Molecular genetic characteristics of the B. pertussis strains isolated in different periods of the pertussis epidemic process

Despite the fact that the mass immunization of the children population with the DPTs vaccine has been carried out in the Russian Federation since 1959, the pertussis infection persists to be one of the pressing problems for the children population. Although the vaccination coverage of the children population with pertussis vaccines is high in Russia, at present time the pertussis incidence rates are increasing among schoolchildren and remain high among infants younger than 12 months old. Many researchers believe that the variability of the genetic structure of the pertussis causative agent may be one of the causes of increasing pertussis incidence rates. This investigation provides the molecular genetic characteristics of 97 B. pertussis strains isolated in pertussis patients in Moscow in different periods of pertussis epidemic process since the 1950s up to present time. It shows the changes in the structures of genes, which are encoding the main protective antigens of the pertussis microbe that are the pertussis toxin (ptxS1) and the pertactin (pm). The structurre of the ptxS1 and pm gene of the B. pertussis vaccine strains was compared with the structures of these genes in the B. pertussis strains isolated from the pertussis patients at present time and also in past years. All B. pertussis strains isolated in the prevaccination period (1948-1959) and most strains (95%) isolated during the first twenty years of the mass immunization in Russia are characterized by the presence of the so called "vaccine" alleles of the pertussis toxin and pertactin genes that are ptxS1 B or ptxS1 D and pm 1 alleles that corresponds to the genetic structure of the vaccine producing strains. In the early 1970s the B. pertussis strains of another toxin and pertactin genetic structures with so-called "non-vaccinal" alleles ptxS1 A and pm 3 (pm 2 since 1980s) began to appear. The B. pertussis strains with "non-vaccinal" alleles have completely displaced the "old" strains. At present time in Moscow the pertussis disease is caused by the B. pertussis strains bearing ptxS1 A and pm 2 or pm 3 alleles of pertussis toxin and pertactin genes. There was no correlation between the genotype and serotype. Thus, the structure of the B. pertussis toxin and pertactin genes in strains which have been isolated since the 1980s up to now differs from the structure of these genes in strains which are used for producing DPTs vaccine. The data obtained in this investigation suggest that the genetic structure specificity of circulating B. pertussis strains that are producing the disease at present time should be used as one of the criteria for selecting vaccine producing strains.     

Bordetella pertussis serotypes in Canada.

A study was done to determine the major antigenic factors of Bordetella pertussis strains isolated throughout Canada and whether these isolates have the same antigenic structure as the bacilli in the currently used vaccines. Testing for the major pertussis antigens, factors 1, 2 and 3, was conducted with 440 freshly isolated strains of B. pertussis received from seven canadian provinces between August 1976 and February 1978 and six batches of pertussis vaccine or immunizing agents containing pertussis vaccine. With the aid of specific antisera prepared in rabbits, the antigenic factors were detected by a slide agglutination technique. Almost all (98.9%) of the pertussis strains examined were serotype 1,3.All six batches of pertussis vaccine or immunizing agents containing pertussis vaccine proved to be rich in each of the three main pertussis agglutinogens.     

Composition of acellular pertussis and combination vaccines: a general review.

Since the development and introduction of the acellular pertussis vaccine in Japan in the early eighties, we have come a long way in using this component in combination with other vaccines. However, the basic problem in development of an effective and safe pertussis vaccine is that the antigens to induce complete protection against clinical pertussis and the precise mechanism by which pertussis vaccine confers immunity is yet unknown. Hence, the composition of future acellular pertussis vaccine remains an open issue. Recently, acellular pertussis vaccine has been licensed for the booster doses in the U.S.A. and for primary immunization of infants in Italy and Germany. A multicentric trial has been carried out to compare the serological response and adverse reactions of 13 acellular pertussis vaccines. These vaccines contained one or more of the four components, i.e. FHA, PT, 69 kDa OMP and fimbriae. All vaccines were associated with substantially fewer and less adverse reactions and were more immunogenic with respect to antibodies against the added antigens. DTP vaccines in the near future will have combinations of other components and the key antigen for combination will be acellular pertussis component which is going to replace whole cell pertussis component in DTP vaccines. In view of this, manufacturers like ourselves from the developing countries are still groping in the dark, uncertain whether we should have a single component acellular pertussis vaccine or multicomponent one. This will have a major impact on the cost of production, the final cost of the combination vaccines and the regulatory issues that we will have to tackle in view of the recent thinking on harmonization in the pharmaceutical industry.     

Restriction analysis of Bordetella pertussis DNA isolated from patients with whooping cough in 1968 and 1995-98 and B. pertussis used for production of national vaccine strains

The genotypic and serotypic analysis of B. pertussis strains isolated from the nasopharynx of children with whooping cough in the years 1968 and 1995-98 and B. pertussis vaccine strains was the aim of this study. The genotyping of the examined strains was done by electrophoretic division of DNA in pulsed field. The 3 types (A, B, C) and 2 subtypes (A1 and A2) of DNA restriction patterns were determined for the B. pertussis strains isolated in 1968. The 2 types (D and E) and 10 subtypes (D1-D10) of DNA restriction patterns were identified for B. pertussis strains from the years 1995-98. The DNA restriction patterns of B. pertussis strains isolated in the years 1968 and 1995-98 were not identical what was the evidence of the fact that in the sixties and nineties whooping cough was caused by different B. pertussis clones. The different DNA profiles were also observed for vaccine strains as well as for vaccine strains and current isolates. Differences in DNA patterns of vaccine strains and B. pertussis strains isolated in the years 1995-98 indicated a relationship possibility in some cases while lack of relationship between these strains in other cases. Serotyping of the examined B. pertussis strains was performed by the agglutination method with the sera against B. pertussis agglutinogens 1, 2 and 3. Most strains--15 (75%) isolated in 1968 possessed only agglutinogens 1 and 3. Serotype 1, 2, 3 was most frequently observed among isolates from the years 1995-98. This study indicates the expediency of periodical change of B. pertussis vaccine strains in the aspect of whooping cough resurgence in the years 1994-95 and 1997-98.     

中国百日咳疫苗的现状及研发趋势初探

百日咳是许多国家的严重公共卫生问题,目前预防百日咳最有效和最经济的方法是接种百日咳疫苗,百日咳组分疫苗由于其保护效果好、不良反应低、质量稳定可控而成为新一代无细胞百日咳疫苗的首选。本文简要介绍百日咳的病原学、传播途径及流行病学特点,并对中国的百日咳疫苗研发趋势进行初步探讨。     

A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.     

Acetone-treated pertussis vaccine--a potent and safer new pertussis vaccine

A vaccine was prepared from the growth of Bordetella pertussis by repeated treatment with acetone. The vaccine has been designated as acetone-treated pertussis vaccine (ATPV). A total of ten batches of ATPV were prepared, five each from B. pertussis strains 134 and 509. These strains are routinely employed at this Institute for the production of conventional whole-cell pertussis vaccine (WCPV) for blending in diphtheria-pertussis-tetanus vaccine. The mouse protective and histamine sensitizing activities of ATPV and WCPV were compared. The ATPV showed 1.5- to 2-fold higher potency than the WCPV. The histamine sensitizing activity of ATPV was much reduced compared with that of the WCPV. No appreciable difference was observed in the results of a mouse weight-gain tests between the ATPV and WCPV. The details of the preparation of ATPV have been described. Because of higher potency and reduced histamine sensitizing activity, the ATPV may prove more acceptable in immunization programmes against pertussis, even in countries where WCPV is unpopular due to its suspected reactogenicity.     

Prophylaxis of pertussis: development and use of acellular pertussis vaccine

Modern data substantiating the expediency of the use of acellular pertussis vaccine were analyzed. Serious postvaccinal complications caused by the action of the corpuscular pertussis component of adsorbed DPT vaccine served as the basis for the development of acellular pertussis vaccine (APV). During the period of 1990-1996 as many as 8 international field trials of the effectiveness of APV were carried out. The results of these trials and studies were evaluated in accordance with the unified programs and criteria. The vaccines under test differed by the composition of Bordetella pertussis purified antigens they contained, the methods of their purification and the detoxification of pertussis toxin. All tested APV, with the exception SKB-2, possessed pronounced prophylactic activity.     

Biofilm forming potential and antimicrobial susceptibility of newly emerged Western Australian Bordetella pertussis clinical isolates

Whooping cough caused by Bordetella pertussis is increasing in several countries despite high vaccine coverage. One potential reason for the resurgence is the emergence of genetic variants of the bacterium. Biofilm formation has recently been associated with the pathogenesis of B. pertussis. Biofilm formation of 21 Western Australian B. pertussis clinical isolates was investigated. All isolates formed thicker biofilms than the reference vaccine strain Tohama I while retaining susceptibility to ampicillin, erythromycin, azithromycin and streptomycin. When two biofilm-forming clinical isolates were compared with Tohama I, minimum bactericidal concentrations of antimicrobial agents increased. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis revealed significant differences in protein expression in B. pertussis biofilms, providing an opportunity for identification of novel biofilm-associated antigens for incorporation in current pertussis vaccines to improve their protective efficacy. The study also highlights the importance of determining antibiograms for biofilms to formulate improved antimicrobial therapeutic regimens.     

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.     

Complete genome sequence of Bordetella pertussis CS, a Chinese pertussis vaccine strain

Bordetella pertussis is the causative agent of pertussis. Here, we report the genome sequence of Bordetella pertussis strain CS, isolated from an infant patient in Beijing and widely used as a vaccine strain for production of an acellular pertussis vaccine in China.     

Prevalence and genetic characterization of pertactin-deficient Bordetella pertussis in Japan

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn(-)) B. pertussis was observed in Japan. The Prn(-) isolate was first discovered in 1997, and 33 (27%) Prn(-) isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn(-) isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn(-) isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn(-) isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn(-) isolates belong to MLVA-186, and 6 and 3 Prn(-) isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn(-) clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn(-) isolates have a higher growth potential than the Prn(+) back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn(-) strains have arisen and that Prn expression is not essential for fitness under these conditions.     

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.     

Consistency of Bordetella pertussis vaccine seed strains and potency of whole-cell pertussis vaccine still in use in Poland

In Poland, where the wP vaccine has been used since 1960, pertussis rates increased in the mid-1990s. In 2012, the rate of pertussis recognised by surveillance was unexpectedly found to be two-fold higher than in the previous decade. Quality measures on potency and vaccine working seeds were introduced, to confirm the possible impact of manufacturing inconsistency or potency lowering on the observed increase in pertussis. Shewhart charts on potency values for lots released between 2001 and 2013 did not reveal any significant fluctuations. Working seeds of three vaccine strains used within last decade for wP manufacturing belong to the PFGE group III and were highly related. According to PFGE and SDS-PAGE data, all vaccine strains were found consistent according profiling on the genomic and protein levels. According to the sequencing data, they harboured ptxA2, ptxC1, prn1, fim2-1, fim3-1, tcfA2, ptxP1 and were assigned as MLST-2 type. Other factors apart from vaccine manufacturing inconsistency might be responsible for the increase in pertussis noted in 2012 in Poland.     

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.     

The development of a new generation of pertussis vaccine

The results of the weight gain test on mice have shown that acellular pertussis vaccine is less toxic than the pertussis component of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine due to a lower content of endotoxin in the acellular vaccine; but the leukocytosis-promoting and histamine-sensitizing activities of JNIH-6 and adsorbed DPT vaccines are indicative of incomplete inactivation of Bordetella pertussis toxin. The content of incompletely inactivated B. pertussis toxin is practically the same in both preparations, constituting 1/100-1/200 of the calculated initial activity. For this reason, the use of the new pertussis vaccine also involves a risk of development of serious postvaccinal reactions and/or complications caused by this toxin. Search for the optimum method of inactivation of B. pertussis main toxin should be continued. As shown by the enzyme immunoassay, acellular pertussis vaccine used in the same immunizing dose as adsorbed DPT vaccine induces a more intensive immune response to hemagglutinin and B. pertussis toxin. This is due to higher residual toxicity of the corpuscular component of adsorbed DPT vaccine. Induction of antibodies to B. pertussis toxin has been shown to decrease in response to injection of acellular pertussis vaccine containing a certain residual amount of incompletely inactivated B. pertussis toxin.     

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.     

Effectiveness of pertussis vaccination and duration of immunity

Background:A resurgence of pertussis cases among both vaccinated and unvaccinated people raises questions about vaccine effectiveness over time. Our objective was to study the effectiveness of the pertussis vaccine and characterize the effect of waning immunity and whole-cell vaccine priming.Methods:We used the test-negative design, a nested case–control study with test-negative individuals as controls. We constructed multivariable logistic regression models to estimate odds ratios (ORs). Vaccine effectiveness was calculated as (1 – OR) × 100. We assessed waning immunity by calculating the odds of developing pertussis per year since last vaccination and evaluated the relative effectiveness of priming with acellular versus whole-cell vaccine.Results:Between Dec. 7, 2009, and Mar. 31, 2013, data on 5867 individuals (486 test-positive cases and 5381 test-negative controls) were available for analysis. Adjusted vaccine effectiveness was 80% (95% confidence interval [CI] 71% to 86%) at 15–364 days, 84% (95% CI 77% to 89%) at 1–3 years, 62% (95% CI 42% to 75%) at 4–7 years and 41% (95% CI 0% to 66%) at 8 or more years since last vaccination. We observed waning immunity with the acellular vaccine, with an adjusted OR for pertussis infection of 1.27 (95% CI 1.20 to 1.34) per year since last vaccination. Acellular, versus whole-cell, vaccine priming was associated with an increased odds of pertussis (adjusted OR 2.15, 95% CI 1.30 to 3.57).Interpretation:We observed high early effectiveness of the pertussis vaccine that rapidly declined as time since last vaccination surpassed 4 years, particularly with acellular vaccine priming. Considering whole-cell vaccine priming and/or boosters in pregnancy to optimize pertussis control may be prudent.Whooping cough, or pertussis, is a highly contagious respiratory infection that has been inadequately controlled compared with other vaccine-preventable diseases. The incidence of pertussis in Canada decreased from 156 cases per 100 000 population during the prevaccination era to a historic low of 2.0 per 100 000 in 2011, increased to 13.9 in 2012, and then decreased to 3.6 in 2013.¹Ontario, Canada’s most populous province with a population of 13.5 million in 2013, experienced a localized outbreak in 2012. This outbreak started in a largely unvaccinated religious community and disproportionately affected infants, but then spread to the general population and mostly involved adolescents.² Consequently, the outbreak raised questions about product-specific vaccine effectiveness and waning immunity.Pertussis vaccines have been available in Ontario for more than 70 years. In 1997, owing to concerns about safety and effectiveness, acellular pertussis vaccine replaced the whole-cell product that had been in use since 1984.^3–5 In Ontario, only the 5-component acellular vaccine (containing pertussis toxoid, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3) has been available for infants and toddlers. Vaccination against pertussis is recommended at 2, 4, 6 and 18 months, and at 4–6 years. In 2003, an adolescent dose at 14–16 years was introduced, and in 2011, a program was started for single-dose adult vaccination against pertussis.⁶Other jurisdictions in Canada, the United States and Australia have reported lower effectiveness with the acellular product and rapidly waning immunity.^7–11 Canada has a unique history of using a whole-cell vaccine with lower effectiveness, and also has different secular trends in pertussis incidence and vaccination coverage, necessitating local evaluation of the effectiveness of the pertussis vaccine to inform vaccination policy. Our objective was to study the effectiveness of the pertussis vaccine in Ontario while characterizing the effect of waning immunity and whole-cell vaccine priming.     

Importance of the degree of antigen polymerization by detoxification in modulating the immunogenicity of acellular pertussis vaccine

For the acellular pertussis vaccine with a high immunogenicity, the concentration, composition and characteristics of acellular pertussis antigens are the crucial points to be considered. Nevertheless, it has not been proved yet whether or not the polymerization degree, one of the characteristics of formalin-detoxified acellular pertussis antigens, has an influence on vaccine potency. Thus, in the present study, the correlations among detoxification conditions of acellular pertussis bulks, their polymerization degrees and their immunogenicities were examined. In addition, the relative importance of pertussis toxoid in vaccine immunogenicity was also investigated. Results show that a lower lysine concentration during detoxification induces highly-polymerized antigens, the immunogenicity has a great dependency on the polymerization degree of antigens, and also pertussis toxoid has a relatively stronger influence on the immunogenicity than other antigens. Accordingly, in the aspect of the potency of detoxified acellular pertussis vaccine, it can be demonstrated that the polymerization of antigens and its degree are the major factors affecting the immunogenicity along with a relatively high content of pertussis toxoid.     

Characterization of Bordetella pertussis strains of recent isolation.

During the clinical trial conducted in Italy to evaluate the efficacy of new acellular pertussis vaccines, the most favorable conditions for the recovery and characterization of Bordetella pertussis strains, isolated from children with cough, were adopted. The nasopharyngeal aspirates were collected and sent to the laboratory in refrigerated conditions within 24 hours. Charcoal agar selective and non selective plates were used, and most of the isolates were recovered after 3-4 days of incubation. Confirmation of all suspected colonies included the use of biochemical tests and specific agglutination reaction with B. pertussis antiserum. Serotyping of fimbriae, susceptibility to erythromycin and DNA fingerprinting by Pulsed Field Gel Electrophoresis (PFGE), were performed to characterize B. pertussis isolates and to determine relatedness among different strains. Serotype 1,3 was the most represented in the bacterial population examined. A predominant pulsetype (PTA) characterized most of the isolates accounting for 71.4% of the strains examined. Eight subclones (23.5%) and three unrelated pulsetypes were also found. No resistant strains to erythromycin were detected.