Comparison of Titrations on the Chorioallantoic

Transcription

1 APPuED MICROBIOLOGY, Mar. 1968, p American Society for Microbiology Vol. 16, No. 3 Printed in U.S.A. Comparison of Titrations on the Chorioallantoic Membrane of Chick Embryos with the Rabbit Scarification Technique for the Potency Assay of Smallpox Vaccines VERNON J. FULLER AND ROBERT W. KOLB Division of Biologics Standards, National Institutes of Health, Bethesda, Maryland Received for publication 30 October 1967 The potency of the U.S. Reference Smallpox Vaccine, Lot 2, the International Reference Preparation of Smallpox Vaccine, and commercial smallpox s was determined by the chorioallantoic membrane (CAM) and rabbit scarification (RS) potency assay methods. The mean titer of the U.S. Reference (based on 107 ampoules) was pock-forming units (PFU) per ml and that of the International Reference (based on 3 ampoules) was 10 PFU/ml. A statistical analysis of the CAM data for the U.S. Reference resulted in the establishment of a table of limits of acceptance for smallpox s. Of the commercial smallpox s tested by the CAM and RS methods, 89% demonstrated potencies comparable to the U.S. Reference. Our results show that the CAM test method has application in the control testing of smallpox s produced by U.S. licensed manufacturers provided it is used within the limits discussed. A titration method for vaccinia virus, based on the number of pocks formed on the chorioallantoic membrane (CAM) of the embryonated chicken egg, was first described by Keogh in 1936 (4). Since then, many reports on the use of the CAM method for smallpox assay have been presented (1-3, 6, 8-11). Recently, the World Health Organization (WHO) adopted the CAM pock-forming method as an official method for determining the potency of smallpox s. A potency of greater than 108 pock-forming units (PFU) per ml, as determined by the CAM test method for smallpox s, has been adopted by an Expert Committee on Smallpox (12, 13). Since the official test for establishing the potency of smallpox s in the United States is the rabbit scarification (RS) test (Minimum Requirements: Smallpox Vaccine, 3rd rev., 1951, and Amendments, 1965, National Institutes of Health), and since no information was available in reference to the titer of the U.S. Reference Smallpox Vaccine, Lot 2, in embryonated eggs, our study was undertaken for the following reasons: (i) to determine the titer of the U.S. Reference in embryonated chicken eggs, (ii) to compare the potency of the U.S. Reference with that of the International Reference by the CAM and RS test methods, and (iii) to compare the potency of commercial smallpox s in relation to that of the U.S. Reference by the CAM and RS test methods. MATERIALS AND METHODS Types of. Liquid and dried smallpox s derived from either calf lymph or the CAM of embryonated chicken eggs (produced by U.S. licensed manufacturers, the International Reference Preparation of Smallpox Vaccine, and the U.S. Reference Smallpox Vaccine, Lot 2) were assayed. Dilution of s. The U.S. Reference used in the RS test was reconstituted with 3 ml of a 50% glycerolwater diluent, and subsequent dilutions were prepared in buffered saline (ph 7.0), following the dilution scheme described in the amended U.S. Minimum Requirements: Smallpox Vaccine (National Institutes of Health, 1965). For the CAM test, dilutions of the reference that were reconstituted, as previously mentioned, were prepared in Beef Heart Infusion broth (ph ). The International Reference Preparation of Smallpox Vaccine was reconstituted and diluted with Mcllvaine buffer in accordance with directions accompanying the. We reconstituted dried commercial s ac- 458 cording to the directions provided by the manufacturer, and subsequent dilutions were prepared in Beef Heart Infusion broth. Liquid s were diluted in a similar manner. CAM assay. The dropped CAM of each 12-day-old

2 VoL. 16, 1968 POTENCY ASSAY OF SMALLPOX VACCINES 459 TABLE 1. Acceptance limits of the choriallantoic membrane assays of U.S. Reference Smallpox Vaccine, Lot 2 No. of tests 99% level of significance Lowera Upper" a PFU log1o per milliliter. embryonated chicken egg was inoculated with 0.2 ml of appropriate dilutions. We tested two dilutions of on five membranes each. The eggs were placed in racks and were neither rocked nor sealed. After 2 days of incubation at 37 C, the membranes of living embryos were removed and placed in a vessel containing physiological saline; the mature pocks (at least 1 mm in diameter) on each membrane were then counted. The number of PFU per milliliter was computed from the direct count of all pocks of all dilutions, and the dilution factor was applied. Experience with lots of smallpox s produced by manufacturers and with the U.S. Reference showed that, for a countable pock range of 20 to 60 per membrane, dilutions of and for the commercial s and and for the U.S. Reference were appropriate. We selected these dilutions for the commercial s to standardize the test procedure. RS assay. The RS test was performed in accordance with the procedures described in Minimum Requirements, Smallpox Vaccine, and with the modifications described by Kolb et al. (5). A potency ratio of 0.7 (relative to the reference ) was acceptable. RESULTS Potency of U.S. Reference in embryonated chicken eggs. A single test performed on each of 107 ampoules of the U.S. Reference gave a mean titer of 108'1 PFU/ml with a standard deviation of 0.2 log. A statistical analysis of the CAM data for the U.S. Reference resulted in the establishment of a table of limits of acceptance for single and repeated sample testing (Table 1) by the CAM test method. At the 99% level of significance, the acceptable value of a single test is 108' PFU log. Comparison of potency of U.S. Reference to that of the International Reference. The potency values that we obtained for the two references are presented in Table 2. The mean value obtained for the International Reference by the CAM method is within the limits indicated above. Also, the potency of this reference was satisfactory by the RS test. Comparison of the potency of commercial smallpox s in relation to the U.S. Reference Smallpox Vaccine. The potency values obtained by the CAM and RS test for the s are given in Table 3. We observed that 89% of the potency values agreed, i.e., the potency ratios were not less than 0.7 for the RS test, and the titers were 1081 PFU/ml i 0.5 log for the CAM test. We considered the CAM test valid if the titer of the reference was within the limits presented in Table 1. Similarly, we regarded the test as satisfactory if its titer was equal to or greater than the low titer given in this table. A comparison of the potency distribution of the s (Table 4) showed that the liquid s meet the accepted 0.7 RS potency ratio and the acceptable CAM value suggested in Table 1. with only one exception. But in the case of dried s of the lots which met the RS potency requirements, only 74% of the lots were able to meet a minimal titer of PFU/ml by the CAM test. In an effort to determine a RS potency ratio which would be "equivalent" to the proposed CAM potency value, we re-evaluated the data. The potency ratios of the lots were then distributed into two new groups, i.e., and potency ratios (Table 4). TABLE 2. Potency values of International and U.S. Reference Smallpox Vaccines as determined by the chorioallantoic membrane and rabbit scarification assay methods Vaccine Rabbit scarifi- cation test Chorioallantoimembrane test Pockforming units (logio/ml) Potency ratio reference) International Reference Smallpox Vaccine (dried), 7.6 XIII , 6-62 Mean a U.S. Reference Smallpox Vaccine (dried), Lot 2 Mean a Satisfactory potency was determined by obtaining a minimum potency ratio between the test and the reference of 0.70.

3 460 FULLER AND KOLB APPL. MICROBIOL. TABLE 3. Potency values of commercial smallpox s as determined by the chorioallantoic membrane and the rabbit scarification assay methods Manufacturer and type of A B C D E F Lot no S Choriallan membrane Ass;ay method Pock-forminjg units LI) Test (logis/m toic estoc toc U.S. Reference U.. (Lot 2) scarificati, Rabbit test.,on Relativ e potenc3by e) referenci a Manufacturer and type of H I (dried) J (dried) TABLE 3.-Continued Assay method Choriallantoic Rabbit membrane tes test Lot no. - Pock-forming units (Log1o/ml) Test U.S. vcie Reference (Lot 2) Relative potency reference) 31 DiscUSSION 32 Data collected in our laboratory indicated that 33 the U.S. Reference Smallpox Vaccine, Lot 2, has 35 a high level of infectivity for the CAM of the 36 embryonated chicken eggs. The titer of the (10 PFU/ml) was of the magnitude re- G cently adopted by the WHO as necessary for 38 successful vaccination in human subjects (12, 13). Satisfactory potency was determined by oh- Successful vaccination of susceptible children taining a minimum potency ratio between the with the U.S. Reference Smallpox Vaccine, by test and the reference of 0.7. either the jet-injection or the percutaneous tech

4 VOL. 16, 1968 POTENCY ASSAY OF SMALLPOX VACCINES 461 TABLE 4. Comparison of the potency distribution of dried and liquid commercial smallpox lots Vaccine Rabbit scarification potency ratio Chorioallantoic membrane titer (107.6 PFU/ml) No. of 0.7a Type lots tested Pass Fail Pass Fail Pass Fail Pass Fail Liquid (98)b 1 (2) 44 (100) 0 (0) 44 (100) 0 (0) 38 (86) 6 (14) Dried (74) 7 (26) 27 (100) 0 (0) 26 (96) 1 (4) 20 (74) 7 (26) a Satisfactory potency was determined by obtaining a minimum potency ratio between the test and the reference of 0.7. The values and represent proposed RS potency ratios. b Numbers in parentheses indicate per cent. nique, has been reported recently by Meyer et al. (7) Ṫhe data from our study showed that the CAM test method was applicable to the estimation of the potency of unknown lots of commercial smallpox s if the titration was performed in comparison with that of the U.S. Reference Smallpox Vaccine. Examination of 27 commercial dried smallpox lots by the CAM test demonstrated that 26% of the lots failed to meet the minimal potency value of PFU/ml. However, of the 44 liquid lots tested, only 2% failed to meet the proposed CAM potency. All of the dried and liquid lots met the present RS test requirement. One factor that may be associated with the presence of a larger number of lower titers for dried s (< PFU/ml) than for liquid s is a change in viral activity during the drying process which may reduce infectivity for the chorioallantoic membrane. Another factor which may account for the difference in viral activity is that the RS test measures skin reactivity, but the CAM test is an estimation of the number of infective viral particles. Because many of the dried s did not meet the minimum proposed CAM potency value, the RS potency ratios of the dried s were re-evaluated to determine what potency value obtained by each of the two test methods would be equivalent. We observed that there was no significant difference between the accepted 0.7 RS potency ratio and a proposed level for dried s, since only one lot had a RS potency ratio of less than during the testing period. However, at the level, four lots failed the RS test and, in addition, did not meet the proposed CAM value. Furthermore, three lots which had a RS potency ratio of, failed to meet the proposed CAM value. Conversely, three other lots which met the proposed CAM value did not meet the RS potency ratio. Perhaps these few discrepancies could be resolved by replicate testing of lots instead of single tests. Should these findings continue to be observed in subsequent testing, it may be necessary to raise the RS potency ratio for dried s to for these s to be equivalent to the potency requirements, when measured by the CAM procedure. Our correlation of the RS test at the level with the proposed CAM value should also satisfy the WHO requirement for correlation of the two test methods (13). It is apparent from our data that the CAM test method has application in the control testing of U.S. licensed smallpox s, provided it is used within the limits discussed. ACKNOWLEDGMENTS We thank Harry T. Aylor and Jack C. Jenkins for technical assistance. We also express our appreciation to Clifford J. Maloney and Sidney S. Spindel, Biometrics Section, Division of Biologics Standards, for assistance in the statistical analysis of the data. LITERATURE CITED 1. BURNET, F. M., AND D. D. FARIS The technique of quantitative chorioallantoic virus titration. J. Bacteriol. 44: COCKBURN, W. C., R. M. CROSS, A. W. DoWNIE, K. R. DUMBELL, C. KAPLAN, D. MCCLEAN, AND A. M.-M. PAYNE Laboratory and vaccination studies with dried smallpox s. Bull. World Health Organ. 16: GHENDON, J. Z., F. B. GENKINA, V. N. MILUSHIN, AND L. S. MUCHNIK Comparative study of methods of evaluating the activity of smallpox. J. Hyg. Epidemiol. Microbiol. Immunol. 8: KEOGH, E. V Titration of vaccinia virus on the chorioallantoic membrane of the chick embryo and its application to immunological studies of neuro-vaccinia. J. Pathol. Bacteriol. 43:

5 462 FULLER A] ND KOLB APPL. MICROBIOL. 5. KOLB, R. W., C. B. Cox, AND H. T. AYLOR Improvements in the rabbit scarification method for the assay of smallpox. Bull. World Health Organ. 25: KRAG, P., AND M. W. BENTZON The international reference preparation of smallpox. Bull. World Health Organ. 29: MEYER, H. M., B. C. BERNHEIM, AND N. G. ROGERs Titration of live measles and smallpox s by jet inoculation of susceptible children. Proc. Soc. Exptl. Biol. Med. 118: ORLANDO, M. D., J. M. RILEY, AND W. C. PATRICK III Studies on the purification of vaccinia virus. Biotechnol. Bioeng. 6: OVERMAN, J. R., AND I. TAMM Quantitative titration of vaccinia virus on the chorioallantoic membrane. J. Immunol. 76: POLAK, M. F., L. M. BRANM, B. J. W. BEUNDERS, AND A. R. VAN DER WERFF Relationship between pock counts on chorio-allantoic membrane and percentages of "takes" in primary vaccination of human beings with two smallpox s. Bull. World Health Organ. 27: WANG, S. P., AND J. T. GRAYSTON Titration of smallpox s from ten countries sent to East Pakistan during the 1958 smallpox epidemic. Bull. World Health Organ. 20 ; WORLD HEALTH ORGANIZATION WHO expert committee on smallpox. World Health Organ. Tech. Rept. Ser. 283, p WORLD HEALTH ORGANIZATION Requirements for biological substances. World Health Organ. Tech. Rept. Ser. 323, p. 68. Downloaded from on May 2, 2018 by guest