A multitude of functional mutations continue to emerge on the N-terminal domain (NTD) of the spike protein in SARS-CoV-2 Omicron subvariants. Understanding the immunogenicity of Omicron NTD and the properties of antibodies elicited by it is crucial for comprehending the impact of NTD mutations on viral fitness and guiding vaccine design. In this study, we find that most of NTD-targeting antibodies isolated from individuals with BA.5/BF.7 breakthrough infection (BTI) are ancestral (wildtype or WT)-reactive and non-neutralizing. Surprisingly, we identified five ultra-potent neutralizing antibodies (NAbs) that can only bind to Omicron but not WT NTD. Structural analysis revealed that they bind to a unique epitope on the N1/N2 loop of NTD and interact with the receptor-binding domain (RBD) via the light chain. These Omicron-specific NAbs achieve neutralization through ACE2 competition and blockage of ACE2-mediated S1 shedding. However, BA.2.86 and BA.2.87.1, which carry insertions or deletions on the N1/N2 loop, can evade these antibodies. Together, we provided a detailed map of the NTD-targeting antibody repertoire in the post-Omicron era, demonstrating their vulnerability to NTD mutations enabled by its evolutionary flexibility, despite their potent neutralization. These results revealed the function of the indels in the NTD of BA.2.86/JN.1 sublineage in evading neutralizing antibodies and highlighted the importance of considering the immunogenicity of NTD in vaccine design.

The recent emergence of a SARS-CoV-2 saltation variant, BA.2.87.1, which features 65 spike mutations relative to BA.2, has attracted worldwide attention. In this study, we elucidate the antigenic characteristics and immune evasion capability of BA.2.87.1. Our findings reveal that BA.2.87.1 is more susceptible to XBB-induced humoral immunity compared to JN.1. Notably, BA.2.87.1 lacks critical escaping mutations in the receptor binding domain (RBD) thus allowing various classes of neutralizing antibodies (NAbs) that were escaped by XBB or BA.2.86 subvariants to neutralize BA.2.87.1, although the deletions in the N-terminal domain (NTD), specifically 15-23del and 136-146del, compensate for the resistance to humoral immunity. Interestingly, several neutralizing antibody drugs have been found to restore their efficacy against BA.2.87.1, including SA58, REGN-10933 and COV2-2196. Hence, our results suggest that BA.2.87.1 may not become widespread until it acquires multiple RBD mutations to achieve sufficient immune evasion comparable to that of JN.1.

Monoclonal antibodies (mAbs) targeting the SARS-CoV-2 receptor-binding domain (RBD) showed high efficacy in the prevention and treatment of COVID-19. However, the rapid evolution of SARS-CoV-2 has rendered all clinically authorized mAbs ineffective and continues to stymie the development of next-generation mAbs. Consequently, the ability to identify broadly neutralizing antibodies (bnAbs) that neutralize both current and future variants is critical for successful antibody therapeutic development, especially for newly emerged viruses when no knowledge about immune evasive variants is available. Here, we have developed a strategy to specifically select for potent bnAbs with activity against both existing and prospective SARS-CoV-2 variants based on accurate viral evolution prediction informed by deep mutational scanning (DMS). By adopting this methodology, we increased the probability of identifying XBB.1.5-effective SARS-CoV-2 bnAbs from ∼1% to 40% if we were at the early stage of the pandemic, as revealed by a retrospective analysis of >1,000 SARS-CoV-2 wildtype (WT)-elicited mAbs. From this collection, we identified a bnAb, designated BD55-1205, that exhibited exceptional activity against historical, contemporary, and predicted future variants. Structural analyses revealed extensive polar interactions between BD55-1205 and XBB.1.5 receptor-binding motif (RBM), especially with backbone atoms, explaining its unusually broad reactivity. Importantly, mRNA-based delivery of BD55-1205 IgG to human FcRn-expressing transgenic mice resulted in high serum neutralizing titers against selected XBB and BA.2.86 subvariants. Together, the ability to identify bnAbs via accurate viral evolution prediction, coupled with the speed and flexibility of mRNA delivery technology, provides a generalized framework for the rapid development of next-generation antibody-based countermeasures against SARS-CoV-2 and potentially other highly variable pathogens with pandemic potential. ### Competing Interest Statement Y.C. is listed as an inventor of provisional patent applications of SARS-CoV-2 RBD-specific antibodies involved in the study, including BD55-1205. The patent of BD55-1205 is licensed to Moderna. Y.C. is a co-founder of Singlomics Biopharmaceuticals. A.Z.W., J.H., D.M.B., D.L., T.S., L.M., T.K., K.W., C.H., S.P., and L.M.W. are full-time employees and holders of equity in Moderna Therapeutics. Other authors declare no competing interests.

The continuous evolution of SARS-CoV-2, particularly the emergence of the BA.2.86/JN.1 lineage replacing XBB, necessitates re-evaluation of vaccine compositions1-3. Here, we provide a comprehensive analysis of the humoral immune response to XBB and JN.1 human exposure. We demonstrate the antigenic distinctiveness of XBB and JN.1 lineages in SARS-CoV-2-naive individuals and show that infection with JN.1 elicits superior plasma neutralization against its subvariants. We highlight the strong immune evasion and receptor-binding capability of KP.3, supporting its foreseeable prevalence. Extensive analysis of the B cell receptor repertoire, in which we isolate approximately 2,000 receptor-binding-domain-specific antibodies, with targeting epitopes characterized by deep mutational scanning, underscores the superiority of JN.1-elicited memory B cells4,5. Class 1 IGHV3-53/3-66-derived neutralizing antibodies (NAbs) are important contributors to the wild-type reactivity of NAbs against JN.1. However, KP.2 and KP.3 evade a substantial subset of these antibodies, even those induced by JN.1, supporting a need for booster updates. JN.1-induced Omicron-specific antibodies also demonstrate high potency across Omicron. Escape hotspots for these NAbs have already been mutated, resulting in a higher immune barrier to escape and indicating probable recovery of escaped NAbs. In addition, the prevalence of IGHV3-53/3-66-derived antibodies and their ability to compete with all Omicron-specific NAbs suggests that they have an inhibitory effect on the activation of Omicron-specific naive B cells, potentially explaining the heavy immune imprinting in mRNA-vaccinated individuals6-8. These findings delineate the evolving antibody response to the antigenic shift of Omicron from XBB to JN.1 and highlight the importance of developing the JN.1 lineage, especially KP.2- and KP.3-based vaccine boosters.

While the BA.2.86 variant demonstrated significant antigenic drift and enhanced ACE2 binding affinity, its ability to evade humoral immunity was relatively moderate compared to dominant strains like EG.5 and HK.3. However, the emergence of a new subvariant, JN.1 (BA.2.86.1.1), which possesses an additional spike mutation, L455S, compared to BA.2.86, showed a markedly increased prevalence in Europe and North America, especially in France. Here, we found that L455S of JN.1 significantly enhances immune evasion capabilities at the expense of reduced ACE2 binding affinity. This mutation enables JN.1 to effectively evade Class 1 neutralizing antibodies, offsetting BA.2.86’s susceptibility and thus allowing it to outcompete both its precursor BA.2.86 and the prevailing variants HV.1 (XBB.1.5+L452R+F456L) and JD.1.1 (XBB.1.5+L455F+F456L+A475V) in terms of humoral immune evasion. The rapid evolution from BA.2.86 to JN.1, similar to the earlier transition from BA.2.75 to CH.1.1, highlights the importance of closely monitoring strains with high ACE2 binding affinity and distinct antigenicity, despite their temporarily unremarkable immune evasion capabilities. Such strains could survive and transmit at low levels, since their large antigenic distance to dominant strains allow them to target distinct populations and accumulate immune-evasive mutations rapidly, often at the cost of receptor binding affinity.

The recently identified SARS-CoV-2 variant, BA.2.86, which carries a substantial number of Spike mutations, has raised a global alarm. An immediate assessment of its antigenic properties and infectivity is necessary. Here, we reveal the distinct antigenicity of BA.2.86 compared with previous variants including XBB.1.5. BA.2.86 significantly evades convalescent plasma from XBB breakthrough infection (BTI) and reinfections. Key mutations that mediate the enhanced resistance include N450D, K356T, L452W, A484K, V483del, and V445H on the RBD, while BA.2.86’s NTD mutations and E554K on SD1 also largely contribute. However, we found that BA.2.86 pseudovirus exhibits compromised efficiency of infecting HEK293T-hACE2 cells compared to XBB.1.5 and EG.5, which may be caused by K356T, V483del, and E554K, and could potentially limit BA.2.86’s transmissibility. In sum, it appears that BA.2.86 has traded its infectivity for higher immune evasion during long-term host-viral evolution. Close attention should be paid to monitoring additional mutations that could improve BA.2.86’s infectivity.

Abstract The continuous emergence of highly immune evasive SARS-CoV-2 variants, like XBB.1.5 and XBB.1.16, highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by wildtype (WT)-based vaccination would compromise the antibody response to Omicron-based boosters. Vaccination strategies that can counter immune imprinting are critically needed. In this study, we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. Our results show that in mice, the efficacy of single Omicron-boosting is heavily limited by immune imprinting, especially when using variants antigenically distinct from WT, like XBB, while the concerning situation could be largely mitigated by a second Omicron booster. Similarly, in humans, we found that repeated Omicron infections could also alleviate WT-vaccination-induced immune imprinting and generate high neutralizing titers against XBB.1.5 and XBB.1.16 in both plasma and nasal mucosa. By isolating 781 RBD-targeting mAbs from repeated Omicron infection cohorts, we revealed that double Omicron exposure alleviates immune imprinting by generating a large proportion of highly matured and potent Omicron-specific antibodies. Importantly, epitope characterization using deep mutational scanning (DMS) showed that these Omicron-specific antibodies target distinct RBD epitopes compared to WT-induced antibodies, and the bias towards non-neutralizing epitopes observed in single Omicron exposures due to imprinting was largely restored after repeated Omicron stimulation, together leading to a substantial neutralizing epitope shift. Based on the DMS profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated the combinations of these mutations could further boost XBB.1.5’s immune-evasion capability while maintaining high ACE2 binding affinity. Our findings suggest the WT component should be abandoned when updating COVID-19 vaccine antigen compositions to XBB lineages, and those who haven't been exposed to Omicron yet should receive two updated vaccine boosters.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) XBB lineages have achieved dominance worldwide and keep on evolving. Convergent evolution of XBB lineages on the receptor-binding domain (RBD) L455F and F456L is observed, resulting in variants with substantial growth advantages, such as EG.5, FL.1.5.1, XBB.1.5.70, and HK.3. Here, we show that neutralizing antibody (NAb) evasion drives the convergent evolution of F456L, while the epistatic shift caused by F456L enables the subsequent convergence of L455F through ACE2 binding enhancement and further immune evasion. L455F and F456L evade RBD-targeting Class 1 public NAbs, reducing the neutralization efficacy of XBB breakthrough infection (BTI) and reinfection convalescent plasma. Importantly, L455F single substitution significantly dampens receptor binding; however, the combination of L455F and F456L forms an adjacent residue flipping, which leads to enhanced NAbs resistance and ACE2 binding affinity. The perturbed receptor-binding mode leads to the exceptional ACE2 binding and NAb evasion, as revealed by structural analyses. Our results indicate the evolution flexibility contributed by epistasis cannot be underestimated, and the evolution potential of SARS-CoV-2 RBD remains high.

The continuous emergence of highly immune evasive SARS-CoV-2 variants, like XBB.1.5 1,2 and XBB.1.16 3,4 , highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by wildtype (WT)-based vaccination would compromise the antibody response to Omicron-based boosters 5-9 . Vaccination strategies that can counter immune imprinting are critically needed. In this study, we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. Our results show that in mice, the efficacy of single Omicron-boosting is heavily limited by immune imprinting, especially when using variants antigenically distinct from WT, like XBB, while the concerning situation could be largely mitigated by a second Omicron booster. Similarly, in humans, we found that repeated Omicron infections could also alleviate WT-vaccination-induced immune imprinting and generate high neutralizing titers against XBB.1.5 and XBB.1.16 in both plasma and nasal mucosa. By isolating 781 RBD-targeting mAbs from repeated Omicron infection cohorts, we revealed that double Omicron exposure alleviates immune imprinting by generating a large proportion of highly matured and potent Omicron-specific antibodies. Importantly, epitope characterization using deep mutational scanning (DMS) showed that these Omicron-specific antibodies target distinct RBD epitopes compared to WT-induced antibodies, and the bias towards non-neutralizing epitopes observed in single Omicron exposures due to imprinting was largely restored after repeated Omicron stimulation, together leading to a substantial neutralizing epitope shift. Based on the DMS profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated the combinations of these mutations could further boost XBB.1.5’s immune-evasion capability while maintaining high ACE2 binding affinity. Our findings suggest the WT component should be abandoned when updating COVID-19 vaccine antigen compositions to XBB lineages, and those who haven’t been exposed to Omicron yet should receive two updated vaccine boosters.

Recently emerged SARS-CoV-2 Omicron subvariant, BA.2.75, displayed a growth advantage over circulating BA.2.38, BA.2.76, and BA.5 in India. However, the underlying mechanisms for enhanced infectivity, especially compared with BA.5, remain unclear. Here, we show that BA.2.75 exhibits substantially higher affinity for host receptor angiotensin-converting enzyme 2 (ACE2) than BA.5 and other variants. Structural analyses of BA.2.75 spike shows its decreased thermostability and increased frequency of the receptor binding domain (RBD) in the "up" conformation under acidic conditions, suggesting enhanced low-pH-endosomal cell entry. Relative to BA.4/BA.5, BA.2.75 exhibits reduced evasion of humoral immunity from BA.1/BA.2 breakthrough-infection convalescent plasma but greater evasion of Delta breakthrough-infection convalescent plasma. BA.5 breakthrough-infection plasma also exhibits weaker neutralization against BA.2.75 than BA.5, mainly due to BA.2.75's distinct neutralizing antibody (NAb) escape pattern. Antibody therapeutics Evusheld and Bebtelovimab remain effective against BA.2.75. These results suggest BA.2.75 may prevail after BA.4/BA.5, and its increased receptor-binding capability could support further immune-evasive mutations.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages have escaped most receptor-binding domain (RBD)-targeting therapeutic neutralizing antibodies (NAbs), which proves that previous NAb drug screening strategies are deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd-immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following these criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS-CoV-2-vaccinated SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities.

Abstract Recent emergence of SARS-CoV-2 Omicron sublineages BA.2.12.1, BA.2.13, BA.4 and BA.5 all contain L452 mutations and show potential higher transmissibility over BA.2. The new variants’ receptor binding and immune evasion capability require immediate investigation, especially on the role of L452 substitutions. Herein, coupled with structural comparisons, we showed that BA.2 sublineages, including BA.2.12.1 and BA.2.13, exhibit increased ACE2-binding affinities compared to BA.1; while BA.4/BA.5 shows the weakest receptor-binding activity due to F486V and R493Q reversion. Importantly, compared to BA.2, BA.2.12.1 and BA.4/BA.5 exhibit stronger neutralization escape from the plasma of 3-dose vaccinees and, most strikingly, from vaccinated BA.1 convalescents. To delineate the underlying evasion mechanism, we determined the escaping mutation profiles, epitope distribution and Omicron sub-lineage neutralization efficacy of 1640 RBD-directed neutralizing antibodies (NAbs), including 614 isolated from BA.1 convalescents. Interestingly, post-vaccination BA.1 infection mainly recalls wildtype-induced humoral memory and elicits antibodies that neutralize both wild-type and BA.1. These cross-reactive NAbs are significantly enriched on non-ACE2-competing epitopes; and surprisingly, the majority are undermined by R346 and L452 substitutions, namely R346K (BA.1.1), L452M (BA.2.13), L452Q (BA.2.12.1) and L452R (BA.4/BA.5), suggesting that R346K and L452 mutations appeared under the immune pressure of Omicron convalescents. Nevertheless, BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1 but do not respond to wild-type SARS-CoV-2, due to the high susceptibility to N501, N440, K417 and E484. However, these NAbs are largely escaped by BA.2 sublineages and BA.4/BA.5 due to D405N and F486V, exhibiting poor neutralization breadths. As for therapeutic NAbs, LY-CoV1404 (Bamlanivimab) and COV2-2130 (Cilgavimab) can still effectively neutralize BA.2.12.1 and BA.4/BA.5, while the S371F, D405N and R408S mutations carried by BA.2/BA.4/BA.5 sublineages would undermine most broad sarbecovirus NAbs. Together, our results indicate that Omicron can evolve mutations to specifically evade humoral immunity elicited by BA.1 infection. The continuous evolution of Omicron poses great challenges to SARS-CoV-2 herd immunity and suggests that BA.1-derived vaccine boosters may not be ideal for achieving broad-spectrum protection.

Omicron sub-lineage BA.2 has rapidly surged globally, accounting for over 60% of recent SARS-CoV-2 infections. Newly acquired RBD mutations and high transmission advantage over BA.1 urge the investigation of BA.2's immune evasion capability. Here, we show that BA.2 causes strong neutralization resistance, comparable to BA.1, in vaccinated individuals' plasma. However, BA.2 displays more severe antibody evasion in BA.1 convalescents, and most prominently, in vaccinated SARS convalescents' plasma, suggesting a substantial antigenicity difference between BA.2 and BA.1. To specify, we determined the escaping mutation profiles1,2 of 714 SARS-CoV-2 RBD neutralizing antibodies, including 241 broad sarbecovirus neutralizing antibodies isolated from SARS convalescents, and measured their neutralization efficacy against BA.1, BA.1.1, BA.2. Importantly, BA.2 specifically induces large-scale escape of BA.1/BA.1.1effective broad sarbecovirus neutralizing antibodies via novel mutations T376A, D405N, and R408S. These sites were highly conserved across sarbecoviruses, suggesting that Omicron BA.2 arose from immune pressure selection instead of zoonotic spillover. Moreover, BA.2 reduces the efficacy of S309 (Sotrovimab)3,4 and broad sarbecovirus neutralizing antibodies targeting the similar epitope region, including BD55-5840. Structural comparisons of BD55-5840 in complexes with BA.1 and BA.2 spike suggest that BA.2 could hinder antibody binding through S371F-induced N343-glycan displacement. Intriguingly, the absence of G446S mutation in BA.2 enabled a proportion of 440-449 linear epitope targeting antibodies to retain neutralizing efficacy, including COV2-2130 (Cilgavimab)5. Together, we showed that BA.2 exhibits distinct antigenicity compared to BA.1 and provided a comprehensive profile of SARS-CoV-2 antibody escaping mutations. Our study offers critical insights into the humoral immune evading mechanism of current and future variants.

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 lineage(1). 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 profiles(2), epitope distribution(3) 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 bebtelovimab(4) and cilgavimab(5) can effectively neutralize BA.2.12.1 and BA.4/BA.5, where as 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.

Omicron sub-lineage BA.2 has rapidly surged globally, accounting for over 60% of recent SARS-CoV-2 infections. Newly acquired RBD mutations and high transmission advantage over BA.1 urge the investigation of BA.2’s immune evasion capability. Here, we show that BA.2 causes strong neutralization resistance, comparable to BA.1, in vaccinated individuals’ plasma. However, BA.2 displays more severe antibody evasion in BA.1 convalescents, and most prominently, in vaccinated SARS convalescents’ plasma, suggesting a substantial antigenicity difference between BA.2 and BA.1. To specify, we determined the escaping mutation profiles[1][1],[2][2] of 714 SARS-CoV-2 RBD neutralizing antibodies, including 241 broad sarbecovirus neutralizing antibodies isolated from SARS convalescents, and measured their neutralization efficacy against BA.1, BA.1.1, BA.2. Importantly, BA.2 specifically induces large-scale escape of BA.1/BA.1.1-effective broad sarbecovirus neutralizing antibodies via novel mutations T376A, D405N, and R408S. These sites were highly conserved across sarbecoviruses, suggesting that Omicron BA.2 arose from immune pressure selection instead of zoonotic spillover. Moreover, BA.2 reduces the efficacy of S309 (Sotrovimab)[3][3],[4][4] and broad sarbecovirus neutralizing antibodies targeting the similar epitope region, including BD55-5840. Structural comparisons of BD55-5840 in complexes with BA.1 and BA.2 spike suggest that BA.2 could hinder antibody binding through S371F-induced N343-glycan displacement. Intriguingly, the absence of G446S mutation in BA.2 enabled a proportion of 440-449 linear epitope targeting antibodies to retain neutralizing efficacy, including COV2-2130 (Cilgavimab)[5][5]. Together, we showed that BA.2 exhibits distinct antigenicity compared to BA.1 and provided a comprehensive profile of SARS-CoV-2 antibody escaping mutations. Our study offers critical insights into the humoral immune evading mechanism of current and future variants. ### Competing Interest Statement X.S.X. and Y.C. are listed as inventors on the provisional patent applications of BD series antibodies. X.S.X. and Y.C. are founders of Singlomics Biopharmaceuticals. The remaining authors declare no competing interests. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5

Continuous evolution of Omicron has led to a rapid and simultaneous emergence of numerous variants that display growth advantages over BA.5 (ref. (1)). Despite their divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots. The driving force and destination of such sudden convergent evolution and its effect on humoral immunity remain unclear. Here we demonstrate that these convergent mutations can cause evasion of neutralizing antibody drugs and convalescent plasma, including those from BA.5 breakthrough infection, while maintaining sufficient ACE2-binding capability. BQ.1.1.10 (BQ.1.1 + Y144del), BA.4.6.3, XBB and CH.1.1 are the most antibody-evasive strains tested. To delineate the origin of the convergent evolution, we determined the escape mutation profiles and neutralization activity of monoclonal antibodies isolated from individuals who had BA.2 and BA.5 breakthrough infections(2,3). Owing to humoral immune imprinting, BA.2 and especially BA.5 breakthrough infection reduced the diversity of the neutralizing antibody binding sites and increased proportions of non-neutralizing antibody clones, which, in turn, focused humoral immune pressure and promoted convergent evolution in the RBD. Moreover, we show that the convergent RBD mutations could be accurately inferred by deep mutational scanning profiles(4,5), and the evolution trends of BA.2.75 and BA.5 subvariants could be well foreseen through constructed convergent pseudovirus mutants. These results suggest that current herd immunity and BA.5 vaccine boosters may not efficiently prevent the infection of Omicron convergent variants.

The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) and humoral immunity requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs identified from SARS-CoV/SARS-CoV-2 convalescents and vaccinees. Based on the results, NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlaps with ACE2-binding motif, are largely escaped by K417N, N440K, G446S, E484A, Q493K, and G496S. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, S371L, and S375F. Furthermore, B.1.1.529 pseudovirus neutralization and RBD binding assay showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, LY-CoV016/LY-CoV555 cocktail, REGN-CoV2 cocktail, AZD1061/AZD8895 cocktail, and BRII-196 were escaped by Omicron, while VIR7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron could cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

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 screening(1,2) 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 classifications(3-5). 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.