Evaluation of two serological methods for potency testing of whole cell pertussis vaccines.

The European Pharmacopoeia (Ph. Eur.) and the World Health Organization (WHO) require the performance of extensive quality control testing including a potency test before a vaccine batch is released for human use. Whole cell pertussis (wP) vaccine potency is assessed by a mouse protection test (MPT) based on the Kendrick test. This test compares the vaccine dose necessary to protect 50% of mice against the effect of a lethal intracerebral dose of Bordetella pertussis and the dose of a suitable reference vaccine needed to give the same protection level. Due to the large variability in the results of this test and the severe distress which is inflicted on the many animals involved, its replacement by an alternative method is highly desirable. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe, in collaboration with the WHO and the In-vitro toxicology Unit/European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission (EC) Joint Research Centre-Institute for Health and Consumer Protection (JRC-IHCP), wP vaccine specialists from all over the world were invited to present an overview of candidate alternatives at a symposium organised in Geneva (Switzerland) in March 2005. Although no alternative method was found suitable for immediate implementation of batch potency control, the Pertussis Serological Potency Test (PSPT), initially developed in mice and recently transferred to guinea pigs (gps), was identified as a model of interest. Using the PSPT in gps to test several components of combined vaccines such as Diphtheria-Tetanus-wP vaccines in the same animal series would allow further implementation of the European 3Rs policy to batch potency control, by additional method refinement and reduction of animal use. The present study evaluated 2 features of the serological response to wP vaccination: 1) the overall antibody response as measured by a "whole cell" ELISA (PSPT-wC-ELISA) which uses the B. pertussis 18323 challenge strain prescribed for the MPT to coat the assay plates and 2) the functional neutralising antibodies to pertussis toxin (PT, one of the main virulence factors of B. pertussis), as measured by the Chinese Hamster Ovary (CHO) cell assay. The results showed that 1) the gp model can be used for wP vaccine potency testing; 2) despite good repeatability and precision, the CHO cell assay did not generate results comparable to the MPT. Moreover, the CHO cell assay showed significant differences in the ability of wP vaccines to induce neutralising anti-PT antibodies, which did not correlate to the overall antibody response evaluated by PSPT-wC-ELISA; 3) comparable potencies were obtained in the MPT and the PSPT-wC-ELISA. This study, supported by the previous ones correlating the PSPT-wC-ELISA in mice with the MPT, confirms that PSPT-wC-ELISA in gps is a promising approach for batch release potency testing of wP vaccines for which consistency in production has already been demonstrated by the MPT. However, a large scale validation study is required prior to the adoption of PSPT-wC-ELISA as a compendial reference method for wP vaccines batch release control.

Decreased sensitivity to diphtheria toxin of Vero cells cultured in serum-free medium.

Vero cell cultures are used in the quality control of Diphtheria vaccines: to estimate vaccine potency and to determine residual toxicity and reversion to toxicity. The impact of replacing foetal calf serum containing medium (SCM) by serum free media (SFM) on the sensitivity of Vero cells to Diphtheria Toxin was studied. Compared to SCM, SFM showed an eight-fold decrease in sensitivity to Diphtheria Toxin. This decrease was almost immediate, indicating that this phenomenon was not caused by a change in membrane structure or protein expression. We investigated the effect of SFM on Diphtheria Toxin in order to determine the cause of the decrease in sensitivity. Our results show that oligopeptides, which are often used in SFM as part of the replacement of foetal calf serum, are the most likely cause.

Evaluation of toxin neutralisation in test systems for diphtheria antibody assessment.

Over the past years, various authors have reported that the amount of toxin used in toxin neutralisation (TN) assays for diphtheria appears to influence the resulting relative antibody titre. Antibody affinity is thought to be an influencing factor. To confirm this observation and study the underlying mechanism of toxin neutralisation, a panel of sera was generated, differing in species of origin (mouse, guinea pig, and rabbit) and in affinity by using different immunisation schedules. The panel was then tested in relevant TN test systems for diphtheria antibody titration, namely the VERO cell test, the Toxin Binding Inhibition (ToBI) assay and the in vivo skin test in guinea pigs. A hyperimmune equine reference serum was used as the standard. Antibody affinity was measured in two different affinity ELISAs, the ammonium thiocyanate elution ELISA and the diethylamine inhibition ELISA. The VERO cell test clearly demonstrates the phenomenon; the higher the toxin dose used in the assay, the higher the resulting relative potency. The difference in relative antibody titre decreases as antibody affinity increases. This is especially evident when an equine hyperimmune reference serum is used as the standard. When a species homologous reference is used, the phenomenon is less distinct. The ToBI test, however, does not show the phenomenon. This discrepancy between these two test systems is being further investigated, and comparison will be made with the in vivo TN test. The findings confirm and support earlier observations. It is still unclear exactly which mechanisms are involved in the toxin neutralisation process. Antibody subclasses and class switching could play a role and will be further studied.

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.

Validation of the toxin-binding inhibition (ToBI) test for the estimation of the potency of the tetanus toxoïd component in vaccines.

A validation study has been performed to determine the suitability of the toxin binding inhibition (ToBI) test for the serological estimation of the potency of the tetanus toxoïd component in vaccines. 37 Murine serum pools over a wide range of antibody levels were titrated in both toxin neutralization (TN) and ToBI test. A good correlation was found between both assays. Sixteen DPT-polio, twelve DT-polio and seven T vaccines were tested in the mouse lethal challenge test and the in vitro serological test, using the ToBI test for determining vaccine-induced tetanus antibodies. For all three types of vaccine a statistically valid correlation between both assays was found. However, for two batches of DPT-polio vaccine an "overestimation" of the tetanus potency was observed in the serological assay compared to the challenge assay. This phenomenon could not be explained by the difference in immunization period nor by misinterpretation of the ToBI test of DPT-polio-induced antibodies. In the LPF test high LPF activity was observed for the deviating DPT-polio vaccines. Therefore, the effect of pertussis toxin (PT) on the potency of the tetanus component in the serological assay was examined. The addition of 2 micrograms of PT to a "normal" DPT-polio vaccine resulted in a nearly twofold increase of the tetanus potency. It was concluded that pertussis toxin has a vaccine dose-dependent adjuvant effect on the potency of tetanus toxoïd resulting in high potency values when determined by ToBI procedure. It is unclear how these findings should be interpreted with respect to the behaviour of such vaccines in man.

Validation of the combined toxin-binding inhibition test for determination of neutralizing antibodies against tetanus and diphtheria toxins in a vaccine field study in Viet Nam.

Determination of seroconversion and measurement of protective antibody levels in children against vaccine components are essential for gauging and monitoring the efficacy of paediatric vaccination programmes. For this purpose, we assessed the combined toxin-binding inhibition (ToBI) test for determining neutralizing antibodies to tetanus and diphtheria in a diphtheria-pertussis-tetanus (DPT) vaccine field trial in Viet Nam. A simple procedure involving collection of blood samples on filter-paper was found to be a suitable alternative to collection by venepuncture, despite a reduction in the sensitivity of the ToBI test as a result of the step necessary to elute the antibodies from the filter-paper. The results obtained demonstrate that the ToBI test can feasibly be carried out under field conditions. Preliminary results obtained with the ToBI test in DPT field trials indicate that a fourth dose of DPT vaccine one year after the third dose should be considered by developing countries.

PIP: In Vietnam, health workers collected blood samples from adults working at the National Institute of Vaccines and Biological Substances in Nha Trang and Dalat and from healthy unvaccinated infants 3-9 months old from the district areas/provinces of Tien Giang and Lam Dong to assess the combined toxin-binding inhibition (ToBI) test for determining neutralizing antibodies to tetanus and diphtheria in a diphtheria-pertussis-tetanus (DPT) vaccine field trial. Researchers also aimed to validate the use of a simple filter-paper blood collection method. There was a necessary step to elute the antibodies from the filter-paper, which reduced the sensitivity of the ToBI test. Nevertheless, in the ToBI test, blood collected on filter-paper yielded similar antibody titer estimations as those obtained by venepuncture. The Biotek method to estimate antibody titers yielded higher correlation coefficients than the OD50 method: tetanus (0.998 vs. 0.98) and diphtheria (0.956 vs. 0.95). One month after the third injection, all the children had antitoxin titers greater than 0.06IU/ml. By one year after the third injection, only 45% had antitoxin titers greater than 0.06IU/ml for diphtheria while all still had antitoxin titer levels above this value for tetanus. This suggests that health providers should consider administering a fourth dose of DPT vaccine one year after the third dose. These findings indicate that the ToBI test can be conducted under field conditions.

Interlaboratory validation of in vitro serological assay systems to assess the potency of tetanus toxoid in vaccines for veterinary use.

An interlaboratory validation study was carried out in seven laboratories to evaluate the suitability of in vitro serological assay systems for the assessment of the potency of tetanus toxoid in single and multicomponent vaccines for veterinary use. Nine commercial vaccines and one experimental tetanus toxoid preparation were selected for immunization purposes according to Method A of the European Pharmacopoeia. Levels of tetanus antibodies in guinea-pig and rabbit serum samples were estimated by indirect ELISA, toxin binding inhibition (ToBI) test, passive haemagglutination (HA) test and by the prescribed standard toxin neutralization (TN) test in mice. Estimates of potency obtained by in vitro tests and by TN test were in good agreement for the various vaccines tested and for antibody levels of individual serum samples in the range 2.6 IU/ml to 266 IU/ml. Significant (P < 0.05) intralaboratory variation occurred less frequently for ELISA and ToBI test than for HA test. The frequency of significant (P < 0.05) interlaboratory variation was acceptable for the ELISA and the ToBI test but greater variation was observed for the HA test. It is concluded that the ELISA and ToBI tests are suitable in vitro assay systems for assessing the potency of tetanus toxoid in batches of single and multicomponent vaccines for veterinary use. Rigid standardization of the HA test is essential before this test can be used for the same quality control purpose.

The use of the in vitro toxin binding inhibition (ToBI) test for the estimation of the potency of tetanus toxoid.

The in vitro toxin binding inhibition (ToBI) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBI test showed good correlation with the in vivo toxin neutralization (TN) test in titration of sera of mice immunized with various doses of DPT-Polio, DT-Polio and a tetanus reference preparation. Estimates of potency of tetanus toxoid obtained in mice by ToBI test correlated significantly with those obtained in mice by the lethal challenge test. In addition, potency values of the European reference preparation, succeedingly estimated by ToBI test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBI test is a promising alternative to the toxic challenge procedure in the potency assay of tetanus toxoid vaccines. A substantial refinement and reduction in the use of animals can be achieved. Additional savings can be made by combining diphtheria and tetanus potency testing.

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)

Combined estimation of tetanus and diphtheria antitoxin in human sera by the in vitro Toxin-Binding Inhibition (ToBI) test.

The use of the principle of inhibition of toxin binding to an antitoxin coated immunoassay plate as described in a previous paper for tetanus antitoxin titration, was adapted for the estimation of diphtheria antitoxin in human sera. With a few modifications, a Toxin-Binding Inhibition (ToBI) test was developed which could be used for a combined estimation of both tetanus and diphtheria antitoxin levels. The application of streptavidin-biotinylated peroxidase complex when using small serum samples (less than 50 microliters) is discussed. Antitoxin titres (both diphtheria and tetanus) of 0.002 IU ml-1 were detectable by the ToBI test, this being far below the level considered to be protective in man. Sera from 140 adults with different vaccination histories were titrated for both tetanus and diphtheria antitoxin. Good correlations were found between the estimates obtained by the ToBI test and those obtained by the toxin-neutralization (TN) test in mice (tetanus antitoxin) and those obtained in the in vitro neutralization test in VERO cells (diphtheria antitoxin). It is concluded that the ToBI test is a simple and reliable alternative to the functional models currently in use for the estimation of diphtheria and tetanus antitoxin levels. In addition, the ToBI test eliminates the need for laboratory-animal or cell-culture facilities and can be performed with small quantities of serum as required in field trials.

The effects of reductions in the numbers of animals used for the potency assay of the diphtheria and tetanus components of adsorbed vaccines by the methods of the European pharmacopoeia.

Forty eight assays of adsorbed diphtheria vaccine and seven assays of adsorbed tetanus vaccine using either a lethal challenge (38 assays) or a serum neutralization test (17 assays) were evaluated for the effects of reductions in the number of animals used at each dilution on the potency values and 95% confidence intervals. The results were assessed in the light of the requirements of the European Pharmacopoeia and the WHO. In the majority of assays, 50% of the number of animals presently required would have sufficed for the determination of a potency within the limits of confidence stipulated by the European Pharmacopoeia and the WHO. Therefore it is concluded that a simplified assay with a reduced number of animals is suitable for the routine potency testing of the diphtheria and tetanus components of the combined vaccines and the monovalent that were examined. Flexibility in the national and international requirements in respect of the numbers of animals used at each dilution is suggested for the routine potency assay of the diphtheria and tetanus components of adsorbed vaccines.

A mouse model to estimate the potency of the diphtheria component in combined vaccines.

A mouse model is presented to estimate the potency of the diphtheria component in combined vaccines. In addition, a simplified mouse test is suggested for routine potency control of diphtheria. It will be important to discuss this approach to replace the lethal challenge test in guinea pigs.

An investigation of a mouse model to estimate the potency of the diphtheria component in vaccines.

A mouse model to estimate the potency of the diphtheria toxoid components in vaccines using Vero cells to detect the neutralizing antibodies in the sera from immunized mice is described. The results obtained with this mouse model correlated significantly with those obtained in the lethal challenge test in guinea-pigs. For this reason it is suggested that the potency test in guinea-pigs be replaced by this mouse model because a considerable reduction in the number of animals used and costs can be achieved by the introduction of this mouse test for the routine control of the potency of the diphtheria component of vaccines.