Mapping and characterization of a sequential epitope on the rabies virus glycoprotein which is recognized by a neutralizing monoclonal antibody, RG719.
We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.
Ni
Y
Y
Department of Molecular Microbiology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Tominaga
Y
Y
Honda
Y
Y
Morimoto
K
K
Sakamoto
S
S
Kawai
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006023:Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D015151:Immunoblotting; D007075:Immunoglobulin M / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011817:Rabbits; D011818:Rabies / Q000276:immunology / Q000517:prevention & control; D011820:Rabies virus / Q000276:immunology*; D014611:Vaccination; D014759:Viral Envelope Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D017931:DNA Primers; D000939:Epitopes; D006023:Glycoproteins; D007075:Immunoglobulin M; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus