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 potent neutralizing antibody provides protection against SARS-CoV-2 Omicron and Delta variants via nasal delivery.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still rapidly spreading worldwide. Many drugs and vaccines have been approved for clinical use show efficacy in the treatment and prevention of SARS-CoV-2 infections. However, the emergence of SARS-CoV-2 variants of concern (VOCs), such as Delta (B.1.617.2) and the recently emerged Omicron (B.1.1.529), has seriously challenged the application of current therapeutics. Therefore, there is still a pressing need for identification of new broad-spectrum antivirals. Here, we further characterized a human antibody (58G6), which we previously isolated from a patient, with a broadly authentic virus-neutralizing activity that inhibits the Delta and Omicron variants with half-maximal inhibitory concentrations (IC50) of 1.69 ng/ml and 54.31 ng/ml, respectively. 58G6 shows prophylactic and therapeutic efficacy in hamsters challenged with the Delta and Omicron variants through nasal delivery. Notably, a very low dosage (2 mg/kg daily) of 58G6 efficiently prevented Omicron variant replication in the lungs. These advantages may overcome the efficacy limitation of currently approved neutralizing antibodies that can be administered only by intravenous injection. In general, 58G6 is a promising prophylactic and therapeutic candidate against current circulating VOCs and even future emerging mutants. To the best of our knowledge, 58G6 is one of the most potent neutralizing antibodies against Omicron, with a broader spectrum than those approved for clinical use. 58G6 could be developed as a nebulized therapy, which would be more cost effective and user friendly and enhance the clinical outcome compared to that obtained with direct nasal delivery.

Structures of Omicron spike complexes and implications for neutralizing antibody development.

The emergence of the SARS-CoV-2 Omicron variant is dominant in many countries worldwide. The high number of spike mutations is responsible for the broad immune evasion from existing vaccines and antibody drugs. To understand this, we first present the cryo-electron microscopy structure of ACE2-bound SARS-CoV-2 Omicron spike. Comparison to previous spike antibody structures explains how Omicron escapes these therapeutics. Secondly, we report structures of Omicron, Delta, and wild-type spikes bound to a patient-derived Fab antibody fragment (510A5), which provides direct evidence where antibody binding is greatly attenuated by the Omicron mutations, freeing spike to bind ACE2. Together with biochemical binding and 510A5 neutralization assays, our work establishes principles of binding required for neutralization and clearly illustrates how the mutations lead to antibody evasion yet retain strong ACE2 interactions. Structural information on spike with both bound and unbound antibodies collectively elucidates potential strategies for generation of therapeutic antibodies.

Reduced neutralization of SARS-CoV-2 B.1.617 variant by convalescent and vaccinated sera.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Spike protein that mediates coronavirus entry into host cells is a major target for COVID-19 vaccines and antibody therapeutics. However, multiple variants of SARS-CoV-2 have emerged, which may potentially compromise vaccine effectiveness. Using a pseudovirus-based assay, we evaluated SARS-CoV-2 cell entry mediated by the viral Spike B.1.617 and B.1.1.7 variants. We also compared the neutralization ability of monoclonal antibodies from convalescent sera and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine) and ZF2001 (RBD-subunit vaccine) against B.1.617 and B.1.1.7 variants. Our results showed that, compared to D614G and B.1.1.7 variants, B.1.617 shows enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization. These findings have important implications for understanding viral infectivity and for immunization policy against SARS-CoV-2 variants.

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.

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.

A non-RBM targeted RBD specific antibody neutralizes SARS-CoV-2 inducing S1 shedding.

Potent neutralizing antibodies (Abs) have been proven with therapeutic efficacy for the intervention against SARS-CoV-2. Majority of these Abs function by directly interfering with the virus entry to host cells. Here, we identified a receptor binding domain (RBD) specific monoclonal Ab (mAb) 82A6 with efficient neutralizing potency against authentic SARS-CoV-2 virus. As most Abs targeting the non-receptor binding motif (RBM) region, 82A6 was incapable to block the RBD-ACE2 interaction. In particular, it actively promoted the S1 subunit shedding from the S protein, which may lead to effective reduction of intact SARS-CoV-2 viruses. Importantly, it could block potential syncytia formation associated with post-infectious cell surface expression of S proteins. Our study evidenced a RBD specific Ab with unique beneficial efficacy against SARS-CoV-2 infection, which might bring informative significance to understand the collective effects of neutralizing Abs elicited in COVID-19 patients.

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.