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.

Moving towards a (microbiological) environmental monitoring programme that can be used to release aseptically produced pharmaceuticals: a hypothesis, a practical programme, and some results.

We present, in detail, the (microbiological) environmental monitoring programme for cleanrooms that is in use at the RIVM. The parameters that are measured are: Total airborne particles of 0.5 micron and larger, airborne colony forming units (cfu's), cfu's on surfaces, cfu's on fingertips of gloves and settling cfu's. Most attention is paid to areas where critical handling is performed (class A areas). In these areas sampling is performed continuously during work. The areas surrounding class A areas are seen as a necessary environment to create a class A area and are called supporting areas. Changes in these supporting areas that will adversely effect the quality of the class A area, will be detected in the continuous sampling of the class A area. Sampling the supporting areas should only bring forward whether the measured parameters of these areas are consistent. The frequency of sampling, either weekly, monthly or three monthly, is based on the data obtained in previous sampling. In addition to the programme some data are presented. These data indicate that the values of the earlier mentioned parameters are not particularly dependent upon the lay-out or classification of areas, but rather on the use of the areas, and how the operators behave in them. The results support the concept of our sampling schedule, that relevant information about the production areas can be gathered with relatively little effort. Evaluating the programme, we conclude that the main missing element is a known relation between the results of environmental monitoring and contamination of products. We strongly advocate research that could result in establishing such a relation.

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.

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.

Monoclonal antibodies to Treponema pallidum.

Three successive fusions of mouse myeloma cells and spleen lymphocytes of a mouse immunized with Treponema Pallidum resulted in one hybridoma producing anti T. pallidum antibodies for each fusion. The mice were immunized with live pallidum cells respectively 1, 3 and 5 months before fusion and with antigen purified on density gradients 4, 3 and 2 days before fusion. Hybridomas cultures were tested for antibody production with an Enzyme Linked Immunosorbent Assay (ELISA) and a Western blotting technique. Two of the three anti T. pallidum antibody producing hybridomas were found with the ELISA, the third was found with a Western blotting technique. These hybridomas were also tested for the production of antibodies to rabbit antigens and T. phagedenis antigens in the ELISA: none appeared to be positive. Two of the hybridomas produce antibodies to a T. pallidum protein antigen of a molecular weight of 46 000: one hybridoma produces antibodies to a T. pallidum protein antigen of a molecular weight of 44 000 as determined by the Western blotting. Antibodies against these antigens are found during almost all stages of syphilis in man. One of the hybridomas produces monoclonal antibodies that react with treponemal antigen from E. coli cells, prepared by recombinant DNA technology as appeared in the Western blotting technique and this antibody will be used for purification of the 44 000 protein.

Monoclonal antibodies for the control of influenza virusvaccines.

Hybridomas producing haemagglutination inhibiting monoclonal antibodies against influenza A/Texas/1/77 H3N2 were developed. One hybridoma producing antibodies reacting with Victoria/3/75, Texas/1/77 Bangkok/1/79 and England/496/80 was selected to determine the potency of influenza virusvaccines. Tests were performed in a newly developed Enzyme Linked Immunosorbent Assay (ELISA). For this purpose microtiter plates were successively coated with the monoclonal antibody, washed, incubated with vaccine or standard, washed, incubated with the peroxidase conjugated monoclonal antibody, washed and finally incubated with a substrate. Samples of the vaccine and of a standard containing 25-100 ng of antigen were assayed in the ELISA and the results were compared with those obtained in a rocket electrophoresis method. Linear regression analysis of the results showed that the correlation coefficients obtained with standards and vaccines for both methods were greater than or equal to 0.96. The comparison of vaccine potencies determined in the ELISA and the rocket electrophoresis method will be discussed.

The effects of nasal drops and their additives on human nasal mucociliary clearance.

A method for measurement of nasal mucociliary clearance in vivo is described. A drop, containing saccharine sodium and indigo carmine is placed on the edge of the ciliary epithelium in the entrance to the nose. The time between placement and the sensing of the sweet taste as well as the appearance of a blue line in the nasopharyngeal cavity is measured and called the transport time. Two preservatives, two nasal drops and one viscosity-increasing substance have been investigated and the results are compared with their effects on the ciliary beat frequency of chicken embryo tracheas in vitro. The more the transport time is increased by a compound the more the ciliary beat frequency is decreased. Chlorbutol 0.5% increases transport time more and decreases ciliary beat frequency more than benzalkonium chloride 0.006% + EDTA 0.1%. Otrivin 0.1% increases transport time more and decreases ciliary beat frequency more than Rhinoguttae xylometazolini 0.1% (F.N.A.). These results support those obtained with the photo-electric registration device applicated on chicken embryo tracheas and human adenoids as described in earlier publications.

Correlation between the sensitivity of the ciliary beat frequency of human adenoid tissue and chicken embryo tracheas for some drugs.

The effects of benzalkonium chloride, chlorbutol,xylometazoline and naphazoline on the ciliary beat frequency of human adenoids and chicken embryo tracheas have been determined and compared. Chlorbutol 0.5% appeared to arrest ciliary motion in both tissues within 5 minutes. Rinsing with Locke Ringer solution (LR) restored the ciliary motion almost completely in both cases. Benzalkonium chloride 0.006% +EDTA 0.1% decreased the ciliary beat frequency 35% for the human tissues and 50% for the chicken tissues after a contact of 20 minutes. In both cases the frequency hardly changed after rinsing with LR. Naphazoline nitrate 0.1% and xylometazoline HCl 0.05% have reversible effects on the ciliary beat frequency of both human adenoids and chicken embryo tracheas. Cilia of human adenoids appeared to be more sensitive for xylometazoline than for naphazoline; whereas cilia of chicken embryo tracheas were more affected by naphazoline than by xylometazoline. The results with human adenoids and chicken embryo tracheas show a correlation (correlation coeff. = 0.82, p less than 0.005). In the initial response the differences in sensitivity to preservatives and drugs were in many cases statistically significant, but the final effects were similar.

Decreases in ciliary beat frequency due to intranasal administration of propranolol.

Recently the intranasal application of 5% propranolol was proposed in order to prevent the extensive first-pass metabolism of this drug. The ciliary epithelium in the nose affects the removal of dust, allergens, and microorganisms. The decreasing effect of propranolol on the ciliary beat frequency of human adenoid tissue and chicken embryo tracheas was measured with a photoelectric registration device. After nasal application of 5% propranolol, the drop is diluted by the nasal mucus. It was found that even 0.1% propranolol had a deleterious effect on the cilia of chicken and human tissue. Ciliary movement was arrested irreversibly within 20 min.

The effects of nasal drops on the ciliary beat frequency of chicken embryo tracheas.

The effects of proprietary preparations in the Netherlands and nasal preparations according to the F.N.A. (Formulary of the Netherlands' Pharmacists Association) on the ciliary beat frequency of chicken embryo tracheas have been determined. In general the preservatives, used in the nasal drops, turned out to have a more decisive influence on the ciliary motion than the pharmacologically active constituents. The average time, necessary to decrease the frequency 50% with 1=5 diluted nasal drops (average t 50%(1=5) containing a mercury compound or chlorbutol, is 0.4 h. Quaternary ammonium compounds containing preparations, however, have less influence, average t 50%(1=5) greater than 1.22 h. They are to be used preferably. Nasal drops, used as decongestants, provided that they are preserved with quaternary ammonium compounds, have little influence on the ciliary motion (average t50%(1=5) greater than 1.38 h). Nasal drops containing antihistamines, in contrast, inhibit the ciliary motion largely and almost independently of the preservatives (average t50%(1=5)=0.22 h); the cromoglycate containing preparation, on the contrary, has only little effect in this respect (t50%(1=5) greater than 2 h). Antimicrobial preparations are not preserved and have an intermediate ciliotoxic effect (average t50%(1=5)=0.7 h).

The effects of preservatives on the ciliary beat frequency of chicken embryo tracheas.

The effects of preservatives on the ciliary beat frequency of chicken embryo tracheas are determined, Polar compounds like benzalkonium chloride in commonly used concentrations, decrease the frequency less than 30% after a 20 minutes' exposure. The effect is not reversible after rinsing with Locke-Ringer solution. Lipophilic compounds however, like chlorbutol, cause an arrest of the cilary movement with 10 minutes. The effect, different from the polar compounds, is reversible; but only after a limited exposure-time. Mercuric compounds, like thiomersal, decrease the frequency non-reversibly 30 to 90% after a 20 minutes' exposure. EDTA decreases the frequency 40 to 50%, independent of the exposure-time and in a reversible way. The combination of benzalkonium chloride 0.01% and EDTA 0.05% is recommended to preserve nasal drops.

The influence of the pH and osmotic pressure upon tracheal ciliary beat frequency as determined with a new photo-electric registration device.

A method for measurement of tracheal ciliary beat frequency in vitro is described. Light transmitted through the cilia is detected by a phototransistor mounted in a microscope, while the frequency is measured instantaneously and the waveform is displayed by an oscilloscope, connected to a transient recorder. Due to the magnification and the method of illumination, the movement of approximately 30 cilia is projected on the phototransistor. In Locke-Ringer solution the waveform shows a very constant amplitude. Interference arises after a noxious influence and is dependent on the frequency of the ciliary movement. The effect of pH and osmotic pressure on chicken embryo and rat tracheal ciliary beat frequency is assessed. The frequency is not influenced between pH = 7 and pH = 10, but higher and lower pH values decrease the frequency. Hypertonic NaCl solutions decrease the frequency of chicken embryo cilia as much as hypotonic NaCl solutions. Rat cilia turned out to be less sensitive for hypotonic NaCl solutions.