| Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),an enveloped single-stranded RNA virus,belongs to the Sarbecovirus subgenus of betacoronaviruses and is acknowledged as the novel coronavirus that causes the global pandemic of COVID-19.The rapid global spread and transmission of SARS-CoV-2 are hypothesized to provide the virus with substantial opportunities for the natural selection of favorable mutations,many of which involved mutations in the spike protein(S).Mutations in the spike protein(S)may enhance viral transmission and show resistant to multiple neutralizing monoclonal antibodies.Therefore,development of antibodies with broad-spectrum activities against SARS-CoV-2 and SARS-CoV-2 variants and synergistic use of antibodies with different epitopes should be investigated for developing future therapeutic antibodies.In this study,12 monoclonal antibodies were screened,and Characterized.Succinctly,two antibodies(F61 and H121)obtained within our study were demonstrated with high affinity and high-neutralizing activity against multiple SARS-CoV-2 variants.In this study,immune antibody phage display libraries were successfully constructed by amplifying the antibody gene from convalescent donors in early outbreaks.The library has high capacity and diversity,which meets the needs of antibody screening.The libraries were panned and screened with purified SARS-CoV-2 RBD protein,S1 protein and S2 protein following the standard panning procedure and 12 high affinity antibodies were generated.Nine antibodies screened with purified RBD and S1 were all positive to RBD.The rest three of them were found attached to S2.However,N terminal domain(NTD)specific antibodies were not screened and identified.Of the nine RBD-binding antibodies,two antibodies(F61 and H121)showed high affinity neutralization against SARS-CoV-2,whereas a high affinity antibody(A199)failed to neutralize SARS-CoV-2,which suggested not all RBD-specific antibodies were neutralizing antibodies.Following structure analysis,nine RBD-binding antibodies can be classified into three types,one of which is mainly represented by F61 to recognize linear epitope located in residues G446-S494 with in angiotensin-converting enzyme 2(ACE-2)binding sites.One is represented by H121 to identify epitopes which not overlap with ACE-2 binding sites,but it has high neutralization activity.Results above suggested that neutralizing capacities of antibodies did not rely on their ability to block RBD-ACE-2 interaction.And the last one is A199 which bound to a non-overlapping epitope with ACE-2 binding sites and had no neutralization activity.The neutralizing activity of nine RBD-binding antibodies against pseudovirus with mutant S protein was evaluated via neutralization assay.Multiple mutations on S protein showed no resistant to F61 and·F163.Importantly,F61 and H121 exhibited efficient neutralizing activity against variants B.1.1.7 and B.1.351.These two antibodies can be candidate to therapeutic antibodies.The cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2,B.1.1.7 and B.1.351,which revealed a broad neutralizing activity against SARS-CoV-2 variants,and mitigated the risk of viral escape.Succinctly,two antibodies(F61 and H121)obtained within our study were demonstrated with high affinity and high-neutralizing activity against distinct RBD epitopes.Meanwhile,Neutralizing activity of F61 and H121 was mostly maintained when tested against multiple mutations and variant B.1.1.7 and B.1.351,which revealed a broad neutralizing activity against SARS-CoV-2 variants.The cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2,B.1.1.7 and B.1.351,which mitigated the risk of viral escape.Our findings defined the basis of therapeutic cocktails of F61 and H121 with broad neutralization and delivered a guideline for the current and future vaccine design,therapeutic antibody development,and antigen diagnosis of SARS-CoV-2 and its novel variants. |
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