Pertussis toxin (PT) is an important protective antigen in vaccines against whooping cough, and a genetically detoxified PT analog is the preferred form of the immunogen. Several amino acids of the Si subunit were identified as functionally critical residues by site-directed mutagenesis, specifically, those at positions 9, 13, 26, 35, 41, 58, and 129. Eighty-three mutated PT operons were introduced into Bordetella parapertussis, and the resultant toxin analogs were screened for expression levels, enzymatic activity, residual toxicity, and antigenicity. While more than half of the mutants were found to be poorly secreted or assembled, the rest were fully assembled and most were highly detoxified. Single mutations resulted in up to a 1,000-fold reduction in both toxic and enzymatic activities, while PT analogs with multiple mutations (Lys-9 Gly-129, Glu-58 Gly-129, and Lys-9 Glu-58 Gly-129) were 106-fold detoxified. Operons coding for stable and nontoxic mutants shown to express a critical immunodominant protective epitope were returned to the chromosome of Bordetella pertussis by allelic exchange. In vivo analysis of the toxin analogs showed a dramatic reduction in histamine sensitization and lymphocytosis-promoting activities, paralleling the reduction in toxic activities. All mutants were protective in an intracerebral challenge test, and the Lys-9 Gly-129 analog was found to be significantly more immunogenic than the toxoid. PT analogs such as those described represent suitable components for the design of a recombinant whooping cough vaccine.
We replaced the wild-type TOX operon of Bordetella pertussis with in vitro mutated, detoxified alleles by electroporetic transformation using unmarked linear DNA. Uptake of DNA was selected by transient ampicillin resistance and two simultaneous recombination events resulted in gene-replacement at the natural locus with no integration of heterologous DNA. TOX alleles were stable without selection and recombinant strains secreted non-toxic, fully assembled, protective pertussis toxin (PT) analogues with kinetics similar to the parental vaccine strain under production-scale fermentation conditions. Strains generated in this way are suitable for the production of recombinant whole-cell or component whooping cough vaccines that require no chemical modification of PT.
The S2, S3, and S4 subunit genes of pertussis toxin (PT) from Bordetella pertussis were subjected to site-directed mutagenesis, and the resultant PT analogs were assayed for altered biological properties. PT analogs S2(T91,R92,N93)A and S2(Y102A,Y103A) exhibited reduced binding to fetuin. Several PT analogs with mutations in the S2, S3, or S4 subunit showed reduced in vitro toxicity, as measured in the Chinese hamster ovary (CHO) cell clustering assay. In particular, PT analogs S3(Y82A) and S3(I91,Y92,K93)A retained 10%o or less residual toxicity. These mutants also exhibited significantly lower mitogenic and hemagglutinating activities and reduced in vivo activities, as measured by the histamine sensitization and leukocytosis assays. The S4(K54A,K57A) PT analog had significantly reduced CHO cell clustering activity, though other biological activities remained unaffected. PT analogs S1(E129G)/S3(Y82A) and S1(E129G)/ S3(191,Y92,K93)A displayed a cumulative effect of the Si and S3 mutations for both in vitro and in vivo toxic activities. These PT analogs, as well as S1(R9K,E129G)/S3(K82A) and S1(R9K,E129G)/S3(I91,Y92,K93)A, still expressed an epitope which elicits a neutralizing antitoxin antibody and were protective in the mouse intracerebral challenge test. Recombinant pertussis vaccines based on PT analogs with detoxifying mutations in multiple subunits may thus represent the next generation of improved whooping cough vaccines.
Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9G1yl29 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox orflaa locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLYs9GIYl29 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.
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