Global variation in prior exposure shapes antibody neutralization profiles of SARS-CoV-2 variants up to BA.2.86 (preprint)

The highly mutated SARS-CoV-2 variant, BA.2.86, and its descendants are now the most frequently sequenced variants of SARS-CoV-2. We analyze antibody neutralization data from eight laboratories from the UK, USA, Denmark, and China, including two datasets assessing the effect of XBB.1.5 vaccines, to determine the effect of infection and vaccination history on neutralization of variants up to and including BA.2.86, and produce antibody landscapes to describe these neutralization profiles. We find evidence for lower levels of immune imprinting on pre-Omicron variants in sera collected from Denmark and China, which may be explained by lower levels of circulation of the ancestral variant in these countries, and the use of an inactivated virus vaccine in China.

Antigenicity assessment of SARS-CoV-2 saltation variant BA.2.87.1 (preprint)

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.

Fast evolution of SARS-CoV-2 BA.2.86 to JN.1 under heavy immune pressure (preprint)

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.2.86s 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.

Antigenicity and infectivity characterization of SARS-CoV-2 BA.2.86 (preprint)

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.

Convergent evolution of SARS-CoV-2 XBB lineages on receptor-binding domain 455-456 enhances antibody evasion and ACE2 binding (preprint)

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 like 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. Specifically, L455F and F456L evades Class 1 NAbs, which could reduce 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. Our results indicate the evolution flexibility contributed by epistasis cannot be underestimated, and the evolution potential of SARS-CoV-2 RBD remains high.

Repeated Omicron infection alleviates SARS-CoV-2 immune imprinting (preprint)

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.

Protective effect of plasma neutralization from prior SARS-CoV-2 Omicron infection against BA.5 subvariant symptomatic reinfection (preprint)

From December 2022 to January 2023, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections caused by BA.5 and BF.7 subvariants of B.1.1.529 (Omicron) swept across mainland China. It is crucial to estimate the protective effect of the neutralizing antibodies generated by such mass infections against the next potential SARS-CoV-2 reinfection wave, especially if driven by CH.1.1 or XBB.1.5. Previously, we recruited and continuously followed a cohort of individuals that experienced Omicron BA.1, BA.2, and BA.5 breakthrough infections, as well as a control cohort with no history of SARS-CoV-2 infection. In the previously uninfected cohort, the total symptomatic infection rate surveyed during the outbreak was 91.6%, while the symptomatic reinfection rate was 32.9%, 10.5%, and 2.8% among individuals with prior Omicron BA.1, BA.2 and BA.5 infection, respectively, with median intervals between infections of 335, 225 and 94 days. Pseudovirus neutralization assays were performed in plasma samples collected from previously Omicron BA.1-infected individuals approximately 3 months before the outbreak. Results indicate a robust correlation between the plasma neutralizing antibody titers and the protective effect against symptomatic reinfection. The geometric mean of the 50% neutralizing titers (NT50) against D614G, BA.5, and BF.7 were 2.0, 2.5, and 2.3-fold higher in individuals without symptomatic reinfection than in those with symptomatic reinfection (p < 0.01). Low plasma neutralizing antibody titer (below the geometric mean of NT50) was associated with an enhanced cumulative risk of symptomatic reinfection, with a hazard ratio (HR) of 23.55 (95% CI: 9.23-60.06) against BF.7 subvariant. Importantly, neutralizing antibodies titers post one month after BF.7/BA.5 breakthrough infections against CH.1.1 and XBB.1.5 are similar to that against BF.7 from individuals with prior BA.1 infection while not experiencing a symptomatic BF.7/BA.5 reinfection (plasma collected 3 months before the outbreak), suggesting that the humoral immunity generated by the current BF.7/BA.5 breakthrough infection may provide protection against CH.1.1 and XBB.1.5 symptomatic reinfection wave for 4 months. Of note, the higher hACE2 binding of XBB.1.5 may reduce the protection period since the potential increase of infectivity.

Imprinted SARS-CoV-2 humoral immunity induces converging Omicron RBD evolution (preprint)

Continuous evolution of Omicron has led to numerous subvariants that exhibits growth advantage over BA.5. Such rapid and simultaneous emergence of variants with enormous advantages is unprecedented. Despite their rapidly divergent evolutionary courses, mutations on their receptor-binding domain (RBD) converge on several hotspots, including R346, R356, K444, L452, N460K and F486. The driving force and destination of such convergent evolution and its impact on humoral immunity established by vaccination and infection remain unclear. Here we demonstrate that these convergent mutations can cause striking evasion of convalescent plasma, including those from BA.5 breakthrough infection, and existing antibody drugs, including Evusheld and Bebtelovimab. BA.2.75.2 is the most evasive strain tested, and only BQ.1.1 could compare. To clarify the origin of the convergent evolution, we determined the escape mutation profiles and neutralization activity of monoclonal antibodies (mAbs) isolated from convalescents of BA.2 and BA.5 breakthrough infection. Importantly, due to humoral immune imprinting, BA.2 and especially BA.5 breakthrough infection caused significant reductions of neutralizing antibody epitope diversity and increased proportion of non-neutralizing mAbs, which in turn concentrated humoral immune pressure and promoted the convergent RBD evolution. Additionally, the precise convergent RBD mutations and evolution trends of BA.2.75/BA.5 subvariants could be inferred by integrating the neutralization-weighted DMS profiles of mAbs from various immune histories (3051 mAbs in total). Moreover, we demonstrated that as few as five additional convergent mutations based on BA.5 or BA.2.75 could completely evade most plasma samples, including those from BA.5 breakthrough infections, while remaining sufficient hACE2-binding affinity. These results suggest herd immunity established by natural infection could hardly stop RBD evolution, and vaccine boosters using BA.5 may not provide sufficiently broad protection. Broad-spectrum SARS-CoV-2 vaccines and NAb drugs development should be in high priority and the constructed convergent mutants could serve to examine their effectiveness in advance.

Further humoral immunity evasion of emerging SARS-CoV-2 BA.4 and BA.5 subvariants (preprint)

Multiple BA.4 and BA.5 subvariants with R346 mutations on the spike glycoprotein have been identified in various countries, such as BA.4.6/BF.7 harboring R346T, BA.4.7 harboring R346S, and BA.5.9 harboring R346I. These subvariants, especially BA.4.6, exhibit substantial growth advantages compared to BA.4/BA.5. In this study, we showed that BA.4.6, BA.4.7, and BA.5.9 displayed higher humoral immunity evasion capability than BA.4/BA.5, causing 1.5 to 1.9-fold decrease in NT50 of the plasma from BA.1 and BA.2 breakthrough-infection convalescents compared to BA.4/BA.5. Importantly, plasma from BA.5 breakthrough-infection convalescents also exhibits significant neutralization activity decrease against BA.4.6, BA.4.7, and BA.5.9 than BA.4/BA.5, showing on average 2.4 to 2.6-fold decrease in NT50. For neutralizing antibody drugs, Bebtelovimab remains potent, while Evusheld is completely escaped by these subvariants. Together, our results rationalize the prevailing advantages of the R346 mutated BA.4/BA.5 subvariants and urge the close monitoring of these mutants, which could lead to the next wave of the pandemic.

Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents (preprint)

SARS-CoV-2 Omicron sublineages have escaped most RBD-targeting therapeutic neutralizing antibodies (NAbs), which proves the previous NAb drug screening strategies 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 the criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from 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. Our screening strategy can also be further applied to identify broad-spectrum NAb drugs against other fast-evolving viruses.

Neutralizing antibody evasion and receptor binding features of SARS-CoV-2 Omicron BA.2.75 (preprint)

The Omicron subvariants BA.2.75 is rapidly raising in India. BA.2.75 also shows a local growth advantage compared to BA.2.38 and BA.4/BA.5. Its immune evasion capability and receptor binding affinity is unclear and requires investigation. Here, we show that BA.2.75 is more neutralization evasive than BA.2.12.1 against the plasma from post-vaccination BA.2 infection, but less compared to BA.4/BA.5. However, as shown in a small sample of plasma from post-vaccination Delta infection, BA.2.75 seems to be more immune evasive than BA.4/BA.5 in Delta-stimulated immune background, which may explain BA. 2.75's growth advantage over BA.4/BA.5 in India. The additional N460K, G446S, D339H and R493Q mutations carried by BA.2.75 allows it to escape BA.2-effective neutralizing antibodies of different RBD epitopes, and BA.2.75 has a distinct antibody escaping profile from BA.4/BA.5. Compared to BA.2, REGN10933 and COV2-2196 partially recovered neutralization against BA.2.75 due to R493Q reversion. However, the efficacy of their corresponding cocktail was not significantly changed, since REGN10987 and COV2-2130 showed reduced neutralizing activity due to G446S. BA.2.75 exhibits higher ACE2-binding affinity than BA.4/BA.5, which should be contributed by R493Q and N460K, according to deep mutational scanning (DMS) results. This affinity-strengthening feature is being further examined and verified, which will be updated soon.

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

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 sublineage 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 (WT) induced humoral memory and elicits antibodies that neutralize both WT 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 WT 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 (Bebtelovimab) 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.

BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron BA.1 infection (preprint)

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.

Comprehensive Epitope Mapping of Broad Sarbecovirus Neutralizing Antibodies (preprint)

Constantly emerging SARS-CoV-2 variants, such as Omicron BA.1, BA.1.1 and BA.2, pose a severe challenge to COVID-19 control. Broad-spectrum antibody therapeutics and vaccines are needed for defending against future SARS-CoV-2 variants and sarbecovirus pandemics; however, we have yet to gain a comprehensive understanding of the epitopes capable of inducing broad sarbecovirus neutralization. Here, we report the identification of 241 anti-RBD broad sarbecovirus neutralizing antibodies isolated from 44 SARS-CoV-2 vaccinated SARS convalescents. Neutralizing efficacy of these antibodies against D614G, SARS-CoV-1, Omicron variants (BA.1, BA.1.1, BA.2), RATG13 and Pangolin-GD is tested, and their binding capability to 21 sarbecovirus RBDs is measured. High-throughput yeast-display mutational screening was further applied to determine each antibody's RBD escaping mutation profile, and unsupervised epitope clustering based on escaping mutation hotspots was performed. A total of 6 clusters of broad sarbecovirus neutralizing antibodies with diverse breadth and epitopes were identified, namely Group E1 (S309, BD55-3152 site), E3 (S2H97 site), F1 (CR3022, S304 site), F2 (DH1047, BD55-3500 site), F3 (ADG-2, BD55-3372 site) and B' (S2K146 site). Members of E1, F2 and F3 demonstrate the highest neutralization potency; yet, Omicron, especially BA.2, has evolved multiple mutations (G339D, N440K, T376A, D405N, R408S) to escape antibodies of these groups. Nevertheless, broad sarbecovirus neutralizing antibodies that survived Omicron would serve as favorable therapeutic candidates. Furthermore, structural analyses of selected drug candidates propose two non-competing antibody pairing strategies, E1-F2 and E1-F3, as broad-spectrum antibody cocktails. Together, our work provides a comprehensive epitope map of broad sarbecovirus neutralizing antibodies and offers critical instructions for designing broad-spectrum vaccines.

B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes (preprint)

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.

B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes (preprint)

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.

A third dose of inactivated vaccine augments the potency, breadth, and duration of anamnestic responses against SARS-CoV-2 (preprint)

Emergence of variants of concern (VOC) with altered antigenic structures and waning humoral immunity to SARS-CoV-2 are harbingers of a long pandemic. Administration of a third dose of an inactivated virus vaccine can boost the immune response. Here, we have dissected the immunogenic profiles of antibodies from 3-dose vaccinees, 2-dose vaccinees and convalescents. Better neutralization breadth to VOCs, expeditious recall and long-lasting humoral response bolster 3-dose vaccinees in warding off COVID-19. Analysis of 171 complex structures of SARS-CoV-2 neutralizing antibodies identified structure-activity correlates, revealing ultrapotent, VOCs-resistant and broad-spectrum antigenic patches. Construction of immunogenic and mutational heat maps revealed a direct relationship between "hot" immunogenic sites and areas with high mutation frequencies. Ongoing antibody somatic mutation, memory B cell clonal turnover and antibody composition changes in B cell repertoire driven by prolonged and repeated antigen stimulation confer development of monoclonal antibodies with enhanced neutralizing potency and breadth. Our findings rationalize the use of 3-dose immunization regimens for inactivated vaccines. One sentence summaryA third booster dose of inactivated vaccine produces a highly sifted humoral immune response via a sustained evolution of antibodies capable of effectively neutralizing SARS-CoV-2 variants of concern.

Structures of potent and convergent neutralizing antibodies bound to the SARS-CoV-2 spike unveil a unique epitope responsible for exceptional potency (preprint)

Understanding the mechanism of neutralizing antibodies (NAbs) against SARS-CoV-2 is critical for effective vaccines and therapeutics development. We recently reported an exceptionally potent NAb, BD-368-2, and revealed the existence of VH3-53/VH3-66 convergent NAbs in COVID-19. Here we report the 3.5-[A] cryo-EM structure of BD-368-2s Fabs in complex with a mutation-induced prefusion-state-stabilized spike trimer. Unlike VH3-53/VH3-66 NAbs, BD-368-2 fully blocks ACE2 binding by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" and "down" positions. BD-368-2 also triggers fusogenic-like structural rearrangements of the spike trimer, which could impede viral entry. Moreover, BD-368-2 completely avoids the common epitope of VH3-53/VH3-66 NAbs, evidenced by multiple crystal structures of their Fabs in tripartite complexes with RBD, suggesting a new way of pairing potent NAbs to prevent neutralization escape. Together, these results rationalize a unique epitope that leads to exceptional neutralization potency, and provide guidance for NAb therapeutics and vaccine designs against SARS-CoV-2.