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 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.     

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.     

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.     

Approaches to the control of acellular pertussis vaccines.

The quality control of acellular pertussis vaccines presents particular problems related to the differences in composition and method of detoxification used in the various type of preparation. These vaccines are not amenable to potency assay by the active mouse protection test used for whole-cell pertussis vaccines and assurance of protective activity is problematic.In contrast, monitoring of these vaccines for safety is relatively straightforward and is centred on assays for the lipooligosaccharide endotoxin, active pertussis toxin and absence of reversion to toxicity of detoxified product. The absence of heat-labile toxin, tracheal cytotoxin and adenyl cyclase toxin is assumed provided that adequate validation of the process has been performed.Confirmation of the antigenic content of the detoxified bulk components is difficult to achieve by conventional binding assays based on monoclonal antibodies because of changes in accessibility of reactive sites post-toxoiding. However, single radial diffusion assay using polyclonal antisera permits estimation of pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (P69). Dot blot immunoassay can be used for the fimbrial agglutinogens 2 and 3 (Fim 2 and 3) and potentially could also be used to check the composition of final filling lots for PT, FHA, P69 and Fim 2 and 3.Gel electrophoresis and immunoblotting can be applied to monitor purity of purified bulk components and the characteristics of these change after chemical detoxification. Electron microscopy provides a useful semi-quantitative supporting method for checking purity of bulk components. Physico-chemical examination, particularly CD and fluorescence spectroscopy, offer a means of monitoring the consistency of detoxified bulk components.No completely satisfactory method is available for monitoring potency. Immunogenicity assays may be useful for checking consistency but do not necessarily correlate with protection. At present, active protection against aerosol challenge offers the best prospect of a functional assay.     

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.     

The Vaccine Containing Recombinant Pertussis Toxin Induces Early and Long-Lasting Protection

Most acellular pertussis vaccines contain a form of pertussis toxin (PT) detoxified by chemical treatment. The Chiron-Vaccines product is unique because it contains a genetically detoxified pertussis toxin. This molecule showed absolute safety, an antigenic profile similar to wild-type PT, and an immunogenicity that is superior to all chemically detoxified PTs. In the efficacy trial, the vaccine containing the genetically detoxified PT demonstrated early and long-lasting protection.     

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.     

Non-pertussis components of combination vaccines: problems with potency testing.

Vaccines comprising combinations of diphtheria, tetanus and pertussis (DTP) with Haemophilus influenzae type b polysaccharide-protein conjugate (Hib), inactivated poliomyelitis virus (IPV) and hepatitis B virus (HBV) are already available, and new combinations using acellular pertussis components in a triple vaccine (DTaP) are under development. Evidence to date has shown that control of the efficacy, safety and stability of combination vaccines cannot be based on information already available on the individual components or existing licensed formulations. Several examples of immunological interference between components of a combination vaccine have been observed both in clinical trials and in laboratory tests. Examples of these for D, T and Hib components in DTP and DTaP combinations have been investigated.     

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.     

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.     

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.     

Single radial immunodiffusion as a method for the assay of the acellular pertussis vaccine components, pertussis toxoid, filamentous haemagglutinin and pertactin.

The development of acellular pertussis vaccines has raised a number of issues relevant to the control of these products. Of particular importance is the need for robust and accurate in vitro assays for the antigen content of the vaccines which might contain up to five different antigen components, each of which needs to be independently assayed. This paper describes a simple method for the quantification of three component antigens. Because relatively high doses of purified antigens are used in those preparations, the elimination of residual toxicity is a major concern. This is achieved by genetic modification of chemical treatment. The latter results in modification of the immunological reactivity of the antigens making direct assay by such methods as ELISA ineffective. A single radial diffusion technique using polyclonal antisera for the assay of pertussis toxoid (PTxd), chemically treated filamentous haemagglutinin (FHA) and pertactin (69 kDa) has been developed. The method uses low concentrations of antisera, allowing accurate and reproducible quantification of antigen content as low as 25 microg/ml of protein for pertussis toxoid and filamentous haemagglutinin and 5 microg/ml for pertactin. Since by the addition of detergent, diffusible subunits are produced irrespective of the original physical state of the antigens, the assay is suitable for assay of these antigens after detoxification/or stabilization by chemical treatment and is able to determine the differences between preparations which have the same protein concentration but different antigenic contents. This provides a means for assuring the consistency of the antigens after detoxification/or chemical stabilization which could be used as an in-process control method for acellular pertussis vaccines.     

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.     

Immunogenicity issues in the quality control of the new acellular pertussis vaccines.

Over the last few years our laboratory has been assessing the consistency of production of different batches of acellular pertussis vaccines to be marketed in Italy. Central to this is immunogenicity assay of the lots under control compared with those of a reference vaccine with documented clinical efficacy.However, the current assays based on the assessment of antibody (Ab) response in the mouse are unrelated to mechanisms of protection in children. The absence of a clear correlation between Ab responses and protection has also been documented in recent clinical trials. On this basis, we are currently considering the possibility of adding to the established criteria of immunogenicity in mice based on Ab responses, information from studies on cell-mediated immune responses to the vaccine constituents.     

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.     

Key Points for the Development of Mouse Immunogenicity Test as Potency Assay for Acellular Pertussis Vaccines

According to WHO and the European Pharmacopoeia, the current potency test for acellular pertussis vaccines is a mouse immunogenicity assay assessing consistency of production from batch to batch. The assay compares the batch under control with a reference vaccine of documented clinical efficacy. This study describes and illustrates critical aspects of the assay, based on our experience on a tricomponent vaccine: validation of immunoassay to quantify mouse antibody response, choice of vaccine immunising doses in the three-doses model, treatment of non-responder mice for calculations, establishment of assay validity criteria.     

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.     

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.     

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.