BINDING OF NEUTRALIZING MONOCLONAL-ANTIBODIES TO REGIONS OF THE FUSION PROTEIN OF RESPIRATORY SYNCYTIAL VIRUS EXPRESSED IN ESCHERICHIA-COLI

cDNA containing the entire coding sequence of the respiratory syncytia l (RS) virus fusion (F) protein gene (574 amino acids) and two large P stI restriction fragments, encoding amino acids 18 to 212 and 214 to 5 74. were expressed in Escherichia coli as C-terminal chimeras with bet a-galactosidase (beta-gal) in the pEX expression vector system. A furt her cDNA fragment. overlapping the PstI restriction site and encoding amino acids 190 to 289, was derived by PCR and expressed in a similar manner. Polyclonal rabbit serum raised against RS virus bound to all f our chimeric proteins but most strongly to those containing C-terminal sequences. Two monoclonal antibodies (MAbs), 1E3 and RS348, capable o f neutralizing the virus and inhibiting the viral fusion function, bou nd to all chimeras except that derived from the N-terminal PstI fragme nt, suggesting that their binding sites were located between amino aci ds 214 and 289. Further analysis of binding to expressed fragments fro m restriction enzyme digests and PCR amplification demonstrated that b oth antibodies bound to amino acids 253 to 289. MAb RS348 bound to 12- mer overlapping synthetic peptides containing the sequence 265 to 272 (PITNDQKK) but MAb 1E3 failed to bind to any 12-mer peptide derived fr om the F protein sequence. Immunization of mice with chimeric proteins containing the whole F protein coding sequence or amino acids 253 to 384, which includes the binding site of the two MAbs identified here, failed to induce antibodies that recognized the native RS virus F prot ein or could neutralize the virus. This suggests that either the beta- gal partner inhibits the immune response to the protein or that elemen ts missing from the protein expressed in E. coli, perhaps conformation al or added post-translation, contribute to the neutralizing antibody epitope.