Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape
IntroductionAmid 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.MethodsHere, we report the...
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1648717/full |
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| author | Casper Marsman Jurgen Heinen Vanessa Clerico Mosina Kelly Maijoor Arjen Q. Bakker Michael Koslowski Alessandra Villa Stefano Gullà |
| author_facet | Casper Marsman Jurgen Heinen Vanessa Clerico Mosina Kelly Maijoor Arjen Q. Bakker Michael Koslowski Alessandra Villa Stefano Gullà |
| author_sort | Casper Marsman |
| collection | DOAJ |
| description | IntroductionAmid 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.MethodsHere, 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.ResultsImmortalization 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.DiscussionOur 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. |
| format | Article |
| id | doaj-art-aa0c706fca4b45178b10a1396bf1c645 |
| institution | Kabale University |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Immunology |
| spelling | doaj-art-aa0c706fca4b45178b10a1396bf1c6452025-08-20T03:46:46ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-08-011610.3389/fimmu.2025.16487171648717Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escapeCasper MarsmanJurgen HeinenVanessa Clerico MosinaKelly MaijoorArjen Q. BakkerMichael KoslowskiAlessandra VillaStefano GullàIntroductionAmid 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.MethodsHere, 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.ResultsImmortalization 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.DiscussionOur 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.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1648717/fullhuman B cellantibody discoveryaffinity maturationdirected evolutionbiparatopic antibodies |
| spellingShingle | Casper Marsman Jurgen Heinen Vanessa Clerico Mosina Kelly Maijoor Arjen Q. Bakker Michael Koslowski Alessandra Villa Stefano Gullà Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape Frontiers in Immunology human B cell antibody discovery affinity maturation directed evolution biparatopic antibodies |
| title | Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape |
| title_full | Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape |
| title_fullStr | Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape |
| title_full_unstemmed | Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape |
| title_short | Immune counter-evolution: immortalized B cell clones can undergo ex vivo directed evolution to counteract viral escape |
| title_sort | immune counter evolution immortalized b cell clones can undergo ex vivo directed evolution to counteract viral escape |
| topic | human B cell antibody discovery affinity maturation directed evolution biparatopic antibodies |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1648717/full |
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