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 Δ42PD1 fusion-expressing DNA vaccine elicits enhanced adaptive immune response to HIV-1: the key role of TLR4.

Since its discovery in the 1990s, the DNA vaccine has been of great interest because of its ability to elicit both humoral and cellular immune responses while showing relative advantages regarding producibility, stability and storage. However, when applied to human subjects, inadequate immunogenicity remains as the greatest challenge for the practical use of DNA vaccines. In this study, we generated a DNA vaccine Δ42PD1-P24 encoding a fusion protein comprised of the HIV-1 Gag p24 antigen and the extracellular domain of murine Δ42PD1, a novel endogenous Toll-like receptor 4 (TLR4) agonist. Using a mouse model, we found that Δ42PD1-P24 DNA vaccine elicited a higher antibody response and an increased number of IFN-γ-producing CD4 and CD8 T cells. Moreover, mice with Δ42PD1-P24 DNA vaccination were protected from a subcutaneous challenge with murine mesothelioma cells expressing the HIV-1 p24 antigen. Importantly, the Δ42PD1-mediated enhancement of immune responses was not observed in TLR4 knockout mice. Collectively, these data demonstrate that the immunogenicity and efficacy of DNA vaccines could be improved by the fusion of the extracellular domain of Δ42PD1 to target the immunogen to dendritic cells.

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.

SARS-CoV-2 Omicron Variants Reduce Antibody Neutralization and Acquire Usage of Mouse ACE2

Striking number of mutations found in the spike protein of recently emerged SARS-CoV-2 Omicron subvariants BA.1, BA.2, BA.3 and BA.4/5 has raised serious concerns regarding the escape from current antibody therapies and vaccine protection. Here, we conducted comprehensive analysis on the extent of two major Omicron lineages BA.1/BA.1.1 and BA.2 to escape neutralization from the therapeutic antibodies approved by the regulatory authorities and convalescent plasma from SARS-CoV-2 patients infected during initial wave of pandemic in early 2020. We showed that Omicron BA.1/BA.1.1 were the most resistant in both magnitude and breadth against antibodies and convalescent plasma, followed by Beta, BA.2, Gamma, Delta and Alpha. While the majority of therapeutic antibodies lost binding and neutralization to Omicron variants, BRII combo (BRII-196 + BRII-198), S309, and AZ combo (COV2-2196 + COV2-2130) maintained neutralization despite of reduction due to either conserved epitope or combinational effect between the two designated antibodies. A single intraperitoneal injection of BRII combo as a prophylactic treatment protected animals from Omicron infection. Treated animals manifested normal body weight, survived infection up to 14 days, undetectable levels of infectious viruses in the lungs, and reduced lung pathology compared to the controls. Analyzing ACE2 from diverse host species showed that Omicron variants acquired ability to use mouse ACE2 for entry. These results demonstrate major antigenic shifts and potentially broadening the host range of two major Omicron lineages BA.1/BA.1.1 and BA.2, posing serious challenges to current antibody therapies and vaccine protection as well as increasing danger of spillover into the wildlife.

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.

Structural and functional analysis of an inter-Spike bivalent neutralizing antibody against SARS-CoV-2 variants.

The different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have attracted most public concern because they caused "wave and wave" COVID-19 pandemic. The initial step of viral infection is mediated by the SARS-CoV-2 Spike (S) protein, which mediates the receptor recognition and membrane fusion between virus and host cells. Neutralizing antibodies (nAbs) targeting the S protein of SARS-CoV-2 have become promising candidates for clinical intervention strategy, while multiple studies have shown that different variants have enhanced infectivity and antibody resistance. Here, we explore the structure and function of STS165, a broadly inter-Spike bivalent nAb against SARS-CoV-2 variants and even SARS-CoV, contributing to further understanding of the working mechanism of nAbs.

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.

SCIGA: Software for large-scale, single-cell immunoglobulin repertoire analysis.

BACKGROUND: B-cell immunoglobulin repertoires with paired heavy and light chain can be determined by means of 10X single-cell V(D)J sequencing. Precise and quick analysis of 10X single-cell immunoglobulin repertoires remains a challenge owing to the high diversity of immunoglobulin repertoires and a lack of specialized software that can analyze such diverse data. FINDINGS: In this study, specialized software for 10X single-cell immunoglobulin repertoire analysis was developed. SCIGA (Single-Cell Immunoglobulin Repertoire Analysis) is an easy-to-use pipeline that performs read trimming, immunoglobulin sequence assembly and annotation, heavy and light chain pairing, statistical analysis, visualization, and multiple sample integration analysis, which is all achieved by using a 1-line command. Then SCIGA was used to profile the single-cell immunoglobulin repertoires of 9 patients with coronavirus disease 2019 (COVID-19). Four neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified from these repertoires. CONCLUSIONS: SCIGA provides a complete and quick analysis for 10X single-cell V(D)J sequencing datasets. It can help researchers to interpret B-cell immunoglobulin repertoires with paired heavy and light chain.

Antibody neutralization of SARS-CoV-2 through ACE2 receptor mimicry.

Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.