Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.
The toxicity of pertussis toxin is mediated by the ADP-ribosyltransferase activity of subunit S1. ADP-ribosylation of the target substrates in eukaryotic cells is a common mechanism of action of many bacterial protein toxins (1-3). The best-studied molecules that adopt this mechanism are diphtheria toxin (4-7), Pseudomonas exotoxin A (8-10), cholera toxin (11-13), and pertussis toxin (14-16), but several other toxins possessing ADP-ribosyltransferase activity have also been described (17,18). The toxins usually contain two functional moieties: A, which is enzymatically active, and B-, which recognizes and binds the receptors on the cell surface facilitating the entry of the enzymatically active subunit into the target cells.The proteins that are ADP-ribosylated by diphtheria, (20)(21)(22).For diphtheria and Pseudomonas toxins, a series of studies, which include nitrosoguanidine mutagenesis of the genes (23), characterization of the mutant genes (5) and their products (24-26), photoaffinity labeling of the toxins with NAD (27, 28), site-directed mutagenesis (29, 30), and crystallographic structure of the Pseudomonas exotoxin A (31) have led to the identification of amino acids essential to the enzymatic activity of the proteins. For example, Glu-148 of diphtheria toxin and Glu-553 of Pseudomonas -exotoxin A, located within the catalytic site of the two enzymes, cannot be replaced even with an aspartic acid without abolishing enzymatic activity (29,30).We used a similar approach to study the structure-function relationship of the S1 subunit ofpertussis toxin. By carboxyland amino-terminal deletion analysis and site-directed mutagenesis, we identified at least three regions of the S1 subunit that are essential for enzyme function. Substitutions within these regions produce enzymatically inactive molecules. MATERIALS AND METHODSConstruction of the Si Deletion Mutants. The S1 subunit of pertussis toxin was expressed in Escherichia coli fused to the 98 amino-terminal amino acids of the MS2 polymerase. This fusion protein (PTE255) contains amino acids 2-235 of the S1 subunit and is enzymatically active (32). The gene coding for the amino acids 2-235 is contained within a BamHI-Xba I fragment flanked by an EcoRI site at the 5' end and a HindIII site at the 3' end. To obtain the plasmids expressing carboxylterminal deletion mutants of the S1 protein, the plasmid pTE255 was digested first with Xba I and then with Nco I, Nru I, Bal I, Sal I, and Sph I, respectively.' The sticky ends generated by the restriction enzymes were then repaired by the large fragment of DNA polymerase, and the plasmids were circularized by DNA ligase. During this process, the natural stop codon of the S1 subunit was lost, and therefore the new proteins contained a few amino acids fused at the carboxyl terminus: NCO, NRU, and SPH proteins had the following carboxyl-terminal unrelated amino acids: Leu-ProArg-Ala-Phe-Arg. BAL and SAL proteins contained, respectively, 50 and 16 amino acids deriving from the sequence of pBR322 (33) fused at th...
Pertussis toxin, a protein composed of five different subunits, is responsible for the pathogenicity of Bordetella pertussis and is the main component of a new vaccine against whooping cough. The genes coding for the five subunits, recently cloned and sequenced, are organized as an operon. We approached the problem of expression of the five genes in Escherichia coli and, although we obtained high levels of transcription of the native pertussis toxin genes, the amount of proteins produced was very low or undetectable. To obtain suitable expression of each of the five subunits, we fused their genes to the gene coding for the DNA polymerase of MS2 in the expression vector pEx31. A total of 5 to 30 mg of purified fusion proteins could be obtained from 1 liter of culture. The purified fusion proteins were used to immunize rabbits to obtain sera against each of the five subunits. These sera, although able to recognize the toxin in an enzyme-linked immunosorbent assay and the corresponding subunits in Western blots, were not able to protect CHO cells from the action of pertussis toxin. Mice immunized with the five subunits were not protected from an intracerebral challenge with B. pertussis. Subunits S2 and S3, which are 67% homologous, were shown to cross-react immunologically. The fused subunit S1 was able to ADP-ribosylate transducin as efficiently as the native pertussis toxin.
Human T lymphocyte clones specific for pertussis toxin (PT) were used to analyze the fine specificity of the response to PT, the basic component of new acellular vaccines against whooping cough. The majority (83%) of the clones specific for PT recognized S1, the subunit that in animal models has been shown to be highly immunogenic. To map T cell epitopes on S1, 18 S1-specific clones were tested for recognition of recombinant fragments representing NH2-terminal and COOH-terminal deletions of S1 and two recombinant S1 subunits containing amino acid substitutions. This approach led to the identification of three regions of the protein as the sequences containing T cell antigenic sites: 1-42, 181-211, and 212-235. Synthetic peptides were eventually used for a finer localization of the T cell epitopes. Two peptides, one of 13 residues (27-39) at the NH2 terminus and one of 24 residues (171-194) at the COOH terminus, stimulated proliferation of three and four clones, respectively. Both peptides are recognized in association with HLA DR1 molecules. These results stress the role of S1 in the immune response to PT and provide data useful for the development of a recombinant or synthetic antipertussis vaccine containing T cell epitopes from S1.
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