Omicron BQ.1.1 and XBB.1 unprecedentedly escape broadly neutralizing antibodies elicited by prototype vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have seriously attacked the antibody barrier established by natural infection and/or vaccination, especially the recently emerged BQ.1.1 and XBB.1. However, crucial mechanisms underlying the virus escape and the broad neutralization remain elusive. Here, we present a panoramic analysis of broadly neutralizing activity and binding epitopes of 75 monoclonal antibodies isolated from prototype inactivated vaccinees. Nearly all neutralizing antibodies (nAbs) partly or totally lose their neutralization against BQ.1.1 and XBB.1. We report a broad nAb, VacBB-551, that effectively neutralizes all tested subvariants including BA.2.75, BQ.1.1, and XBB.1. We determine the cryoelectron microscopy (cryo-EM) structure of VacBB-551 complexed with the BA.2 spike and perform detailed functional verification to reveal the molecular basis of N460K and F486V/S mutations mediating the partial escape of BA.2.75, BQ.1.1, and XBB.1 from the neutralization of VacBB-551. Overall, BQ.1.1 and XBB.1 raised the alarm over SARS-CoV-2 evolution with unprecedented antibody evasion from broad nAbs elicited by prototype vaccination.

Additional mutations based on Omicron BA.2.75 mediate its further evasion from broadly neutralizing antibodies

SARS-CoV-2 Omicron BA.2.75 subvariant has evolved to a series of progeny variants carrying several additional mutations in the receptor-binding domain (RBD). Here, we investigated whether and how these single mutations based on BA.2.75 affect the neutralization of currently available anti-RBD monoclonal antibodies (mAbs) with well-defined structural information. Approximately 34% of mAbs maintained effective neutralizing activities against BA.2.75, consistent with that against BA.2, BA.4/5, and BA.2.12.1. Single additional R346T, K356T, L452R, or F486S mutations further facilitated BA.2.75-related progeny variants to escape from broadly neutralizing antibodies (bnAbs) at different degree. Only LY-CoV1404 (bebtelovimab) displayed a first-class neutralization potency and breadth against all tested Omicron subvariants. Overall, these data make a clear connection between virus escape and antibody recognizing antigenic epitopes, which facilitate to develop next-generation universal bnAbs against emerging SARS-CoV-2 variants. Graphical

Additional mutations based on Omicron BA.2.75 mediate its further evasion from broadly neutralizing antibodies.

SARS-CoV-2 Omicron BA.2.75 subvariant has evolved to a series of progeny variants carrying several additional mutations in the receptor-binding domain (RBD). Here, we investigated whether and how these single mutations based on BA.2.75 affect the neutralization of currently available anti-RBD monoclonal antibodies (mAbs) with well-defined structural information. Approximately 34% of mAbs maintained effective neutralizing activities against BA.2.75, consistent with those against BA.2, BA.4/5, and BA.2.12.1. Single additional R346T, K356T, L452R, or F486S mutations further facilitated BA.2.75-related progeny variants to escape from broadly neutralizing antibodies (bnAbs) at different degree. Only LY-CoV1404 (bebtelovimab) displayed a first-class neutralization potency and breadth against all tested Omicron subvariants. Overall, these data make a clear connection between virus escape and antibody recognizing antigenic epitopes, which facilitate to develop next-generation universal bnAbs against emerging SARS-CoV-2 variants.

Infection with wild-type SARS-CoV-2 elicits broadly neutralizing and protective antibodies against omicron subvariants.

The omicron variants of SARS-CoV-2 have substantial ability to escape infection- and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants.

Omicron variants escape the persistent SARS-CoV-2-specific antibody response in 2-year COVID-19 convalescents regardless of vaccination.

With the ongoing COVID-19 pandemic and the emergence of various SARS-CoV-2 variants, a comprehensive evaluation of long-term efficacy of antibody response in convalescent individuals is urgently needed. Several longitudinal studies had reported the antibody dynamics after SARS-CoV-2 acute infection, but the follow-up was mostly limited to 1 year or 18 months at the maximum. In this study, we investigated the durability, potency, and susceptibility to immune evasion of SARS-CoV-2-specific antibody in COVID-19 convalescents for 2 years after discharge. These results showed the persistent antibody-dependent immunity could protect against the WT and Delta variant to some extent. However, the Omicron variants (BA.1, BA.2, and BA.4/5) largely escaped this preexisting immunity in recovered individuals. Furthermore, we revealed that inactivated vaccines (BBIBP-CorV, CoronaVac, or KCONVAC) could improve the plasma neutralization and help to maintain the broadly neutralizing antibodies at a certain level. Notably, with the time-dependent decline of antibody, 1-dose or 2-dose vaccination strategy seemed not to be enough to provide immune protection against the emerging variants. Overall, these results facilitated our understanding of SARS-CoV-2-induced antibody memory, contributing to the development of immunization strategy against SARS-CoV-2 variants for such a large number of COVID-19 survivors.

Antigenic sin of wild-type SARS-CoV-2 vaccine shapes poor cross-neutralization of BA.4/5/2.75 subvariants in BA.2 breakthrough infections.

With declining SARS-CoV-2-specific antibody titers and increasing numbers of spike mutations, the ongoing emergence of Omicron subvariants causes serious challenges to current vaccination strategies. BA.2 breakthrough infections have occurred in people who have received the wild-type vaccines, including mRNA, inactivated, or recombinant protein vaccines. Here, we evaluate the antibody evasion of recently emerged subvariants BA.4/5 and BA.2.75 in two inactivated vaccine-immunized cohorts with BA.2 breakthrough infections. Compared with the neutralizing antibody titers against BA.2, marked reductions are observed against BA.2.75 in both 2-dose and 3-dose vaccine groups. In addition, although BA.2 breakthrough infections induce a certain cross-neutralization capacity against later Omicron subvariants, the original antigenic sin phenomenon largely limits the improvement of variant-specific antibody response. These findings suggest that BA.2 breakthrough infections seem unable to provide sufficient antibody protection against later subvariants such as BA.2.75 in the current immunization background with wild-type vaccines.

A key F27I substitution within HCDR1 facilitates the rapid maturation of P2C-1F11-like neutralizing antibodies in a SARS-CoV-2-infected donor.

Although thousands of anti-SARS-CoV-2 monoclonal neutralizing antibodies (nAbs) have been identified and well characterized, some crucial events in the development of these nAbs during viral infection remain unclear. Using deep sequencing, we explore the dynamics of antibody repertoire in a SARS-CoV-2-infected donor, from whom the potent and broad nAb P2C-1F11 (the parent version of Brii-196) was previously isolated. Further analysis shows a rapid clonal expansion of some SARS-CoV-2-specific antibodies in early infection. Longitudinal tracing of P2C-1F11 lineage antibodies reveals that these elite nAbs were rare. Using sequence alignment, structure modeling, and bioactivity analysis based on site-mutated assay, we demonstrate that a key substitution F27I in heavy chain contributes significantly to the maturation of P2C-1F11-like antibodies. Overall, our findings elucidate the developmental process and maturation pathway of P2C-1F11, providing some important information for the design of novel immunogens to elicit more potent nAbs against SARS-CoV-2 infection.

Identification and application of a pair of noncompeting monoclonal antibodies broadly binding to the nucleocapsid proteins of SARS-CoV-2 variants including Omicron.

The SARS-CoV-2 nucleocapsid protein (NP) is an important indicator for the virus infection, highlighting the crucial role of NP-specific monoclonal antibodies (mAbs) used in multiple biochemical assays and clinical diagnosis for detecting the NP antigen. Here, we reported a pair of noncompeting human NP-specific mAbs, named P301-F7 and P301-H5, targeting two distinct linear epitopes on SARS-CoV-2 or SARS-CoV. We evaluated the application of P301-F7 in the analysis of enzyme linked immunosorbent assay, western blot, flow cytometry, immunofluorescence, and focus reduction neutralization test. We for the first time report a broad mAb effectively recognizing various live viruses of SARS-CoV-2 variants including Alpha, Beta, Delta, and Omicron, indicating a wide range of application prospects.

Increased resistance of SARS-CoV-2 Lambda variant to antibody neutralization.

A recently identified SARS-CoV-2 variant, Lambda, has spread to many countries around the world. Here, we measured and evaluated the reduced sensitivity of Lambda variant to the neutralization by plasma polyclonal antibodies elicited by the natural SARS-CoV-2 infection and inactivated vaccine. The combination of two substitutions appearing in the RBD of spike protein (L452Q and F490S) resulted in noticeably reduced neutralization against Lambda variant. F490S contributed more than L452Q in affecting the neutralization. In addition, the neutralization test with 12 published nAbs binding to RBD of SARS-CoV-2 with defined structures suggested that Lambda variant resisted the neutralization by some antibodies from Class 2 and Class 3. Overall, these results suggest that pre-existing antibody neutralization established by natural infection from non-Lambda variants or immunization could be significantly decreased, re-emphasizing the importance of ongoing viral mutation monitoring.

A fourth dose of Omicron RBD vaccine enhances broad neutralization against SARS-CoV-2 variants including BA.1 and BA.2 in vaccinated mice.

The SARS-CoV-2 vaccines have been widely used to build an immunologic barrier in the population against the COVID-19 pandemic. However, a newly emerging Omicron variant, including BA.1, BA.1.1, BA.2, and BA.3 sublineages, largely escaped the neutralization of existing neutralizing antibodies (nAbs), even those elicited by three doses of vaccines. Here, we used the Omicron BA.1 RBD as a fourth dose of vaccine to induce potent Omicron-specific nAbs and evaluated the broadly neutralizing activities against SARS-CoV-2 variants. The BA.1-based vaccine was indeed prone to induce a strain-specific antibody response substantially cross-reactive with BA.2 sublineage, and yet triggered broad neutralization against SARS-CoV-2 variants when it was used in the sequential immunization with WT and other variant vaccines. These results demonstrated that the booster of Omicron RBD vaccine could be a rational strategy to enhance the broadly nAb response.

Sequential immunization with SARS-CoV-2 RBD vaccine induces potent and broad neutralization against variants in mice.

The current COVID-19 pandemic caused by constantly emerging SARS-CoV-2 variants still poses a threat to public health worldwide. Effective next-generation vaccines and optimized booster vaccination strategies are urgently needed. Here, we sequentially immunized mice with a SARS-CoV-2 wild-type inactivated vaccine and a heterologous mutant RBD vaccine, and then evaluated their neutralizing antibody responses against variants including Beta, Delta, Alpha, Iota, Kappa, and A.23.1. These data showed that a third booster dose of heterologous RBD vaccine especially after two doses of inactivated vaccines significantly enhanced the GMTs of nAbs against all SARS-CoV-2 variants we tested. In addition, the WT and variants all displayed good cross-immunogenicity and might be applied in the design of booster vaccines to induce broadly neutralizing antibodies.

Potent and protective IGHV3-53/3-66 public antibodies and their shared escape mutant on the spike of SARS-CoV-2.

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.

Impact of the N501Y substitution of SARS-CoV-2 Spike on neutralizing monoclonal antibodies targeting diverse epitopes.

The emergence and rapid spread of the B.1.1.7 lineage (VOC-202012/01) SARS-CoV-2 variant has aroused global concern. The N501Y substitution is the only mutation in the interface between the RBD of B.1.1.7 and ACE2, raising concerns that its recognition by neutralizing antibodies may be affected. Here, we assessed the neutralizing activity and binding affinity of a panel of 12 monoclonal antibodies against the wild type and N501Y mutant SARS-CoV-2 pseudovirus and RBD protein, respectively. We found that the neutralization activity and binding affinity of most detected antibodies (10 out of 12) were unaffected, although the N501Y substitution decreased the neutralizing and binding activities of CB6 and increased that of BD-23. These findings could be of value in the development of therapeutic antibodies.

Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies.

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus disease 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2-infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). However, the structural basis for their potent neutralizing activity remains unclear. Here, we report cryo-EM structures of the ten most potent nAbs in their native full-length IgG-form or in both IgG-form and Fab-form bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBDs in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of a large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides a structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and its correlation with more potent neutralization and the shedding of S1 subunit.

Human neutralizing antibodies elicited by SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.