Clinical evaluation of a new measles-mumps-rubella combined live virus vaccine in the Dominican Republic*

VACCINE EVALUATION Clinical evaluation of a new measles-mumps- rubella combined live virus vaccine in the Dominican Republic * N. JOEL EHRENKRANZ,' ARNOLDO K. VENTURA,2 EDWARD M. MEDLER,3 JOSEPH E. JACKSON,4 & MICHAEL T. KENNY5 Over 900 children were enrolled in a double-blind placebo-controlled clinical study of measles (Schwarz strain), mumps (Jeryl Lynn strain), and rubella (Cendehill strain) trivalent vaccine. The trivalent vaccine caused about the same degree of reactivity as is generally associated with the Schwarz strain measles vaccine. Paired sera from triple- susceptible vaccinees had seroconversion rates of 99 % for measles, 94 % for mumps, and 93 % for rubella. The results of this study show that this trivalent vacci-le is as well tolerated and as effective as its component vaccines. The growing list of virus diseases becoming ame- nable to control by vaccination necessitates improve- ments in the system for delivery of this type of preventive medical care. The efficiency of immuniza- tion programmes, both private and public, can be greatly increased by the use of polyvalent vaccines provided that the individual components were all intended for the same populations. The polyvalent combinations should be no more reactive and no less effective than the components used singly. For live, attenuated virus vaccines, both the practical and technical requirements have been met by a variety of double and triple combinations (1, 2) each prepared for use in certain countries or populations. A double-blind, placebo-controlled study was con- ducted to evaluate clinically a new measles-mumps- rubella trivalent live virus vaccine. Consultation with health authorities in the Dominican Republic re- vealed that country to be an appropriate site for a project of this type. In intercity and rural popula- tions the use of measles vaccine had been so recently introduced that it had not yet made an impact on the * From Cedars of Lebanon Hospital, Miami, FL 33125, USA, and the Human Health Research Laboratories, Dow Chemical Co., Indianapolis, IN 46268, USA. Requests for reprints should be addressed to Dr Ehrenkranz. I Chief of Medicine, Cedars of Lebanon Hospital. 2 Department of Medicine, Cedars of Lebanon Hospital. Associate Clinical Investigator, Dow Chemical Co. 'Director, Biological Clinical Research, Dow Chemical Co. 5 Senior Research Immunologist, Dow Chemical Co. age-specific susceptibility rates. In these same popu- lations, mumps and rubella vaccines had not yet come into use. MATERIALS AND METHODS Vaccines The vaccine was a combination of live attenuated measles virus vaccine, Schwarz strain (3, 4), live mumps virus vaccine, Jeryl Lynn strain (5-7), and live rubella virus vaccine, Cendehill strain (8, 9). Three lots of this trivalent vaccine were used in these studies. Each lot was made from separate mono- valent components and contained a minimum of 1 000 TCID50 (50% tissue culture infective doses) of measles and rubella vaccine viruses and 5 000 TCIDF0 of mumps vaccine virus per dose. The trivalent vaccine was dispensed into single dose vials, lyophil- ized, and stored at 5°C until use. Single-dose vials were reconstituted with sterile diluent from pre- filled syringes just prior to subcutaneous inocula- tion. The vaccine and placebo were produced at the Biological Laboratories of the Dow Chemical Co. The placebo was prepared in a similar manner but without the addition of any virus, and was indistin- guishable from the vaccine whether lyophilized or reconstituted. Serology Antibodies to measles and rubella viruses were determined by the hemagglutination-inhibition (HI) 3326 - 81 - BULL. WORLD HEALTH ORGAN., Vol. 52, 1975 N. JOEL EHRENKRANZ ET AL. method (10, 11). Antibodies to mumps virus were determined by the serum neutralization (SN) method. Serum for HI testing was pretreated with kaolin (for measles) or with manganese chloride and heparin (for rubella) and then adsorbed with the appropriate test erythrocytes (i.e., African green monkey erythrocytes for measles, newly-hatched chick erythrocytes for rubella). The HI assays were done in microtitre plates, in which 0.025-ml volumes of serial 2-fold dilutions of treated serum were tested against 4 hemagglutination (HA) units of Norrby- type measles HA antigen, or against 4 HA units of commercial rubella HA antigen. The highest dilution of serum completely inhibiting hemagglutination was taken as the antibody endpoint. Although this microtitre system yields considerably lower titres than the tube hemagglutination technique, its con- venience for large-scale work outweighed this dis- advantage. Pairs of serum samples that failed to show obvious seroconversion in the microtitre sys- tem were retested by the conventional tube technique (12). Mumps antibody titres were determined by the VERO cell microtitre serum neutralization (SN) technique (13). If postinoculation antibody was not detected by this technique, paired serum samples were retested undiluted and at 1: 2 dilution by a plaque reduction technique in VERO cells (14). Children were considered susceptible to a disease if antibody to that disease was not detectable (measles HI titre <1: 2, mumps SN titre <1: 2, rubella HI titre <1: 8) in the preinoculation serum. Seroconversion after inoculation was determined by the appearance of antibody in the postinoculation serum sample of a previously susceptible child. Serum samples were always tested as pairs. All serologic assays were double blind. Results were then entered into a computer for automated decod- ing and calculation and tabulation of seroconversion rates and geometric mean titres. Study design A randomized double-blind design was used to distribute the study population among the 3 lots of vaccine and 1 lot of placebo in the ratio of 1 placebo subject to every 5 vaccinees. Each vial of vaccine or placebo was labelled with a unique serial number that became the subject number of the child inocu- lated from that vial. A blood sample was obtained prior to vaccination. Clinical observations for pos- sible vaccine reactions and intercurrent illnesses were made approximately 3 times per child during the study period from about day 7 to day 21. The results of these observations were decoded and tabulated by computer. A second blood sample was taken 8 weeks after vaccination. The children who had originally received the placebo were then vaccinated. Study population The children admitted to this study were residents of the Herrera District of Santo Domingo, Domin- ican Republic. Requirements for inclusion in the study were: (a) informed consent of the parents; (b) absence of any history of natural measles, mumps, or rubella, or of immunization against these diseases; and (c) absence of all the usual medical contraindications to immunization with live, at- tenuated virus vaccine. RESULTS Clinical findings A total of 926 children participated in the study. Their age distribution and age-specific susceptibility rates are shown in Table 1. The sharp drop in triple susceptibility rate in those aged 2 and 3 years is due mainly to natural measles infection. The apparently higher triple susceptibility rate in those 5 and 6 years of age is an artifact probably caused by the small number of that age sampled. Other serologic surveys of children in and around Santo Domingo revealed no such unevenness in the decline of triple suscepti- bility rate with age (15). There were no reports of any reactions at the injection site, and no immediate or early adverse systemic effects of the injection. Table 1. Age distribution and age-specific triple susceptibility rates in the study population Age in years Number Tripleof subjects susceptible (%) 1 181 50.3 2 179 26.8 3 209 14.3 4 247 7.3 5 94 10.6 6 4 50.0 7 1 0.0 unknown 11 9.1 total 926 21.6 82 NEW MEASLES-MUMPS-RUBELLA COMBINED LIVE VIRUS VACCINE Table 2. Distribution of maximum axillary temperature readings from the 7th to the 21 st day after vaccination Temperature Triple-susceptible Placebo range (°C) vaccinees (all lots) subjects 36.7 6 (3.7) a 6 (4.7) a 36.7-37.2 122 (74.8) 96 (75.6) 37.3-37.7 27 (16.6) 22 (17.3) 37.8-38.3 3 (1.8) 2 (1.6) 38.4-38.8 1 (0.6) 1 (0.8) 38.9-39.4 2 (1.2) 0 39.5-40.0 2 (1.2) 0 total 163 127 a Figures in parentheses are percentages. The distribution of axillary temperature readings from the 7th to the 21st day after vaccination are shown in Table 2. The vaccinees showed slightly higher frequencies than those given placebos at the upper limits of the temperature range, but these differences in distribution were not statistically signi- ficant (Fisher's Exact Probability: P = 0.22 for temperatures >37.8°C and P = 0.18 for tempera- tures >38.3°C). The frequencies of clinical findings other than fever during the 2nd and 3rd weeks after vaccination are shown in Table 3. Rash was slightly more Table 3. The frequencies of positive clinical findings from the 7th to the 21 st day after vaccination Triple-susceptible Susceptible vaccinees placebo (all lots) subjects rash 11 (6.7) a 6 (4.7) a lymphadenopathy 1 (0.6) 2 (1.6) conjunctivitis 7 (4.3) 5 (3.9) otitis media 4 (2.5) 1 (0.8) coryza 3 (1.0) 1 (0.8) rhinitis 65 (39.9) 55 (43.3) pharyngitis 2 (1.2) 2 (1.6) cough 1 (0.6) 0 headache 1 (0.6) 1 (0.8) a Figures in parentheses are percentages. frequent in triple-susceptible vaccinees (6.7%) than in susceptible placebo subjects (4.7 %). Although this slight excess of rash in vaccinees is similar to that which may occur with measles vaccine used singly, the difference between vaccinees and placebo con- trols in this study was not statistically significant (X2 = 0.53, P >0.1). None of the other clinical findings showed any statistically significant difference be- tween triple-susceptible vaccinees and placebo con- trols. The most frequent finding was rhinitis, which was reported more frequently in placebo subjects (43.3 %) than in triple-susceptible vaccinees (39.9 %). Symptoms that were specifically looked for but not found in this study population included bronchitis, parotitis, orchitis, and paraesthesia. Joint pain was reported for only one child, a 4- year-old triple-susceptible male vaccinee. It was described as minimal, and occurred on the 14th day after vaccination at which time the child also had rhinitis, pharyngitis, and an axillary temperature of 37.8°C. This child was free of symptoms on sub- sequent observations. Miscellaneous clinical findings unrelated to vac- cination, but characteristic of this tropical intercity population, included a wide variety of enteric, respi- ratory, and dermatologic symptoms. Such findings were as prevalent in placebo subjects as in triple- susceptible vaccinees. Serologic results Postvaccination serum samples were obtained from 439 children, of whom 84 were triple-suscep- tible. The serologic results obtained with these triple- susceptible subjects are shown in Table 4. In 72 susceptible vaccinees the seroconversion rates were 99% for measles, 94% for mumps, and 93% for rubella. The geometric mean antibody titres were 35.6 for measles, 3.9 for mumps, and 34.4 Table 4. Immune responses of 72 triple-susceptible children inoculated with the trivalent vaccine and of 12 triple-susceptible children inoculated with placebo Sero- triple-susceptiblevaccinees Placeboconversion subjects to: No. % GMTa measles 71 99 35.6 0 mumps 68 94 3.9 0 rubella 67 93 34.4 0 a Geometric mean titre. 83 N. JOEL EHRENKRANZ ET AL. for rubella. These values are no different from those usually found following the use of the same virus strains as monovalent vaccines. There were no seroconversions among the 12 triple-susceptible placebo subjects, or among 47 others with various single and double susceptibilities. DISCUSSION The occurrence of transient fever within the range 38.90C-40.00C in 4 (2.3%) of the triple-susceptible vaccinees and none of the placebo controls is con- sistent with that which would be expected following administration of the Schwarz strain measles vaccine alone (4). This, together with the lack of any statisti- cally significant difference between the distributions of temperature readings in triple-susceptible vac- cinees and placebo subjects, indicates that combin- ing the vaccines did not enhance their known minor febrile effects. The same may be said of rash, the frequency of which was only 2% higher in triple- susceptible vaccinees than in placebo subjects, and was well within the range generally reported for the Schwarz strain measles vaccine (4, 16). Comparison of the frequencies of these and other symptoms in triple-susceptible trivalent vaccinees and placebo subjects indicates that there is no enhancement of side effects when the three vaccines are combined to make a trivalent vaccine. The evidence also indicates that the 3 vaccines are as effective when given together as when used in- dividually. Since the seroconversion rates and geometric mean titres induced by the trivalent vac- cine are quantitatively equivalent to those generally induced by the component vaccines used singly, it is to be expected that the qualitative aspects of the immune response, particularly its long duration, will also be the same for the trivalent vaccine. ACKNOWLEDGMENTS Thanks are especially due to the Secretary of Health of the Dominican Republic and to members of his staff for their support. This work would not have been possible without the cooperation of the people of Santo Domingo and the help of many local physicians. We also acknow- ledge the work of our field teams, and esp:cially that of Mrs Joan Ventura whose skill enabled us to obtain repeated blood samples. The manuscript was prepared by Mrs Doris Brown. RItSUMI2 EVALUATION CLINIQUE EN RfPUBLIQUE DOMINICAINE D'UN NOUVEAU VACCIN VIVANT MIXTE ANTIROUGEOLEUX, ANTIOURLIEN ET ANTIRUBE'OLEUX Un vaccin trivalent antirougeoleux (souche Schwartz), antiourlien (souche Jeryl Lynn) et antirubeoleux (souche Cendehill) a fait l'objet, en Republique Dominicaine, d'une etude clinique portant sur 926 enfants et visant a determiner le taux de r6actions cliniques ainsi que l'efficacit6 de ce vaccin comme agent immunisant. Trois lots du vaccin en question ont ete compares a un placebo, la methode du double insu dtant employee de telle fagon qu'un enfant sur six, designe au hasard, regoive le pla- cebo. Un echantillon de sang a et preleve sur chaque enfant avant la vaccination. Du septieme ou vingt et unieme jour inclus suivant la vaccination, on a procede, toujours selon la methode du double insu, a environ trois examens cliniques de chaque enfant pour detecter les r6actions postvaccinales et les maladies intercurrentes possibles. Un dernier echantillon de sang a e't preleve' huit semaines apres la vaccination et les enfants qui avaient recu le placebo ont alors ete vaccines. Des donnees cliniques ont etd obtenues chez les 290 enfants suivis, dont 163 vaccines sensibles aux trois virus en cause et 127 sujets ayant requ le placebo. On a observ6 de la fievre un peu plus fr6quemment parmi les vaccines que parmi les autres, mais la difference n'est pas statistiquement significative. Une eruption s'est produite chez 6,7% des vaccines sensibles aux trois virus et chez 4,7% des sujets ayant regu le placebo. La proportion legerement plus elevee d'eruptions dans le groupe des vaccines sensibles correspond a ce qu'on aurait pu escomp- ter avec un vaccin antirougeoleux monovalent, la diff&e rence n'etant pas statistiquement significative. La conjonc- tivite a egalement ete plus fr6quente parmi les vaccines sensibles, mais la difference est inferieure a 1 %. Un seul enfant, vaccine' sensible, a souffert de douleurs articu- laires; elles ont et6 minimes et de courte duree. L'analyse des echantillons de sang preleves avant et apres la vaccination sur 84 enfants sensibles aux trois 84 NEW MEASLES-MUMPS-RUBELLA COMBINED LIVE VIRUS VACCINE 85 virus, dont 72 sujets vaccines et 12 sujets ayant requ le placebo, a donn6 les r6sultats suivants: parmi les vac- cines, acquisition d'une immunit6 a la rougeole chez 99 %, la moyenne geometrique des titres etant de 35,6, d'une immunit6 aux oreillons chez 94% (moyenne: 3,9), et d'une immunite a la rubeole chez 93% (moyenne: 34,4). Ces donnees cliniques et serologiques indiquent que les trois vaccins consid6res presentent la meme securit6 d'emploi et la meme efficacite lorsqu'ils sont administres ensemble que lorsqu'ils sont utilis6s separement. REFERENCES 1., MEYER, H. M. El AL. In: First International Confer- ence on Vaccines against Viral and Rickettsial Diseases of Man, Washington, DC, November, 1966. Washington, DC, Pan American Health Orga- nization, 1967, pp. 336-342. 2. REY, M. In: Proceedings of the International Confer- ence on the Application of Vaccines against Viral, Rickettsial, and Bacterial Diseases of Man, Wash- ington, DC, December, 1970. Washington, DC, Pan American Health Organization, 1971, pp. 407-412. 3. SCHWARZ, A. J. F. Annales de pediatrie, 202: 241-252 (1964). 4. ScHWARZ, A. J. F. ET AL. Journal of the American Medical Association, 199: 26-30 (1967). 5. WEIBEL, R. E. ET AL. New England journal of medicine, 276: 245-251 (1967). 6. HILLEMAN, M. R. ET AL. New England journal of medicine, 276: 252-258 (1967). 7. HELLEMAN, M. R. ET AL. New England journal of medicine, 278: 227-232 (1968). 8. DUPAN, R. M. ET AL. American journal ofdiseases of children, 115: 658-662 (1968). 9. PINziE, A. ET AL. American journal of diseases of children, 118: 172-177 (1969). 10. LiNNEMANN, C. C. JR ET AL. American journal of epidemiology, 95: 238-246 (1972). 11. COOPER, L. Z. ET AL. Journal ofthe American Medical Association, 207: 89-93 (1969). 12. ROSEN, L. Virology, 13: 139-141 (1961). 13. KENNY, M. T. ET AL. Applied microbiology, 20: 371- 373 (1970). 14. KrENNY, M. T. ET AL. Journal of biological standard- ization, 3: 291-306 (1975). 15. KENNY, M. T. ET AL. American journal of epidemi- ology, 103 (1976) (in press). 16. ANDELMAN, M. B. ET AL. American journal ofpublic health, 56: 1891-1897 (1966).