Immunological characterization of a rotavirus-neutralizing epitope fused to the cholera toxin B subunit.
A highly conserved neutralizing epitope from the surface protein VP4 (amino acids 296-313) of human rotaviruses was genetically fused to the B subunit of cholera toxin (CTB). Synthetic oligodeoxyribonucleotides encoding the VP4 peptide were inserted between the 3' end of the DNA that codes for the leader peptide, and the 5' end of the gene encoding mature CTB. The hybrid protein synthesized in Escherichia coli was found to maintain the ability of CTB to pentamerize, and to adhere to its cell receptor, the GM1 ganglioside. The chimera was efficiently recognized by a monoclonal antibody (mAb) directed at CTB and by a virus-neutralizing mAb against the VP4 peptide. The hybrid polypeptide was shown to induce high titers of serum antibodies (Ab) against CTB and the synthetic VP4 peptide following subcutaneous immunization; paradoxically, however, the Ab obtained did not recognize the virus by an enzyme-linked immunosorbent assay method, nor had detectable neutralizing activity. Potential implications of these results for future design and evaluation of fusion proteins as immunogens are discussed.
González
R A
RA
Departamento de Biología Molecular, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
Sánchez
J
J
Holmgren
J
J
López
S
S
Arias
C F
CF
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins; D002772:Cholera Toxin / Q000276:immunology*; D003001:Cloning, Molecular; D004279:DNA, Viral; D000939:Epitopes / Q000276:immunology*; D000922:Immunotoxins / Q000276:immunology*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D012401:Rotavirus / Q000235:genetics / Q000276:immunology*
D036022:Capsid Proteins; D004279:DNA, Viral; D000939:Epitopes; D000922:Immunotoxins; C060339:VP4 protein, Rotavirus; D002772:Cholera Toxin