Conversion of vaccines from low to high immunogenicity by antibodies with epitope complementarity.

Existing antibodies (Abs) have varied effects on humoral immunity during subsequent infections. Here, we leveraged in vivo systems that allow precise control of antigen-specific Abs and B cells to examine the impact of Ab dose, affinity, and specificity in directing B cell activation and differentiation. Abs competing with the B cell receptor (BCR) epitope showed affinity-dependent suppression. By contrast, Abs targeting a complementary epitope, not overlapping with the BCR, shifted B cell differentiation toward Ab-secreting cells. Such Abs allowed for potent germinal center (GC) responses to otherwise poorly immunogenic sites by promoting antigen capture and presentation by low-affinity B cells. These mechanisms jointly diversified the B cell repertoire by facilitating the recruitment of high- and low-affinity B cells into Ab-secreting cell, GC, and memory B cell fates. Incorporation of small amounts of monoclonal Abs into protein- or mRNA-based vaccines enhanced immunogenicity and facilitated sustained immune responses, with implications for vaccine design and our understanding of protective immunity.

Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability.

While neutralizing antibodies are highly effective against ebolavirus infections, current experimental ebolavirus vaccines primarily elicit species-specific antibody responses. Here, we describe an immunization-elicited macaque antibody (CA45) that clamps the internal fusion loop with the N terminus of the ebolavirus glycoproteins (GPs) and potently neutralizes Ebola, Sudan, Bundibugyo, and Reston viruses. CA45, alone or in combination with an antibody that blocks receptor binding, provided full protection against all pathogenic ebolaviruses in mice, guinea pigs, and ferrets. Analysis of memory B cells from the immunized macaque suggests that elicitation of broadly neutralizing antibodies (bNAbs) for ebolaviruses is possible but difficult, potentially due to the rarity of bNAb clones and their precursors. Unexpectedly, germline-reverted CA45, while exhibiting negligible binding to full-length GP, bound a proteolytically remodeled GP with picomolar affinity, suggesting that engineered ebolavirus vaccines could trigger rare bNAb precursors more robustly. These findings have important implications for developing pan-ebolavirus vaccine and immunotherapeutic cocktails.

Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting.

The continuing emergence of SARS-CoV-2 variants highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by vaccination based on the ancestral (hereafter referred to as WT) strain would compromise the antibody response to Omicron-based boosters1-5. Vaccination strategies to counter immune imprinting are critically needed. Here we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. In mice, the efficacy of single Omicron boosting is heavily limited when using variants that are antigenically distinct from WT-such as the XBB variant-and this concerning situation could be mitigated by a second Omicron booster. Similarly, in humans, repeated Omicron infections could alleviate WT vaccination-induced immune imprinting and generate broad neutralization responses in both plasma and nasal mucosa. Notably, deep mutational scanning-based epitope characterization of 781 receptor-binding domain (RBD)-targeting monoclonal antibodies isolated from repeated Omicron infection revealed that double Omicron exposure could induce a large proportion of matured Omicron-specific antibodies that have distinct RBD epitopes to WT-induced antibodies. Consequently, immune imprinting was largely mitigated, and the bias towards non-neutralizing epitopes observed in single Omicron exposures was restored. On the basis of the deep mutational scanning profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated that these mutations could further boost the immune-evasion capability of XBB.1.5 while maintaining high ACE2-binding affinity. Our findings suggest that the WT component should be abandoned when updating COVID-19 vaccines, and individuals without prior Omicron exposure should receive two updated vaccine boosters.

Imprinting of serum neutralizing antibodies by Wuhan-1 mRNA vaccines.

Immune imprinting is a phenomenon in which prior antigenic experiences influence responses to subsequent infection or vaccination1,2. The effects of immune imprinting on serum antibody responses after boosting with variant-matched SARS-CoV-2 vaccines remain uncertain. Here we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike protein minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2-infected and uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses.

Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses.

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.

HIV-1 VRC01 Germline-Targeting Immunogens Select Distinct Epitope-Specific B Cell Receptors.

Activating precursor B cell receptors of HIV-1 broadly neutralizing antibodies requires specifically designed immunogens. Here, we compared the abilities of three such germline-targeting immunogens against the VRC01-class receptors to activate the targeted B cells in transgenic mice expressing the germline VH of the VRC01 antibody but diverse mouse light chains. Immunogen-specific VRC01-like B cells were isolated at different time points after immunization, their VH and VL genes were sequenced, and the corresponding antibodies characterized. VRC01 B cell sub-populations with distinct cross-reactivity properties were activated by each immunogen, and these differences correlated with distinct biophysical and biochemical features of the germline-targeting immunogens. Our study indicates that the design of effective immunogens to activate B cell receptors leading to protective HIV-1 antibodies will require a better understanding of how the biophysical properties of the epitope and its surrounding surface on the germline-targeting immunogen influence its interaction with the available receptor variants in vivo.

Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation.

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.

Antigenicity and receptor affinity of SARS-CoV-2 BA.2.86 spike.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant, BA.2.86, has emerged and spread to numerous countries worldwide, raising alarm because its spike protein contains 34 additional mutations compared with its BA.2 predecessor1. We examined its antigenicity using human sera and monoclonal antibodies (mAbs). Reassuringly, BA.2.86 was no more resistant to human sera than the currently dominant XBB.1.5 and EG.5.1, indicating that the new subvariant would not have a growth advantage in this regard. Importantly, sera from people who had XBB breakthrough infection exhibited robust neutralizing activity against all viruses tested, suggesting that upcoming XBB.1.5 monovalent vaccines could confer added protection. Although BA.2.86 showed greater resistance to mAbs to subdomain 1 (SD1) and receptor-binding domain (RBD) class 2 and 3 epitopes, it was more sensitive to mAbs to class 1 and 4/1 epitopes in the 'inner face' of the RBD that is exposed only when this domain is in the 'up' position. We also identified six new spike mutations that mediate antibody resistance, including E554K that threatens SD1 mAbs in clinical development. The BA.2.86 spike also had a remarkably high receptor affinity. The ultimate trajectory of this new SARS-CoV-2 variant will soon be revealed by continuing surveillance, but its worldwide spread is worrisome.

Antibody feedback regulates immune memory after SARS-CoV-2 mRNA vaccination.

Feedback inhibition of humoral immunity by antibodies was first documented in 19091. Subsequent studies showed that, depending on the context, antibodies can enhance or inhibit immune responses2,3. However, little is known about how pre-existing antibodies influence the development of memory B cells. Here we examined the memory B cell response in individuals who received two high-affinity anti-SARS-CoV-2 monoclonal antibodies and subsequently two doses of an mRNA vaccine4-8. We found that the recipients of the monoclonal antibodies produced antigen-binding and neutralizing titres that were only fractionally lower compared than in control individuals. However, the memory B cells of the individuals who received the monoclonal antibodies differed from those of control individuals in that they predominantly expressed low-affinity IgM antibodies that carried small numbers of somatic mutations and showed altered receptor binding domain (RBD) target specificity, consistent with epitope masking. Moreover, only 1 out of 77 anti-RBD memory antibodies tested neutralized the virus. The mechanism underlying these findings was examined in experiments in mice that showed that germinal centres formed in the presence of the same antibodies were dominated by low-affinity B cells. Our results indicate that pre-existing high-affinity antibodies bias germinal centre and memory B cell selection through two distinct mechanisms: (1) by lowering the activation threshold for B cells, thereby permitting abundant lower-affinity clones to participate in the immune response; and (2) through direct masking of their cognate epitopes. This may in part explain the shifting target profile of memory antibodies elicited by booster vaccinations9.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody1. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab2, S2X2593 and S2H974. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

Long-primed germinal centres with enduring affinity maturation and clonal migration.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies.

The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here we use high-throughput yeast display screening1,2 to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A-F)-a grouping that is highly concordant with knowledge-based structural classifications3-5. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A-D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)6 and group F (for example, CR3022)7, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.

BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages BA.2.12.1, BA.4 and BA.5 exhibit higher transmissibility than the BA.2 lineage1. The receptor binding and immune-evasion capability of these recently emerged variants require immediate investigation. Here, coupled with structural comparisons of the spike proteins, we show that BA.2.12.1, BA.4 and BA.5 (BA.4 and BA.5 are hereafter referred collectively to as BA.4/BA.5) exhibit similar binding affinities to BA.2 for the angiotensin-converting enzyme 2 (ACE2) receptor. Of note, BA.2.12.1 and BA.4/BA.5 display increased evasion of neutralizing antibodies compared with BA.2 against plasma from triple-vaccinated individuals or from individuals who developed a BA.1 infection after vaccination. To delineate the underlying antibody-evasion mechanism, we determined the escape mutation profiles2, epitope distribution3 and Omicron-neutralization efficiency of 1,640 neutralizing antibodies directed against the receptor-binding domain of the viral spike protein, including 614 antibodies isolated from people who had recovered from BA.1 infection. BA.1 infection after vaccination predominantly recalls humoral immune memory directed against ancestral (hereafter referred to as wild-type (WT)) SARS-CoV-2 spike protein. The resulting elicited antibodies could neutralize both WT SARS-CoV-2 and BA.1 and are enriched on epitopes on spike that do not bind ACE2. However, most of these cross-reactive neutralizing antibodies are evaded by spike mutants L452Q, L452R and F486V. BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1. Nevertheless, these neutralizing antibodies are largely evaded by BA.2 and BA.4/BA.5 owing to D405N and F486V mutations, and react weakly to pre-Omicron variants, exhibiting narrow neutralization breadths. The therapeutic neutralizing antibodies bebtelovimab4 and cilgavimab5 can effectively neutralize BA.2.12.1 and BA.4/BA.5, whereas the S371F, D405N and R408S mutations undermine most broadly sarbecovirus-neutralizing antibodies. Together, our results indicate that Omicron may evolve mutations to evade the humoral immunity elicited by BA.1 infection, suggesting that BA.1-derived vaccine boosters may not achieve broad-spectrum protection against new Omicron variants.

An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus.

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells, the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical, and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden.

Antibody epitopes in vaccine-induced immune thrombotic thrombocytopaenia.

Vaccine-induced immune thrombotic thrombocytopaenia (VITT) is a rare adverse effect of COVID-19 adenoviral vector vaccines1-3. VITT resembles heparin-induced thrombocytopaenia (HIT) in that it is associated with platelet-activating antibodies against platelet factor 4 (PF4)4; however, patients with VITT develop thrombocytopaenia and thrombosis without exposure to heparin. Here we sought to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine-scanning mutagenesis5, we found that the binding of anti-PF4 antibodies from patients with VITT (n = 5) was restricted to eight surface amino acids on PF4, all of which were located within the heparin-binding site, and that the binding was inhibited by heparin. By contrast, antibodies from patients with HIT (n = 10) bound to amino acids that corresponded to two different sites on PF4. Biolayer interferometry experiments also revealed that VITT anti-PF4 antibodies had a stronger binding response to PF4 and PF4-heparin complexes than did HIT anti-PF4 antibodies, albeit with similar dissociation rates. Our data indicate that VITT antibodies can mimic the effect of heparin by binding to a similar site on PF4; this allows PF4 tetramers to cluster and form immune complexes, which in turn causes Fcγ receptor IIa (FcγRIIa; also known as CD32a)-dependent platelet activation. These results provide an explanation for VITT-antibody-induced platelet activation that could contribute to thrombosis.

Anti-SARS-CoV-2 receptor-binding domain antibody evolution after mRNA vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection produces B cell responses that continue to evolve for at least a year. During that time, memory B cells express increasingly broad and potent antibodies that are resistant to mutations found in variants of concern1. As a result, vaccination of coronavirus disease 2019 (COVID-19) convalescent individuals with currently available mRNA vaccines produces high levels of plasma neutralizing activity against all variants tested1,2. Here we examine memory B cell evolution five months after vaccination with either Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) mRNA vaccine in a cohort of SARS-CoV-2-naive individuals. Between prime and boost, memory B cells produce antibodies that evolve increased neutralizing activity, but there is no further increase in potency or breadth thereafter. Instead, memory B cells that emerge five months after vaccination of naive individuals express antibodies that are similar to those that dominate the initial response. While individual memory antibodies selected over time by natural infection have greater potency and breadth than antibodies elicited by vaccination, the overall neutralizing potency of plasma is greater following vaccination. These results suggest that boosting vaccinated individuals with currently available mRNA vaccines will increase plasma neutralizing activity but may not produce antibodies with equivalent breadth to those obtained by vaccinating convalescent individuals.

Hybrid immunity improves B cells and antibodies against SARS-CoV-2 variants.

The emergence of SARS-CoV-2 variants is jeopardizing the effectiveness of current vaccines and limiting the application of monoclonal antibody-based therapy for COVID-19 (refs. 1,2). Here we analysed the memory B cells of five naive and five convalescent people vaccinated with the BNT162b2 mRNA vaccine to investigate the nature of the B cell and antibody response at the single-cell level. Almost 6,000 cells were sorted, over 3,000 cells produced monoclonal antibodies against the spike protein and more than 400 cells neutralized the original SARS-CoV-2 virus first identified in Wuhan, China. The B.1.351 (Beta) and B.1.1.248 (Gamma) variants escaped almost 70% of these antibodies, while a much smaller portion was impacted by the B.1.1.7 (Alpha) and B.1.617.2 (Delta) variants. The overall loss of neutralization was always significantly higher in the antibodies from naive people. In part, this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in people who were convalescent and generated potent and broadly neutralizing antibodies. Our data suggest that people who are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control emerging SARS-CoV-2 variants.

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.