{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,8,21]],"date-time":"2025-08-21T17:10:23Z","timestamp":1755796223436,"version":"3.44.0"},"reference-count":22,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T00:00:00Z","timestamp":1755475200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Amid the persistent threat of future pandemics, the continuous evolution of SARS-CoV-2 exposed critical challenges for vaccine efficacy and therapeutic interventions, highlighting the need for rapid and adaptable approaches to respond to immune escape variants.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Here, we report the use of immortalized B cell libraries from human peripheral blood mononuclear cells (PBMCs) and tonsil tissues to uncover B cell clones exhibiting cross-reactive neutralization against various SARS-CoV-2 variants and perform directed evolution of immortalized B cell clones to produce antibodies with improved binding and neutralization against emerging SARS-CoV-2 variants.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Immortalization of PBMC and tonsil-derived human B cells was achieved through transduction with retroviral vectors encoding apoptosis inhibitors, yielding transduction efficiencies of 67.5% for PBMCs and 50.2% for tonsil-derived cells. Analysis revealed that immortalized B cell libraries produced with this method retain diverse immunoglobulin isotype representations. Through high-throughput functional screening of approximately 40,000 B cells per library, we identified 12 unique clones with neutralization activity for SARS-CoV-2, leading to selection of monoclonal antibodies with robust neutralization activity against Delta and BA.5 variants. We applied our directed evolution approach to libraries generated by ex vivo AID-induced somatic hypermutation (SHM) of immortalized B cell clones to enhance the affinity and cross-reactivity, resulting in improved binding and neutralization potency to escape variants such as EG.5.1 and JN.1. Furthermore, we engineered a bi-paratopic antibody combining KBA2401, a broadly neutralizing antibody binding to highly conserved epitope on Spike-RBD, and KBA2402, a broadly binding non-neutralizing antibody, resulting in enhanced potency against SARS-CoV-2 variant JN.1 and KP.3.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>Our findings illustrate the use of immortalized B cell libraries for development of therapeutics that adapt to viral evolution and highlight the application of ex vivo directed evolution in refining antibody responses against emerging immune escape SARS-CoV-2 variants. The approach here described offers a promising pathway for rapid therapeutic development in the face of evolving viral threats.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2025.1648717","type":"journal-article","created":{"date-parts":[[2025,8,18]],"date-time":"2025-08-18T05:27:49Z","timestamp":1755494869000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape"],"prefix":"10.3389","volume":"16","author":[{"given":"Casper","family":"Marsman","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jurgen","family":"Heinen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vanessa","family":"Clerico 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