Two influenza A virus-specific fabs neutralize by inhibiting virus attachment to target cells, while neutralization by their IgGs is complex and occurs simultaneously through fusion inhibition and attachment inhibition

Mabs H36 (IgG2a) and H37 (IgG3) recognize epitopes in antigenic sites Sb an d Ca2, respectively, in the HA1 subunit of influenza virus A/PR/8/34 (H1N1) . Their neutralization was complex. Our aim here was to investigate the mec hanism of neutralization by the IgGs and their Fabs. In MDCK and BHK cells, both IgGs neutralized primarily by inhibiting virus-cell fusion, although at higher IgG concentrations virus attachment to target cells was also inhi bited. In contrast, the Fabs neutralized entirely by inhibiting virus attac hment, although a higher concentration of Fab than IgG was required to brin g this about. Both H36 and H37 exerted a concentration-dependent spectrum o f neutralization activity, with virus-cell fusion inhibition and virus-cell attachment inhibition being the predominant mechanisms at low- and high-an tibody concentration, respectively, and both mechanisms occurring simultane ously at intermediate concentrations. However, it may be that attachment in hibition was a secondary event, occurring to virus that had already been ne utralized through inhibition of its fusion activity. Neutralization by H36 and H37 Fabs was a simple process. Both inhibited virus attachment but requ ired much higher (>100-fold) molar concentrations for activity than did IgG . The functional affinities of the IgGs were high (0.4-0.6 nM) and differen ces between these and the affinity of their Fabs (H36, nit; H37, 23-fold) w ere not sufficient to explain the differences observed in neutralization. S imilar neutralization data were obtained in two different cell lines. The d ose-response curve for neutralization by H36 F(ab')(2) resembled that for I gG, although eightfold more F(ab')(2) was required for 50% neutralization. Overall, neutralization mechanisms of H36 and H37 antibodies were similar, and thus independent of antigenic site, antibody isotype, and target cell. (C) 2000 Academic Press.