are co-inventors on patent applications describing the neutralizing mAbs (patent application number: CN202210313522.1). 8HC7, respectively. Cryo-EM density maps for the SARS-CoV-2 Omicron BA.1 spike (S-6P) in complex with YB13-292 Fab have been deposited in the Electron Microscopy Data Lender (EMDB) with accession codes EMD-34655, EMD-34656, EMD-34657 and EMD-34658. The related atomic models have been deposited in Protein Data Lender (PDB) under accession codes 8HC8, 8HC9, 8HCA and Harmine hydrochloride 8HCB.?Source data are provided with this paper. Abstract SARS-CoV-2 Omicron variants feature highly mutated spike proteins with remarkable abilities in evading antibodies isolated earlier in the pandemic. Investigation of memory B cells from patients primarily with breakthrough infections with the Delta variant enables isolation of a number of neutralizing antibodies cross-reactive to heterologous variants of concern (VOCs) including Omicron variants (BA.1-BA.4). Structural studies identify altered complementarity determining region (CDR) amino acids and highly unusual heavy chain CDR2 insertions respectively in two representative cross-neutralizing antibodiesYB9-258 and YB13-292. These features are putatively launched by somatic hypermutation and they are heavily involved in epitope acknowledgement to broaden neutralization breadth. Previously, insertions/deletions were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. These data provide molecular mechanisms for cross-neutralization of heterologous SARS-CoV-2 variants by antibodies isolated from Delta variant infected patients with implications for future vaccination strategy. Subject terms: SARS-CoV-2, Somatic hypermutation, Cryoelectron microscopy, Inactivated vaccines In this study, authors identified neutralizing antibodies by isolating B cells from SARS-CoV-2 Delta infected patients and detect altered structural features, likely introduced by somatic hypermutation, that Harmine hydrochloride are involved in epitope binding and increase neutralization Hepacam2 breadth against virus variants. Introduction Evasion of vaccine-induced neutralizing antibodies is believed to be one contributing factor of breakthrough infections caused by SARS-CoV-2 variants1. Omicron variants effectively evade most characterized antibodies2 likely contributing to an increased rate of breakthrough infections3. Studies of antibody responses in naive individuals infected by the original SARS-CoV-2 strain revealed that germline-like antibodies are widely and rapidly induced4C6. Antibody isolation and informatics studies revealed that germline antibodies of different clonotypes preferentially target specific spike protein surfaces generating antibodies of 4 different classes as define by epitope locations7. For examples, IGHV(VH)3-53/VH3-66 and VH1-2 germline antibodies are predominantly class 1 and class 2 antibodies respectively8,9. We recently identified that characterized VH1-69 antibodies are predominantly class 2 antibodies10. The Omicron BA.1 variant emerged with a highly mutated spike protein bearing more than 30 substitutions with 15 located in the receptor binding domain (RBD) alone. Some of the RBD substitutions have been repeatedly observed in many variants, Harmine hydrochloride among these, substitutions at 417 and 484 are known to be highly efficient in evading currently isolated germline class 1 and class 2 antibodies respectively11. It has been reported that repeated exposures of SARS-CoV-2 antigens by either infections or vaccinations elicit superior humoral responses with neutralizing activities against the highly mutated Omicron BA.1 variant12,13, however, the molecular mechanisms underlaying such superior immune responses remained incompletely characterized. In this study, SARS-CoV-2 antigen specific memory B cells isolated from patients primarily with breakthrough Harmine hydrochloride infections of Delta variant are shown to exhibit higher level of somatic hypermutation (SHM). We identify a number of cross-reactive neutralizing antibodies (nAbs), with neutralizing activities towards wildtype (WT), Beta and Delta strains. In addition, a subset of these antibodies maintains neutralization towards the Omicron BA.1 variant. Two representative Omicron BA.1 variant neutralizing antibodies YB9-258 and YB13-292, encoded by commonly induced VH3-53 and VH3-21 antibody genes respectively, are identified as class 1 and class 2 RBD antibodies. These two antibodies feature residues introduced by somatic hypermutation or highly unusual heavy chain complementarity determining region 2 (HCDR2) loop insertions rendering them highly resistant to known RBD substitutions including substitutions at 417, 452, 484, and 501. By antibody informatics and structural analysis, we investigate structural and genetic basis of cross-neutralizing activities of YB9-258 and YB13-292. These data provide molecular mechanisms for cross-neutralization of SARS-CoV-2 variants by YB9-258 and YB13-292 isolated from Delta-infected patients, providing important data informing future vaccination strategy. Results High-throughput single B-cell sequencing revealed elevated SHM in Delta variant infection patients Four pooled samples of peripheral blood mononuclear cells (PBMCs) were derived from blood samples of 15 COVID-19 convalescent patients primarily with breakthrough infections (13 have been confirmed vaccinated) of SARS-CoV-2 Delta variant (patient details are shown in Supplementary Table?1). Their plasmas show binding to various SARS-CoV-2 RBDs by ELISA (Supplementary Fig.?1). We isolated CD19+ CD27+ memory B cells which bind the homologous Delta variant RBD and S1 by FACS (Supplementary Fig.?2a) and subjected them to 10 Chromium 5mRNA and V(D)J single-cell sequencing (Fig.?1a)14. After standard quality control, from the sorted CD19+CD27+RBD+S1+ B cells we obtained single-cell transcriptome data for 3286 CD19+CD27+RBD+S1+ B cells and.
