Pertussis Toxin Subunit Research Articles

Role of histidine 35 of the S1 subunit of pertussis toxin in the ADP-ribosylation of transducin

Molecular modeling of the S1 subunit (S1) of pertussis toxin with other ADP-ribosylating bacterial exotoxins predicted that histidine 35 (His-35) would residue within the active site of S1. Recombinant derivatives of S1 (rS1 and the C180 peptide) which contained either a H35Q or H35P mutation were analyzed to determine the role of His-35 in ADP-ribosylation. C180 peptide is a recombinant peptide composed of the amino-terminal 180 amino acids of S1. Under linear velocity conditions, C180H35Q possessed 2% of wild type C180 peptide activity and C180H35P possessed no detectable activity in the ADP-ribosylation of transducin. The H35Q mutation did not change the affinity of recombinant peptides for NAD or two targets for ADP-ribosylation, transducin, or alpha i3C20, but did lower the kcat in the NAD glycohydrolase and ADP-ribosyltransferase reactions. Neither the H35Q nor H35P mutation reduced the ability of recombinant proteins to be photocross-linked with NAD which was consistent with the His-35 mutations not reducing the affinity for NAD. These data indicate that His-35 does not reduce the affinity of S1 for NAD or transducin, but functions as a catalytic residue in the ADP-ribosylation reaction possibly in a hydrogen bonding capacity.

The NAD-glycohydrolase activity of the pertussis toxin S1 subunit. Involvement of the catalytic HIS-35 residue.

Pertussis toxin is a member of ADP-ribosylating bacterial toxins that are capable of catalyzing the cleavage of the N-glycosidic bond of NAD+ and the transfer of its ADP-ribose moiety to G proteins. The catalytic S1 subunit of pertussis toxin uses signal transducing G proteins as acceptor substrates but can also catalyze the transfer of the ADP-ribose moiety to water in the absence of G proteins. Site-directed mutagenesis followed by kinetic analyses of truncated soluble mutant proteins revealed that His-35 of S1 is a catalytic residue because alterations of this residue affect the turnover rate of NAD-glycohydrolysis by approximately two orders of magnitude without significantly affecting substrate binding. Replacement of the imidazole of His-35 by the side chain of glutamine maintained the highest residual activity. The pH dependence of the enzyme activity showed only slight variations over the experimental range with an optimum at pH 7.5 and an approximate pKa of 6.5 to 7. This pH dependence was abolished by the Gln substitution, which still retained significant activity, suggesting that His-35 probably does not act as a true base but rather as a proton acceptor. Direct catalytic roles for several other residues were ruled out. Ser-52 substitutions resulted in slight alterations of both kcat and Km for NAD+ suggesting an involvement in maintaining the local geometry of the active site rather than a direct role in catalysis for this residue. Kinetic studies on mutants with substitutions of Ser-40 indicate a role in NAD+ binding for this residue. In conjunction with previous findings, these studies suggest that the NAD-glycohydrolase activity of S1 utilizes 2 catalytic residues, His-35 and the previously identified Glu-129. The enzyme mechanism could therefore proceed through an activation by polarization of the acceptor substrate water or G protein by His-35, and the stabilization of an oxocarbonium-like transition state intermediate by Glu-129.

Peptides from pertussis toxin interfere with neutrophil adherence in vitro and counteract inflammation in vivo

Selectins on the surface of endothelial cells initiate leukocyte rolling along the capillary walls during inflammation. The amino acid sequence 19-52 of pertussis toxin subunit S3 is strikingly similar to the sequence 15-46 of the selectins. The S3 subunit inhibits the binding of neutrophils to selectin-coated surfaces and a peptide spanning the 28-45 sequence of S3 reduces leukocyte binding to endothelial cells in vitro and inhibits leukocyte recruitment to the subarachnoid space in vivo. To identify sequences within the 28-45 S3 peptide responsible for these activities, 27 peptides derived by successive truncation of amino acids from either the amino or the carboxyl terminus were tested for anti-inflammatory activity. Truncation at five residues ablated the ability to inhibit neutrophil adherence to endothelial monolayers: valine 32, alanine 33, arginine 36, asparagine 38, and threonine 43. The most active peptides were either full-length molecules (28-44, 30-45) or short peptides from both ends of the full sequence (39-45, 40-45, 41-45, 28-32). Three peptides with the strongest ability to prevent neutrophil adherence in vitro (28-44, 30-45, 40-45) reduced the cerebrospinal fluid leukocytosis in a pneumococcal meningitis model when administered intravenously. We conclude that peptides derived from a prokaryotic lectin have anti-inflammatory properties consistent with inhibition of selectin participation in leukocyte recruitment during inflammation.

Structural relationship between the S1 and S4 subunits of pertussis toxin.

Pertussis toxin, the most important protective antigen of Bordetella pertussis, is a 106-kDa hexameric protein composed of an A-protomer (subunit S1) and a pentameric B-oligomer (S2 + S3 + 2S4 + S5). The most potent mouse-protective monoclonal antibodies against both respiratory and intracerebral infections were specified for either S1 or S4 and competed with each other in binding to epitopes of native pertussis toxin captured by haptoglobin or in solution, although they did not compete on unfolded pertussis toxin. These data suggest that the protective epitope(s) of S1 and S4 are very closely correlated; they are probably close together sterically. Non-protective anti-S1 and anti-S4 monoclonal antibodies recognized inner antigenic determinants which are not exposed on the surface of native pertussis toxin and interfered with association of the A-protomer and the B-oligomer. These data suggest that the A-protomer and the S4 subunit of the B-oligomer may be closely associated in the native hexameric pertussis toxin molecule.

Antiinflammatory effects in experimental meningitis of prokaryotic peptides that mimic selectins.

The lectin domains of two subunits of pertussis toxin, S2 and S3, share amino acid sequence similarity with the lectin domains of the eukaryotic selectin family. During inflammation, selectins appear on endothelial cells and promote recruitment of leukocytes by reversibly binding carbohydrates. Synthetic peptides representing the carbohydrate recognition domains of S2 and S3 competitively inhibited adherence of neutrophils to endothelial cells in vitro. For some peptides, this antiinflammatory effect occurred without up-regulation of the function of the leukocyte integrin CD11b/CD18. Intravenous administration of peptides to animals with meningitis disrupted recruitment of leukocytes into the cerebrospinal fluid. These findings indicate that peptides derived from prokaryotic members of the selectin family have therapeutic antiinflammatory potential.

Evidence for a catalytic role of glutamic acid 129 in the NAD-glycohydrolase activity of the pertussis toxin S1 subunit.

The S1 subunit of pertussis toxin is an ADP-ribosyl-transferase capable of transferring the ADP-ribose moiety of NAD+ to nucleotide-binding signal-transducing proteins of the Gi/G(o) family. In the absence of G proteins, the enzyme also catalyzes the hydrolysis of NAD+. Glu-129 was previously shown to be critical for both enzymatic activities. In this study, site-directed mutagenesis was used to make the conservative substitution of aspartate for Glu-129. The recombinant wild type and mutant proteins were purified to near homogeneity and used for enzymatic analyses. Kinetic experiments showed that the kcat of the mutant protein was about 200 times less than that of the wild type enzyme, whereas the Km for NAD+ of the two proteins were very similar, suggesting that Glu-129 is a catalytic residue for the NAD-glycohydrolase reaction of S1. This hypothesis was confirmed by a less than 2-fold change in Kd as measured by fluorescence quenching studies, indicating that the binding of NAD+ is not affected in the mutant protein in any important way. In another experiment, the replacement of Glu-129 by cysteine resulted in a disulfide bridge between Cys-129 and Cys-41 in rS1d-E129C, suggesting that the folding of the polypeptide chain is such that the catalytic Glu-129 residue is close to the amino-terminal NAD-binding site of S1. These findings imply that Glu-129 plays a key role in catalysis of the NAD-glycohydrolase reaction, possibly by electrostatically stabilizing a cationic transition state intermediate, or by serving as a general base to deprotonate the ADP-ribosyl acceptor substrates.

Conformational study of a short Pertussis toxin T cell epitope incorporated in a multiple antigen peptide template by CD and two-dimensional NMR. Analysis of the structural effects on the activity of synthetic immunogens.

The immunogenic efficacy of multiple antigen peptides, MAPs, i.e. branched molecules in which multiple copies of a given immunogenic peptide are attached on a scaffold of lysine residues via both alpha and epsilon linkages, has been repeatedly demonstrated, but little is known about the structural arrangement of these peptide constructs. A conformational characterization was therefore performed for a known T cell epitope of the S1 subunit of Pertussis toxin, whose sequence is predicted to form alpha-helix. The peptide DNVLDHLTGR, its N-acetylated and C-amidated analogue and a tetrabranched MAP based on the N-acetylated peptide were prepared and studied by CD and two-dimensional 1H-NMR. No evidence of helical structure was obtained in water for the isolated peptides. In contrast, in triflouroethanol, the isolated epitopes fold into a helical structure spanning the segment Val3-Thr8 in the uncapped molecule and encompassing also the N-terminal region in the capped analogue. The mobile C-terminal region tends to adopt a distorted turn arrangement in both peptides due to the folding of Arg10 guanidinium over the backbone. No distortion of the helix structure was observed for the single-copy epitope in the four-branched MAP molecule in trifluoroethanol: each peptide chain is equivalent within the MAP and shows an even more regular helical pattern than the isolated end-blocked sequence. A slight difference was located at the junction with the lysine scaffold: the peptide bond to epsilon NH was found in a much more extended conformation than the corresponding link to alpha NH. These structural results correlate with in vitro T cell stimulatory activity of the three compounds examined and provide arguments supporting the previous suggestion that MAP tetramers are unlikely to elicit an immune response specific for the synthetic template assembly, a feature necessary to retain the advantage of the polymeric epitope presentation.

Evaluation of pooled and individual components of Bordetella pertussis as antigens in an enzyme immunoassay for diagnosis of pertussis.

Six different antigen preparations for use in an enzyme immunoassay (EIA) to detect IgM, IgA and IgG antibodies to Bordetella pertussis were evaluated using sera from 13 randomly selected culture-positive patients and from 87 patients with suspected pertussis during a pertussis outbreak. Based on results in 80 healthy control sera a specificity limit of 99.9% was selected. Sera from all culture-positive patients reacted with at least one of the antigens. The sensitivity of the EIA using the individual antigen preparations was 85% for filamentous hemagglutinin, 92% for pertussis toxin, 62% for 69 kDa outer membrane protein, 85% for a pool of these three antigens, 54% for sonicated whole bacteria and 69% for 21 kDa pertussis toxin subunit S1. In the outbreak patient group 49 (56%) of the initial sera reacted with at least one of five antigen preparations. The EIA using sonicated bacteria detected only 41% of all seropositive cases compared with 51% using filamentous hemagglutinin, 61% using pertussis toxin, 65% using 69 kDa OMP and 65% using pooled antigen. It is concluded that either the pooled antigen or pertussis toxin antigen are suitable antigen preparations for use in the EIA for diagnosis of pertussis.

Molecular characterization of the in vitro activation of pertussis toxin by ATP

Pertussis toxin (PT)-catalyzed ADP-ribosylation of transducin (Gt) is stimulated by ATP. In the absence of ATP, PT exhibited an approximately 20-fold lower linear velocity than the recombinant S1 subunit (rS1) in catalyzing the ADP-ribosylation of Gt. In the presence of 0.1 mM ATP, the linear velocities of rS1 and PT were essentially identical. ATP increased the kcat of PT-catalyzed ADP-ribosylation of Gt without altering the Kmapp for either Gt or NAD. Further, in the presence of ATP, PT exhibited similar kinetic constants under conditions of variable Gt and variable NAD as rS1 in catalyzing the ADP-ribosylation of Gt. The S1 subunit of PT was cleaved by chymotrypsin to a single immunoreactive peptide in the absence of ATP, while three immunoreactive peptides were generated in the presence of ATP. The S1 subunit of PT was not cleaved by trypsin in the absence of ATP, at the concentrations of trypsin used, while two immunoreactive peptides were produced in the presence of ATP. The immunoreactive peptides produced either by chymotrypsin or trypsin cleavage of the S1 subunit of PT in the presence of ATP were indistinguishable from those produced by cleavage of rS1 with the same protease. Chymotryptic and tryptic cleavage of rS1 was not altered by ATP. When PT was incubated with ATP prior to Bio-Gel P-100 gel filtration, approximately 8% of the S1 subunit dissociated from the B oligomer, as determined by ADP-ribosyltransferase assays of the column eluant. This increased to 20% when ATP was included in the column buffer. The presence of dithiothreitol and NAD in addition to ATP did not affect the amount of dissociated S1 subunit. Our data further indicated that activation of PT by ATP was a reversible process. Together, these data showed that ATP quantitatively converted the S1 subunit of PT to a form which was kinetically and conformationally identical with rS1, while only a fraction of the S1 subunit was dissociated from the B oligomer. These results indicate that both S1 subunit which is bound to the B oligomer as well as dissociated S1 subunit are capable of catalyzing the ADP-ribosylation of Gt.

Identification of B-cell epitopes on the S4 subunit of pertussis toxin

The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT) by the synthetic peptide approach. Two strategies were followed: (i) screening of two series of overlapping peptides (12- and 25-residue peptides) covering the entire S4 sequence by a panel of murine monoclonal anti-PT antibodies and various polyclonal anti-PT antisera in an enzyme-linked immunosorbent assay (ELISA), and (ii) analysis of the S4 amino acid sequence by a predictive algorithm followed by synthesis and immunization of mice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide conjugate antisera were tested in an ELISA for cross-reactivity with native PT, B oligomer, and S4. Screening of the free peptides in an ELISA by the PT antisera indicated the presence of six B-cell epitope-containing domains covered by residues 18 to 32, 33 to 46, 39 to 52, 51 to 65, 71 to 84, and 91 to 106. None of the peptides, however, were recognized by the monoclonal anti-PT antibodies in an ELISA. Immunization with six computer-predicted peptides (B1 to B6) and three potential T-cell epitopes (T1 to T3) gave rise to very high antibody responses towards the homologous conjugates. With the exception of the anti-T1/diphtheria toxoid antisera, all anti-peptide conjugate antisera cross-reacted with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however, showed any PT-neutralizing effect as measured by the Chinese hamster ovary cell assay and the leukocytosis-promoting activity test. The results of the present study suggest that discontinuous epitopes are predominant in the S4 subunit of native PT.

Evidence that lipopolysaccharide and pertussis toxin bind to different domains on the same p73 receptor on murine splenocytes.

In previous studies, we used a photoactivable, radioiodinated lipopolysaccharide (LPS) derivative to define and characterize a specific bacterial endotoxic LPS-binding protein (p73) on mammalian lymphoreticular cells, including B and T lymphocytes and macrophages. More recently, using the same methodology, we characterized a specific interaction of LPS with the S2 subunit of Bordetella pertussis pertussis toxin (PT) in the fluid phase (M.-G. Lei and D. C. Morrison, J. Biol. Chem., 268:1488-1493, 1993). Furthermore, we showed that lysozyme (LZM) but not polymyxin B can compete with PT for binding to LPS in the fluid phase, a result suggesting that these two molecules compete for the same binding site on LPS. In this report, we demonstrate that the binding of PT to murine splenocytes (cell-bound PT) reduces the ability of the LPS photo-cross-linking probe to bind to the p73 receptor. The reduction can also be demonstrated with the PT B oligomer, a result indicating that the observed reduction of LPS binding to the p73 receptor by PT is A-protomer (S1-subunit) independent. More importantly, our studies document that cell-bound PT can be radiolabelled by the LPS probe, coincident with the observed reduction in p73 photoaffinity labelling. The preferential interaction of LPS with the PT S2 subunit in the fluid phase was, however, not observed with cell-bound PT. The reduction in radiolabelling of the p73 receptor by the LPS probe and in radiolabelling of cell-bound PT was shown to be concentration dependent. The data presented here document, however, that LZM does not reduce the ability of the LPS probe to bind to the p73 receptor on mouse splenocytes, nor does the presence of LZM bound to LPS influence the observed reduction in photoaffinity labelling of p73 by the LPS probe or radiolabelling of cell-bound PT by the LPS probe. Collectively, these results support the concept that the ability of LPS to interact with PT in the fluid phase is not responsible for the ability of cell-bound PT to influence the binding of the LPS probe to the p73 receptor. Thus, it is suggested that PT and LPS bind to different sites on the p73 molecule and that this same p73 protein may recognize both LPS and PT.

Identification of Immunogenic and Antigenic Epitopes of the S4 Subunit of Pertussis Toxin

Identification of Immunogenic and Antigenic Epitopes of the S4 Subunit of Pertussis Toxin

Localization of a Receptor Recognition Domain on the S3 Subunit of Pertussis Toxin by Peptide Mapping

Localization of a Receptor Recognition Domain on the S3 Subunit of Pertussis Toxin by Peptide Mapping

Localisation of a receptor-recognition domain on the S3 subunit of pertussis toxin by peptide mapping.

Overlapping 10-amino-acid peptides, which consecutively span the amino acid sequence of the S3 subunit of pertussis toxin, were synthesised on polyethylene pins and screened for their ability to bind the glycoprotein fetuin. Fetuin binding was localised to a single peptide comprising amino acids 46-55. A free peptide, (E)S3c, of longer sequence (S3 amino acids 44-58) was also found to bind alpha-1-acid glycoprotein, mixed brain gangliosides and fetuin. (E)S3c also recognised asialofetuin but with a lower apparent affinity relative to fetuin. The single tryptophan residue of the peptide yielded a fluorescence-emission maximum of 355 nm. In the presence of either ganglioside or the phospholipid L-alpha-lysolecithin, but not N-acetylneuramin-lactose or lactosylceramide, the emission intensity of (E)S3c was enhanced and the emission maximum blue-shifted to 340 nm by ganglioside, or to 345 nm by L-alpha-lysolecithin. Monosialogangliosides, disialogangliosides, and trisialogangliosides, when fluorescence-titrated, were each found to bind the peptide with a similar dissociation constant of 4.4 +/- 2.8 microM. These findings demonstrate that region 44-58 of the pertussis-toxin S3 subunit is likely to be involved in the recognition of both glycosylated and phospholipid constituents of target-cell membranes.

Lipopolysaccharide interaction with S2 subunit of pertussis toxin.

Using radioiodinated, photoactivable, reducible cross-linker conjugated bacterial endotoxic lipopolysaccharide (125I-ASD-LPS), we have demonstrated that LPS selectively binds to the S2 subunit of pertussis toxin (PT). Since LPS also interacts with the S2 subunit of the B-oligomer of the toxin, the binding of LPS to PT is not A-protomer (S1 subunit) dependent. The binding can be inhibited with native underivatized LPS and with purified lipid A, suggesting that the binding is mediated through the lipid A moiety of the LPS molecule. The binding of PT to LPS can be inhibited by bovine fetuin glycoprotein. Since PT has been demonstrated to interact specifically with N-linked oligosaccharide side chains of fetuin, the interaction of LPS with the S2 subunit of PT may involve carbohydrate-dependent interactions of the disaccharide backbone of lipid A with S2. Additional studies have documented that LPS binding to PT may be competitively inhibited by lysozyme but not by polymyxin B. Sequence analysis has allowed identification of a high degree of amino acid sequence similarity between the S2 subunit of PT and hen egg white lysozyme at the N-terminal 80-residue regions. Shared N-terminal sequence similarity between lysozyme, PT-S2, and a third LPS-binding protein alpha-lactalbumin allows tentative identification of a second family of LPS binding proteins.

Identification of murine T-cell epitopes on the S4 subunit of pertussis toxin.

The aim of the present study was to identify murine T-cell epitopes on pertussis toxin subunit S4. Six mouse strains with five different haplotypes at the H-2 locus were immunized with the pertussis toxin B oligomer. Lymph node lymphocytes were isolated and stimulated in an in vitro proliferation assay with pertussis toxin components and 11 overlapping synthetic peptides synthesized on the basis of the primary sequence of S4. In vitro proliferative responses to the synthetic peptides revealed the presence of four distinct murine T-cell epitopes on subunit S4. The recognition of the peptides was major histocompatibility complex restricted. Immunizing four of the six mouse strains with the synthetic peptides showed that the peptides which were demonstrated to contain T-cell epitopes following immunization with the B oligomer were able to induce proliferative responses to detoxified pertussis toxin and pertussis toxin components containing subunit S4. One of the identified murine T-cell epitopes corresponded to one of the major human T-cell epitopes previously identified on subunit S4. It is hoped that this murine model system will facilitate the development of a synthetic immunogen mimicking the protective properties of pertussis toxin.

Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin.

Synthetic peptides corresponding to selected sequences in the S2 and S3 subunits of pertussis toxin were prepared and evaluated for their ability to inhibit the binding of biotinylated pertussis toxin and three biotinylated sialic acid specific plant lectins to fetuin and asialofetuin. The screening results indicated that two regions in the S2 subunit corresponding to amino acids 78-98 and 123-154 inhibited pertussis toxin binding to fetuin at submillimolar concentrations, while S3 sequences corresponding to amino acids 87-108 and 134-154 inhibited pertussis toxin-biotin binding to asialofetuin albeit with lower affinity. These results confirm earlier findings, which suggest that the S2 subunit is responsible for binding sialylated glycoconjugates. This was further confirmed by the ability of S2 peptides to inhibit the binding of the lectins from Maackia amurensis and wheat germ to fetuin. Two additional peptides from the S2 subunit of pertussis toxin corresponding to sequences 9-23 and 1-23 were found to contain within their sequences a 6-amino acid fragment which has strong homology with a sequence in wheat germ agglutinin that has been shown to be a component of the sialic acid binding site as determined by x-ray crystallography. One of these sequences from S2 (9-23) was biotinylated and evaluated for its ability to bind to carbohydrate. Through a series of experiments using fetuin, asialofetuin, asialoagalactofetuin, and simple saccharides, the biotinylated peptide was shown to bind with high affinity to sialic acid-containing glycoconjugates indicating that these sequences within the S2 subunit of pertussis toxin also play an important role in binding sialic acid.

Construction of a diphtheria toxin A fragment-C180 peptide fusion protein which elicits a neutralizing antibody response against diphtheria toxin and pertussis toxin.

A genetically engineered gene fusion was constructed which encoded a nontoxic derivative of the A fragment of diphtheria toxin joined to the C180 peptide of the S1 subunit of pertussis toxin. The product of this gene fusion, termed the DTA-C180 protein, was purified from the periplasm of Escherichia coli to approximately 80% purity. The DTA-C180 protein possessed an apparent molecular weight of 43,000 by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DTA-C180 protein was cleaved into two tryptic peptides, which migrated with apparent molecular weights of approximately 22,000. One tryptic peptide reacted with diphtheria antitoxin, while the other tryptic peptide reacted with anti-C180 peptide immunoglobulin G. The DTA-C180 protein did not inhibit protein synthesis or stimulate clustering morphology in Chinese hamster ovary cells. The DTA-C180 protein elicited an immune response, in guinea pigs, against both the DTA and C180 peptide components of the fusion protein, with alum being a more efficient adjuvant than Freund's adjuvant for eliciting neutralization titers. Neutralization titers elicited by DTA-C180 protein were weaker than those elicited by diphtheria toxoid and pertussis toxin 9K/129G, a genetically engineered double mutant of pertussis toxin. Three doses of DTA-C180 protein yielded a neutralization titer of 1/750 against pertussis toxin in Chinese hamster ovary cells and a neutralization titer of 1/50 against diphtheria toxin in Vero cells. This is the first report of a protein derived from a recombinant S1 subunit that elicits a neutralizing titer against pertussis toxin.

Identification of T- and B-cell epitopes of the S2 and S3 subunits of pertussis toxin by use of synthetic peptides.

To design an optimized synthetic vaccine against whooping cough, we have studied the biological and immunological properties of three peptides of the S2 subunit and nine overlapping synthetic peptides covering the entire sequence of the S3 subunit of pertussis toxin (PT). Synthetic peptides corresponding to sequences 18 to 41, 78 to 108, 134 to 154, and 149 to 176 of S3 were found to be consistently capable of stimulating the proliferation of PT-specific T-cell lines primed with pertussis toxoid in both BALB/c and A/J strains of mice. All synthetic peptides were recognized by rabbit antisera raised against PT or pertussis toxoid. Both S2 and S3 peptide-keyhole limpet hemocyanin (KLH) conjugates in the presence of complete Freund's adjuvant induced peptide-specific antibody responses in rabbits, and the antisera raised against S2(1-23), S3(18-41), S3(37-64), and S3(149-176) peptide-KLH conjugates cross-reacted with both subunits in the immunoblots. All antisera except those against S2(123-154) and S3(103-127) reacted with native PT in an enzyme-linked immunosorbent assay (ELISA) with PT directly coated onto microtiter wells. In contrast, antisera raised against S2(123-154), S3(1-23), S3(18-41), S3(37-64), S3(60-87), and S3(103-127) peptide-KLH conjugates recognized native PT in a fetuin-PT capture ELISA. S2(78-98), S3(1-23), and S3(149-176) peptide-KLH conjugates elicited good PT-neutralizing antibody responses as judged by the antitoxin CHO cell assay. Identification of these B-cell neutralization epitopes and T-cell immunodominant determinants represents a first step towards the rational design of a synthetic vaccine against whooping cough.

Identification of human T-cell epitopes on the S4 subunit of pertussis toxin.

Ten adult humans were vaccinated with the Japanese acellular pertussis vaccine JNIH-3, containing detoxified pertussis toxin (PT), formaldehyde, and filamentous hemagglutinin. The vaccination induced a specific antibody response to PT and filamentous hemagglutinin, and a Western blot (immunoblot) analysis of the antibody response to PT revealed antibodies to PT subunits S1, S2, S3, S4 and S5. The response of peripheral lymphocytes to PT was assessed in an in vitro proliferation assay. A proliferative response to detoxified PT and PT dimers S2-S4 and S3-S4 was found, and it was further demonstrated that the proliferative response to detoxified PT and dimer S2-S4 was mediated by T cells of the CD4+ phenotype. The specificity of the proliferative response to subunit S4 was analyzed with a range of synthetic peptides synthesized on the basis of the primary sequence of subunit S4. The proliferative response to the peptides revealed two major and one minor T-cell epitope located in the NH2-terminal end of subunit S4.