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[PMC free article] [PubMed] [Google Scholar] 2. unique function and epitope acknowledgement were selected to understand SARS\CoV\2 neutralization. High\affinity RBD\specific antibodies exhibited high potency in neutralizing both live and pseudotype SARS\CoV\2 viruses and the SARS\CoV\2 pseudovirus particle made up of the spike protein S\RBDV367F mutant (SARS\CoV\2(V367F)). These results demonstrated that these antibodies recognize four unique groups (ICIV) of epitopes around the RBD and that Oxantel Pamoate mAbs targeting group I epitope can be Oxantel Pamoate used in combination with mAbs realizing groups II and/or IV epitope to make mAb cocktails against Oxantel Pamoate SARS\CoV\2 and its mutants. Moreover, structural characterization reveals that groups I, III, and IV Colec11 epitopes are closely located to an RBD hotspot. The identification of RBD\specific antibodies and cocktails may provide an effective therapeutic and prophylactic intervention against SARS\CoV\2 and its isolates. genus and shares high sequence homology with SARS\CoV (82%). 1 The emergence of SARS\CoV\2 posed a serious global health emergency; it spread rapidly worldwide, leading to a COVID\19 pandemic that infected more than 79 million people and killed over 1.7 million. Several studies have shown the protective functions of neutralizing antibodies against SARS\CoV\2. Plasma from convalescent individuals, which contains neutralizing antibodies, inhibited computer virus infection and has a potential for therapeutic interventions. 2 , 3 Given the limitations of plasma for therapeutic use, several research groups have recognized neutralizing antibody candidates from humanized mice and convalescent individuals. 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 SARS\CoV\2 and SARS\CoV infect host cells in a similar mechanism. These viruses apply angiotensin\transforming enzyme 2 (ACE2) as a cell receptor for viral access Oxantel Pamoate through their transmembrane spike glycoprotein (S). 12 , 13 , 14 , 15 , 16 , 17 The SARS\CoV\2 S protein have two functional subunits: an N\terminal S1 domain name and a C\terminal S2 domain name (S2 domain name protruding from your viral surface). RBD domain name of the S1 tightly binds to the peptidase domain name of ACE2, playing key functions in determining host range, tropism, and infectivity. The coronavirus RBD is the main target of neutralizing antibodies and has been a focus of vaccine design and therapeutic efforts. 18 , 19 , 20 , 21 SARS\CoV and the Middle East respiratory syndrome coronavirus RBD\based antibodies have shown neutralization activities in the previous studies. 22 The SARS\CoV\2 RBD shares 50% sequence identity with that of SARS\CoV, explaining why formerly SARS\CoV antibodies could not neutralize SARS\CoV\2. 23 The neutralizing anti\RBD monoclonal antibodies (mAbs) are believed to disrupt the virusCreceptor engagement. Several potent neutralizing antibodies from convalescent patients, which identify the SARS\CoV\2 RBD, have been recently reported. 5 , 6 , 7 , 8 , 9 , 10 , 11 This study explained the identification of potent neutralizing RBD\specific mAbs from mice vaccinated by a recombinant SARS\CoV\2 RBD. These antibodies exhibited a higher binding affinity to the RBD and high potency to neutralize both live and pseudotype SARS\CoV\2 viruses and SARS\CoV\2(V367F) pseudovirus. Overall, these antibodies identify four unique epitopes around the RBD, and cocktails made up of mAbs targeting different antigenic sites showed higher potency to neutralize the SARS\CoV\2 computer virus and SARS\CoV\2(V367F) pseudovirus particle. 2.?RESULTS 2.1. Characterization of high\affinity RBD\specific mAbs As the RBD mediates access into host cells through direct conversation with ACE2, and it is the primary target of neutralizing mAbs, a recombinant RBD\Fc was used to immunize mice to isolate RBD\targeted mAbs (Figures?1A and S1.) Using enzyme\linked immunosorbent assay (ELISA) competition and surface plasmon resonance (SPR) assays, 17 mAbs binding to the SARS\CoV\2 RBD were identified (Figures?1B, 1C, and S1\S3). Open in a separate window Physique 1 Characterization of RBD\specific mAbs inhibiting binding of the S to ACE2 receptor. (A) Serological antibody responses to the SARS\CoV\2 RBD\mFc evaluated by ELISA. (B) Diagram shows the percentage of hybridoma clones with different competition levels against ACE2 binding to the SARS\CoV\2 RBD. Inhibition rate Q 0% indicated approximately 39% of total clones could not compete with ACE2. (C) Inhibitory curves of representative mAbs competing with ACE2. (D) Representative mAbs block SARS\CoV\2 S binding to ACE2 in FACS\based assay. (E) The inhibition rate of all selected mAbs was measured by circulation cytometry with 10?g/ml of each antibody, and experiments were performed three times. (F) Binding kinetics of MA1 and ACE2 with SARS\CoV\2 RBDV367F. The purified soluble SARS\CoV\2 RBDV367F was covalently immobilized onto a CM5 sensor chip followed by injection of ACE2 or MA1 with five different concentrations. The black collection indicates the experimentally derived curves, and the colored lines represent the fitted curves based on the experimental data To identify mAbs that inhibit the conversation between Oxantel Pamoate the SARS\CoV\2 RBD and ACE2, 74 of 220 positive clones were finally selected for any competition assay in the presence of ACE2 for.