A single amino acid substitution in a recombinant G protein vaccine drastically curtails protective immunity against respiratory syncytial virus (RSV).
Recent studies have indicated a dominant T cell epitope located approximately between amino acids 184 and 203 on the respiratory syncytial virus (RSV) G protein. Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128-229) of the RSV G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids on vaccine efficacy. By changing two amino acids Arg 188 and Lys 192 to alanine, the ability of the Trx-G 128-229 fusion protein to protect mice against RSV challenge was virtually abolished. Mice immunized with the double mutant protein showed low levels of neutralizing antibodies and no pulmonary eosinophilic infiltrate, in contrast to that observed in mice immunized with wild type protein prior to RSV challenge. While less effective than the double mutant, mutation of either Arg 188 or Lys 192 to Ala drastically impaired the ability of immunized Trx-G 128-229 to induce neutralizing antibodies and to elicit pulmonary eosinophilia associated with RSV challenge. Despite low levels of virus-neutralizing antibodies, G protein-specific antibodies were detected by Western blotting in the sera from mice immunized with either of the single mutants (Arg 188 or Lys 192) but not the double mutant. Finally, immunization of mice with truncated forms of the Trx-G protein, showed partial protection against RSV challenge with Trx-G 128-188 but not with Trx-G 189-229. Taken together, the results indicate an important role for Arg 188 and Lys 192 in the induction of protective immunity and priming for eosinophilia against RSV. Furthermore, while the dominant protective linear epitope on the RSV G protein requires an intact sequence around Arg 188, there are additional, but less potent, protective epitopes upstream of Arg 188.
Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Yan
Y
Huang
Robert
R
Anderson
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D017931:DNA Primers; D004337:Drug Carriers; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D007958:Leukocyte Count; D008081:Liposomes; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology*; D022261:Respiratory Syncytial Virus Vaccines / Q000276:immunology*; D012136:Respiratory Syncytial Viruses / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured; D014614:Vaccines, Synthetic / Q000276:immunology*
D017931:DNA Primers; D004337:Drug Carriers; D000939:Epitopes; D008081:Liposomes; D022261:Respiratory Syncytial Virus Vaccines; D014614:Vaccines, Synthetic; D019204:GTP-Binding Proteins