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In many infectious diseases, control or progression of the infection depends on differential expression of CD4⁺ T-cell subsets and their associated lymphokines. In mice, T-helper-1 (Th1) cells produce gamma interferon (IFN-), interleukin-2 (IL-2), and tumor necrosis factor , which are important for the induction of cell-mediated immunity (CMI). Th1 cells are associated with delayed-type hypersensitivity (DTH), macrophage activation, synthesis of complement-fixing immunoglobulin G2a (IgG2a) antibodies, and antibody-dependent, cell-mediated cytotoxicity. In contrast, Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 and are proficient at providing B-cell help and stimulating production of IgE and non-complement-fixing IgG1 antibodies (7). Whereas many factors influence differentiation of CD4⁺ T cells, the cytokine microenvironment present during initiation of the immune response has a major influence. IFN- and IL-12 are key cytokines for the differentiation of Th1 cells, whereas IL-4 and IL-10 promote Th2-cell development (5, 8, 10).

Protective immunity to infections with endemic fungi, including Blastomyces dermatitidis, is thought to require Th1-dependent CMI responses. In a previous study, we investigated the immunogenicity and protective efficacy of WI-1, a surface protein adhesin of B. dermatitidis, against murine experimental pulmonary blastomycosis (11). WI-1 administration evoked antibody and CMI responses, but immunized mice were only modestly protected against lethal experimental infection. The goal of the present study was to investigate whether IL-12 as an adjuvant could enhance the protective efficacy of WI-1. IL-12 was first described as a vaccine adjuvant in experimental leishmaniasis (1). Soluble leishmania antigen administered to mice led to antigen-specific immune responses with a Th2 phenotype and progressive infection after challenge. The addition of IL-12 converted soluble leishmania antigen from a nonprotective antigen to a protective antigen by enhancing differentiation of CD4⁺ T cells toward a Th1 subset and cytokine profile needed to foster DTH responses.

Development of DTH is associated with the ability to resist a lethal experimental infection with B. dermatitidis (3, 9). We previously reported that WI-1 immunization evokes DTH responses (11). We wondered whether addition of IL-12 as an adjuvant to WI-1 immunization could increase those DTH responses. Mice were immunized either with 100 µg of WI-1 and complete Freund adjuvant (CFA) as described previously (11) or with WI-1, CFA, and IL-12 (0.5 µg/mouse). Mice receiving IL-12 were boosted intraperitoneally with 0.5 µg of IL-12 at 1, 3, 5, and 7 days after initial immunization. Two weeks after the first immunization, mice were boosted either with antigen and incomplete Freund adjuvant (IFA) or with antigen, IFA, and IL-12 as indicated for the first immunization. This immunization protocol was used for all experiments presented in this study. Two weeks after boosting, DTH responses were assessed by measuring the footpad swelling of immunized and control mice (n = 24 mice per group) as described elsewhere (11). Mice immunized with WI-1 and IL-12 showed significantly greater footpad swelling in response to WI-1 administration (mean ± the standard error of the mean [SEM] of 0.9 ± 0.1 mm) than did mice immunized with WI-1 alone (0.6 ± 0.06 mm) (P = 0.0015) when analyzed statistically using the Wilcoxon rank test for nonparametric data (4). Virtually no footpad swelling was observed in mice immunized with either IL-12 or bovine serum albumin (BSA) alone as a control (0.03 ± 0.01 mm).

WI-1 immunization evokes humoral immune responses that illustrate a bias toward a Th2 phenotype, based on the subclass distribution of anti-WI-1 IgG antibodies (11). We tested whether recombinant murine IL-12 as an adjuvant together with WI-1 may alter the phenotype of Th cells, as determined by the subclass distribution of WI-1-specific IgG antibodies. Two weeks after immunization, mice (n = 10 per group) were bled and anti-WI-1 IgG subclasses were assessed by enzyme-linked immunosorbent assay as described previously (11). Figure 1 shows that the subclass profile of anti-WI-1 IgG in mice immunized with WI-1 alone was dominated by IgG1 and IgG2b, which is indicative of a Th2 phenotype. The addition of IL-12 as an adjuvant shifted the IgG subclasses toward mainly IgG2a and IgG3, which is indicative of a Th1 phenotype. Mean reciprocal endpoint titers of antibody subclasses were significantly different between the two groups of immunized mice (P = 0.0001).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   IgG subclass distribution of anti-WI-1 antibodies in mice immunized with WI-1 alone or together with IL-12 as an adjuvant. The bars represent mean reciprocal titers ± the SEM of each antibody subclass. Reciprocal endpoint titers were defined as the maximal dilution of a sample that resulted in a value 2 times greater than the background. Differences in endpoint titers for each subclass were analyzed statistically using the Wilcoxon rank test for nonparametric data (4). P values are for comparison of immunization with WI-1 alone or together with IL-12 adjuvant: IgG1, P = 0.0001; IgG2a, P = 0.0001; IgG2b, P = 0.0001; and IgG3, P = 0.0001. The data shown represent one experiment out of at least three with similar results.

The increase in DTH and shift in the Th phenotype raised the possibility that the use of IL-12 as an adjuvant in WI-1 immunization might also augment resistance against experimental infection. Following immunization, mice (n = 10 per group) were infected intratracheally with a lethal dose of 5 × 10 organisms of B. dermatitidis ATCC 60636. WI-1 used for immunization was purified from ATCC 60636 yeast as described elsewhere (2). Mice immunized with WI-1 alone, with WI-1 in the presence of IL-12, or with IL-12 alone showed a significant reduction in the burden of lung infection at 13 days postinfection compared to mice immunized with the control antigen BSA (P = 0.0211, P = 0.0006, and P = 0.0022, respectively) (Fig. 2). WI-1 immunization in the presence of IL-12 led to a greater reduction of lung infection than did WI-1 immunization alone (P = 0.0017) or IL-12 administration alone (P = 0.0139). These results indicate that IL-12 used as an adjuvant for WI-1 immunization enhances resistance in a murine model of pulmonary blastomycosis, as assessed by reduction in the burden of lung infection. We monitored similar groups of immunized mice (n = 10 per group) for survival after infection. IL-12 as an adjuvant prolonged the survival of WI-1 immune mice significantly (P = 0.0005) (Table 1). WI-1 or IL-12, when used alone for immunization, did not prolong survival after infection (P = 0.6992) and (P = 0.2942), respectively, even though they did reduce the burden of lung infection. In a previous report (11), WI-1 alone did prolong survival modestly, but those mice were infected intranasally. In the present study the mice were infected intratracheally, which may have contributed to the lower protective efficacy of immunization with WI-1 alone. We have found that only ca. 10% of the yeast inoculum reaches the alveoli after intranasal infection (unpublished observations).

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Burden of lung disease after experimental infection with B. dermatitidis in mice immunized with WI-1 alone or together with IL-12 as an adjuvant. Two weeks after immunization, mice were infected intratracheally with 5 × 10 yeast of ATCC strain 60636. Two weeks postinfection, the mice were sacrificed and analyzed for burden of lung infection, assessed by CFU of yeast on brain heart infusion agar plates. The CFU values are the geometric mean ± the SEM for 10 mice per group. Differences in CFUs were analyzed statistically using the Wilcoxon rank test for nonparametric data (4). The data shown represent one experiment out of at least three with similar results.

In summary, the use of IL-12 as an adjuvant for WI-1 immunization significantly augmented DTH, shifted the phenotype of Th cells, and enhanced resistance against experimental B. dermatitidis infection. Nevertheless, although the protective efficacy of WI-1 was enhanced, the level of resistance to experimental pulmonary blastomycosis continued to be modest, if not marginal. However, we cannot exclude that another form of WI-1, as in a DNA vaccine, another adjuvant combination, or an increase in the number of boosters might provide a higher level of protection. Alternatively, antigens other than, or in addition to, WI-1 will be needed for a more effective fungal vaccine. Such antigens have recently come under investigation in a recombinant vaccine strain of B. dermatitidis which lacks WI-1 and is correspondingly nonpathogenic (M. Wüthrich, H. I. Filutowicz, and B. S. Klein, submitted for publication).