Human cellular immune responses to Bordetella pertussis infection

Abstract We have compared the responses of peripheral blood leucocytes from three groups (i) patients suffering from pertussis (whooping cough), (ii) clinical staff caring for those patients and laboratory staff working with Bordetella pertussis , and (iii) staff with no known recent contact with B. pertussis . In vitro stimulation with filamentous haemagglutinin (FHA) caused significant increases in proliferation of only the patient group's lymphocytes. In vitro stimulation with pertussis toxin (PT) caused a large increase in proliferation of lymphocytes from all three groups and in the patient group the increase in proliferation was related to the dose of PT. Interleukin 2 (IL-2) production by leucocytes from all three groups was significantly increased following challenge with FHA or PT. The increases in IL-2 production were greatest in lymphocytes from patients with pertussis. Challenge with toxoided pertussis toxin had no effect on either proliferation or IL-2 production in any of the groups.     

Human cellular immune responses to Bordetella pertussis infection

We have compared the responses of peripheral blood leucocytes from three groups (i) patients suffering from pertussis (whooping cough), (ii) clinical staff caring for those patients and laboratory staff working with Bordetella pertussis, and (iii) staff with no known recent contact with B. pertussis. In vitro stimulation with filamentous haemagglutinin (FHA) caused significant increases in proliferation of only the patient group's lymphocytes. In vitro stimulation with pertussis toxin (PT) caused a large increase in proliferation of lymphocytes from all three groups and in the patient group the increase in proliferation was related to the dose of PT. Interleukin 2 (IL-2) production by leucocytes from all three groups was significantly increased following challenge with FHA or PT. The increases in IL-2 production were greatest in lymphocytes from patients with pertussis. Challenge with toxoided pertussis toxin had no effect on either proliferation or IL-2 production in any of the groups.     

A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that products possessed a wide range of ribosylation activities. The pertussis toxin S1 subunit catalyzed ADP- ribosylation of the FAC-Galpha(i3)C20 peptide substrate and its subsequent quantitation by HPLC was demonstrated to be a sensitive and quantitative method for measuring intrinsic pertussis toxin activity. This methodology not only has the potential to be an alternative physicochemical method to replace existing bioassay methodology, but has the added advantage of being a universal method applicable to the assay of pertussis toxin in both whole cell and acellular vaccines as well as bulk and final formulated vaccine products. Acceptance of this method by regulatory agencies and industry as a credible alternative to existing methods would, however, require validation in an international collaborative study against the widely accepted bioassay methods.     

The cell mediated and humoral immune response to vaccination with acellular and whole cell pertussis vaccine in adult humans.

The cell mediated immune response (CMI) against pertussis antigens following vaccination with the traditional Danish whole cell pertussis vaccine (WC-P) and the Japanese acellular pertussis vaccine (A-PV) JNIH-3 was studied in four adult human volunteers. Vaccination with the A-PV induced an in vitro proliferative response of peripheral blood lymphocytes to pertussis toxin (PT) subunits S2-S4, S3-S4 and S5 and the filamentous hemagglutinin (FHA), and a better serological response to native PT, detoxified PT (dPT) and FHA than the WC-PV. The induced CMI and serological response were followed over a period of 17 weeks, and were not seen to decline during this period. Further, an in vitro proliferative response to Bordetella pertussis agglutinogen 2 and 3 were demonstrated using lymphocytes from recently and not-so-recently pertussis-vaccinated adults.     

Verification of components of acellular pertussis vaccines that have been distributed solely, been in routine use for the last two decades and contributed greatly to control of pertussis in Japan.

An ideal acellular pertussis vaccine is now under investigation worldwide. We have had acellular pertussis vaccines available for the last 22 years, which contributed greatly to the control of pertussis in Japan, although it has not been known whether they are one of ideal acellular pertussis vaccines or not. Moreover, the formulations of acellular pertussis vaccines that we have been using have not been widely recognized. Serum samples were taken from recipients of the T type, B type, and two-component acellular pertussis vaccine and assayed by ELISA for anti-PT, anti-FHA, and anti-69 kD OMP antibody levels and by the agglutination test. Although it was shown that T type vaccine contained four components (PT, FHA, 69 kD OMP, agglutingen), B type vaccine contained three components (PT, FHA, 69 kD OMP) and the two-component vaccine contained PT and FHA, it was concluded that PT and FHA were essential and common antigens contained in all three acellular pertussis vaccines in Japan. The national monitoring system for adverse effects of routine immunization demonstrated low reactogenicity of DTaP in Japan. This resulted in high acceptance rates of DTaP and in virtual control of pertussis.     

Humoral reaction to Bordetella pertussis antigens: pertussis toxin, filamentous hemagglutinin and lipopolysaccharide in children with clinical symptoms of whooping cough. II. Occurence and level of B. pertussis antigens in children with suspected whooping cough

The aim of this study was to determine and evaluate IgG, IgM and IgA levels to pertussis toxin (PT), filamentous hemagglutinin (FHA) and endotoxin (LPS) of B. pertussis in children with clinical symptoms of whooping cough. The serum samples obtained from 265 children (age range: 2 months-16 years) suspected of pertussis were examined by indirect haemagglutination (IH) and ELISA tests. Higher antibody level was most frequently observed in IgA class to PT, FHA and LPS in 45.3%, 35.1% and 66% of pertussis patients sera respectively. The least positive results were obtained in IgM class to PT and FHA (in 9.8% and 2.6% of children sera respectively) but in the case of LPS applied as the antigen in ELISA, higher IgM level was determined in 46.8% of pertussis patients sera. The four times increase of antibody level to LPS determined by IH was observed in 86.7% of children suspected of pertussis. Humoral response to B. pertussis infection is mainly connected with higher IgA level to PT, FHA, LPS and IgM to LPS in children with clinical symptoms of whooping cough.     

Essential role of the consensus nucleotide-binding site of PtlH in secretion of pertussis toxin from Bordetella pertussis.

PtlH is a member of a specialized set of transport proteins that is essential for secretion of pertussis toxin (PT) from Bordetella pertussis. Previously, PtlH was shown to contain a consensus nucleotide-binding motif. Here, we demonstrate that introduction of plasmids containing mutant forms of ptlH, altered in the putative nucleotide-binding region, into a wild-type strain of B. pertussis resulted in inhibition of PT secretion. Thus, this region of PtlH appears to be essential for protein function. Moreover, the observed dominant negative phenotype suggests that PtlH either functions as a multimer or interacts with another component necessary for secretion of PT.     

Antigenic heterogeneity in subunit S1 of pertussis toxin

Culture supernates containing pertussis toxin (PT) from four strains of Bordetella pertussis were examined for both immunological reactivity and biological activity. PT from all four strains sensitized mice to histamine and toxin was detectable in supernates of all strains when examined by Western blotting with polyclonal antiserum to PT. In supernates of three of the four strains, PT was detectable by an enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody to subunit S1 of PT as the third antibody layer. However, supernates from one strain, 18323, failed to react in ELISA. Electroblots probed with the monoclonal antibody labelled subunit S1 of PT from all strains except that of strain 18323. PT of strain 18323, whilst retaining histamine-sensitizing activity, differed antigenically from that of other strains.     

Development of acellular pertussis vaccines.

In 1974, the authors reported the isolation and characterization of protective antigens of Bordetella pertussis in mice. With this information, an acellular pertussis vaccine was developed, composed mainly of pertussis toxin (PT) and filamentous haemagglutinin (FHA). Substances causing side effects, especially lipopoly sacahoride (LPS) or endotoxin that cause fever, were removed, and detoxification of the PT by formaldehyde with retention of potency was achieved. In 1981, an acellular pertussis vaccine called the "Adsorbed Purified Pertussis Vaccine" was approved in Japan, in place of the whole-cell pertussis vaccine. The acellular pertussis vaccine has been widely accepted as safer and more efficacious in Japan. Since 1981, intense surveillance has shown that there are only rare adverse reactions and that pertussis has virtually been eliminated in Japan. Evaluation of active immunization with highly purified and pharmacologically inert PT and FHA and passive immunization with polyclonal and monoclonal antibodies, provide quantitative data about the vaccine-induced immunity in mice. Finally, it was discovered that the PT toxoid in the vaccine is the major and essential protective antigen. The toxoid of PT should be sufficient for protection against pertussis.     

DTaP vaccines from north american vaccine (NAVA): composition and critical parameters.

NAVA's acellular pertussis vaccine is based on highly purified pertussis toxin (PT) inactivated with H(2)O(2). PT was analysed using advanced biochemical methodology including mass spectroscopy (LC/MS), yielding mass and peptide mapping information on the subunits. Pertactin, adenylate cyclase, and Fim 1, 2 were below detection levels and only trace amounts of filamentous haemagglutinin (FHA) have been identified as a minor impurity. The vaccine does not induce anti-FHA antibodies during the course of a 3-dose primary immunization series in infants. B and T cell epitopes are preserved to a higher extent after H(2)O(2)detoxification when compared with chemical inactivation with formaldehyde, thus providing new information explaining why vaccines employing formaldehyde detoxified PT may need additional pertussis components added to induce high levels of protection. Anti-PT antibodies generated by NAVA diphtheria, tetanus, and acellular pertussis vaccine (DTaP) showed a positive correlation with protection against WHO-defined pertussis. The safety profiles for these vaccines showed low reactogenicity with no serious adverse events due to the vaccines.     

Increase in intradermal vascular permeability caused by pertussis toxin from Bordetella pertussis

Rabbits that were injected intradermally with pertussis toxin (PT), produced from Bordetella pertussis, showed slight edema and erythema at the injection sites, but not hemorrhage nor necrosis. The edema lesions were stained blue by the intravenous injection of Pontamine Sky Blue 6B dye, suggesting that PT caused increased vascular permeability, similarly to the permeability factor (PF) of cholera toxin. The reaction of the PF of PT could be determined by measuring the diameter of the blue area. The diameter of the blue area bore a good linear relationship to the logarithm of the dose of PT. The activity of the PF was neutralized by anti-PT rabbit serum. Detoxification of PT with formalin did not increase the vascular permeability, but reverted pertussis toxoid showed a PF reaction in proportion to the reverted leukocytosis-promoting and histamine-sensitizing activities of PT. The supernate of a Bordetella pertussis culture also induced a PF reaction and the reaction could be made clear by heating the supernate at 56 C for 30 min, but the supernate of Bordetella bronchiseptica did not induce the reaction at all.     

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

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 to 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.     

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.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

溶液环境对百日咳疫苗分离纯化的影响

针对百日咳疫苗在低盐条件下不稳定, 容易聚集而导致层析过程收率低、分离度低的难题, 实验中选择脲作为稳定剂来改善百日咳疫苗所处的溶液环境, 并采用离子交换层析和凝胶过滤层析进行百日咳疫苗的分离纯化, 通过ELISA抗原活性测定和还原性SDS-PAGE等方法研究了脲对百日咳疫苗分离纯化的影响。结果表明, 在流动相中加入 2 mol/L脲作为稳定剂, 能显著提高离子交换层析和凝胶过滤层析中的PT和FHA活性回收率、凝胶过滤层析的分离度、PT和FHA的纯度。这些结果对百日咳疫苗的分离纯化和层析工艺优化提供了重要的依据和参考。     

Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter.

Pertussis toxin (PT) is an essential component of accellular vaccines against whooping cough. However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields. To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer. The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg. Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized. The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B. pertussis grown for 24 h. The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure. The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression. Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography. The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test. These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in a nonvirulent and fast-growing B. bronchiseptica background.     

百日咳杆菌69KDa外膜蛋白的分离纯化及生物学特性研究

本文发展了一种从百日咳杆菌Ⅰ相菌株中纯化６９ＫＤａ外膜蛋白的简易方法,将细菌体经加热浸提、乙醇沉淀蛋白、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析精制而成。用ＳＤＳ－ＰＡＧＥ、免疫印迹、光密度仪扫描分析,证明纯化制剂为均一的、特异性的６９ＫＤａ外膜蛋白,其收率为５４．２％,纯度达９９．２％,每微克６９ＫＤａ蛋白制剂中的内毒素含量低于０．８５ＥＵ;ＰＴ残留量小于０．１０５ｎｇ。抗６９ＫＤａ蛋白抗血清能     

Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity

Pertussis (whooping cough) is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8) infection in mouse models and the role of pertussis toxin (PT) in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT) and subsequently (up to 14 days later) infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT) and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs) in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.