THE ANTIBODY RESPONSE OF GUINEA PIGS TO EPIDEMIC TYPHUS

THE ANTIBODY RESPONSE OF GUINEA PIGS TO EPIDEMIC TYPHUS VACCINES OF VARIOUS ANTIGENICITIES RICHARD DONOVICK, MARY FARRELL, AND FLORENCE SMITH Reichel Laboratories, Inc., Kimberton, Pennsylvania Received for publication November 24, 1943 In previous studies it was indicated that vaccines showing especially high protective powers in the "guinea pig test" also induce in these animals high titers of the complement-fixing antibodies (Donovick and Wyckoff, 1942a, TABLE 1 The antibody response of guinea pigs vaccinated with epidemic typhus vaccines having antigenic titers of LOT NO.? VACCINE 27 31 32 34 35 39 4 41 43* 44* 7 43 6* 4+ Fixation 1:4 Complement fixation ANTIBODY TITERS OF POOLED SERA 3+ Fixation Complete * These lots show 3+ complement fixation at dilution as antigens. t At least two out of four mice remain alive at given serum dilution. 1942b). During the past year it has been possible to compare the antigenic titers of various lots of epidemic typhus vaccine with the complement-fixing and "mouse neutralizing" (Bengston et at., 1942) antibody titers induced in vaccinated guinea pigs. All of the pools of vaccine studied were made from yolk sacs infected with the Breinl strain of Rickettsia prowazeki. The lots of vaccine varied somewhat in size, but the smallest were made of infected yolk sacs from about 2, eggs, whereas the larger lots contained about 5,. For each pool of material studied 12 to 2 guinea pigs were given two.5-ml subcutaneous injections one week apart. The animals were bled from the heart 14 days after the second injection. Equal amounts of serum from the guinea pigs injected with a given lot of vaccine were pooled and the pooled serum subjected to complement fixation and 241 Mouse Partlalt

TABLE 2 The antibody response of guinea pigs vaccinated with epidemic typhus vaccines having antigenic titers of ANTIBODY TITERS O POOLED SERA LOT NO. VACCI Complement fixation Mouse 28 29 3 37 38 42 45 46 5a 5b 5c 51* 53* 55a 55b 56* 58* 61 65 4+ fixation 3+ fixationcmltepril 1:4 1:256 * These lots show 3+ complement fixation at dilution as antigens. t At least two out of four mice remain alive at given serum dilution. t There is reason to believe that these mice received an overdose of virus. t TABLE 3 The antibody response of guinea pigs vaccinated with epidemic typhus vaccines having antigenic titers of 2 ANTIODY TITERS OF POOLED SERA LOT NO. O VACCIN Complement fixation Mouse 4+ fixation 3+ fixation Cotplete Partialt neutrallzation 25* 26* 36 49 52 54a 54b 16 54c 64 57* 59 62* 63 64 1:256 66 1:256 67 68* * These lots show 3+ complement fixation at dilution as antigens. *At least two out of four mice remain alive at given serum dilution. 242

ANTIBODY RESPONSE OF GUINEA PIGS TO EPIDEMIC TYPHUS VACCINES 243 Complement Fixation Titers Mouse Neutralization Titers 4 8 16 32 64 128 256 4 8 16 32 64 128 256 SERUM TITER FIG. 1. GUINEA PIG RESPONSE TO VACCINES. ANTIGENIC TITERS, Complement Fixation Titers Mouse Neutralization Titer4 Pooled Guilea Pig Sera 4J E-4. o VO '4 ID. co h co '4 4 8 16 S2 64 128 256 4 8 16 32 64 128 25CO SERUM TITER FIG. 2. GUINEA PIG RESPONSE TO VACCINES. ANTIGENIC TITERS,

244 EXOARD 13DONOVICK, MARY FARRELL, AND FLORENCE SMITH mouse tests. The results of these tests are listed in tables 1, 2, and 3. It will be noted that the lots of vaccine are grouped according to the antigenic titer in the complement fixation test. 54 4a E-4 to Complement Fixation Titers Mouse Neutralization Titers 1 9o 8 7 2 H 54 V4. 6 5 4 3 2..o 4 8 16 32 64 128 256 4 8 16 32 64 128 256 SERUM TITER FIG. 3. GuiNEA PIG RESPONSE TO VACCINES. ANTIGENIC TITERS, TABLE 4 Ditribution of antibody titers of pooled sera from guinea pigs vaccinated with epidemic typhus vaccines of various antigenic titers A=IGTONIC TITERS PERCENTAGE OF POOLED SERA GIVIG X ANTIBODY TITERS OPF VACCINES Complement fixation titers No. of 4+ ltf ment D-Degree of Mouse titers vaciei fixation gree, of 1:4 1:256 neutraliza- 1:4 1:256 tetd titer fxa- tion tion 13 4+ 7.7 3.8 46.2 15.4 Complete 3.8 46.2 15.4 7.7 3+ 3.8 23.2 7.7 Paxtial 23.2 15.4 7.7 19 4+ 5.126 26.53 42.1 1.*5 1.25 5.126 Complete 31.6 26.3 26.3 1.5 5.26 3+ 5.26 21.2 21.2 1.5 5.26 5.26 Partial 1.5 1.5 5.26 1.5 16 4+ 6.25 25. 7.5 18.75 6.25 6.25 Complete 12 5 25. 37.5 25. 3+ 6.25 18.75 25. 12.5 6.25 Partial 6.25 12.5 12.5 12.5 Plots (1943) mentions the purification of rickettsial antigens for use in the complement fixation test. We find that our present ether-treated epidemic typhus vaccines are very satisfactory antigens without further purification.

ANTIBODY RESPONSE OF GUINEA PIGS TO EPIDEMIC TYPHUS VACCINES 245 However, Plotz (personal communication) and his coworkers indicate that some cross reactions with endemic typhus antisera are eliminated by their procedure. Since our present work deals only with the epidemic strain, this factor has not entered. In general, as can be seen in tables 1, 2, and 3, the titers of the complementfixing and mouse-neutralizing antibodies in the pooled guinea pig sera are parallel for given lots of vaccine. However, the antibody titers (both complement fixing and mouse neutralizing) of the sera may differ greatly from the antigenic titers of the vaccine used. Lots having antigenic titers of have yielded pooled sera showing neutralizing antibody titers ranging from 1: 8 up to 1: 64. The ranges for the and antigens were similar. The distribution of antibody titers induced by vaccines of equal antigenic titers are shown in table 4 and in figures 1, 2, and 3. In each figure, curve 1 signifies the highest dilution giving 4+ complement fixation, or complete mouse as the case may be. The 3+ fixations and partial s, which occur occasionally at one dilution higher, are shown in curve 2. The third curve is the arithmetical summation of the other two curves. DISCUSSION Although the exact percentages shown in table 4 may have little significance, we feel that in general they are indicative of the manner in which guinea pigs respond to epidemic typhus vaccines of various antigenic titers. Since the method of production of the different lots of vaccine has remained essentially constant throughout the past nine months, and since there has been a large number of yolk sacs per lot, we feel that lots giving equal antigenic titers in the complement fixation test are very similar. It is significant that great variations may sometimes occur in the antibody titers induced when a given lot of vaccine is injected into two groups of guinea pigs. (See data in table 2 for lots 5 a, b, and c and 55 a and b; table 3 for lot 54 a, b, and c.) Thus far we have found that epidemic typhus vaccines having 1 : 8 antigenic titers induce antibody titers as follows: 38.5 per cent of the pooled sera fix complement at or less, 46.2 per cent at, and 15.3 per cent above. The mouse titers are similar. From lots having antigenic titers of, antibody end points obtained in the complement fixation tests are 31.5 per cent at or less, 42.1 per cent at, and 26.2 per cent above. The distribution of titers is 31.6 per cent at, 26.3 per cent at, and 42.1 per cent above. We have no explanation for thq reversal in percentages here. Perhaps a greater number of sera would bring the results into closer agreement. Only a low percentage of the pooled sera from guinea pigs injected with antigens show neutralizing titers lower than. The majority of these pools neutralize at or better. The complement fixation titers are similar. The causes for the wide range of antibody responses to antigens of like titer

2496 RICHARD DONOVICK, -MARY FARRELL, AND FLORENCE SMITH were not at once apparent to us. As mentioned above, we saw no reason to believe that two lots of vaccine having equal antigenic titers were as different as the resulting antibody contents of pooled sera would indicate. We therefore ran tests on the individual guinea pig sera as well as on the pools. It immediately became evident that the wide discrepancies between the antigen and antibody titers could be explaiued by the variation of response of individual pigs. Of course such idiosyncrasies are not limited to response to rickettsial antigens. Mueller et al. (1943a, 1943b), for example, speak of this problem with regard to the response of guinea pigs to tetanal toxoids. The claim may be made that using pools of sera from a "number" of guinea pigs compensates for these individual variations. We feel that the number of animals required to give an "average" result would have to be very large, perhaps one hundred or more. An admittedly extreme example arose in our work recently. Samples of a lot of epidemic typhus vaccine which had an antigenic titer of were injected into two groups of guinea pigs, 2 pigs in each group. At the usual time the animals were bled and separate pools were made of the two groups of sera. It is to be noted that the 4 pigs were chosen at random. Of course, none of them had been previously exposed to typhus, and they all appeared to be in good health. Although they had all received equal amounts of the same vaccine, one pool of serum showed a titer of, whereas the titer of the other pool was. (See data for lot 55 a and b, table 2.) Subsequent tests on the sera of the individual guinea pigs involved fully justified these findings. This may lead one to question the efficacy of using the titers of pooled sera as a means of measuring the protective value of epidemic typhus vaccmes. We have found that usually the complement-fixing antibody content of a serum parallels the mouse-neutralizing content. We hope soon to present the results of further studies on the relationship between these antibodies. In the examination of human blood, Bengston, Topping, and Henderson (1942) showed very wide discrepancies between the end points of these two tests. Perhaps the titers are no longer parallel in the blood stream several months after vaccination. Whatever the case may eventually prove to be, we have thus far found that when guinea pigs are bled 14 days after a second injection of.5 ml of vaccine, the complement-fixing and mouse-neutralizing antibody titers are usually similar. We do niot know whether it is possible to destroy the complement-fixing antigen in a vaccine without also removing the protective quality, or vice versa, but in the work we have done up to the present, both antigens (if they are indeed different) seem to be subject to the same factors pf concentration or destruction. If eventually they are proved to be one and the same, a much simpler method of assay than the mouse test would be available, i.e., the determination of the antigenic titer of the vaccine itself in the complement fixation test. We have found this procedure of great aid in the production of considerablequantities of this vaccine.

ANTIBODY RESPONSE OF GUINEA PIGS TO EPIDEMIC TYPHUS VACCINES 247. SUMMARY Forty-eight groups of guinea pigs, varying from 12 to 2 animals per group, were inoculated with samples of 43 large lots of epidemic typhus vaccine. The' antigenic titers of the lots of vaccine fell into three groups:,, and. Broad antibody responses were found to result upon vaccination with any of the antigenic groups. The complement-fixing antibody titers of the various pooled sera were usually similar to the mouse-neutralizing titers. Pools of sera from two groups of animals, all of which received the same vaccine, may vary greatly in their antibody content. Hence, the antibody content of vaccinated guinea pig serum is an inconsistent and, consequently, poor measure of the antigenic value of a given vaccine. It is pointed out that under present industrial procedures, direct determination of the antigenic titer of epidemic typhus vaccine by means of the complement fixation test appears to be an accurate measure of its protective value in guinea pigs. REFERENCES BENGTSON, I. A., TOPPING, N. H., AND HENDERSON, R. G. 1942 Demonstration of a substance lethal for mice in the yolk sac of eggs infected with Rickettsia prowazeki. Publication withheld for the duration. See circular letters of July 11, 1942, and August 1, 1942, of the Division of Biologics Control, National Institute of Health, Bethesda, Maryland. DONOVICK, R., AND WYCKOFF, R. W. G. 1942a Tests of epidemic typhus vaccines. Public Health Reports. Publication withheld for the duration. See circular letter of September 28, 1942, of the Division of Biologics Control, National Institute of Health, Bethesda, Maryland. DONOVICK, R., AND WYCKOFF, R. W. G. 1942b Comparative potencies of several typhus vaccines. See circular letter of December 11, 1942, of the Division of Biologics Control, National Institute of Health, Bethesda, Maryland. MUELLER, J. H., AND MILLER, P. A. 1943a Large-scale production of tetanal toxin on a peptone-free medium. J. Immunol., 47, 15-21. MUELLER, J. H., SEIDMAN, L. R., AND MILLER, P. A. 1943b A comparison of antigenicities of hydrolyzate and peptone tetanus toxoids in the guinea pig. J. Clin. Investigation, 22, 321-324. PLOTZ, H. 1943 Complement fixation in rickettsial diseases. Science, 97, 2-21.