CONFORMATION-DEPENDENT RECOGNITION OF BACULOVIRUS-EXPRESSED EPSTEIN-BARR VIRUS-GP350 BY A PANEL OF MONOCLONAL-ANTIBODIES

The Epstein-Barr virus (EBV) major membrane protein, gp350, induces an tibodies that neutralize virus infectivity in vitro and is a potential candidate for an EBV vaccine. Full-length EBV gp350 and five protein fragments, encompassing the entire protein sequence, were generated in a baculovirus expression system. The recombinant proteins were analys ed using a panel of 14 monoclonal antibodies (MAbs) (13 prepared again st native gp350 derived from virus-producing cells and one prepared ag ainst an Escherichia coli recombinant protein). All 14 MAbs, including a virus-neutralizing antibody, reacted with the full-length recombina nt gp350 in a dot blot immunoassay, but only four of the 14 MAbs react ed with polypeptides expressed by the five subclones. indicating that the full-length protein, but not the protein fragments. was antigenica lly similar to native gp350. Treatment of the six recombinant proteins with peptide-N-glycosidase F (PNGase F) indicated that the full-lengt h gp350 protein and the N-terminal fragment were glycosylated and that the four internally initiated polypeptides were not glycosylated. PNG ase F treatment of the full-length glycosylated gp350 did not eliminat e its reactivity with all of the 10 MAbs examined (including the neutr alizing MAb) in a dot blot immunoassay; however, denatured glycosylate d gp350 lost reactivity with all but four of the 14 MAbs when analysed by either dot blot or Western blot immunoassay. The data suggest that conformational epitopes are more important in recognition of gp350 by this panel of MAbs than glycosylation sites, and that the epitope on gp350 recognized by the neutralizing MAb is conformation- and not glyc osylation-dependent.