Characterization of pertussis toxin analogs containing mutations in B-oligomer subunits.
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) delta 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) delta retained 10% 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(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 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.
Connaught Centre for Biotechnology Research, Willowdale, Ontario, Canada.
S
S
Loosmore
G
G
Zealey
S
S
Cockle
H
H
Boux
P
P
Chong
R
R
Yacoob
M
M
Klein
D000595:Amino Acid Sequence; D000818:Animals; D001886:Bordetella pertussis / Q000235:genetics*; D016466:CHO Cells; D006224:Cricetinae; D005798:Genes, Bacterial; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D037342:Pertussis Toxin; D010567:Pertussis Vaccine / Q000276:immunology; D013329:Structure-Activity Relationship; D014614:Vaccines, Synthetic / Q000276:immunology; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity*
D010567:Pertussis Vaccine; D014614:Vaccines, Synthetic; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin