Delayed Hypersensitivity Responses of Experimental ... - Europe PMC

Vol. 21, No. 3

INFECON AND IMMUNITY, Sept. 1978, p. 705-713 0019-9567/78/0021-0705$02.00/0 Copyright i 1978 American Society for Microbiology

Printed in U.S.A.

Delayed Hypersensitivity Responses of Experimental Animals to Histoplasmin from the Yeast and Mycelial Phases of Histoplasma capsulatum G. M. SCALARONE,l* H. B. LEVINE,' AND S. D. CHAPARAS2 Naval Biosciences Laboratory, University of California, School of Public Health, Berkeley, California 94720,' and Bureau of Biologics, Food and Drug Administration, Rockville, Maryland 208522 Received for publication 30 March 1978

Controlled yeast lysate (CYL) and controlled mycelial lysate (CML) histoplasmins were produced from Histoplasma capsulatum grown in a nutritionally lean, chemically defined medium. The lysates were assayed for skin-test activity in guinea pigs sensitized by infection with the homologous organism. In some studies, nonliving vaccine preparations were employed also. Inter-lot biological variation was animal, and 20 lots of the CYL reagent elicited strong dermal reactions with high specificity. Further, CYL reagents were nonreactive in guinea pigs infected with Coccidioides immitis, whereas the commercial Food and Drug Administration preparations cross-reacted to some degree. The CML histoplasnins were generally less reactive than the CYL preparations and exhibited somewhat more inter-lot variation in sensitivity and specificity. No correlation between potency and protein:polysaccharide ratios were observed with either reagent. An intradermal test with the CYL reagent did not induce significant changes in the complement-fixing titer of sensitized guinea pigs. Such changes in sensitized animals were elicited by a skin test with commercial histoplasmin. Presently, most reagents in

use

for detecting

previous experience with Histoplasma capsulatum by delayed dermal sensitivity are prepared from the mycelial growth phase. These reagents are reliably reactive, but have two ma-

a

jor limitations or disadvantages: First, they may give falsely positive reactions in humans or animals sensitized by fungi other than H. capsulatum (3, 5, 7, 27), and, second, a single histoplasmin skin test administered to a sensitive individual may induce complement-fixing (CF) antibodies (4, 8, 10, 17, 19, 24). This latter attribute limits the use of serological change for charting histoplasmal episodes. Earlier observations on the sensitivity (12, 30) and specificity (13) of spherulin, prepared by controlled autolysis of washed Coccidioides immitis spherules in an aqueous menstruum (11, 22, 14), prompted us to evaluate analogous methods for the production of yeast and mycelial histoplasmins. An initial report (15) described the application of this method for the preparation of histoplasmin CYL (controlled yeast lysate) and histoplasmin CML (controlled mycelial lysate) from different strains of H. capsulatum grown in a nutritionally lean, chemically defined medium. The investigation was concerned with the influence of strain and morphological phase on histoplasmin potency. Histo-

plasmin CYL from strain G-217A was more sensitive and specific than the CML preparation from the same strain, and both reagents were generally more efficacious than commercial histoplasmin or Food and Drug Administration (FDA) reference histoplain. Neither reagent presented problems in detecting sensitivity induced by heterologous strains of H. capsulatum. An extension of the previous work is described in this report. Twenty lots each of CYL and CML, as well as a pooled preparation from each growth phase, were compared with respect to chemical and biological properties. MATERIALS AND METHODS Histoplasmin production. Twenty lots of CYL and CML histoplasmins were produced from H. capsulatum G-217A (albino strain; obtained through the courtesy of C. C. Campbell, Southern Illinois University School of Medicine, Springfield, l.) as previously described (15). A nutritionally lean, chemically defined

medium, which was a modification of the medium of McVeigh and Morton (18), was employed as the menstruum for strain G-217A growth in both the yeast or mycelial form (15). Washed-cell suspensions were autolyzed for CYL/CML production by shaking in sterile pyrogen-free water for 1 day at 370C (15). The supernatant liquors were sterilized by passage through HA 0.45- and 0.20-Itm Nalgene filter units (Nalgene Labware Div., Nalge/Sybron Corp., Rochester, N.Y.).

705

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SCALARONE, LEVINE, AND CHAPARAS

NaCl was added to a final concentration of 0.9%, and 1:10,000 merthiolate was incorporated as a preservative. Pooled skin-testing reagents were also prepared from 18 lots each of CYL and CML histoplasmin. These are designated as either CYL or CML (lot C-T pooled). The dry weight and gross chemical composition of each of the pooled reagents were calculated from individual lot data. Testing in experimental animals. CYL and CML histoplasmins were assayed for delayed skin-test activity in previously sensitized Hartley strain albino guinea pigs. The animals had been infected earlier with approximately 5 x 105 viable H. capsulatum G217A or G-17M yeast cells by the subcutaneous route. Guinea pigs sensitized by vaccination with Formalinkilled organisms prepared from strain G-217A were also employed in certain assays. The animals were vaccinated intramuscularly with killed yeast or mycelial cells (either 3 or 6 mg, total dosage; 1/3 this amount at weekly intervals). Reactivity was determined at intervals after the third vaccine dose. Commercial histoplasmin (Parke, Davis & Co., Detroit, Mich., lot 903673L) and FDA reference histoplasmin (lot H-42) were also included for purposes of comparison. The capacity of different preparations to elicit a delayed dermal hypersensitivity reponse was determined in parallel in the same animals by measuring the induration at 24 h resulting from the intracutaneous injections of 6 ,zg of the reagents (0.1-ml volume). The results were expressed either as the longest and shortest axes of induration (millimeters) or the mean diameter of induration (mean of the shortest and longest axes). All reactions were read "blind" by an individual who was not one of the investigators. Crossreactivity was determined in guinea pigs previously infected subcutaneously with ca. 103 C. immitis Silveira arthrospores. Nonsensitized (control) animals were employed in each comparison. Chemical determinations. All chemical determinations were performed on CYL and CML aliquots that contained no merthiolate or NaCl. Polysaccharide (as mannose) in the different lots of histoplasmins CYL and CML was determined by the anthrone reaction (6). Mannose was used as the standard because other investigators had shown that yeast and mycelial extracts contained a high mannose content in the polysaccharide moiety (1, 2, 20, 21). Protein was determined by the method of Lowry et al. (16) with bovine serum albumin as a standard, and the total nitrogen was determined by using the micro-Kjeldahl method described by Hoffman and Osgood (9) as modified by Sippel et al. (25). An estimate of the nucleic acid content of the CYL (lot C-T pooled), commercial histoplasmin, and FDA reference was determined. The three preparations were digested with a combination of deoxyribonuclease I (0.1 mg/ml), crystalline ribonuclease (0.1 mg/ml), and purified phosphodiesteron from venom, in the presence of 0.001 M MgC12 at pH 7.4. The digests were subjected to automatic ion-exchange chromatography at pH 2.72 by using 0.015 M lithium citrate as eluant and a column of Hamilton-resin at 370C as stationary phase.

INFECT. IMMUN.

Serological changes after a single skin test. Hartley strain albino guinea pigs, previously sensitized by vaccination with killed yeast cells or by infection with yeast cells, were skin tested with a single dose (6 #g/0.1 ml) of either histoplasmin CYL or commercial histoplasmin (six animals per group). Dermal reactivity was determined at 24 h as described above. Blood was obtained by cardiac puncture from all animals at three intervals. The first bleeding was performed just before the administration of the skin test (zero time); the second and third samples were obtained at 12 and 25 days, respectively, after the single skin test. AU sera were preserved with merthiolate (1:10,000) and stored at -300C. Complement fixation tests were performed on all serum samples by the Kolmer method as described by Smith et al. (26, 28), except that overnight binding at 40C was utilized throughout. Complement was obtained from Microbiological Associates (Los Angeles, Calif.), sheep cell hemolysin was from Hyland Div., Travenol Laboratories, Inc. (Costa Mesa, Calif.), and sheep erythrocytes in modified Alsevers solution was from Microbiological Media (Concord, Calif.). Histoplasmin CYL (290 ,ug/ml) was employed as the CF antigen. The CF titer was expressed as the reciprocal of the dilution showing 3+ or greater fixation. The adequacy of histoplasmin CYL as a CF antigen was ascertained by prior studies on the serum of 10 histoplasmosis patients and 3 control human serums. CYL was examined in parallel with the Center for DiseaseControl (CDC) yeast and mycelial CF antigens. It gave titers at least as high as the more reactive CDC reagent in each instance.

RESULTS Although untreated control animals were tested for delayed dermal hypersensitivity reactions in each assay to be described, none of these animals showed nonspecific reactions. Control reactions are therefore not further mentioned below.

Activity and cross-reactivity of histoplasmins. Table 1 shows the influence of dose of CYL and CML on the delayed hypersensitivity responses and also the responses obtained with graded doses of the CYL pooled preparation. The reagents were tested in groups of homologously and heterologously infected animals at different intervals after infection. The variation in mean dermal reactivity shown was related to the time interval between initiation of the infection and skin testing (15). In most instances with both CYL and CML the dernal response plateau was reached at a dose of approximately 5 pg, and increasing it to 10 pg or 25 pg did not markedly increase the magnitude of induration (Table 1). We used 6 ug in the comparative studies to be described on mycelial histoplasmin from FDA and Parke Davis histopasmin. Commercial histoplasinn also contained 6 pg/dose (determined gravimetrically), and this reagent was bioequivalent to FDA his-

DELAYED HYPERSENSITIVITY TO HISTOPLASMINS

VOL. 21, 1978

707

TABLE 1. Delayed hypersensitivity reactions in guinea pigs infected with H. capsulatuma Trial (1) Infected with strain G-17M 92 days earlier (1 and 12 Ag) and 94 days earlier (6 jg)

(2) Infected with strain G-217A 42 days earlier

Reagent

CYL (lot A) CML (lot A)

CYL (lot C-T pooled)

No. of dose (J~g/Ol ml)

Guinea pigs

c/total re((nog/0.1actors)

Mean axis of induration Range (mm)

(num)

1 6 12 1 6 12

3/8 4/4 8/8 1/8 4/4 8/8

1.8 7.9 8.9 0.4 7.3 6.9

0-6.5 6.5-9.0 6.0-13.5 0-3.5 5.0-10.0 5.0-8.5

1 5

10

10/10 10/10 10/10

7.0 9.5 9.6

4.5-9.5 7.0-12.0 5.5-12.5



1 5 10

6/10 10/10 10/10

3.9 6.4 6.5

0-8.5 4.0-9.5 4.5-10.0



0.5 5 25

10/10 10/10 10/10

4.5 9.3 10.1

2.5-6.0 5.5-11.5 7.5-12.0



0.5

1/5 5/5 5/5

0.6

0-3.0 5.0-8.0 4.5-10.5

5 25

6.4 8.3

a'Dose responses to histoplasmins CYL and CML.

toplasmin diluted 1:100. The 6-pug dose of CYL, as will be shown, gave reactions marginally larger than either commercial or FDA histoplasmin. We considered this to be desirable in studies on the specificity of CYL and in determining whether serological changes might follow its use in skin testing. Figure 1 shows the dermal response spectrum of 20 successive lots of CYL histoplasmin (at a dose of 6 Itg); preliminary findings on the first 10 lots have been presented elsewhere (15). Commercial histoplasrnin (6 jig) and FDA reference histoplasmin (1:100) were also employed. In all instances the CYL product at 6 ,tg reacted more sensitively than the FDA reference reagent (P =