title: The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes.
abstract: Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363-371 and the A epitope (TGEV-A) aa 522-531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG. Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process. Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding CIpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context. The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202-204 and 202-218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.
authors.affiliation: Laboratoire de Microbiologie, Institut National de la Recherche Agronomique, Centre de Recherches de Clermont-Ferrand-Theix, Saint-Genès-Champanelle, France.
authors.forename: M C
authors.initials: MC
authors.lastname: Méchin
authors.forename: M
authors.initials: M
authors.lastname: Der Vartanian
authors.forename: C
authors.initials: C
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics; D005760:Transmissible gastroenteritis virus / Q000235:genetics* / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D000939:Epitopes; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C071730:clpG protein, E coli