Large-scale use of freeze-dried smallpox vaccine prepared in primary cultures of rabbit kidney cells

Large-scale use of freeze-dried smallpox vaccine prepared in primary cultures of rabbit kidney cells A. C. HEKKER,' J. M. BOS,2 N. KUMARA RAI,3 J. KEJA,4 G. CUBONI,4 B. EMMET,4 & J. DJALINS 5 A lyophilized smallpox vaccine madefrom infected monolayer cultures ofprimary rabbit kidney cells was used together with a calf lymph vaccine in a field trial in Lombok, Indonesia, in 1973. About 60 000 children below 15 years of age were vaccinated: some 50 000 with the tissue culture vaccine and about 10 000 with calf lymph vaccine. Similar results were obtained with both vaccines in primary vaccinees and in revaccinees as regards the take rate, pock reactions, and serious secondary reactions. Smallpox vaccines prepared on the skin of living animals have been used for the vaccination of human beings for many decades. These vaccines have been shown to be immunogenic as well as to protect against smallpox. Although the introduction of the seed virus principle, the use of a better defined strain of vaccinia virus, and, in many places, the use of freeze-dried vaccine instead of liquid glycerinated vaccine improved safety and stability, certain recent developments in virology have not yet been applied to the production of smallpox vaccine. Hitherto the results of the use of vaccines produced in cell cultures have been unsatisfactory, since passaging the vac- cinia virus through cell cultures only a few times results in a vaccine that is less immunogenic. For this reason WHO does not accept vaccines made in tissue cultures if there have been more than 5 passages of the vaccinia virus (8). In the Netherlands a freeze-dried smallpox vaccine was prepared in primary rabbit kidney cell cul- tures (5). The seed virus for the production of this vaccine was prepared in calves and was the same as that used for the production of calf lymph vaccine. The virus was thus not more than one passage I Head, Laboratory of Virology (formerly Head, Small- pox Vaccine Laboratory), Rijks lnstituut voor de Volksge- zondheid, Postbus 1, Bilthoven, Netherlands. I Assistant virologist, Smallpox Vaccine Laboratory, Rijks Instituut voor de Volksgezondheid, Bilthoven. 3 Directorate General, Department of Health, P.O. Box 223, Jakarta, Indonesia. 4 World Health Organization, P.O. Box 302, Jakarta, Indonesia. 5 Directorate of the Health Service, Propinsi Nusa Tenggara Barat, Mataram, Lombok, Indonesia. removed from animal skin. Satisfactory potency was obtained by concentration through ultrafiltration. Intensive laboratory and field studies with this vaccine have been conducted in the Netherlands. No differences as regards the development of vaccination illness, the take rate, and antibody-inducing capacity were observed between the tissue culture vaccine and calf lymph vaccine in approximately 1600 persons subjected to either primary vaccination or revaccina- tion (4, 6). In order to supplement and confirm these results on a larger scale under field conditions, the island of Lombok, Indonesia, was chosen for the mass vaccination of 50 000 children below 15 years of age with the tissue culture vaccine. The take rate and adverse reactions were observed. For compara- tive purposes, 10 000 other children were vaccinated with calf lymph vaccine. Both vaccines were pro- duced in the Rijks Instituut voor de Volksgezond- heid, Bilthoven, were freeze-dried, and had a log potency of 8.5 pock-forming units per millilitre. The studies were conducted in May and June 1973. The Indonesian government authorities were involved in the discussions about the study from the beginning and fully agreed to it. The results are presented in this report. LOCATION, MATERIALS, AND METHODS Lombok Lombok is situated east of Java, between Bali and Sumbawa. The island has an area of approximately 5000 km2. The northern half is mountainous and the southern half flat. The great majority of the about 1.5 million inhabitants live in a strip 15-20 km wide, 3519 - 279 BULL. WORLD HEALTH ORGAN., Vol. 54, 1976 280 A. C. HEKKER ET AL. which traverses the island from west to east just below the centre. There are five towns in this strip: Ampenan, Mataram (capital of the island and of the west district, Lombok Barat), Cakranegara, Selong (capital of the east district, Lombok Timur), and Praja (capital of the central district, Lombok Tengah). The total population of these five towns is approximately 100 000. The other 1.4 million people live mostly in small villages scattered over the island. The people live on agricultural products, mainly rice. Design of the study On the basis of a scar survey, carried out by the Provincial Health Services in July 1972 on 7000 children, at least 20% (12 000) primary vaccinees could be expected in a sample of 60 000 children. This number was considered to be sufficient for the study. Fifteen experienced vaccinators were available for 8 weeks for vaccination and follow-up by house- to-house visit of some 60 000 children below 15 years of age. The tissue culture smallpox vaccine was pre- pared from the Elstree strain as described earli- er (5). The calf lymph vaccine was prepared in the same laboratory by a technique similar to that described in "' Methodology of freeze-dried smallpox vaccine production ".u Both types of vaccine were freeze-dried in 0.2-mI quantities. The titrations were done as described by Hekker & Bos.b The log titre of both vaccines was 8.5 pock-forming units per milli- litre. During the trial and at the end, vaccine samples were taken in the field and titrated in the laboratory in Bilthoven. The virus titre was the same as had been found before. For practical purposes it was decided to vaccinate on 5 days a week with tissue culture vaccine and on I day a week with calf lymph vaccine. Preparatory phase After the central and local government authorities had agreed to the proposed study, the local staff, vaccinators, and recorders were selected and in- structed. Out of a total of 206 villages and 5 towns, 55 villages and 4 towns were randomly chosen for inclusion in the study. The cooperation of the village heads and village population was sought and ob- tained. Forms were prepared to record the family name, first name, age, sex, and vaccination history. A schedule was made for the vaccination and follow-up a Unpublished WHO document SE/68.3, Rev.i, 1968. b Hekker, A. C. & Bos, J. M. Potency testing of smallpox vaccine. Unpublished WHO document SE/72.43, 1972. of 60 000 children with the tissue culture vaccine and 10 000 with the calf lymph vaccine within 6-8 weeks. Vaccination The vaccinations were carried out by 15 teams during May and June 1973. Each team consisted of one vaccinator, one recorder, and one inhabitant of the village where the team was working. An average of 1700 vaccinations were performed each day with a bifurcated needle. Four medical officers and one technical officer assisted in the instruction, organiza- tion, coordination, and supervision. Two Landrovers and three jeeps were available for transport. Readings For the reading of vaccination reactions the vac- cinees were revisited 13-14 days after vaccination. For this purpose 3 or 4 teams were used. The teams were instructed to consider a vaccination result as positive only if the child showed a so-called " major reaction " as described in the " Handbook for small- pox eradication programmes in endemic areas ".c If the children were absent on the day of the inspection, further attempts were made to identify the primary vaccinees and to record the results of vaccination at a second visit within a couple of days of the first. An extra form was designed to obtain as much informa- tion as possible about the vaccinees who died during the 13-14 days following vaccination. RESULTS A total of 61 808 children were vaccinated: 51 268 (82.9°,) with tissue culture vaccine and 10 540 (17.1%) with calf lymph vaccine; 15 390 (30.0%O) of those vaccinated with tissue culture vaccine and 2992 (28.4%) of those vaccinated with calf lymph vaccine were primary vaccinees. The vaccinated children were distributed into 4 age groups: < 1, 1-4, 5-6, and 7-14 years of age. The last two age groups were fixed because, during 1965, 1966, and early 1967 Lombok suffered a smallpox epidemic of some 21 000 cases. Children of 7 years and over had therefore lived through this epidemic. Comparison of the two vaccine groups Sex distribution. Since no sex differences were found, the results for boys and girls are presented together. c Unpublished WHO document SE67/5 Rev.I, 1967. SMALLPOX VACCINE Table 1. Distribution by age group of children subjected to primary vaccination and revaccination with tissue culture or calf lymph vaccine Primary vaccination Revaccination Age group Tissue culture Calf lymph Tissue culture Calf lymph (years) vaccine vaccine vaccine vaccine No. % No. % No. % No. % < 1 4 014 26.1 729 24.4 201 0.6 51 0.7 1-4 9 572 62.2 1 890 63.2 14 696 41.0 3 036 40.2 5-6 1 180 7.7 251 8.4 9 192 25.6 1 940 25.7 7-14 624 4.1 122 4.1 11 789 32.9 2 521 33.4 Total 15 390 100 2 992 100 35 878 100 7 548 100 Age group distribution. Table 1 gives the distribu- tion according to age group of children who received primary vaccination and revaccination with each of the two vaccines. For both vaccines, the distribution of primary vaccinees was not significantly different from that of revaccinees (P >0.10). Unrecorded vaccination results. Table 2 gives the numbers and percentages of unrecorded results of primary vaccination and revaccination with each of the two vaccines and in each of the four age groups. The percentages of unrecorded results varied some- what among the different age groups and the per- centages for calf lymph vaccine were somewhat higher than those for tissue culture vaccine. This can be explained by the fact that absenteeism in different villages varied between 0%0 and 35%4 depending on the presence of a school, a market, or work in the rice fields. Calf lymph vaccine was used in only 15 of the 55 villages in which children were vaccinated, and in 3 of these 15 villages extremely high percentages of absenteeism were found. Success rate. Table 3 shows the success rates for primary vaccination and revaccination according to type of vaccine for each of the four age groups. There were no significant differences in the success rate between the two types of vaccine, either after pri- mary vaccination or after revaccination in the cor- responding age groups. The success rate in the age group 7-14 years in primary vaccinees and in revac- cinees for both types of vaccine was a little lower Table 2. Number and proportion (%) of unrecorded results of primary vaccination and revaccination with each of the two vaccines in each of the four age groups Primary vaccination Revaccination Age group Tissue culture Calf lymph Tissue culture Calf lymph (years) vaccine vaccine vaccine vaccine No. % No. % No. % No. % < 1 120 3.0 34 4.7 12 6.0 8/51 a 1-4 436 4.6 111 5.9 1 286 8.8 343 11.3 5-6 72 6.1 22 8.8 1 102 12.0 292 15.1 7-14 85 13.6 29 23.8 2 712 23.0 640 25.4 Total 713 4.6 196 6.6 5112 14.2 1 283 17.0 a Numerator = number of unrecorded vaccination results; denominator = number of vaccinees in the group. 281 282 A. C. HEKKER ET AL. Table 3. Success rate for primary vaccination and revaccination with each of the two vaccines in each of the four age groups Primary vaccination Revaccination Tissue culture Calf lymph Tissue culture Calf lymph Age group vaccine vaccine vaccine vaccine (years) No. in Success No. in Success No. in Success No. in Success group rate (%) group rate (%) group rate (%) group rate (%) recorded recorded recorded recorded < 1 3 894 96.8 695 96.4 189 73.0 26/43 a 1-4 9 136 97.8 1 779 98.0 13410 78.6 2693 75.8 5-6 1 108 96.6 229 93.5 8 090 77.4 1 648 74.9 7-14 539 88.1 93 88.2 9077 65.8 1 881 61.7 Total 14 677 97.1 2 796 96.9 30 766 74.7 6 265 71.2 a Numerator = number of successful vaccinations; denominator = number of vaccinees in the group. than the success rate in the other age groups for the corresponding type of vaccine. Complications after vaccination. All reported ill- nesses that occurred subsequent to vaccination were carefully evaluated. None of the children in either group experienced disseminated vaccinia, eczema vaccinatum, or vaccinia necrosum. Although tran- sient allergic skin reactions may have occurred, none was serious enough to be brought to the attention of the investigators. A number of cases of severe malaria, gastroenteritis, and pneumonia occurred in both vaccinated and unvaccinated children. Among those in the study group, illnesses were randomly distributed according to the time of onset and their occurrence appeared to bear no relationship to vaccination. One case of encephalitis occurred that may have been vaccine-related. The patient-a 5-month-old girl -- underwent primary vaccination with tissue culture vaccine on 7 May, the first day of the campaign. One week later the mother brought the child to the doctor because it had fever. The child had a major reaction at the vaccination site. A nurse injected penicillin and streptomycin intramuscularly and advised the mother to come back if the fever did not disappear. Four days later the mother returned. The fever had persisted and the child had experi- enced convulsions and had been unconscious for several days. The body temperature was 41°C and the child showed neck stiffness. The corneal reflex was negative. Late at night barbiturate and anti- pyretic injections were given. The next morning the body temperature was 38.5°C and the neck stiffness had disappeared, but the child was still unconscious. A blood smear showed no malaria parasites. The same morning the child died from asphyxia during a convulsion. DISCUSSION Smallpox vaccine made in tissue culture has been used on a very limited scale. This may be due to difficulties in preparing a vaccine that is satisfactory in potency and stability. Ehrengut (2) mentions the long-term use of smallpox vaccine made in mono- layer cultures of primary bovine embryonic muscle cells. The vaccinia virus in this vaccine was passaged 10 times in cultures of these cells. In order to fulfil the WHO requirements (8), the number of passages was reduced to five. However, in order to obtain, in primary vaccinees and in revaccinees, a take rate comparable to that obtained with dermovaccine, the titre of the tissue culture vaccine had to be at least 0.5 logs higher than the titre of the dermovaccine. Undoubtedly, in the present trial, some who had faint or imperceptible vaccination scars were errone- ously assigned to the unvaccinated group. Since sma!lpox vaccination scars become less evident in time, such errors of assignment may be expected to be more frequent with older than with younger children. Moreover, it may be assumed a that about a Foster S. Persistence of facial scars of smallpox in West African populations. Unpublished WHO document SE/72.34, 1972. SMALLPOX VACCINE one-third of those who had experienced smallpox during the 1965-1966 smallpox epidemic in Lombok, no longer bore the destructive facial pockmarks and so were erroneously assigned to the unvaccinated group. Others who were already protected against clinical illness by prior vaccination, undoubtedly experienced reinforcement of their immunity, thus accounting for a lower proportion of major reactions among revaccinees in the older age groups. The patient with the symptoms described on page 282 apparently had encephalitis. The disease started to develop 6-7 days after primary vaccination. Approximately 90%O of cases of post-vaccination encephalitis start to develop in the second week after vaccination (1). However, it is impossible to rule out with certainty the possibility that this child had post- vaccination encephalitis. In Indonesia and other tropical areas, there are various causes of encephali- tis, especially in very young children. Even if this case was vaccine-related, post-vaccination encephalitis is a recognized complication of smallpox vaccination and occurs with a certain frequency (2, 3, 7). The incidence of encephalitis in mass vaccination cam- paigns-which include primary vaccination and re- vaccination at any age-varies between 1 in 8300 or less and 1 in 150 000 or more (1). The conclusion is that lyophilized smallpox vac- cine prepared from the Elstree strain, with only one passage in monolayer cultures of primary rabbit kidney cells and meeting the WHO requirements for safety, potency, and stability, is comparable with calf lymph vaccine as regards the success rate, pock reaction, and serious secondary reactions. ACKNOWLEDGEMENTS The statistical evaluation of the data was done by the Biometrical Section, Rijks Instituut voor de Volksgezondheid (Dr V. M. Sekhuis). The cooperation of the Ministry of Health, Indonesia, and the Provincial Inspectorate of Health, Lombok, is gratefully acknowledged. The World Health Organization gave financial assistance and helped to facilitate arrangements for the study. RItSUMI UTILISATION A GRANDE ECHELLE DE VACCIN ANTIVARIOLIQUE LYOPHILISE, PREPARE EN PRIMOCULTURE DE CELLULES RENALES DE LAPIN Pendant de nombreuses decennies, on a utilise pour vacciner l'homme des vaccins antivarioliques prepares sur la peau d'animaux vivants, et ces vaccins se sont montres immunogenes de meme que protecteurs. Bien que l'introduction du principe du virus de semence, l'utilisation de souches de virus vaccinal mieux definies et, en maints endroits, 1'emploi de vaccin lyophilise A la place du vaccin liquide glycerine aient accru la securite et la stabilite, certains progres recents de la virologie n'ont pas encore ete appliques A la production du vaccin antivariolique. Jusqu'ici les resultats obtenus avec des vaccins produits en culture de cellules n'ont pas et satisfaisants car un petit nombre de passages du virus vaccinal en culture cellulaire suffit pour abaisser le pou- voir immunogene du vaccin. C'est pourquoi l'OMS n'accepte pas ce type de vaccin si le nombre de passages du virus depasse cinq. Aux Pays-Bas, un vaccin antivariolique lyophilise a et prepare en primoculture de cellules renales de lapin. Le virus de semence utilise pour la production de ce vaccin a et prepare sur des veaux, et il etait identique a celui qui est utilise pour la production du vaccin cons- titue de lymphe de veaux. Ainsi le virus vaccinal n'avait pas subi plus d'un passage a partir de son prelevement de la peau de l'animal. Une activite satisfaisante a et obtenue grace A la concentration par ultrafiltration. Ce vaccin a ete soumis A des etudes intensives au labo- ratoire et en pratique, aux Pays-Bas, en mai et juin 1973. Chez environ 1600 enfants soumis A la primovaccination ou A une revaccination, on n'a observe aucune difference entre le vaccin classique et le vaccin prepare en culture de tissu au point de vue de l'apparition de la maladie vaccinale, du taux de (( prises et de la capacite de sus- citer des anticorps. Afin de completer et confirmer ces resultats sur une plus grande echelle dans les conditions pratiques, on a choisi d'appliquer le vaccin de culture de tissu pour une vaccination de masse, dans l'ile de Lombok, Indonesie, sur environ 50 000 enfants de moins de 15 ans. On a surveille les taux de ((prises * et les reactions adverses; il y a eu un cas d'encephalite suscep- tible d'etre en rapport avec le vaccin. A des fins de comparaison, environ 10 000 enfants ont ete vaccines 283 284 A. C. HEKKER ET AL. avec de la lymphe vaccinale de veau. Les deux vaccins ont e produits au Rijks Instituut voor de Volksgezond- heid, Bilthoven, ont e lyophilises et avaient une activite (log) de 8,5 unites de formation de pustules par millilitre. Des resultats similaires ont et obtenus avec les deux vaccins chez les primovaccines et les revaccines, du point de vue du taux de e prises *, de la reaction pustuleuse et des reactions secondaires graves. REFERENCES 1. DIXON, C. W. Smallpox. London, Churchill, 1962. 2. EHRENGUT, W. Tissue culture vaccine in clinical practice. In: Proceedings of the Symposium on Smallpox, Zagreb, 2-3 September 1969. Zagreb, Yugoslav Academy of Sciences and Arts, 1969, pp. 135-143. 3. EHRENGUT, W. & EHRENGUT-LANGE, J. Postvaccinal convulsions: age disposition and prognosis. In: Pro- ceedings of the Symposium on Smallpox, Zagreb, 2-3 September 1969. Zagreb, Yugoslav Academy of Sciences and Arts, 1969, pp. 205-213. 4. HEKKER, A. C. ET AL. Field work with a stable freeze- dried smallpox vaccine prepared in monolayers of primary rabbit kidney cells. In: Proceedings of the 37th Symposium of the International Association of Biological Standardization, Bilthoven, Netherlands. Basle, Karger, 1973, pp. 187-195 (Symposia series in immunobiological standardization, vol. 19). 5. HEKKER, A. C. ET AL. A stable freeze-dried smallpox vaccine made in monolayer cultures of primary rabbit kidney cells. Journal of biological standardization, 1: 21-32 (1973). 6. HEKKER, A. C. ET AL. Pokkenvaccin geproduceerd in culturen van primaire konijneniercellen [Smallpox vaccine prepared in primary cultures of rabbit kidney cells]. Berichten Rijks Instituut voor de Volksgezond- heid, pp. 39-46 (1975). 7. LANE, J. M. Complications following smallpox vac- cination. In: Proceedings of the 37th Symposium of the International Association of Biological Standard- ization, Bilthoven, Netherlands. Basle, Karger, 1973, pp. 217-226 (Symposia series in immunobiological standardization, vol. 19). 8. WHO EXPERT GROUP ON REQUIREMENTS FOR BIOLOCG- CAL SUBSTANCES. Report. Geneva, 1966 (WHO Tech- nical report series, No 323).