Immortalized Human B Cells

Unbiased Screening of Immortalized B Cell Libraries Derived from Human PBMCs and Tonsils Produce B Cell Clones with Cross Reactive Neutralization to SARS-Cov-2

The continuous evolution of SARS-CoV-2 poses significant challenges to vaccine efficacy and therapeutic interventions, necessitating rapid, adaptable approaches to combat immune escape variants. Marsman et al. aimed to develop a robust platform using immortalized human B cell libraries from PBMCs and tonsil tissues to discover cross-reactive neutralizing antibodies.

They generated immortalized B cell libraries from fresh human PBMCs and frozen tonsil tissue by retroviral transduction with BCL-6, BCL-xl, and GFP. Transduction efficiencies were 67.5% for PBMCs and 50.2% for tonsils (Fig. 1A), with the difference likely due to fresh versus frozen sample handling. Flow cytometry revealed diverse Ig isotypes in both libraries, with IgM predominating-particularly in tonsils, consistent with their role as naive B cell reservoirs. IgG comprised 26% (PBMC) and 19% (tonsils), while IgA represented 15% and 13%, respectively (Fig. 1B), indicating representative Ig repertoire capture.

'Minipools' (MPs) were generated by sorting 25 cells per well into ten 384-well plates, followed by 3-week expansion. MPs were screened for SARS-CoV-2 reactivity by binding to Spike and envelope proteins, yielding 474 positive MPs from PBMCs and 123 from tonsils. MPs with >45% neutralization against Delta and BA.5 variants (Fig. 1C) were single-cell sorted to establish monoclonal populations. From 440 monoclonal supernatants screened against XBB.1.5 and BA.5.5, most showed >50% neutralization, with 26 demonstrating >60% neutralization against both variants (blue box, Fig. 1D). Tonsil-derived B cells exhibited lower binding and hit rates but enriched cross-neutralizing clones (Fig. 1D). Sequencing of clones with >60% dual-variant neutralization identified 12 unique VH/VL pairs.