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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 authorization 1 – 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 antibodies 4 . 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.

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 (R 2  = 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.

Previous studies have linked the evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic variants to persistent infections in people with immunocompromising conditions, but the processes responsible for these observations are incompletely understood. Here we use high-throughput, single-genome amplification and sequencing (HT-SGS) to sequence SARS-CoV-2 spike genes from people with HIV (PWH, n  = 22) and people without HIV (PWOH, n  = 25). In PWOH and PWH with CD4 T cell counts (i.e., CD4 counts) ≥ 200 cells/μL, we find that most SARS-CoV-2 genomes sampled in each person share one spike sequence. By contrast, in people with advanced HIV infection (i.e., CD4 counts < 200 cells/μL), HT-SGS reveals a median of 46 distinct linked groupings of spike mutations per person. Elevated intra-host spike diversity in people with advanced HIV infection is detected immediately after COVID-19 symptom onset, and early intra-host spike diversity predicts SARS-CoV-2 shedding duration among PWH. Analysis of longitudinal timepoints reveals rapid fluctuations in spike sequence populations, replacement of founder sequences by groups of new haplotypes, and positive selection at functionally important residues. These findings demonstrate remarkable intra-host genetic diversity of SARS-CoV-2 in advanced HIV infection and suggest that adaptive intra-host SARS-CoV-2 evolution in this setting may contribute to the emergence of new variants of concern. High-throughput, single-copy sequencing of SARS-CoV-2 spike in a longitudinal cohort of people with and without HIV infection demonstrates striking intra-host diversity and adaptive evolution of SARS-CoV-2 in people with advanced HIV infection.

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

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

Background/Objectives: Vaccine elicitation of antibodies with high HIV-1 neutralization breadth is a long-standing goal. Recently, the induction of such antibodies has been achieved at the fusion peptide site of vulnerability. Questions remain, however, as to how much anti-fusion peptide antibodies can be improved and whether their neutralization breadth and potency are sufficient to prevent HIV-1 infection. Methods: Here, we use yeast display coupled with deep mutational screening and biochemical and structural analyses to study the improvement of the best fusion peptide-directed, vaccine-elicited antibody, DFPH_a.01, with an initial 59% breadth. Results: Yeast display identified both single and double mutations that improved recognition of HIV-1 envelope trimers. We characterized two paratope-distal light chain (LC) mutations, S10R and S59P, which together increased breadth to 63%. Biochemical analysis demonstrated DFPH-a.01_10R59P-LC, and its component mutations, to have increased affinity and stability. Cryo-EM structural analysis revealed elbow-angle influencing by S10R-LC and isosteric positioning by S59P-LC as explanations for enhanced breadth, affinity, and stability. Conclusions: These results, along with another antibody with enhanced performance (DFPH-a.01_1G10A56K-LC with 64% breadth), suggest that mutations improving DFPH_a.01 are plentiful, an important vaccine insight.

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 μg, 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.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a primary target of neutralizing antibodies and a key component of licensed vaccines. Substantial mutations in RBD, however, enable current variants to escape immunogenicity generated by vaccination with the ancestral (WA1) strain. Here, we produce and assess self-assembling nanoparticles displaying RBDs from WA1 and BA.5 strains by using the SpyTag:SpyCatcher system for coupling. We observed both WA1- and BA.5-RBD nanoparticles to degrade substantially after a few days at 37 °C. Incorporation of nine RBD-stabilizing mutations, however, increased yield ~five-fold and stability such that more than 50% of either the WA1- or BA.5-RBD nanoparticle was retained after one week at 37 °C. Murine immunizations revealed that the stabilized RBD-nanoparticles induced ~100-fold higher autologous neutralization titers than the prefusion-stabilized (S2P) spike at a 2 μg dose. Even at a 25-fold lower dose where S2P-induced neutralization titers were below the detection limit, the stabilized BA.5-RBD nanoparticle induced homologous titers of 12,795 ID50 and heterologous titers against WA1 of 1767 ID50. Assessment against a panel of β-coronavirus variants revealed both the stabilized BA.5-RBD nanoparticle and the stabilized WA1-BA.5-(mosaic)-RBD nanoparticle to elicit much higher neutralization breadth than the stabilized WA1-RBD nanoparticle. The extraordinary titer and high neutralization breadth elicited by stabilized RBD nanoparticles from strain BA.5 make them strong candidates for next-generation COVID-19 vaccines.

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

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-Å 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.