The Pertussis Serological Potency Test. Collaborative study to evaluatereplacement of the Mouse Protection Test.

The Pertussis Serological Potency Test (PSPT)--based on in vitro assessment of the humoral immune response against Bordetella pertussis--was developed as an alternative for the Mouse Protection Test (MPT). A small-scale collaborative study was carried out in five laboratories to evaluate the relevance and reliability of the PSPT. The study has been divided into three separate phases, each with its own objective. A pilot-phase study of the antibody detection assay, the 18323-whole cell ELISA (WCE), was included for training purposes. Significant differences in absorbance and antibody concentrations between the laboratories were found. In the Phase I study, the intra-assay, inter-assay and inter-laboratory precisions of the 18323-WCE were assessed. Although a precision of less than 20% was not always established and significant differences in antibody concentrations were found at random throughout the Phase I study, the ranking of the antibody concentrations corresponded well between the laboratories and should warrant a reliable potency estimation of whole cell vaccines (WCV's) in the PSPT. Phase II was a comparative study of the PSPT and the MPT to evaluate the implementation of the PSPT, to demonstrate correlation and to compare the reproducibility and reliability of both tests. The mean antibody concentrations per vaccine dose in the PSPT and the survival of mice in the MPT differed significantly within and between the laboratories. Nevertheless, the potencies of the vaccines under test estimated in both test models did not differ significantly (P>0.05). The PSPT and MPT correlated well in chi2-test of homogeneity within and between the laboratories. The potencies were similar (overall ratio=0.877), but the PSPT is more reproducible and reduces the chance of re-testing due to the smaller 95% confidence intervals. We have demonstrated that the PSPT is a valid model to estimate the potencies of pertussis WCV's from different manufacturers. Moreover, the 18323-WCE is easy to carry out and the intra-assay precision and antibody ranking warrants a reliable potency testing of pertussis WCV's in the PSPT.

Collaborative study on test systems to assess toxicity of whole cell pertussis vaccine.

A collaborative study has been carried out to establish the precision and accuracy of five test systems for the assessment of the toxicity of whole cell pertussis vaccine. To this end, six vaccines, including both "normal" and "abnormal" products with respect to arbitrary levels of Pertussis toxin and/or potency were tested. The study included in vivo test systems as the Mouse Weight Gain (MWG) test; the current WHO-recommended bioassay to evaluate overall pertussis toxicity and four specific test systems; the Leukocytosis Promotion (LP) test, the Histamine Sensitization (HS) test and in vitro the Chinese Hamster Ovary (CHO) clustering test to estimate pertussis toxin (PT) levels, and the Limulus Amoebocyte Lysate (LAL) test to evaluate endotoxin levels. In addition, participants were also asked to estimate potency by the Mouse Protection test according to Kendrick (MP). Fourteen laboratories in various countries participated in the study. In almost all participating laboratories, the MWG test was not very accurate in evaluating the overall toxicity of whole cell pertussis vaccines. In addition, statistical significant interlaboratory variation was frequently seen. The specific toxicity tests (LP, HS, CHO and LAL test) appeared to be more accurate, but large interlaboratory variation was seen, statistically significant at P < 0.05 for LP test, CHO test and LAL test. Significant variation in test results also occurred in the potency test. Furthermore, the discriminative power of the MP test between different levels of potency was low. It was concluded that, on the condition of optimization and stringent standardisation, HS and CHO test and in particular LP test might be more appropriate to assess PT activity than the MWG test provided that the tests are optimised and stringently standardized. An inhibition ELISA was used to estimate levels of PT. This test could be of value for prescreening purposes. The LAL test should be used to estimate endotoxin activity. The value of the MP test, as a model to assess potency, is disputed.

Pertussis serological potency test as an alternatively to the intracerebral mouse protection test.

The current potency test for pertussis vaccines, the intracerebral protection test (MPT), is still the only mandatory laboratory model available. This test, however, is a valid, but inhumane and imprecise test and therefore a good candidate for replacement. Recently we have developed the Pertussis Serological Potency Test (PSPT) as an alternative for the MPT. The PSPT is based on in vitro assessment of the humoral immune response against the whole range of surface -antigens of B. pertussis in mice after immunisation with Whole Cell Vaccine (WCV). We have demonstrated a relationship between the mean pertussis antibody concentration at the day of challenge and the proportion of surviving mice at each vaccine dose in the MPT (R = 0.91). The PSPT is a model in which mice (20-24 g) are immunised i.p. with graded doses of vaccine and bled after four weeks. Sera are titrated in a whole cell ELISA and potency based on the vaccine dose-dependent antibody response is estimated by means of a parallel line analysis. In an in-house validation study 13 WCVs were tested in the PSPT and MPT. Homogeneity of both tests was proven by means of the chi-square test; potencies were significantly similar (p = 0.95). Compared to the MPT, the PSPT is more reproducible as is indicated by its smaller 95% confidence intervals. Moreover, by using the PSPT the animal distress can be reduced to an acceptable level and the PSPT also results in a reduction of more than 25% in use of mice. Additional experiments showed that estimation of WCV-potency in the PSPT based on specific antibody responses against protective antigens (PT, FHA, 69- and 92-kDa OMPS) was not possible or did not correlate with protection in MPT. Sera obtained from the PSPT showed a correlation between pertussis antibody levels and complement-mediated killing by pertussis antibodies in in vitro assays. In conclusion, the PSPT is a promising substitute for the MPT though further validation and additional studies on functional validity should finally warrant replacement of the MPT by this serological model.

Development of pertussis serological potency test. Serological assessment of antibody response induced by whole cell vaccine as an alternative to mouse protection in an intracerebral challenge model.

The current potency test for pertussis vaccines, the mouse protection test (MPT), has many disadvantages. However, no alternative is yet available. The purpose of this study is to develop a serological alternative for the MPT based on in vitro assessment of the humoral immune response against pertussis in mice. After immunization with pertussis whole cell vaccine, the MPT shows a normal primary and secondary antibody response. Moreover, the i.c. challenge has a distinct booster effect on the pertussis IgG response. The relationship between the concentration of IgG antibodies against the surface-antigens of pertussis bacteria and the survival of mice after the i.c. challenge was demonstrated in a modified MPT (R = 0.91). To this end a protecting antibody level of > or = 45 EU/ml was selected as a level at which concentration most of the mice survived. Survival of mice in the MPT could be predicted, based on the antibody concentration at the day of challenge. Potencies estimated with the predicted and actual survival corresponded well (P = 0.990). This confirmed the essential role of vaccine induced pertussis antibodies in the protection against a lethal i.c. challenge and offered a possibility to develop a pertussis potency test based on serology. We developed a model in which mice (20-24 g) are immunized (i.p.) with graded doses of vaccine and bled after four weeks. Sera are titrated in Bordetella pertussis whole cell ELISA and potency based on vaccine dose dependent antibody response is estimated by means of a parallel line analysis. The potency of vaccines tested in the Pertussis Serological Potency Test (PSPT) and MPT are significantly similar, a P-value of 0.92 was found by means of the chi 2 test. Compared to the MPT, the PSPT is more reproducible as is indicated by its smaller 95% confidence intervals. Moreover, by using the PSPT the animal distress can be reduced to an acceptable level and the PSPT also results in a reduction of more than 25% in use of mice.

The modified leukocytosis promoting factor (LPF)-test: a valuable supplement to the mouse weight gain (MWG)-test in toxicity control of whole cell pertussis vaccine.

For the safety testing of pertussis vaccine, many in vivo assays have been developed, but none of these assays, except the Mouse Weight Gain (MWG)-test, are obligatory. Leukocytosis Promoting Factor (LPF) test, performed in mice, is one of the tests to examine the toxicity. However, due to lack of standardization, this test has not been implemented in the regular safety testing of the vaccine. Our investigations demonstrate that the LPF-test becomes more reproducible and sensitive if preparations are administered subcutaneously on day 0 and and counting of the leukocytes are done on day 6. Therefore, it is suggested to include the revised LPF-test in the quality control panel for the assessment of the toxicity of whole-cell pertussis vaccine.

Acellular and whole cell pertussis vaccines protect against the lethal effects of intracerebral challenge by two different T-cell dependent humoral routes.

Athymic (nu/nu) and euthymic (+/nu) BALB/c mice were immunized with a whole cell pertussis vaccine or with an acellular vaccine which contained detoxified pertussis toxin (PT) and filamentous hemagglutinin (FHA). Only the euthymic mice were protected against intracerebral challenge with virulent Bordetella pertussis which implies involvement of T-cells. As a cell transfer from mice immunized with whole cell or acellular vaccine prior to the challenge did not protect naive euthymic recipients, cellular immunity seems to be non-protective as an effector mechanism. Mice could be protected passively against a challenge by administration of immune sera. Therefore, T-cell dependent humoral immune responses to B. pertussis appear to be crucial for protection. The humoral response was further studied with athymic and euthymic mice. In euthymic mice the whole cell vaccine induced antibodies to FHA, pililipopolysaccharides (LPS) and an outer membrane protein (OMP) preparation, whereas the acellular vaccine induced antibodies to PT, FHA and OMP. Both IgM and IgG could be detected. From the nude mice only those immunized with the whole cell vaccine showed an antibody response which consisted of low titres of IgM directed to LPS. Sera from both +/nu and nu/nu mice immunized with the whole cell vaccine were bactericidal in vitro. These data demonstrate that in the mouse model protection to intracerebral challenge with B. pertussis is T-cell dependent as is the humoral response to PT, FHA, OMP and pili. The T-independent B-cell activation by the whole cell preparation is due to the presence of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

A biphasic serum glucose response in mice to inoculation with pertussis vaccine.

The effect of B. pertussis vaccine on the serum glucose level of mice was investigated. The results show that at least two components in the vaccine interfere with glucose metabolism. A heat-stable component which is assumed to be LPS induced hypoglycemia 3-5.5 h after inoculation, especially in LPS-sensitized mice. A heat-labile component which is possibly equivalent with the LPF/HSF/IAP complex, is responsible for persistence of the hypoglycaemia for at least 6 days. If hypoglycaemia contributes to the neurological side effects after pertussis vaccination both components have to be considered as being responsible for these effects.