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Requisite Elements in Vaccine Immunity to Blastomyces dermatitidis: Plasticity Uncovers Vaccine Potential in Immune-Deficient Hosts^{1 ,2}

Marcel Wüthrich^*, Hanna I. Filutowicz^*, Tom Warner and Bruce S. Klein^3,*,,,¶

Departments of ^* Pediatrics, Pathology and Laboratory Medicine, Internal Medicine, and Medical Microbiology and Immunology, and ^¶ Comprehensive Cancer Center, University of Wisconsin Medical School, Madison, WI 53792

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Understanding fundamental mechanisms of vaccine immunity will allow proper use and optimization of vaccines. Vaccination with a genetically engineered, live, attenuated strain of Blastomyces dermatitidis carrying a targeted deletion at the BAD1 locus confers sterilizing immunity against experimental lethal pulmonary infection. We found in this study that T cells are requisite for durable vaccine immunity, whereas other T and B cells are dispensable. In immune-competent animals, CD4⁺ T-cell derived cytokines TNF- and IFN- mediate vaccine immunity. Surprisingly, these factors are dispensable in immune-deficient animals, which rely on alternate mechanisms for robust vaccine immunity, yet still require O₂^- production rather than generation of NO. Our results clarify the cellular and molecular bases behind the first genetically engineered fungal vaccine. They also illustrate a sharp difference in vaccine mechanisms between immune-competent and immune-deficient hosts, which underscores the plasticity of residual immune elements in compromised hosts, and points to the feasibility of developing vaccines against invasive fungal infection in this fast growing patient population.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Despite a growing awareness of the need for vaccines against global infectious diseases, relatively little is known about the fundamental mechanisms behind vaccine-induced immunity, particularly against facultative intracellular pathogens (1, 2). Few efficacious vaccines are available against such pathogens due to the unique requirements for evoking cell-mediated immunity and the difficulties in doing so without live attenuated vaccines.

Systemic infections with fungal pathogens that exhibit intracellular lifestyles have increased sharply in worldwide incidence among patients with impaired immunity due to cancer, AIDS, and other immune-based disorders (3, 4, 5). This fact emphasizes the need for developing fungal vaccines and understanding their mechanism(s) of action, particularly if they can harness residual, nonclassical elements of host immunity and be effective in immune-compromised patients that need them most.

To our knowledge, we have developed one of the first genetically engineered, live, attenuated fungal vaccines (6, 7). The recombinant vaccine strain of Blastomyces dermatitidis, a dimorphic fungus and the causative agent of the worldwide systemic mycosis blastomycosis, carries a targeted gene deletion of the BAD1 adhesin, an indispensable virulence factor. B. dermatitidis is a primary pathogen capable of causing progressive and fatal pneumonia in immune-competent hosts, and also an opportunistic pathogen that can reactivate after latent infection and produce widely disseminated disease in immune-compromised hosts (8, 9). The biology of disease with this agent thus represents a gamut of pathogenesis events typical of systemic fungal disease. Importantly, vaccination of experimental animals with the attenuated fungus confers long-term survival and sterilizing immunity in nearly 90% of vaccine recipients exposed to a uniformly lethal pulmonary infection (7).

In the present study, we sought to elucidate the cell and molecular mechanisms of vaccine immunity to B. dermatitidis, as conferred by the genetically engineered vaccine strain, to enhance strategies for vaccine development to fungi and other facultative intracellular pathogens. We report in this study that CD4⁺ T cells are largely responsible for vaccine resistance to B. dermatitidis infection, whereas T and B cells are dispensable. CD4⁺ T cells mediate resistance by production of TNF-, and to a lesser extent, IFN-. Surprisingly, although these regulatory cytokines contribute significantly to the expression of vaccine immunity in immune-competent, wild-type mice, each of them was shown to be dispensable for vaccine immunity in IFN-^-/- and TNF-^-/- mice. Whereas CD4⁺ T cells showed remarkable plasticity in their capacity to alternately regulate robust vaccine immunity, reactive oxygen species were demonstrated to be indispensable for clearing the fungus from vaccinated mice, and reactive nitrogen intermediates were unexpectedly nonessential.

Sharp differences observed in vaccine mechanisms between immune-competent hosts and immune-deficient hosts underscore the capacity of residual immune elements for vaccine resistance in compromised hosts, and point to the feasibility of developing vaccines against invasive fungal infections and other facultative intracellular infections in this growing patient population.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fungi

Strains used were American Type Culture Collection (ATCC, Manassas, VA) 26199 (10) and the isogenic mutant lacking BAD1, designated strain 55 (6). Isolates were maintained as yeast on Middlebrook 7H10 agar with oleic acid-albumin complex (Sigma-Aldrich, St. Louis, MO) at 37°C.

Mouse strains

Mice from The Jackson Laboratory (Bar Harbor, ME) included C57BL/6, µ-deficient C57BL/6-Igh-6^tm1Cgn (stock 002288) (11, 12), TCR-^-/- B6.129S2-TCR-^{tm1 Mom} (stock 002116) (13), IFN--deficient C57BL/6-Ifng^tm1Ts (stock 002287) (14, 15, 16), gp91 phox^-/- B6.129S6-Cybb^tm1 mice (stock 002365) (17), inducible NO synthase (iNOS)⁴-deficient B6.129P2-NOS2^tm1Lau (stock 002609) (18), TNF--deficient B6,129-Tnf^tm1Gkl (stock 003008) (19), and control B6129SF2 (stock 101045), and BALB/c and nu/nu BALB/cByJ-Hfh11^nu (stock 000711). TNF--deficient mice were made with stem cells of 129S6/SvEv mice from Taconic (Germantown, NY). In contrast to other transgenic mouse strains, which had been backcrossed with the C57BL/6 parent strain at least 10 times, TNF--deficient B6,129-Tnf^tm1Gkl (stock 003008) had been backcrossed only twice. We confirmed that 129S6/SvEv mice could acquire vaccine resistance to B. dermatitidis infection (data not shown). Male mice 6–7 wk of age at the time of purchase were housed and cared for according to guidelines of the University of Wisconsin Animal Care Committee.

In vivo cell depletion and neutralization of IFN-, TNF-, and GM-CSF

CD4⁺ and CD8⁺ T cells were depleted by mAb treatment. mAb GK1.5 (rat IgG2b anti-CD4) was purchased from ATCC. mAb 2.43 (rat IgG2b anti-CD8) was provided by A. Rakhmilevich (University of Wisconsin); mAb XMG1.2 (rat IgG1 anti-IFN-) was provided by R. Seder (National Institutes of Health, Bethesda, MD); and mAbs XT22.1 and MP1-22E9 (rat IgG2a anti-TNF- and rat IgG2a anti-GM-CSF, respectively) were provided by G. Deepe, Jr. (University of Cincinnati, Cincinnati, OH) with permission of J. Abrams (DNAX Research Institute, Palo Alto, CA). Ascites was made in BALB/c Nu/Nu males. Rat IgG in ascites was ammonium-sulfate precipitated and quantified by measuring OD₂₈₀. For depletion, mice received 250 µg anti-CD4 mAb or anti-CD8 mAb i.v. 1 day before infection and weekly afterward. Cell depletion analyzed by FACS showed >95% depletion of desired subsets in the peripheral blood and lung (data not shown). For neutralization of IFN-, TNF-, and GM-CSF, mice were injected i.v. with 1, 0.5, and 0.5 mg mAb, respectively, 4–6 h before infection, and then i.p. every other day (IFN-) or every 3 days (TNF- and GM-CSF) afterward with mAb doses above. Controls were given 500 µg of rat IgG (Sigma-Aldrich) by a similar schedule.

Cytokine measurements

Cell culture supernatants were generated in 24-well plates in 1 ml containing 5 x 10⁶ splenocytes or lung mononuclear cells and 5 µg/ml of Con A or 12.5 µg/ml of Blastomyces yeast cell wall/membrane (CW/M) Ag (7). CW/M Ag contained 0.1 endotoxin U/ml. Supernatants were collected after 96 h of coculture. IL-2 (BD PharMingen, San Diego, CA) and TNF- and IFN- (R&D Systems, Minneapolis, MN) were measured by ELISA, according to manufacturer specifications.

Intracellular cytokine staining

Lung cells were obtained by crushing the organs in 40-µM cell strainers (BD Biosciences, Lincoln Park, NJ) to obtain single cell suspensions. Erythrocytes were lysed with NH₄Cl-Tris solution and washed twice. An aliquot of isolated cells was stained for surface CD4 using anti-CD4 CyChrome mAb (clone H129.19; BD PharMingen) to determine the percentage of CD4⁺ T cells. The rest of the cells were stimulated for 4–6 h with anti-CD3 (clone 145-2C11; 0.1 µg/ml) and anti-CD28 (clone 37.51; 1 µg/ml) in the presence of 2 µM monensin (Sigma-Aldrich) to halt egress of cytokines from the cells. After cells were washed, they were stained for surface CD4 as above and fixed in 2% paraformaldehyde at 4°C overnight. Fixed cells were permeabilized with 0.1% saponin in PBS containing 0.1% BSA and 0.1% sodium azide. Permeabilized cells were stained with PE-conjugated mAb and isotype controls (BD PharMingen) for IFN- (clone XMG1.2) and TNF- (clone MP6-XT22) in 20% mouse serum for 30 min at 4°C, washed, and analyzed by FACS.

Inhibition of xanthine oxidase and iNOS

Mice received 4-amino-6-hydroxypyrazolo-pyrimidine (AHPP) to inhibit xanthine oxidase (Sigma-Aldrich) and L-N⁶-iminoethyl-lysine (L-NIL) to inhibit iNOS (Alexis, San Diego, CA). A total of 50 mg AHPP was dissolved in 1 ml of 1 M NaOH and diluted with H₂O to 0.5 mg AHPP in 0.2 ml of 0.05 M NaOH. A total of 0.2 ml of AHPP or 0.05 M NaOH as control was given per oral on days 0, 1, 2, 3, 5, 7, 9, 11, and 13 after infection. This AHPP dose and schedule were nontoxic in preliminary studies (data not shown). L-NIL was given i.p. (2.5 mg L-NIL in 0.5 ml PBS twice daily) starting the day of and continuing throughout infection (20). Mice received 0.5 ml of PBS i.p. as a control.

NO₂^- and O₂^- were measured using resident peritoneal cells of mice (21). Cells were collected from vaccinated mice before and after infection, suspended in ice-cold DMEM at 2 x 10⁶ cells/ml, with 100 µl/well in 96-well plates, and incubated at 37°C/5% CO₂. After 1 h, wells were washed free of nonadherent cells. Cells were cultured with 100 ng/ml murine IFN- (R&D Systems) to stimulate NO production and nitrite (NO₂^-) detection, 5 ng/ml murine GM-CSF (R&D Systems) to stimulate O₂^- production, or medium as control. To measure NO₂^-, supernatants were harvested after 72 h of incubation. A total of 100 µl of sample or NaNO₂ standard (range 1–250 µM) was added to 100 µl of Griess reagent. Absorbance was measured at 550 nm. To measure O₂^- release, macrophages incubated with GM-CSF or medium for 72 h were stimulated for 1 h with 110 µl HBSS containing 9 ng/ml PMA, 6.1 µg/ml Fe³⁺ cytochrome c, and 2.3 µg/ml superoxide dismutase. Absorbance was measured at 550 nm, and O₂^- release was calculated as described (22).

Vaccination and experimental infection

Mice were vaccinated twice, as described (7), 2 wk apart, each time receiving an s.c. injection of 10⁵ 55 yeast at each of two sites, dorsally and at the base of the tail, unless otherwise stated. Mice were infected intratracheally with 2 x 10³ yeast, as described (7), and sacrificed 2 wk later to analyze extent of lung infection, which was determined by plating of homogenized lung and enumeration of yeast CFU on brain heart infusion (Difco, Detroit, MI) agar. To distinguish wild-type strain 26199 from vaccine strain 55 (containing a selectable marker) in tissue homogenates, samples were plated on brain heart infusion with and without 100 µg/ml hygromycin B (6).

Histology

Lung tissue was fixed in 10% Formalin and embedded in paraffin wax. Sections 5 µm thick were stained with H&E and Gomori’s methenamine silver. Areas of pneumonic consolidation were measured at a final projected magnification of 8.8 and expressed as a percentage of total lung areas in sections. The number of yeast was counted in 20 fields with a x60 objective, projected on a TV screen, and expressed as yeast/high power field. To evaluate host control and dissemination of the vaccine strain, we biopsied tissue at the site of inoculation, and collected the draining lymph nodes for histopathological examination.

Statistical analysis

Kaplan Meier survival curves were generated (23). Survival times of infected mice alive by the end of the study were regarded as censored. Time data were analyzed by the log rank statistic (Mantel-Haenszel test) (24), and exact p values were computed using the statistical packaged Stat Xact-3 by Cytel Software (Cambridge, MA). Levels of NO, O₂^-, IL-2, IFN-, and TNF- produced in vitro were compared by pair-wise analysis using Student’s t test (25). Differences in number of CFU were analyzed using the Wilcoxon rank test for nonparametric data (23). A p value of < 0.05 is considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The absolute requirement for T cells and TCR-

To define cell population(s) responsible for vaccine resistance, we vaccinated athymic nude mice, TCR-^-/- mice, µ-chain-deficient, and wild-type mice. Vaccinated nude and TCR-^-/- mice were unable to resist lethal pulmonary infection with virulent wild-type yeast. In contrast, vaccinated µ-chain-deficient mice and wild-type littermates resisted reinfection (Fig. 1, A and B); 80–90% had sterilizing immunity.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. T cells mediate vaccine immunity. Athymic nude mice and µ-chain-deficient mice (A) or TCR--/- mice (B) and controls were vaccinated or not and then infected with wild-type yeast. Lung CFU are depicted as geometric mean ± SEM of 8–10 mice per group analyzed 16 days postinfection. *, p < 0.0001 vs unvaccinated mice. Results are representative of two experiments. All experiments were done in C57BL/6 mice and on this genetic background, except for the use of BALB/c nude mice (nu/nu) and wild-type (wt) littermates in A. Unvaccinated wild-type BALB/c mice and C57BL/6 mice had similar lung CFUs postinfection (data not shown) and acquire similar levels of vaccine immunity (7 ).

The role of T cell subsets

To define the role of T cell subsets in vaccine immunity, we depleted CD4⁺ and CD8⁺ cells alone or in combination during the expression phase of the vaccine immune response, defined as the period after infection. Mice depleted of CD4⁺ cells had 100-fold more lung CFU than rat IgG-treated controls, and died 32 ± 2 days after infection, whereas the controls all appeared healthy 50 days after infection when the experiment was terminated (Fig. 2A). In contrast, mice depleted of CD8⁺ cells did not differ significantly from rat IgG-treated controls as measured by lung CFU and survival. CD4⁺ and CD8⁺ double-depleted mice had 18-fold more lung CFU and a shorter mean survival (25 ± 1 days) than CD4⁺-depleted mice, and were nearly as vulnerable as unvaccinated mice. Hence, CD4⁺ cells are chiefly responsible for vaccine resistance, but CD8⁺ cells can contribute when CD4⁺ cells are absent.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Role of CD4+ T cells and CD8+ T cells during the expression of vaccine immunity. A, Effect of T cell depletion. Vaccinated mice were depleted of T cell subsets shown throughout infection and then followed for survival or analyzed for lung CFU 16 days postinfection. A, Shows one representative survival experiment and an average of three experiments for lung CFU. Values depicted are survival curves and geometric mean CFU ± SEM. For CFU data, *, p < 0.0001 vs vaccinated (rat IgG control) mice and CD4-- and CD8-depleted mice; and **, p = 0.08 vs unvaccinated mice. For survival data, p < 0.0074 for comparison of CD4-depleted mice vs IgG control mice and CD4-- and CD8-depleted mice. B, Cytokine responses in T cell-depleted mice. When mice were analyzed for lung CFU, splenocytes were cultured in vitro with medium or CW/M Ag. Mean ± SEM of four experiments is shown; medium control values were <0.05 ng/ml for IL-2 and below the detection limit for IFN-, and were subtracted from responses shown. *, p = 0.10 for products in CD4 depletion vs control rat IgG and CD8 depletion. **, p = 0.10 for products in double depletion vs control rat IgG or CD8 depletion.

CD4⁺ cells mediate vaccine immunity by production of TNF- and IFN-

Levels of vaccine immunity correlated with IFN-, TNF-, and IL-2 production by splenocytes cultured with B. dermatitidis CW/M Ag in vitro (Fig. 2B). These type 1 cytokines were produced maximally in the presence of immune CD4⁺ cells, for example, in rat IgG control mice and CD8⁺-depleted mice. Conversely, production was greatly reduced in vitro in the absence of CD4⁺ cells. For example, in CD4⁺ cell-depleted or double-depleted mice, type 1 cytokine production was comparable with that in unvaccinated mice. Lung cells of vaccinated mice also produced type 1 cytokines in response to CW/M Ag: 4 days after infection, the cells released 780 ± 33 pg/ml IFN- and 320 ± 65 pg/ml TNF- in vitro specifically in response to the Ag, whereas lung cells of unvaccinated mice or of vaccinated mice depleted of CD4 cells produced no IFN- or TNF in response to the Ag. Lung cells from all three groups failed to produce IL-2 in vitro.

We tested the role of type 1 cytokines in functional studies. Neutralization of IFN- or TNF- during expression of vaccine immunity increased lung CFU by 30- and 270-fold, respectively, compared with treatment with rat IgG control (Fig. 3A). Neutralization of IFN- and TNF- together had an additive effect and abolished vaccine immunity. In survival studies of these vaccinated mice, neutralization of TNF- alone or together with IFN- sharply reduced duration and percentage of survival compared with treatment with rat IgG control. Thus, both cytokines independently contribute to vaccine immunity. Because vaccine immunity was found in separate, parallel experiments to require CD4⁺ cells and expression of IFN- and TNF-, respectively, and lung cells produced IFN- and TNF- in response to Ag principally when CD4⁺ cells were present, we postulated that CD4⁺ cells mediate their effect by production of these regulatory cytokines. Indeed, neutralization of IFN- and TNF- either alone or together sharply reduced resistance adoptively transferred by immune CD4⁺ cells, compared with controls that received these CD4⁺ cells with rat IgG control Ab, CD4⁺ cells from hen egg lysozyme-vaccinated mice, or no transferred cells (Fig. 3b). Because the Abs could also neutralize TNF- or IFN- from non-T cells, we monitored by intracellular cytokine staining the expression of these products ex vivo in lung T cells. The number of lung CD4⁺ T cells that produced IFN- and TNF- rose sharply between days 2 and 8 after infection in vaccinated mice vs unvaccinated mice (Table I). Thus, vaccine-induced, protective CD4⁺ cells most likely mediate their effect(s) via production of IFN- and TNF-.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. CD4+ T cells mediate vaccine immunity by production of IFN- and TNF-. A, Neutralization of type 1 cytokines after infection in vaccinated mice. Vaccinated C57BL/6 mice received anti-IFN-, anti-TNF-, both, or rat IgG during the expression phase of vaccine immunity, i.e., after infection. Data are geometric mean CFU ± SEM of three experiments and survival analysis of one representative experiment; n = 10 mice/group. %, Percentage of mice alive 50 days postinfection when the experiment was terminated. *, p < 0.0001 vs control rat IgG or neutralization of both IFN- and TNF-. **, p = 0.40 vs unvaccinated mice. B, Neutralization of type 1 cytokines after adoptive transfer of protective CD4+ T cells. Purified CD4+ T cells from spleens and lymph nodes of vaccinated (Blastomyces YCE-Ag (7 ) boosted) mice were transferred (7 x 106 cells/mouse) in the presence or absence of neutralizing mAb against IFN-, TNF-, both, or rat IgG. Other controls were naive mice that did not receive cells, and mice that received lymph node cells from mice vaccinated with hen egg lysozyme (HEL) (5 x 107 total cells/mouse; 8.5 x 106 CD4+ T cells/mouse). Mice were analyzed for lung CFU 16 days postinfection. Values shown are geometric mean CFU ± SEM of 10 mice/group. *, p < 0.0004 vs all other groups.

IFN- and TNF- are dispensable in vaccine immunity

To assess whether IFN- and TNF- are dispensable in vaccine immunity, we vaccinated transgenic mice lacking these cytokines. Five of ten TNF-^-/- mice died within 2 wk after an initial vaccination and had >10⁷ vaccine yeast disseminated to their lungs. By 4 wk postvaccination, two more TNF-^-/- mice had vaccine yeast that had disseminated to the lung (10³ and 10⁴ yeast); the three remaining vaccinated mice appeared healthy and had no vaccine yeast in the lung. In contrast to TNF-^-/- mice, IFN-^-/- mice controlled vaccine yeast as effectively as wild-type mice. Hence, TNF- is needed for innate immunity to B. dermatitidis infection, priming of vaccine immunity, or both. To explore dispensability of TNF- in the expression of vaccine immunity, we circumvented the difficulty of controlling the vaccine strain by using 10-fold less yeast for vaccination (10⁴/site) and mice that were 4 wk older than in the first experiment (26). With these modifications, TNF-^-/- mice controlled the vaccine strain as effectively as wild-type mice. Moreover, TNF-^-/- and IFN-^-/- mice acquired vaccine immunity; lung CFU was sharply reduced in vaccinated vs unvaccinated transgenic mice (Fig. 4, A and B). Surprisingly, lung CFU was significantly lower in vaccinated transgenic mice than in vaccinated wild-type mice, even though lung CFU was significantly higher in unvaccinated transgenic mice than in unvaccinated wild-type mice. To exclude that vaccinated cytokine knockout mice cleared the infection only temporally, we monitored survival. Vaccinated IFN--deficient or TNF--deficient mice all survived infection and appeared healthy during observation over 50 days, whereas unvaccinated littermates all died rapidly in 2–3 wk. Hence, although both IFN- and TNF- contribute significantly to control of primary pulmonary infection and are crucial mediators of vaccine expression in wild-type mice, each of these cytokines becomes dispensable for vaccine expression when absent during the induction phase, as illustrated in the genetically deficient mouse strains.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. IFN- and TNF- are dispensable in vaccine immunity. A and B, Cytokine-deficient mice and wild-type littermates were analyzed for lung CFU 2 wk after infection or followed for survival. Values shown are geometric mean CFU ± SEM and survival time ± SEM. %, Percentage of mice alive 50 days postinfection when the experiment was terminated. *, p < 0.0007 vs unvaccinated controls. **, p < 0.012 vs vaccinated transgenic mice. ***, p < 0.005 vs unvaccinated wild-type mice. C, Vaccinated IFN--/- mice received mAb against CD4+ or CD8+ cells alone or together, TNF-, or rat IgG throughout infection. Lung CFU was analyzed 2 wk after infection. Values depicted are mean CFU ± SEM. *, p < 0.0002 vs control rat IgG. **, p < 0.009 vs CD4 depletion. ***, p < 0.0001 vs control rat IgG. D, Vaccinated TNF--/- mice received anti-IFN- or anti-GM-CSF mAb throughout infection and were analyzed for lung CFU 2 wk postinfection. Data are geometric mean CFU ± SEM of two experiments. *, p = 0.0002 vs control rat IgG.

To determine cellular and molecular sources of compensation, vaccinated transgenic mice were depleted of CD4⁺ and CD8⁺ cells or, alternatively, treated with neutralizing Ab against TNF- (IFN-^-/- mice) or IFN- or GM-CSF (TNF-^-/- mice) during expression of vaccine immunity. In IFN-^-/- mice, depletion of CD4⁺ cells increased lung CFU 63-fold compared with rat IgG control (Fig. 4C). Depletion of CD8⁺ T cells alone did not significantly increase lung CFU. However, in mice depleted of CD4⁺ cells, concomitant depletion of CD8⁺ cells further increased lung CFU 5-fold. Most strikingly, neutralization of TNF- in vaccinated IFN-^-/- increased lung CFU 563-fold compared with rat IgG-treated controls. Hence, TNF- alone, most likely produced by CD4⁺ cells, regulates expression of vaccine immunity efficiently in the absence of IFN-.

Neutralization of GM-CSF, but not IFN-, in vaccinated TNF-^-/- mice during infection sharply increased lung CFU compared with rat IgG control treatment (Fig. 4D). These findings suggest complexity and plasticity in vaccine immunity to fungal infection mediated by CD4⁺ cells in the absence of type 1 cytokines IFN- or TNF-.

To see whether GM-CSF serves as the chief regulatory cytokine of vaccine immunity in our model, we neutralized GM-CSF in vaccinated wild-type mice during the expression phase of vaccine immunity. Neutralization increased lung CFU 219-fold compared with rat IgG control treatment (p < 0.0001), but lung CFU in anti-GM-CSF-treated mice remained 3.5 log lower than that in unvaccinated mice (p < 0.0001). Thus, GM-CSF does not seem to serve as the sole or primary upstream regulator of TNF- and IFN- expression in vaccine immunity.

Regulatory cytokines mediate their effects via superoxide anion (O₂^-) and NO production

We investigated downstream effector mechanisms of regulatory cytokines using three complementary approaches. First, we assessed a correlation between levels of IFN- and TNF- production, and those of O₂^- and NO during the expression phase of vaccine immunity. In vitro stimulated resident macrophages produced O₂^- levels that correlated with the amounts of IFN- and TNF- in vivo and the extent of vaccine immunity (Figs. 5A and 3A). Vaccinated mice treated with rat IgG control Ab or neutralizing mAb against IFN- produced the highest levels of O₂^-. Levels of O₂^- fell in TNF--neutralized mice, and especially in mice neutralized for both TNF- and IFN-, in which levels were similar to those in unvaccinated mice. A similar association was found between cytokine presence and absence, resistance in vivo, and the amount of NO produced by stimulated peritoneal macrophages in vitro (Fig. 5A).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Role of NO2- and O2- production in vaccine immunity. A, In vitro production of NO2- and O2- by resident peritoneal macrophages. Macrophages were isolated from unvaccinated mice or vaccinated mice that received mAb against cytokine shown or rat IgG. Cells were cultured with IFN- to stimulate NO production, GM-CSF to stimulate O2- production, or medium as control. Data show a representative experiment of three performed. Mean ± SEM is for triplicate samples. *, p < 0.03 vs anti-TNF--treated mice, anti-IFN-- and anti-TNF--treated mice, and unvaccinated mice. **, p < 0.01 vs vaccinated mice that got control rat IgG. B, Effect of L-NIL and AHPP on resistance of vaccinated mice. Mice were treated with L-NIL, AHPP, both, or vehicle control beginning with infection and continuing afterward. Lung CFU were analyzed 2 wk postinfection. Values depicted are geometric mean CFU ± SEM. Vehicle control of 0.05 M NaOH alone did not significantly increase lung CFU (data not shown). *, p < 0.002 vs vaccinated mice that got vehicle control. **, p < 0.004 vs either inhibitor alone.

Finally, we used phox^-/- and iNOS^-/- mice to establish whether O₂^- and NO production, respectively, are requisite or dispensable in vaccine immunity. Vaccinated phox^-/- mice had just 14-fold less lung CFU than did unvaccinated phox^-/- mice, whereas vaccinated wild-type mice had 770-fold less lung CFU and vaccinated iNOS^-/- mice had 34,000-fold less lung CFU than their respective, unvaccinated controls (Fig. 6A). L-NIL treatment of vaccinated phox^-/- mice markedly elevated lung CFU, compared with untreated controls, suggesting that compensatory iNOS activity may mediate residual vaccine resistance in phox^-/- mice. When vaccinated iNOS^-/- mice were treated with AHPP, lung CFU increased by 4 logs, compared with untreated controls, indicating that vaccine resistance in iNOS^-/- mice is largely mediated by O₂^- production. Hence, O₂^- production is requisite for expression of vaccine immunity, but iNOS activity is dispensable.

View larger version (30K): [in this window] [in a new window]   FIGURE 6. Vaccine immunity to B. dermatitidis in iNOS-/-, gp91phox-/-, and wild-type mice. A, Extent of lung infection. Mice (n = 6–8/group) were vaccinated and infected, and iNOS-/- mice were treated with AHPP, and gp91phox-/- mice were treated with L-NIL as in Fig. 5. Lung CFU were analyzed 2 wk postinfection. Data are geometric mean CFU ± SEM of two experiments. *, p < 0.0005 vs unvaccinated phox-/- mice or vaccinated phox-/- mice that received L-NIL. **, p < 0.0001 vs unvaccinated wild-type (wt) mice. ***, p < 0.005 vs unvaccinated iNOS-/- mice or vaccinated iNOS-/- mice treated with AHPP. B, T cell priming. After vaccination (and before infection), splenocytes were cultured in vitro in the presence of medium alone or CW/M Ag to analyze cytokines produced. Medium control values ranged from 0.02 to 0.04 ng/ml for cytokines measured and were subtracted from values shown. *, p < 0.01 for products in unvaccinated wild-type mice vs vaccinated wild-type mice, phox-/- mice, and iNOS-/- mice. C, In vitro production of NO2- and O2- by resident peritoneal macrophages. Production was measured before infection. Cells were cultured and stimulated as in Fig. 5A. *, p < 0.02 vs vaccinated phox-/- mice and unvaccinated wild-type mice; **, p < 0.002 for O2- levels (over background) vs vaccinated iNOS-/- mice. D, Histological examination of lung tissue. Tissue was analyzed postinfection when lung CFU was measured. Lungs were stained with H&E, or Gomori’s methenamine silver (inset). Large panel images, x20; inset images, x200. Unvaccinated wild-type (WT) mice and vaccinated phox-/- mice both had extensive inflammation (large panel; 46% vs 77% of lung, respectively); however, phox-/- mice had 10-fold less yeast in lung tissue (3.9 yeast vs 24 yeast per high power field, respectively).

Resident peritoneal macrophages of vaccinated phox^-/- mice produced 4-fold more NO than vaccinated wild-type mice and 16-fold more NO than unvaccinated wild-type mice (Fig. 6C). Vaccinated iNOS^-/- mice produced 50% more O₂^- than vaccinated wild-type mice before infection, 2-fold more than them after infection, and 3-fold more O₂^- than unvaccinated mice. These in vitro results, together with in vivo studies using transgenic mice and chemical inhibitors, suggest that O₂^- production is largely responsible and indispensable for vaccine clearance of B. dermatitidis. Although phox^-/- mice made excess NO in vitro, and NO mediates the residual vaccine immunity in these mice, elevated NO levels do not compensate. In fact, vaccinated phox^-/- mice had accelerated illness after infection, and few yeast were seen in their heavily inflamed lungs (Fig. 6D). In vaccinated phox^-/- mice, 77% of the lung was replaced by granulomatous inflammation and 3.9 yeast were detected per high power field. In contrast, in unvaccinated wild-type mice, 46% of the lung showed inflammation, and 24 yeasts were detected per high power field. For the remaining groups, the extent of tissue inflammation corresponded closely with the number of visualized yeast. Thus, NO is dispensable, compensatory O₂^- elevation in iNOS^-/- mice may be responsible for enhanced vaccine immunity, and excessive NO production is associated with dysregulated inflammation and a poor outcome.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Current-day vaccines licensed for use in humans are generally believed to induce protective immunity through induction of neutralizing Abs (1, 2). For a number of globally important pathogens, however, including bacteria such as Mycobacterium tuberculosis, parasites such as Leishmania major, and fungi such as Histoplasma capsulatum and B. dermatitidis, cellular immune responses are thought to be more important or essential for protection (1, 7, 27, 28, 29, 30). Several factors have stood in the way of vaccines against facultative intracellular pathogens. First, it has been difficult to create safe and reliable attenuated strains that recapitulate the natural biology of infection. Second, even so, it remains unclear how such vaccines work, for example, which elements of host immunity are essential and indispensable for vaccine induced immunity. Third, persons that are at risk for infection with these pathogens may have compromised immune systems, raising the practical concerns that they may either respond poorly to vaccine, or be at risk for adverse effects. These challenges are evident in the global epidemic of invasive fungal infections that are becoming increasingly common in patients with impaired immunity due to cancer and AIDS. There are currently no licensed vaccines available against fungal pathogens despite the growing number of fungal infections worldwide.

To our knowledge, we have developed the first genetically engineered, fungal vaccine, which protects against lethal infection with B. dermatitidis (7), a pathogen of global importance. We analyzed in this study the cellular and molecular bases of vaccine immunity. We show that vaccine immunity pivots on elements of cellular immunity, distinguishing this vaccine and its mechanism from many others currently available. Vaccination of nude mice indicated that T cells are essential for clearance of vaccine yeast from the site of administration and that, in the absence of T cells, the vaccine also failed to induce immunity. The requisite role of T cells was confirmed in TCR-^-/- mice, which further illustrated a crucial role for T cells. Inability of TCR-^-/- mice to control infection implies that T cells are unable to compensate for T cells, in contrast to their role in Candida albicans immunity (31). Using µ-chain-deficient mice, we found that B cells also are dispensable in vaccine immunity, although B cells may promote clearance of this or related fungi in other models of infection (32, 33).

Depletion of distinct T cell subsets during the expression phase of vaccine immunity showed that resistance in an immune-competent animal depends primarily on CD4⁺ T cells. Depletion of CD8⁺ cells impaired resistance only if CD4⁺ cells were concomitantly depleted. This finding suggests a minor contribution of CD8⁺ cells to vaccine immunity in the presence of CD4⁺ cells, but a potentially greater role for CD8⁺ cells in the absence of CD4⁺ cells. Thus, the hierarchical contribution of CD4⁺ and CD8⁺ cells toward resistance to primary and secondary infection with other fungi is similar to the resistance mechanism to B. dermatitidis induced by vaccination with our live, recombinant, attenuated strain (28, 29, 30).

Production of type 1 cytokines in vitro was associated with the presence of CD4⁺ cells in vaccinated mice and the loss of vaccine resistance in T cell-depleted mice. Lung CD4⁺ cells showed enhanced expression of these products after vaccination and infection. Independent and combined neutralization of IFN- and TNF- also impaired resistance adoptively transferred by CD4⁺ cells, offering further evidence that CD4⁺ cells mediate protection by production of IFN- and TNF-. Similarly, in vaccinated wild-type mice, in which CD4⁺ cells are largely responsible for vaccine resistance, neutralization of IFN- and TNF- during the expression phase of vaccine immunity greatly impaired resistance, although TNF- neutralization reduced vaccine immunity 10-fold more than IFN- neutralization. Although CD4⁺ cells are one important source of these products, we cannot exclude additional cellular sources.

Because IFN- and TNF- were key regulators of vaccine resistance, it was surprising that robust vaccine immunity could be induced and expressed in both IFN-^-/- and TNF-^-/- mice. Either the reciprocal cytokine or alternatively GM-CSF could compensate, illustrating that CD4⁺ cells show plasticity in their capacity to regulate vaccine immunity. Hence, IFN- and TFN- are independently regulated and dispensable in vaccine immunity to B. dermatitidis infection. Our findings confirm and extend those in a systemic histoplasmosis model, which demonstrated that IFN- is nonessential in secondary immunity (34). Our recent studies have shown that CD4⁺ cells and class II MHC too are dispensable in vaccine immunity (35), and that CD8⁺ cells alone are sufficient. These findings, together with the cytokine plasticity observed in this study, illustrate the capacity of a compromised immune system for recruitment of residual elements in vaccine resistance to fungal respiratory infection.

Vaccine resistance raised by the attenuated strain required reactive oxygen intermediates (ROI) as the effector molecule. T cell depletion was linked with reduced ROI production; ROI inhibition by AHPP treatment of vaccinated mice impaired their resistance; and phox^-/- mice failed to acquire vaccine immunity. In contrast to the pivotal role of NO in host immunity to many obligate and facultative intracellular infections (36, 37), NO played an unexpectedly minor role in vaccine immunity to B. dermatitidis. This fact was underscored by the ferociously effective response of vaccinated iNOS^-/- to lethal fungal challenge.

In sum, our study offers proof-of-concept that live attenuated fungal vaccines can be engineered by disruption of key virulence genes. Such a vaccine is capable of inducing potent and durable cellular immunity, features essential in control of facultative intracellular pathogens (27). Our study also illustrates that the parameters that contribute to protection in normal hosts may not be the same in immune-deficient hosts, and that vaccines may elicit different protective responses depending on immune competency. Hence, vaccines designed to protect against a pathogen based on what we know about normal immune function may not translate to immune-deficient hosts. Although it is presumed that vaccines will not protect immune-deficient hosts, our study shows that an attenuated fungal vaccine can protect a compromised host, but that the mechanisms are different from in a normal host. These findings provide encouragement that vaccines can be designed that will protect immune-deficient hosts.

	Footnotes

 
¹ This work was supported by Grants AI40996 and AI35681 from the U.S. Public Health Service, by a grant from the Morris Animal Foundation (to B.S.K.), and by Fellowship Grant 823A-56729 from the Swiss National Science Foundation (to M.W.). B.S.K. is the recipient of a Burroughs Wellcome Fund Scholar Award in Molecular Pathogenic Mycology.

² This work was presented in part at the 101st General Meeting of the American Society for Microbiology, Orlando, FL, May 2001. Portions of the work appeared in abstract form (Session 63, Abstract E-41, p. 337).

³ Address correspondence and reprint requests to Dr. Bruce S. Klein, University of Wisconsin-Madison, 600 Highland Avenue, K4/434, Madison, WI 53792. E-mail address: bsklein{at}facstaff.wisc.edu

⁴ Abbreviations used in this paper: iNOS, inducible NO synthase; AHPP, 4-amino-6-hydroxypyrazolo-pyrimidine; CW/M, cell wall membrane; L-NIL, L-N⁶-iminoethyl-lysine; ROI, reactive oxygen intermediates.

Received for publication August 8, 2002. Accepted for publication October 4, 2002.

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