Development of canine parvovirus-neutralizing monoclonal antibodies from natural host and their germline gene usage.

Canine parvovirus 2 (CPV-2) remains a leading cause of acute infectious gastroenteritis with high global morbidity in dogs. While murine neutralizing monoclonal antibodies (mAbs) are widely used for antiviral therapy, their efficacy is limited by immune rejection in canine recipients. Here, we developed an efficient single B-cell cloning platform to generate canine-derived neutralizing mAbs against CPV-2 and characterized their germline gene usage patterns. Specifically, using biotinylated CPV-2 virions as bait, CPV-2-binding B cells were singly isolated via fluorescence-activated cell sorting (FACS) from peripheral blood mononuclear cells of immunized dogs. The heavy and light chain variable region (VH/VL) sequences were amplified through nested RT-PCR from single B cells, and cloned into canine immunoglobulin heavy/light chain (IgH/IgL) expression vectors. A total of 22 canine-derived mAbs were successfully expressed and purified from suspended ExpiCHO-S cells, and 20 of which demonstrated CPV-2-binding reactivity in enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay (IFA). Among these, 13 mAbs exhibited neutralizing activity (IC50 < 25 μg/mL) against a CPV-2c strain in F81 cells by virus micro-neutralization assays. Notably, the clone B11 showed potent virus neutralization activity with an IC50 of 0.06 μg/mL. Furthermore, germline gene usage analysis revealed preferential utilization of IGHV3-5, IGHD1, and IGHD3 in the heavy chain, and IGLV1-46, IGLV1-48, and IGLJ4/9 in the light chain in these CPV-2-specific canine antibodies. These canine-derived mAbs show promise for clinical diagnostics and therapeutics, overcoming the limitations of murine antibodies. Our platform establishes a framework for developing canine mAbs against other pathogens. KEY POINTS: Efficient single B-cell platform developed for rapid canine mAb generation. Potent canine mAb identified with an IC50 of 0.06 μg/mL against CPV-2c. Preferential germline gene usage revealed in canine antibody response to CPV-2.