1887

Abstract

cDNA containing the entire coding sequence of the respiratory syncytial (RS) virus fusion (F) protein gene (574 amino acids) and two large I restriction fragments, encoding amino acids 18 to 212 and 214 to 574, were expressed in as C-terminal chimeras with -galactosidase (-gal) in the pEX expression vector system. A further cDNA fragment, overlapping the I 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 four chimeric proteins but most strongly to those containing C-terminal sequences. Two monoclonal antibodies (MAbs), 1E3 and RS348, capable of neutralizing the virus and inhibiting the viral fusion function, bound to all chimeras except that derived from the N-terminal I fragment, suggesting that their binding sites were located between amino acids 214 and 289. Further analysis of binding to expressed fragments from restriction enzyme digests and PCR amplification demonstrated that both 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 from 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 protein or could neutralize the virus. This suggests that either the -gal partner inhibits the immune response to the protein or that elements missing from the protein expressed in , perhaps conformational or added post-translation, contribute to the neutralizing antibody epitope.

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1993-12-01
2021-10-16
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