Studies of combined quadruple vaccines against diphtheria, pertussis, tetanus, and typhoid fever: reactogenicity and antigenicity

Bull. Org. mond. Santeh 1972, 46, 47-52 Bull. Wld Hlth Org. Studies of combined quadruple vaccines against diphtheria, pertussis, tetanus, and typhoid fever: reactogenicity and antigenicity B. CVJETANOVIC,1 D. IKIC,2 W. R. LANE,3 T. MANHALTER,4 & S. TAPA5 Different DPT + typhoid vaccines were produced by two laboratories and tested in the field for untoward local or systemic reactions and in the laboratory for immunogenic properties. The combined DPT and acetone-inactivated and dried typhoid antigen was found to cause more numerous and more severe local and systemic reactions than the DPT and DPT + typhoid (heat-thiomersal) vaccines. The antibody responses to all four antigens in both of the combined DPT + typhoid vaccines were very satisfactory. From the results of these studies it is concluded that quadruple DPT + typhoid vaccine is suitable for use in areas where typhoid fever is a problem and where logistic and other considerations require the use of combined vaccines. Combined vaccines are much needed in order to carry out immunization programmes in public health practice without increasing either the number of inoculations or the cost. In scattered populations, which cannot be visited frequently by vaccination teams, as many people as possible have to be immu- nized with as few inoculations as possible. In order to protect children against typhoid fever in areas where this infection is an important problem, combined diphtheria, pertussis, and tetanus (DPT) vaccine with typhoid antigen has been used as a quadruple vaccine. The vaccines were prepared in Melbourne, Aus- tralia, by the Commonwealth Serum Laboratories, and field studies were carried out in To*hga. Identical studies were carried out in Yugoslavia with vaccines prepared by the Institute of Immunology, Zagreb. DPT vaccine combined with acetone-inactivated, dried, and reconstituted typhoid vaccine and another DPT vaccine with heat-killed, thiomersal-preserved typhoid vaccine were produced and studied in Yugo- slavia, while DPT vaccine combined with heat-killed, 1 Chief Medical Officer, Bacterial Diseases, World Health Organization, Geneva, Switzerland. 2Director, Institute of Immunology, Zagreb, Yugoslavia. 3Director, Commonwealth Serum Laboratories, Mel- bourne, Australia. 'Institute of Immunology, Zagreb, Yugoslavia. 'Minister of Health, Nuku'alofa, Tonga. thiomersal-preserved typhoid vaccine was prepared in Australia and studied in Tonga. The vaccines were administered intramuscularly to infants and children in two doses four weeks apart. The primary aim of the study was to ascertain whether such vaccines cause mild or severe reactions and, accordingly, whether they are suitable for use in public health practice. The antibody responses to the four components in the combined vaccines were also measured. MATERIALS AND METHODS Composition of the vaccines The vaccines were prepared according to the me- thods for DPT and typhoid vaccine production re- commended by the WHO Expert Committee on Biological Standardization (1964a, 1964b, 1967). The composition of the vaccines is given in Table 1. The typhoid vaccines were prepared from Salmo- nella typhi, strain Ty2. The pertussis vaccines were also prepared from fully antigenic strains. Safety and toxicity tests proved satisfactory. As a control vaccine, triple DPT antigen of the same composition as above was used in doses of 0.5 ml given intramuscularly. In Tonga, 1 595 chil- dren from 3 months to 3 years of age were studied, while in Yugoslavia 789 children aged 3-7 months, 2781 47- B. CVETANOVIC AND OTHERS Table 1. Composition of the vaccines used in these studies I____________________________________________________ Origin Components of vaccine (per ml) Commonwealth Serum Institute of Immunology, Institute of Immunology, Laboratories, Melbourne Zagreb Zagreb diphtheria toxoid 50 Lf 50 Lf 50 Lf(alum-precipitated) tetanus toxoid 10 Lf 10 Lf 1o0U(alum-precipitated) pertussis vaccine 25 000 million organisms 25 000 million organisms 25 000 million organisms typhoid vaccine heat-killed: 500 million organisms heat-killed: 500 million organisms acetone-inactivated, dried: 500 million organisms preservative thiomersal, 0.01 % thiomersal, 0.01 % thiomersal, 0.01 % 5-12 months, and 6-7 years were studied. Using the system of random allocation, two similar groups of children were formed; these were found to be com- parable in age, sex, and other characteristics. In each country, one group received a quadruple DPT plus typhoid vaccine and the other (control) group re- ceived DPT vaccine only. The vaccines were given code numbers so that their composition was not known to the staff engaged in the trials. Assessment of reactions The reactions were observed daily for 3 days and, if necessary, for a longer period. The children were visited by medical auxiliaries 24, 48, and 72 hours after vaccination and the reactions were recorded and graded according to the method described by Pollock (1966). While all the reactions and their specific characteristics were noted and classified ac- cording to severity, only those signs and symptoms that could be objectively measured-namely, tem- perature, swelling, and erythema-are compared in this report. The rectal temperature was measured in young children and the axillary temperature in schoolchildren. A temperature higher than 37.5°C (rectal) or 37°C (axillary) was taken as abnormal. In spite of the fact that the same protocol and instruc- tions were used for recording the results in both Tonga and Yugoslavia, it would be difficult to make a proper comparison of the data from these two countries. We have, therefore, compared the different groups of immunized children in each country sepa- rately. Designs of the studies The study of DPT+Ty (acetone-dried) vaccine, prepared by the Institute of Immunology, Zagreb, was carried out on 396 children, aged 3-7 months, in the town of Cakovec, Yugoslavia, in 1968. The children were divided at random into 3 groups similar in age and sex and the vaccines were given intra- muscularly in the gluteal region. One group received the usual dose of DPT plus 250 million organisms of S. typhi Ty2; another small group of 63 children received DPT plus 125 million organisms of S. typhi Ty2; the third group (controls) received only DPT antigen. Local and general reactions in children of the groups that received 250 million and 125 million S. typhi organisms were strictly comparable. Thus it was concluded that the dose of 250 million organisms of S. typhi Ty2 was well tolerated by infants and there was no need to decrease the dose. The propor- tion of children with febrile reactions was higher in the quadruple vaccine group than in the DPT group. The children in the quadruple vaccine group also had more significant local reactions, in particular, more frequent erythema, swelling, and pain. The children who received quadruple vaccine showed levels of antibodies against diphtheria, tetanus, and pertussis comparable with those in the children who received the DPT vaccine. The study of DPT+Ty (heat-killed, thiomersal- preserved) vaccine, containing 250 million S. typhi TY2 organisms per dose and prepared by the Com- monwealth Serum Laboratories, Melbourne, Austra- 48 VACCINES AGAINST DIPHTHERIA, PERTUSSIS, TE-rANUS, AND TYPHOID FEVER lia, was carried out, by two of the authors (S. T. & B. C.), on 1 595 children in Tonga in 1968. About one-quarter (417) of the children were less than 1 year old and about three-quarters (1 178) of them were 1-2 years old. One-half of these children were given the quadruple vaccine and the other half were given DPT vaccine intramuscularly in the deltoid area of the upper arm. The reactions in both groups were more or less identical in every respect. Since these two studies, carried out in Yugoslavia and Tonga, respectively, indicated that the combined DPT+Ty acetone-dried vaccine caused more fre- quent and more severe reactions than the DPT+Ty heat-phenol-inactivated vaccine, further studies were carried out to compare more directly the degree of reaction produced when these two different typhoid components were incorporated in the quadruple vaccine. The study of DPT+Ty (heat-killed, thiomersal- preserved) vaccine and DPT+Ty (acetone-inacti- vated and preserved) vaccine prepared by the Insti- tute of Immunology, Zagreb, and each containing 250 million S. typhi Ty2 organisms per dose, was carried out, by two of the authors (D. I. & T. M.), on two groups, children aged 6-7 years and infants aged 5-12 months, in Krapina, Yugoslavia, in 1970. The vaccines were given intramuscularly in the gluteal region in the infants and in the deltoid area of the upper arm in the older children. The results confirmed that the quadruple vaccine containing the acetone- inactivated typhoid vaccine caused more severe local and general reactions than that containing the heat- thiomersal-inactivated component. There was no difference in the reactions following administration of the DPT prophylactic and DPT+Ty heat-thio- mersal quadruple vaccine. RESULTS The reactions to vaccination were studied accord- ing to the methods described above and the results of the study are shown in summary form in Tables 2, 3, and 4. We have taken into consideration only the more or less objective symptoms-namely, fever (> 37°C axillary, or > 37.5°C rectal), swelling (> 10 cm diameter), and severe pain. From the study carried out with the quadruple vaccine containing the heat-killed typhoid compo- nent (manufactured by the Commonwealth Serum Laboratories), it is seen that reactions to this vaccine differed very little from those to the DPT vaccine (Table 2). On the contrary, the study in which the quadruple vaccine prepared by the Institute of Immunology, Zagreb, was used (i.e., the vaccine that contained the acetone-inactivated typhoid component) indicated clearly that reactions to that quadruple vaccine were more severe than those to DPT vaccine (Table 2). The two studies in which the reactions to the quadruple vaccines with typhoid components prepa- red from acetone-inactivated or heat-killed organisms were compared show that reactions to the acetone- inactivated vaccine were more severe (Table 3). A closer study of the febrile reactions shows that the vaccine with acetone-inactivated typhoid com- ponent tended to give a higher proportion of febrile reactions than the vaccine with heat-thiomersal-in- activated typhoid component. These differences are presented in Table 4. It was observed that those who developed fever after the first injection tended to develop fever again after the second dose. Those who did not develop fever after the first dose usually did not do so after the second dose. There were, however, some children who developed fever only once after either the first or second dose. The immunogenic properties of the bacterial com- ponents of the DPT+Ty vaccines used in the trials in Yugoslavia were tested in the laboratories of the Institute of Immunology, Zagreb, using the active mouse protection test. The antigenic properties of both toxoids were assessed by the two-stimulant method. The results of these showed the contents of the diphtheria and tetanus components to be 7 IU and 17 IU, respectively, several times higher than the WHO requirements (2 IU), and the content of the pertussis component was almost twice as high as the WHO requirement of 8 IU, while the typhoid com- ponent showed better protection in the active mouse protection test than did our reference vaccine. Meth- ods and criteria for these tests have been published elsewhere (Ikic, 1965a, 1965b, 1965c; Mandi6-Higy, 1964). It was found in earlier studies that children who received the above amounts of DPT antigens had satisfactory levels of antibodies against diphtheria, tetanus, and pertussis (Ikic, 1965b, 1965c; Mandic- Higy, 1965). It was established in previous studies in man that S. typhi acts as an adjuvant on the tetanus antigen and increases antibody titre against tetanus (Maclean & Holt, 1940). The influence of S. typhi on other components in combined vaccines has not been studied in man. 49 50 B. CVETANOVIC AND OTHERS Table 2. Percentage of vaccinated children with reactions after administration of either the quadruple (DPT + Ty) or the triple (DPT) vaccines in 1968 DPT+Ty, heat-thiomersal vaccine (Commonwealth Serum Laboratories, Melbourne) Nuku'alofa, Tonga 3-1 1 months (417) 12-35 months (1 178) fever swelling pain fever swelling pain 27.2 37.1 80.3 14.9 32.9 50.3 7.5 14.5 47.3 10.6 14.7 50.6 2.8 2.3 4.2 2.2 1.7 4.7 2.2 0 2.2 2.4 0.6 2.6 0.9 0 0.5 1.7 0 0.3 2.2 0 1.1 0.4 0 0.4 24.0 34.3 65.7 15.1 30.3 60.2 8.3 16.0 42.0 7.0 14.4 45.8 6.4 3.4 6.4 3.4 3.6 6.3 3.3 0.6 2.8 1.5 0.7 1.7 3.4 0 1.5 2.2 0.3 0.7 2.8 0 1.7 0.6 0.4 0.4 DPT+Ty, acetone-inactivated vaccine(Institute of Immunology, Zagreb) Iakovec, fever 35.7 33.3 4.8 5.3 0 0 21.2 30.4a 12.1 6.5al 0 Oatakovec' 3-7 months Yugoslavia (396) swelling 51.6 49.1 11.1 14.0 0 0 30.3 47.8a 15.2 1 o.9a 0 oa pain 35.7 35.1 9.5 10.5 0 0 15.2 23.9a 0 4.3a 0 oa a The second dose given to these children was DPT+Ty vaccine. Table 3. Percentage of vaccinated children with reactions after administration of the acetone-inactivated or the heat-thiomersal vaccines prepared at the Institute of Immunology, Zagreb: the tests were carried out at Krapina, Yugoslavia in 1970 Percentage with reactions Age group 2 8h 7 *accine (no. Symptoms 24 h 48 h 72 h of children) 1st 2nd 1st 2nd 1st 2nd dose dose dose dose dose dose fever 51.9 50.0 8.3 11.5 1.9 0 DPT+Ty, acetone-inactivated 5-12 months swelling 70.4 63.5 13.9 11.5 0 0 (108) pain 48.1 45.8 13.0 13.5 0 0 fever 42.2 39.5 4.9 8.8 0 0 DPT+Ty, heat-thiomersal 5-12 months swelling 53.9 46.2 11.8 8.8 0 0 (102) pain 41.2 41.8 7.8 11.0 0 0 fever - 43.2 - 17.9 - 0 DPT+Ty, acetone-inactiv- 6-7 years swelling - 35.8 - 5.3 - 0 ated: booster after DPT (95) pain - 27.4 - 2.1 - 0 fever - 35.2 - 11.4 - 0 DPT+Ty, heat-thiomersal: 6-7 years booster after DPT (88) swelling - 29.5 - 3.4 - 0 pain - 23.9 - 1.1 - 0 VACCINES AGAINST DIPHTHERIA, PERTUSSIS, TETANUS, AND TYPHOID FEVER Table 4. Body temperatures observed in children in Krapina, Yugoslavia, in 1970 after immunization with either the quadruple DPT + Ty acetone-inactivated or the heat- thiomersal-preserved vaccines D PT+Ty acetone-inactivated DPT+Ty heat-thiomersal Temperature(0c) I 1 st 2nd booster dose 1 st 2nd booster dose dose dose after D PT dose dose after D PT 37.5-37.9 42 34 21 36 30 18 38.0-38.4 1 1 9 1 7 5 2 8 38.5-38.9 4 5 2 1 1 2 39.0-39.4 1 0 1 1 1 0 39.5-39.9 0 0 0 0 0 0 CONCLUSIONS From the results of studies on the reactions and responses of infants and children to two combined DPT plus typhoid vaccines with differently prepared typhoid components, it may be concluded that both of these quadruple vaccines cause local and systemic reactions in a proportion of the vaccinated children but that these are not sufficiently severe to preclude their wider use. The frequency and severity of the reactions were rather greater after the vaccine containing DPT plus the acetone-inactivated typhoid component than after the vaccine containing DPT plus the heat-phenol- killed typhoid component. Although the acetone- inactivated typhoid vaccine gave better protection than the heat-phenol-inactivated vaccine in earlier controlled trials (Cvjetanovid & Uemura, 1965), the latter vaccine is easier to prepare and administer and is also cheaper. Incorporated in a quadruple vaccine, it would probably be more acceptable because of the milder reactions. The antibody responses to all four components of both quadruple vaccines were very satisfactory. ACKNOWLEDGEMENTS The authors thank the workers at the Commonwealth Serum Laboratories, Melbourne, Australia, and the Institute of Immunology, Zagreb, Yugoslavia, for prepar- ing and testing the vaccines, and the health authorities and workers in Tonga and Yugoslavia who carried out the field studies for their co-operation. RUESUM12 ETUDE DE PRtPARATIONS ASSOCIANT DES VACCINS CONTRE LA DIPHTtRIE, LA COQUELUCHE, LE TtTANOS ET LA FItVRE TYPHOIDE: POUVOIR RtACTOGtNE ET ANTIGtNICITt Pour des raisons de commodit6 et de couit, il est avantageux de recourir aux associations de vaccins pour l'ex6cution de programmes d'immunisation de grande envergure. Une 6tude collective a e menee en Australie, a Tonga et en Yougoslavie en vue d'evaluer le pouvoir reactogene et l'antigenicite de preparations renfermant quatre vaccins: antidipht6rique-anticoquelucheux-anti- tetanique(DPT) et antityphoidique. Deux types d'associations ont ete utilise's. Le compo- sant DPT comportait des anatoxines diphterique (50 Lf/ ml) et tetanique (10 Lf/ml) et un vaccin anticoquelucheux (25 milliards de germes par millilitre). On lui a ajoute le composant Ty (vaccin antityphoidique prepare & partir de la souche Ty2 de Salmonella typhi) tue par la chaleur ou inactive par l'acetone. La premiere etude a ete effectuee en 1968 en Yougo- 51 52 B. CVJETANOVIC AND OTHERS slavie sur 396 enfants ages de 3 A 7 mois. Repartis en trois groupes, ils ont requ soit l'association DPT+Ty inac- tive par l'acetone (250 millions de germes) soit 1 'associa- tion DPT+Ty inactive par P'acktone (125 millions de germes), soit F'antigene DPT seul. Les reactions generales et locales (erytheme, tum6faction, douleur) ont et stric- tement comparables dans les deux premiers groupes, ce qui prouve la bonne tol6rance d'une dose 6levee de Ty, mais plus frequentes et plus accentuees que dans le 3e groupe (DPT seul). L'association DPT+Ty tue par la chaleur (250 millions de germes) a e etudiee A Tonga en 1968 sur 1595 enfants ages de moins de 3 ans. La moitie d'entre eux a ete vacci- nee par cette preparation, I'autre moitie ne recevant que le vaccin DPT. Les reactions dans les deux groupes n'ont pas differ6 sensiblement. Ces deux etudes ayant revele que l'association DPT+Ty inactive par l'acetone provoquait des reactions plus fr6quentes et plus graves que l'association DPT+Ty tue par la chaleur, une comparaison directe entre ces deux preparations a et jugee necessaire. Elle a et6 faite en 1970 en Yougoslavie sur deux groupes d'enfants ages de 5 A 12 mois ou de 6 a 7 ans et a confirme le pouvoir reacto- gene plus eleve du composant Ty inactive par l'acetone. Les investigations serologiques menees en Yougoslavie n'ont fait apparaitre aucune difference entre la reponse immunitaire aux differents antigenes apres vaccination par le vaccin DPT ou par l'association DPT+Ty. Les auteurs concluent que si les associations vaccinales DPT+Ty provoquent des reactions generales et locales dans certains cas, ces incidents ne sont pas suffisamment graves et frequents pour contre-indiquer leur emploi. Ces preparations suscitent une reponse en anticorps tres satisfaisante. Pour des raisons de commodite, il con- viendrait d'employer, comme composant antityphoidique, le vaccin Ty tue par la chaleur. REFERENCES Cvjetanovic, B. & Uemura, K. (1965) Bull. Wld Hlth Org., 32, 29-36 Ikic, D. (1965a) In: Proceedings of the Ninth International Congress for Microbiological Standardization, Lisbon, 1964, Basel, Karger, pp. 175-182 (Progress in immuno- biological standardization, vol. 2) lkic, D. (1965b) Rad. Immunol. Zav., Zagreb, 34, 9-14 Lki6, D. (1965c) Rad. Immunol. Zav., Zagreb, 34, 15-19 MacLean, I. H. & Holt, L. B. (1940) Lancet, 2, 581 Mandic-Higy, Lj. (1965) Rad. Immunol. Zav., Zagreb, 34, 43-52, 143-152 Pollock, T. M. (1966) Trials ofprophylactic agents for the control of communicable diseases, Geneva (World Health Organization: Monograph Series, No. 52) WHO Expert Committee on Biological Standardization (1964a) Wld Hlth Org. techn. Rep. Ser., No. 274 WHO Expert Committee on Biological Standardization (1964b) Wld HIth Org. techn. Rep. Ser., No. 293 WHO Expert Committee on Biological Standardization (1967) Wld Hlth Org. techn. Rep. Ser., No. 361