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 antibodies 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 analysed using a panel of 14 monoclonal antibodies (MAbs) (13 prepared against native gp350 derived from virus-producing cells and one prepared against an Escherichia coli recombinant protein). All 14 MAbs, including a virus-neutralizing antibody, reacted with the full-length recombinant gp350 in a dot blot immunoassay, but only four of the 14 MAbs reacted with polypeptides expressed by the five subclones, indicating that the full-length protein, but not the protein fragments, was antigenically similar to native gp350. Treatment of the six recombinant proteins with peptide-N-glycosidase F (PNGase F) indicated that the full-length gp350 protein and the N-terminal fragment were glycosylated and that the four internally initiated polypeptides were not glycosylated. PNGase F treatment of the full-length glycosylated gp350 did not eliminate its reactivity with all of the 10 MAbs examined (including the neutralizing MAb) in a dot blot immunoassay; however, denatured glycosylated 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 glycosylation-dependent.
Zhang
P F
PF
Division of Viral Products, Food and Drug Administration, Rockville, Maryland 20892.
Marcus-Sekura
C J
CJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000956:Antigens, Viral / Q000096:biosynthesis / Q000276:immunology*; D001483:Base Sequence; D002460:Cell Line; D000939:Epitopes / Q000032:analysis; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000276:immunology*; D004854:Herpesvirus 4, Human / Q000276:immunology*; D008969:Molecular Sequence Data; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D000939:Epitopes; D006023:Glycoproteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins