RESISTANCE INDUCED AGAINST HISTOPLASMA CAPSULA TUM: QUANTITATIVE ASPECTS* GILBERT A. HILLt AND STANLEY MARCUS From the Department of Bacteriology, College of Medicine, University of Utah, Salt Lake City I t has been shown by several investigators that resistance to Histoplasma capsulatum maybe increased by prior infection or by immunization. Successful active immunization against histoplasmosis has been reported by Salvin (1, 2), Schaefer and Saslaw (3), and Marcus and Rambo (4). These investigators (2, 3, 5) have also reported that mice infected intravenously with sublethal doses of H. ca.psulatum showed marked increase in resistance to subsequent challenge with numbers of organisms lethal to previously unexposed animals. The adaptation of a statistical method for evaluating dose-effect experiments with drugs (6) to dose-effect results with infectious organisms has allowed quantitative comparison of the degree of resistance engendered by immunization with killed vaccine and that induced by sublethal chronic infection. Experiments were also carried out to compare the effect of immunization via different routes and with strains of different virulence as sources of vaccine. MATERIALS AND METHODS LD,o determinations.-adult albino mice (Mus Received for publication December 30, 1958. * Presented in part at the 41st annual meeting of The American Association of Immunologists, 1957. Work reported in this paper was carried out under research grants from the United States Department of the Navy, Office of Naval Research (1310-00) and National Institute of Allergy and Infectious Diseases (E-922), Public Health Service. This paper is taken, in part, from thesis material submitted by G. A. Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School, University of Utah. 26 musculus) obtained from local sources were used in all determinations. The organisms used were grown at 37 C upon a medium containing tryptose phosphate broth (Difco), 2% agar and 20% human blood. A final concentration of 25 to 50 units of penicillin and 25 to 50 micrograms of streptomycin per ml of medium was added. The inoculum was made by suspending yeast phase organisms in infusion broth. Total numbers of cells were estimated from counts made on the stock suspension in a hemocytometer (7). The concentration was adjusted with broth so that each dose of organism was injected intravenously in a 0.5 ml volume. Varying numbers of organisms were injected depending upon the strain employed. Deaths were recorded on the day they occurred, and the mortality ratio results after 21 days were analyzed by the use of the methods of Wilcoxon and Litchfield (6). Immunization.-Albino mice were injected with a formalin-killed suspension of yeast phase cells grown in the following liquid culture medium: tryptose phosphate broth (Difco) 29.4 grams, yeast extract 4.0 grams, maltose 10.0 grams, cystine 0.5 grams, water 1000 m\. The medium was sterilized in low form culture flasks of 2500 ml capacity, inoculated with 1.0 ml of a broth suspension of yeast phase organisms and incubated at 37 C until maximum growth was obtained (3 to 7 days). The cultures were mechanically shaken during the incubation period. Formalin was added to a final concentration of 0.5% and the flasks were kept at 37 C overnight. The sterility of the vaccine was determined by culture on blood agar mediums. The culture material was concentrated by centrifugation and washed with saline. After the final wash the cells were suspended (2 X 10 7/ml) in saline and used as a vaccine. The groups of mice were immunized by injection of the formalin-killed yeast phase organisms. Each animal received two injections of 0.5 ml one week apart. Challenge.-Thirty days after the second injection with vaccine, the randomly selected animals were challenged intravenously with various amounts of yeast phase H. capsulatum, by the method described under LD,o determinations. RESULTS LD 60 determinations.-in order to study the resistance of mice to H.
RESISTANCE TO H. CAPSULATUM IN MICE 27 TABLE 1.-Determination of the virulence of Histoplasma capsulatum intravenously injected into adult white mice. Number of organisms 21-day mortality ratio LDoo determination for H. capsulatum strain 6651 I XIO' 5/20 2 XIO' 7/20 5 XIO' 10/20 LDoo =4.5 XIO' (2.14 XIO' -9.25 XI()6) LD,o determination for H. capsulatum strain GI7M O.IXIO' 1/12 0.5 XIO' 3/11 1.0 XIO' 7/12 2.5XIO' 8/14 LD" =0.92 X 10' (.042 X 10' -2.06 X 10 6 ) ca.psulatum, it was desirable to have available a strain of relatively high virulence and a strain of relatively low virulence. The LDso by intravenous challenge for each of two previously studied strains of H. capsulatum was redetermined. It was observed that strain 6651 was less virulent than strain G 17M. The LD so for each strain has not changed significantly during the past four years, during which time determinations similar to those described by the data in table 1 have been carried out. Table 1 gives the data and the calculated LD so values for strains 6651 and G17M. It is seen that the LD so for strain 6651 is between 2.14X10 6 and 9.25 X 10 6 organisms, and that the LD so for strain G17M is between 0.042X10 6 and 2.06 X 10 6 organisms at the 95% confidence level. Active immunization.-groups of mice were immunized by intraperitoneal injection of the formalin-killed yeast phase organisms.vstrain 6651 or strain G17M. Each animal received two injections one week apart. Thirty days after the second injection the randomly selected animals were challenged intravenously with varying numbers of yeast phase H. capsulatum; strain G17M. At this time a group of normal animals received a like challenge employing the same suspension for challenge inoculum. The results obtained are presented in table 2. These data were analyzed by the method of Litchfield and Wilcoxon (1949), and the LD 50 and potency ratio are given. It was recognized, however, that the dose-mortality response in the two vaccinated groups was considerably flattened. Therefore, the mortality ratios were analyzed also by the chisquare method. The calculated chisquare for comparison of the three groups is 13.70. This indicates that there is a difference between the populations of the three groups that is significant with a probability of <0.01. The chi-square value obtained for comparison of the two immunized groups indicates that there is no significant difference in the mortality ratios of these two groups. Therefore, immunization with either one of the two strains of H. capsulatum is as effective as the other when compared to nonimmunized animals. From the potency ratios calcu- TABLE 2.-Vaccination of mice against histoplasmosis. Mice vaccinated with formalin-killed Y.P. G17M and 6651 challenged with G17M. Number of organisms 5 XIO' I X10' 2.5 X 10' Total LDoo 21-day mortality ratios and percents among Normal G 17M vaccinated 6651 vaccinated 3/11 (27) 7/12 (58) 8/11 (73) 18/34* (53).94X1()6 (2.0 X 10' -.04 X10') 0/12 (0) 3/12 (25) 3/12 (25) 6/36 (19) 5 X10' (17 X 10' -1.4 X1()6) 2/12 (17) 1/12 (8) 4/12 (33) 7/36 (17) 5X10' (12 X10' -2.0 X10') LD,o, 5X1()6 Potency Ratio=--=---=5.3 (1.21-23.0). LD'o2.94X10' * Chi-square for total mortality ratios = 13.70. Value for chi-square with two degrees of freedom is 5.99 at 95% percentile, p = <.01.
28 GILBERT A. HILL AND STANLEY MARCUS TABLE 3.-Ejfect of variation in route of immunization against H. capsulatum on induced resistance. Route of immunization Challenge dose 5Xl0' 1 Xl0' 2.5Xl0' 5Xl0' Normal 2/20 5/21 7/20 19/20 1.8 Xl0' (1.2-2.8 Xl0') 1M 6/20 6/20 8/27 4.3 Xl0' (3.4-6.2 X 10') IP 1/19 6/19 10/23 4.3 XlO' (2.5-7.2 Xl0') SC 3/18 6/20 12/26 4.3 X 10' (2.7-6.9 X 10') IV 3/19 7/20 11/24 4.3 X 10' (2. 7-6.6X10') Potency ratio, comparing normal with the other 4 groups is 2.4 with 95% confidence limits of 1.25-4.5. LD60 lated it can be concluded that the true potency ratio is between 1.21 and 23.0 95% of the time, or that the vaccinated mice are resistant to between 1.21 to 23 times the number of organisms that will kill 50% of the untreated animals. Route oj immunization.-it was desired to determine the effect of route of immunization upon the resistance of mice to subsequent challenge with H. capsulatum, Therefore groups of mice were injected with 0.5 ml of a killed yeast phase suspension of H. capsulatum by either oral, intramuscular, intraperitoneal, subcutaneous, or intravenous inoculations. Thirty days later these mice were challenged with graded doses of H. capsulatum strain G17M. The results obtained are shown in table 3. It may be seen that while there is an increased resistance among those groups immunized, with the exception of the oral group, over the normal animals, there were no significant differences among the animals immunized by the different routes. The orally immunized animals were not different from the controls. The potency ratio for the immunized groups, when compared to the normal group is 2.4 with 95% confidence limits of 1.25 to 4.5. InJection-immunity.-In order to study the effect of infection upon a subsequent challenge, groups of mice were infected by intravenous inoculation with a suspension of yeast phase H. aipsulatum strain 6651 that had been shown to produce a sublethal infection. Approximately 90 days later all animals were challenged intravenously with varying amounts of strain G17M. At the time of challenge a sample of 10 mice was taken and spleen cultures were made upon antibiotic containing blood agar plates. All cultures were positive and it can, therefore, be suggested with confidence that the entire group was chronically infected with strain 6651. The results obtained are summarized in table 4. From the mortality ratios it can be seen that the animals that had been injected with a sublethal number of the less virulent strain were significantly more resistant to a subsequent challenge with the more virulent strain G17M than were normal animals. The potency ratio indicates that 50% of the previously infected animals were resistant to 2.78 to 21.3 times the number of organisms that would kill 50% of normal animals. The group infected with strain 6651 prior to challenge showed the same type of dose-inde- TABLE 4.-Infection-immltnity in histoplasmosis. Mice infected with strain 6651 (5X10 5/i.v.) and challenged with strain G17M. No. of organisms 5Xl0' 1 Xl0' 2.5 Xl0' 5 X10' Total 21-day mortality ratio among Normal 4/19 11/20 11/12 16/20 42/71 Sublethally infected (6651) 3/21 4/21 3/21 8/22 18/85 LD60 1.3Xl0' 10 Xl0' (.72 X10' -2.3 X 10') (4.3 Xl0' -23 Xl0') Potency ratio 10 X 10' 1.3 X10' 7.69 (2.78-21.30)
RESISTANCE TO H. CAPSULATUM IN MICE 29 pendent mortality relationship that had been observed in other groups of immunized animals. DISCUSSION The concept that a state of increased resistance could be induced in experimental animals by chronic infection ("infection-immunity") was first clearly enunciated by Kolle (8). He found that resistance to inoculation site (chancre) lesion formation in experimental syphilis increased with duration of infection, and lasted for a period of time after the animals had been treated. The study of experimental tuberculosis has yielded essentially similar information. Koch made the first observation of resistance induced by infection. The sequence of events that occur on reinoculation of an infected animal are known collectively as the "Koch phenomenon." Subsequent work by others has shown that tuberculous infection is not nearly as extensive in reinoculated animals as in primarily infected ones when the two groups are compared at any stage of infection. In recent years more quantitative work has been carried out employing antibiotic-resistant and sensitive strains of tubercle bacilli. Kanai and Yanagisawa (9) employed streptomycin-resistant and sensitive strains to show that animals infected with either virulent tubercle organisms or BCG were able to limit the extent of disease resulting from subsequent injections of virulent organisms. Similar experiments have been carried out with brucella organisms. Pollack et al (10) have reported that superinfection in guinea pigs was not only less extensive but that the lesions that did develop were not as severe as in primary infection. The potential significance of this phenomenon of infection-immunity becomes apparent when it is recalled that in all the chronic infections cited there is little evidence for induction of artificial immunity in the absence of infection. All practical immunization procedures in current use depend on an artificially induced chronic infection. Attempts to induce immunity by various killed preparations fall short of that obtained by live vaccine. In this report data are presented which permit calculation of an estimate of the quantitative extent of resistance induced by sublethal infection as well as by immunization. This estimate is defined by the "potency ratio," that is, the ratio of the LD so estimates for the control and infected groups. The graphic method employed was modified by Litchfield and Wilcoxon (6) from a method previously described for estimating median effective dose and standard error. The procedure involves fitting dose-response lines by eye and then calculating errors by means of graphs and nomograms. Goodness of fit of the dose-response lines as well as parallelism are tested by use of nomograms. Provided these tests are satisfactory, the potency ratio and its error may be calculated. Challenge of the accuracy of the method (11) has been effectively answered (12). Since the method of analysis is based on the assumption of a dose-response relationship, the adaptation to data presented here is valid only if there exists a direct relationship between challenge dose of organisms and mortality. Although the dose-response characteristic is clearly evident for normal animals, it is less apparent for the immunized animals. Still, our experiences have indicated that challenge dosemortality response relationship does exist with immunized as well as normal mice: therefore the application of the method of analysis is justified. However, the shallow slopes of dose-response
30 GILBERT A. HILL AND STANLEY MARCUS lines leads to a wide spread in the confidence limits. The extent of resistance engendered either by killed yeast phase vaccine immunization or by chronic sublethal infection is comparable when the data are analyzed by the methods used. When normal mice and mice immunized with killed organisms, strain 6651, were challenged with strain G 17M, a potency ratio of 5.3 (1.21 to 23.0) was obtained. The potency ratio for normal mice and mice sublethally infected with strain 6651, when both groups were challenged with strain G17M, was 7.69 (2.78 to 21.30). There is no significant difference in the comparable potency ratios and it may be inferred that under the experimental circumstances no advantage lies with either procedure employed to increase resistance. The experimental design lends itself to quantitative evaluation of immunization against any chronic infection. Such knowledge may be of significance in the development and evolution of nonviable vaccines to replace the living, and in some cases dangerous, vaccines that are being used at present as a prophylaxis for some of the chronic infectious diseases. SUMMARY Mice can be immunized against the lethal effect of systemic histoplasmosis by killed yeast phase organisms or by sublethal infections with such organisms. No difference in immunizing efficacy is achieved by employing a more virulent strain of organisms for vaccine than a less virulent strain. When mice were immunized by subcutaneous, intramuscular and intravenous routes it was found that similar resistance levels were achieved by each route. Oral immunization yielded no increase in resistance. The increase in resistance can be described quantitatively. The extent of resistance engendered by infectionimmunity was not significantly different from that induced by killed vaccine. REFERENCES 1. Salvin, S. B. 1953, J. Immunol. 70: 267-270. 2. Salvin, S. B. 1955, J. Immunol. 74: 214-221. 3. Schaefer, J. and Saslaw, S. 1954, Proc. Soc. Exper. BioI. & Med. 85: 223-225. 4. Marcus, S. and Rambo, F. 1955, Bact. Proc. p.92. 5. Marcus, S. and Rambo, F. 1952, Bact. Proc. p.88. 6. Litchfield, J. I. and Wilcoxon, F. 1949, J. Pharmacol. & Exper. Therap. 96: 99-113. 7. Marcus, S., Nielson, B. and Rambo, F. 1956, J. Bact. 72: 473-477. 8. Kolle, W. 1922, Deutsche med. Wchnschr. 48: 1301-3102. 9. Kanai, K. and Yanagisawa, K. 1955, Jap. J. Med. Sc. & BioI. 8: 115-127. 10. Pollack, A. D., Kelly, E. H., Gorelick, A. N., Braun, W. and Victor, J. 1952, J. Infect. Dis. 90: 267-272. 11. Finney, D. J. 1952, J. Pharmacol. & Exper. Therap. 104: 440-444. 12. Litchfield, J. I. and Wilcoxon, F. 1953, J. Pharmacol. & Exper. Therap. 108: 18-25.