Anatoxin

ANATOXIN, anatoxine, a name first proposed by the Frenchman Ramon in 1924 for diphtheria toxin, which Ramon succeeded in modifying with formaldehyde and heating so that, having lost its toxic function, it retained its antigenic properties, i.e., continued to cause in animals a state of active insusceptibility to diphtheria poison when introduced parenterally. Ramon's work on transforming diphtheria toxin into anatoxin, both in concept and methodology, is closely related to the research of his predecessors. Thus, it had long been established by systematic observations of Roux and Yersin that diphtheria toxin in liquid state loses its poisonous properties when heated to 58-60°C. Furthermore, Kitasato proved that tetanus toxin is almost completely destroyed when heated to 60-65°C for 20-25 minutes. These observations prompted attempts by various methods to weaken bacterial toxins used for laboratory immunization of large animals to obtain antitoxic serums from them. Special difficulties were presented by the immunization of horses with tetanus poison, therefore the attention of bacteriologists was first concentrated mainly on methods of weakening tetanus toxin. Here acids and alkalis, oxidases, potassium permanganate, trichlorine iodine, Lugol's solution, etc., were tested. Good results from such immunization were first obtained at the end of 1890 by Behring and Kitasato. Behring in 1892 recommended for immunizing horses a tetanus broth culture to which trichlorine iodine (ICl₃) was added in decreasing amounts (from 0.25%) to weaken its poisonous effect. Vaillard successfully immunized horses by first heating the tetanus toxin at 55-60°C, Roux and Martin - by adding Lugol's solution to the toxin in a ratio of 1:500, etc. In 1908, Loewenstein proposed for the initial immunization of horses against tetanus the preliminary treatment of tetanus toxin with formalin while heating, as a result of which the tetanus poison loses its toxic function but retains its antigenic function. Loewenstein named his preparation a 'toxoid', adhering to the terminology of P. Ehrlich, according to which a toxin that has lost its poisonous properties but retained its antigenic properties and ability to combine with antitoxic serum is called a 'toxoid'. The experiments of Eisler and Loewenstein, as well as Sordelli with immunization of horses with tetanus toxoid gave good results. However, Loewenstein's attempts to apply his technique to transform diphtheria toxin into the corresponding diphtheria toxoid, in which the combined action of heat, formalin, as well as a quartz lamp, X-rays and radium on the toxin was tested, did not give the desired results. This task was successfully solved in 1924 by Ramon at the Pasteur Institute in Paris. To obtain diphtheria A., Ramon uses a sufficiently strong diphtheria toxin prepared on Martin's broth (see Diphtheria). To such toxin Ramon adds 0.3-0.4% commercial formalin containing approximately 40% pure formaldehyde. The formalized toxin is kept in a hermetically sealed vessel in an incubator at 40-42°C until it completely loses its toxic properties, which usually occurs after approximately 4 weeks. The gradual loss of poisonous properties by such a toxin is tested on guinea pigs. A good A. should not cause either local or general reaction when injected under the skin to guinea pigs even in doses up to 5 cubic cm. In addition, it must preserve its antigenic function intact, i.e., in the same doses and in the same period of time as the original toxin from which it is prepared, give flocculation reactions with antitoxic antidiphtheric serum. Such an A., as shown by the observations of Ramon and others, possesses high immunizing properties for animals and humans. Thus, for example, according to the experiments of Barykin and his colleagues (Klyukhin, etc.), the subcutaneous administration twice to pigs weighing 250-300 g of A. in doses of 0.5 and 1.0 already after 3 weeks makes them insusceptible to 100 lethal doses of toxin. The double injection of A. to horses already after 12 days, according to Ramon's observations, gives their serum clear antitoxic properties that rapidly increase with further immunization (for example, up to 1,000 or more antitoxic units in 1 cubic cm of serum). One of the most valuable qualities of A., along with its complete harmlessness and excellent immunizing properties, is the stability of this preparation during storage. According to Barykin's data, A., kept in the dark and on ice for 1¹/2 years, invariably retains its activity and harmlessness. All this makes A. a very valuable means for vaccinating children against diphtheria. The initial experiments on immunizing children with Ramon's anatoxin were carried out in 1924 in France (Martin, Darre, etc.). The anatoxin was injected under the skin in doses of 0.5 cubic cm for the first and 1 cubic cm for the second injection, which was made 3 weeks after the first. Such immunization after 5-6 weeks gave in 90-95%, and after 2 months in 98-100% of cases the conversion of a positive Schick reaction to negative (see Diphtheria). In the USSR, the first experiment of mass immunization of children with diphtheria antitoxin was carried out in 1924-25 (Barykin with colleagues). The experiment was completely successful; at the same time, observations on the immunizing properties of A. in animals were reported (Maslakovets, Patevich and Arkhipov, Zdrodovsky and Brenn, Shutzer, Grigorovich), also with quite favorable results. From this time begins in the USSR the widespread use of A. as a reliable preparation for immunizing children against diphtheria intoxication. These experiments, as a rule, give good results. When widely using A., it must be borne in mind that injections of it should be given only to groups of children with a positive Schick reaction, indicating their susceptibility to diphtheria. The results of vaccination should be controlled by repeated Schick test. Small children aged 1 to 6 years tolerate injections of A. very easily, almost without local and general reactions. In older children (8-15 years), injections of A. in doses of 0.5-1 cubic cm sometimes cause slight swelling, redness and rapidly resorbing infiltrate at the injection site; in rare cases, general malaise and a slight increase in temperature to 38-39°C are observed. In general, the reaction to A. in even older children is noted no more frequently and no more strongly than to Behring's mixture, and cannot serve as a contraindication to the widespread use of A. Individual contraindications, as with any vaccination, are febrile processes, in particular tuberculosis (Dencks), and acute nephritis. Kraus, Glenny and others are engaged in improving the existing methods of preparing diphtheria anatoxin and accounting for its individual immunizing properties, i.e., those properties that are called avidity (see). The Microbiological Institute of the People's Commissariat of Health (Kulikov and Smirnov, 1927) developed a technique that allows concentrating the immunizing dose of purified diphtheria A. to 0.00005 g. In conclusion, it should be mentioned that Glenny disputes Ramon's priority in the discovery of the method of obtaining diphtheria A., that German, American and some Russian researchers call A. 'toxoid' of P. Ehrlich, and that it was possible to transfer not only tetanus and diphtheria toxins but also a number of other poisons into the state of A.: botulism and gas gangrene poison (Weinberg, Goy, Prevot), abrin and snake venom (Ramon), dysentery toxin (Lesbe, Verdeau, Glotova), etc.