THE AMERICAN JOURNAL OF HYGIENE

VOL. 74 JULY, 1961 NO. 1

AVIAN EMBEYO RABIES II. A COMPARISON OF THE ANTIGENICITY OF. HIGH EGG-PASSAGE AND DUCK-EMBRYO ADMINISTERED INTRADERMALLY Downloaded from https://academic.oup.com/aje/article/74/1/1/136363 by guest on 30 September 2021 IN MAN

BY PAUL R. SCHNURRENBERGER, GEORGE R. ANDERSON, JACK H. RUSSELL AND FREDERICK H. WENTWORTH* (Received for publication November 22, 1961)

Several earlier studies have demon- been possible to make valid conclusions strated the feasibility of avian embryo concerning their relative efficiency. pre-exposure rabies in the It was the purpose of the study re- human population (1-4). The chick ported here to make such a direct com- embryo (HEP), which contains parison in a selected pre-exposure hu- high egg-passage attenuated rabies vi- man group. It was also our intent to rus, had been administered primarily in compare the results of 3 participating a series of 3 intradermal of laboratories performing parallel serum 0.2 ml each at 5-day intervals, with the neutralization studies in an effort to recommendation that a booster inocula- gain information relative to the sensi- tion of 0.2 ml be given subsequently. tivity and reproducibility of this test. Such a procedure has two advantages Freshman and sophomore veterinary over those presently employed for rabies students at the Ohio State University vaccination: it reduces the risk of Veterinary College were selected for test anaphylactic reactions and provides subjects.2 The vaccines used were desig- minimal inconvenience to the recipient. nated A and B.3 The students were In a recent report describing experi- assigned these letters at random and ence with the duck-embryo rabies vac- received the corresponding vaccine cine (DEV) (5), administered in the throughout the study. The subjects, same fashion as described for the HEP inoculators and laboratories were not vaccine, it was suggested that further informed of the true identity of the investigation of this material in high 2 risk groups might be profitable. Studies Our sincere appreciation is extended to Dr. John H. Helwig and the other faculty members reported to date were not designed to of the College of Veterinary Medicine, The compare the antigenicity of the two Ohio State University, who made this study vaccines directly and it has thus not possible. 3 We are grateful to Dr. J. M. Ruegsegger i Division of Communicable Diseases and Di- of Lederle Laboratories and Dr. F. B. Peck, Jr., vision of Laboratories, Ohio Department of of Eli Lilly & Company for furnishing the Health, Columbus, Ohio. vaccines used in this study.

AM.J.HYG. 1961, VOL. 74: 1-6 SCHNURRENBEKGEB, ANDERSON, RUSSELL AND WENTWORTH

vaccines until after the laboratory re- The sera were inactivated at 56 C sults were recorded. for 30 minutes. Four-fold dilutions of Two persons did the inoculating. This serum were mixed with an equal volume necessitated a study design which would of CVS fixed rabies virus and the mix- differentiate the effect of the vaccine ture was incubated in a water bath at and inoculator technique. This was ac- 37 C for 90 minutes. The challenge complished by alternating inoculators virus was diluted in 10 per cent inacti- so that no subject was given 3 inocula- vated guinea-pig serum in saline. After tions by the same person. incubation the sera were placed in ice

Vaccines and vaccination procedures and 5 or 6 Swiss white mice weighing Downloaded from https://academic.oup.com/aje/article/74/1/1/136363 by guest on 30 September 2021 were the same as those described in approximately 10 grams each were in- previous communications (4, 5). The oculated intracerebrally with 0.03 ml of high egg-passage vaccine is a live prod- the serum virus mixture. The mice were uct, Flury strain, attenuated by 180 to observed for 15 days and 50 per cent 210 passages on chick embryos. Duck- mortality end points were calculated ac- embryo vaccine, however, is the CVS cording to the method of Eeed and strain of fixed virus, grown on duck Muench (6). Appropriate controls were embryos and inactivated with beta included with each test group. propiolactone. Both vaccines were ad- Laboratory EL used approximately 100 ministered in a series of 4 intradermal LD50 (5) of challenge virus; Laboratory inoculations of 0.2 ml on the medial as- L used 100 to 300 LD50 (7) ; and Lab- pect of the forearm. The right and left oratory O used approximately 300 LD50 forearms were alternated on successive of virus. inoculations. The primary series of 3 The antibody titer of the serum was inoculations was administered at 5-day expressed as the reciprocal of the final intervals while the fourth or booster serum dilution protecting 50 per cent dose was given 171 days after the of the mice. original . Blood samples were obtained prior to vaccination, 31 RESULTS days after the first inoculation, prior to the booster, and 7 and 30 days after the Pre-immunization sera from 119 sub- booster. jects with no history of prior antirabic treatment were examined. Three of Each serum specimen was divided into these had demonstrable neutralizing 3 aliquots and placed into screw-capped antibody. tubes. Aliquots obtained from each A total of 111 subjects with negative specimen were sent to the two pharma- pre-immunization sera received the pri- ceutical houses supplying the vaccine mary series of inoculations at the pre- while the remaining portions were stored scribed intervals and their postim- at — 20 C by our own laboratory until munization sera were tested. In each tested. of the three laboratories, antibody was The serum neutralization test (SN) demonstrated in a higher percentage of used by the participating laboratories 4 the subjects who had received the duek- was performed as follows: embryo vaccine than of those who had * We would like to acknowledge our thanks been given the high egg-passage vaccine to H. M. Powell of Eli Lilly and Company and (table 1). On the basis of the end- Jack Black of Lederle Laboratories who so graciously performed the serum neutralization point titers, this difference was statisti- studies. cally significant (P < 01) for the re- AVIAN EMBRYO RABIES IMMUNIZATION

TABLE 1 Serum neutralizing liters follomng 3 intradermal inoculations of , as determined by 3 laboratories on serum, aliquots

Lab EL LabL LabO

Vaccine HEP DEV HEP DEV HEP DEV

Number of subjects 5C 55 56 52 21 24

Per cent Per cent Per cent Per cent Per cent Per cent Downloaded from https://academic.oup.com/aje/article/74/1/1/136363 by guest on 30 September 2021

Titer Negative 33.9 11.1 41.1 23.1 37.5 23.8 (reciprocal) 1-3 17.9 10.7 15.9 30.8 37.5 33.4 4-15 35.6 60.0 35.6 26.9 8.3 28.5 16-128 12.6 18.2 7.4 19.2 16.7 14.3

Total responding 66.1 88.9 58.9 76.9 62.5 76.2 suits obtained by Laboratory EL, but sults of Laboratory EL, which showed was not significant at this level for those the greatest apparent advantage for obtained by the other two laboratories. HEP, the statistical difference was ques- This apparent advantage did not per- tionable (P = .02). sist, for the results obtained by all 3 As may be seen in figure 1, there was laboratories on the pre-booster sera no essential difference between titers ob- failed to demonstrate a significant dif- tained on the 77 individuals with sera ference between the two groups. available 7 and 30 days after the fourth According to the results from all 3 inoculations. In view of this, it is not laboratories with reference to sera ob- surprising that no significant difference tained 7 days after the booster inocula- in response could be detected between tion, HEP vaccine appeared to be the two vaccines 30 days after the slightly more antigenic than DEV vac- booster (table 2). cine. However, judging from the re- Analysis of the results in terms of

TABLE 2 Serum neutralizing titers SO days after a fourth intradermal injection of rabies vaccine, as determined by 8 laboratories on serum aliquots

Lab EL Lab L LabO

Vaccine HEP DEV HEP DEV HEP DEV

Number of subjects 51 47 51 51 10 15

Per cent Per cent Per cent Per cent Per cent Per cent

Titer Negative 7.7 14.9 11.8 21.6 10.0 20.0 (reciprocal) 1-3 2.0 4.3 9.8 7.8 20.0 26.6 4-15 17.5 19.1 15.7 27.5 20.0 26.7 16 or greater 72.8 61.7 62.7 43.1 50.0 26.7

Total responding 92.3 85.1 88.2 78.4 90.0 80.0 SCHNURRENBEHGER, ANDERSON, RUSSELL AND WENTWORTH

• significant titer (1 in 5, Laboratory EL) / failed to exhibit any neutralizing ca- rf> pacity when tested in another labora- / / • tory. The subject from whom this was (> / ?8 o° obtained showed no antibody response to ( / o • / £• / °o° / 3 injections of the HBP vaccine. ( o ' o Q The relative sensitivity of the tech- o / niques as employed by the different 1 / V > O / laboratories is demonstrated by the 8 8 < / geometric mean antibody titers reported Downloaded from https://academic.oup.com/aje/article/74/1/1/136363 by guest on 30 September 2021 • / ft ••' / by the 3 laboratories on the post-pri- / / / mary sera. For Laboratory EL it was S 1 in 5.2; for L, 1 in 3.6; and 0, 1 in 3.1. 16 3264128256512 As might be expected, the height of the 7 Do, Tito Reciprocal titer varied inversely with the dosage of FIGURE 1. Serum neutralization titers of challenge virus. Despite these differ- 77 individuals, 7 and 30 days after a booster inoculation of rabies vaccine, laboratory EL. ences, the results were quite comparable. For example, Laboratories L and EL differences that might have occurred be- agreed within a 4-fold dilution in 84.7 cause two persons were involved in the per cent of 374 sera, Laboratories EL inoculating showed that the antibody and 0 had 71.7 per cent agreement titers to each vaccine did not vary sig- on 109 sera, and Laboratories L and 0 nificantly (P > .05) with the inoculator. on 90.2 per cent of 101 sera. With this serum neutralization technique, it was DISCUSSION not uncommon for one laboratory to re- port antibody and another to report The discovery of serum neutralizing none on the same serum sample. This antibody against rabies in unimmunized may account for some reports of low animals as well as possible explanations level antibody in pre-immunization sera. for this finding have been reported pre- It would be extremely valuable if, on viously (8-11). Similar findings have the basis of serologic results after a been observed in man (12, 13, 14). primary series, one could predict the Some doubt might be cast on the ability to respond to a booster with validity of this observation with respect undisputed production of antibody. to the 3 pre-immunization sera reported Disregarding the question of the cor- here. In none of these instances was relation between titer and , the serum tested in 3 different labora- two problems remain: tories. In the one instance in which antibody was demonstrated by two lab- 1. What is the minimal antibody titer oratories, the subject failed to respond that can be invariably detected by to 3 injections of duck-embryo vaccine. other laboratories? Had this pre-exposure titer been due to 2. What minimal titer must an indi- actively induced, specific antibody vidual attain after the primary se- against rabies, one would have antici- ries that will result in further re- pated an anamnestic type of response sponse to subsequent inoculations? (4, 5, 15). In this study, of the 232 sera reported The only one of these 3 sera with a by at least one laboratory as having a AVIAN EMBRYO RABIES IMMUNIZATION titer greater than 1 in 5, only 15 (6.5 demonstrate that such titers must per cent) were reported to have no de- be interpreted cautiously; tectable antibody by another laboratory. 3. Since HEP vaccine is a live prod- Thirteen of these 15 sera were examined uct, there might be other immuno- in all 3 laboratories. Of these, 10 had logic factors in operation such as demonstrable antibody reported from 2 the interference phenomenon. of the 3 laboratories. On arbitrary selection of a titer of 1 SUMMARY in 16, it was found that 156 sera had By means of a double blind technique, such a titer reported from at least one high egg-passage and duck-embryo Downloaded from https://academic.oup.com/aje/article/74/1/1/136363 by guest on 30 September 2021 laboratory. Four (2.6 per cent) of this rabies vaccines were compared on the group were reported negative in at least basis of antibody production subsequent one laboratory, but only one of these to a series of 3 intradermal injections, 4 was found negative in two laboratories. followed in 6 months by a fourth injec- This suggests that with any of the tion. Serum aliquots were tested by 3 3 test procedures described here, a titer different laboratories using variations of greater than 1 in 5 can be reasonably of the serum neutralization test. expected to represent true virus neu- Both vaccines elicited a response in a tralizing capacity. satisfactory percentage of the individu- There were 87 individuals with neu- als injected. There was no apparent tralizing antibody demonstrated by at quantitative difference in the antigenic least one laboratory after the first 3 in- response produced by the two vaccines. oculations who received a booster injec- Persons receiving these vaccines as a tion. Of this group there were only 3 in pre-exposure measure should have post- which postbooster antibody could not be immunization sera examined for the detected at a titer of greater than 1 in presence of neutralizing antibody. 5. One subject with a postprimary titer of 1 in 4.4 had a postbooster titer of 1 Quantitative results of serum neutrali- in 2 reported from 2 of the 3 labora- zation tests from different laboratories tories. The other two subjects with post- using basically similar techniques were primary titers of 1 in 2 and 1 in 4.7, comparable but not identical. End-point respectively, had no demonstrable anti- titers in this test seem to vary inversely body by any of the 3 laboratories. An- with the challenge dose of virus. When derson et al. (5) report only one indi- a titer greater than 1 in 5 was reported vidual with a neutralizing titer of 1 in by one laboratory, at least one of the 4.9 after the primary series who did other two laboratories found demon- not have demonstrable antibody after strable antibody in 98.7 per cent of the boostering. instances. Persons with any demonstrable anti- Conclusions drawn from these findings body following the primary series may must be tempered by 3 points; reasonably be expected to respond to a 1. Above a certain minimum titer, the single intradermal injection 6 months level of antibody might be of no later. This fourth injection should in significance; all probability be considered an integral 2. The quantitative difference of the part of the immunization protocol and data obtained by 3 laboratories not a booster. using aliquots of the same sera Titer determinations 7 and 30 days SCHNURRENBEHGEE, ANDERSON, RUSSELL AND WENTWORTH after the fourth inoculation do not dif- 8. Gomez, C, Black, J., and Koprowski, H. fer markedly. Babies in cattle. III. Comparative stud- ies on vaccination of cattle in Colombia with Flury virus and chloroform-inacti- REFERENCES vated vaccine. Jour. Amer. Vet. Med. 1. Schwab, M. P., Fox, J. P., Conwell, D. P., Ass'n, 1955, 127: 360-363. and Eobinson, T. A. Avianized rabies 9. Koprowski, H., Black, J., and Johnson, virus vaccination in man. Bull. World W. P. Babies in cattle. IV. Vaccina- Health Org., 1954, 10: 823-835. tion of cattle with high egg-passage, 2. Peck, F. B., Jr., Powell, H. M., and chicken embryo-adapted rabies virus. Culbertson, C. G. A new antirabies Jour. Amer. Vet. Med. Ass'n, 1955, 127: vaccine for human use. Clinical labora- 363-366. Downloaded from https://academic.oup.com/aje/article/74/1/1/136363 by guest on 30 September 2021 tory results using rabies vaccine made 10. Tierkel, E. S. Introduction to recent de- from embryonated duck eggs. Jour. velopments in the of Lab. and Clin. Med., 1955, 45: 679-683. rabies. Ann. New York Aead. Sci., 3. Peck, F. B., Jr., Powell, H. M., and 1958, 70: 445-448. Culbertson, C. G. Duck-embryo rabies 11. Kleckner, M. Babies ecological problems. vaccine. Study of fixed virus vaccine The disease in sylvatic fauna in the grown in embryonated duck eggs and southeast. Proc. C.D.C. Conf. Teach. killed with beta-propiolaetone (BPL). Vet. Pub. Health and Prev. Med. and Jour. Amer. Med. Ass'n, 1956, 162: Pub. Health "Work. 1958, 204-212. 1373-1376. 12. Ohio Department of Health. Unpublished 4. Fox, J. P. Prophylaxis against rabies in data. humans. Ann. New York Aead. Sei., 13. Buegsegger, J. M., Black, J., and Sharpless, 1958, 70: 480-494. G. B. Primary antirabies immunization 5. Anderson, G. B., Schnurrenberger, P. E., of man with HEP flury virus vaccine. Masterson, E. A., and Wentworth, F. H. Amer. Jour. Pub. Health, 1961, 51: (In Avian embryo rabies immunization. I. press.) Duck-embryo vaccine administered intra- 14. Greenberg, M., and Childress, J. Vaccina- dermally in man. Amer. Jour. Hyg., tion against rabies with duck-embryo and 1960, 71: 158-167. Semple vaccines. Jour. Amer. Med. 6. Beed, L. J., and Muench, H. A simple Ass'n, 1960, 173: 333-337. method of estimating fifty per cent end- 15. World Health Organization. Expert Com- points. Amer. Jour. Hyg., 1938, 27: mittee on Babies. Third Beport. Techn. 493-497. Bep. Ser. no. 121. Geneva: World 7. Sharpless, G. B. Personal communication. Health Organization, 1957.