Calibration of pertussis toxin BRP batch 1 in a standardised CHO cell-based clustering assay.

The European Pharmacopoeia (Ph. Eur.) pertussis toxin (PT) Biological Reference Preparation (BRP) is used as a working standard for safety testing of acellular pertussis vaccines as prescribed in the Ph. Eur. monographs 1356 "Pertussis vaccine (acellular, component, adsorbed)" and 1595 "Pertussis vaccine (acellular, co-purified, adsorbed)". The BRP was calibrated in 2006 in the murine histamine sensitisation test (HIST) against the World Health Organization (WHO) 1st International Standard (IS) for PT. In recent years, there have been increasing efforts to replace the in vivo test with in vitro methods. The Chinese hamster ovary (CHO) cell clustering assay has been used for many years by manufacturers to monitor residual PT activity in detoxified non-adjuvanted bulks. More recently a standardised protocol has been developed for this assay and a PT reference preparation was needed. Due to low stocks, the WHO 1st International Standard for Pertussis Toxin (JNIH-5) needed to be replaced and therefore a joint study between the European Directorate for the Quality of Medicines & HealthCare (EDQM) and WHO was initiated to calibrate the PT BRP for the CHO clustering assay and to replace the IS. The collaborative study involved 14 laboratories from Europe, North America and Asia. The outcome of the study confirmed that the BRP is suitable for use as a reference preparation in the CHO clustering assay. The material was assigned a potency of 1360 IU per vial for the CHO clustering assay.

Safety testing of acellular pertussis vaccines: Use of animals and 3Rs alternatives.

The current test of acellular Bordetella pertussis (aP) vaccines for residual pertussis toxin (PTx) is the Histamine Sensitization test (HIST), based on the empirical finding that PTx sensitizes mice to histamine. Although HIST has ensured the safety of aP vaccines for years, it is criticized for the limited understanding of how it works, its technical difficulty, and for animal welfare reasons. To estimate the number of mice used worldwide for HIST, we surveyed major aP manufacturers and organizations performing, requiring, or recommending the test. The survey revealed marked regional differences in regulatory guidelines, including the number of animals used for a single test. Based on information provided by the parties surveyed, we estimated the worldwide number of mice used for testing to be 65,000 per year: ∼48,000 by manufacturers and ∼17,000 by national control laboratories, although the latter number is more affected by uncertainty, due to confidentiality policies. These animals covered the release of approximately 850 final lots and 250 in-process lots of aP vaccines yearly. Although there are several approaches for HIST refinement and reduction, we discuss why the efforts needed for validation and implementation of these interim alternatives may not be worthwhile, when there are several in vitro alternatives in various stages of development, some already fairly advanced. Upon implementation, one or more of these replacement alternatives can substantially reduce the number of animals currently used for the HIST, although careful evaluation of each alternative's mechanism and its suitable validation will be necessary in the path to implementation.

Effectiveness of experimental whole-cell pertussis vaccines in murine model.

INTRODUCTION: Since the 1990s pertussis re-emergence has been observed in many highly immunized countries. Genetic divergence between circulating B. pertussis isolates and vaccine strains has been suggested as one of the reasons responsible for the resurgence of pertussis. This divergence was observed in some studies to affect the effectiveness of pertussis vaccine when tested in murine model. In the study, using the murine intranasal challenge model we evaluated the effectiveness of four experimental wP vaccines, prepared with B. pertussis isolates belonging to different PFGE groups, in the elimination of the bacterial infection induced with mixture of the four B. pertussis isolates. METHODS: The experimental wP vaccines were prepared with clinical isolates belonging to PFGE groups V, IVγ and C, used individually or together. The mixture of four isolates classified to PFGE groups V, IVγ, III and C was used as intranasal mice challenge. The chosen strains represent PFGE groups characteristic for isolates currently circulating in Europe (PFGE groups IV and V), specific for Poland (PFGE group C) and vaccine strains of Polish wP vaccine (PFGE group III). Additionally, to study bacterial fitness, changes in the proportions of four isolates used as the challenge within the course of infection in mice lungs were monitored. RESULTS AND CONCLUSIONS: All experimental wP vaccines were found to be equally effective in eliminating B. pertussis from mice lungs. Their effectiveness was independent on PFGE group of vaccine strain. The results on bacterial fitness during mixed infections induced in the non-immunized mice found the isolate of PFGE group IVγ dominating among the other isolates used in the mixture belonging to PFGE group III, V, and C. This data might suggest that the isolates belonging to PFGE group IV, so commonly seen in Europe, might be more fitted to explore in conditions of waning immunity.

Characterization of the carbohydrate binding and ADP-ribosyltransferase activities of chemically detoxified pertussis toxins.

Pertussis toxin (PTx) is an essential component of the acellular pertussis (aP) vaccine. However, because PTx in its native form is considered too toxic for human vaccine use, it must be inactivated into a stable, nontoxic form by treatment with chemical detoxifying agents or by genetic modification. Therefore, testing for the residual PTx in the aP vaccine is a major quality control step for vaccine manufacturers and regulatory authorities. The histamine sensitization test is currently the standard safety test method for all aP vaccines, regardless of the vaccine formula or the detoxification process, except for those with genetically modified PTx. However, test result variability and ethical concerns regarding animal use necessitate an alternative method. In vitro assays based on the biochemical properties of PTx have been considered as potential alternatives to the histamine sensitization test. In this study, the suitability of assays based on the ADP-ribosyltransferase and carbohydrate binding activities of PTx was assessed for PTx after treatment with formaldehyde, glutaraldehyde or both denaturants in sequence. The results indicated a distinctive pattern of the biochemical activities depending on the detoxification methods and storage conditions. These results suggest that although a more careful study is needed, these in vitro biochemical assays can be considered potential alternatives to the histamine sensitization test, as they might provide more specific safety information of aP vaccines.

Collaborative study for the standardisation of the histamine sensitizing test in mice and the CHO cell-based assay for the residual toxicity testing of acellular pertussis vaccines.

The European Pharmacopoeia (Ph. Eur.) and the World Health Organisation (WHO) require the performance of extensive quality and safety control testing before the release on the market of vaccine products for human use. Safety testing with regard to residual pertussis toxin (PT) in acellular pertussis combination vaccines is performed through assessment of fatal sensitisation of mice to histamine challenge by the vaccine product under test. Currently, use of different in-house procedures and no requirement for the inclusion of a standard reference in each assay render comparisons of results obtained for identical vaccine batches between different control laboratories very difficult. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM), an international collaborative study was organised for the standardization of the Histamine Sensitizing Test (HIST) in mice and the Chinese Hamster Ovary (CHO)-cell-based assay (performed at the bulk product level) for the residual toxicity testing of acellular pertussis vaccines or acellular pertussis-based combination vaccines. The study was run under the aegis of the Biological Standardisation Programme, jointly supported by the Council of Europe and the European Commission under the project code BSP076. Ten (10) laboratories participated in the study and were requested to perform 3 independent Histamine Sensitizing Tests in mice and to report results of the lethal end-point measurement as prescribed by the Ph. Eur. monographs. Some of them also reported data from an in-house validated CHO-cell-based assay. In addition, some of the laboratories reported concomitantly data obtained by measurement of the drop in temperature induced after the histamine challenge, a method currently under investigation to be added as an alternative end-point for the HIST in the Ph. Eur. monographs for acellular pertussis-based combination vaccines in order to alleviate animal suffering (in application of the 3Rs principle). Based on the results of the collaborative study, a potency of 7500 IU/vial (International Units per vial) was assigned to the current Ph. Eur. Biological Reference Preparation (BRP) for PT. The results of the study also show that 1) intra- and inter-laboratory variations can be improved by the use of a validated standard operating procedure; 2) inclusion in each assay of a standard reference sample, calibrated in IU, can increase comparability of results among laboratories and thus help to reduce repeat testing; 3) a correlation between mortality data and temperature data was observed although, due to the limited number of data sets and the lack of a common method for the temperature end-point, further investigation of this point is required; 4) the CHO-cell-based assay did not yield comparable results and further standardisation of the assay procedure may be investigated in a follow-up project.

An alternative temperature-based histamine-sensitisation test for absence of residual pertussis toxin in acellular pertussis vaccines.

As an extension of the BSP076 study (see the article 'Collaborative Study for the Standardisation of the Histamine Sensitizing Test in Mice and the CHO Cell-based Assay for the Residual Toxicity Testing of Acellular Pertussis Vaccines (BSP076)', page 51 of this issue of Pharmeuropa Bio & Scientific Notes), it was decided to publish the following experimental method for the temperature-based histamine-sensitisation test, validated at the SSI (Statens Serum Institut, Denmark), as a working basis for the growth of the method in individual laboratories.

[New evaluation methods of vaccine safety].

In this study, we showed, using comprehensive gene expression analysis, that pertussis and pandemic influenza vaccine affected many lung genes. The 13 and 76 genes were extracted from the pertussis vaccine- and pandemic influenza vaccine-treated lung at day 1, respectively. Furthermore, QuantiGene Plex methods, quantitative analysis of the expression of these genes could detect the toxicity from samples containing various concentrations of reference pertussis vaccines. Our findings and methods provide the first assessment for the influence of vaccine toxicity with an approach using experimental biology and genome science.

Application of quantitative gene expression analysis for pertussis vaccine safety control.

Although vaccines are routinely used to prevent infectious diseases, little is known about the comprehensive influences caused by vaccines. In this study, we showed, using comprehensive gene expression analysis, that pertussis vaccine affected many genes in multiple organs of vaccine-treated animals. In particular, lung was revealed to be the most suitable target to evaluate pertussis vaccine toxicity. The 13 genes identified from the analysis of vaccine-treated lung at day 1 showed a clear dendrogram corresponding to pertussis vaccine toxicity. Furthermore, quantitative analysis of these genes revealed a positive correlation between their respective expression levels and the degree of toxic effects observed in samples that had been treated with various doses of reference pertussis vaccines. The quantification of this 13 gene-set is an indicator of the vaccine toxicity-related reaction.

[Acellular pertussis vaccine].

Protective, immunogenic, toxic, and sensitizing properties of acellular pertussis vaccine (aPV) developed according to original technology were studied, aPV had marked protective activity which lasted more than 2 years. Sera of mice immunized by aPV also possess protective properties, and they were more prominent than in sera of mice immunized by pertussis bacteria suspension (PS). Immune sera to aPV neutralized cytopathogenic effect of pertussis toxin (PT) on ovarian Chinese hamster cells in 1:250 dilution, whereas neutralizing activity of sera to PS was very low. Level of antibodies to PT was higher in rabbits immunized, according to schedules and dosage recommended for children, by aPV than by PS. High immunogenicity of aPV was proved also by levels of IgG to PT in sera of mice immunized three times by aPV in human dosage. During experiments on mice and guinea pigs aPV had mild toxicity, did not induce autoimmune process, did not have anaphylactogenic properties compared with bacterial suspension characterized by high anaphylactogenic activity. Histamine-sensitizing abilityof aPVwas 40 times lower than that of PS. Assessment of pyrogenic properties of aPV and PS performed on rabbits showed that aPV was 1,000 times less pyrogenic than PS. Obtained results demonstrate high protective and immunogenic properties of domestic acellular pertussis vaccine and its low toxic and sensitizing characteristics.

Formulation of the adenylate cyclase toxin of Bordetella pertussis as protein-coated microcrystals.

Adenylate cyclase toxin (CyaA) is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and, in its detoxified form, a potential component of acellular pertussis vaccines. This study reports the application of a novel technology, formulation of CyaA as protein-coated microcrystals (PCMC), to improve the performance of CyaA as a vaccine component. CyaA is normally stored in a high urea concentration to prevent aggregation and to maintain stability of the protein. The aim of the work was to stabilise CyaA on a crystalline support to create a dry powder that could be reconstituted in aqueous buffer, free of urea. CyaA, formulated as PCMC with microcrystals of dl-valine, retained full adenylate cyclase (AC) and cell invasive (cytotoxic) activities after solubilistion in urea buffer. After storage as a dry powder at 37 degrees C for 2 weeks, the AC activity recovered from the CyaA-PCMC was only marginally reduced when solubilised in urea buffer. No AC activity was detected after attempts to solubilise CyaA-PCMC in aqueous buffer alone, in the absence of urea. Inclusion of various ionic, non-ionic or zwitterionic detergents in the aqueous buffer had little effect on recovery of CyaA activities. However, preparation of PCMC with CyaA plus calmodulin (CaM) or bovine serum albumin (BSA) or with both proteins allowed restoration of AC and cytotoxic activities of CyaA upon solubilisation in aqueous buffer. Incorporation of BSA and CaM with CyaA allowed essentially full recovery of AC activity but lower recovery of cytotoxicity. CyaA-CaM-BSA-PCMC, after reconstitution in aqueous buffer, induced a strong serum IgG response to CyaA when injected subcutaneously into mice.

Highly sensitive histamine-sensitization test for residual activity of pertussis toxin in acellular pertussis vaccine.

Histamine-sensitization test method based on histamine-sensitizing death is widely used for controlling residual activity of pertussis toxin in acellular pertussis vaccines. The test method evaluates the residual activity according to the death of mice injected with a test vaccine after histamine challenge and the test result, therefore, depends on the sensitivity of mice. A highly sensitive test method based on change in rectal temperature of mice has been used in Japan for many years but has limited feasibility in other countries. We examined the possibility of a test method using dermal temperature measured by infrared thermometer to reduce animal suffering instead of rectal temperature. The dermal temperature method was shown to be as sensitive as the rectal temperature method. Furthermore, the dermal as well as rectal temperature methods can evaluate the activity of a test vaccine in relative to a reference preparation so as to allow direct comparison of the test results among different laboratories. The activity by means of the dermal temperature method was also found to be well consistent with that by the rectal temperature method.

Two vaccine toxicity-related genes Agp and Hpx could prove useful for pertussis vaccine safety control.

Conventional animal tests such as leukocytosis promoting tests have been used for decades to evaluate toxicity of pertussis vaccine. Here, we examined gene expression in relation to the vaccine toxicity using a DNA microarray. Comparison of conventional animal test data with the DNA microarray-based gene expression data revealed a gene expression pattern highly correlated with leukocytosis in animals. Of 10,490 rat genes analyzed, two genes, alpha1-acid-glycoprotein (Agp) and hemopexin (Hpx), were found up-regulated by the toxin administration in a dose-dependent manner (assayed by a quantitative PCR based on the microarray). Variation of the gene expression was very small amongst the test animals, and the results were highly reproducible. These findings suggest that gene expression analysis of vaccine-treated animals can be used as an accurate and simple method of pertussis vaccine safety assessment.

Lung pathology and immediate hypersensitivity in a mouse model after vaccination with pertussis vaccines and challenge with Bordetella pertussis.

While evaluating vaccine efficacy against clinical Bordetella pertussis isolates in mice, after challenge vaccinated mice showed increased lung pathology with eosinophilia, compared to challenged, non-vaccinated animals. This led us to study bacterial clearance, lung pathology, lung TNF-alpha expression, and parameters of immediate hypersensitivity (IH), being serum IgE levels, eosinophil numbers in the bronchoalveolar lavage fluid, and ex vivo IL-4, IL-5, IL-10, IL-13, and IFN-gamma production by the bronchial lymph node cells. BALB/c mice received a combined Diphtheria (D), Tetanus (T), Poliomyelitis, and whole-cell Pertussis vaccine (WCV), a combined D, T, and three-component acellular Pertussis vaccine (ACV), aluminium hydroxide adjuvant, or PBS, 28 and 14 days before B. pertussis infection. Similarly treated non-infected mice were taken as a control. Infection induced pathology; this induction was stronger after (especially WCV) vaccination. WCV but not ACV vaccination induced TNF-alpha expression after challenge. After challenge, IH parameters were strongly increased by (especially ACV) vaccination. Vaccinated IL-4 KO mice showed similar clearance and pathology, in the absence of IgE and with reduced numbers of eosinophils. Vaccinated (Th1-deficient) T-bet KO mice showed reduced clearance and similar pathology. In summary, after challenge vaccination increased lung pathology, TNF-alpha expression (only WCV), and IH parameters. Th1 cells were critical for clearance.

[Evaluation of the stability of pertussis vaccine OCO-3 standard]. / Otsenka stabil'nosti standarta kokliushnoi vaktsiny OCO-3.

The evaluation of the immunogenic activity and residual toxicity of the National Standard of pertussis vaccine (OCO-3) was carried out. As shown by observations lasting for a period of 25 years, the preparation possesses stable immunogenicity and its toxicity remained unchanged, which makes it possible to use OCO-3 for the routine control of the pertussis component of commercial lots of adsorbed DPT vaccine.

Toxicity and potency evaluation of pertussis vaccines.

Current methods for determining the potency and toxicity of pertussis vaccines are outdated and require improvement. The intracerebral challenge test is effective for determining the potency of whole-cell vaccines but is objectionable on animal welfare and technical grounds. The same applies to its modification for assaying acellular pertussis vaccines. Respiratory challenge methods offer an interim solution pending establishment of validated in vitro correlates of protection, for example nitric oxide induction. Their evaluation is being promoted by the World Health Organization through the Pertussis Vaccines Working Group. Current toxicity assays based on weight gain and histamine sensitization of mice are imprecise and need replacement. Limits need to be established for specific toxin content of both acellular and whole-cell vaccines and should be supported by specific assays. More precise methods based on determination of ribosyltransferase activity in tandem with receptor-binding assays are under evaluation. Genome sequence data and the use of gene microarrays to screen responses triggered by vaccine components may also provide leads to improved methods for assessing both toxicity and immunogenicity.

Developments in reduction and replacement of in vivo toxicity and potency tests for pertussis vaccines.

The regulatory control of pertussis vaccines, as for other biological products, requires that they conform to specified standards of safety and efficacy. The current potency test for whole cell vaccines, the intracerebral mouse protection test (AMPT) is still the only such assay that has shown a correlation with protection in children. An alternative in vivo assay based on non-lethal aerosol challenge of mice has been assessed as a replacement for the current AMPT. An in vitro assay based on determination of reactive nitrogen/oxygen intermediates produced as a result of macrophage activation has also been investigated as a potential replacement for the in vivo challenge test. On the other hand, for safety testing, an enzymatic-HPLC coupled assay using a fluorescein-labelled G alpha(i3)C20 peptide to measure the enzymatic ribosylation activity of active pertussis toxin was evaluated for its suitability as a replacement for the current histamine sensitisation test (HIST). An assay for adenylate cyclase toxin (ACT)-related toxicity, based on measuring the ACT-induced oxidative burst in macrophage-like cell cultures has also been investigated. Although some questions still need to be answered in relation to the development of suitable replacements for in vivo tests of pertussis vaccines, the prospects for further improvements are promising.

Disturbance of cardiovascular circadian rhythms by pertussis vaccine in freely-moving rats.

Vaccination of young children with diphtheria, tetanus, poliomyelitis and pertussis (DTPoP) vaccine is effective in preventing outbreaks of whooping cough but adverse events sometimes occur. This pilot study shows that in freely-moving rats, multiple treatment with DTPoP (at day 0 and day 5, 6 ml/kg i.v.) increased heart rate (HR) for 5 days after the first treatment and decreased diastolic blood pressure (DBP) for at least 26 days after the first treatment and inhibited the circadian rhythm of HR and DBP for at least 10 days. DTPo vaccine, containing no pertussis vaccine, was free of such effects. Thus, in rats, the pertussis component of DTPoP acts on the cardiovascular system and disturbs its circadian rhythm. The contribution of these findings to clinical adverse effects is as yet unknown and needs further research.

Toxicity tests on native and recombinant Bordetella pertussis adenylate cyclase toxin preparations.

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Active and inactive CyaA proteins were prepared in crude and purified form from B. pertussis or from recombinant E. coli expressing both cyaA and cyaC genes or the cyaA gene alone, respectively. The specific AC activities of all the crude or all the purified toxins were similar. The toxins produced in the absence of CyaC activity had no cell invasive, haemolytic or cytotoxic activity. The cell invasive and cytotoxic activities of native and recombinant active CyaA preparations were similar, but the haemolytic activity of the recombinant toxin was lower than that of the native protein. As part of mouse protection tests, mice were injected subcutaneously with 15 microg per mouse of crude or purified CyaA preparations plus alhydrogel or with 1/5 human dose of adsorbed DPT vaccine (Wellcome Trivax-AD) using two doses at a two week interval. Control groups of mice were injected with alhydrogel in PBS alone or left unvaccinated. The toxin preparations had little or no effect on mouse weight gain, and there were no marked differences between mice vaccinated with the active toxin and those given the inactive form. Thus, at the dose used, there was no clear toxic physiological response caused specifically by active CyaA.