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.     

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.     

Evaluation of whole-cell and acellular pertussis vaccines effectiveness in clearance of experimental B. pertussis infection in mice

The study is based on assumption that B. pertussis strains harbouring different allele variants of genes encoding subunit S1 of pertussis toxin and pertactin might be eliminated with different efficiency from lung tissue of mice which were immunized with whole-cell and acellular pertussis vaccines. It has been assumed that strains containing combinations of genes alleles which were not prevalent since 1990-ties are consisting of mutated strains in respect to pertussis toxin subunit S1 and pertactin, and are capable to decrease efficiency of pertussis vaccines. Experiments performed in vivo dealt with activity of tested vaccines against B. pertussis strains of different combinations of ptxS1/prn. The study indicated for lowered efficiency of whole-cell and acellular pertussis vaccines in elimination of mutated strains of B. pertussis from animal lung tissue in comparison with strains currently used for vaccine production.     

Evaluation of respiratory model employing conventional NIH mice to access the immunity induced by cellular and acellular pertussis vaccines

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.     

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.     

Evaluation of transmission electron microscopy for examination of components of acellular pertussis and combination vaccines.

New acellular pertussis and combination vaccines vary in the concentration and presence or absence of specific components and in extent of adsorption to adjuvants. There is a pressing need to develop new control methods for acellular pertussis vaccines. Negative staining electron microscopy has been evaluated as a method for assessing the purity of individual vaccine components and the amount of adsorption to aluminium gels. Negative staining showed the characteristic morphology of vaccine components and permitted detection of contaminants and morphological changes. Reproducible results were obtained by use of a standardized negative staining technique and confidence in the technique was increased by comparison of previously unexamined specimens with a specimen that had been characterized by repeated observations. The degree of adsorption to aluminium adjuvants could only be assessed by observation of the amount of non-adsorbed material in the specimen. Negative staining electron microscopy can be used as one of a number of techniques for control of acellular pertussis combination vaccines.     

Pertussis control: time for something new?

Childhood acellular pertussis vaccines were licensed and implemented in the US in the 1990s following an effort to improve on the safety profile of whole-cell vaccines. However, waning of immunity from acellular vaccines may be driving the recent resurgence of pertussis, raising the need to consider new prevention strategies.     

A common vaccination strategy to solve unsolved problems of tuberculosis and pertussis?

Respiratory pathogens are amongst the world's most successful killers. Tuberculosis kills approximately 2 million individuals each year, and pertussis is responsible for roughly 300,000 annual deaths. Although the two diseases are fundamentally different in the expression of their pathogenesis and in the biology of their causative agents, a common heterologous prime/boost vaccination strategy is proposed, using live attenuated vaccines against tuberculosis (the already existing BCG) and pertussis (a novel attenuated Bordetella pertussis strain) early in life for priming, followed by a booster with acellular vaccines, based on the novel heparin-binding haemagglutinin for tuberculosis, and already available acellular vaccines against pertussis.     

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.     

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.     

Isolation of hemolytic cultures of Bordetella pertussis intended for the production of acellular pertussis preparations and the characterization of their biological properties

The heterogeneity of the population of B. pertussis laboratory strains with respect to the capacity of individual clones to lyse erythrocytes has been established. The complete or partial reduction of the antigens under study in nonhemolytic bacteria has been shown. The use of hemolytic cultures for the preparation of acellular pertussis vaccine makes it possible to increase the content of B. pertussis toxin in these vaccines.     

Standardization of acellular pertussis vaccines.

In comparison with the current whole cell pertussis vaccine, the new generation of acellular pertussis vaccines opens new opportunities to improve the standardization of the product, because well defined and characterized components are used in these new products.However, different compositions, purification and inactivation methods are used by different manufacturers. Consequently the various acellular pertussis vaccines in the world are difficult to compare in a meaningful manner using simple laboratory tests. In addition, the absence of a reliable animal model and serological correlates with protection in children are other complicating factors.For that reason it seems that the consistency in manufacturing based on a clinically validated production process is the best way to ensure the safety and efficacy of routinely produced acellular pertussis vaccines.Laboratory tests to monitor the antigen content, purity, safety and immunogenicity seem to be the best approach to standardize this new generation of pertussis vaccines against homologous standard vaccines with known clinical efficacy and safety and to support the consistency in manufacture.     

The effect of pertussis whole cell and acellular vaccines on pulmonary immunology in an aerosol challenge model

Recent clinical trials have shown that the new generation of acellular pertussis vaccines (Pa) can confer protection against whooping cough with negligible adverse reactions. We have compared the effects of pertussis whole cell and acellular vaccines on pulmonary immune responses after aerosol challenge in a murine model of infection. Mice were vaccinated with PBS, Pw or Pa and challenged with Bordetella pertussis by the aerosol route. Cytokine gene expression was analysed from lung tissue and cells; lung lymphocytes were re-stimulated in vitro and cytokines produced measured. The results obtained are consistent with the proposal that a strong Th-1 response is associated with bacterial clearance in both the non-vaccinated and Pw vaccinated mice. The acellular vaccine treated mice cleared the bacterial challenge (with an intermediate efficacy) in the presence of low levels of any of the cytokines assessed. This suggests that Pa protects via a Th-2 independent mechanism.     

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.     

Overview of recent clinical trials of acellular pertussis vaccines.

The evidence from pre-licensure studies does not suggest that there are clinically important differences in reactogenicity between acellular vaccines. The merits of different acellular products will therefore have to be compared on efficacy criteria. Ideally, acellular vaccines with the minimum antigen content necessary to ensure optimum protection should be used in order to avoid administration of superfluous antigens to children and to simplify licensing and batch release procedures.On the basis of the evidence so far available it seems unlikely that monocomponent pertussis toxin (PT) vaccines provide optimal protection and that multicomponent vaccines are needed to achieve a level of disease control that approaches that of a good whole-cell vaccine. It is unclear whether all two component vaccines containing PT and filamentous haemagglutinin (FHA) have similar efficacy but on the available evidence the safest option for policy makers would seem to be to use a vaccine with at least three components, PT+FHA+pertactin. There is now good evidence that the five component vaccine which contains agglutinogens 2 and 3 in addition to PT/FHA and pertactin provides the best protection and is the only acellular vaccine whose efficacy matches that of a good whole cell vaccine. However, the public health advantage of the five component vaccine over other acellular vaccines may not become apparent until they have been in routine use for some decades and their ability to protect against transmission as well as clinical pertussis has emerged.The decision to replace an effective whole-cell vaccine by an acellular vaccine for primary immunisation needs careful consideration. Apart from the probable sacrifice of efficacy for reduced reactogenicity (at least for vaccines which do not contain agglutinogens 2 and 3) there is the question of value for money and the ease with which acellular DTP vaccines can be combined with conjugate polysaccharide vaccines such as Haemophilus influenzae type b.Whatever the decision of policy makers, the need for continued follow up of trial cohorts and active surveillance of the efficacy and safety of those acellular vaccines that are introduced into routine use must be accorded a high priority.     

The effect of bacterial and synthetic peptidoglycans on the toxicity of a cell-free pertussis preparation

The influence of bacterial and synthetic peptidoglycans on the toxicity of acellular pertussis preparations (APP) has been studied in the mouse weight gain test and in the endotoxic shock development test on mice treated with Actinomycin D. The data obtained in these tests indicate that the immunomodulators (IM) under study are capable of changing (increasing or decreasing, depending on the dose) the toxic properties of APP. The antitoxic action of IM, established in this study, depends on the combination of the doses of IM and APP and the time elapsed from the time of immunization. The possibility of using these IM in a dose of 0.0001 microgram for reducing the LD50 of APP has been established. The use of IM belonging to the group of bacterial synthetic peptidoglycans for the development of acellular pertussis vaccines with reduced reactogenicity has been shown to hold much promise.     

The action of pertussis vaccines and GMDP on the change in the enzymatic activity of macrophages from peritoneal exudate

The influence of whole-cell and acellular pertussis vaccines, introduced both alone and in combination with N-acetylglucosaminylmuramyl-2-alanine-D-isoglutamine (GMDP) on the activity of two enzymes of peritoneal exudate macrophages (5'-nucleotidase and Na+K(+)-adenosine triphosphatase) was studied. The study revealed that both pertussis vaccines exhibited immunomodulating properties, these properties being most pronounced in whole-cell pertussis vaccine. The use of GMDP in combination with pertussis vaccines led to changes in the enzymatic activity of peritoneal exudate macrophages, which was indicative of a decrease in the immunomodulating action of pertussis preparations.     

Acellular pertussis vaccines: evaluation of reversion in a nude mouse model

An animal model has been developed to assess the safety of acellular pertussis vaccines in terms of reversion to toxicity. Adsorbed pertussis toxoid preparations, alone or combined in a DTP formulation, were administered to nude mice intraperitoneally. In parallel, groups of positive and negative control mice received pertussis toxin and buffer, respectively. The circulating white blood cells of the animals were monitored for 28 days. Mice immunized with glutaraldehyde toxoid preparations did not develop a lymphocytosis during the observation period, whereas mice immunized with an experimental formalin pertussis toxoid vaccine exhibited a high lymphocytosis six days after vaccine administration, demonstrating, in this model, a reversion of the toxoid. The nude mouse model thus appears to reveal the in-vivo reversion of pertussis toxoids and could be included in the quality control panel for the assessment of the safety of acellular pertussis vaccine.     

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.     

Acellular pertussis vaccines: neutralization by immune sera of the lethality of pertussis toxin and viable Bordetella pertussis for chick embryos.

Vaccines containing acellular pertussis components, either separate or combined with other microbial antigens, were evaluated for specific immune responses in guinea-pigs and mice. The capacity of sera to protect chick embryos from the lethal effect of pertussis toxin was independent of the Chinese hamster ovary cell clumping neutralization titre and the antigen binding ELISA anti-toxin titre. Direct correlations did not exist between ELISA titres to Pt, FHA, fimbria or 69 kDa and capacity to prevent killing of embryos by different strains of Bordetella pertussis. With the exception of one combination vaccine product, addition of foreign microbial antigens to acellular pertussis vaccines did not significantly alter capacity of the sera to protect embryos against toxin or bacteria.