title: Immunization with an alphatoxin variant 121A/91-R212H protects mice against Clostridium perfringens alphatoxin.
abstract: As shown previously, a recombinant alphatoxin variant (rAT121A/91) constructed from the naturally occurring Clostridium perfringens mutant strain 121A/91, was devoid of enzymatic (PLC), hemolytic and lethal activity (18). In the present study, the recombinant variant was altered by an oligonucleotide-directed reversion of an arginine in position 212 for a histidine residue, corresponding to the sequence of the wild-type alphatoxin. The new variant rAT121A/91R212H proved to be negative in enzymatic, hemolytic and lethal activity as well. RAT121A/91 as well as rAT121A/91R212H was used for i.p. immunization of balb/c mice. The immune response was studied in ELISA as well as in the mouse neutralization test. Furthermore, immunized mice were challenged by i.p. application of active C. perfringens alphatoxin. In all immunized groups, mice developed high anti-alphatoxin titers (up to 1:128000). Antisera of both groups were able to reduce the hemolytic effect of native alphatoxin with predominance of anti-rAT121A/91R212H sera. During neutralization experiments, mice receiving a mixture of anti-rAT121A/91R212H and wild-type toxin were protected completely, whereas an anti-rAT121A/91/toxin mixture prolonged time until death but failed in protection. I.p immunization with rAT121A/91R212H yielded a significant protection rate (76%) when mice were challenged intraperitoneal with wild-type toxin. Our cumulative data indicates that the reversion of arginine in position 212 to histidine for rAT121A/91R212H was necessary to induce production of protective antibodies against wild-type alphatoxin of C. perfringens.
authors.affiliation: Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany. Heike.Schoepe@vetmed.uni-giessen.de
authors.forename: Heike
authors.initials: H
authors.lastname: Schoepe
authors.forename: Axel
authors.initials: A
authors.lastname: Neubauer
authors.forename: Tobias
authors.initials: T
authors.lastname: Schlapp
authors.forename: Lothar H
authors.initials: LH
authors.lastname: Wieler
authors.forename: Georg
authors.initials: G
authors.lastname: Baljer
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology* / Q000633:toxicity; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology* / Q000633:toxicity; D003016:Clostridium perfringens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D007114:Immunization; D015151:Immunoblotting / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D010738:Type C Phospholipases / Q000235:genetics / Q000276:immunology* / Q000633:toxicity; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens