title: A continuous epitope from transmissible gastroenteritis virus S protein fused to E. coli heat-labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity.
abstract: Antigenic site D from the spike protein of transmissible gastroenteritis virus (TGEV), which is a continuous epitope critical in neutralization, has been expressed as a fusion protein with E. coli heat-labile toxin B subunit (LT-B) in attenuated S. typhimurium. Synthetic peptides containing the sequence of site D induced TGEV neutralizing antibodies when inoculated subcutaneously in both rabbits and swine. A synthetic oligonucleotide encoding residues 373-398 of TGEV S protein, including antigenic site D, was cloned in frame with the 3' end of LT-B gene, into a plasmid used to transform S. typhimurium delta asd chi 3730. A collection of 6 recombinant plasmids designated pYALTB-D I-VI encoding LTB-site D fusions with a variable number of site D sequences were selected. Four of the 6 LTB-site D fusion products expressed in S. typhimurium chi 3730 formed oligomers (pentamers) that dissociated at > 70 degrees. S. typhimurium chi 3730 (pYALTB-D) V and VI expressed the oligomer forming products with higher antigenicity. Partially purified LTB-site D fusion product expressed from S. typhimurium chi 3730 (pYALTB-D) V induced anti-TGEV neutralizing antibodies in rabbits. Recombinant vaccine strain S. typhimurium delta cya delta crp delta asd chi 3987 transformed with plasmid pYALTB-D V expressed constitutively products that formed oligomers presumably containing 20 copies of site D, and showed a high stability in vitro. This recombinant strain was orally inoculated in rabbits and induced TGEV specific antibodies in both serum and intestinal secretion.
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnologia, CSIC, Madrid, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Smerdou
authors.forename: I M
authors.initials: IM
authors.lastname: Anton
authors.forename: J
authors.initials: J
authors.lastname: Plana
authors.forename: R
authors.initials: R
authors.lastname: Curtiss
authors.forename: L
authors.initials: L
authors.lastname: Enjuanes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004279:DNA, Viral; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology*; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012486:Salmonella typhimurium; D064370:Spike Glycoprotein, Coronavirus; D013552:Swine; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D001427:Bacterial Toxins; D004279:DNA, Viral; D004768:Enterotoxins; D018985:Epitopes, B-Lymphocyte; D029968:Escherichia coli Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; C027538:heat-labile enterotoxin, E coli