Simultaneous administration of smallpox, measles, yellow fever, and diphtheria—pertussis—tetanus antigens to Nigerian children*

Bull. Org. mond. Sante 1973, 48, 175-181Bull. Wid Hlthi Org.f Simultaneous administration of smallpox, measles, yellow fever, and diphtheria-pertussis-tetanus antigens to Nigerian children * F. L. RUBEN, E. A. SMITH, S. 0. FOSTER, H. L. CASEY, J. M. PIFER, R. B. WALLACE, A. I. ATTA, W. L. JONES, R. B. ARNOLD, B. E. TELLER, Z. Q. SHAIKH, B. LOURIE, D. L. EDDINS, S. M. DOKO, & W. H. FOEGE Children receiving smallpox, measles, and yellow fever vaccines simultaneously at separate sites responded adequately to all three vaccines. In those children, 9 months of age and older, who received these three vaccines in addition to diphtheria-pertussis- tetanus vaccine, there was a decrease in measles seroconversion ratesfrom 89 % to 70 %. Possible interactions between live and killed vaccines should be considered when the adninistration of multiple antigens is planned. The simultaneous administration of multiple anti- gens overcomes many of the practical and logistic obstacles to providing complete immunization ser- vices to children. A single immunzing session to protect against several diseases reduces the cost and increases the number of diseases for which prophy- laxis can be obtained during a limited number of visits. This is especially true in developing countries where most of the population live in rural areas inaccessible to fixed health installations and depend on mobile teams for preventive health services. In urban areas in both developed and developing coun- tries where return rates for preventive services are low, simultaneous administration of several antigens has similar advantages. Multiple vaccine administration is a common practice in many parts of the world. Serologic studies to support such practices are limited except for those dealing with frequently used vaccine combinations: diphtheria-pertussis-tetanus (DPT) Miller & Ryan (1944), DPT and mono- (Benson et al., 1963; Kele- mon et al., 1961), and trivalent poliomyelitis (Hardy et al., 1970). The studies of combined vaccines * From the Epidemiologic Unit, Federal Ministry of Health, Lagos, and the North Central State Ministry of Health, Kaduna, Nigeria, and from the Center for Disease Control, US Department of Health, Education, and Welfare, Atlanta, Ga., USA. Requests for reprints should be addressed to Dr E. Ademola Smith at the first or to Dr Stanley V. Foster at the last address. against smallpox and measles (Budd et al. 1967; Sherman et al., 1967; Kalabus et al. 1967; Weibel et al. 1969), smallpox and yellow fever (Peltier, 1947; Dick & Horgan, 1952), and smallpox, measles, and yellow fever (Meyer et al., 1964) are conclusive that these vaccine combinations can be administered safely and effectively. DPT has not been studied in combination with these live virus vaccines except by Winter et al. (1963), who studied the simultaneous administration of smallpox, oral poliomyelitis, and DPT antigens. Limited serologic data indicated a satisfactory immunologic response to smallpox, poliomyelitis, diphtheria, and tetanus antigens. Response to pertussis vaccine was not measured. No data have been collected on the addition of measles vaccine to this combination. In 1968, a study in the Gambia with smallpox, measles, yellow fever, DPT, and oral poliomyelitis vaccines showed that this combination was safe when administered simultaneously but separately (Teller & Helmholz, unpublished data). In that study, four groups of children were given either placebo or vaccines against smallpox-measles-yellow fever (SMY), SMY and poliomyelitis (P), or SMYP plus DPT. Children were followed on alternate days for 14 consecutive days and temperatures were recorded. Children in the vaccine groups had a higher inci- dence of temperatures of 37.8°C and above than the placebo group (Table 1). No complications or deaths occurred among the study participants. 2993 -175- 176 F. L. RUBEN ET AL. Table 1. Frequency during 14 days after immunization and percentage distribution of temperature readings on alternate days in children receiving vaccine combina- tions Temperature (°C) Treat- No. of ment sub- <37.8 37.8-38.6 38.7-39.7 39.8-40.6 group a tects No. (%) No. (%) No. (%) No. (%) Placebo 69 499 (91.1) 42 (7.7) 5 (0.9) 2 (0.4) SMY 70 460 (83.2) 85 (15.4) 7 (1.3) 1 (0.2) SMYP 71 488 (84.9) 76 (13.2) 10 (1.7) 1 (0.2) SMY DPT 68 455 (84.4) 70 (13.0) 13 (2.4) 1 (0.2) a See text p. 176. The present study measures the smallpox vaccina- tion take rates as assessed at 7 days and the serologic responses to smallpox, DPT, yellow fever, and measles vaccines when given by jet injector at four separate sites and followed after two months by a second dose of DPT. MATERIALS AND METHODS The area Daura Emirate in North Central State, Nigeria, was selected for the study because it had reported no cases of measles since a mass campaign 2 years previously. The excellent cooperation and assistance provided by the Emir, his counsellor, and the people of Daura in previous campaigns were also taken into consideration. Blood collection and immunization began in early May at the height of the dry season and at a time when the temperature was 32-430C in the daytime and the humidity was very low. Final specimens were collected in August at the beginning of the rainy season, when the daytime temperatures were 27-32°C and when the humidity had increased. The study population Children 6 months to 2 years of age were selected from three separate villages. Infants under 6 months were rejected because of possible interference to measles seroconversion by maternal antibody. Child- ren who had acute or chronic illness, a history of measles or measles immunization, smallpox or a smallpox vaccination scar, or who did not have a neck or waist band to which to attach a metal identification disk, were excluded from the study. Some children were accepted and then later ex- cluded if a blood specimen could not be obtained after reasonable effort. Finally, only children from whom a satisfactory second blood sample was ob- tained were included in the final analysis. Vaccine groups Subjects in each village were randomly assigned to one of the four study groups. Cards, previously shuffled, were used to randomize the subjects. Group 0 received placebo, group SMY received smallpox, measles, and yellow fever vaccines; group SMYT received smallpox, measles, yellow fever, and DPT vaccines; and group T received DPT vaccine. The two groups receiving DPT were given a booster dose at 2 months. At the conclusion of the study, all children who had received placebo or DPT alone were vaccinated with smallpox and measles vaccines. Vaccines and their administration 1 Lyophilized smallpox vaccine,' further attenuated measles vaccines,3 and 17D yellow fever vaccine ' were used exclusively. These vaccines were sampled for potency before the trial and after exposure to field trial conditions (Table 2). Commercial DPT was used but the titre was not determined. All vaccines were kept frozen or refrigerated and were reconsti- tuted as recommended by the manufacturer. Diluted vaccines were discarded after a day's use, except for Table 2. Dosage and pre- and poststudy titration for vaccine potency Titre a Vaccine Dosage (ml) before study after study smallpox 0.1 10 6.7 10 6.0 measles 0.5 10 3.5 10 3.5 yellow fever 0.5 10 5-3 10 5.48 DPT (2 doses) 0.5 NT NT a NT = titre not determined. measles vaccine, which was discarded after 4 hours, and yellow fever vaccine, which was discarded after 1 hour. The arms of the persons to be vaccinated were not cleaned before vaccination and all vaccines were given by jet injector.' An intradermal nozzle was 1 Inclusion of trade names is for identification only and does not imply endorsement by the Public Health Service of the US Department of Health, Education and Welfare. 2 Wyeth, Lot 275-703, Dryvax. 3 Pitman-Moore, Lot 185420. ' National Drug, Lot 6986N9. 5 Ped-O-Jet. MULTIPLE VACCINATION used for smallpox vaccine and for placebo; other vaccines were given subcutaneously through a stan- dard nozzle. Excess smallpox vaccine was wiped from the skin with cotton-wool. Vaccine dosages for the four separately administered vaccines were 0.5 ml for measles, yellow fever, and DPT, and 0.1 ml for smallpox and the saline placebo. Serologic testing Blood samples were taken before immunization and 3 months after the initial vaccine administration. Whole blood was collected by venepuncture, was placed in preservative-free tubes, and allowed to clot in a cooler. The sera were separated by centrifuga- tion each evening and were frozen during storage and shipment. Haemagglutination inhibition (HI) tests were run on all paired sera for antibodies against measles virus (Hierholzer & Suggs, 1969). Yellow fever neutraliza- tion (N) tests were performed by a plaque reduction technique in Vero cell cultures using the Asibi strain (Monath et al., 1970). Cell monolayers were inocu- lated with a preincubated mixture of serum and virus containing 100 plaque-forming units. A 90-100% reduction in plaques compared with the controls was considered positive, and a reduction of less than 90% negative. Pertussis agglutinins were determined by the rapid tube test (Kendrick et al., 1970). Antitoxin titres to diphtheria and tetanus were determined by a haemagglutination method (Schubert & Cornell, 1958). Although paired sera were identified, the initial laboratory studies were carried out without knowledge of the vaccine group to which the sera belonged. Individuals in this study were classified as suscep- tible to smallpox when no scars of vaccination or of smallpox were noted on physical examination; to measles when HI antibody was less than 5 in undi- luted serum; to yellow fever when neutralization in undiluted serum was negative; to pertussis when agglutinin titres were less than 8; and to tetanus and diphtheria when the HI titre was less than 0.01 antitoxin units. Positive response to injected antigens was defined by the following criteria: vaccinia-if a major reaction as defined by theWHO Scientific Group on Smallpox Eradication (1968) developed. measles-if HI antibody increased from < 5 to >5. yellow fever-if the neutralization test converted from negative to positive. Table 3. Age distribution of subjects by study group 6-11 months 12-24 months Group Total No. % No. % 0 28 (29.5) 67 (70.5) 95 SMY 36 (40.0) 54 (60.0) 90 SMYT 30 (38.5) 48 (61.5) 78 T 25 (30.9) 56 (69.1) 81 total 119 (34.6) 225 (65.4) 344 diphtheria and tetanus-if antitoxin levels increased from <0.01 units/ml to >0.01 units/ml. pertussis-if agglutinins increased from < 8 to > 8. RESULTS Distribution ofsubjects Table 3 shows the age distribution of the subjects: 35% of the subjects were between 6 and 11 months of age. The subjects were evenly distributed by age in the four study groups. Preimmunization data Table 4 shows the preimmunization serologic titres of the study subjects. Diphtheria was the only disease to which the children had any substantial prestudy protection: 20% had antitoxin levels of 0.01 or greater. For all other antigens, 94-100% of the study population were classified as susceptible. No small- pox vaccination scars were found. Table 4. Preimmunization vaccination scar rate and results of serologic tests Percentage positive Group ~~YellowGroup Measles fever Per- Tetanus c Diphthe- HI a neutrali- tussis b ria c zation 0 1.0 0 4.1 0.6 17.6 SMY 2.2 2.2 SMYT 1.3 1.3 1.3 0 19.2 T 2.5 2.5 21.0 a Titre >5. b Agglutination >8. c Antitoxin >0.01. 177 F. L. RUBEN ET AL. Table 5. Postvaccination serologic titres in susceptible subjects Measles (HI) Titre levels PretgGroup Total Percentage <5 5 10 20 40 80 160 320 pstv saline 92 0 1 0 0 1 0 0 94 2.1 SMY 15 0 6 2 4 16 21 24 88 83.0 SMY DPT 25 0 3 3 9 8 10 19 77 67.5 Yellow fever (neutralization) Group Positive Negative Total Percentage positive] saline 5 89 94 5.3 SMY 85 3 88 96.6 SMY DPT 73 4 77 94.8 Pertussis (agglutination) litre levels PercentageGroup Total percetage <8 8 16 32 64 128 256 512 positive saline & SMY 41 5 1 0 0 0 0 0 47 12.8 SMY DPT 23 11 8 8 10 10 5 2 77 70.1 DPT 16 11 10 8 10 9 13 2 79 79.7 Tetanus (antitoxin) Percentage Group >0.01 >0.1 Total >0.01 >0.1 saline & SMY 16 3 176 9.1 1.7 SMY DPT 78 78 78 100.0 100.0 DPT 77 75 79 97.5 94.9 Diphtheria (antitoxin) Group >0.01 >0.1 Total >0.01 >0.1 saline & SMY 18 5 145 12.4 3.4 SMY DPT 55 49 63 87.3 77.8 DPT 57 49 64 89.1 76.6 Postimmunization results dence rates for major reactions and seroconversions Table 5 includes the postimmunologic-serologic were similar for all vaccine groups except for the titres in susceptible subjects. The rates of major SMYT group, which showed a significantly lower reactions to smallpox vaccination and of serologic measles conversion rate (67.5 %) than the SMY conversions are summarized in Table 6. The inci- group (83.0°) (x2= 4.51, P = <0.05). 178 MULTIPLE VACCINATION Table 6. Major reaction rates to smallpox vaccination and seroconversion rates in susceptible subjects Percentage positive Group Vaccina- Measles Yellow fever Pertussis Tetanus Diphtheria tion (HI)a (neutralization) >1 :8 b >0.01 c >0.1 c >0.01 c >0.1 c 0 4.6 2.1 5.3 12.7 9.0 1.7 12.4 3.4 SMY 97.6 83.0 96.6 I SMYT 97.3 67.6 94.8 70.1 100.0 100.0 87.3 77.8 T 79.7 97.4 94.9 89.1 76.5 a Titre > 1: 5. b Agglutination titre. c Antitoxin units. In the SMY and SMYT groups, the measles seroconversion rates were, as expected, lower in children under 9 months of age: 64% and 59%, respectively (Table 7). In children 9 months and older, measles conversion rates for the SMY group (89.5%) were greater than for the SMYT group (70.9%) (X2= 5.51, P = <0.025). The geometric mean titres for the groups were 62 and 32, respec- tively. DISCUSSION Smallpox-measles mobile vaccination teams from the Nigerian Ministry of Health administered the antigens to the study participants. The teams wil- lingly assumed the additional responsibility of the multiple antigen programme, including maintenance of the extra jet injectors, mixing of vaccines, vaccine administration, and supervision of vaccine use to ensure its use within the defined time period. The second dose of DPT at 2 months required a single person and one jet injector. Simultaneous adminis- Table 7. Measles seroconversions by age group SMY SMYT Age group Converters Converters(months) Total Total No. % No. % 6-8 22 14 63.6 22 13 59.1 9-24 66 59 89.4 a 55 39 70.9 a a Significant difference, P <0.025; x' = 5.51. tration of four vaccines was well within the capability of the vaccination teams. The levels of preexisting humoral immunity to the diseases studied were low, except for diphtheria. The relatively large number of children with preimmuniz- ation diphtheria protection is striking and most probably represents cutaneous infection (Barr, 1962). Subjects in the SMYT groups tolerated the mul- tiple injections well. Since careful follow-up for febrile reactions had been carried out previously in the Gambia, no attempt was made in Nigeria to detect fever. At 7 days, 93% of the children returned for the reading of the smallpox vaccination reac- tions, and most of them were followed up at 2 months. All subjects were accounted for when the second serum specimen was collected. No vaccine- associated mortality was detected. The smallpox major reactions in the placebo group might have been false positive readings or might indicate incorrect vaccine administration. Serocon- versions in the placebo groups to measles, yellow fever, and DPT could indicate either the occurrence of concurrent disease in the community, incorrect vaccine administration, or false positive laboratory results. Pertussis agglutinins showed a good response to two doses of DPT administered 2 months apart. Similar findings have been reported by Bell (1941) and Wilkins (1971). Other epidemiological studies have shown protection despite the lack of a signifi- cant agglutinin titre (Sako, 1947; Volk, 1949). Diphtheria and tetanus antitoxin titres reached protective levels in about 90% of both antigen groups after two doses. No attempt was made to 179 F. L. RUBEN ET AL. determine the duration of these levels, but from the experience of others it is expected that they will last at least 36 months (Volk, 1949). The low measles seroconversion rates in children under 9 months of age are probably a result of interference from technically unmeasurable levels of maternal antibody. In children over 9 months of age the measles conversion rate in the SMY group (89.4%) is similar to the 88.0% conversion rate obtained by Teller et al. (unpublished observations) using measles vaccine alone in the same geographic area. The major reaction and seroconversion rates of the SMY group are similar to those reported by Meyer et al. (1964) who administered smallpox, measles, and yellow fever vaccines in combination. The higher yellow fever seroconversion rates seen in the simul- taneously but separately vaccinated SMY group (98%) as compared with that of the combination group in the study of Meyer et al. (85 %) supports the reported data on interference of yellow fever seroconversion in combined vaccines (Dick & Hor- gan, 1952). Measles seroconversion rates in the SMY group were, however, lower than the 97% reported by Meyer et al. (1964). This could be related to one or more of the differences between the SMY group and the Meyer et al. study, including virus strain (further attenuated versus attenuated), dose (TCID50 of 4 000 versus TCID50 of 12 000) and the timing of postimmunization serologic samples (90 versus 21 days). The 19% difference between the measles serocon- version rates in children 9 months of age and older in the SMY and SMYT groups is difficult to explain. Although failures could have occurred in the measles vaccine administration or individual vials of vaccine could have lost their potency, it is unlikely that such failures would have been limited to one group. From the literature, there are few reports of studies in man to support the concept of vaccine interference or of a limited capacity for immunologic responsiveness to simultaneous antigen exposure. Chen et al. (1956, 1957), in studying responses to DPT antigens, show- ed that, in the special circumstance of preexisting diphtheria immunity, the response to tetanus was reduced. MacGregor & Barr (1962) suggested that chronic parasitaemia with malaria reduced the re- sponse to tetanus toxoid in Gambian children The decrease in measles seroconversion rates for the SMYT group has not been explained. Although some unrecognized change in the field conditions could possibly explain the difference, the possible interaction between simultaneously but separately administered killed and live vaccines must be consid- ered. Interferon induction by one of the components of the killed vaccines has been postulated as a possible explanation for the observed interference. Until this question is resolved, clinical use of antigen combinations should be limited to those of proved effectiveness. In this study it was decided not to mix vaccines. By giving each of the four vaccines separately and at separate sites, the problem of vaccines interacting locally was not encountered. Parents accepted the multiple injections with appreciation and without complaint. ACKNOWLEDGEMENTS The authors express their gratitude to Dr S. L. Adesuyi, Chief Medical Adviser to the Federal Government of Nigeria, for permission to publish the paper; the Emir of Daura, Alhaji Mohmmadu Bashar; the village chiefs and citizens in Daura Emirate; the vaccinating team, the health workers, and the Ministry of Health of North Central State, Nigeria; Dr Pearl Kendrick of the Univer- sity of Michigan School of Public Health, for perfor- mance of the pertussis agglutinin studies; Dr Geoffrey Edsall, of the Massachusetts Department of Public Health; Dr David Karzon of Vanderbilt Medical Center; Dr Brian Henderson, Dr Joel Breman, Mr Thomas Kimmel, Miss Donna Sasso, Mrs Norma Jordan, and Mr William DesPrez of the Center for Disease Control; Dr Peter N'Dow, Chief Medical Officer, Director of Health Service, the Gambia; Dr Jack Bunn and Mr Robert Helmholz for the safety data collected in the Gambia; and Dr Michael Spector for his assistance in carrying out the studies in Nigeria. These studies were partly financed by the US Agency for International Development as part of the West African Smallpox Eradication and Measles Control Program. 180 MULTIPLE VACCINATION 181 Rt]SUME ADMINISTRATION SIMULTANPE D'ANIGtNES VARIOLIQUE, ROUGEOLEUX, AMARIL ET DIPHT2RIQUE-COQUELUCHEUX-TgTANIQUE CHEZ DES ENFANTS NIGMRIANS On a determine le taux de prise (reactions majeures a la vaccination antivariolique) et les r6actions serologiques dans quatre groupes d'enfants nigerians, ages de 6 a 35 mois, vaccines suivant l'un ou l'autre des schemas suivants: groupe 0: placebo (solut6 salin); groupe SMY: vaccins antivariolique, antirougeoleux et antiamaril; groupe SMYT: vaccins antivariolique, antirougeoleux, antiamaril et vaccins antidipht6rique-anticoquelucheux- antit6tanique (DPT), puis aprbs 2 mois, deuxieme dose de DPT; groupe T: deux doses de DPT a 2 mois d'inter- valle. Tous les vaccins ont et6 administres par injection sous pression et les injections simultanees ont ete faites a des endroits differents. Chez les enfants Ag6s de 9 mois et plus, on a note 90% de reponses immunitaires apr&s inoculation de chacun des trois antigenes vivants (variole, rougeole et fievre jaune) injectes simultanement. Chez les enfants vaccin6s en outre par le DPT (groupe SMYT), la proportion des s6roconversions induites par le vaccin antirougeoleux est tomb6e a 70,9% (contre 89,4% dans le groupe SMY). Ces observations suggerent la possibilit6 d'une inter- f6rence entre vaccins vivants et vaccins tues. Les auteurs conseillent de ne recourir qu'aux seules associations vaccinales qui ont fait la preuve de leur efficacit6. REFERENCES Barr, M. (1962) Trans. roy. Soc. trop. Med. Hyg., 56, 368-370 Bell, J. A. (1941) Pub. Hlth Rep. (Wash.), 56, 1535-1546 Benson, P. F. et al. (1963) Brit. med. J., 1, 641-643 Budd, M. et al. (1967) Amer. J. publ. Hlth, 57, 80-86 Chen, B. et al. (1956) J. Immunol., 77, 144-155 Chen, B. et al. (1957) J. Immunol., 79, 3945, 393 400 Dick, G. W. A. & Horgan, E. S. (1952) J. Hyg. (Lond.), 50, 376-383 Hardy, G. E. et al. (1970) Pediatrics, 45, 444-448 Hierholzer, J. C. & Suggs, M. T. (1969) Appl. Microbiol., 18, 816-833 Kalabus, F. et al. (1967) Amer. J. Epidem., 86, 95-111 Kelemon, G. et al. (1961) Lancet, 1, 456 Kendrick, P. L. et al. (1970) In: Diagnostic procedures for bacterial, mycotic and parasitic infections, 5th ed., New York, American Public Health Association, p. 106 MacGregor, 1. A. & Barr, M. (1962) Trans. roy. Soc. trop. Med. Hyg., 56, 364-367 Meyer, H. M. et al. (1964) Bull. Wld Hlth Org., 30, 783-794 Miller, J. J. & Ryan, M. L. (1944) J. Pediat., 1, 8-22 Monath, T. P. et al. (1970) Amer. J. Epidem., 92, 40-50 Peltier, M. (1947) Amer. J. publ. Hlth, 37, 1026-1032 Sako, W. (1947) J. Pediat., 30, 29-40 Schubert, J. H. & Cornell, R. C. (1958) J. Lab. clin. Med., 52, 737-743 Sherman, P. et al. (1967) Brit. med. J., 2, 672-676 Volk, V. K. (1949) Amer. J. publ. Hith, 39, 1299-1313 Weibel, R. E. et al. (1969) Pediatrics, 43, 567-572 WHO Scientific Group on Smallpox Eradication (1968) Wld Hlth Org. techn. Rep. Ser., No. 393, p. 30 Wilkins, J. (1971) J. Pediat., 79, 197-202 Winter, P. A. D. et al. (1963) S. Afr. med. J., 37, 513-515