Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein.
Expression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the 10 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.
Mason
P W
PW
Arbovirus Research Unit, Yale University School of Medicine, New Haven, Connecticut 06510.
Dalrymple
J M
JM
Gentry
M K
MK
McCown
J M
JM
Hoke
C H
CH
Burke
D S
DS
Fournier
M J
MJ
Mason
T L
TL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D000914:Antibodies, Viral / Q000032:analysis; D000956:Antigens, Viral / Q000276:immunology; D001666:Binding Sites, Antibody; D002872:Chromosome Deletion; D004220:Disulfides; D004664:Encephalitis Virus, Japanese / Q000276:immunology*; D004672:Encephalitis, Japanese / Q000517:prevention & control; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011487:Protein Conformation; D014759:Viral Envelope Proteins / Q000276:immunology / Q000302:isolation & purification*; D014760:Viral Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D004220:Disulfides; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins