Search

SummaryDeveloping broad coronavirus vaccines requires identifying and understanding the molecular basis of broadly neutralizing antibody (bnAb) spike sites. In our previous work, we identified sarbecovirus spike RBD group 1 and 2 bnAbs. We have now shown that many of these bnAbs can still neutralize highly mutated SARS-CoV-2 variants, including the XBB.1.5. Structural studies revealed that group 1 bnAbs use recurrent germline encoded CDRH3 features to interact with a conserved RBD region that overlaps with class 4 bnAb site. Group 2 bnAbs recognize a less well-characterized "site V" on the RBD and destabilize spike trimer. The site V has remained largely unchanged in SARS-CoV- 2 variants and is highly conserved across diverse sarbecoviruses, making it a promising target for broad coronavirus vaccine development. Our findings suggest that targeted vaccine strategies may be needed to induce effective B cell responses to escape resistant subdominant spike RBD bnAb sites.

In a phase I clinical trial, the chimeric hemagglutinin (cHA) immunogen induced antibody responses against the conserved HA stalk domain. However, the landscape of the B cell specificities and subsets induced by this vaccine remain undetermined. Here, we paired single cell RNA-sequencing and B cell receptor repertoire sequencing to analyze the relationship between transcriptome and B cell specificity following cHA immunization. We show that the cHA inactivated vaccine with a squalene-based adjuvant induced a robust activated B cell and memory B cell phenotype against two broadly neutralizing epitopes of the stalk domain. The overall specificities of the acute plasmablast and memory B cell responses were distinct, with the plasmablast compartment largely targeting non-neutralizing epitopes of the HA stalk. Altogether, these data indicate the B cell landscape following cHA vaccination includes diverse B cell subsets that are differentially induced by distinct vaccine formulations, including memory and de novo B cell responses against diverse broadly conserved epitopes.

The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that evade immunity elicited by vaccination has placed an imperative on the development of countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent monoclonal antibodies (mAbs) that neutralized multiple sarbecoviruses from macaques vaccinated with AS03-adjuvanted monovalent subunit vaccines. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells (MBCs) for at least 1 year after primary vaccination. Antibodies generated from these antigen-specific MBCs at 5 to 12 months after vaccination displayed greater potency and breadth relative to those identified at 1.4 months. Fifteen of the 338 (about 4.4%) antibodies isolated at 1.4 to 6 months after the primary vaccination showed potency against SARS-CoV-2 BA.1, despite the absence of serum BA.1 neutralization. 25F9 and 20A7 neutralized authentic clade 1 sarbecoviruses (SARS-CoV, WIV-1, SHC014, SARS-CoV-2 D614G, BA.1, and Pangolin-GD) and vesicular stomatitis virus–pseudotyped clade 3 sarbecoviruses (BtKY72 and PRD-0038). 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1, and XBB. Crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved sites within the RBD. Prophylactic protection of 25F9, 20A7, and 27A12 was confirmed in mice, and administration of 25F9 particularly provided complete protection against SARS-CoV-2, BA.1, SARS-CoV, and SHC014 challenge. These data underscore the extremely potent and broad activity of these mAbs against sarbecoviruses.

The protective efficacy of neutralizing antibodies (nAbs) elicited during natural infection with SARS-CoV-2 and by vaccination based on its spike protein has been compromised with emergence of the recent SARS-CoV-2 variants. Residues E484 and K417 in the receptor-binding site (RBS) are both mutated in lineages first described in South Africa (B.1.351) and Brazil (B.1.1.28.1). The nAbs isolated from SARS-CoV-2 patients are preferentially encoded by certain heavy-chain germline genes and the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2) can each bind the RBS in two different binding modes. However, their binding and neutralization are abrogated by either the E484K or K417N mutation, whereas nAbs to the cross-reactive CR3022 and S309 sites are largely unaffected. This structural and functional analysis illustrates why mutations at E484 and K417 adversely affect major classes of nAbs to SARS-CoV-2 with consequences for next-generation COVID-19 vaccines.

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting broadly neutralizing antibody responses to CoVs. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein with a two-shot protocol generated potent serum receptor binding domain cross-neutralizing antibody responses to both SARS-CoV-2 and SARS-CoV-1. Furthermore, responses were equally effective against most SARS-CoV-2 variants of concern (VOCs) and some were highly effective against Omicron. This result contrasts with human infection or many two-shot vaccination protocols where responses were typically more SARS-CoV-2 specific and where VOCs were less well neutralized. Structural studies showed that cloned macaque neutralizing antibodies, particularly using a given heavy chain germline gene, recognized a relatively conserved region proximal to the angiotensin converting enzyme 2 receptor binding site (RBS), whereas many frequently elicited human neutralizing antibodies targeted more variable epitopes overlapping the RBS. B cell repertoire differences between humans and macaques appeared to influence the vaccine response. The macaque neutralizing antibodies identified a pan-SARS–related virus epitope region less well targeted by human antibodies that could be exploited in rational vaccine design.

Influenza virus hemagglutinin (HA) has been the primary target for influenza vaccine development. Broadly protective antibodies targeting conserved regions of the HA unlock the possibility of generating universal influenza immunity. Two group 2 influenza A chimeric HAs, cH4/3 and cH15/3, were previously designed to elicit antibodies to the conserved HA stem. Here, we show by X-ray crystallography and negative-stain electron microscopy that a broadly protective antistem antibody can stably bind to cH4/3 and cH15/3 HAs, thereby validating their potential as universal vaccine immunogens. Furthermore, flexibility was observed in the head domain of the chimeric HA structures, suggesting that antibodies could also potentially interact with the head interface epitope. Our structural and binding studies demonstrated that a broadly protective antihead trimeric interface antibody could indeed target the more open head domain of the cH15/3 HA trimer. Thus, in addition to inducing broadly protective antibodies against the conserved HA stem, chimeric HAs may also be able to elicit antibodies against the conserved trimer interface in the HA head domain, thereby increasing the vaccine efficacy.

Many neutralizing antibodies (nAbs) elicited to ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through natural infection and vaccination have reduced effectiveness to SARS-CoV-2 variants. Here, we show that therapeutic antibody ADG20 is able to neutralize SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize a broad range of VOCs, albeit with reduced potency against Omicron. Thus, this conserved and vulnerable site can be exploited for the design of universal vaccines and therapeutic antibodies.

NOX5 (NADPH oxidase 5) is a homolog of the gp91 phox subunit of the phagocyte NOX, which generates reactive oxygen species. NOX5 is involved in sperm motility and vascular contraction and has been implicated in diabetic nephropathy, atherosclerosis, and stroke. The function of NOX5 in the cardiac hypertrophy is unknown. Because NOX5 is a Ca 2+ -sensitive, procontractile NOX isoform, we questioned whether it plays a role in cardiac hypertrophy. Studies were performed in (1) cardiac tissue from patients undergoing heart transplant for cardiomyopathy and heart failure, (2) NOX5-expressing rat cardiomyocytes, and (3) mice expressing human NOX5 in a cardiomyocyte-specific manner. Cardiac hypertrophy was induced in mice by transverse aorta coarctation and Ang II (angiotensin II) infusion. NOX5 expression was increased in human failing hearts. Rat cardiomyocytes infected with adenoviral vector encoding human NOX5 cDNA exhibited elevated reactive oxygen species levels with significant enlargement and associated increased expression of ANP (atrial natriuretic peptides) and β-MHC (β-myosin heavy chain) and prohypertrophic genes ( Nppa , Nppb , and Myh7 ) under Ang II stimulation. These effects were reduced by N-acetylcysteine and diltiazem. Pressure overload and Ang II infusion induced left ventricular hypertrophy, interstitial fibrosis, and contractile dysfunction, responses that were exaggerated in cardiac-specific NOX5 trangenic mice. These phenomena were associated with increased reactive oxygen species levels and activation of redox-sensitive MAPK (mitogen-activated protein kinase). N-acetylcysteine treatment reduced cardiac oxidative stress and attenuated cardiac hypertrophy in NOX5 trangenic. Our study defines Ca 2+ -regulated NOX5 as an important NOX isoform involved in oxidative stress- and MAPK-mediated cardiac hypertrophy and contractile dysfunction.

A common theme in antibody responses In the fight against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), antibodies are a key tool, both as potential therapeutics and to guide vaccine development. Yuan et al. focused on finding shared antibody responses, in which multiple individuals develop antibodies against the same antigen using the same genetic elements and modes of recognition. The authors identified the immunoglobulin heavy-chain variable region 3-53 gene as the most frequently used among 294 antibodies that target the receptor-binding domain (RBD) of the viral spike protein. These antibodies have few somatic mutations, and crystal structures of two neutralizing antibodies bound to the RBD show that mostly germline-encoded residues are involved in binding. The minimal affinity maturation and high potency of these antibodies is promising for vaccine design. Science , this issue p. 1119

Current seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Several factors, including immunodominance of hemagglutinin over neuraminidase, instability of neuraminidase in vaccine formulations, and variable, nonstandardized amounts of neuraminidase in the vaccines, may contribute to this effect. However, vaccines that induce strong antineuraminidase immune responses would be beneficial, as they are highly protective. Furthermore, antigenic drift is slower for neuraminidase than for hemagglutinin, potentially providing broader coverage. Here, we designed stabilized recombinant versions of neuraminidase by replacing the N-terminal cytoplasmic domain, transmembrane, and extracellular stalk with tetramerization domains from the measles or Sendai virus phosphoprotein or from an Arabidopsis thaliana transcription factor. The measles virus tetramerization domain-based construct, termed N1-MPP, was chosen for further evaluation, as it retained antigenicity, neuraminidase activity, and structural integrity and provided robust protection in vivo against lethal virus challenge in the mouse model. We tested N1-MPP as a standalone vaccine, admixed with seasonal influenza virus vaccines, or given with seasonal influenza virus vaccines but in the other leg of the mouse. Admixture with different formulations of seasonal vaccines led to a weak neuraminidase response, suggesting a dominant effect of hemagglutinin over neuraminidase when administered in the same formulation. However, administration of neuraminidase alone or with seasonal vaccine administered in the alternate leg of the mouse induced robust antibody responses. Thus, this recombinant neuraminidase construct is a promising vaccine antigen that may enhance and broaden protection against seasonal influenza viruses. IMPORTANCE Influenza virus infections remain a high risk to human health, causing up to 650,000 deaths worldwide every year, with an enormous burden on the health care system. Since currently available seasonal vaccines are only partially effective and often mismatched to the circulating strains, a broader protective influenza virus vaccine is needed. Here, we generated a recombinant influenza virus vaccine candidate based on the more conserved neuraminidase surface glycoprotein in order to induce a robust and broader protective immune response against a variety of circulating influenza virus strains.

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID‐19) pandemic is used to discover three highly potent antibodies that selectively bind SARS‐CoV‐2 spike protein and neutralize authentic SARS‐CoV‐2 virus. Compared to neutralizing antibodies from COVID‐19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13–22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS‐CoV‐2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre‐pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS‐CoV‐2., Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the causative agent of the coronavirus disease 2019 pandemic. From a 20‐year‐old, combinatorial library, the authors isolate three neutralizing antibodies with high somatic hypermutations that are involved in antigen recognition, suggesting cross‐reactivity with a related antigen present in the pre‐pandemic‐era.

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use.ONE SENTENCE SUMMARYBroadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the naïve macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.

Inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation has emerged as a promising target for the treatment of nonalcoholic steatohepatitis (NASH). Multiple forms of posttranslational modifications determine the activity of ASK1. In addition to phosphorylation, recent studies revealed that ubiquitination is essential for ASK1 activation. However, the endogenous factor that regulates ASK1 ubiquitination and activation remains poorly defined. In this study, we identified the E3 ligase Skp1-Cul1-F-box (SCF) protein F-box/WD repeat-containing protein 5 (FBXW5) as a key endogenous activator of ASK1 ubiquitination. FBXW5 is the central component of the SCF complex (SCFFbxw5 ) that directly interacts with and ubiquitinates ASK1 in hepatocytes during NASH development. An in vivo study showed that hepatocyte-specific overexpression of FBXW5 exacerbated diet-induced systemic and hepatic metabolic disorders, as well as the activation of ASK1-related mitogen-activated protein kinase (MAPK) signaling in the liver. Conversely, hepatocyte-specific deletion of FBXW5 significantly prevented the progression of these abnormalities. Mechanically, FBXW5 facilitated the addition of Lys63-linked ubiquitin to ASK1 and thus exacerbated ASK1-c-Jun N-terminal kinase/p38 MAPK signaling, inflammation, and lipid accumulation. Furthermore, we demonstrated that the N-terminus (S1) and C-terminus (S3) of FBXW5 respectively and competitively ablate the function of FBXW5 on ASK1 activation and served as effective inhibitors of NASH progression. Conclusion: This evidence strongly suggests that SCFFbxw5 is an important activator of ASK1 ubiquitination in the context of NASH. The development of FBXW5(S1) or FBXW5(S3)-mimicking drugs and screening of small-molecular inhibitors specifically abrogating ASK1 ubiquitination-dependent activation are viable approaches for NASH treatment.

The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses (β-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 Å resolution and found that the peptide adopted a mainly helical structure. Conserved residues in β-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on β-CoV spike proteins for protective antibodies that may facilitate the development of pan-β-CoV vaccines.SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-β-CoV vaccine design strategies

Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short complementarity-determining region (CDR) H3. Germline-encoded sequence motifs in heavy chain CDRs H1 and H2 have a major function, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, is not clear. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that seem to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. These results advance understanding of the antibody response to SARS-CoV-2., Public antibody clonotypes that recognize SARS-CoV-2 spike protein are important for protection against COVID-19. Here, the authors characterize sequence motifs in the heavy chain complementarity-determining region (CDR) H3s of two public clonotypes and their association with light chain identity.

Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short CDR H3. Germline-encoded sequence motifs in CDRs H1 and H2 play a major role, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, have not been elucidated. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that appear to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. Overall, our results advance fundamental understanding of the antibody response to SARS-CoV-2.

A double punch against SARS-CoV-2 Monoclonal antibodies are an important weapon in the battle against COVID-19. However, these large proteins are difficult to produce in the needed quantities and at low cost. Attention has turned to nanobodies, which are aptly named, single-domain antibodies that are easier to produce and have the potential to be administered by inhalation. Koenig et al. describe four nanobodies that bind to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and prevent infection of cells (see the Perspective by Saelens and Schepens). Structures show that the nanobodies target two distinct epitopes on the SARS-CoV-2 spike protein. Multivalent nanobodies neutralize virus much more potently than single nanobodies, and multivalent nanobodies that bind two epitopes prevent the emergence of viral escape mutants. Science, this issue p. eabe6230; see also p. 681, SARS-CoV-2–neutralizing nanobodies were combined to design potent multivalent nanobodies., INTRODUCTION The global scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose unprecedented challenges to society, health care systems, and science. In addition to effective and safe vaccines, passive immunization by antibody-related molecules offers an opportunity to harness the vertebrate immune system to fight viral infections in high-risk patients. Variable domains of heavy-chain–only antibodies (VHHs), also known as nanobodies, are suitable lead molecules in such efforts, as they are small, extremely stable, easy to engineer, and economic to produce in simple expression systems. RATIONALE We engineered improved multivalent nanobodies neutralizing SARS-CoV-2 on the basis of two principles: (i) detailed structural information of their epitopes and binding modes to the viral spike protein and (ii) mechanistic insights into viral fusion with cellular membranes catalyzed by the spike. RESULTS Nanobodies specific for the receptor binding domain (RBD) of SARS-CoV-2 spike were identified by phage display using nanobody libraries from an alpaca and a llama immunized with the RBD and inactivated virus. Four of the resulting nanobodies—VHHs E, U, V, and W—potently neutralize SARS-CoV-2 and SARS-CoV-2–pseudotyped vesicular stomatitis virus. X-ray crystallography revealed that the nanobodies bind to two distinct epitopes on the RBD, interfaces “E” and “UVW,” which can be synergistically targeted by combinations of nanobodies to inhibit infection. Cryo–electron microscopy (cryo-EM) of trimeric spike in complex with VHH E and VHH V revealed that VHH E stabilizes a conformation of the spike with all three RBDs in the “up” conformation (3-up), a state that is typically associated with activation by receptor binding. In line with this observation, we found that VHH E triggers the fusion activity of spike in the absence of the cognate receptor ACE2. VHH V, by contrast, stabilizes spike in a 2-up conformation and does not induce fusion. On the basis of the structural information, we designed bi- and trivalent nanobodies with improved neutralizing properties. VHH EEE most potently inhibited infection, did not activate fusion, and likely inactivated virions by outcompeting interaction of the virus with its receptor. Yet evolution experiments revealed emergence of escape mutants in the spike with single–amino acid changes that were completely insensitive to inhibition by VHH EEE. VHH VE also neutralized more efficiently than VHH E or VHH V alone; stabilized the 3-up conformation of spike, as determined by cryo-EM; and more strongly induced the spike fusogenic activity. We conclude that the premature activation of the fusion machinery on virions was an unexpected mechanism of neutralization, as enhanced neutralization could not be attributed simply to better blocking of virus-receptor interactions. Activation of spike in the absence of target membranes likely induces irreversible conformational changes to assume the energetically favorable postfusion conformation without catalyzing fusion per se. Simultaneous targeting of two independent epitopes by VHH VE largely prevented the emergence of resistant escape mutants in evolution experiments. CONCLUSION Our results demonstrate the strength of the modular combination of nanobodies for neutralization. Premature activation of spike by nanobodies reveals an unusual mode of neutralization and yields insights into the mechanism of fusion. Bivalent nanobodies neutralize by inducing postfusion conformation of the SARS-CoV-2 spike. On virions, SARS-CoV-2 spike trimers are mostly in an inactive configuration with all RBDs in the down conformation (left). Binding of bivalent nanobody VE stabilizes the spike in an active conformation with all RBDs up (middle), triggering premature induction of the postfusion conformation, which irreversibly inactivates the spike protein (right)., The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo–electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

Most antibodies isolated from individuals with coronavirus disease 2019 (COVID-19) are specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, COVA1-16 is a relatively rare antibody that also cross-neutralizes SARS-CoV. Here, we determined a crystal structure of the COVA1-16 antibody fragment (Fab) with the SARS-CoV-2 receptor-binding domain (RBD) and negative-stain electron microscopy reconstructions with the spike glycoprotein trimer to elucidate the structural basis of its cross-reactivity. COVA1-16 binds a highly conserved epitope on the SARS-CoV-2 RBD, mainly through a long complementarity-determining region (CDR) H3, and competes with the angiotensin-converting enzyme 2 (ACE2) receptor because of steric hindrance rather than epitope overlap. COVA1-16 binds to a flexible up conformation of the RBD on the spike and relies on antibody avidity for neutralization. These findings, along with the structural and functional rationale for epitope conservation, provide insights for development of more universal SARS-like coronavirus vaccines and therapies., Graphical Abstract, Highlights • X-ray and EM structures of cross-neutralizing antibody COVA1-16 with SARS-CoV-2 RBD • COVA1-16 binding to SARS-CoV-2 RBD is dominated by CDR H3 • COVA1-16 binds to a highly conserved non-RBS epitope but still competes with ACE2 • IgG avidity is the key for the cross-neutralization activity of COVA1-16, COVA1-16 is a SARS-CoV-2 antibody from an individual with COVID-19 that cross-neutralizes SARS-CoV. Liu et al. reveal that COVA1-16 binds to a highly conserved epitope using a long CDR H3, where its approach angle sterically blocks ACE2 from engaging the RBS. Virus neutralization by COVA1-16 is driven by IgG avidity.

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses., Author summary The ongoing COVID-19 pandemic is caused by SARS-CoV-2. Due to the genetic similarity of SARS-CoV-2 and SARS-CoV, which caused an epidemic in 2003, a few of the SARS-CoV antibodies have now been found to also cross-react with SARS-CoV-2. One such antibody is CR3022, which was isolated from a convalescent SARS patient 14 years ago. However, the 100-fold lower binding to SARS-CoV-2 does not enable neutralization of SARS-CoV-2 compared to SARS-CoV. This study shows that one (P384A) of the four mutational differences in the CR3022 epitope between SARS-CoV and SARS-COV-2 fully accounts for the differences in CR3022 binding affinity and neutralization. These findings advance our understanding of antibody cross-reactivity among SARS-like CoVs with implications for vaccine and therapeutic design.

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The SARS-CoV-2 pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, we used a combinatorial human antibody library constructed 20 years before the COVID-19 pandemic to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in crystal structures with SARS-CoV-2 spike RBD. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of human immune responses to SARS-CoV-2.

IGHV3-53-encoded neutralizing antibodies are commonly elicited during SARS-CoV-2 infection and target the receptor-binding domain (RBD) of the spike (S) protein. Such IGHV3-53 antibodies generally have a short CDR H3 due to structural constraints in binding the RBD (mode A). However, a small subset of IGHV3-53 antibodies to the RBD contain a longer CDR H3. Crystal structures of two IGHV3-53 neutralizing antibodies here demonstrate that a longer CDR H3 can be accommodated in a different binding mode (mode B). These two classes of IGHV3-53 antibodies both target the ACE2 receptor binding site, but with very different angles of approach and molecular interactions. Overall, these findings emphasize the versatility of IGHV3-53 in this common antibody response to SARS-CoV-2, where conserved IGHV3-53 germline-encoded features can be combined with very different CDR H3 lengths and light chains for SARS-CoV-2 RBD recognition and virus neutralization, Graphical Abstract, Highlights • Crystal structures of IGHV3-53 antibodies that frequently bind SARS-CoV-2 RBD • Binding modes (A and B) of these IGHV3-53 antibodies depend on CDR H3 length • Germline-encoded CDR H1 and H2 motifs dominate the two binding poses • CDR H3 length of IGHV3-53 antibodies is associated with light chain preference, Antibodies to the SARS-CoV-2 receptor-binding domain are commonly encoded by IGHV3-53 and most have a short CDR H3. Wu et al. show that IGHV3-53 antibodies with a long CDR H3 adopt an alternative binding mode, demonstrating that IGHV3-53 is even more versatile than previously thought in targeting SARS-CoV-2.

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from ten COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb CV07-209 neutralized authentic SARS-CoV-2 with IC50 of 3.1 ng/ml. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2 neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy., HIGHLIGHTS • Characterization of potent human monoclonal SARS-CoV-2 neutralizing antibodies • Some SARS-CoV-2 antibodies reacted with mammalian self-antigens in different organs • Crystal structures of two antibodies in complex with SARS-CoV-2 RBD at 2.55/2.70 Å • Post-exposure antibody treatment protected from lung damage in infected hamsters, Kreye et al. report the isolation and characterization of monoclonal antibodies isolated from COVID-19 patients, some of which were found to display autoreactivity with mammalian self-antigens in different organs. Crystal structures of two antibodies in complex with SARS-CoV-2 Spike RBD reveal antibody engagement with the ACE2 binding site from different approach angles. One antibody is further evaluated for in vivo efficacy and was found to be both protective and efficacious post-challenge in a hamster infection model.