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.

Spike N354 glycosylation augments SARS-CoV-2 fitness for human adaptation through multiple mechanisms (preprint)

Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity could be recovered in the presence of heparin sulfate, which targets the N354 pocket to ease restrictions of conformational transition resulting in a RBD-up state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms.

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.

Enhanced transmissibility of XBB.1.5 is contributed by both strong ACE2 binding and antibody evasion (preprint)

SARS-CoV-2 recombinant subvariant XBB.1.5 is growing rapidly in the United States, carrying an additional Ser486Pro substitution compared to XBB.1 and outcompeting BQ.1.1 and other XBB sublineages. The underlying mechanism for such high transmissibility remains unclear. Here we show that XBB.1.5 exhibits a substantially higher hACE2-binding affinity compared to BQ.1.1 and XBB/XBB.1. Convalescent plasma samples from BA.1, BA.5, and BF.7 breakthrough infection are significantly evaded by both XBB.1 and XBB.1.5, with XBB.1.5 displaying slightly weaker immune evasion capability than XBB.1. Evusheld and Bebtelovimab could not neutralize XBB.1/XBB.1.5, while Sotrovimab remains its weak reactivity and notably, SA55 is still highly effective. The fact that XBB.1 and XBB.1.5 showed comparable antibody evasion but distinct transmissibility suggests enhanced receptor-binding affinity would indeed lead to higher growth advantages. The strong hACE2 binding of XBB.1.5 could also enable its tolerance of further immune escape mutations, which should be closely monitored.

Monkeypox virus quadrivalent mRNA vaccine induces antibody responses and cellular immunity and protects mice against Vaccinia virus (preprint)

There is an urgent need for efficient and safe vaccines against the monkeypox virus (MPXV) in response to the rapidly spreading monkeypox epidemic. In the age of COVID-19, mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation. Here, we have developed two MPXV quadrivalent mRNA vaccines, named mRNA-A-LNP and mRNA-B-LNP, based on two IMVs (A29L and M1R) and two EEVs (A35R and B6R). By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice, mice have induced MPXV-specific IgG antibodies and potent Vaccinia virus (VACV)-specific neutralizing antibodies. Additionally, it elicited durable MPXV-specific killer memory T-cell immunity as well as memory B-cell immunity in mice. Furthermore, the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protected nude mice against the VACV challenge. In addition, two doses of mRNA-A-LNP and mRNA-B-LNP were also protective against the VACV challenge in mice. Overall, our results demonstrated that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics, as well as against outbreaks caused by other orthopoxviruses, including the smallpox virus.

Inhalable SARS-CoV-2 vaccines for single-dose dry-powder aerosol immunization and orchestrated mucosal/systemic immune responses (preprint)

The ongoing coronavirus disease pandemic has fostered major advances in vaccination technologies; however, there are urgent needs of mucosal immune responses and single-dose, non-invasive administration. Here, we develop a SARS-CoV-2 vaccine for single-dose, dry-powder aerosol inhalation that induces potent systemic and mucosal immune responses. Our vaccine encapsulates proteinaceous cholera toxin B subunit-assembled nanoparticles displaying the SARS-CoV-2 RBD antigen (R-CNP) within microcapsules of optimal aerodynamic size, and such unique nano-micro coupled structure supports efficient alveoli delivery, sustained R-CNP release, and antigen presenting cell uptake, which are favorable for invocation of immune responses. Moreover, our vaccine successfully induces robust serological IgG and secretory IgA production, collectively conferring effective protection from SARS-CoV-2 challenge (including pseudovirus and the authentic virus) in mice, hamsters, and non-human primates. Finally, we also demonstrate a “mosaic iteration” of our vaccine that co-displays ancestral and Omicron’s antigens, thus extending the breadth of antibody response against co-circulating strains and transmission of Omicron variant. These findings support our inhalable vaccine as a promising candidate to prevent SARS-CoV-2 infection, disease, and transmission.

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.

Spike-specific CXCR3+ TFH cells play a dominant functional role in supporting antibody responses in SARS-CoV-2 infection and vaccination (preprint)

CD4+ T follicular helper (TFH) cells are required for high-quality antibody generation and maintenance. However, the longevity and functional role of these cells are poorly defined in COVID-19 convalescents and vaccine recipients. Here, we longitudinally investigated the dynamics and functional roles of spike-specific circulating TFH cells and their subsets in convalescents at the 2nd, 5th, 8th, 12th and 24th months after COVID-19 symptom onset and in vaccinees after two and three doses of inactivated vaccine. SARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, of which spike-specific CXCR3+ TFH cells but not spike-specific CXCR3- TFH cells and neutralizing antibodies were persistent for at least two years in more than 80% of convalescents who experienced symptomatic COVID-19, which was well coordinated between spike-specific TFH cell and antibody responses at the 5th month after infection. Inactivated vaccine immunization also induced spike-specific TFH cell and antibody responses; however, these responses rapidly declined after six months with a two-dose standard administration, and a third dose significantly promoted antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells exhibited better responsiveness than spike-specific CXCR3- TFH cells upon spike protein stimulation in vitro and showed superior capacity in supporting spike-specific antibody secreting cell (ASC) differentiation and antibody production than spike-specific CXCR3- TFH cells cocultured with autologous memory B cells. In conclusion, spike-specific CXCR3+ TFH cells played a dominant functional role in antibody elicitation and maintenance in SARS-CoV-2 infection and vaccination, suggesting that induction of CXCR3-biased spike-specific TFH cell differentiation will benefit SARS-CoV-2 vaccine development aiming to induce long-term protective immune memory.

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.

A subset of Memory B-derived antibody repertoire from 3-dose vaccinees is ultrapotent against diverse and highly transmissible SARS-CoV-2 variants, including Omicron (preprint)

Omicron, the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. We examined whether sera from individuals who received two or three doses of inactivated vaccine, could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2/60) and 95% (57/60) for 2- and 3-dose vaccinees, respectively. For three-dose recipients, the geometric mean neutralization antibody titer (GMT) of Omicron was 15, 16.5-fold lower than that of the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in 3-dose vaccinees, half of which recognize the receptor binding domain (RBD) and show that a subset of them (24/163) neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron, potently. Therapeutic treatments with representative broadly neutralizing mAbs individually or antibody cocktails were highly protective against SARS-CoV-2 Beta infection in mice. Atomic structures of the Omicron S in complex with three types of all five VOC-reactive antibodies defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to one major class of antibodies bound at the right shoulder of RBD through altering local conformation at the binding interface. Our results rationalize the use of 3-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are a rational target for a universal sarbecovirus vaccine. One sentence summary A sub-set of antibodies derived from memory B cells of volunteers vaccinated with 3 doses of an inactivated SARS-CoV-2 vaccine work individually as well as synergistically to keep variants, including Omicron, at bay.

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.