Monovalent single-chain Fv fragments and bivalent mini antibodies bound to vesicular stomatitis virus protect against lethal infection

Several antibody-dependent mechanisms have been postulated to mediate neutralization of different animal viruses, including blocking of docking to receptors, induction of conformational changes in the virus coat, and Fc-dependent opsonization. We have studied the molecular requirements for antibody-mediated neutralization of vesicular stomatitis virus (VSV) in vitro and protection against lethal disease in vivo with a single-chain Fv fragment (scFv) and the corresponding bivalent miniantibody (scFv-dHLX) generated from a VSV-neutralizing monoclonal antibody. Both monovalent scFv and bivalent scFv-dHLX miniantibodies were able to neutralize VSV in vitro and to protect interferon-alpha beta receptor-deficient (IFN-alpha beta R(-/-)) mice against lethal disease after intravenous injection of 50 plaque-forming units (pfu) VSV pre-incubated with the scFv reagents. Similarly, severe-combined immunodeficient (SCID) mice infected with immune complexes of 10(8) pfu VSV and bivalent scFv-dHLX were protected against lethal disease; however, mice infected with immune complexes of 10(8) pfu VSV and monovalent scFv were not. Although repeated scFv-dHLX treatment reduced virus quantities in the blood, neither SCID nor IFN-alpha beta R(-/-) mice were protected against lethal disease after passive immunization and subsequent VSV infection. This was due to the short half-life of 17 min of scFv-dHLX in the circulation. These data demonstrate that neutralization of VSV and protection against lethal disease do not require Fc-mediated mechanisms and that cross-linking is not crucial for protection against physiologically relevant virus doses in vivo.