Neutralizing human monoclonal antibodies against Puumala virus, causative agent of nephropathia epidemica: a novel method using antigen-coated magnetic beads for specific B cell isolation.

Abstract

Human monoclonal antibodies (MAbs) against Puumala (PUU) virus were generated and characterized. Human spleen B lymphocytes were preselected for specific surface immunoglobulin (Ig) using magnetic beads coated with the viral glycoproteins, and subsequently immortalized by Epstein-Barr virus transformation. Four IgG-positive monoclonal lymphoblastoid cell lines (LCLs) were established and have remained stable MAb secretors for over 12 months. Analyses of the antigen and epitope specificities recognized by the MAbs showed overlapping binding patterns of four anti-glycoprotein 2-specific clones. Identical isotypes (IgGl lambda) and isoelectric points (9.2) of the four MAbs suggested that they were derived from the same original clone. The MAbs reacted with eight PUU virus-like strains, but were negative for Hantaan, Seoul, and Prospect Hill viruses in an immunofluorescence assay, indicating binding to a conserved epitope unique for strains associated with the European form of haemorrhagic fever with renal syndrome, nephropathia epidemica. The MAbs neutralized all investigated PUU virus-like strains in a focus reduction neutralization test. The MAb neutralizing activity was significantly enhanced in the presence of human or guinea-pig complement. To stabilize and increase antibody secretion and to reduce the demand for culture medium supplements (e.g. fetal calf serum), three of the monoclonal LCLs were fused with the non-secreting human x mouse partner SPAM-8. Several of the established human x (human x mouse) monoclonal triomas grew faster and produced larger amounts of MAbs when compared with the original LCLs.