Current state of methods for control the safety and potency of diphtheria toxoid and tetanus toxoid in combined vaccines

Abstract

Relevance. Diphtheria toxoid (DT) and tetanus toxoid (TT) manufacturing appears as many steps process. On every stage of proceeding vaccine the control of critical points is being provided. The Parke Williams 8 strain of Corynebacterium diphtheriae used in Russia for producing DT, Clostridium tetani strain Harvard – for TT. Each culture's supernatant proceed being estimated in relevance of toxoid potency via in vivo and/or in vitro methods. To produce DT, the activity of the toxoid must be not less than 50 Lf/ml and 40 Lf/ml for TT. Toxoids must fit in the main safety conditions – absence of toxin and reversion to toxicity impossibility. In accordance with WHO recommendations, five guinea pigs are injected subcutaneously with at least 500 Lf/ml of purified diphtheria toxoid, animals are observed for 42 days. By the end of observation period not the least than 80% of animals must remain alive without diphtheria intoxication (red adrenals). In Russia WHO approach was modified: guinea pigs are injected subcutaneously with purified diphtheria toxoid at a dose of at least 1500 Lf. During 42 days long observation period weight loss and animals dies must not appear. In case of death purified DT is not applicable. TT specific safety control is also carried out on guinea pigs. In accordance with WHO recommendations, five animals are injected subcutaneously with 500 Lf of purified TT. The animals are observed for 21 days daily, noting clinical signs of tetanus. If during the entire observation period no tetanus symptoms are observed in a single guinea pig and after the entire observation period 80% of the animals survives, tetanus toxoid is considered suitable for use. In Russia, the test for the absence of tetanus toxin is carried out similarly, excepting tetanus toxoid dose, which is 1500 Lf. The suitability criteria for purified TT are the absence of clinical signs of tetanus intoxication, weight loss and death of animals throughout entire observation period. Toxicity reversion tests are also provided. WHO considers guinea pig intradermal test to be suitable method for detecting diphtheria toxin, while guinea-pig methods are preferred for tetanus toxin, due to mice less sensitivity to tetanus toxin. In Russia, the most sensitive methods are used to detect the presence of toxins: intradermal administration to two guinea pigs in a volume of 0.1 ml or to one rabbit in a volume of 0.2 ml when testing diphtheria toxoid. Within four days, local reactions must not appear at the injection site. In the event of reactions, hence reversal of toxicity, the substance is rejected. When testing tetanus toxoid, five guinea pigs are injected subcutaneously into both sides of ten single human doses. Animals should be free of clinical signs of tetanus for 21 days after injection. Diphtheria and tetanus toxoids, after adsorption to a suitable adjuvant are monitored for specific safety. In accordance with WHO recommendations, at least 5 single human doses are administered subcutaneously to five guinea pigs. In case of testing adsorbed diphtheria toxoid, animals are observed for 42 days, tetanus toxoid – 21 days. The criteria for evaluating the suitability of adsorbed DT and TT are similar: during the entire observation period, animals should not show signs of diphtheria or tetanus intoxication; at the end of the observation period, at least 80% of the animals remain alive. In Russia, to test adsorbed DT and TT, five guinea pigs are injected subcutaneously with 10 single human doses. The duration of observation of animals in the DT test is 21 days, in the TT test – 30 days. The drug is considered to have passed the test if, during the entire observation period, the animals did not experience weight loss, signs of tetanus or, respectively, diphtheria intoxication, and all animals remained alive. In the event of the death of at least one animal in both cases from specific intoxication, the drug is considered not to have passed the test. Modern identifying potency (immunogenicity) of diphtheria toxoid and tetanus toxoid tests are based on determining immunized animals resistance for administration challenge toxin or evaluation of protective antibodies level in serum. In Russia to assess the potency of diphtheria toxoid (DT) and tetanus toxoid the challenge lethal method has been used for more than 60 years, challenge is based on determination of potency via its possibility to defend immunized animals from lethal doses of toxins. This method is used as «golden standard». The analysis of normative documents and guidelines of the World Health Organization, the European Union, the USA and Japan, concerning the issues of safety assessment and methods for determining the immunogenicity of diphtheria and tetanus toxoids at all stages of production, was carried out. It has been established that the approach adopted in the Russian Federation meets all international requirements. Moreover, with regard to methods for detecting reversion of toxicity, the most sensitive methods and more stringent criteria for the acceptance of experience are applied. The review presents data on methods for determining the immunogenicity of vaccines for the prevention of diphtheria and tetanus in the world. The advantages and disadvantages of some methods are reflected. The results of the analysis of these methods allow us to conclude that it is necessary to harmonize domestic and international methods for assessing the safety and immunogenicity of diphtheria and tetanus toxoids, which will make it possible not only to facilitate the registration of foreign vaccines in Russia, but also to speed up the registration of domestic vaccines in other countries.