| Co-Investigator(Kenkyū-buntansha) |
KEITH Jerry M. Laboratory of Microbial Ecology, National Institutes of Health, Chief, Laboratory, Chief
SATO Yuji Department of Bacteriology, National Institute of Health, Chief, 細菌部, 室長 (40072889)
|
|---|
| Research Abstract |
Pertussis toxin (PT) is a typical virulence factor of Bordetella pertussis and the most important protective antigen which must be essential to a pertussis vaccine. As a component of the vaccine, non-toxic and immunoprotective pertussis toxoid is required. Although formaldehyde (FA) treatment is a popular method for toxoiding of bacterial protein toxins, the detoxification of PT, especially inactivation of ADP-ribosyltransferase activity of S1, is not necessarily complete. Since S1 does not contain lysine residue, mainly B oligomer part (S2-S5) of PT is considered to be modified by the FA treatment. Furthermore, some toxicity can be reversed by incubation of the toxoid at high temperature. We have attempted to construct mutant strains of B. pertussis which produce pertussis toxoid by the introduction of site-directed mutations in the PT gene, S1 gene which have profound effects on ADP-ribosyltransferase activity was mutated to construct pertussis toxoid producing strains. Arginine at p
… More
osition 9 was converted to lysine and doubly mutated S1 genes were constructed by additional changes of glutamic acid to aspartic acid, glutamine or glycine at position 129. The pertussis toxoid operons were reassembled and inserted into the B. pertussis chromosome. These toxoids were assembled and expressed to approximately wild type levels. PT activities such as ADP-ribosylation, CHO-cell clusterization, leukocytosis promotion and histamine sensitization activities were greatly reduced. Particularly, the toxicities of the double mutant toxoids were under detectable level. Furthermore, virulence in mice by intracerebral or aerosol challenge with these toxoid producing B. pertussis strains was also reduced. Mice immunized with the toxoids or with mouse-antibody against the toxoid were protected from the intracerebral or aerosol challenge with virulent pertussis organisms, respectively. Since immunoprotective potency of the toxoid produced by the mutants was the same level to that of FA-treated pertussis toxoid, mutant strains would be good candidates for the vaccine production. Less
|
