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.     

Development of pertussis vaccine production and control in the National Institute of Public Health in the Netherlands during the years 1950–1962

The development of the pertussis vaccine production in the National Institute of Public Health in the Netherlands since 1953, and the results with the consecutive lots of vaccine in the mouse protection test and the U.S.A. toxicity test are described. The results in the latter test are compared with the results of a locally developed guinea pig toxicity test. Special attention is given to the difficulties encountered when the U.S.A. toxicity test is used for adsorbed DPT vaccines. The potency data of all lots of DPT vaccines produced since 1958 fall within the limits of the potency test as prescribed in the U.S.A. Minimum Requirements. There are indications that the increased potency of the vaccine may have led to a lower mortality rate of pertussis.     

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.     

Potency control of diphtheria component in adsorbed vaccines by in vitro neutralization tests.

Samples from 20 lots of dT vaccine and from 20 lots of DTP vaccine were used to standardize and validate the Vero cell and the toxin binding inhibition (ToBI) tests for the potency control of diphtheria component. For the Vero cell method, violet crystal solution was used to stain the cells and estimate the endpoint of diluted diphtheria antitoxin. Diphtheria anatoxin was used for performing the ToBI test instead of toxin. The results obtained by both in vitro tests were similar to those obtained by in vivo toxin neutralization test in guinea pigs. The various analysis and the chi(2) test applied to evaluate the reproducibility and homogeneity, respectively, among in vitro tests and in vivo toxin neutralization test did not detect statistical significant difference for both analysed vaccines. An excellent correlation among in vitro tests and in vivo neutralization test was observed by Spearman's correlation coefficient.     

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.     

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.     

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.     

Neonatal immunization with a single dose of recombinant BCG expressing subunit S1 from pertussis toxin induces complete protection against Bordetella pertussis intracerebral challenge

The currently used pertussis vaccines are highly efficacious; however, neonates are susceptible to whooping cough up to the sixth month. In agreement, DTP-immunized neonate mice were not protected against intracerebral challenge with Bordetella pertussis. Neonate mice immunized with either DTP or a recombinant-BCG strain expressing the genetically detoxified S1 subunit of pertussis toxin do not show a humoral immune response against PT. On the other hand, rBCG-Pertussis induces higher PT-specific IFN-gamma production and an increase in both IFN-gamma(+) and TNF-alpha(+)-CD4(+)-T cells than the whole cell pertussis vaccine and confers protection against a lethal intracerebral challenge with B. pertussis.     

The purification and characterization of an acellular pertussis vaccine

An acellular pertussis vaccine manufactured by Biken was investigated for purity, potency and toxicity. The vaccine was composed of almost equal proportions of pertussis toxin (PT) and filamentous hemagglutinin (FHA). The purity of the vaccine was 97-99%. The protective effects of component vaccines containing various ratios of PT and FHA were tested and it was found that the ratio of 1:1 provided the most effective vaccine.     

Modern strains of Bordetella pertussis: immunobiological properties and vaccine improvement

Strains of B. pertussis isolated from patients in Moscow in 2001-2005 as well as strains included in locally produced diphtheria-tetanus-whole cell pertussis (DTP) vaccine were studied. Nucleotide sequences in genes of pertactin and S1-subunit of pertussis toxin of isolated strains, their immunobiological properties and opportunity to use for producing of the acellular pertussis vaccine were determined. Genes of pertactin and S1-subunit of pertussis toxin in the isolated wild strains differed from the same genes in strains included in the local DTP vaccine. Majority of the isolated strains belonged to serotype 1.0.3 and were markedly virulent.     

A review of the effectiveness of vaccine potency control testing

The use of potency control testing is a valuable tool for testing the actual relative strength of manufactured assembly lots of vaccine. Biological-based manufacturing methods are inherently variable and potency testing is a tool to ensure lot-to-lot consistency of commercial vaccines. A strong historical link to clinical efficacy has been established where correlation to efficacy and adequate test validation have been achieved. The link to immunogenicity and efficacy has traditionally been strongest with attenuated vaccines and toxoids. Control potency test failure does predict that a serial or batch of vaccine would most likely provide insufficient immunogenicity in typical field applications. Because of the complexity of pathogenic processes and associated immune responses, potency tests may not always directly predict the effectiveness of a vaccine. Thus, vaccines that pass control potency testing may not always provide adequate efficacy. This is particularly true of adjuvanted, inactivated vaccines. In the development of vaccine formulations and control tests for vaccines, the nature of the desired protective immune responses to the targeted pathogen (when known) should be considered. These considerations could provide better alternatives in the assays chosen as correlates of immunity and may more accurately predict efficacy and assure batch-to-batch consistency. Also, the effects of the dose and duration of antigen exposure as well as the nature of antigen presentation and generation of extrinsic cytokines could be characterised and correlated to vaccine potency as additional indicators of vaccine efficacy.     

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.     

Analysis of chosen parameters of immuno response in mice immunized with whole-cell or acellular pertussis vaccines and challenged with B. pertussis strains harbouring different ptxS1/prn allele genes combinations

Studies concerned evaluation of differences between parameters of cell-mediated immunity in mice, induced with whole-cell and acellular pertussis vaccines with subsequent challenge with B. pertussis strains harbouring different ptxS1/prn allele genes. In the study, concentrations of IFN-gamma/Il-2 and 1l-4/Il-5 in supernatants of cultured mice splenocytes have been determined to evaluate differences in Th1 or Th2 lymphocytes subpopulation response. Simultaneously, studies of intracellular expression of genes encoding of Il-2, Il-12, IFN-gamma and Il-4, Il-5, Il-10, Il-13 in mice splenocytes, and genes encoding factors involved in inflammatory process in the lung tissue (GM-CSF, TNF-alpha, Il-1beta, Il-6 i TGF-beta) have been performed on RNA level. The obtained results, confirmed high polarization of immunological response toward Th1 in mice immunized with DTP vaccine with whole-cell pertussis component, and toward Th2 in mice immunized with acellular pertussis vaccine. Inflammatory process in the lung tissue was more pronounced in animals immunized with whole-cell pertussis vaccine. There were no quantitative differences of analysed factors involved in the immune response among mice challenged B. pertussis strains containing different ptxS1/prn composition.     

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.     

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.     

Relative Stability of Pertussis Vaccine Preserved with Merthiolate, Benzethonium Chloride, or the Parabens

When stored at 4 C, or heated at 22 or 35 C followed by storage at 4 C, the potency of pertussis vaccines preserved with Merthiolate was more stable than the potency of vaccines preserved with benzethonium chloride or the parabens (methyl- and propyl-p-hydroxybenzoate). Without preservative, potency was more stable than in the presence of benzethonium chloride or the parabens, but less stable than when Merthiolate was present. The histamine-sensitizing factor of the vaccines likewise decreased with the loss of potency. The deleterious effect of benzethonium chloride and the absence of the stabilizing effect of Merthiolate were contributing factors, if not the sole cause, for the instability of pertussis vaccine in quadruple antigen vaccine (diphtheria and tetanus toxoids and pertussis and poliomyelitis vaccines).     

Advantages of Aluminium Hydroxide Adsorbed Combined Diphtheria,Tetanus, and Pertussis Vaccines for the Immunization of Infants

Three combined triple antigen vaccines were used to inoculate infants receiving primary immunization at 3 to 6 months of age. Laboratory potency and toxicity tests and clinical evaluation again showed that the mouse weight gain test is able to predict which vaccines will give reactions in children. The addition of aluminium hydroxide to the vaccine both increased potency and reduced the tendency to cause reactions. Assays on sera showed that almost all children produced agglutinins to Bordetella pertussis types 1, 2, and 3 when the vaccine contained aluminium hydroxide.     

Results of a study of the reactogenicity and epidemiological effectiveness of a 2d revaccination against whooping cough]

The reactogenicity and epidemiological effectiveness of the second revaccination against pertussis were studied in conformity with all the conditions of a controlled epidemiological trial. The character of the distribution of local and fever reactions in children aged 6 years after the second revaccination with adsorbed DTP vaccine suggests the presence of high sensitivity to the pertussis component of absorbed DTP vaccine in children of this age group. The results obtained from the study of epidemiological effectiveness (in 15,621 children) indicated that the second revaccination of children aged 6 years (at an interval of 3 or more years after the first revaccination) was not advisable as it did not influence noticeably the pertussis incidence.     

吸附精制百日咳菌苗质量控制的研究

本文就吸附精制百日咳菌苗制检过程中使用的菌株、有效组分ＬＰＦ和ＦＨＡ提纯工艺以及毒性,效力等检定方法阳相应的质量控制指标等进行了研究。结果表明,中国Ｃ、Ｓ菌株产毒优于日本Ｔｏｈａｍａ株。ＬＰＦ和ＦＨＡ─ＥＬＩＳＡ用作菌苗工艺各步骤中质控有效组分的产量,是一种快速、敏感和特异的方法。根据５年中菌苗批量生产每ｍｌ培养基ＬＰＦ和ＦＨＡ产量逐年上升趋势,说明生产工艺不断改进完善和操作技术的熟练是提高菌苗产量和质量的关键。制检规程中用作毒性和效力试验的方法,是菌苗安全有效质控的有效实用方法。     

In vitro induction of a diphtheria toxoid specific antibody response in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid

In comparison with the presently used potency test for diphtheria vaccine, in vitro examination of the immunogenicity of the vaccine would have great advantages. For this reason in vitro induction of diphtheria toxoid specific antibody synthesis in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid was investigated. The results showed that a dose dependent synthesis of diphtheria antibody was induced by adsorbed diphtheria toxoid and combined vaccines containing the diphtheria toxoid component. Plain diphtheria toxoid appeared to be less immunogenic in comparison with adsorbed toxoid. There is some indication that the pertussis component had a stimulating effect on the diphtheria antibody synthesis. In conclusion, these results are promising for in vitro examination of the immunogenicity of diphtheria vaccines. The model will be validated for the routine control of diphtheria vaccine.