Corrêa, Alcino

Lopes, Oscar Souza

Veronesi, Ricardo

Zuccas, Waldemar A.

English

Cadernos Espinosanos (E-Journal)

Rev. Inst. Med. trop. São Paulo 8(2):83-88, março-abril, 1966 UDC 615.371 616.981.551 TET ANUS IMMUNIZATION WITH A SINGLE DOSE OF V ACCINE. IMPORT ANCE ANO POSSIBILITIES FOR MASS-IMMUNIZATION PRO-GRAMS. PRELIMINARY REPORT ON EXPERIMENTAL STUDIES Ricardo VERONESI (1), Alcino CoRRÊA (2), Waldemar A. ZuccAs (3) and Oscar Souza LoPES ( 4 ) SUMMARY Humans and animals experiments had already demonstrated the possibilities of protection against Tetanus with a single dose of toxoid. The Authors studied different preparations of vaccines and concluded that the more potent one was the mixture of a potent natural anatoxin plus the adjuvant 65. This vaccine was tested in animals and humans and showed to be more potent than the classical toxoids. The method of injection under pression (jet-injection) was considered important to accomplish the immunization because it allows wider contact of the antigen with elements able to produce antitoxins besides others advantages such as rapidity, economy of people and materials of injection. The single-dose-scheme of protection against Tetanus was advised to countries that cannot afford enough money to fight the disease and intend to decrease the high index of mortality among their inhabitants. INTRODUCTION The classical schemes for Tetanus immu-nization includes 3 or, at least, 2 dosis of toxoid and the accomplishment of such schemes does not carry too much problems when we are dealing with well developed countries. But, when we are drawing a mass-immunization program to cover millions of individuals living in vast areas of under-developed countries, things change tremen-dously and all kinds of difficulties appear to obstruct the accomplishment of such a plan. Besides the lack of a preventive medicine mentality among these populations, the go-vernments, for a lot of reasons, do not include in their budgets enough amounts of money to fight preventable diseases and mil-lions of people pay, every year, with their own lives, the toll for such a situation. Considering all these difficulties and as an effort to contribute to make simpler the schemes of immunization against Tetanus, we carried out some experiments to prove that it is possible to afford full protection against the disease with a single dose of an adequate vaccine. Many experiments had already shown that a single dose of toxoid confer to animals a full protection against Tetanus and that they may survive the challenging with many lethal dosis of toxin 1 • 2 • SMITH 7 demonstrated that a single dose of toxoid is enough to protect mice or guinea-pigs when challenged with tetanic spores, 10 or 12 days later. He also showed that a single dose of toxoid is enough to determine, m humans, the appea-(1) Professor of Infectious and Parasitic Diseases (Faculdade de Ciências Médicas da Santa Casa de Misericórdia de São Paulo), São Paulo, Brasil (2) Chief of the Section of Biological Controls (Instituto Pinheiros), São Paulo, Brasil (3) Pediatrician (Créche Catharina Labouré), São Paulo, Brasil (4) Chief of Laboratory of ArbOYÍr\.l~ (Instituto Adolfo Lutz), São Paulo, Brasil 83 VERONESI, R.; CORRÊA, A.; ZUCCAS, W. A. & LOPES, O. S. - Tetanus immunization with a single dose of vaccine. Importance and possibilities for mass-immunization programs. Prelimi-nary report on experimental studies. Rev. Inst. Med. trop. São Paulo 8:83-88, 1966. rence of the mm1mum protective level of antitoxins at the end of the fourth week. The experiments of NEWELL et al.5 on the prevention of Tetanus neonatorum sho-wed that a single dose of aluminum phos-phate adsorbed tetanus toxoid was able to determine an immediate and marked diffe-rence in the incidence of Tetanus neonato-rum among the babies born from mothers vaccinated during the pregnancy. Based on the experiments of NEWELL et al." we are authorized to admit that a much better pro-tection should be afforded by actively im-munized individual, since the humoral im-munity gained by the babies through the placenta is less effective than the cellular im-munity confered by the toxoid to the rnother. W e believe that, if a single dose of toxoid is able to rnarkedly diminish the incidence of Tetanus neonatorum, we have strong rea-sons to admit that the sarne single dose of vaccme should be more efficient in dirni-nishing the incidence of others forrns of Tetanus which, very probably, are less seve-re than neonatorum Tetanus, in the vast rna-jority of instances. Another important fact to discuss, when we are going to admit the possibility of immunization with a single dose of toxoid is the necessity or not of the presence of the minimum title of antitoxin in the blood to testify the grade of protection attained by the animal actively immunized with one dose of toxoid. HEINIG 3 demonstrated that a single dose of toxoid was enough to full protect 15 horses and 38 sheeps when chal-lenged with up to 120 M.L.D. of tetanus toxin, despite the absence of measurable antitoxin in the blood of the animais. The experiments of WüLTERS and DEHMEL ( cit. 2) demonstrated that the title of 0.01 unit, considered as the minimum protective levél of antitoxin in the blood, is much above the "real" protective level, which can be determined only by challenging the ani-mal with toxin. Titers as low as 0.005 units were able to full protect WüLTERS and DEHMEL when they were inoculated with many lethal dosis of toxin. This two re-searchers were protected by a high degree of cellular immunity, acquired years before through the injection of Tetanus toxoid. 84 The grade of protection afforded by a single dose of toxoid is much higher than indi-cated by the level of antitoxins in the blood Under the influence of such a knowledge we tried to obtain a potent antitetanic vacci-ne which, in a single dose, could determine full protection against the disease, in a short period of time and the level of antitoxin produced could be high enough to rnerit the confidence of those who evaluate the potence of the vaccines only by the classical standards that includes, necessarily, the determination of the amounts of antitoxin elaborated by the body under toxoid stimulus. MATERIAL AND METHODS With preliminary experirnents we tested different adjuvants which, joined to the anatoxin, could determine the higher and faster production of antitoxin in guinea-pigs. Seventeen groups ( each one with 10 ani-rnals) of guinea-pigs weighing ± 350 g were submited to the experiment. The follow-ing preparations were inoculated in a single dose of 0.5 or 1 ml: natural anatoxin; na-tural anatoxin plus peanut oil ( 1: 1) ; na-tural lyophylized anatoxin plus peanut oil ( 1 :1), natural anatoxin plus adjuvant 65 ( 1: 1) ; aluminum precipitated anatoxin plus peanut oil ( 1: 1) ; aluminum precipitated anatoxin plus adjuvant 65 (1 :1); lyophy-lized natural anatoxin; aluminum precipi-tated anatoxin; natural anatoxin plus peanut oil and 0.1 ml of Tween 1% (1:1). The adjuvant 65 was prepared according to our citations 6 and 11. Titrations of antitoxins were carried out at 30, 60 and 120 days from the injection of the different preparations and the results were cornpared. The potence of our natural anatoxin was tested in guinea-pigs and showed the follow-ing results: 1 ml of the natural anatoxin was able to protect 100% of the animais ( 10 animais) when challenged against 10 M.L.D. of toxin at the end of the sixth week. Another test of protection showed that 100% of the animais were protect against 2 M.L.D. of toxin at the end of the 15th day; with the sarne dose of natural anatoxin 90% of the animais were protected VERONESI, R.; CORRÊA, A.; ZUCCAS, W. A. & LOPES, O. S. - Tetanus immunization with a single dose o! vaccine. Importance and possibilities for mass-immunization programs. Prelimi-nary report on experimental studies. Rev. Inst. Med. trop. São Paulo 8:83-88, 1966. against 5 and 10 M.L.D. at the end of the 15th day. However, when challenged with 5 and 10 M.L.D. of toxin 50% of the animais showed symptoms of Tetanus ( 1 to 2 + ) which disappeared at the end of 1 week and no deaths occurred among the 30 animais submited to this triai of earlier pro-tection. The title of antitoxin at the 15th day was of 0.01 unit per mi of serum. The four more potent preparations were separated according to the antigenic power demonstrated through the titrations of anti-toxins and the results are shown in the Fig. 1. e ' ., e ::, z X o 1-i== z <( 10 9 8 7 6 5 4 3 2 30 60 90 120 DA~ AFTER VACCINE Fig. 1 - The antigenic power of the four more potent vaccines are represented here. The prepa-rations were inoculated in guinea-pigs, in a sin-gle dose. The four groups were chosen among those showing the most satisfactory results in our experiments which included 17 groups of +++++ animais. Alum. ppt. anat. + peanut oi!. Dose: 2 ml Alum. ppt. anat. Dose: 1 ml Nat. anat. + adjuv. 65. Dose: 2 ml Alum. ppt. anat. + adjuv. 65. Dose: 2 ml Proportion of components: 1:1 RESULTS W e can see in the Fig. 1 that the more potent preparation was the mixture of a potent natural anatoxin plus the adju-vant 65. This preparation should be our vaccine of choice for further experiments in humans. Our preparation of choice showed to be, in a single dose, 10 to 15 times more potent than the single dose of precipitated anatoxin used by D'ANTONA 1 in his expe-riments in animais and more efficient than the toxoid used by SMITH 7, also in a sm-gle dose, in humans. Our experiment in animais showed that a single dose of an adequate vaccine is able to sensitize the body to elaborate antitoxins and, at the sarne time, to work as a booster for a period of 2 or 3 months. This booster effect is clearly demonstrated by following the curve of antitoxins at 1 to 4 months from the injection of the vaccine. Further experiments were carried out in humans in order to confirm the results we obtained in animais. Some alterations were clone in these humans experiments: a) The proportion of the components changed to 2 parts of natural anatoxin to 1 part of adjuvant 65. This had to be clone because the proportion of 1 :1 rendered the preparation too thick and very difficult to pass through the orifices of the jet-gun. b) Instead of injection with the classi-cal needle and syringe we used the jet-in-jector appareil because we were interested to know how this appareil works when using thick toxoids and to study the reactions of the body to the jet inoculations. Despite rendered the preparations less thick we still have doubts on the exact proportion of the components in each injection. The first human experiment we carried out in ten volonteers of the military police of the State of São Paulo. Seven of the men were injected with 0.5 mi of the vacci-ne using the jet-injector appareil. Three others men were injected with 1 ml of the sarne vaccine but using the classical needle and syringe. The route of inoculation was intramuscular. The results of the experiment are shown in Table I. As we see in Table I, 100% of the indi-viduais of the jet-injector group showed se-rological conversions at the end · of 30 or 60 days. Surprisingly, only one individual, among the three that received 1 ml of the vaccine through the needle, showed conver-85 VERONESI, R.; CORRÊA, A.; ZUCCAS, W. A. & LOPES, O. S. - Tetanus immunization with a single dose of vaccine. Importance and possibilities for mass-immunization programs. Prelimi-nar:y report on experimental studies. Rev. Inst. Med. trop. Siío Paulo 8:83-88, 1966. TABLE I Ident. 1 2 3 4 5 6 7 8 9 10 Units/ml before -C.01 -C.01 -0.01 -0.01 -0.01 -0.01 -0.01 -0.01 -0.01 -0.01 Units/ml 30 days +0.01 -0.05 -C.01 ---C.01 -C.01 -· -0.01 -C.01 -C.01 Units/ml 60 days 0.05 +ü.05 -0.1 +0.01 0.05 0.01 +ü.01 -0.1 0.05 -0.01 0.01 -0.01 Note - Individuais numbers 1 to 7 were inoculated with the jet-injector and numbers 8 to 10 with the classical needle. sion at the end of 60 days. One expla-nation for this phenomenon would be that the jet-system allows the formation of mil-lions of particles of the vaccine and the anti-genic stimulus under this condition is greater. W e already know that small amounts of smallpox vaccine diluted to 1: 100 or 1 :50 are enough to determine serological conver-sion to 90 to 100% of the vacinées, when we use the jet-injector appareil 4 • 10 • Like we observed in animals, the vaccine showed to be still active after 1 month from the m1ection. No untoward effects were obser-ved among the vaccinated individuals. · Despite convincing the results of this first human trial, we thought that better results could be obtained with 1 ml of the vaccine instead of 0.5 ml. The amount of 1 ml of vaccine contains 0.66 ml of natural anatoxin and, obviously, should determine a greater production of antitoxin and a better grade of protection. So, ten children at the age of 2 to 5 years were selected in one nur-sery among those not previously vaccinated against Tetanus and 1 ml of the vaccine was injected with the jet-injector appareil. Samples of blood were taken before and at the end of the 30th day and 60th day after the vaccination with a single dose. No untowards effects were observed among the chidren and the results are represented in Table II. TABLE II Ident. Units/ml before 1 0.01 2 +0,01 -0,1 3 -0.01 4 -0.01 5 +0.1 6 -0.01 7 -0.01 8 -0.01 9 -0.01 10 0.01 Results· of antitoxin titrations in the blood of children vaccinated with 1 ml of the vac-cine showed that this should be the dose of choice, since 100% of serological con-86 Units/ml Units/ml 30 days 60 days 0.5 0.7 0.1 0.05 +0.01 -0.1 +0.01 -0.05 +0.05 +0.5 -0.7 +5 +12 +0.01 -0.1 0.05 0.1 0.1 +0.1 -1 0.15 +1 +1 -1.5 +5 +12 versions were obtained at the 30th day after the vaccination as we can see at the Table II. The levei of antitoxins are, also, higher with this dose ( compare with Table I). VERONESI, R.; CORR:E:A; A.; ZUCCAS, W. A. & LOPES, O. S. - Tetanus immunization with a single dose of vaccine. Importance and possibilitles for mass-immunization programs. Prelimi-nary report on experimental studies. Rev. Inst. Med. trop. São Paulo 8:83-88, 1966. DISCUSSION The results obtained with this vaccine allow us to state that a single dose of an adequate vaccine is able to confer a high grade of protection against Tetanus and millions of individuals should afford the be-nefit of it in countries where it is very difficulty to inject, in such a large population, 2 or 3 dosis of toxoid, in a short period of time. W e believe that the single-dose-scheme accomplished with the jet-injectors of mul-tiple dosis (2,500 injections per hour) is the more rapid method of protection that could be carried out to decrease the tre-menduous index of mortality by Tetanus in many countries all o ver the world. W e agree, on the other hand, that the single-dose-scheme is important only for countries where there is no conditions to plan a mass immunization program which includes two or three dosis of toxoid. W e are convin-ced also, that there is much more scientific reasons to believe on the efficacy of a sin-gle dose of vaccine in the protection against Tetanus than on the world-wide belief in the protection afforded by the use of antite-tanic serum with the classical dosis o.f 1.500 or even 3.000 I.U. 9 • With the expenses required for the pro-duction and use of A.T.S. we can easily offer, to millions of people, a single dose of vaccine which is much safer, not hazar-dous and long-lasting. The animals experiments showed also that a single dose of an adequate toxoid, even when affording only a partial immunity, this is enough to, significantly, diminish the lethality ( deatsh are very unusual when the animals are challenged against many lethal dosis of tetanus toxin). All in all, we strongly believe that a "bath" of mass-immunization with a single dose of an adequate Tetanus vaccine would be the only plan which could be offered to many countries to fight the disease and ra-pidly recover the time they lost in pre-venting the deaths by Tetanus which should be prevented since 1924, when RAMON dis-covered the anatoxin. People immunized with a single-dose must be advised to use a booster dose of toxoid whenever necessary and possible. With this precautions we be-lieve that a full protection is afforded and, surely, the index of mortality and lethality should decrease significantly. RESUMO 1 munização contra o tétano com dose única de vacina. 1 mportância e possibilidade em programas de imunização em massa. Nota preliminar sôbre estudos experimentais. Experimentações humanas e em animais já haviam demonstrado as possibilidades de proteção contra o tétano com dose única de toxóide. Os Autores estudaram diferentes preparações de vacina e concluiram que a. preparação mais potente era constituída de uma mistura de potente anatoxina mais o adjuvante 65. Esta vacina quando testada em animais e sêres humanos demonstrou ser mais potente que os clássicos toxóides. O método de injeção sob pressão ( a j acto) foi considerado importante nesse esquema de imunização porque êle permite contato mais amplo entre o antígeno e os elementos pro-dutores de antitoxinas, além de outras van-tagens, tais como rapidez, economia de pes-soal e de material de injeção. O esquema de dose única foi aconselhado para países que não possuem verbas sufi-cientes para combater a doença e pretendem diminuir os altos índices de mortalidade en-tre os seus habitantes. ACKNOLEDGMENTS The Authors are very greatfull to the direction of the "Instituto Pinheiros de São Paulo" by offering the facilities to carry out this work. REFERENCES 1. D' ANTONA, D. - La vacination centre le tetanos. II partie: L'anatoxine tetanique et son action préventive centre le tetanos. Rev. Immun. 16:33085, 1952. 2. ECKMANN, L. - Tetanus. Prophylaxis and Therapy. New York, Grune & Stratton, 1963. 3. HEINIG, A. - Experimentelle untersuchun-gen über den entritt der immunitat nach einmaliger Tetanus-Schutzimpflung. Arch. Exp. Vet. Med. 8:394-403, 1954. 87 VERONESI, R.; CORRll:A, A.; ZUCCAS, W. A. & LOPES, O. S. - Tetanus immunization with a single dose of vaccine. Importance and possibilities for mass-immunization programs. Prelimi-nary report on experimental studies. Rev. Inst. Med. trop. São Paulo 8:83-88, 1966. 4. MILLAR, J. D. & ROBERTO, R. R. - Va-cunacion intradermica contra la viruela por inyecion a pression. Bol. 0/ic. Sanit. Pa-namer. 57: 537-554, 1964. 5. NEWELL, K. W.; DUENAS LEHMAN, A.; LE BLANC, D. R. & GARCEZ OSORIO, N. - The use of toxoid for the prevention of Tetanus Neonatorum: preliminary report of double-blind centrolled field trial. Bull. W.H.0. 30:439-444, 1964. 6. PECK, H. M.; WOODHOUR, A. F.; METZGAR, D. P.; McKINNEY, S. E. & HILLEMAN, M. R. - New metabolizable immunologic adju-vant for human use. II - Short term animal toxicity tests. Proc. Soe. Exp. Biol. & Med. 116:523-530, 1964. 7. SMITH, J. W. G. - Tetanus prophylaxis. Brit. Med. J. 1:373, 1964. 8. VERONESI, R. - Contribuiçiío para o es-tudo clínico e experimental do tétano. Tese de docência. São Paulo, 1960. 88 9. VERONESI, R. & GUIDOLIN, R. - Pre-venção do tétano: papel da terramicina de ação prolongada (micronizada) e da terra-micina de ação rápida. Resultados no téta-no experimental. Rev. Hosp. Clín. Fac. Med. Univ. São Paulo 19:311-320, 1964. 10. VERONESI, R.; SALLES GOMES, L. F.; SOARES, M. A. & CORREIA, A. - Impor-tância do "jet-injector" (injeção sem agu-lha) em programas de imunização em mas-sa no Brasil. Resultados experimentais na vacinação anti-variólica e antitetânica. Tra-balho apresentado ao IV Congresso da Asso-ciação Médica Brasileira em Pôrto Alegre, 24-29 de outubro de 1965. 11. WOODHOUR, A. F.; METZGAR, D. P.; STIM, T. B.; TYTELL, A. A. & HILLEMAN, M. R. - New metabolizable immunologic adjuvant for human use. I - Development and animal immune response. Proc. Soe. Exp. Biol. & Med. 116:516-523, 1964. Recebido para publicação em 20/12/1965. 

Tetanus immunization with a single dose of vaccine. Importance and possibilities for mass-immunization programs. Preliminary report on experimental studies

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Tetanus immunization with a single dose of vaccine. Importance and possibilities for mass-immunization programs. Preliminary report on experimental studies

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