INTRODUCTION

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Blastomyces dermatitidis is a thermal dimorphic fungus and the causal agent of blastomycosis, which is one of the principal endemic systemic mycoses of humans and other mammals. Inhaled conidia of B. dermatitidis initiate the infection, and at body temperature they convert to invasive yeast forms that produce a chronic, progressive pneumonia, which often disseminates to extrapulmonary organs (25). Infections that go undiagnosed or untreated may progress and become fatal even in immunocompetent hosts. Patients with AIDS or other immunosuppressive conditions are prone to disseminated, often lethal infections (22, 23). Dogs that reside in the zone where the disease is endemic are a common victim of blastomycosis; incidence rates approach 1 to 2% of susceptible animals (4).

Innate and adaptive mechanisms that limit infection and promote clearance of the fungus that have been characterized include polymorphonuclear leukocytes, mononuclear phagocytes, and antigen-specific T lymphocytes (14). However, the antigens of B. dermatitidis that stimulate clearance of the fungus have not been identified. We previously identified a 120-kDa protein on the surface of B. dermatitidis yeasts, designated WI-1 (17). WI-1 is an adhesin that binds the fungus to complement and CD14 receptors on host cells (21) and an immunodominant antigen (17). Most infected patients develop strong humoral and cell-mediated immune responses to WI-1 during the course of illness (17, 18). Despite the fact that WI-1 is consistently recognized as an antigen by infected patients, the value of these immune responses in resistance to B. dermatitidis infection has not been studied.

In this study we investigated the immunogenicity of WI-1 and its protective efficacy in an experimental infection of mice. The goals of our study were to (i) raise immune responses to WI-1 in inbred strains of mice, (ii) characterize humoral and cellular anti-WI-1 responses elicited by the immunization, and (iii) assess the protective efficacy of these immune responses in a murine model of lethal pulmonary blastomycosis. Our findings demonstrate that administration of WI-1 elicits immune responses that significantly enhance resistance against a lethal pulmonary infection.

	MATERIALS AND METHODS

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Fungal strains and growth. Strains of B. dermatitidis used here include ATCC (American Type Culture Collection) 60636, originally isolated from soil and patients during an outbreak of blastomycosis in Wisconsin (19), and ATCC 26199, originally isolated from a human patient in South Carolina (5). Isolates were maintained in the yeast form on Middlebrook 7H10 agar slants with oleic acid-albumin complex (Sigma Chemical Co., St. Louis, Mo.) at 37°C. Histoplasma capsulatum 184 AS ura 5-11 is a uracil auxotroph of a smooth variant of the parental isolate 184 AR (28, 29). The variant is highly attenuated in virulence for mice due to two independent alterations, including loss of surface -(1,3)-glucan and uracil auxotrophy. This isolate was grown in Histoplasma macrophage medium supplemented with uracil (50 µg/ml) as described previously (28, 29).

Mouse strains. Male C57BL/6 and BALB/c strains of mice were 5 to 6 weeks old at the time of purchase from The Jackson Laboratory. They were housed and cared for throughout these experiments according to guidelines of the University of Wisconsin Animal Care Committee, which approved all aspects of this work.

Antigens. Secreted WI-1 was purified from the ATCC 60636 yeasts as previously described (3). Briefly, yeasts were grown in liquid Histoplasma macrophage medium in a gyratory shaker at 37°C for 2 weeks. Supernatants enriched for WI-1 were collected and purified in a two-step process using anion-exchange chromatography followed by hydrophobic interaction chromatography. The homogeneity of purified WI-1 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining with silver nitrate.

Immunizations. Purified WI-1 or bovine serum albumin (BSA) as a control was administered to mice in Freund's adjuvant subcutaneously at the base of the tail. The immunogens were diluted to the desired concentration in phosphate-buffered saline (PBS) and emulsified in an equal volume of either complete Freund's adjuvant (for the first immunization) or incomplete Freund's adjuvant (for the booster). Each immunogen was administered in a volume of 0.2 ml of emulsion.

Measurement of immune responses. Mice were tested 2 weeks after the second immunization to assess humoral and cellular immune responses to WI-1. For measurement of antibody responses, mice were bled from the tail vein to obtain serum samples. Anti-WI-1 antibody was detected and titered in serum by using a previously described solution-phase radioimmunoassay (17). A positive test in this assay is defined as at least 20% specific binding of the radiolabeled WI-1 antigen target at a serum dilution of 1:40 or greater. The endpoint titer of a serum sample is defined as that dilution of serum yielding 20% specific binding of the radiolabeled WI-1 antigen target.

Experimental infection. Two weeks after mice received a second immunization, they were infected with B. dermatitidis yeasts intranasally. After mice were anesthetized with inhaled Metafane (Mallinckrodt Veterinary Inc., Mundelein, Ill.), a suspension of yeast cells was administered in a volume of 25 µl dropwise into their nares. The number of yeasts needed to achieve a lethal pulmonary infection was established in preliminary studies as 10⁴ yeasts for both ATCC 60636 and ATCC 26199. Unimmunized mice that received a lethal dose died from a progressive pulmonary infection 14 to 21 days after they were challenged. To evaluate the protective efficacy of immunization, WI-1-immunized mice and control mice were given a lethal dose of yeast cells, and their survival was assessed daily over 30 days after challenge.

Statistical analysis. Kaplan-Meier curves were generated for mice that survived a lethal challenge with B. dermatitidis. The survival times of mice that were alive at the end of the study were regarded as censored. Time data were analyzed by the log rank statistic, which summarizes the extent to which the observed survival times in the two groups of data (immunized versus control) deviate from those expected under the null hypothesis of no group differences. Since the number of mice per group is considered small, the exact P values were computed by using the statistical package StatXact-3 (CYTEL Software Corporation). The survival rates of two groups are considered to be significantly different if the two-sided P value is less than 0.05. When multiple comparisons were made simultaneously, the P values were adjusted according to Bonferroni's correction in order to protect the overall significance level of 0.05.

	RESULTS

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Delayed-type hypersensitivity responses in immunized mice. Previous work in a murine model of blastomycosis has demonstrated that the development of delayed-type hypersensitivity correlates temporally with the ability to resist a lethal experimental infection (10, 27). To investigate whether WI-1 immunization evoked delayed-type hypersensitivity responses, we measured the swelling of footpads injected with WI-1. C57BL/6 mice immunized with WI-1 showed substantial amounts of footpad swelling upon recall with WI-1, whereas mice immunized with BSA showed no footpad swelling in response to WI-1 (Fig. 1). The size of the delayed-type hypersensitivity response in WI-1-immunized mice increased in a concentration-dependent manner with the immunizing dose of antigen. Mice that received a subcutaneous infection with 4 × 10⁴ live B. dermatitidis yeasts had weaker delayed-type hypersensitivity responses to WI-1.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Immunization with WI-1 induces delayed-type hypersensitivity responses in a concentration-dependent manner. Groups of three C57BL/6 mice were immunized with various doses of WI-1 or live yeast according to the protocol described in Materials and Methods. Mice were tested 2 weeks after the second immunization for delayed-type hypersensitivity by administration of 10 µg of WI-1 in saline into one footpad and saline alone into the other footpad. Measurements were taken 24 h later. Error bars represent standard errors of the mean.

Antibody responses in immunized mice. Natural infection with B. dermatitidis is accompanied by a strong antibody response to WI-1. To determine whether immunization evokes an immune response that resembles the natural one, we investigated the antibody response. C57BL/6 mice immunized with 100 µg of WI-1 and boosted 2 weeks later with the same dose demonstrated strong anti-WI-1 antibody responses. Sera from the immunized mice showed an average of 80% specific binding of ¹²⁵I-WI-1 by radioimmunoassay and an endpoint titer of 1:814,013. Mice that received either heat-killed yeasts or viable yeasts also had detectable anti-WI-1 antibodies; however, the average endpoint titers were 16-fold lower (1:49,799) and 358-fold lower (1:2,274) in these two groups of mice, respectively.

Anti-WI-1 IgG subclass profile in immunized mice. Because cell-mediated immunity is important in developing acquired resistance to B. dermatitidis infection, we characterized aspects of the cellular immune response in WI-1-immunized mice. To investigate the phenotype of T helper cells that arise in immunized mice, we investigated the subclass of serum IgG antibodies specific for WI-1. This parameter has been used as a surrogate marker to indicate T helper 1 responses (IgG2a and IgG3 antibodies) and T helper 2 responses (IgG1 and IgG2b antibodies) (12, 26).

View larger version (45K): [in this window] [in a new window]   FIG. 2.   Anti-WI-1 IgG subclasses in four C57BL/6 mice per group immunized with either 100 µg of WI-1, 106 heat-killed yeasts (HK Yst), or 4 × 104 live yeast (Live Yst) subcutaneously (A) and three C57BL/6 mice per group immunized with various doses of WI-1 as shown (B). In each experiment, sera were obtained 2 weeks after the second immunization and analyzed for anti-WI-1 IgG subclass by ELISA. Error bars indicate standard error of the mean. OD, optical density. NMS, normal mouse serum.

Protective efficacy of WI-1. The above results indicated that WI-1 is immunogenic and raises both humoral and cell-mediated immune responses. We next sought to determine whether these anti-WI-1 immune responses confer any protective benefit on the host following a lethal pulmonary challenge with yeasts. WI-1 immunization of C57BL/6 mice and BALB/c mice significantly protected these animals against death after infectious challenge (Fig. 3 and Table 1). The immunizing dose of WI-1 was found to be important since mice immunized with 200 µg of antigen fared significantly better than those immunized with only 10 µg (Table 1, experiment 3). In other experiments (Table 1, experiments 1, 2, and 4), mice were immunized with 100 µg of WI-1 protein according to the protocol described in Materials and Methods. This vaccination significantly prolonged the lives of the mice in each of the experiments and protected a proportion of them from dying during the observation interval. Figure 3 shows a representative survival analysis of C57BL/6 mice immunized with either WI-1 or BSA as a control (P = 0.0002).

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Protective efficacy of WI-1 immunization against lethal pulmonary blastomycosis. Groups of C57BL/6 mice were immunized subcutaneously either with 100 µg of WI-1 or with BSA according to the protocol described in Materials and Methods. Two weeks after a second immunization, the mice were challenged with a lethal dose of 104 B. dermatitidis ATCC 60636 yeasts and observed for survival. Kaplan-Meier survival curves were generated for WI-1-immunized mice and control mice during an observation interval of 30 days after experimental infection.

	DISCUSSION

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In prior work, WI-1, an abundant surface protein on B. dermatitidis yeasts, has been shown to be a chief antigen of both humoral and cellular immune responses in people and dogs infected with the fungus. Antibody to WI-1 was detected in 85% of infected patients (17) and almost 90% of infected dogs (15). Peripheral blood mononuclear cells from human blastomycosis patients also react strongly in an in vitro proliferation assay after stimulation with WI-1 (18).

In this study, we sought to investigate the immunogenicity of purified WI-1, characterize features of the humoral and cellular response to the immunogen, and analyze whether these anti-WI-1 immune responses are able to enhance resistance against experimental infection. Our results demonstrate that WI-1 administration prompts antibody and cell-mediated immune responses in two inbred strains of mice. The immune responses after administration of WI-1 were qualitatively similar to those after administration of live or dead yeasts but were quantitatively much stronger. Thus, WI-1 is immunogenic and evokes immune responses that resemble the ones observed following exposure to the intact yeast, whether it is dead or alive.

The immune responses engendered by WI-1 administration enhanced the resistance of mice against a lethal experimental infection. Mice immunized with WI-1 lived significantly longer than control mice that were unimmunized or received BSA. Ultimately, only a small proportion of the immunized mice survived the infection. Thus, it would be desirable to investigate methods of enhancing the protective efficacy of WI-1. Alternatively, our model of experimental infection may be overly rigorous. A lethal dose of yeast from cells, as delivered in this study, might overwhelm even a robust, effective immune response. Under circumstances of natural infection, the host is exposed to conidia rather than yeasts, and the infection evolves slowly as a subacute or chronic process. In contrast, animals infected in this study developed a progressive pulmonary infection and died of overwhelming pneumonia 14 to 21 days after the infection. In the related mycosis, histoplasmosis, hsp-80 immunization protects mice against a sublethal infection but not a lethal one. Thus, WI-1 immunization might be more protective in a model of blastomycosis producing a sublethal infection or one that is less rapidly lethal.

The profile of the immune response to WI-1 administration may help explain its modest protective benefit and offer a clue on how to improve the protective efficacy of WI-1. WI-1 evokes a robust immune response, with a humoral component illustrating a mixed T helper phenotype that is biased toward a T helper 2 phenotype. Antibodies offer no established benefit in conferring resistance to B. dermatitidis infection (7) and could harm the host more than help it, as reported for Coccidioides immitis infection (9). Conceivably, some antibodies might benefit the host, as in experimental infections with Cryptococcus neoformans (20) and C. albicans (13). Nonetheless, a T helper 2 response, as suggested by the distribution of anti-WI-1 IgG subclasses, is probably not optimally protective.

Our inferences about T helper phenotype responses to WI-1 immunization were based on analyzing the subclass of WI-1-specific antibodies rather than on directly measuring the cytokines themselves. This approach is indirect, and our data should be interpreted cautiously. However, the approach is supported by a substantial body of literature (12, 26). Moreover, Brummer et al. (6) has shown that mice infected with B. dermatitidis yeasts develop features of a T helper 2 immune response during chronic progression of the infection and features of a T helper 1 immune response during a healing phase of the infection after antifungal therapy. Those findings indicate that both elements of T helper immunity can be observed during infection and suggest that T helper 2 responses to WI-1 may not help in clearing B. dermatitidis infection and may even retard its clearance.

The protective efficacy of WI-1 might be enhanced by modifying the composition of the immunogen. For example, the immunogen might be delivered with adjuvant that preferentially drives T helper 1 immune responses, a possibility that had been a consideration in immunizing mice with H. capsulatum yeasts transfected with WI-1. This approach showed some benefit. However, the protective efficacy was not that different from what we observed with WI-1 protein alone, and we were unable to assess whether the T helper phenotype had been altered using IgG subclass analyses since only IgM antibody was present.

Interleukin-12 (IL-12) as an adjuvant together with WI-1 may offer the most direct way to alter the phenotype of T helper cells that arise after vaccination. IL-12 was first described as a vaccine adjuvant in experimental leishmaniasis (1). Soluble leishmania antigen administered to BALB/c mice led to antigen-specific immune responses with a T helper 2 phenotype and progressive infection after challenge. The addition of IL-12 to soluble leishmania antigen converted it from a nonprotective antigen to a protective antigen by enhancing the differentiation of CD4⁺ T cells toward a T helper 1 subset and cytokine profile needed to promote delayed-type hypersensitivity responses. Administration of IL-12 together with WI-1 is presently under investigation.

We chose not to investigate various schedules of WI-1 administration in this study. Immunized mice evinced strong antibody and delayed hypersensitivity responses with the protocol described, and their immune responses and survival appeared to plateau with doses of 100 to 200 µg per immunization. Consequently, we judged that improved protection is more likely to be achieved by changing the profile, rather than the intensity, of the response.

It will be informative to dissect the correlates of resistance after immunization with WI-1, in view of the current reexamination of the contributions of humoral and cellular immunity in resistance to medical fungi (8). This reevaluation has been prompted by work involving the passive transfer of protective monoclonal antibodies against experimental cryptococcosus, candidiasis, and Pneumocystis infection (8). The prevailing view in the literature is that resistance to B. dermatitidis is mediated by cellular immunity, but the roles of humoral immunity and anti-WI-1 antibodies have not been studied systematically. Although antigen-specific T cells are likely to be important in resistance to this fungus, and WI-1 reactive T cells are likely to help in this regard, the host mounts a strong antibody response to WI-1. We speculate that WI-1-reactive antibodies may benefit the host, possibly by promoting opsonic clearance of fungus from tissue or by enhancing the formation of granulomas, as reported for experimental Cryptococcus infection (11). Alternatively, the antibodies might interfere with adhesive properties of WI-1, as many of them are directed at the tandem repeat (16), which mediates binding to complement and CD14 receptors (21). The exact role and action of anti-WI-1 antibodies and T cells require further studies.