title: Antigenicity of amino-acid sequences from Clostridium difficile toxin B.
abstract: Clostridium difficile toxins A and B cause antibiotic-associated colitis. Whereas antigenic determinants specifying neutralisation of toxin A have been partially elucidated, those of toxin B remain unknown. To define antigenic determinants of toxin B, synthetic peptides were prepared for five linear sequences selected by computer analysis for putative T and B epitopes. Peptides spanning the carboxy terminal region (aa 2155-2283) were also selected because this region contains repetitive units thought to bind the toxin to cell receptors. Multiple antigenic peptides were synthesised by linking four peptide copies to a core of four lysine residues (tetraMAP). Outbred mice were given four doses of each tetraMAP by intraperitoneal injection and specific immunoglobulins G and A were measured by enzyme-linked immunosorbent assay (ELISA) in serum, ascitic fluid and faeces. All 14 MAPs induced strong IgG responses against the homologous peptide; peptides representing aa 2155-2179 and 2246-2270 induced the strongest responses, of 592 and 493 ELISA units, respectively - although, to a lower extent, all 14 MAPs induced serum and faecal IgA responses against the homologous peptide. All MAPs induced IgG1 and IgG2b subclasses, documenting their capacity to elicit Th2-dependent mucosal immunity. IgG anti-MAPs were assayed for reaction with native toxins A and B; most anti-MAPs recognised the toxins only weakly or did not recognise them. Antibodies against peptide representing aa 2168-2192 recognised both native toxin B (19 ELISA units) and toxin A (2 ELISA units). None of the antibodies neutralised cytotoxicity of either toxin in cell culture. In contrast, four MAPs (aa 2080-2095, 2168-2192, 2220-2244 and 2233-2257) inhibited cytotoxicity when mixed with toxin B before addition to cells; inhibition was mediated by a direct interaction with toxin B.
authors.affiliation: OraVax Inc., Cambridge, MA, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Torres
authors.forename: T P
authors.initials: TP
authors.lastname: Monath
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology*; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology*; D016360:Clostridioides difficile / Q000276:immunology*; D003603:Cytotoxins / Q000737:chemistry / Q000276:immunology; D004761:Enterocolitis, Pseudomembranous / Q000276:immunology / Q000382:microbiology; D004768:Enterotoxins / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D007070:Immunoglobulin A / Q000096:biosynthesis; D007071:Immunoglobulin A, Secretory / Q000096:biosynthesis; D007074:Immunoglobulin G / Q000096:biosynthesis; D007136:Immunoglobulins / Q000096:biosynthesis*; D051379:Mice; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D004768:Enterotoxins; D000939:Epitopes; D007070:Immunoglobulin A; D007071:Immunoglobulin A, Secretory; D007074:Immunoglobulin G; D007136:Immunoglobulins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile