biostudies-arrayexpressHimanshu BatraMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16106During V(D)J recombination antibody diversity is enhanced by non-templated junctional modifications that generate immensely diverse heavy chain (HC) and light chain (LC) complementarity-determining 3 antigen-contact regions (CDR3s). We previously developed a mouse model that generates diverse antibody repertoires by rearranging a single human VH1-2 and Vk1-33, associated with highly diverse CDR3s generated by V(D)J recombination with mouse Ds and/or Js. Immunization of this model with SARS-CoV-2 D614G spike elicited an antibody that potently neutralized SARS-CoV-2 variants through Omicron BA.2.754. Here, we report a next generation mouse model in which a single VH1-2 rearranges to human D3-3 and JH6, generating diverse HC-CDR3s much longer on average than those of our prior model. Omicron BA.4/.5 spike ferritin nanoparticle-immunization of the new model elicited four highly-related humanized antibodies that potently neutralize downstream Omicron sub-variants. All four antibodies had 12 AA HC-CDR3s with two aromatic amino acids that engage an epitope comprising a hydrophobic patch opened-up by early omicron lineage mutations and conserved in subsequent variants. Immunization of our prior, shorter CDR3-based model, elicited slightly less potent neutralizing antibodies that bound the same Omicron epitope, and were similar in all other aspects to those from the long, fully-human CDR3 model. One tested antibody from each set reduced lung viral titers in a mouse-adapted BQ1.1 challenge. The antibodies we describe are highly related to, but more potent than, recently described antibodies from omicron-infected humans. These studies validate the utility of single human VH- and Vk-rearranging mice for discovering humanized antibodies that neutralize emerging pathogens.biostudies-arrayexpressSample Collection - Splenic B cells used for HTGTS-rep-seq were purified from unimmunized 5-8 weeks old mice by MACS® Microbeads according to the manufacturer’s protocol. In brief, spleens were dissected out from unimmunized mice, prepared into single cell suspensions and stained with anti-B220 Microbeads for 20 minutes at 4°C. The splenic B cells were collected using the LS column and MACSTM Separater.Sequencing - HTGTS-Rep-seq, using MiSeq 300bp paired-end reads for IgH, or NextSeq 550 151bp paired-end reads for IgKNucleic Acid Extraction - Genomic DNA was extracted from purified splenic B cells using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s protocol.Library Construction - The libraries of HTGTS-Rep-seq was constructed as previous described (Lin et al., PNAS 2016)OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - HTGTS-Rep-seq data analysis was done by the LAM-HTGTS-Rep-SHM-Seq bioinformatics pipeline (Github address: https://github.com/Yyx2626/HTGTSrep), using IgBLAST with default parameters for qualified reads mapping.Data Transformation - For downstream analysis, percentages in each library were calculated.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina MiSeqDNA-seqMus musculusAdam Yongxin YeSai LuoFrederick AltQingchen J ZhouHimanshu BatrafalseHTGTS-Rep-Seq for Humanized Antibodies that Broadly Neutralize Omicron Sub-variantsDuring V(D)J recombination antibody diversity is enhanced by non-templated junctional modifications that generate immensely diverse heavy chain (HC) and light chain (LC) complementarity-determining 3 antigen-contact regions (CDR3s). We previously developed a mouse model that generates diverse antibody repertoires by rearranging a single human VH1-2 and Vk1-33, associated with highly diverse CDR3s generated by V(D)J recombination with mouse Ds and/or Js. Immunization of this model with SARS-CoV-2 D614G spike elicited an antibody that potently neutralized SARS-CoV-2 variants through Omicron BA.2.754. Here, we report a next generation mouse model in which a single VH1-2 rearranges to human D3-3 and JH6, generating diverse HC-CDR3s much longer on average than those of our prior model. Omicron BA.4/.5 spike ferritin nanoparticle-immunization of the new model elicited four highly-related humanized antibodies that potently neutralize downstream Omicron sub-variants. All four antibodies had 12 AA HC-CDR3s with two aromatic amino acids that engage an epitope comprising a hydrophobic patch opened-up by early omicron lineage mutations and conserved in subsequent variants. Immunization of our prior, shorter CDR3-based model, elicited slightly less potent neutralizing antibodies that bound the same Omicron epitope, and were similar in all other aspects to those from the long, fully-human CDR3 model. One tested antibody from each set reduced lung viral titers in a mouse-adapted BQ1.1 challenge. The antibodies we describe are highly related to, but more potent than, recently described antibodies from omicron-infected humans. These studies validate the utility of single human VH- and Vk-rearranging mice for discovering humanized antibodies that neutralize emerging pathogens.2026-02-23T00:00:00Z2026-05-27T15:54:23.601Z2025-11-12T20:41:21.257ZE-MTAB-16106ERP184331EFO_0002944EFO_0004170EFO_0002693EFO_0004917EFO_0005518EFO_0003816EFO_0004184