THE MAJOR SUBUNIT CLPG OF ESCHERICHIA-COLI CS31A FIBRILLAE AS AN EXPRESSION VECTOR FOR DIFFERENT COMBINATIONS OF 2 TGEV CORONAVIRUS EPITOPES

Previously, two B-cell epitopes from the entero-pathogenic transmissib le gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino a cids (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 fibrill ae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGE V-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different ta ndem association with TGEV-A (A::C or C::A) in twelve putatively expos ed regions of ClpG. Among the 28 resulting engineered proteins only 15 , carrying up to 51 extra aa, had not essentially disturbed the correc t CS31A fibrillae formation process. Six partially permissive sites ac cepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacter ia or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the f oreign parent protein, providing a direct argument for exposure of the corresponding ClpG 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 the re 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 rele vant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, w hatever its position in ClpG, mediated the partial proteolytic degrada tion of the hybrid proteins, suggesting that it functions as a substra te for a cellular protease, and thereby that its suitability as a vacc ine antigen candidate is doubtful.