ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines.

Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared with conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for 3 months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.

A naturally arising broad and potent CD4-binding site antibody with low somatic mutation.

The induction of broadly neutralizing antibodies (bNAbs) is a potential strategy for a vaccine against HIV-1. However, most bNAbs exhibit features such as unusually high somatic hypermutation, including insertions and deletions, which make their induction challenging. VRC01-class bNAbs not only exhibit extraordinary breadth and potency but also rank among the most highly somatically mutated bNAbs. Here, we describe a VRC01-class antibody isolated from a viremic controller, BG24, that is much less mutated than most relatives of its class while achieving comparable breadth and potency. A 3.8-Å x-ray crystal structure of a BG24-BG505 Env trimer complex revealed conserved contacts at the gp120 interface characteristic of the VRC01-class Abs, despite lacking common CDR3 sequence motifs. The existence of moderately mutated CD4-binding site (CD4bs) bNAbs such as BG24 provides a simpler blueprint for CD4bs antibody induction by a vaccine, raising the prospect that such an induction might be feasible with a germline-targeting approach.

ESCRT recruitment to mRNA-encoded SARS-CoV-2 spike induces virus-like particles and enhanced antibody responses.

Prime-boost regimens for COVID-19 vaccines elicit poor antibody responses against Omicron-based variants and employ frequent boosters to maintain antibody levels. We present a natural infection-mimicking technology that combines features of mRNA- and protein nanoparticle-based vaccines through encoding self-assembling enveloped virus-like particles (eVLPs). eVLP assembly is achieved by inserting an ESCRT- and ALIX-binding region (EABR) into the SARS-CoV-2 spike cytoplasmic tail, which recruits ESCRT proteins to induce eVLP budding from cells. Purified spike-EABR eVLPs presented densely-arrayed spikes and elicited potent antibody responses in mice. Two immunizations with mRNA-LNP encoding spike-EABR elicited potent CD8+ T-cell responses and superior neutralizing antibody responses against original and variant SARS-CoV-2 compared to conventional spike-encoding mRNA-LNP and purified spike-EABR eVLPs, improving neutralizing titers >10-fold against Omicron-based variants for three months post-boost. Thus, EABR technology enhances potency and breadth of vaccine-induced responses through antigen presentation on cell surfaces and eVLPs, enabling longer-lasting protection against SARS-CoV-2 and other viruses.

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies.

Many anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) neutralizing antibodies target the angiotensin-converting enzyme 2 (ACE2) binding site on viral spike receptor-binding domains (RBDs). Potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly emergent zoonotic sarbecoviruses and variants, but they usually show only weak neutralization potencies. Here, we characterize two class 4 anti-RBD antibodies derived from coronavirus disease 2019 (COVID-19) donors that exhibit breadth and potent neutralization of zoonotic coronaviruses and SARS-CoV-2 variants. C118-RBD and C022-RBD structures reveal orientations that extend from the cryptic epitope to occlude ACE2 binding and CDRH3-RBD main-chain H-bond interactions that extend an RBD ß sheet, thus reducing sensitivity to RBD side-chain changes. A C118-spike trimer structure reveals rotated RBDs that allow access to the cryptic epitope and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.

Affinity maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, and resilience to viral escape mutations.

Antibodies elicited by infection accumulate somatic mutations in germinal centers that can increase affinity for cognate antigens. We analyzed 6 independent groups of clonally related severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) Spike receptor-binding domain (RBD)-specific antibodies from 5 individuals shortly after infection and later in convalescence to determine the impact of maturation over months. In addition to increased affinity and neutralization potency, antibody evolution changed the mutational pathways for the acquisition of viral resistance and restricted neutralization escape options. For some antibodies, maturation imposed a requirement for multiple substitutions to enable escape. For certain antibodies, affinity maturation enabled the neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies.

Many anti-SARS-CoV-2 neutralizing antibodies target the ACE2-binding site on viral spike receptor-binding domains (RBDs). The most potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly-emergent zoonotic sarbecoviruses and variants, but usually show only weak neutralization potencies. We characterized two class 4 anti-RBD antibodies derived from COVID-19 donors that exhibited broad recognition and potent neutralization of zoonotic coronavirus and SARS-CoV-2 variants. C118-RBD and C022-RBD structures revealed CDRH3 mainchain H-bond interactions that extended an RBD ß-sheet, thus reducing sensitivity to RBD sidechain changes, and epitopes that extended from the cryptic epitope to occlude ACE2 binding. A C118-spike trimer structure revealed rotated RBDs to allow cryptic epitope access and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.

B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV.

Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies.

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.

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

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-21-4. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection5,6. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors5-8. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these 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 a potential loss of clinical efficacy.

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 vaccines 1,2 . These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known 3-6 . 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 titers 3,5,6 . Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection 7,8 . 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.

The structures of secretory and dimeric immunoglobulin A.

Secretory (S) Immunoglobulin (Ig) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here, we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a ß-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen-binding fragments (Fabs) and preserves steric accessibility to receptor-binding sites, likely influencing antigen binding and effector functions.

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

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

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.

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

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

Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals.

During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies.

Neutralizing antibody responses to coronaviruses focus on the trimeric spike, with most against the receptor-binding domain (RBD). Here we characterized polyclonal IgGs and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their degree of focus on RBD epitopes, recognition of SARS-CoV, MERS-CoV, and mild coronaviruses, and how avidity effects contributed to increased binding/neutralization of IgGs over Fabs. Electron microscopy reconstructions of polyclonal plasma Fab-spike complexes showed recognition of both S1A and RBD epitopes. A 3.4Å cryo-EM structure of a neutralizing monoclonal Fab-S complex revealed an epitope that blocks ACE2 receptor-binding on "up" RBDs. Modeling suggested that IgGs targeting these sites have different potentials for inter-spike crosslinking on viruses and would not be greatly affected by identified SARS-CoV-2 spike mutations. These studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions.

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.