Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion

The nine ptl genes (A-I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA-H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5- to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele.     

A rapid and sensitive assay for pertussis toxin based on pigment aggregation within the melanophores of an isolated fish scale

The noradrenaline-induced pigment aggregation within the melanophores of the cuckoo wrasse ( Labrus ossifagus L.) has been shown to be mediated by postsynaptic alpha₂-adrenoceptors which in turn act via an inhibitory control of adenylate cyclase. In a previous paper it was shown that pertussis toxin (PT) caused a blockade of noradrenaline-induced pigment aggregation. Based on these findings, an assay for PT has been developed. The method involves toxin titration directly on isolated fish scales from L. ossifagus , takes about 2 h to perform and needs no sophisticated equipment. The method allows detection of femtogram quantities of toxin per ml. The effect of purified PT was neutralized by antiserum. Some preliminary results indicate that it is possible to detect PT-like activity in sputum samples from patients with suspected whooping cough, whereas no PT-like activity could be detected in samples from healthy volunteers.     

Optimal conditions for the toxoiding of pertussis toxin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide · HCl

Abstract The optimal conditions for toxoiding a pertussis toxin (PT) preparation with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide · HCl (EDAC) were determined. The prime factor affecting the toxoiding of PT was the EDAC to protein ratio. A ratio of 40–80 : 1 EDAC to protein by weight was optimal for abolishing the acute toxicity, histamine-sensitising and leucocytosis-promoting activities associated with PT, whilst maintaining the antigenicity of the vaccine antigens. An EDAC-toxoid also manifested no late histamine-sensitising activity. Duration of exposure to EDAC, temperature and pH value of the reaction were found not be be critical for toxoiding. The data indicated that the use of EDAC for toxoiding PT in a B. pertussis extract is a simple and reproducible procedure and should be considered as a method for the production of acellular pertussis vaccines.     

Effect of hydromechanical forces on the production of filamentous haemagglutinin and pertussis toxin ofBordetella pertussis

Summary The production ofBordetella pertussis extracytoplasmic filamentous haemagglutinin (FHA) and pertussis toxin (PT) in a bioreactor under stirring conditions was studied in order to investigate the effect of hydromechanical forces on yields of both antigens. It was shown that FHA loses its haemagglutinin activity when the power transmitted by the agitator and the aerator per unit volume increases, whereas PT production is not affected. The loss of FHA activity can be explained by the action of shear forces on the filamentous structure of this antigen.Nomenclature C^* dissolved oxygen saturation concentration - C₁ dissolved oxygen concentration - D impeller diameter - power transmitted by the agitator and the aerator per unit of liquid volume - E_m maximum local energy dissipation rate per unit of liquid volume - K_La volumetric oxygen transfer coefficient - N impeller speed - Pg power input in aerated system - qO₂m maximum specific oxygen consumption rate - R_e Reynold number (D²N /) - VVM volume of air per volume of fermentation broth per minute - Xm maximum of biomass concentration - o Kolmogorov-microscale - fermentation broth viscosity - fermentation broth kinematic viscosity - fermentation broth density - expt experiment     

A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids

Abstract Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) at doses as low as 0.8 ng·ml⁻¹, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertusis , containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for PT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.     

重组百日咳毒素Sl亚单位的纯化及免疫原性

将重组百日咳毒素S1亚单位基因的质粒在DH5α和TOPl0两株大肠杆菌中进行表达,并对表达产物进行了初步纯化。粗制的rSl免疫家兔所得血清,用HP—PT及1B7-PT包被的ELISA测定效价,并做被动免疫保护试验。抗PT的抗体滴度为1：32000。该抗rSl血清在小鼠被动免疫保护试验中,对百日咳杆菌18323毒株进行脑腔攻击的被动免疫保护作用达到100％。证明rSl亚单位具有良好的抗原性和免疫原性,并与天然S1具有相同的抗原表位。     

Recombinant acellular pertussis vaccine—from the laboratory to the clinic: improving the quality of the immune response

Abstract Vaccination is the most effective way to prevent infectious diseases. Recombinant DNA technologies have provided powerful new tools to develop vaccines that were previously impossible or difficult to make, and to improve the vaccines that were already available but had been developed using old technology. In the case of whooping cough, an effective vaccine (composed of killed bacterial cells) is available, but its use is controversial because of the many side effects that have been associated with it. An improved vaccine against this disease should contain pertussis toxin, a molecule that needs to be detoxified in order to be included in the vaccine. Classical methods of detoxification, such as formaldehyde treatment have been used to inactivate this toxin. We have used recombinant DNA technologies to clone the pertussis toxin gene, express it in bacteria, map the B and T cell epitopes of the molecule, and to identify the amino acids that are important for enzymatic activity and toxicity. Finally, we have used this information to mutate the gene in the chromosome of Bordetella pertussis in order to obtain a strain that produces a molecule that is already non-toxic. This genetically inactivated pertussis toxin was tested extensively in animal models and clinical trials and was found to induce an immune response that is superior in quality and quantity to that induced by the vaccines produced by conventional technologies.     

Immune responses to the pertussis toxin in Balb/c and C57B1/6 mice

Abstract Immunization of Balb/c and C57B1/6 mice with the pertussis toxin (Ptx), purified from the culture supernatant of Bordetella pertussis (the whooping cough bacillus) resulted in different immune reactions in these genetically different strains of mice. Antibody responses to Ptx were detected only in Balb/c, whereas both Balb/c and C57B1/6 produced anti-Ptx antibodies when immunized with detoxified Ptx. Also, delayed-type hypersensitivity reactions differ strongly according to the use of Ptx or detoxified Ptx as eliciting antigen.     

An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines

Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose–response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.     

Antibodies to Bordetella pertussis adenylate cyclase toxin in neonatal and maternal sera

Abstract To investigate the high prevalence among infants of antibodies to Bordetella pertussis adenylate cyclase toxin (ACT), cord-blood sera were examined for antibodies to ACT, filamentous hemagglutinin (FHA) and pertussis toxin (PT) using immunoblot analysis. Antibodies reactive with ACT were the most prevalent in neonatal sera. Similar reactivity of IgG with ACT was found in each sample of a given neonatal-maternal pair, yet IgM reactive with ACT was virtually absent in neonatal sera, suggesting that antibodies to ACT are maternally derived. Antibodies to ACT might come from infection or childhood vaccination of the mothers since pertussis vaccines from all US manufacturers elicited antibodies to ACT in mice. Alternatively, these antibodies may have been elicited by a cross-reactive antigen such as Escherichia coli α-hemolysin, since all of the neonatal and maternal sera contained antibodies reactive with α-hemolysin.     

Effects of antibodies to the filamentous haemagglutinin and to the pertussis toxin of Bordetella pertussis on adherence and toxic effects to 3T3 cells

Abstract Adherence of B. pertussis to NIH3T3 mouse fibroblasts was efficiently inhibited by a mouse immune serum reacting specifically with the filamentous haemagglutinin (FHA), whereas a mouse immune serum reacting specifically with the pertussis toxin (Ptx) produced partial inhibition only significant after 3 h infection. Protection against cytopathic effects on infected 3T3 cells with anti-FHA antibodies was at least as effective (83.3%± 7.5) as with anti-Ptx antibodies (75%± 4). This suggests that adherence of B. pertussis to eukaryotic receptors is a primary mechanism determining both bacterial proliferation and toxic effects in susceptible cells, and that prevention of B. pertussis attachment to cell receptors might be sufficient to protect against both infectious and toxic processes in whooping cough.     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Effect of hydromechanical stress on cellular antigens ofBordetella pertussis

Cells ofBordetella pertussis grown in a bioreactor under stirring conditions were studied to investigate the effect of shear stress on cellular-bound filamentous haemagglutinin (FHA). FHA attached to the bacterial surface, unlike extracellular FHA, was not affected at the shear levels tested. Moreover, no other cellular immunogen involved in the whole-cell protective activity seemed to be affected by hydromechanical forces.     

Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.     

Lectin domains in the toxin ofBordetella pertussis: selectin mimicry linked to microbial pathogenesis

The pathogenesis of many infectious diseases is critically determined by prokaryotic lectins which enable differential recognition and activation of targeted eukaryotic cells. Some bacterial adhesins mimic and co-opt eukaryotic cell-cell adhesion motifs. This is illustrated by the toxin ofBordetella pertussis. Pertussis toxin mediates intoxication of eukaryotic cells by elevation of cAMP and it serves as an adhesin binding the bacteria to ciliated cells and respiratory macrophages. These activities are mediated by the lectin-like properties of the binding oligomer of the toxin. A comparison of pertussis toxin and the selectins involved in leukocyte trafficking indicates that these prokaryotic and eukaryotic C-type lectins share some element of primary sequence similarity, three dimensional structure, and biological activities. Such mimicry suggests a link between eukaryotic cell-cell adhesion motifs and microbial pathogenesis.     

The Bvg accessory factor (Baf) enhances pertussis toxin expression in Escherichia coli and is essential for Bordetella pertussis viability

Pertussis toxin expression in the Gram-negative respiratory pathogen, Bordetella pertussis, is regulated by the BvgAS two-component system. Previous studies suggested that an additional gene encoding a Bvg accessory factor (Baf) was required, along with BvgAS, for expression of a ptx-lacZ fusion in Escherichia coli grown in rich medium. However, other studies showed that BvgAS is sufficient for ptx-lacZ expression in minimal medium. Here we show that Baf acts with BvgAS to further increase ptx-lacZ expression in E. coli grown in minimal media and this is concomitant with a two-fold increase in BvgA protein levels. Gene replacement experiments show that baf is essential for viability of B. pertussis, suggesting that Baf affects the expression of other genes in addition to ptx.     

Characterization of the N-terminal structure of pertussis toxin subunit S1 and hybridization of oligodeoxyribonucleotide probes with a Bordetella pertussis DNA fragment

Abstract N-terminal amino acid sequence analysis of the enzymatic subunit S1 of pertussis toxin from Bordetella pertussis showed the structure of the first 25 residues. 2 different oligodeoxyribonucleotide probes were synthesized as mixed 32-mers corresponding to the DNA sequence deduced. Southern blot analysis of pertussis DNA digested with restriction endonuclease Cla I showed that both mixed probes hybridized with a DNA fragment of about 10 kb, thus identifying a B. pertussis gene corresponding to the protein structure determined.     

Pertactin antigens extracted from Bordetella pertussis and Bordetella bronchiseptica differ in the isoelectric point

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica. Received: 9 April 1997 / Accepted: 20 May 1997     

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.