Human live anthrax vaccine in the former USSR

Human live anthrax vaccine in the former USSR E.N. Shlyakhov* and E. Rubinstein The h&tory o f the development and use o f the Soviet live spore human anthrax vacc&e is described. Results o f mass field trials on this vaccine following administration by scarification, by subcutaneous injection route or by aerosol exposure are presented. For the immunological assessment o f these vaccinations a skin test with an original product 'Anthraxin' was used. Keywords: Anthrax;fieldtrials; human anthrax vaccine;efficacy;history

In their review on anthrax vaccines, Hambleton et al. ~ state that 'unfortunately, there appears to be little or no information on the relative efficacy in humans of the Russian live (anthrax) spore vaccine ...'. This paper is intended to remedy that situation. In the 1930s, Soviet specialists were propelled into developing an anthrax vaccine for human use as a result, first, of the persistently high incidence of human anthrax in the southern European and Middle Asian Soviet republics, and second, of the fear that in the impending war anthrax spores might be used as biological weapons. Before World War 2 the incidence of human anthrax in the endemic Soviet republics ranged from 40 per 100 000 population (Azerbaidjan) to 60 per 100000 (Moldavia (then Bessarabia)) 2'3. The task of development of a human anthrax vaccine was given in 1935 to military microbiologists working in the Red Army Research Institute of Epidemiology and Hygiene, also known as the SanitaryTechnical Institute (STI) at Kirov (now Viatka). A live anthrax vaccine was envisaged along the lines of the vaccines being developed by Stamatin and Stamatin4 and Sterne 5. In 1940 two avirulent non-capsulating Bacillus anthracis strains, STI-16 and strain no. 37, were derived from virulent parents by culture in coagulated horse serum s. A suspension of 3 x 107 STI-1 spores in 30% glycerolsaline produced local oedema following administration by the subcutaneous route in almost all of 115 guinea-pigs and protected 69 (60%) of them when challenged subcutaneously 3 weeks later with 200 LDso of a virulent B. anthracis strain. Of 73 rabbits immunized by the subcutaneous route with 25 x 107 spores of STI-1 and challenged also subcutaneously 6 weeks later with 250 LDso of a virulent B. anthracis strain, 51 rabbits (70%) survived. When tested on sheep, 77 (97 %) of 80 vaccinated sheep (25 x 106 spores) inoculated by the subcutaneous Infectious Diseases Unit, Sheba Medical Centre, Sackler School of Medicine, TeI-Aviv University, TeI-Hashomer, Israel 52621. *To whom correspondence should be addressed. (Received 12 May 1993; revised 15 September 1993; accepted 27 September 1993) 0264-410YJ94/08/0727-04 © 1994 Butterworth-Heinemann Ltd

route survived after challenge 6 weeks later with 200 LDso of a virulent B. anthracis strain as compared with 10 (26%) of 38 non-vaccinated controls. This vaccine was subsequently administered to over 2 million domestic animals 6,7,9. After World War 2 the number of domestic animals in the former USSR vaccinated with the live anthrax vaccine increased from 38.4 million head in 1947 to 140 million in 1960, while the anthrax morbidity in livestock decreased from 30 500 cases in 1947 to 3500 cases in 1960, or 8.8 times lower, thus demonstrating a notable reduction in incidence 1°. The prototype of a human live anthrax vaccine was subsequently developed by the same workers using a mixture of spores of the STI-1 and no. 3 strains suspended in 50% glycerol-saline6'7. Two methods of administration were recommended: skin scarification using two drops of the vaccine containing 1.3 x 10s spores, or subcutaneous injection of 5 x 107 spores. In May 1943 a preliminary trial on 12 volunteers showed the vaccine to be safe when administered by scarification10. Subsequently, from 1943 to 1950, 3500 volunteers were vaccinated by subcutaneous inoculation. Complete safety and a lack of local side-effects were reported 1°. The protective potency of the vaccine remained unknown, however, and in 1951-1952 a field trial was carried out in 14 anthraxendemic rural districts of the Moldavian Republic ~°. A total of 141 663 individuals aged 14-60 (the majority 20-50) years old were vaccinated, 92 150 by scarification and 49513 by subcutaneous inoculation; 416010 nonvaccinated residents in the same districts and from the same age groups represented controls. Close examination of 1010 persons immunized by scarification disclosed a temporary (1-2 days) rise in body temperature to 38°C in three cases (0.3%) without any other adverse effects. Among 5402 individuals vaccinated subcutaneously, a rise in body temperature and local erythema with a slight induration at the site of inoculation occurred in 14 cases (0.26%); these included four cases (0.074%) in whom a moderate transient swelling of the regional lymph nodes was noted. For the subsequent statistical analysis, Student's t test,

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Z 2 distribution and confidence limits for p = 9 5 % were used. Follow-up surveillance from July 1951 to December 1952 revealed three cases of cutaneous anthrax in the scarification group (3.2 per 100 000 vaccinees), no anthrax cases in the subcutaneously immunized group and 47 cases of cutaneous anthrax in the control group. This represented an overall case rate of 2.1 per 100 000 among vaccinated persons compared with 11.3 per 100000 among unvaccinated persons (p<0.001). The risk of contracting anthrax in this population was thus estimated to be reduced by a factor of 5.4 in the 18 months following vaccination (Z2 < 0.001). The human live anthrax vaccine was licensed by the Soviet Ministry of Health for administration by scarification in 1953 and by subcutaneous inoculation in 1959. Although officially for immunization of persons in at-risk occupations, administration to members of the general public was permitted in certain endemic areas. In the 1960s two million individuals were immunized annually2,9 11. The initial immunization consisted of a single scarification or subcutaneous injection; subsequent annual boosters using the same doses and routes were also recommended. Further studies on live human anthrax vaccine were conducted by a second team of military microbiologists headed by N.I. Aleksandrov 1°'12, who, in 1957, brought out an aerosolized dry spore vaccine composed of a mixture of strains STI-1 and no. 3. Thirty-two volunteers were exposed for 15 min to 17.4-53 × 106 viable spores dispersed in a room of 1 0 m 3. No adverse general reactions were recorded except in one individual who developed transient symptoms of tracheitis. Subsequently, the effects of exposure levels ranging from t5-63 x 106 to 440 640× l06 viable spores were studied by daily follow-up examinations on 263 adult volunteers for 7 days, and on 100 of them for 21 days. None of the volunteers experienced fever or other ill effects and all were able to continue their normal daily occupations I2 Also in 1957, a skin test for monitoring immunity to anthrax following infection or vaccination using an original product 'Anthraxin' was introduced i o, ~3-15. The test, which was based on the cell-mediated response as confirmed histologically ~6, reliably identified vaccineinduced immunity in guinea-pigs, sheep and humans and patients with histories of anthrax 1°. In an experiment involving 29 guinea-pigs immunized with a veterinary ST1 vaccine and tested by the Anthraxin skin test 15 33 days after vaccination, 27 (93%) animals developed positive response to the Anthraxin skin test (confidence limits 77 99%). All animals survived when challenged with 200 LDso of a virulent strain of B. anthracis, while all of 18 non-vaccinated guinea-pigs succumbed to this challenge (/2 <0.001)1o. To the present day the Anthraxin test remains widely used for assessing postvaccinal immunity in humans and livestock vaccinated against anthrax with live or chemical vaccines in the former Soviet Union 1v 26. The test formed the basis of a field trial in 1959-1960 in civilians of a rural district of Moldavia and organized by the Soviet Ministry of Health to assess and compare the aerosol vaccine and the routine STI vaccine as administered by scarification or subcutaneously ~°'lz. The aerosol vaccination was carried out in a room 3.7 m x 3.6 m × 2.9 m (40 m3). The aerosol cloud was dispersed from a height of 2.4m from a suspension containing 20-2500 × l09 spores per gram dry weight. Inhalation

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time varied from 5 to 15 min. There were 143 individuals vaccinated by aerosol vaccine with a dose of 43 63 × 10" spores during three sessions (groups of 40, 51 and 52 persons), and 109 individuals with a dose of 440 640 million spores also during three sessions, in groups of 24, 43 and 42 persons. A second group of individuals was vaccinated by scarification and a third group subcutaneously with the human STI vaccine. As an additional control a group of patients with cutaneous anthrax was also included. All the immunized individuals, the persons who experienced anthrax itself and the non-immunized controls, were tested by the Anthraxin skin test at various time intervals after vaccination or after the onset of the disease. The results are summarized in Table / 10'12. Despite the satisfactory results obtained by using relatively high doses (660 x 106 spores/individual) of the aerosol vaccine, this method, described as a 'brilliant achievement in bacteriology '27, was not recommended for general purpose vaccination but was reserved as an emergency method to be employed in 'critical situations'. Although the results of the Anthraxian skin test indicated that the scarification method induced relatively lower immunogenicity than the subcutaneous and aerosol methods, the Ministry of Health continued to recommend the use of scarification with the condition that the individual dose be increased to 1.6 3 x 108 spores 10. In 1973--1975, renewed studies on the immunological efficacy of the human STI vaccine were undertaken in which subcutaneous injection was replaced by 'ped-o-jet'

Table 1 Summarized data of Anthraxin skin test results in humans vaccinated with a live anthrax vaccine and in patients with cutaneous anthrax Days after vaccination or after the onsetofthe disease

Groups

Vaccinated a 15 Aerosol-1 Aerosol-2 Subcutaneous Scarification

No. of persons tested

No. positive

Rates {%)

Confidence limits ( p - 95%)

17 9 49 48

12 7 23 22

70.8 78.0 47.0 46.0

44.0-89.7 40.0-97.2 28.6-85.9 31.4-61.4

90

Aerosol-1 Aerosol-2 Subcutaneous Scarification

25 26 50 22

18 18 27 9

72.0 69.1 54.0 40.9

50.6-87.9 42.2-89.1 35.0-72.0 20.7-83.6

180

Aerosol-1 Aerosol-2 Subcutaneous Scarification

26 10 42 28

13 4 17 6

50.0 40.0 39.6 21.4

29.9-70.1 8.0-81.0 21.8-61.4 8.3-41.0

365

Aerosol-1 Aerosol-2 Subcutaneous Scarification

24 38 46 49

9 13 16 11

37.4 34.2 34.8 22.4

18.8-59.4 19.6-51.4 18.0-54.8 11.7-36.7

27 29 50 39

93.1 93.5 92.6 90.7

77.2-99.1 78.6-99.2 82.1-97.9 77.8-97.4

2

2.8

0.3-9.7

Patients with cutaneous anthrax 15 29 90 31 180 54 365 43 Non-vaccinated/ healthy persons

72

a Doses: Aerosol-I, ~ x 106 spores; Aerosol-2, 440---640 x 106 spores; subcutaneous, 50 x 106 spores; scarification, 1.3 x 108 spores/dose from a suspension of 2 × 109 spores/ml

Human live anthrax vaccine: E.N. Shlyakhov and E. Rubinstein

administration 2a. In a field trial in Middle Asia, 107 285 persons were vaccinated with the STI vaccine, 52763 individuals by the ped-o-jet method, and 54 522 persons by scarification. The control group included 49974 unvaccinated individuals. In a 2-year surveillance period, three cases of cutaneous anthrax were recorded among those vaccinated by ped-o-jet (5.7 per 100 000 vaccinated persons), five cases were recorded among those vaccinated by scarification (9.2 per 100000) and 18 cases were recorded in the control group (36 per 100000 persons). The combined preventive efficacy of the vaccine was thus shown to be highly significant (p < 0.001), and the risk of acquiring anthrax to be 4.9 times lower among vaccinated as compared with unvaccinated individuals (Z2 < 0.001). These results were similar to those obtained in the first trial in Moldavia (see above), but the ped-o-jet method was apparently less effective than subcutaneous administration. In a subsequent attempt to evaluate the efficacy of the STI vaccine administered by scarification, Marchuk and Cherkaski 29 analysed data collected throughout the former USSR and found annual postvaccinal anthrax morbidity to be as high as 2.3-2.5 per 100 000 vaccinated persons; 74% of the cutaneous anthrax cases observed occurred during the first 5 months after vaccination. In addition, an analysis 3° of 1445 persons vaccinated by scarification in 1975-1980 showed that there were 151 (10.4%) cases of cutaneous anthrax. Of these, 86 cases were recorded in 310 vaccinated individuals belonging to a risk group (27.7%), and 65 cases occurred in 1135 vaccinated persons (5.7%) not belonging to a risk group. These 1445 vaccinated persons lived in different regions of the former USSR, mostly in the Middle Asian republics. A similar analysis of data collected in Moldavia (1964-1973) showed that the morbidity of cutaneous anthrax was 30.6 per 100000 in unvaccinated persons while in those vaccinated by scarification it was 2.5 per 100 0003. Similar rates were also obtained in the Kirghizia Republic in 1969 (1.8 cases of cutaneous anthrax per 100 000 vaccinated by scarification as compared with 30.7 per 100000 unvaccinated individuals) 1°. In more than 30 years' experience no instances of adverse affects following use of the STI human vaccine were recorded :°. CONCLUDING

REMARKS

In the light of these data and of our own experience, the safety and the preventive efficacy of the human STI anthrax vaccine have been well established. We believe that the non-acceptance of live anthrax vaccine for human use on the basis of lack of safety 31 should be reconsidered. Evidence of the preventive efficacy of the human chemical vaccine is poorly documented and is largely confined to the paper of Brachman et a1.32. Results of tests in animals have shown the superior efficacy of the live vaccine derived from the Sterne strain (34F2) as compared to chemical vaccines 31'33-36 and that the latter do not provide an adequate protective response against some isolates of B. anthracis 37. Since it was demonstrated experimentally 33 that increased levels of immunity were achieved following a combination of protective antigen with the live vaccine, it seems logical to conduct further studies to assess the efficacy of such a combination in humans. The best

approach might be preliminary immunization with a chemical vaccine followed by boosters with the live vaccine. Such a tactic might also avoid the undesirable side-effects reported in association with repeated boosters using the chemical vaccines 32. Insofar as Anthraxin reveals the cell-mediated immunity in individuals vaccinated both with a live or a chemical anthrax vaccine, it seems to be useful to assess the postvaccinal reactions using EITB/ELISA tests along with the Anthraxin skin test in order to obtain finally a more comprehensive picture of the two sides of anthrax immunity: humoral and cell-mediated. REFERENCES 1 Hambleton, P., Carmon, J.A. and Melling, J. Anthrax: the disease in relation to vaccines. Vaccine 1984, 2, 125-132 2 Shlyakhov, E.N., Gruz, E.V. and Prisakari, V.I. Anthrax Shtiintza, Kishinev, 1975, p. 164 (in Russian) 3 Shlyakhov, E.N. and Prisakari, V.I. Epidemiological Surveillance of Anthrax. Shtiintza, Kishinev, 1989, p. 240 (in Russian) 4 Stamatin, N. and Stamatin, L. Le pouvoir immunisant de souches acapsulogenes du B. anthracis. C.R. Soc. Biol. 1936, 122, 491-493 5 Sterne, M. The use of anthrax vaccines prepared from avirulent (unencapsulated) variants of Bacillus anthracis. Onderstepoort J. Vet. Sci. Anim. Ind. 1939, 13, 307-312 6 Schaefer, W. Dissociation de la bacteridie charbonneuse. C.R. Soc. Biol. 1936, 122, 1178-1181 7 Ginsburg, N.N. Anthrax vaccine STI. Collected Works of the Red Army Institute of Epidemiology and Hygiene 1946, 1, 5-90 (in Russian) 8 Tamarin, A.L. and Spitzyn, N.A. Development of the anthrax vaccine STI-I. Collected Works of the Red Army Institute of Epidemiology and Hygiene 1946, 1, 153-170 (in Russian) 9 Shlyakhov, E.N. Products for active immunization against anthrax. In: Problems of Experimental and Applied Immunology, Kartia Moldoveniaska, Kishinev, 1967, pp. 171-197 (in Russian) 10 Shlyakhov, E.N. Allergy in Anthrax, Kartia Moldoveniaska, Kishinev, 1968, p. 188 (in Russian) 11 Knudson, G.B. Treatment of anthrax in man: history and current concepts. Mil. Med. 1986, l s l , 71-77 12 Aleksandrov, N.I., Gefen, N.E., Garin, N.S., Gapochko, K.G., Sergeev, V.M, Smirnov, M.S. et al. Experience in massive aerogenic vaccination of humans against anthrax. Voen. Mad. Zh. 1959, 8, 27-32 13 Shlyakhov, E.N. Development and experimental study of a product to reveal an allergic reaction in anthrax. In: Interinstitute Scientific Conference on Strains, Standards and Diagnostic Products. Abstracts. Leningrad, 1957, pp. 53-55 (in Russian) 14 Shlyakhov, E.N. Milzbrand: Biologische und immunologische Grundlagen der Diagnostik und Prophylaxe. 1. Gewinnung und experimentelle Untersuchung des Milzbrand-Allergen. J. Hyg. Epidemiol. (Praha) 1958, 11,266-273 15 Shlyakhov, E.N. Epidemiology, Diagnostics and Prevention of Anthrax. Kartia Moldoveniaska, Kishinev, 1960, p. 115 (in Russian) 16 Shlyakhov, E.N. and Shroit, I.G. Anthrax. Biological and immunological principles of diagnosis and prevention. 2. Pathomorphological changes in skin in the anthraxin allergy test. J. Epidemiol. (Praha) 1964, 8, 307-312 17 Aleksandrov, N.I., Gefen, N.E., Gapochko, K.G., Garin, N.S., Sergeyev, V.M., Lasareva, E.S. et al. Aerosol immunization with dry live vaccines and toxoids. VI. A study of postvaccination reactions and immunological efficacy of aerosol immunization with brucellosis, tularemia, anthrax and plague vaccines. Zh. Mikrobiol. Immunol. (English edn) 1951, 32, 1245-1252 18 Knop, A.G. Reactogenicity, immunogenicity and epidemiological efficacy of the STI vaccine by needle-free method of immunization. PhD Dissertation, Moscow, 1975 19 Boyakhchian, A.B. and Ambardanian, L.A. A study of comparative reactogenicity and immunological efficacy of anthrax STI vaccine in sheep immunized by aerosol or subcutaneously. In: Current Problems of Anthrax Prevention in the USSR, Moscow, 1971, pp. 101-103 20 Adylov, D.A. Anthrax in Uzbekistan, Meditsina, Tashkent, 1977, p. 183 21 Derbin, M.I., Sadovoy, N.V., Tarumov, V.S. and Garin, N.S. Comparative study of the humoral anthrax immunity in those

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