Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States
Alicia Gonzalez-Martin
Department of Biochemistry, Universidad Autónoma de Madrid (UAM) and Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain
Raiees Andrabi
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States
Roberta P Fuller
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States
Ben Murrell
Department of Medicine, University of California, San Diego, San Diego, United States; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
Laura E McCoy
Division of Infection and Immunity, University College London, London, United Kingdom
Katelyn Porter
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States
Deli Huang
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
Wenjuan Li
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
Devin Sok
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States
Khoa Le
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States
Bryan Briney
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States
Morgan Chateau
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, United States
Geoffrey Rogers
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, United States
Lars Hangartner
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
Ann J Feeney
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
David Nemazee
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States
Paula Cannon
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, United States
Dennis R Burton
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States; Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID), The Scripps Research Institute, La Jolla, United States; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard, Cambridge, United States
We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.
