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The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although significant progress has been made in understanding the structures of various Env conformations and transition intermediates that occur within the millisecond timescale, faster transitions in the microsecond timescale have not yet been observed. In this study, we employed time-resolved, temperature-jump small angle X- ray scattering to monitor structural rearrangements in an HIV-1 Env ectodomain construct with microsecond precision. We detected a transition correlated with Env opening that occurs in the hundreds of microseconds range and another more rapid transition that preceded this opening. Model fitting indicated that the early rapid transition involved an order-to-disorder transition in the trimer apex loop contacts, suggesting that conventional conformation-locking design strategies that target the allosteric machinery may be ineffective in preventing this movement. Utilizing this information, we engineered an envelope that locks the apex loop contacts to the adjacent protomer. This modification resulted in significant angle-of-approach shifts in the interaction of a neutralizing antibody. Our findings imply that blocking the intermediate state could be crucial for inducing antibodies with the appropriate bound state orientation through vaccination.

Antibody affinity maturation enables adaptive immune responses to a wide range of pathogens. In some individuals broadly neutralizing antibodies develop to recognize rapidly mutating pathogens with extensive sequence diversity. Vaccine design for pathogens such as HIV-1 and influenza has therefore focused on recapitulating the natural affinity maturation process. Here, we determine structures of antibodies in complex with HIV-1 Envelope for all observed members and ancestral states of the broadly neutralizing HIV-1 V3-glycan targeting DH270 antibody clonal B cell lineage. These structures track the development of neutralization breadth from the unmutated common ancestor and define affinity maturation at high spatial resolution. By elucidating contacts mediated by key mutations at different stages of antibody development we identified sites on the epitope-paratope interface that are the focus of affinity optimization. Thus, our results identify bottlenecks on the path to natural affinity maturation and reveal solutions for these that will inform immunogen design aimed at eliciting a broadly neutralizing immune response by vaccination.

Langya virus (LayV) is a paramyxovirus in theHenipavirusgenus, closely related to the deadly Nipah and Hendra viruses, that was identified in August 2022 through disease surveillance following animal exposure in eastern China. Paramyxoviruses present two glycoproteins on their surface, known as attachment and fusion proteins, that mediate entry into cells and constitute the primary antigenic targets for immune response. Here, we determine cryo-EM structures of the uncleaved LayV fusion protein (F) ectodomain in pre- and post-fusion conformations. The LayV-F protein exhibits pre- and post-fusion architectures that, despite being highly conserved across paramyxoviruses, show differences in their surface properties, in particular at the apex of the prefusion trimer, that may contribute to antigenic variability. While dramatic conformational changes were visualized between the pre- and post-fusion forms of the LayV-F protein, several domains remained invariant, held together by highly conserved disulfides. The LayV-F fusion peptide is buried within a highly conserved, hydrophobic, interprotomer pocket in the pre-fusion state and is notably less flexible than the rest of the protein, highlighting its “spring-loaded” state and suggesting that the mechanism of pre-to-post transition must involve perturbations to the pocket and release of the fusion peptide. Together, these results offer a structural basis for how the Langya virus fusion protein compares to its Henipavirus relatives and propose a mechanism for the initial step of pre- to post-fusion conversion that may apply more broadly to paramyxoviruses.ImportanceThe Henipavirus genus is quickly expanding into new animal hosts and geographic locations. This study compares the structure and antigenicity of the Langya virus fusion protein to other henipaviruses, which has important vaccine or therapeutic development implications. Furthermore, the study proposes a new mechanism to explain the early steps of the fusion initiation process that can be more broadly applied to theParamyxoviridaefamily.

Prophylactic and therapeutic drugs are urgently needed to combat coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Over the past year, SARS-CoV-2 neutralizing antibodies have been developed for preventive or therapeutic uses. While neutralizing antibodies target the spike protein, their neutralization potency and breadth vary according to recognition epitopes. Several potent SARS-CoV-2 antibodies have shown degrees of success in preclinical or clinical trials, and the US Food and Drug Administration has issued emergency use authorization for two neutralizing antibody cocktails. Nevertheless, antibody therapy for SARS-CoV-2 still faces potential challenges, including emerging viral variants of concern that have antibody-escape mutations and the potential for antibody-mediated enhancement of infection or inflammation. This review summarizes representative SARS-CoV-2 neutralizing antibodies that have been reported and discusses prospects and challenges for the development of the next generation of COVID-19 preventive or therapeutic antibodies. Expected final online publication date for the Annual Review of Medicine, Volume 73 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Recent estimates have placed the number of new human immunodeficiency virus type 1 (HIV-1) infections at approximately 1.5 million per year, emphasizing the ongoing and urgent need for an effective vaccine. The envelope (Env) glycoprotein is the main focus of HIV-1 vaccine development, but, due to its inherent metastability, many Env variants are difficult to recombinantly express in the relatively large quantities that are required for biochemical studies and animal trials.

SUMMARYThe CD4 binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the CD4bs is occluded by glycans, immunogen expansion of appropriate naïve B cells, and selection of functional antibody mutations. Here, we demonstrate immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAbs bound to HIV-1 Env demonstrated binding angles similar to human bnAbs and heavy chain second complementarity determining region-dependent binding characteristic of all known human CD4-mimicking bnAbs. Macaque nAbs were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAbs. This vaccine study initiated the B cells from which derive CD4bs bnAbs in primates, accomplishing the key first step in development of an effective HIV-1 vaccine.

A major goal of HIV-1 vaccine development is induction of broadly neutralizing antibodies (bnAbs). While success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knock-in mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof-of concept and milestone towards development of an HIV vaccine.One-Sentence SummaryA vaccine strategy for selecting key rare antibody mutations is shown to induce HIV broadly neutralizing antibodies in mice.

Antibody affinity maturation enables adaptive immune responses to a wide range of pathogens. In some individuals broadly neutralizing antibodies develop to recognize rapidly mutating pathogens with extensive sequence diversity. Vaccine design for pathogens such as HIV-1 and influenza have therefore focused on recapitulating the natural affinity maturation process. Here, we determined structures of antibodies in complex with HIV-1 Envelope for all observed members and ancestral states of a broadly neutralizing HIV-1 antibody clonal B cell lineage. These structures track the development of neutralization breadth from the unmutated common ancestor and define affinity maturation at high spatial resolution. By elucidating contacts mediated by key mutations at different stages of antibody development we have identified sites on the epitope-paratope interface that are the focus of affinity optimization. Thus, our results identify bottlenecks on the path to natural affinity maturation and reveal solutions for these that will inform immunogen design aimed at eliciting a broadly neutralizing immune response by vaccination.SummarySomatic hypermutation drives affinity maturation of germline-encoded antibodies leading to the development of their pathogen neutralization function1. Rational vaccine design efforts that aim to recapitulate affinity maturation rely on information from antibodies elicited and matured during natural infection. High-throughput next generation sequencing and methods for tracing antibody development have allowed close monitoring of the antibody maturation process. Since maturation involves both affinity-enhancing and affinity-independent diversification, the precise effect of each observed mutation, their role in enhancing affinity to antigens, and the order in which the mutations accumulated are often unclear. These gaps in knowledge most acutely hinder efforts directed at difficult targets such as pan-HIV, pan-Influenza, and pan-Coronavirus vaccines. In HIV-1 infection, antibody maturation over several years is required to achieve neutralization breadth. Here, we determined structures of antibodies in complex with HIV-1 Envelope trimers for all observed members and ancestral states of a broadly neutralizing HIV-1 antibody clone to examine affinity maturation as neutralization breadth developed from the unmutated common ancestor. Structural determination of epitope-paratope interfaces revealed details of the contacts evolving over a timescale of several years. Structures along different branches of the clonal lineage elucidated differences in the branch that led to broad neutralization versus off-track paths that culminated in sub-optimal neutralization breadth. We further determined structures of the evolving Envelope revealing details of the virus-antibody co-evolution through visualization of how the virus constructs barriers to evade antibody-mediated neutralization and the mechanisms by which the developing antibody clone circumvents these barriers. Together, our structures provide a detailed time-resolved imagery of the affinity maturation process through atomic level descriptions of virus-antibody co-evolution leading to broad HIV neutralization. While the findings from our studies have direct relevance to HIV-1, the principles of affinity optimization and breadth development elucidated in our study should have broad relevance to other pathogens.

A successful HIV-1 vaccine will require induction of a polyclonal neutralizing antibody (nAb) response, yet vaccine-mediated induction of such a response in primates remains a challenge. We found that a stabilized HIV-1 CH505 envelope (Env) trimer formulated with a Toll-like receptor 7/8 agonist induced potent HIV-1 polyclonal nAbs that correlated with protection from homologous simian-human immunodeficiency virus (SHIV) infection. The serum dilution that neutralized 50% of virus replication (ID 50 titer) required to protect 90% of macaques was 1:364 against the challenge virus grown in primary rhesus CD4 + T cells. Structural analyses of vaccine-induced nAbs demonstrated targeting of the Env CD4 binding site or the N156 glycan and the third variable loop base. Autologous nAb specificities similar to those elicited in macaques by vaccination were isolated from the human living with HIV from which the CH505 Env immunogen was derived. CH505 viral isolates were isolated that mutated the V1 to escape both the infection-induced and vaccine-induced antibodies. These results define the specificities of a vaccine-induced nAb response and the protective titers of HIV-1 vaccine–induced nAbs required to protect nonhuman primates from low-dose mucosal challenge by SHIVs bearing a primary transmitted/founder Env.

SARS-CoV-2 spike (S) protein ectodomain purification can be challenging, with engineered and natural variations often resulting in lower yields. Here, we present a detailed transfection and purification protocol for the SARS-CoV-2 S ectodomain. We describe how to trace protein yields during purification using highly sensitive and characteristic changes in S ectodomain intrinsic fluorescence upon thermal denaturation. Additionally, we detail several optimized aspects of the purification including timing and temperature. This protocol facilitates consistent, high-quality preparations of the SARS-CoV-2 S ectodomain. For complete details on the use and execution of this protocol, please refer to Stalls et al. (2022), Gobeil et al. (2022), Edwards et al. (2021), and Henderson et al. (2020).

The impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine development. Here we report an unexpected cold sensitivity of a stabilized SARS-CoV-2 ectodomain construct currently being widely used for immunogen design. We found that when stored at 22 or 37 °C for 1 week, the S-protein displayed well-ordered trimeric spikes by negative stain electron microscopy. However, storage at 4 °C reduced the trimeric spikes to

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sub-lineage has gained in proportion relative to BA.1. Because spike (S) protein variations may underlie differences in their pathobiology, here we determine cryoelectron microscopy (cryo-EM) structures of the BA.2 S ectodomain and compare these with previously determined BA.1 S structures. BA.2 receptor-binding domain (RBD) mutations induce remodeling of the RBD structure, resulting in tighter packing and improved thermostability. Interprotomer RBD interactions are enhanced in the closed (or 3-RBD-down) BA.2 S, while the fusion peptide is less accessible to antibodies than in BA.1. Binding and pseudovirus neutralization assays reveal extensive immune evasion while defining epitopes of two outer RBD face-binding antibodies, DH1044 and DH1193, that neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the closed state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant and show differences in key functional regions in the BA.1 and BA.2 S proteins.

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor binding domain (RBD) and neutralizing antibody epitope presentation affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.

Severe acute respiratory syndrome coronaviruses 1 (SARS-CoV) and 2 (SARS-CoV-2), including SARS-CoV-2 variants of concern, can cause deadly infections. The mortality associated with sarbecovirus infection underscores the importance of developing broadly effective countermeasures against them, which could be key in the prevention and mitigation of current and future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV; bat coronaviruses WIV-1 and RsSHC014; and SARS-CoV-2 variants D614G, B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, B.1.617.1, and B.1.617.2 by a receptor binding domain (RBD)–specific human antibody, DH1047. Prophylactic and therapeutic treatment with DH1047 was protective against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B.1.351 infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among sarbecoviruses. Thus, DH1047 is a broadly protective antibody that can prevent infection and mitigate outbreaks caused by SARS-related strains and SARS-CoV-2 variants. Our results also suggest that the conserved RBD epitope bound by DH1047 is a rational target for a universal sarbecovirus vaccine.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has triggered myriad efforts to understand the structure and dynamics of this complex pathogen. The Spike glycoprotein of SARS-CoV-2 is a significant target for immunogens as it is the means by which the virus enters human cells, while simultaneously sporting mutations responsible for immune escape. These functional and escape processes are regulated by complex molecular-level interactions. Our study presents quantitative insights on domain and residue contributions to allosteric communication, immune evasion, and local- and global-level control of functions through the derivation of a weighted graph representation from all-atom MD simulations. Focusing on the ancestral form and the D614G-variant, we provide evidence of the utility of our approach by guiding the selection of a mutation that alters the Spike's stability. Taken together, the network approach serves as a valuable tool to evaluate communication "hot-spots" in proteins to guide design of stable immunogens.

SUMMARYBreast milk secretory IgA antibodies provide a first line of defense against enteric infections. Despite this and an effective vaccine, human rotaviruses (RVs) remain the leading cause of severe infectious diarrhea in children in low- and middle-income countries (LMIC) where vaccine efficacy is lower than that of developed nations. Therapeutic strategies that deliver potently neutralizing antibodies into milk could provide protection against enteric pathogens such as RVs. We developed a murine model of maternal protective-transfer using systemic administration of a dimeric IgA (dIgA) monoclonal antibody. We confirmed that systemically-administered dIgA passively transferred into milk and stomach of suckling pups in a dose-dependent manner. We then demonstrated that systemic administration of an engineered potent RV-neutralizing dIgA (mAb41) in lactating dams protected suckling pups from RV-induced diarrhea. This maternal protective-transfer immunization platform could be an effective strategy to improve infant mortality against enteric infections, particularly in LMIC with high rates of breastfeeding.GRAPHICAL ABSTRACT

SummaryNew SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes.HighlightsCryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion.Adaptation to mink resulted in increased ACE2 binding and spike destabilization.B.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding.E484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation.S protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning.

SUMMARYBetacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

SummarySARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infectionin vitro, while five non-neutralizing NTD antibodies mediated FcγR-independentin vitroinfection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, whilein vitroantibody-enhanced infection does not necessarily herald enhanced infectionin vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.

The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage altered the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design.HighlightsSARS-CoV-2 S ectodomains with or without the K986P, V987P mutations have similar structures, antigenicity and stability.The D614G mutation alters S protein conformational dynamics.D614G enhances protease cleavage susceptibility at the S protein furin cleavage site.Cryo-EM structures reveal allosteric effect of changes at the S1/S2 junction on RBD dynamics.

Understanding and eliciting protective immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent priority. To facilitate these objectives, we profile the repertoire of human leukocyte antigen class II (HLA-II)-bound peptides presented by HLA-DR diverse monocyte-derived dendritic cells pulsed with SARS-CoV-2 spike (S) protein. We identify 209 unique HLA-II-bound peptide sequences, many forming nested sets, which map to sites throughout S including glycosylated regions. Comparison of the glycosylation profile of the S protein to that of the HLA-II-bound S peptides reveals substantial trimming of glycan residues on the latter, likely induced during antigen processing. Our data also highlight the receptor-binding motif in S1 as a HLA-DR-binding peptide-rich region and identify S2-derived peptides with potential for targeting by cross-protective vaccine-elicited responses. Results from this study will aid analysis of CD4+ T cell responses in infected individuals and vaccine recipients and have application in next-generation vaccine design., Graphical Abstract, Parker et al map the HLA-II-bound peptides and glycopeptides presented by SARS-Cov-2 spike protein-pulsed monocyte-derived dendritic cells. They observe that complex glycans on the spike immunogen are trimmed during antigen processing, revealing a signature for HLA-II presentation, and highlight congruence between the HLA-II-bound peptides identified and T cell epitopes.

The glycan shield of the beta-coronavirus (β-CoV) Spike (S) glycoprotein provides protection from host immune responses, acting as a steric block to potentially neutralizing antibody responses. The conformationally dynamic S-protein is the primary immunogenic target of vaccine design owing to its role in host-cell fusion, displaying multiple receptor binding domain (RBD) ‘up’ and ‘down’ state configurations. Here, we investigated the potential for RBD adjacent, N-terminal domain (NTD) glycans to influence the conformational equilibrium of these RBD states. Using a combination of antigenic screens and high-resolution cryo-EM structure determination, we show that an N-glycan deletion at position 234 results in a dramatically reduced population of the ‘up’ state RBD position. Conversely, glycan deletion at position N165 results in a discernable increase in ‘up’ state RBDs. This indicates the glycan shield acts not only as a passive hinderance to antibody meditated immunity but also as a conformational control element. Together, our results demonstrate this highly dynamic conformational machine is responsive to glycan modification with implications in viral escape and vaccine design.

SummaryThe HIV-1 envelope (Env) is comprised by mass of over 50% glycans. A goal of HIV-1 vaccine development is the induction of Env glycan-reactive broadly neutralizing antibodies (bnAbs). The 2G12 bnAb recognizes an Env glycan cluster using a unique variable heavy (VH) domain-swapped conformation that results in fragment antigen-binding (Fab) dimerization. Here we describe Fab-dimerized glycan (FDG)-reactive antibodies without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques that neutralized heterologous HIV-1 isolates. FDG precursors were boosted by vaccination in macaques, and were present in HIV-1-naïve humans with an average estimated frequency of one per 340,000 B cells. These data demonstrate frequent HIV-1 Env glycan-reactive bnAb B cell precursors in macaques and humans and reveal a novel strategy for their induction by vaccination.HighlightsDiscovery of Fab-dimerized HIV-1 glycan-reactive antibodies with a non-domain-swapped architectureFab-dimerized antibodies neutralize heterologous HIV-1 isolates.Antibodies with this architecture can be elicited by vaccination in macaques.Fab-dimerized antibodies are found in HIV-1 naïve humans.

The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single−molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env. Structural analysis by single−particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins shows rearrangements in the gp120 topological layer contacts with gp41. Displacement of a conserved tryptophan (W571) from its typical pocket in these Env mutants renders the Env insensitive to CD4 binding. These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design., Here, using cryo-EM and smFRET, Henderson et al. show how tryptophan 571 in the HIV-1 Env acts as a conformational switch during receptor-mediated viral entry and design HIV-1 Env proteins that cannot undergo conformational changes. This has important implications for HIV-1 vaccine design.

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ~3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

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.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques., Graphical abstract, Convalescent human-derived SARS-CoV-2 RBD and NTD antibodies mediated neutralization as well as infection enhancement in vitro, yet infusion of these antibodies in mice or cynomolgus macaques resulted in suppression of virus replication.

BackgroundThe DHVI Division of Structural Biology seeks to use atomic level structural information for design of an effective HIV-1 vaccine. Through visualization of the HIV-1 envelope (Env) and its interactions with the human immune system, we obtain structural information that we translate into the rational development vaccine immunogensMethodsWe use negative stain electron microscopy (NSEM), cryo-electron microscopy (cryo-EM), and x-ray crystallography as the major structural techniques for visualization of HIV-1 Env, and combine these with biochemical and biophysical studies, as well as computational methods to obtain a basic understanding of the functions and interactions of the HIV-1 Env.Results:1.The DHVI NSEM pipeline runs on a daily basis to quality control vaccine immunogens for animal studies and other applications. Offering rapid sample turnover and economical operations, the NSEM pipeline is the most widely utilized resource of the DHVI Division of Structural Biology. Over the last year, the NSEM team has focused efforts on improving operational speed and data processing allowing high-quality visualization of a large variety of samples including HIV-1 Env immunogens, antibodies, nanoparticles, and VLPs. In the last year we have also expanded our NSEM studies to the analyses of serum samples and mucosal fluids.2.To understand the mechanism of HIV-1 entry we have determined structures of HIV-1 entry intermediates. We have determined a 3.8 Å resolution structure of a single CD4 bound to a closed HIV-1 Env trimer revealing new contacts of CD4 with Env. We have also structurally characterized an Env designed to prevent CD4-induced rearrangements by targeted disruption of an allosteric network modulating Env conformational changes.3.We have structurally characterized the HIV-1 glycan-V3 targeting DH270 Broadly Neutralizing Antibody Lineage. The structures revealed movements in the V1 loop and interactive glycans, shifts in antibody orientations, antibody VH-VL orientations, and antibody elbow angles, as the lineage progressed to maturation.4.We have solved a structure in complex with the HIV-1 Env immunogen Man5-enriched CH505.N279K.G458Y.SOSIP.664 of the unmutated common ancestor (UCA) of the HIV-1 CD4-binding site targeting CH235 Broadly Neutralizing Antibody Lineage. The structure revealed interactions of the N279K and G458Y mutations with the CDR L3 loop of CH235 UCA thus providing a structural understanding of the role of these mutations in facilitating binding to the CH235 UCA. (see also Henderson et al abstract)5.Using NSEM and cryo-EM we have characterized the structural properties of a novel class of 2G12-mimetic, yet non domain-swapped Fab dimer glycan-reactive (FDG) antibodies. These studies showed that the Fab-dimerized 2G12-like motif is more common than previously thought, and that creation of a Fab-dimerized paratope for an HIV-1 neutralizing antibody does not require VH domain-swapping.6.Finally, the structural team is an integral part of the CHAVD Kalma Immunogen Design Team, wherein we are defining the structural basis of bnAb affinity maturation to guide sequential immunogen design.Conclusions:These results highlight the power of structural information on HIV-1 vaccine design, from leveraging a basic understanding of HIV-1 entry mechanism for immunogen design, to rapid visualization of Env immunogens by NSEM for quality control, discovery of novel antibody interactions, and atomic level visualization of antibody/Env interactions.

INTRODUCTION A major goal of HIV-1 vaccine development is the design of immunogens that induce broadly neutralizing antibodies (bnAbs). However, vaccination of humans has not resulted in the induction of affinity-matured and potent HIV-1 bnAbs. To devise effective vaccine strategies, we previously determined the maturation pathway of select HIV-1 bnAbs from acute infection through neutralizing antibody development. During their evolution, bnAbs acquire an abundance of improbable amino acid substitutions as a result of nucleotide mutations at variable region sequences rarely targeted by activation-induced cytidine deaminase, the enzyme responsible for antibody mutation. A subset of improbable mutations is essential for broad neutralization activity, and their acquisition represents a key roadblock to bnAb development. RATIONALE Current bnAb lineage–based vaccine strategies can initiate bnAb lineage development in animal models but have not specifically elicited the improbable mutations required for neutralization breadth. Induction of bnAbs requires vaccine strategies that specifically engage bnAb precursors and subsequently select for improbable mutations required for broadly neutralizing activity. We hypothesized that vaccination with immunogens that bind with moderate to high affinity to bnAb B cell precursors, and with higher affinity to precursors that have acquired improbable mutations, could initiate bnAb B cell lineages and select for key improbable mutations required for bnAb development. RESULTS We elicited serum neutralizing HIV-1 antibodies in human bnAb precursor knock-in mice and wild-type macaques vaccinated with immunogens designed to select for improbable mutations. We designed two HIV-1 envelope immunogens that bound precursor B cells of either a CD4 binding site or V3-glycan bnAb lineage. In vitro, these immunogens bound more strongly to bnAb precursors once the precursor acquired the desired improbable mutations. Vaccination of macaques with the CD4 binding site–targeting immunogen induced CD4 binding site serum neutralizing antibodies. Antibody sequences elicited in human bnAb precursor knock-in mice encoded functional improbable mutations critical for bnAb development. In bnAb precursor knock-in mice, we isolated a vaccine-elicited monoclonal antibody bearing functional improbable mutations that was capable of neutralizing multiple HIV-1 global isolates. Structures of a bnAb precursor, a bnAb, and the vaccine-elicited antibody revealed the precise roles that acquired improbable mutations played in recognizing the HIV-1 envelope. Thus, our immunogens elicited antibody responses in macaques and knock-in mice that exhibited the mutational patterns, structural characteristics, or neutralization profiles of nascent broadly neutralizing antibodies. CONCLUSION Our study represents a proof of concept for targeted selection of improbable mutations to guide antibody affinity maturation. Moreover, this study demonstrates a rational strategy for sequential immunogen design to circumvent the difficult roadblocks in HIV-1 bnAb induction by vaccination. We show that immunogens should exhibit differences in affinity across antibody maturation stages where improbable mutations are necessary for the desired antibody function. This strategy of selection of specific antibody nucleotides by immunogen design can be applied to B cell lineages targeting other pathogens where guided affinity maturation is needed for a protective antibody response.

Parainfluenza virus types 1–4 (PIV1–4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1–4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

An antibody to block viral fusion A small fraction of HIV-1–infected individuals develop broad and potent antibodies that bind the HIV-1 envelope protein (Env). These antibodies recognize a limited set of conserved epitopes on Env, such as Env's host receptor-binding site. Kong et al. now report a neutralizing antibody isolated from an HIV-1–infected individual that binds to the fusion peptide of Env. This is unexpected because viruses often try to mask such key components of their cell entry machinery from antibody attack. Crystal structures of the antibody bound to the fusion peptide and to Env itself define the epitope, provide insight into the specific mechanism of antibody binding, and may inform HIV-1 vaccine design. Science , this issue p. 828

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.

Over the past decade, structures have been determined for broadly neutralizing antibodies that recognize all major exposed surfaces of the prefusion-closed HIV-1-envelope (Env) trimer. To understand this recognition and its implications, we analyzed 206 antibody-HIV-1 Env structures from the Protein Data Bank with resolution suitable to define interaction chemistries and measured antibody neutralization on a 208-strain panel. Those with25% breadth segregated into almost two dozen classes based on ontogeny and recognition and into six epitope categories based on recognized Env residues. For paratope, the number of protruding loops and level of somatic hypermutation were significantly higher for broad HIV-1 neutralizing antibodies than for a comparison set of non-HIV-1 antibodies (p 0.0001). For epitope, the number of independent sequence segments was higher (p 0.0001), as well as the glycan component surface area (p = 0.0005). The unusual characteristics of epitope and paratope delineated here are likely to reflect respectively virus-immune evasion and antibody-recognition solutions that allow effective neutralization of HIV-1.

Antigen multimerization on a nanoparticle can result in improved neutralizing antibody responses. A platform that has been successfully used for displaying antigens from a number of different viruses is ferritin, a self-assembling protein nanoparticle that allows the attachment of multiple copies (24 monomers or 8 trimers) of a single antigen. Here, we design two-component ferritin variants that allow the attachment of two different antigens on a single particle in a defined ratio and geometric pattern. The two-component ferritin was specifically designed for trimeric antigens, accepting four trimers per particle for each antigen, and was tested with antigens derived from HIV-1 envelope (Env) and influenza hemagglutinin (HA). Particle formation and the presence of native-like antigen conformation were confirmed through negative-stain electron microscopy and antibody-antigen binding analysis. Immunizations in guinea pigs with two-component ferritin particles, displaying diverse Env, HA, or both antigens, elicited neutralizing antibody responses against the respective viruses. The results provide proof-of-principle for the self-assembly of a two-component nanoparticle as a general technology for multimeric presentation of trimeric antigens.

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

The HIV-1 envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and coreceptor-bound conformations by transitioning into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1 Env trimer to its prefusion-closed state as this state is recognized by most broadly neutralizing, but not nonneutralizing, antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env trimer mimic developed by Sanders, Moore, and colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-Å resolution and of an additional DS-SOSIP.6mut variant at 4.3-Å resolution, and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier 2 autologous titers versus tier 1 V3-sensitive titers than BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggest utility as an immunogen or a serologic probe; moreover, the specific four alterations identified here, M154, M300, M302, and L320 (4mut), can also be transferred to other HIV-1 Env trimers of interest to improve their properties. IMPORTANCE One approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the structurally flexible HIV-1 envelope (Env) trimer in a conformation that displays predominantly broadly neutralizing epitopes and few to no nonneutralizing epitopes. The prefusion-closed conformation of HIV-1 Env has been identified as one such preferred conformation, and a current leading vaccine candidate is the BG505 DS-SOSIP variant, comprising two disulfides and an Ile-to-Pro mutation of Env from strain BG505. Here, we introduced additional mutations to further stabilize BG505 DS-SOSIP in the vaccine-preferred prefusion-closed conformation. In guinea pigs, our best mutant, DS-SOSIP.4mut, elicited a significantly higher ratio of autologous versus V3-directed neutralizing antibody responses than the SOSIP-stabilized form. We also observed an improvement in thermostability and a reduction in CD4 affinity. With improved antigenicity, stability, and immunogenicity, DS-SOSIP.4mut-stabilized trimers may have utility as HIV-1 immunogens or in other antigen-specific contexts, such as with B-cell probes.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Delineation of antibody epitopes at the residue level is key to understanding antigen resistance mutations, designing epitope-specific probes for antibody isolation, and developing epitope-based vaccines. Ideally, epitope residues are determined in the context of the atomic-level structure of the antibody-antigen complex, though structure determination may in many cases be impractical. Here we describe an efficient computational method to predict antibody-specific HIV-1 envelope (Env) epitopes at the residue level, based on neutralization panels of diverse viral strains. The method primarily utilizes neutralization potency data over a set of diverse viral strains representing the antigen, and enhanced accuracy could be achieved by incorporating information from the unbound structure of the antigen. The method was evaluated on 19 HIV-1 Env antibodies with neutralization panels comprising 181 diverse viral strains and with available antibody-antigen complex structures. Prediction accuracy was shown to improve significantly over random selection, with an average of greater-than-8-fold enrichment of true positives at the 0.05 false-positive rate level. The method was used to prospectively predict epitope residues for two HIV-1 antibodies, 8ANC131 and 8ANC195, for which we experimentally validated the predictions. The method is inherently applicable to antigens that exhibit sequence diversity, and its accuracy was found to correlate inversely with sequence conservation of the epitope. Together the results show how knowledge inherent to a neutralization panel and unbound antigen structure can be utilized for residue-level prediction of antibody epitopes.