The minimum protective level of antibodies in smallpox*

SMALLPOX The minimum protective level of antibodies in smallpox* J. K. SARKAR,1 A. C. MITRA,2 & M. K. MUKHERJEE8 Blood samples from 57 contacts of6 smallpox cases were testedfor haemagglutination- inhibiting (HI) and neutralizing antibodies. All 6 contacts who subsequently developed smallpox were unvaccinated and had neutralizing antibody titres of 10 or less. However, 6 unvaccinated contacts with similar antibody levels did not develop smallpox. None of the 41 vaccinated contacts, regardless of their antibody level, contracted the disease. As in many other infectious diseases, the minimum level of humoral antibody necessary to prevent small- pox is not known. It is generally thought that a high level of antibody, specially neutralizing antibody, circulating in the blood protects an individual against smallpox, although the disease has been reported to occur in persons with a high level of neutralizing antibody (1). In the present paper, an attempt is made to examine this question. The study is incom- plete, but because the rapid disappearance of small- pox from Calcutta and nearby areas has meant that subjects can no longer be found for study, it was thought worth while to document whatever data have been collected, and to try if possible to draw some conclusions. Work of this nature does not appear to have been done previously. MATERLALS AND METHODS The procedure followed was to collect blood samples from voluntary donors amongst the contacts of smallpox cases in the Calcutta area, test the samples for haemagglutination-inhibiting (HI) and neutralizing antibodies, and follow up the donors to see if they developed smallpox. * From the Department of Virology, School of Tropical Medicine, Chittaranjan Avenue, Calcutta-12, India. This work was supported by a research grant from the World Health Organization. 1 Professor of Virology and Officer-in-Charge, WHO Research Project on Smallpox, School of Tropical Medicine, Calcutta, India. * Research Officer, WHO Research Project on Smallpox, School of Tropical Medicine, Calcutta, India. 3 Demonstrator ofVirology, School ofTropical Medicine, Calcutta, India. Smallpox patients Only virologically proved cases were included in the study. The patients were clinically examined, and the presence or absence of vaccination marks was recorded. The smallpox patients whose contacts were studied were designated as " index cases ". The criteria for classifying cases in descending order of severity as " haemorrhagic ", " confluent " and " discrete " and the definition of the word " con- tact" were the same as adopted previously (5-7). The presence of a vaccination scar was taken as evidence of previous vaccination. Vaccinations done after the day of our first visit, and histories of pre- vious vaccinations or revaccinations, were not recorded. The day of onset of fever was taken as the date of onset of the disease. The contacts were visited every 3rd or 4th day for 3 weeks to ascertain if any of them developed smallpox. Whether the contacts lived in the same room or in the same com- pound as the index cases was recorded. Collection of blood On the first day of our visit after an index case was traced, blood specimens were collected from con- tacts. Venous blood was drawn whenever possible. In 15 instances when permission for this was refused, blood was collected on filter paper discs by finger tip puncture (2). The discs were air-dried and preserved at -20°C. Before testing they were soaked in saline overnight in the refrigerator; the fluid was then tested for antibodies. The technique was shown to be satisfactory by simultaneous testing of the venous blood and finger tip blood of the same subjects (3 vaccinated volunteers and 3 smallpox patients). 3354 - 307 - BULL. WORLD HEALTH ORGAN., Vol. 52, 1975 SMALLPOX HI and neutralizing tests The techniques were the same as described previ- ously (8). The sensitivity of the neutralizing tests was checked from time to time by incorporating a refer- ence serum obtained from the Statens Seruminstitut, Copenhagen, in the same tests together with a hyper- immune serum prepared locally. RESULTS Altogether 57 contacts of 30 index cases (16 dis- crete, 9 confluent, and 5 haemorrhagic) were studied; 15 of the contacts had no primary vaccination mark, but the others had. The age of the contacts varied from 4 to 70 years; 34 were males and 23 females. Twenty-seven contacts lived in the same room as the index case concerned, and 30 in the same compound. Only 6 of the 57 contacts developed the disease. The details are shown in Table 1, while the HI and neutralizing antibody titres of the 57 contacts are shown graphically in Fig. 1. DISCUSSION As the number of contacts studied was rather small, any deductions drawn from the results should be taken as provisional. All 6 contacts who contracted smallpox were previously unvaccinated, and all had HI antibody titres (expressed as the reciprocal of the highest dilution of serum inhibiting haemagglutination or pock formation) of 20 or less and neutralizing anti- body titres of 10 or less. None of the contacts, vac- cinated or unvaccinated, with a pre-existing neutral- izing titre of 20 or more developed the disease. However, other contacts who were unvaccinated and had antibody levels as low as the 6 who con- tracted smallpox did not develop the disease. It is worth noting that none of the vaccinated contacts, even without detectable neutralizing antibody, developed smallpox. These findings indicate that the humoral antibody level at the time of infection may not be the only factor in determining whether a person exposed to infection will develop the disease, and that other factors such as tissue immunity may play some role. Secondly, the results show that the rapid formation of antibody after infection and not its initial level in the blood may be an important factor in deciding whether the infecting organism is able to produce clinical illness. Four of the 6 contacts who developed the disease lived in the same room as an index case, but the other 2 did not, although they lived in the same compound. It has previously been shown (4) that the infection rate is practically the same among room contacts and compound contacts, because in any case the people mix freely. Of the 6 secondary cases, 4 contracted the disease from discrete index cases, 1 from a confluent case, and 1 from a haemorrhagic case, suggesting that the mildness of a case does not in any way diminish its ability to spread infection. All the secondary cases were discrete, although the clinical form of the index cases varied. As pointed out previously (5), the expression of clinical illness in an individual depends on the interaction between the virulence of the invading organism and the immunity developed in the individual. Five of the 6 secondary cases developed the disease 10-12 days after the onset of fever of the index cases, and the other 7 days after. It may be that the short incubation period of the latter case was attributable to a massive inoculum from the index case concerned (haemorrhagic cases are known (6) to excrete a very high titre of virus in the throat) or to his acquiring the infection during the incubation period of the index case (7). The sera of 2 unvaccinated contacts (serial Nos. 1 and 2) showed a fair level of antibody. The explana- tion might be that they had earlier had subclinical infections, successful vaccinations without a scar persisting, or mild smallpox without detectable pock marks (3). 80 40 N titre HI titre 160 80 xx x x 20 40 x xxx XXxx xx xx XX xx x xxXXXX x x x x x 10 x x x x 20 x x x x xxXxx xxxxx xxxxxxx xxxx xo x x <10 x x x x 10 x x x x xx xx xxse x x x x x x se) xxxxxxx xXxxxxxx VAC. UNVAC. VAC. UNVAC. Fig. 1. HI and neutralizing (N) antibody titres of the 57 contacts (shown by crosses). Circles round the crosses denote contacts who developed smallpox. 308 J. K. SARKAR ET AL. 309 Table 1. Antibody titres and other details of contacts of smallpox cases Whether developed Living in D n f Antibody titre bsmallpox; if so, Serial No. Age (years) Primary same room Type of Duraitonof number of days after of contact and sex vaccination as index index case a contact onset of index case, case or not (days) H Netaing and type ofaHI eu alizing secondary casea 40 10 <10 10 10 10 10 10 10 <10 10 <10 20 10 <10 10 10 40 40 10 <10 10 <10 <10 10 40 10 <10 <10 <10 <10 20 10 <10 No No No No No No No No No No Yes; 13 days; D No No No No No No No No No No No No No No No No No No No No No Yes; 13 days; D Yes; 11 days; D 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35/M 40/ F 23/M 38/M 60/M 24/M 25/M 17/F 25/F 18/M 13/F 14/F 29/F 40/M 30/F 30/ F 40/F 13/F 25/M 29/M 70/F 17/F 29/M 18/M 23/M 45/F 18/M 21/M 30/F 22/F 35/M 4/M 35/M 50/M - Yes - Yes - No + Yes + Yes + Yes + Yes + Yes - Yes + Yes - Yes + No + No + Yes - Yes - Yes + No + No + Yes + No + No + No + No No + No + No + Yes + Yes - Yes Yes + No + Yes - Yes - No C C C C C H H D D D D D D D D D D D D D D D H D D D C C c C c D D D 3 3 3 7 7 2 2 8 8 8 8 8 8 5 5 5 5 5 7 7 7 7 2 6 6 6 7 7 4 4 4 5 6 6 80 40 10 10 40 20 20 20 10 10 20 10 40 20 10 10 20 80 40 40 10 40 10 10 20 80 20 20 20 10 20 40 20 20 a H = haemorrhagic, C = confluent, and D = discrete. b Expressed as reciprocal of highest dilution of serum inhibiting haemagglutination or pock formation. In the neutralizing test, the comparable titre for the international standard antismallpox serum was 320. Table 1. Antibody titres and other details of contacts of smallpox cases (continued) Whether developed Living in D of Antibody titre b smallpox; if so,Serial No. Age (years) Primary same room Type of uration o number of days after of contact and sex vaccination as index index case a (dacn) onset of index case, case or not ays H Neutralizing secondrye ofaHi ~~ ~~~eonaycasea 35 25/M + No D 11 10 < 10 No 36 30/M + No D 11 160 80 No 37 6/F + No D 11 10 < 10 No 38 5/F + No D 11 20 < 10 No 39 11/F + Yes D 6 10 < 10 No 40 12/M + Yes D 6 20 < 10 No 41 31/M + Yes C 5 10 < 10 No 42 14/M + No D 5 80 20 No 43 29/M + No D 6 40 20 No 44 38/M + No D 6 10 < 10 No 45 17/M + Yes D 8 20 20 No 46 16/F - No C 5 20 10 Yes; 10 days; D 47 25/M + No D 7 20 10 No 48 28/M - Yes H 3 10 < 10 Yes; 7 days; D 49 30/F + No D 7 40 40 No 50 38/F + No C 5 40 20 No 51 40/M + No H 2 10 10 No 52 35/M + No D 8 40 20 No 53 13/M + Yes D 7 iO < 10 No 54 30/F + No C 5 40 20 No 55 24/M + No H 3 40 10 No 56 27/F - Yes D 7 10 < 10 Yes; 12 days; D 57 25/M + No C 5 20 10 No ACKNOWLEDGEMENTS The authors are indebted to Dr D. A. Henderson and Dr I. Arita of the Smallpox Eradication Unit, World Health Organization, Geneva, Switzerland, for their advice, help and constructive criticism; to the UNICEF East India Office for the loan of a vehicle to carry out the field work; and to Mr A. Pal for technical assistance. R2SUMIt NIVEAU MlNIMAL D'ANTICORPS NiCESSAIRE POUR CONFiRER UNE PROTECTION CONTRE LA VARIOLE Afin de d6terminer approximativement le titre minimal sur 57 contacts de 6 cas de reference pour la recherche d'anticorps necessaire pour conf6rer une protection d'anticorps inhibant l'hemagglutination (HI) et d'anti- contre la variole, des 6chantillons de sang ont ete preleves corps neutralisants (N). Six de ces contacts ont contracte 310 SMALLPOX J. K. SARKAR ET AL. 311 la variole. Ces 6 personnes n'avaient pas ete vaccinees et presentaient des titres d'anticorps N egaux ou infe- rieurs A 10. Toutefois, 6 autres contacts non vaccin6s presentant des titres d'anticorps N egaux ou inferieurs A 10 n'ont pas contracte la maladie. Tous les contacts chez lesquels les titres d'anticorps N etaient egaux ou superieurs A 20 sont rest6s indemnes, mais les contacts vaccines chez lesquels la recherche d'anticorps N est restee negative n'ont pas non plus contracte la maladie. REFERENCES 1. DOWNIE, A. W. ET AL. Virus and virus antigen in the blood of smallpox patients. Their significance in early diagnosis and prognosis. Lancet, 265: 164-166 (1953). 2. DUCA, M. ET AL. Sampling of blood on filter-paper disks for serological investigations. Microbiol. Parazit. Epidemiol., 13: 459-464 (1968). 3. HEINER, G. G. ET AL. A study of inapparent infection in smallpox. Am. J. Epidemiol., 94: 252-268 (1971). 4. MUKHERJEE, M. K. ET AL. Pattern of intrafamilial transmission of smallpox in Calcutta, India. Bull. World Health Organ., 51: 219-225 (1974). 5. SARKAR, J. K. & MITRA, A. C. Virulence of variola virus isolated from smallpox cases of varying severity. Indian J. med. Res., 55: 13-20 (1967). 6. SARKAR, J. K. ET AL. Virus excretion in smallpox. 1. Excretion in the throat, urine and conjunctiva of patients. Bull. World Health Organ., 48: 517-522 (1973). 7. SARKAR, J. K. ET AL. Virus excretion in smallpox. 2. Excretion in the throats of household contacts. Bull. World Health Organ., 48: 523-527 (1973). 8. SARKAR, J. K. ET AL. Antibody response in haemorrha- gic smallpox. Indian J. med. Res., 55: 1143-1149 (1967).