Biology subjects

Abernathy, M. E.

Publications and source records attributed to Abernathy, M. E..

10 recordsLinked to original sources

bioRxiv2026

Structural basis of diverse antibody recognition of conserved coronavirus spike S2 epitopes that contribute to protective immunity

Conserved epitopes within the coronavirus spike S2 domain elicit broadly reactive antibodies, yet many characterized responses show limited neutralizing and variable protective activity, leaving their contribution to antiviral immunity unclear. Building on our previous mapping of evolutionarily conserved spike coldspots, we isolated human monoclonal antibodies targeting four conserved epitopes in the spike S2 domain: the internal fusion peptide (iFP), the central helix (CH), the connector domain (CD), and a membrane-proximal epitope in the heptad repeat 2 that we term the lower stalk (LS). A crystal structure of an LS-directed antibody defined a previously unresolved mode of antibody recognition of this membrane-proximal epitope, while cryogenic electron microscopy (cryo-EM) structures revealed that genetically diverse CH-specific antibodies use distinct binding modes to converge on conserved features of the prefusion S2 apex. Despite minimal neutralizing activity, CH- and LS-directed antibodies exhibited distinct antiviral functions. LS-directed antibodies mediated Fc{gamma} receptor-dependent effector activity in vitro, whereas the broadly reactive CH-directed antibody ch.007 lacked detectable antibody-dependent cellular cytotoxicity (ADCC) or cellular phagocytosis (ADCP) activity yet protected mice from lethal SARS-CoV-2 MA10 challenge, with protection abrogated by Fc{gamma} receptor-silencing mutations. Together, these findings expand the genetic, structural and functional landscape of human antibody responses to conserved coronavirus S2 epitopes and demonstrate that CH-directed antibodies can contribute to protective immunity through Fc-dependent mechanisms not predicted by in vitro neutralization or conventional in vitro Fc effector assays.

biophysics↗

bioRxiv2026

Structural and mutational analyses define distinct molecular routes to broad SARS-CoV-2 receptor-binding domain recognition

Broadly reactive antibodies elicited by SARS-CoV-2 infection or vaccination can reveal conserved viral vulnerabilities and inform vaccines with broad coronavirus coverage. Here, we characterize two human-derived monoclonal antibodies, B2014 and C5078, that recognize conserved epitopes on the SARS-CoV-2 RBD and retain activity across antigenically distinct variants. Notably, C5078 also recognizes diverse sarbecoviruses and remains active against currently circulating variants, including XFG and NB.1.8.1. Cryo-EM structures reveal that B2014 recognizes an epitope adjacent to the class 3 antibody site, whereas C5078 targets the highly conserved, cryptic site V epitope. Structural analysis defines how C5078 uses affinity-matured interactions to engage conserved RBD residues, providing a molecular basis for its exceptional breadth. Deep mutational scanning across multiple SARS-CoV-2 variant backgrounds further defines potential pathways of antibody escape, explaining the loss of B2014 activity against antigenically evolved variants while revealing a high barrier to escape from C5078. Together, these findings define distinct structural solutions for broad RBD recognition and highlight conserved, mutationally constrained epitopes that may serve as targets for vaccines designed to elicit antibody responses resilient to ongoing SARS-CoV-2 evolution and future sarbecovirus emergence.

biophysics↗

bioRxiv2026

Dissociation kinetics and avidity gate SARS-CoV-2 neutralization by HR2 stem helix antibodies

SARS-CoV-2 evolution has reduced the efficacy of clinical monoclonal antibodies, underscoring the need for therapeutics targeting conserved viral regions. The Spike (S) heptad repeat 2 (HR2) stem helix is highly conserved across SARS-CoV-2 variants and related betacoronaviruses. Although antibodies to this region can neutralize infection, their natural occurrence and evolution remain poorly understood. We previously identified human neutralizing antibodies to a conserved peptide within this region (HR2 coldspot). Here, we show that plasma IgG reactivity to this region remains rare, even after repeated antigen exposure. Longitudinal analysis over 30 months revealed continued somatic hypermutation of HR2-specific antibodies, yet none surpassed the potency or breadth of hr2.016, which emerged shortly after primary infection. Crystal structures of four HR2 stem helix antibodies revealed convergent recognition across distinct antibody lineages. Comparison of hr2.016 with its non-neutralizing clonal relative hr2.086 showed that structural convergence masks distinct binding kinetics. Surface plasmon resonance and molecular dynamics simulations revealed a more stable interaction network for hr2.016, with slower dissociation and prolonged S residence time. Neutralization required the IgG format, supporting an avidity-driven mechanism. Together, these findings define kinetic and avidity constraints governing neutralization at the HR2 stem helix and position hr2.016 as a resilient therapeutic candidate.

immunology↗

bioRxiv2024

Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern

The ongoing emergence of SARS-CoV-2 variants of concern (VOCs) that reduce the effectiveness of antibody therapeutics necessitates development of next-generation antibody modalities that are resilient to viral evolution. Here, we characterized N-terminal domain (NTD) and receptor binding domain (RBD)-specific monoclonal antibodies previously isolated from COVID-19 convalescent donors for their activity against emergent SARS-CoV-2 VOCs. Among these, the NTD-specific antibody C1596 displayed the greatest breadth of binding to VOCs, with cryo-EM structural analysis revealing recognition of a distinct NTD epitope outside of the site i antigenic supersite. Given C1596s favorable binding profile, we designed a series of bispecific antibodies (bsAbs) termed CoV2-biRNs, that featured both NTD and RBD specificities. Notably, two of the C1596-inclusive bsAbs, CoV2-biRN5 and CoV2-biRN7, retained potent in vitro neutralization activity against all Omicron variants tested, including XBB.1.5, EG.5.1, and BA.2.86, contrasting the diminished potency of parental antibodies delivered as monotherapies or as a cocktail. Furthermore, prophylactic delivery of CoV2-biRN5 significantly reduced the viral load within the lungs of K18-hACE2 mice following challenge with SARS-CoV-2 XBB.1.5. In conclusion, our NTD-RBD bsAbs offer promising potential for the design of resilient, next-generation antibody therapeutics against SARS-CoV-2 VOCs. One Sentence SummaryBispecific antibodies with a highly cross-reactive NTD antibody demonstrate resilience to SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2022

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2 site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants. One sentence summaryBroadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

immunology↗

bioRxiv2022

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

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4-binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Envs N276gp120 N-glycan, and rare 5-residue light chain complementarity determining region 3s (CDRL3s). As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in animals containing polyclonal antibody repertoires. Thus, germline-targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

immunology↗

bioRxiv2022

Human anti-ACE2 monoclonal antibodies as pan-sarbecovirus prophylactic agents

Human monoclonal antibodies from convalescent individuals that target the SARS-CoV-2 spike protein have been deployed as therapeutics against SARS-CoV-2. However, nearly all of these antibodies have been rendered obsolete by SARS-CoV-2 variants that evolved to resist similar, naturally occurring antibodies. Here, we describe the development of human monoclonal antibodies that bind the ACE2 receptor rather than the viral spike protein. These antibodies block infection by all ACE2 binding sarbecoviruses, including emergent SARS-CoV-2 variants. Structural and biochemical analyses revealed that the antibodies target an ACE2 epitope that engages SARS-CoV-2 spike. Importantly, the antibodies do not inhibit ACE2 enzymatic activity, nor do they induce ACE depletion from cell surfaces. The antibodies exhibit favorable pharmacology and protect human ACE2 knock-in mice against SARS-CoV-2 infection. Such antibodies should be useful prophylactic and treatment agents against any current and future SARS-CoV-2 variants, as well as ACE2-binding sarbecoviruses that might emerge as future pandemic threats.

microbiology↗

bioRxiv2021

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

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

immunology↗

bioRxiv2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease 2019 (COVID-19) including two novel mRNA-based vaccines1,2. These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known3-6. Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S) and receptor binding domain (RBD) binding titers3,5,6. Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection7,8. However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.

immunology↗

bioRxiv2020

Structural classification of neutralizing antibodies against the SARS-CoV-2 spike receptor-binding domain suggests vaccine and therapeutic strategies

The COVID-19 pandemic presents an urgent health crisis. Human neutralizing antibodies (hNAbs) that target the host ACE2 receptor-binding domain (RBD) of the SARS-CoV-2 spike1-5 show therapeutic promise and are being evaluated clincally6-8. To determine structural correlates of SARS-CoV-2 neutralization, we solved 8 new structures of distinct COVID-19 hNAbs5 in complex with SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed classification into categories: (1) VH3-53 hNAbs with short CDRH3s that block ACE2 and bind only to "up" RBDs, (2) ACE2-blocking hNAbs that bind both "up" and "down" RBDs and can contact adjacent RBDs, (3) hNAbs that bind outside the ACE2 site and recognize "up" and "down" RBDs, and (4) Previously-described antibodies that do not block ACE2 and bind only "up" RBDs9. Class 2 comprised four hNAbs whose epitopes bridged RBDs, including a VH3-53 hNAb that used a long CDRH3 with a hydrophobic tip to bridge between adjacent "down" RBDs, thereby locking spike into a closed conformation. Epitope/paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally-occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects, suggesting combinations for clinical use, and providing insight into immune responses against SARS-CoV-2.

molecular biology↗