Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

Neutralizing antibodies from the rare convalescent donors elicited antibody-dependent enhancement of SARS-CoV-2 variants infection.

Currently, neutralizing antibody and vaccine strategies have been developed by targeting the SARS-CoV-2 strain identified during the early phase of the pandemic. Early studies showed that the ability of SARS-CoV-2 RBD or NTD antibodies to elicit infection enhancement in vivo is still controversial. There are growing concerns that the plasma and neutralizing antibodies from convalescent patients or people receiving vaccines mediate ADE of SARS-CoV-2 variants infections in immune cells. Here, we constructed engineered double-mutant variants containing an RBD mutation and D614G in the spike (S) protein and natural epidemic variants to gain insights into the correlation between the mutations in S proteins and the ADE activities and tested whether convalescent plasma and TOP10 neutralizing antibodies in our laboratory mediated the ADE effects of these SARS-CoV-2 variants. We found that one out of 29 convalescent plasma samples caused the ADE effect of pandemic variant B.1.1.7 and that the ADE effect of wild-type SARS-CoV-2 was not detected for any of these plasma samples. Only one antibody, 55A8, from the same batch of convalescent patients mediated the ADE effects of multiple SARS-CoV-2 variants in vitro, including six double-mutant variants and four epidemic variants, suggesting that ADE activities may be closely related to the antibody itself and the SARS-CoV-2 variants' S proteins. Moreover, the ADE activity of 55A8 depended on FcγRII on immune cells, and the introduction of LALA mutations at the Fc end of 55A8 eliminated the ADE effects in vitro, indicating that 55A8LALA may be a clinical drug used to prevent SARS-CoV-2 variants. Altogether, ADE may occur in rare convalescent patients or vaccinees with ADE-active antibodies who are then exposed to a SARS-CoV-2 variant. These data suggested that potential neutralizing antibodies may need to undergo ADE screening tests for SARS-CoV-2 variants, which should aid in the future design of effective antibody-based therapies.

Extended SARS-CoV-2 RBD booster vaccination induces humoral and cellular immune tolerance in mice.

The repetitive applications of vaccine boosters have been brought up in face of continuous emergence of SARS-CoV-2 variants with neutralization escape mutations, but their protective efficacy and potential adverse effects remain largely unknown. Here, we compared the humoral and cellular immune responses of an extended course of recombinant receptor binding domain (RBD) vaccine boosters with those from conventional immunization strategy in a Balb/c mice model. Multiple vaccine boosters after the conventional vaccination course significantly decreased RBD-specific antibody titers and serum neutralizing efficacy against the Delta and Omicron variants, and profoundly impaired CD4+ and CD8+T cell activation and increased PD-1 and LAG-3 expressions in these T cells. Mechanistically, we confirmed that extended vaccination with RBD boosters overturned the protective immune memories by promoting adaptive immune tolerance. Our findings demonstrate potential risks with the continuous use of SARS-CoV-2 vaccine boosters, providing immediate implications for the global COVID-19 vaccination enhancement strategies.

A Highly Conserved Peptide Vaccine Candidate Activates Both Humoral and Cellular Immunity Against SARS-CoV-2 Variant Strains.

Facing the imminent need for vaccine candidates with cross-protection against globally circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants, we present a conserved antigenic peptide RBD9.1 with both T-cell and B-cell epitopes. RBD9.1 can be recognized by coronavirus disease 2019 (COVID-19) convalescent serum, particularly for those with high neutralizing potency. Immunization with RBD9.1 can successfully induce the production of the receptor-binding domain (RBD)-specific antibodies in Balb/c mice. Importantly, the immunized sera exhibit sustained neutralizing efficacy against multiple dominant SARS-CoV-2 variant strains, including B.1.617.2 that carries a point mutation (SL452R) within the sequence of RBD9.1. Specifically, SY451 and SY454 are identified as the key amino acids for the binding of the induced RBD-specific antibodies to RBD9.1. Furthermore, we have confirmed that the RBD9.1 antigenic peptide can induce a S448-456 (NYNYLYRLF)-specific CD8+ T-cell response. Both RBD9.1-specific B cells and the S448-456-specific T cells can still be activated more than 3 months post the last immunization. This study provides a potential vaccine candidate that can generate long-term protective efficacy over SARS-CoV-2 variants, with the unique functional mechanism of activating both humoral and cellular immunity.

T Cell Immunity Evaluation and Immunodominant Epitope T Cell Receptor Identification of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Glycoprotein in COVID-19 Convalescent Patients.

A better understanding of the role of T cells in the immune response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is helpful not only for vaccine development but also for the treatment of COVID-19 patients. In this study, we determined the existence of SARS-CoV-2-specific T cells in the blood of COVID-19 convalescents. Meanwhile, the specific T cell response in the non-RBD region was stronger than in the RBD region. We also found that SARS-CoV-2 S-specific reactive CD4+ T cells exhibited higher frequency than CD8+ T cells in recovered COVID-19 patients, with greater number of corresponding epitopes presented. Importantly, we isolated the SARS-CoV-2-specific CD4+ T cell receptors (TCRs) and inserted the TCRs into allogenic CD4+ T cells. These TCR-T cells can be activated by SARS-CoV-2 spike peptide and produce IFN-γ in vitro. These results might provide valuable information for the development of vaccines and new therapies against COVID-19.

Potent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants.

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

Identification of cross-reactive CD8+ T cell receptors with high functional avidity to a SARS-CoV-2 immunodominant epitope and its natural mutant variants.

Despite the growing knowledge of T cell responses in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here, with a predicted peptide library from SARS-CoV-2 S and N proteins, we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein, N361-369 (KTFPPTEPK), was the most dominant epitope from our selected peptide library. Importantly, we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants, as to the wild-type epitope. Further, the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally, this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains.

A Rapid and Efficient Screening System for Neutralizing Antibodies and Its Application for SARS-CoV-2.

After the pandemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 have been developed for the prophylactic and therapeutic purposes. However, few methodologies are described in detail on how to rapidly and efficiently generate effective NAbs to SARS-CoV-2. Here, we integrated and optimized a strategically screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific NAbs within 6 days, followed by additional 9 days for antibody production and function analysis. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited advanced neutralizing potency and high affinity, with the top two NAbs showing half-maximal inhibitory concentration (IC50) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides an effective methodology with high applicable value for discovering potential preventative and therapeutic NAbs for the emerging infectious diseases.

Immune characteristics analysis reveals two key inflammatory factors correlated to the expressions of SARS-CoV-2 S1-specific antibodies.

The pandemic of COVID-19 caused by SARS-CoV-2 has made serious threats to the public health. Antibodies have been considered as promising therapeutics for the prevention and treatment of pathogens. So far, effectors that can influence the sustainability of SARS-CoV-2 specific antibodies in COVID-19 patients are still unclear. In this paper, we attempted to find potential key factors correlated with SARS-CoV-2 specific antibodies. Transcriptional analysis with the peripheral blood mononuclear cells (PBMCs) revealed proportional changes of immune cell subsets in COVID-19 convalescent patients, including a substantial decrease of monocytes and evident increase of dendritic cells (DCs). Moreover, we found that the gene expressions of chemokines associated with monocyte/macrophage were significantly up-regulated during the COVID-19 recovery phase. Most importantly, we found a set of 27 immune genes corresponding to a comparatively lower amount of SARS-CoV-2 specific antibodies, and identified two hub genes, IL1ß and IL6, the protein expressions of which exhibited negative correlation with the immunoglobulin G (IgG) levels in COVID-19 convalescent sera. In addition, we found that high expressions of these 2 hub genes during the convalescent stage were negatively associated with the plasma cell marker CD138. Our study presented two key inflammatory factors correlated to the low level of SARS-CoV-2 specific antibodies, which indicated the potential regulatory process of plasmatic antibodies levels in some COVID-19 convalescent patients.