S10). To determine if SW186 could neutralize a divergent coronavirus, such as SARS-CoV-1, we generated a pseudovirus displaying the spike protein of SARS-CoV-1. the viral spike protein. Administration of SW186 in mice after they were infected with SARS-CoV-2 Alpha, Beta, or Delta variants reduced the viral loads in Rabbit Polyclonal to FRS2 the lung. These results exhibited that SW186 neutralizes diverse SARS coronaviruses by binding to a conserved RBD epitope, which could serve as a target for further antibody development. A SARS-CoV-2 antibody with broad neutralizing activity against SARS-CoV-1 and SARS-CoV-2. == Introduction == SARS-CoV-2 has caused one of the worst pandemics in human history (1). The computer virus is usually airborne and enters cells in the airway through the conversation between the receptor binding domain name (RBD) of the viral spike (S) protein and angiotensin-converting enzyme 2 (ACE2) around the host cell membrane (2). SARS-CoV-2 neutralizing monoclonal antibodies (mAbs), together with vaccines and antiviral drugs, are important tools to control the PFI-3 COVID-19 pandemic. Although many mAbs have been developed, most were developed for the early SARS-CoV-2 strains (3). The SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), Gamma (P.1), Lambda (C.37), Mu (B.1.621), and Omicron (B.1.1.529 and its sublineages, including BA.1, BA.2, BA.2.12.1, BA.3, BA.4 and BA.5), developed resistance to therapeutic mAbs to varying extents (36). Significantly, the recently emerged Omicron variants harbor more than 15 mutations in the RBD of the spike protein and exhibit strong resistance to most therapeutic mAbs and even the plasma of vaccinated individuals (5,6). Most therapeutic antibodies were developed based on their ability to compete with the binding of ACE2 to the spike protein’s receptor-binding motif (RBM). This strategy allows for fast antibody development, but most if not all therapeutic mAbs directed against the RBM lost their activities against the Omicron variant (7). Therefore, there is an urgent need to develop new broadly neutralizing antibodies against current and future coronaviruses. Here, we isolated a panel of SARS-CoV-2 antibodies from mice immunized with the viral spike protein and recognized that SW186 monoclonal antibody as showing the best neutralizing activity against all variants tested, including the Omicron variants, and SARS-CoV-1. Using cryo-electron microscopy (cryo-EM) reconstruction, we decided the structure of the fragment antigen-binding region (Fab) of SW186 bound to the outer surface of RBD, which is usually distinct from your RBM that binds to ACE2. The SW186 antibody reduced SARS-CoV-2 viral loads in the lungs of mice, demonstrating a therapeutic PFI-3 effect. Thus, SW186 is usually a broadly neutralizing antibody against SARS coronaviruses and PFI-3 binds to a unique and conserved epitope of the spike protein. == RESULTS == == Identification of monoclonal antibodies against SARS-CoV-2 spike protein == To obtain antibodies targeting the viral spike protein, we immunized C57BL/6J mice with the spike ectodomain (S-ecto) or RBD protein of the Wuhan-hu-1 strain, using the STING agonist 23-cGAMP as an adjuvant (8). After improving twice, splenic memory B cells displaying IgGs against S-ecto or RBD were isolated (Fig. 1A, Fig. S1). To reduce non-specific binding, we used LIBRA-seq (linking B cell receptor to antigen specificity through sequencing) (9). In brief, we stained splenic B cells with S-ecto or RBD protein that was biotinylated and labeled with DNA barcodes. Cells were then stained with PE-streptavidin, and the IgG+PE+ cells were sorted and processed for single-cell RNA sequencing (scRNA-seq). The DNA barcode on each B cell indicated its antigen specificity. To further reduce background noise, we selected B cells positive for both PE-streptavidin and DNA PFI-3 barcodes as antigen-specific B cells (Fig. S2A). This strategy removed clonally expanded B cells without DNA barcodes (Fig. S2C). In total, 533 B cells with paired heavy chains and light chains as well as DNA barcodes (UMI > 100) were isolated (Table S1). B cells from your same V-D-J lineage share comparable CDR3 sequences and epitopes. Therefore, we selected BCRs with unique V-D-J lineage and clonally expanded B cell clones (frequency 3) for further analyses to maximize the epitope diversity (Fig. S2A and Fig. S2B). == Fig. 1. Identification of broadly neutralizing antibodies against SARS-CoV-2 variants. == (A) A pipeline of antibody identification and characterization. Splenocytes.
