INTRODUCTION

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Candida albicans is part of the microbial flora that colonizes the mucocutaneous surfaces of the oral cavity and gastrointestinal tract of many mammals and other animals (12). Although the yeast rarely causes infections in healthy humans without predisposing factors, immunocompromised patients can suffer from mucosal, cutaneous, or systemic candidiasis. Recently, the frequent use of immunosuppressants and chemotherapeutic drugs for cancers has caused a decrease in immune function in patients and has increased the frequency of life-threatening systemic candidiasis (3, 8).

Medical treatment for fungal infections is generally carried out with chemotherapeutic drugs. Such treatment may cause the emergence of resistant cells, which is a cause for concern because it has long interfered with the treatment of bacterial infections. Immunotherapy for prevention and treatment has less possibility of producing resistance, and it will be beneficial in managing fungal infections (10). In order to obtain clues to developing an immunotherapy, it is important to know the mechanism of resistance to candidiasis. Experimental murine models of acquired resistance to systemic candidiasis by intravenous (i.v.) injection of live attenuated C. albicans cells have shown that the development of such resistance is associated with CD4⁺ T helper cells (2, 13, 14). In contrast, antigens that are useful for active immunization to induce resistance include mannoprotein, a major component of surface cell walls. Resistance induced by cell wall mannoprotein is mediated mainly by cell-mediated immunity (9), but the resistance is inferior to that induced by i.v. injection of live attenuated cells (9). In order to discover an antigen that induces resistance as high as that induced by live cells, we searched for antigens recognized in mice immunized with live cells. Here we show that an insoluble membrane fraction, whose antigenicity had not been studied before, caused cell-mediated immune responses and that the membrane fraction induced high resistance comparable to that induced by live cells.

	MATERIALS AND METHODS

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Mice. Specific-pathogen-free female BALB/c mice, 6 to 8 weeks old, were purchased from Japan SLC, Inc. (Shizuoka, Japan). C.B-17 female SCID mice were obtained from Clea Japan, Inc. (Osaka, Japan).

Preparation of C. albicans cells for immunization and infection. C. albicans TIMM 1768 and TIMM 0136 were provided by Teikyo University Research Center for Medical Mycology (Tokyo, Japan). We used C. albicans TIMM 1768, a highly virulent strain that kills all mice within 14 days after i.v. injection of 5 × 10⁵ or more cells, and C. albicans TIMM 0136, a low-virulence strain that colonizes the kidneys of mice without killing the animals after i.v. injection of 10⁶ cells. After culture in Sabouraud dextrose broth at 35°C overnight, C. albicans cells were harvested by centrifugation, washed three times with sterile saline, counted using a hemocytometer, and adjusted to a cell density appropriate for injection into mice.

Preparation of subcellular fractions. We prepared protoplasts of C. albicans cells and fractionated them as follows. C. albicans TIMM 1768 was cultured in YPD medium (yeast extract, 1%; polypeptone, 2%; and glucose, 2%) at 35°C overnight with shaking. Cells harvested by centrifugation were washed and suspended in sterile 50 mM potassium phosphate buffer (pH 7.5) containing 1 M NaCl as a stabilizer and treated with 0.3 mg of Zymolyase-20T (Seikagaku Corp., Tokyo, Japan)/ml and then with 1 mg of Trichoderma lysing enzymes (Sigma, St. Louis, Mo.)/ml to remove the cell walls. The cell suspension was centrifuged, and the supernatant was collected as the cell wall (CW) fraction. Other subcellular fractions were prepared according to the method described by Nishikawa and Nakano (11) with some modifications. Briefly, the protoplasts obtained were carefully washed three times with the same buffer containing 1 M NaCl. The protoplasts were then suspended in sterile saline to lyse them osmotically, and the suspension was homogenized, centrifuged at low speed (10,000 × g) for 30 min, and separated into the precipitate and supernatant. The precipitate, a membrane fraction, was washed, suspended in saline, boiled in a water bath for 15 min, and resuspended to form a uniform homogenate using a sonicator (Insonator Model 200 M; Kubota, Tokyo, Japan), yielding a preparation of C. albicans membrane antigen (CMA). The supernatant was centrifuged at 100,000 × g for 30 min and separated into high-speed (HS) supernatant, containing soluble cytoplasmic materials, and HS precipitate, containing insoluble cytoplasmic materials. From 1.9 × 10¹² cells harvested from a 4-liter culture, we obtained CW fraction (1,810 ml; 3.94 mg of protein/ml), contaminated with the cell wall-digesting enzymes used, and CMA, HS supernatant, and HS precipitate containing 1.5, 5.5, and 0.5 g of protein, respectively.

Immunization. BALB/c mice were immunized on day 0 with live C. albicans TIMM 1768 cells (5 × 10⁴, 5 × 10⁵, or 5 × 10⁶ cells/mouse) by subcutaneous (s.c.) injection of 0.1 ml (total volume) of a 1:1 (vol/vol) mixture of a suspension of the cells and incomplete Freund adjuvant (IFA; Difco, Detroit, Mich.). Similarly, mice were immunized with the subcellular fraction (20 µg of protein/mouse) mixed with IFA. To determine the dose response of CMA, mice were immunized at doses of 20, 2, and 0.2 µg of protein/mouse using IFA as the adjuvant. The mice received one booster s.c. injection of the same dose of the same immunogen emulsified in IFA on day 7. Control mice received a mixture of saline and IFA on the schedule described above.

Resistance to candidiasis. Seven days or 1 month after the second immunization, the mice were infected with C. albicans TIMM 1768 (5 × 10⁵ cells) or TIMM 0136 (1 × 10⁵ cells) by i.v. injection of 0.5 ml of a cell suspension. Resistance was assessed by determining survival days or viable cells in the kidneys as follows. TIMM 1768 was used to determine survival prolongation. We observed the mice daily for 30 days after infection and determined their survival days. TIMM 0136 was used to measure the numbers of viable cells in the kidneys 7 days after infection. The mice were killed, and both kidneys were removed aseptically and homogenized in a glass tissue grinder with 6 ml of saline. Then, 10-fold serial dilutions of each homogenate were prepared, the preparations were plated on Sabouraud dextrose agar, and the colonies that had grown after 48 h of incubation at 30°C were counted. The number of viable C. albicans cells was expressed as the mean ± standard deviation (SD) of log₁₀ CFU per homogenate of the two kidneys of five to seven mice per group.

Assay for antibodies. Antibodies against the CW fraction, CMA, and HS (a mixture of HS supernatant and precipitate) were determined by enzyme-linked immunosorbent assay as follows. Microtiter wells were coated with a portion of the CW fraction, CMA, or HS (all diluted to 10 µg of protein/ml) for 16 h at 4°C. The plates were then treated with 0.1% BSA in phosphate-buffered saline (PBS). Tenfold dilutions of sera from mice immunized with whole cells or control mice were added to the wells, the plates were incubated for 1 h at 37°C, and peroxidase-conjugated rabbit anti-mouse immunoglobulin G (H+L) (Zymed Laboratories, Inc., San Francisco, Calif.) was used as the secondary antibody. The reaction products were treated with a mixture of tetramethylbenzidine and hydrogen peroxide, and the absorbance was measured at 450 nm. Factor serum 1 (diluted 60:1; Iatron Laboratories, Tokyo, Japan), rabbit antiserum to C. albicans whole cells, was used as a positive control, and peroxidase-conjugated goat anti-rabbit immunoglobulin G (H+L) (Zymed Laboratories, Inc.) was used as the secondary antibody.

DTH assay. For measurement of DTH, a solution (50 µl) of the CW fraction, CMA, HS supernatant, or HS precipitate (all diluted to 200 µg of protein/ml) was injected into the left footpads of the mice. The footpad swelling at 24 h after the injection of antigen was measured with calipers, and the difference in thickness from the right footpad was expressed as the mean ± SD of the five to seven mice per group.

Splenocyte proliferation. The spleens of immunized mice were removed aseptically, and the spleens from each group were pooled and then homogenized in RPMI 1640 medium. The homogenate was filtered through nylon mesh (70-µm pore size), and the filtrate was centrifuged and washed with the same medium. The spleen cells were suspended in RPMI 1640 medium containing 10% fetal calf serum and 50 µM 2-mercaptoethanol to 5 × 10⁷/ml, and the suspension was then passed through a nylon wool column to enrich T cells. The splenocytes, which showed 90 to 95% viability, were used as responders. Spleen cells from nonimmunized mice were irradiated (7,500 rads) using an X-ray irradiator (Hitex Co., Ltd., Osaka, Japan) and used as stimulator cells. A 100-µl aliquot of a suspension of responder cells (1.5 × 10⁶/ml) was mixed with 100 µl of a suspension of stimulator cells (3.0 × 10⁶/ml) in 96-well flat-bottom microtiter plates. Then, 10 µl of a dilution of the CW fraction, CMA, HS supernatant, HS precipitate (all diluted to 100 µg of protein/ml), or saline was added. The plates were incubated in a CO₂ incubator at 37°C, and the cells were harvested after 7 days of culture. [³H]Thymidine (Amersham International, Little Chalfont, United Kingdom) was added to a final concentration of 37 kBq/well 18 h before harvest. Experiments were performed in triplicate, and the results were expressed as mean counts per minute.

Cytokine release by splenocytes. Nylon wool-filtered splenocytes from immunized mice and irradiated spleen cells from nonimmunized mice prepared as described above were used as responder and stimulator cells, respectively. A suspension (1.0 ml) of responder cells (1.5 × 10⁶/ml) was mixed with 1.0 ml of a suspension of stimulator cells (3.0 × 10⁶/ml) in a 24-well flat-bottom plate for tissue culture, and then 100 µl of a dilution of the CW fraction, CMA, HS supernatant, HS precipitate (all diluted to 100 µg of protein/ml), or saline was added. The plate was incubated in a CO₂ incubator for 7 days, and the culture supernatants were assayed for gamma interferon (IFN-) (R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions.

Depletion of CD4⁺ or CD8⁺ cells by MAbs. Hybridomas GK1.5 (anti-CD4) and 53-6.72 (anti-CD8) were purchased from the American Type Culture Collection (Manassas, Va.). The hybridomas were injected into C.B-17 SCID mice, and the ascites fluid was collected and brought to 50% saturation with ammonium sulfate. The protein that precipitated was dialyzed against PBS. The protein concentration of the dialyzed solution was determined by measuring the optical density at 280 nm.

Adoptive transfer of resistance to SCID mice. Spleen cells of BALB/c mice immunized with CMA or control mice given saline were passed through a nylon column and treated by Ficoll gradient centrifugation. The T-cell-enriched splenocytes were then suspended in PBS containing 5 mM EDTA and 0.5% BSA, and the suspension was incubated at 4°C for 15 min in a mixture with microbeads conjugated with MAb to CD4 or CD8 (Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany) according to the manufacturer's instructions. The suspension was then washed by centrifugation and passed through a column inserted into a magnetic cell-sorting system (MACS; Miltenyi) so that splenocytes without CD4⁺ or CD8⁺ cells were obtained. These splenocytes (T cell enriched and either CD4⁺ or CD8⁺-cell-depleted) were suspended in RPMI 1640 medium, and 5.4 × 10⁷, 2.1 × 10⁷, or 1.9 × 10⁷ cells were administered to C.B-17 SCID mice by i.p. injection; these numbers of cells are equivalent to the numbers of corresponding cells in a single spleen. One day after injection, the mice were infected with C. albicans TIMM 0136, and 7 days after infection, the CFU in both kidneys were counted as before. CMA was injected into a footpad of each mouse 6 days after infection, and the increase in footpad thickness was calculated as described above.

Statistical analysis. The results in different groups were compared by Student's t test with correction for unequal variance. The Kaplan-Meier test was used for comparison of survival days, and the results were statistically evaluated by the generalized Wilcoxon test. Statistical significance was established at a P value of <0.05 (two-tailed test).

	RESULTS

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Immune responses of mice immunized with live C. albicans cells to subcellular fractions. Immunization of BALB/c mice with two s.c. injections of live C. albicans cells emulsified in IFA caused strong resistance to systemic C. albicans infection (Fig. 1). The resistance was mediated by CD4⁺ cells as shown below, indicating that the mechanism of resistance of the immunized mice is similar to that of the mice immunized by i.v. injection of live attenuated cells by Cenci, Romani, et al. (2, 14). To examine the antigens involved in the immune reactions of the mice, we fractionated C. albicans cells into four subcellular fractions, the CW fraction, CMA, HS supernatant, and HS precipitate, and tested each of the fractions for cell-mediated immune reactions and antibodies in sera. The protein and carbohydrate contents of the subcellular fractions are shown in Table 1. Most cell surface carbohydrates were recovered in the CW fraction, which contained a much higher amount of mannan than the other fractions. The protein content of CMA was about 60%, and its carbohydrate content was about 7% (dry weight), and the HS supernatant and precipitate also had high protein contents. These results indicate sufficient separation of cell walls from other fractions by the treatment.

View larger version (8K): [in this window] [in a new window]   FIG. 1.   Resistance to systemic candidiasis after immunization with live C. albicans cells. Mice (n = 8 or 9) were immunized with different numbers of live C. albicans cells (circles, 5 × 104; triangles, 5 × 105; squares, 5 × 106 cells/mouse) emulsified in IFA on days 0 and 7. Control mice received saline (diamonds) emulsified in IFA on the same days. Seven days after the second immunization, the mice were infected by i.v. injection of C. albicans TIMM 1768 cells and observed for 30 days for determination of survival days. Statistical differences versus the control group, shown as P values, were determined by the generalized Wilcoxon test.

Immune responses caused by individual subcellular fractions. To determine the immunogenicities of the individual subcellular fractions, we tested for induction of DTH reaction and resistance to C. albicans infection in mice immunized with each fraction emulsified in IFA. The membrane fraction, CMA, was immunogenic in induction of DTH response, as was the cell surface CW fraction (Table 3). In addition, CMA induced resistance to systemic candidiasis, which was as effective as that induced by live whole cells and the CW fraction, as indicated by the CFU counts in the kidneys (Table 3) and survival prolongation (data not shown) by immunization. CMA showed dose-dependent immunogenicity and induced significant DTH response and significant resistance to systemic candidiasis even at a dose of 0.2 µg of protein/mouse (Table 4).

Effect of depletion of CD4⁺ or CD8⁺ cells by respective MAbs. We revealed that the membrane fraction was immunogenic and could induce resistance to systemic candidiasis which was as effective as that induced by live cells and cell wall components. To determine the effector cells involved in the resistance to systemic infection induced by immunization with the membrane fraction, CMA, we examined the effects of depletion of CD4⁺ or CD8⁺ T cells by their respective MAbs on the immune reactions and compared them with the resistance induced by immunization with live cells. DTH reaction induced by CMA entirely disappeared upon administration of anti-CD4 MAb (Fig. 2A). Furthermore, the reduction of CFU in the kidneys induced by CMA was inhibited by depletion of CD4⁺ T cells (Fig. 2B). These results indicate that CD4⁺ T cells are involved in the immune responses, DTH reaction and resistance to systemic candidiasis, of the CMA-immunized mice. DTH reaction and resistance induced by live cells were also inhibited by depletion of CD4⁺ T cells (Table 5), indicating that the immune responses induced by CMA are similar to those induced by live cells.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Effect of depletion of CD4+ or CD8+ cells on the DTH and the reduction in CFU of mice immunized with CMA. Mice (n = 5 to 7) were immunized with CMA (20 µg of protein) or saline (control) emulsified in IFA and were infected with C. albicans TIMM 0136 1 month after the second immunization. Each MAb was administered three times as described in Materials and Methods. The control group and one CMA-immunized group received saline instead of MAb. All mice received an intrafootpad injection of CMA 6 days after infection, and CFU in the kidneys were counted 7 days after infection. The data are expressed as means ± SD. P values were calculated by Student's t test versus the group of CMA-immunized mice (A) and versus the control group (B).

Adoptive transfer of resistance by infusion of splenocytes from CMA-immunized mice. To determine the effectors that confer the protective cell-mediated immunity induced by CMA, we prepared T-cell-enriched, CD4⁺-cell-depleted, or CD8⁺-cell-depleted splenocytes from spleen cells of the BALB/c mice immunized with CMA and transferred these splenocytes to C.B-17 SCID mice by i.p. injection. The transfer of T cell-enriched splenocytes caused DTH reaction (Fig. 3A) and conferred reduction of CFU in the kidneys (Fig. 3B) on SCID mice. However, CD4⁺-cell-depleted splenocytes gave no significant reduction of CFU in the kidneys or DTH reaction, whereas CD8⁺-cell-depleted splenocytes conferred significant reduction of CFU in the kidneys and DTH reaction (Fig. 3). These results indicate that CD4⁺ T helper lymphocytes are major effectors or mediators of the induction of resistance as well as the DTH reaction in the cell-mediated immunity of mice immunized with CMA.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Adoptive transfer of DTH response and resistance to systemic candidiasis from CMA-immunized mice to SCID mice. SCID mice (n = 5) received T-cell-enriched (No depletion), CD4+-cell-depleted, or CD8+-cell-depleted splenocytes from CMA-immunized BALB/c mice. Control SCID mice received T-cell-enriched splenocytes from mice given saline emulsified in IFA. The kidneys were removed 7 days after infection, and CFU of C. albicans in the kidneys were counted the next day. The data are expressed as means ± SD. P values were calculated by Student's t test versus the group of CMA-immunized mice.

	DISCUSSION

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Antigens that induce resistance to systemic candidiasis include cell wall components and cytosol-soluble proteins (5, 9, 17). In the present study, we searched subcellular fractions of Candida cells for antigens using mice that had acquired high resistance to systemic candidiasis by immunization with two s.c. injections of live cells and IFA. The resistance was mediated by CD4⁺ cells, indicating that the mechanism of resistance of the immunized mice is similar to that of the mice immunized by i.v. injection of live attenuated cells by Cenci, Romani, et al. (1, 14). Interestingly, we revealed that the mice showed strong cell-mediated immune responses to an insoluble membrane fraction composed of plasma membranes and cytoplasmic organelles, including mitochondria, whose antigenicity and immunogenicity have not been studied. We also found that the membrane fraction, CMA, induced resistance to systemic candidiasis which was as effective as that induced by live cells or the cell surface CW fraction and that the resistance was mediated by CD4⁺ T cells similarly to that induced by live cells.

CW fraction prepared by digestion with cell wall-lysing enzymes was recognized by lymphocytes of live-cell-immunized mice and conferred strong resistance to systemic infection by immunization, although it caused an inflammatory response when injected into the footpads of nonimmunized mice. Mencacci et al. and Torosautucci et al. have reported that the crude cell wall mannoprotein extract of C. albicans and its purified preparation, MP-F2, are antigens of Candida-sensitized mice and human subjects and sufficiently immunogenic to confer resistance (9, 19). It is unknown whether our CW fraction contains the mannoprotein because the methods for preparation differ (16), but there is a high possibility that MP-F2 or its hydrolysates are contained in the fraction. In contrast, the active ingredient of CMA differs from those of the CW fraction and is derived from plasma membranes and cytosol organelles according to the following results. That is, CMA showed a ratio of carbohydrates to proteins distinctly different from those of the CW fraction and MP-F2 (18). Also, CMA caused immune responses different from those caused by the CW fraction (Table 2). However, there is no clear evidence of the absence of MP-F2 or its hydrolysates from CMA. The similar responses, including the induction of resistance by CMA and the CW fraction, suggest that a common ingredient or active determinant that contributes to the responses is present among these antigen preparations. Li and Cutler have shown that a MAb, 10G, prepared from sera of mice immunized with a membrane fraction recognizes antigens that are present in both the cell wall and the plasma membrane and that the epitope of 10G is -1,2-tetramannose (6, 7).

The resistance to systemic candidiasis of mice immunized by s.c. injection of live cells or CMA was proved to be mediated by CD4⁺ cells based on the disappearance of resistance by depletion of CD4⁺ cells. DTH responses induced by immunization not only with live cells but also with CMA positively correlated with resistance in experiments with administration of MAbs and adoptive transfer of splenocytes. The DTH response is mediated by CD4⁺ T helper type 1 (Th1) cells, which produce IFN-. Splenocytes from the immunized mice showing resistance produced large amounts of IFN- in response to CMA. These results suggest Th1 predominance in the live-cell-immunized mice and also in CMA-immunized mice (2, 13, 14).

The resistance induced in mice by CMA was effective against systemic candidiasis even 1 month after the last immunization. The results of the present study suggest a potential for the membrane fraction to act as an antigen conferring resistance to systemic candidiasis in place of live cells and also as a source for the isolation of a new antigen. Purification of antigens contained in our CMA preparation has enabled the isolation of mitochondrial superoxide dismutase, and studies of its antigenicity and immunogenicity are being carried out (K. Takesako et al., Prog. Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2162, 1999). In humans, frequent use of antifungal chemotherapeutic drugs, including fluconazole, has evoked an emergence of resistant strains of Candida and fear of cross-resistance to azoles and other chemotherapeutic drugs (15, 20). This situation argues strongly for the development of immunotherapy using a vaccine for the treatment of fungal infections, including candidiasis.