CHARACTERIZATION OF PERTUSSIS TOXIN ANALOGS CONTAINING MUTATIONS IN B-OLIGOMER SUBUNITS

The S2, S3, and S4 subunit genes of pertussis toxin (PT) from Bordetel la pertussis were subjected to site-directed mutagenesis, and the resu ltant PT analogs were assayed for altered biological properties. PT an alogs S2(T91,R92,N93)DELTA and S2(Y102A,Y103A) exhibited reduced bindi ng to fetuin. Several PT analogs with mutations in the S2, S3, or S4 s ubunit showed reduced in vitro toxicity, as measured in the Chinese ha mster ovary (CHO) cell clustering assay. In particular, PT analogs S3( Y82A) and S3(191,Y92,K93)DELTA retained 10% or less residual toxicity. These mutants also exhibited significantly lower mitogenic and hemagg lutinating activities and reduced in vivo activities, as measured by t he histamine sensitization and leukocytosis assays. The S4(K54A,K57A) PT analog had significantly reduced CHO cell clustering activity, thou gh other biological activities remained unaffected. PT analogs S1(E129 G)/S3(Y82A) and S1(E129G)/S3(I91,Y92,K93)DELTA displayed a cumulative effect of the S1 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)DELTA, still expressed an epitope which eli cits a neutralizing antitoxin antibody and were protective in the mous e intracerebral challenge test. Recombinant pertussis vaccines based o n PT analogs with detoxifying mutations in multiple subunits may thus represent the next generation of improved whooping cough vaccines.