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Antibody-Framework-to-Antigen Distance (AFAD) – the distance between the body of an antibody and a protein antigen – is an important parameter governing antibody recognition. Here, we quantify AFAD for ~2,000 non-redundant antibody-protein-antigen complexes in the Protein Data Bank. AFADs showed a gaussian distribution with mean of 16.3 Å and standard deviation (σ) of 2.4 Å. Notably, antibody-antigen complexes with extended AFADs (>3σ) were exclusively human immunodeficiency virus-type 1 (HIV-1)-neutralizing antibodies. High correlation (R2 = 0.8110) was observed between AFADs and glycan coverage, as assessed by molecular dynamics simulations of the HIV-1-envelope trimer. Especially long AFADs were observed for antibodies targeting the glycosylated trimer apex, and we tested the impact of introducing an apex-glycan hole (N160K); the cryo-EM structure of the glycan hole-targeting HIV-1-neutralizing antibody 2909 in complex with an N160K-envelope trimer revealed a substantially shorter AFAD. Overall, extended AFADs exclusively recognized densely glycosylated surfaces, with the introduction of a glycan hole enabling closer recognition., Here, the authors analyse the distance between the body of an antibody and a protein antigen denoted as the Antibody-Framework-to-Antigen Distance (AFAD) for about 2000 non-redundant antibody-protein antigen complexes in the Protein Data Bank. They observe that antibodies with exceptionally long AFADs were all broad HIV-1-neutralizing antibodies that targeted densely glycosylated regions on the HIV-1-envelope trimer. The connection between long AFAD and dense glycan was further validated by the cryo-EM structure of antibody 2909 recognizing a glycan hole and by glycan shielding analyses based on molecular dynamics simulations.

Snake envenomation is a neglected tropical disease, causing >100,000 deaths and 300,000 permanent disabilities in humans annually. Could monoclonal antibody technology provide a solution? Here, we recover Centi-3FTX-D09, a potent broadly neutralizing antivenom antibody from the B-cell memory of a human subject with snake venom exposure. Centi-3FTX-D09 recognized a conserved neutralizing epitope on long 3-finger toxins (3FTXs), a dominant snake neurotoxin. Crystal structures of Centi-3FTX-D09 in complex with 3FTXs from mamba, taipan, krait, and cobra revealed epitope mimicry of the interface between these neurotoxins and their host target, the nicotinic acetylcholine receptor. Centi-3FTX-D09 providedin-vivoprotection against diverse recombinant long 3FTXs,in-vivorescue from whole venom challenge from cobras, black mamba, and king cobra, and, when combined with the phospholipase inhibitor varespladib,in-vivoprotection extending to a majority of tested elapid venoms. Thus, a single antibody can broadly neutralize long neurotoxins and contribute to broad protection from envenomation.

Lectins, carbohydrate-binding proteins of nonimmune origin, bind to carbohydrates and glycan shields present on the surfaces of cells and viral spike proteins. Lectins thus hold great promise as therapeutic and diagnostic proteins, exemplified by their potent antiviral activities and the desire to engineer synthetic carbohydrate receptors based on lectin recognition principles. Here, we describe a new carbohydrate-binding architectural motif—namely, a C(3)-symmetric tyrosine-based aromatic core, present in the therapeutic lectin griffithsin (GRFT). By using structure-based amino acid substitutions, X-ray crystallography, molecular dynamics (MD) simulations, and HIV-1 neutralization assays, we show that this core is critical for potent (pM) antiviral activity and nanomolar binding to the glycan shield largely consisting of high mannose glycans. Crystal structures and MD simulations show that CH–π interactions stabilize the aromatic cluster to maintain the three pseudo-symmetric carbohydrate-binding sites, nonaromatic amino acid substitutions (Tyr to Ala) abrogate antiviral activity, and increasing the aromatic CH–π edge-to-centroid interface via a Tyr to Trp substitution yields a GRFT variant with improved potency and increased residence time of Man-9 observed in MD simulations. NMR titrations of a Tyr-to-Ala variant indicate that disruption of the aromatic prevents the intermolecular crosslinking between two equivalents of Man-9 and one carbohydrate-binding face observed in wild-type GRFT and known to be critical for picomolar potency of this lectin. This C(3)-symmetric aromatic core defines a new recognition motif for the design of carbohydrate receptors and suggests principles for engineering known lectins to have increased affinity and stability.

Lassa virus (LASV) is responsible for 100,000-300,000 zoonotic infections annually and poses a threat to public health. Development of antibody-based therapeutics or vaccines has been challenging because neutralizing antibodies – even among Lassa hemorrhagic fever survivors – are generally of low titer, and the target of neutralizing antibodies, the trimeric glycoprotein complex (GPC), a type 1-fusion machine with GP1 and GP2 subunits, has been difficult to produce. Here, we use structure-based design to obtain a soluble LASV GPC by engineering an inter-protomer disulfide (R207GCGP1-L326CGP2) and appending the T4-fibritin trimerization domain. We verified the antigenicity of this prefusion-stabilized LASV GPC against a panel of human antibodies and used electron microscopy (EM) to confirm its trimeric association. We panned the prefusion-stabilized LASV GPC against single domain ‘nanobody’ libraries and identified one of camel origin, which we named D5, which bound GPC with 27 nM affinity and neutralized the Josiah strain of LASV with an IC50 of 12 µg/ml when formatted into a bivalent IgG2a context. The cryo-EM structure of a ternary complex of the D5 nanobody, the antigen-binding fragment of human antibody 8.11G, and LASV GPC revealed D5 to recognize a site-of-vulnerability at the trimer apex. The recognized site appeared to be specific to GPC lacking cleavage of between GP1 and GP2 subunits. Collectively, our findings suggest that GPC-cleavage intermediates may be targets for LASV neutralization and define an apex-situated site of vulnerability for vaccine development.SignificanceLassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a biothreat to the world. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into requirements for antibody-mediated neutralization of LASV, we developed a prefusion-stabilized LASV glycoprotein trimer (GPC), panned it against phage libraries comprised single-domain antibodies or nanobodies from shark and camel, and identified one, D5, which – when placed into bivalent IgG2a context – could neutralize LASV. Cryo-EM analysis revealed D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. We propose this apex-situated site to be an attractive target for LASV vaccine and therapeutic development.

Simian immunodeficiency viruses (SIVs) are lentiviruses that naturally infect non-human primates of African origin and seeded cross-species transmissions of HIV-1 and HIV-2. Here we report prefusion stabilization and cryo-EM structures of soluble envelope (Env) trimers from rhesus macaque SIV (SIV

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1–3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD–ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and—to our knowledge—rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised., Multivalent nanobodies against SARS-CoV-2 from mice engineered to produce camelid nanobodies recognize conserved epitopes that are inaccessible to human antibodies and show promise as a strategy for dealing with viral escape mutations.

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization1–3. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies4. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD–ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and—to our knowledge—rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised. Multivalent nanobodies against SARS-CoV-2 from mice engineered to produce camelid nanobodies recognize conserved epitopes that are inaccessible to human antibodies and show promise as a strategy for dealing with viral escape mutations.

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 &mu, g, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.

The HIV-1 envelope (Env) spike is a conformational machine that transitions between prefusion (closed, CD4- and CCR5-bound) and postfusion states to facilitate HIV-1 entry into cells. Although the prefusion closed conformation is a potential target for inhibition, development of small-molecule leads has been stymied by difficulties in obtaining structural information. Here, we report crystal structures at 3.8-Å resolution of an HIV-1-Env trimer with BMS-378806 and a derivative BMS-626529 for which a prodrug version is currently in Phase III clinical trials. Both lead candidates recognized an induced binding pocket that was mostly excluded from solvent and comprised of Env elements from a conserved helix and the β20-21 hairpin. In both structures, the β20-21 region assumed a conformation distinct from prefusion-closed and CD4-bound states. Together with biophysical and antigenicity characterizations, the structures illuminate the allosteric and competitive mechanisms by which these small-molecule leads inhibit CD4-induced structural changes in Env.

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and—in half the subjects—multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.

Cryo-EM analyses of the initial contact of the HIV-1 Env trimer with the CD4 receptor reveal that CD4 interacts with two gp120 protomers; these quaternary contacts are important for viral infectivity. Binding of the gp120 envelope (Env) glycoprotein to the CD4 receptor is the first step in the HIV-1 infectious cycle. Although the CD4-binding site has been extensively characterized, the initial receptor interaction has been difficult to study because of major CD4-induced structural rearrangements. Here we used cryogenic electron microscopy (cryo-EM) to visualize the initial contact of CD4 with the HIV-1 Env trimer at 6.8-A resolution. A single CD4 molecule is embraced by a quaternary HIV-1–Env surface formed by coalescence of the previously defined CD4-contact region with a second CD4-binding site (CD4-BS2) in the inner domain of a neighboring gp120 protomer. Disruption of CD4-BS2 destabilized CD4-trimer interaction and abrogated HIV-1 infectivity by preventing the acquisition of coreceptor-binding competence. A corresponding reduction in HIV-1 infectivity occurred after the mutation of CD4 residues that interact with CD4-BS2. Our results document the critical role of quaternary interactions in the initial HIV-Env-receptor contact, with implications for treatment and vaccine design.

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ~90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, which encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans amongst known broadly neutralizing antibodies that target the glycan-shielded trimer.

Nat. Struct. Mol. Biol. 24, 370–378 (2017); published online 20 February 2017; corrected after print 27 April 2017 In the version of this article initially published, funding information for B.C. and C.S.P. was missing NIH grant S10 OD019994-01. In addition, there was an incorrect comma in the introduction (after “glycoprotein” in the sentence “Upon binding to the primary cellular receptor, CD4, the external gp120 Env glycoprotein undergoes major conformational changes.