The present regulation on Pertussis Vaccine in Japan requires the following method for the “safety test ”: Each of two guinea pigs should be inoculated intraperitoneally with 5.0 cc and 5 mice 0.5 cc of a test vaccine. The animals should survive for a specified period without any pathological signs due to the vaccine, and their body weight should regain to its starting level within a specified time. This method is essentially equal to those used in many other countries.This type of method belongs to the so-called “minimum requirement” type in which an absolute value of toxicity is defined. The definition of an absolute toxicity in terms of animal reactions (animal units) is unsatisfactory, because the animal unit is highly variable due not only to the variation of animals, but also to that in the mean response of stocks of animals in different laboratories and at different times. Even if the assay conditions are controlled as strictly as possible, it is not possible that the experimental conditions are quite the same or different only in minor factors, therefore another source of variation occurs. Thus, when the toxicity of a vaccine is sufficiently far from the specified level, no matter whether it is above or below the level there will be no trouble. It is obvious, however, that the results of assay are variable when the difference in toxicities between a test preparation and the specified level is within the range of variation of the response in the system, as we have often experienced in the routine assay for toxicity of commercial pertussis vaccines.In contrast to the failure to make accurate measurements in terms of animal reactions, much greater success has been attained by the introduction of a method estimating relative potency, in which estimation of an unknown preparation is made by comparison with a standard (or a reference) preparation. The standards in a stable form, which was introduced by Ehrlich, made this comparative methods possible.Besides, by the method of minimum requirement type neither quantitative determination of toxicity nor statistical analysis of the accuracy of the results are possible.For these reasons we investigated the comparative method in order not only to establish a more reliable method in the “safety test” of pertussis vaccine, but also to reinvestigate the relationship between the toxicity of pertussis vaccine and the magnitude of reactions by the vaccine in human beings.As there had never appeared reports concerning biological assay for toxicity of pertussis vaccine by the comparative method, the first step of this study was to select the response of animals or its metameter (Bacharach et al., 1942) biologically reasonable and suitable for the statistical analysis. Since the final step of the study should be the comparison of results between human beings and test animals, it would be desirable to select such a response as fever, which is common and measurable both in human beings and in such an experimental animal as the rabbit. However it was considered impractical to select fever as the response because of the difficulty in using adequately large numbers of rabbits for the routine work. We have long been making assays measuring the body weight changes of animals according to the current regulation. Measuring the body weight is relatively easy in practice and a large number of mice are available for the test under more or less controlled condition. Therefore, the assay method by the body weight change in mice was first investigated.If we find no significant correlation between the magnitude of reaction in human beings and the toxicity of vaccines estimated by the method proposed, then we will have to further search for other animal responses suitable for the purpose.
In the titration of Dlm of a diphtheria toxin it is necessary to use a large number of guinea pigs, for example, several tens as many guinea pigs for an accurate estimation. Therefore, it is natural that attempts have been made to find an alternative and less expensive method which would be of value, at least for a preliminary work.Jensen (1933) proposed a method by which a fairly precise estimation of Dlm was thought to be possible. His method was to compute Dlm from (DRM) m actually measured by the intracutaneous rabbit method, basing on the evidence obtained in his experiments that the Dlm/DRM ratio of diphtheria toxin of every kind was constant.Lately, Pope (1954) offered an opinion that the intracutaneous method was an alternative for the subcutaneous method in the titration of diphtheria toxin and was advantageous in the point that many titrations might be made on one animal.Gerwing (1957) recently proposed a more reasonable method of Drm titration, in which the parallel line assay method was introduced, saying that the minimal reacting dose was preferable to the Dlm to define a Schick test dose. However, they declared nothing as to the Dlm/DRM or Dlm/Drm ratio.A good number of experiments carried out in our laboratory with various kinds of diphtheria toxins have shown that the assumption that the Dlm/DRM or Dlm/Drm ratio of diphtheria toxin of every kind was constant did not hold in the majority of cases; particularly with fresh toxins, the Dlm/DRM ratio frequently being proved to be far from constant (Kurokawa, Kondo and Kondo, to be published) . Therefore, it is clear that the method proposed by Jensen cannot be used as a method estimating Dlm.As we were compelled to do a good number of fairly accurate titrations of Dlm in experiments pursuing the factor or factors concerning the Dlm/DRM ratio of diphtheria toxin (Kurokawa, Kondo and Kondo, to be published), an alternative method, less expensive and fairly precise, for the classical subcutaneous guinea pig method was looked for.Two methods which may meet this demand are found in the literatures.Frobisher and Parsons (Frobisher and Parsons, 1940; Frobisher, 1942) reported that mice inoculated intracerebrally with living culture of toxigenic diphtheria bacilli or toxic culture filtrates showed characteristic responses, which could be specifically neutralized with diphtheria antitoxin. According to their brief description, the minimal dose that killed almost all mice fairly corresponded with 1 guinea pig Dlm when the toxin was intracerebrally inoculated into mice. Shortly later, Suzuki (1944) confirmed Frobisher's report basing on his extensive experiments and showed that there was a certain relationship between Dlm and the minimal dose killing mice with certainty.Meanwhile, Hosoya and his collaborators (Hosoya, Ozawa and Tanaka, 1934; Ozawa, 1935) described that domestic fowls and 3 to 4 weeks old chicks of White Leghorn having received intramuscularly diphtheria toxin developed a characteristic paralysis of wings and of legs, and died for the most parts. Inoue (1936) confirmed the report of Hosoya and his collaborators and reported that approximately one week old chicks were the most susceptible to diphtheria toxin and that the paralysis developed in chicks with the toxin of one-several tensth of Dlm. Frobisher and his collaborators (Frobisher, 1940; Frobisher, Parsons and Tung, 1942), probably independently from Japanese authors' works, reported briefly that White Leghorn or Plymouth Rock chicks could be used for toxigenicity test of diphtheria bacillus. Branham and Wormald (1954) used the chicks in titration of diphtheria antitoxin.These methods may provide a new tool which enables the quantitative titration of diphtheria toxin, less expensively and fairly precisely.However, few report directed to this purpose using mice or chicks has yet appeared.
Kurokawa, Masami (National Institute of Health, Tokyo, Japan), Motoichi Hatano, Noboru Kashiwagi, Tateo Saito, Setsuji Ishida, and Reiko Homma. A new method for the turbidimetric measurement of bacterial density. J. Bacteriol. 83:14-19. 1962.-It was demonstrated that by a combination of two types of metameter of transmittance (T), -log T (0.0
The effect of bacterial endotoxin on the change in peripheral leukocyte population in mice due to the lymphocytosis-promoting factor (LPF) was investigated. Endotoxin affected not only the total leukocyte count but also the leukocyte proportions at any observation time. Both the coefficient and the intercept of regression of the leukocytic response on dose of LPF were modified by endotoxin. Therefore, in a valid biological assay for LPF using the peripheral leukocyte count as a response, a common reference preparation available for any test materials, irrespective of presence or absence of endotoxin, will be impracticable. Two reference preparations were tentatively established, one being a vaccine and the other an LPF preparation containing little endotoxin. A unitage of LP activity was assigned to each reference preparation.The results also showed that an LPF material to be tested for its possible effects on the lymphatic tissues or the reticuloendothelial system should be free from endotoxin.
Antibody production in mice after immunization with diphtheria-pertussis-tetanus (DPT) vaccine was investigated. Six lots of the vaccine produced in the same year by six manufacturers in Japan were chosen. Production of IgE antibody specific to either diphtheria or tetanus toxoid varied among vaccine preparations used, although there was no apparent difference in IgG antibody production after immunization. In addition, the level of IgE antibody specific to diphtheria toxoid was correlated with that of IgG1 antibody and inversely with that of IgG2 antibody. These results suggest that each vaccine preparation may induce a distinct pattern of antibody production and, therefore, the type of immune response induced by vaccination may vary among vaccine preparations.
Purity of the parent toxin influenced greatly the immunogenicity in guinea pigs of HR1 component of Habu-venom toxoid. The potency of HR1 toxoid in terms of the immunizing unit (ImU) appeared to be related to the antigen and aluminum contents. The immune response of the animals differed depending on the purity of the toxoid when the dose-response curve was examined over a wide range of the antigen dosage. Anti-HR1 titers of the guinea pigs immunized with crude toxoid (adsorbed) reached a plateau at about 10-20 U/ml ane further rise was not remarkable even when the amout of the antigen was increased several times, while 200 U/ml (maximum 400 U/ml or higher) of annti-HR1 was produced with a large amount of highly purified HR1 toxoid. Purified toxoid showed an excellent immunogenicity also in monkeys. The amounts of antigens necessary for the basic immunization to resist the challenge with a certainly lethal dose of crude venom were 18 and 0.5 mg as protein, for a crude and a highly purified toxoid, respectively. The total amount of aluminum was also reduced from 8.1 (crude toxoid) to 0.5 mg (purified toxoid). Multiple injections for the primary immunization enhanced early production of anti-HR1 in monkeys. However, at later stage of immunization, significant correlation was observed between the amount of HR1 toxoid (in ImU) for the primary immunization and the circulating anti-HR1 titer irrespective of the schedule of immunization. Anti-HR2 higher than 10 U/ml was produced consistently in monkeys with HR2 toxoid of 1.0 ImU for the basic immunization. However, excess HR2 toxoid of more than 10 ImU seemed rather unfavorable for anti-HR2 production.
To establish a quantitative method for titration of the heatlabile toxin of Bordetella pertussis by the skin reaction, various conditions were examined and their relative merits assessed.The skin reaction of the rabbit was characterized by a pale, ischemia-like spot with or without petechial hemorrhage, while hemorrhage was prominent in the guinea pig and suckling mouse. Mean diameters of the pale spot in the rabbit and of the hemorrhagic spot in the other species provided a linear regression on log dose over a relatively wide range. The regression co-efflcient was not affected by endotoxin or the lymphocytosis-promoting factor, though endotoxin intensified the reaction in the guinea pig and suckling mouse. These facts enabled us to construct a statistically valid assay method. Animal variations were controled by expressing the toxicity relative to that of a reference.An absolute unit of the toxicity was defined. Normalization of toxicities determined under different conditions is possible.
A table was constructed for use in estimating the mean of distribution of logarithms of titers based on data obtained with a pooled material instead of those with individuals in a sample. A table of standard errors of the estimator was also constructed. Examples showing the utility and applicability of the tables were presented. Several relating problems were discussed.
Precise and reproducible quantitative determination of hepatitis B surface antigen by applying the parallel line bioassay method was proposed. In this method, the value of any given sample can be expressed in terms of a relative value to a fixed standard or reference preparation.Decrease in antigenic reactivity due to the lowered protein content could be prevented by using 0.02% human albumin in physiological saline solution as the diluent. The use of such stabilizer (s) should be considered in the dilution or storage of samples containing HBs antigen.
