The sensitizing effect of various typhoid vaccines as revealed under experimental conditions*

Bull. Org. mond. Sante' 1969, 40, 395-399 Bull. Wld Hlth Org. The Sensitizing Effect of Various Typhoid Vaccines as Revealed under Experimental Conditions* E. N. MELIKOVAI & S. V. LESNJAK 2 Typhoid vaccines, both heat-inactivated vaccines prepared in liquid and solid media and alcohol-inactivated dry vaccines, frequently possess sensitizing properties. Heat-inactivated vaccines prepared in liquid media have higher sensitizing activities than those prepared in agar and alcohol-inactivated vaccines grown in liquid media under aeration. By inducing a Shwartzman phenomenon in rabbits, it is possible to characterize the sensitizing activity ofeach vaccine lot, and ofits components, in comparison with a reference vaccine. The sensitizing activity of the supernatant fluid is as high as that of the whole vaccine but the washed microbic cells have a low activity. There is a marked correlation between the immunogenic activity, the toxicity and the sensitizing properties of the whole vaccine and its components-supernatant fluid and washed cells. Minimum requirements for permitted amounts of toxicity and sensitizing activity of typhoid vaccines can be formulated only after further information is obtained on the corre- lation between data from laboratory tests and assays of toxicity of vaccines in human sub- jects. Previous studies (Melikova, Stepanova & Gutman, 1953) have shown that typhoid micro-organisms possess sensitizing properties which can be de- monstrated in experimental animals by means of the Shwartzman phenomenon. Bernard et al. (1963) have confirmed that the Sanarelli-Shwartzman phenomenon may develop in people receiving typhoid-paratyphoid vaccines as- sociated with diphtheria and tetanus toxoids. It is known that the presence of a high con- centration of soluble antigens in the supernatant fluid of typhoid vaccines, particularly of those that are prepared by growing the microbes in reactors in liquid media under aeration, is responsible for the development of immediate, and more marked, general reactions in vaccinated persons. It was there- fore of interest to test the capacity of soluble antigens to provoke a Shwartzman phenomenon. This capacity may be used as a criterion to characterize individual types and lots of vaccines as well as different fractions of the preparation such as * From the L. A. Tarasevic State Control Institute for Medical Biological Preparations, Moscow, USSR. 1 Late Chief, Vaccine Department, L. A. Tarasevic State Control Institute for Medical Biological Preparations. ' Chief, Unit of Bacterial Vaccines, Tarasevic State Con- trol Institute for Medical Biological Preparations. carefully washed microbic cells and supernatant fluid. It was necessary to find out which of these 2 components of a vaccine possesses greater sensitiz- ing activity and whether there is any correlation (parallelism) between the capacity to produce allergic reactions, the toxicity and the immunogenicity of either component of the vaccine. METHODS The experiments were performed in chinchilla rabbits weighing 1.5 kg-2 kg (if albino rabbits are available, they should be used for preference) and the sensitizing (preparative) dose of the test material was injected into the skin of abdomen which previously had been thoroughly shaven. The challenge dose was injected 24 hours later into the marginal ear vein of the rabbit. Optimal doses for the immunizing and challenge injections had been established in preliminary tests; the sensitizing dose was 0.2 ml and the challenge dose was 3 ml. A 3-ml dose when injected intravenously did not cause the death of normal non-sensitized rabbits. Continu- ous observation of the animals was made during the first 6 hours and the final results were read 16-18 hours after the challenge injection. 2309 395- E. N. MELIKOVA & S. V. LESNJAK The Shwartzman phenomenon was considered positive if 2 or more hours (up to 24 hours) after the intravenous injection discrete and confluent haemor- rhages with dark necroses, having the appearance of brownish blue, slightly prominent spots, developed at the sites of sensitizing injections. Heat-inactivated vaccine belonging to lot 7, which uniformly induced a Shwartzman phenomenon; was used as a reference vaccine to determine the suscepti- bility of rabbits to sensitization. In order to characterize the test lots in a more reliable way they were compared with the Inter- national Reference Preparation of Typhoid Vaccine (Heat-phenol-inactivated) (vaccine L) which, as was shown by the field trials sponsored by WHO and field trial No. 5 carried out in the USSR (Cvjetanovic & Uemura, 1965; Hejfec, 1965; Spaun & Uemura, 1964), possessed low reactivity and was sufficiently immunogenic. Under our conditions of assay, low titres of this vaccine induced the development of a Shwartzman phenomenon in rabbits. Since we have no complete data concerning the toxicity in man for all the lots studied, it was convenient to use vaccine L as the reference preparation in order to study all lots by titrating their sensitizing capacity in parallel with this reference vaccine which induced the develop- ment of the Shwartzman phenomenon and at the same time did not possess high toxicity for humans. In such a way it was possible to determine indirectly the permissible toxicity of those lots of vaccine whose allergic capacity was equal to, or lower than, that of the reference preparation. The toxicity of the former should not exceed that of the latter. RESULTS The comparative studies of the sensitizing and challenging activity of washed microbic cells, super- natant fluids and whole vaccines performed with several production lots of heat-inactivated and alcohol-inactivated vaccines grown in broth under aeration showed that neither sensitization nor challenge with washed microbic cells induced the development of a Shwartzman phenomenon. This means that the washed sediments of vaccines pre- pared in reactors did not possess sensitizing capacity or adequate challenge properties. Thus, further studies were performed using only whole vaccine and its supematant fluid (SF). The data obtained in tests performed on 58 test rabbits and 29 control rabbits are summarized in Table 1. In this table it can be seen that the heat- inactivated vaccine prepared from micro-organisms grown in reactor-vessels in liquid culture medium under aeration possesses the greatest sensitizing activity. It should be noted that the activity of the supernatant fluid is equal to, and sometimes higher than, the activity of the whole vaccine. The alcohol- treated vaccine, though prepared from microbic cells grown under the same conditions (in a reactor- vessel), possesses the lowest sensitizing activity. It is likely that, as the result of the washing with alcohol, soluble antigens are partly removed, while in heat- inactivated vaccines further heating of microbic cells grown in a reactor-vessel greatly promotes the release of antigens in the liquid phase of the vaccine. Heat-inactivated vaccines grown in solid agar media occupy an intermediate position between the 2 types of vaccines mentioned above. Having determined the sensitizing action oftyphoid vaccines, we tested it on 4 lots of heated reactor- produced vaccine which had given untoward re- actions in vaccinated persons. All the 4 lots induced the development of highly marked Shwartzman phenomena when sensitizing and challenge injections, using whole vaccine and supernatant fluid, were given. Altogether, 3 lots out of the 4 caused the death of animals from 1 to 5 hours after the challenge injection. Control animals receiving intravenously the same dose (3 ml) of each of the 4 lots of vaccine survived. For a more complete characterization of sensitizing activity of the production lots they were tested in parallel with the International Reference Preparation of Typhoid Vaccine (Heat-phenol-inactivated) and the International Reference Preparation of Typhoid Vaccine (Acetone-inactivated) (known in the field trial as vaccine K). The results of these tests are presented in Table 2. As seen in Table 2, the sensitizing activity of the heat-inactivated vaccine grown in broth is con- siderably higher than that of heat-inactivated vac- cine L (reference vaccine). The sensitizing activity of the heat-inactivated vaccine grown in agar is practically equal to that of reference vaccine L. The sensitizing activity of only 6 lots out of 14 of alcohol- treated vaccine exceeds that of the reference pre- paration but is considerably lower than that of the heated vaccine prepared in broth. Six lots of alcohol-inactivated vaccine, tested in the same assays with heat-inactivated vaccine L and acetone-inactivated vaccine K, were 2 or more times weaker in their sensitizing capacity in comparison with the latter 2 vaccines. There was no significant 396 SENSITIZING EFFECT OF VARIOUS TYPHOID VACCINES SUMMARY OF DATA ON TABLE I THE SHWARTZMAN PHENOMENON IN VACCINATED RABBITS Number Number Number of surviving Number of dead of of animals animals Material Total rabbits rabbits Number for intra- number with with With With With With of lots Type of vaccine venous of positive negative positive negative positive negative tested in- of Shwartz- Shwartz- Shwartz- Shwartz- Total Shwartz- Shwartz-jection a rabbits man man Total man man T aa man man pheno- pheno- pheno- pheno- pheno- pheno- menon menon menon menon menon menon 11 Heat-inactivated Wv 11 6 5 5 2 3 6 4 2 typhoid-para- typhoid B, pre- Sf 11 10 1 9 9 0 2 1 1 pared in broth under aeration Wv 11 11 0 12 Heat-inactivated Wv 12 6 6 10 6 4 2 0 2 typhoid-para- typhoid B, pre- Sf 12 7 5 12 7 5 0 pared in agar (control) Wv 12 12 0 6 Dry, alcohol- inactivated typhoid- Wv 6 2 4 5 2 3 1 0 1 paratyphoid B, prepared in broth Sf 6 1 5 4 1 3 2 2 under aeration (control) Wv 6 6 0 Total (experimental rabbits) 58 32 26 45 27 18 13 5 8 Total (control rabbits) 29 29 0 a Wv = whole vaccine; Sf = supernatant fluid. TABLE 2 SENSITIZING ACTIVITY OF HEAT-INACTIVATED AND ALCOHOL-INACTIVATED TYPHOID VACCINES AS COMPARED WITH THE REFERENCE VACCINE Type of vaccine Heat-inactivated, prepared in broth with aeration Heat-inactivated, prepared in agar Alcohol-inactivated, prepared in broth with aeration Acetone-inactivated vaccine K a Number of lots tested 8 8 14 Number of lots with sensitizing activity that is: 2 times lower than that of reference vaccine 1 4 8 equal to that of reference vaccine 3 2 higher than that of reference vaccine by: 2 4 8 16 times times times times 2 4 2 (a The acetone-inactivated vaccine K was estimated in 3 assays in comparison with heat-vaccine L, the sensitizing activity of which was taken as unity. 397 1 E. N. MELIKOVA & S. V. LESNJAK TABLE 3 RELATIVE IMMUNOGENIC ACTIVITY OF THE COMPONENTS OF AND ALCOHOL-INACTIVATED VACCINES a HEAT-INACTIVATED Lot IWhole Super- TWashedType of vaccine No. vaccine natant microbic Heat-inactivated, prepared in reactor-vessel 71 2.4 2.0 <0.04 76 2.8 2.0 <0.04 87 1.7 1.0 <0.04 Heat-inactivated, prepared in agar at the State 1 0.8 0.6 0.1 Control Institute 3 1.6 1.2 0.1 6 1.4 0.5 0.16 Dry, alcohol-inactivated, production lots 70 3.3 1.1 0.22 99 2.5 1.5 0.21 a Challenge was made with 3 LD5o of Ty2 4446 was taken as unity. difference between heat-inactivated vaccine L and acetone-inactivated vaccine K in these tests. To make sure that the data obtained were consist- ent and were reproducible within the limits of deviation allowed for biological tests, we twice tested the sensitizing activity of several lots of heat-inactivated and alcohol-inactivated vaccines in parallel with the reference preparation. The results were closely similar. The active mouse-protection test and the haem- agglutination-inhibition test were used to establish the degree of correlation between the sensitizing activity, the immunogenicity and the tQxicity of the whole vaccine and its supernatant fluid, and washed microbic cells. The immunogenicity of the pre- parations under test was estimated on the basis of The EDso of heat-inactivated reference vaccine the ED50 value in comparison with the ED,o value of whole standard vaccine that was tested in parallel in each experiment. The ED50 value of the latter was taken as unity (1). These data are shown in Table 3; they prove that whole vaccine and the supernatant fluid possess the highest activity. Washed microbic cells, especially those of reactor-produced vaccines, are characterized by poor immunogenic activity. The results of further (repeated) tests confirmed the results of the original test. The toxic properties of vaccines were determined in haemagglutination-inhibition (HI) tests. Data obtained in repeated tests showed that the inhibition of haemagglutination is similar with whole vaccine and its supematant fluid. Sediments of TABLE 4 SUMMARIZED DATA FOR THE DETERMINATION IN Hi TESTS OF SOLUBLE ANTIGENS IN SUPERNATANT FLUIDS OF TYPHOID-PARATYPHOID B VACCINES PREPARED BY DIFFERENT METHODS Dilutions of supernatant fluid: Type of vaccine lo I_-_of ots diluted 1 2 1:4 1:8 1:16 1:32 1:64 1:128 1 :256 Heat-inactivated, prepared in reactor- vessel 15 4 5 5 1 Heat-inactivated, prepared in solid medium 11 4 5 2 Dry, alcohol-inactivated, prepared in reactor-vessel 20 13 6 1 398 SENSITIZING EFFECT OF VARIOUS TYPHOID VACCINES 399 microbic cells contain practically no soluble antigens. Taking this into account, further experiments were performed with supernatant fluid only. The data of tests on 35 production lots and 11 experimental lots of vaccine are given in Table 4. From Table 4 it can be seen that in supernatants of production lots of heat-inactivated vaccine pre- pared in reactors using broth, soluble antigens reacting with 0-serum are detectable in a much higher dilution as compared with supematants of alcohol-inactivated vaccine prepared in the same way and with supernatants of heat-inactivated vac- cine prepared in agar. It is of interest to note that high titres of soluble antigens were also revealed when supernatant fluids of the 4 above-mentioned lots of heat-inactivated vaccine, which had induced untoward reactions in vaccinated persons, were subjected to HI tests. One lot had a titre of 1: 128, another read 1: 64 and 2 lots had titres of 1: 32. We have no data concerning any untoward reactions with the vaccine lots having low titres of soluble antigens (not more than 1: 16) as determined in HI tests. However, there were vaccine lots with relatively high titres of soluble antigens which did not induce such reactions; this may be due to the fact that not all cases of severe reactions in vaccinated persons are reported. RtSUME ETUDE EXPERIMENTALE DU POUVOIR SENSIBILISATEUR DE DIVERS VACCINS ANTITYPHOYDIQUES On observe parfois un phdnomene de Shwartzman chez les sujets qui reroivent outre la vaccination anti- typho-paratyphoidique des injections d'anatoxines teta- nique ou diphterique. L'apparition de reactions generales immediates et plus ou moins intenses est attribuee a la forte teneur en antigenes solubles du surnageant des vaccins antityphoidiques. Pour mettre en evidence les facteurs sensibilisants presents dans les differentes fractions de ces vaccins, les auteurs provoquent chez le lapin un phenomene de Shwartzman par injection sous-cutanee d'une premiere dose vaccinale suivie 24 heures plus tard d'une injection d'epreuve par voie intraveineuse. Le pouvoir sensibilisa- teur de differentes preparations vaccinales peut de la sorte etre evalue en prenant comme element de compa- raison la preparation intemationale de reference de vaccin antityphoidique (vaccin L) qui, quoique peu toxique, suscite reguli&ement un phenomene de Shwartz- man chez le lapin. Les vaccins inactives par la chaleur ou par l'alcool sensibilisent tous deux le lapin a l'injection ulterieure de l'antigene homologue. Le vaccin inactive par la chaleur prepare sur milieu liquide a une activite maximale, de loin superieure a celle de la preparation de reference. Le vaccin inactive par l'alcool est le moins actif. Les vaccins complets et les surnageants font preuve du pouvoir allergisant le plus eleve; les cellules microbiennes lavees ont une activite sensibilisatrice faible. On note par ailleurs une correlation tres nette entre les proprietes allergisantes, la toxicite et le pouvoir immunogene des preparations etudiees. Les exigences minimales en matiere de toxicite et d'effet sensibilisateur des vaccins antityphoidiques ne pourront etre definies qu'apres une etude comparative des donnees du laboratoire et des resultats des essais de vaccination chez l'homme. REFERENCES Bernard, J.-G., Grossetete, G., Couture, H., Perrot, G.-J. & Tourette, C. (1963) Presse med., 71, 1467-1470 Cvjetanovic, B. & Uemura, K. (1965) Bull. Wid Hith Org., 32, 15-27 Hejfec, L. B. (1965) Bull. Wid Hlth Org., 32, 1-14 Melikova, E. N., Stepanova, S. L. & Gutman, N. R. (1953) Z. Microbiol. (Mosk.), No. 1, pp. 72-73 Spaun, J. & Uemura, K. (1964) Bull. Wld Hlth Org., 31, 761-791