs. Intraperitoneal injections of CD4-CCR5-VLP produced only subneutralizing plasma concentrations in HIV-1-infected humanized mice but elicited CD4-binding site mutations that reduced viral fitness. All mutant viruses showed reduced sensitivity to sCD4 and CD4-Ig but remained sensitive to neutralization by CD4-VLPs in vitro. In vitro evolution studies demonstrated that CD4-VLPs effectively controlled HIV-1 replication at neutralizing concentrations, and viral escape was not observed. Moreover, CD4-VLPs potently neutralized viral swarms that were completely resistant to CD4-Ig, suggesting that escape pathways that confer resistance against conventional CD4-based inhibitors are ineffective against CD4-VLPs. These findings suggest that therapeutics that mimic HIV-1 target cells could prevent viral escape by exposing a universal vulnerability of HIV-1: the requirement to bind CD4 on a target cell. We propose that therapeutic and delivery strategies that ensure durable bioavailability need to be developed to translate this concept into a clinically feasible functional cure therapy.

Structural basis for Zika envelope domain III recognition by a germline version of a recurrent neutralizing antibody.

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs.

Cryo-EM structure of a CD4-bound open HIV-1 envelope trimer reveals structural rearrangements of the gp120 V1V2 loop.

The HIV-1 envelope (Env) glycoprotein, a trimer of gp120-gp41 heterodimers, relies on conformational flexibility to function in fusing the viral and host membranes. Fusion is achieved after gp120 binds to CD4, the HIV-1 receptor, and a coreceptor, capturing an open conformational state in which the fusion machinery on gp41 gains access to the target cell membrane. In the well-characterized closed Env conformation, the gp120 V1V2 loops interact at the apex of the Env trimer. Less is known about the structure of the open CD4-bound state, in which the V1V2 loops must rearrange and separate to allow access to the coreceptor binding site. We identified two anti-HIV-1 antibodies, the coreceptor mimicking antibody 17b and the gp120-gp41 interface-spanning antibody 8ANC195, that can be added as Fabs to a soluble native-like Env trimer to stabilize it in a CD4-bound conformation. Here, we present an 8.9-Šcryo-electron microscopy structure of a BG505 Env-sCD4-17b-8ANC195 complex, which reveals large structural rearrangements in gp120, but small changes in gp41, compared with closed Env structures. The gp120 protomers are rotated and separated in the CD4-bound structure, and the three V1V2 loops are displaced by ∼40 Šfrom their positions at the trimer apex in closed Env to the sides of the trimer in positions adjacent to, and interacting with, the three bound CD4s. These results are relevant to understanding CD4-induced conformational changes leading to coreceptor binding and fusion, and HIV-1 Env conformational dynamics, and describe a target structure relevant to drug design and vaccine efforts.

Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway.

Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3-effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.

Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting.

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.

Crystal structure of mitochondrial fission complex reveals scaffolding function for mitochondrial division 1 (Mdv1) coiled coil.

The mitochondrial fission machinery is best understood in the yeast Saccharomyces cerevisiae, where Fis1, Mdv1, and Dnm1 are essential components. Fis1 is a mitochondrial outer membrane protein that recruits the dynamin-related GTPase Dnm1 during the fission process. This recruitment occurs via Mdv1, which binds both Fis1 and Dnm1 and therefore functions as a molecular adaptor linking the two molecules. Mdv1 has a modular structure, consisting of an N-terminal extension that binds Fis1, a central coiled coil for dimerization, and a C-terminal WD40 repeat region that binds Dnm1. We have solved the crystal structure of a dimeric Mdv1-Fis1 complex that contains both the N-terminal extension and coiled-coil regions of Mdv1. Consistent with previous studies, Mdv1 binds Fis1 through a U-shaped helix-loop-helix motif, and dimerization of the Mdv1-Fis1 complex is mediated by the antiparallel coiled coil of Mdv1. However, the complex is surprisingly compact and rigid due to two additional contacts mediated by the surface of the Mdv1 coiled coil. The coiled coil packs against both Fis1 and the second helix of the Mdv1 helix-loop-helix motif. Mutational analyses showed that these contacts are important for mitochondrial fission activity. These results indicate that, in addition to dimerization, the unusually long Mdv1 coiled coil serves a scaffolding function to stabilize the Mdv1-Fis1 complex.

CD4 binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wild-type animals.

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3. As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

HIV-1 CD4-binding site germline antibody-Env structures inform vaccine design.

BG24, a VRC01-class broadly neutralizing antibody (bNAb) against HIV-1 Env with relatively few somatic hypermutations (SHMs), represents a promising target for vaccine strategies to elicit CD4-binding site (CD4bs) bNAbs. To understand how SHMs correlate with BG24 neutralization of HIV-1, we report 4.1 Å and 3.4 Å single-particle cryo-EM structures of two inferred germline (iGL) BG24 precursors complexed with engineered Env-based immunogens lacking CD4bs N-glycans. Structures reveal critical Env contacts by BG24iGL and identify antibody light chain structural features that impede Env recognition. In addition, biochemical data and cryo-EM structures of BG24iGL variants bound to Envs with CD4bs glycans present provide insights into N-glycan accommodation, including structural modes of light chain adaptations in the presence of the N276gp120 glycan. Together, these findings reveal Env regions critical for germline antibody recognition and potential sites to alter in immunogen design.

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer.

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope.

HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. We report characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7 Å cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, raising the possibility that only two fusion peptides per trimer are required for viral-host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.

Sequential immunization of macaques elicits heterologous neutralizing antibodies targeting the V3-glycan patch of HIV-1 Env.

Broadly neutralizing antibodies (bNAbs) against HIV-1 develop after prolonged virus and antibody coevolution. Previous studies showed that sequential immunization with a V3-glycan patch germline-targeting HIV-1 envelope trimer (Env) followed by variant Envs can reproduce this process in mice carrying V3-glycan bNAb precursor B cells. However, eliciting bNAbs in animals with polyclonal antibody repertoires is more difficult. We used a V3-glycan immunogen multimerized on virus-like particles (VLPs), followed by boosting with increasingly native-like Env-VLPs, to elicit heterologous neutralizing antibodies in nonhuman primates (NHPs). Structures of antibody/Env complexes after prime and boost vaccinations demonstrated target epitope recognition with apparent maturation to accommodate glycans. However, we also observed increasing off-target antibodies with boosting. Eight vaccinated NHPs were subsequently challenged with simian-human immunodeficiency virus (SHIV), and seven of eight animals became infected. The single NHP that remained uninfected after viral challenge exhibited one of the lowest neutralization titers against the challenge virus. These results demonstrate that more potent heterologous neutralization resulting from sequential immunization is necessary for protection in this animal model. Thus, improved prime-boost regimens to increase bNAb potency and stimulate other immune protection mechanisms are essential for developing anti­HIV-1 vaccines.

Publisher Correction: Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.