A cost-effective approach for comprehensive analysis of human memory B Cell repertoire: fast isolation of broad neutralizing mAbs to SARS-CoV-2 variants

The SARS-CoV-2 pandemic highlighted the urgent need for innovative methods to study humoral immune responses and identify monoclonal antibodies (mAbs) with diagnostic and therapeutic potential. Memory B cells (MBCs), pivotal to adaptive immunity, generate high-affinity antibodies upon antigen re-encounter. While single-cell high-throughput sequencing has revolutionized antibody repertoire studies, it has critical limitations: the inability to simultaneously determine antigen-binding specificities and immunoglobulin gene sequences, and high resource demands that limit accessibility in low-resource settings. Here, we present a cost-effective single-cell culture (SCC) platform enabling comprehensive analysis of human MBC repertoires, including epitope-specific responses, cross-reactivity studies, and mAb isolation. Using SARS-CoV-2 convalescent and vaccinated donor samples, we optimized MBC SCCs with NB21 feeder cells, R848, and IL-2 stimulation, achieving high cloning efficiency and a 30-fold enrichment of antigen-specific MBCs compared to bulk cultures. Among 592 isolated mAbs, 52.7% exhibited specificity to the Wuhan strain Spike protein, with 27.9% targeting the receptor-binding domain (RBD), 15.4% the N-terminal domain (NTD), and 56.7% other regions, likely the S2 domain. Comparative analysis revealed distinct cross-reactivity patterns: 40.5% of all anti-Spike mAbs recognized all tested SARS-CoV-2 variants (Wuhan, Beta, Delta, Gamma and Omicron BA.2), while 29.6% showed recognition of only four variants, the majority not including Omicron BA.2; 56 single strain reactive mAbs (14.9%) were also identified. Notably, all screening and neutralization assays were performed directly with culture supernatants, eliminating the need for large-scale sequencing and transfection. Desired clones were selected for recombinant mAb production. The SCC platform also enabled unbiased immunoglobulin repertoire profiling, revealing convergent V-region rearrangements, including public V3-30 and V3-53/V3-66 antibodies consistent with prior SARS-CoV-2 studies. Two public RBD-targeting clones with broad neutralization potential were validated in pseudovirus neutralization assays. This streamlined platform delivers simultaneous antigen-specific mAb isolation, V-region sequencing, and functional studies within seven days, empowering researchers in low-resource settings to address global health inequities and enhance preparedness for future pandemics.