Marcel Wüthrich

Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice

Worldwide rates of systemic fungal infections, including three of the major pathogens responsible for such infections in North America (Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis), have soared recently, spurring interest in developing vaccines. The development of Th1 cells is believed to be crucial for protective immunity against pathogenic fungi, whereas the role of Th17 cells is vigorously debated. In models of primary fungal infection, some studies have shown that Th17 cells mediate resistance, while others have shown that they promote disease pathology. Here, we have shown that Th1 immunity is dispensable and that fungus-specific Th17 cells are sufficient for vaccine-induced protection against lethal pulmonary infection with B. dermatitidis in mice. Further, vaccine-induced Th17 cells were necessary and sufficient to protect against the three major systemic mycoses in North America. Mechanistically, Th17 cells engendered protection by recruiting and activating neutrophils and macrophages to the alveolar space, while the induction of Th17 cells and acquisition of vaccine immunity unexpectedly required the adapter molecule Myd88 but not the fungal pathogen recognition receptor Dectin-1. These data suggest that human vaccines against systemic fungal infections should be designed to induce Th17 cells if they are to be effective.

Vaccine Immunity to Pathogenic Fungi Overcomes the Requirement for CD4 Help in Exogenous Antigen Presentation to CD8+ T Cells Implications for Vaccine Development in Immune-deficient Hosts

Systemic fungal infections with primary and opportunistic pathogens have become increasingly common and represent a growing health menace in patients with AIDS and other immune deficiencies. T lymphocyte immunity, in particular the CD4+ Th 1 cells, is considered the main defense against these pathogens, and their absence is associated with increased susceptibility. It would seem illogical then to propose vaccinating these vulnerable patients against fungal infections. We report here that CD4+ T cells are dispensable for vaccine-induced resistance against experimental fungal pulmonary infections with two agents, Blastomyces dermatitidis an extracellular pathogen, and Histoplasma capsulatum a facultative intracellular pathogen. In the absence of T helper cells, exogenous fungal antigens activated memory CD8+ cells in a major histocompatibility complex class I–restricted manner and CD8+ T cell–derived cytokines tumor necrosis factor α, interferon γ, and granulocyte/macrophage colony-stimulating factor–mediated durable vaccine immunity. CD8+ T cells could also rely on alternate mechanisms for robust vaccine immunity, in the absence of some of these factors. Our results demonstrate an unexpected plasticity of immunity in compromised hosts at both the cellular and molecular level and point to the feasibility of developing vaccines against invasive fungal infections in patients with severe immune deficiencies, including those with few or no CD4+ T cells.

Immunity to fungi

The global increase in fungal disease burden, the emergence of novel pathogenic fungi, and the lack of fungal vaccines have focused intense interest in elucidating immune defense mechanisms against fungi. Recent studies in animal models and in humans identify an integrated role for C-type lectin and Toll-like receptor signaling in activating innate and adaptive responses that control medically relevant fungi. Beyond the critical role of phagocytes in host defense, the generation and balance of specific T helper subsets contributes to sterilizing immunity. These advances form a basis for the development of fungal vaccines and immune-based therapeutic adjuncts.

Adaptive Immunity to Fungi

Only a handful of the more than 100,000 fungal species on our planet cause disease in humans, yet the number of life-threatening fungal infections in patients has recently skyrocketed as a result of advances in medical care that often suppress immunity intensely. This emerging crisis has created pressing needs to clarify immune defense mechanisms against fungi, with the ultimate goal of therapeutic applications. Herein, we describe recent insights in understanding the mammalian immune defenses deployed against pathogenic fungi. The review focuses on adaptive immune responses to the major medically important fungi and emphasizes how dendritic cells and subsets in various anatomic compartments respond to fungi, recognize their molecular patterns, and signal responses that nurture and shape the differentiation of T cell subsets and B cells. Also emphasized is how the latter deploy effector and regulatory mechanisms that eliminate these nasty invaders while also constraining collateral damage to vital tissue.

Mutation of the WI-1 gene yields an attenuated blastomyces dermatitidis strain that induces host resistance.

Systemic fungal infections are becoming more common and difficult to treat, and vaccine prevention is not available. Pulmonary infection with the dimorphic fungus Blastomyces dermatitidis often progresses and requires treatment to prevent fatality. We recently created a recombinant strain of the fungus lacking the WI-1 adhesin and pathogenicity. We show here that administration of viable yeast of this attenuated strain vaccinates against lethal pulmonary experimental infection due to isogenic and nonisogenic strains from diverse geographic regions. To our knowledge, this is the first example of a recombinant attenuated vaccine against fungi. The vaccine induces delayed-type hypersensitivity and polarized type 1 cytokine responses, which are linked with resistance. A cell-wall/membrane (CW/M) antigen from the vaccine strain also induces polarized and protective immune responses. Lymph node cells and CD4(+) T-cell lines raised with CW/M antigen transfer protective immunity when they release type 1 cytokine IFN-gamma, but not when they release IL-4, and neutralization of IFN-gamma confirmed its role in vivo. Thus, by mutating a pathogenetic locus in a dimorphic fungus, we have created an attenuated vaccine strain and have begun to elucidate fungal and host elements requisite for vaccine immunity.

Requisite Elements in Vaccine Immunity to Blastomyces dermatitidis : Plasticity Uncovers Vaccine Potential in Immune-Deficient Hosts

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 αβ 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-α and IFN-γ 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.

C-Type Lectin Receptors Differentially Induce Th17 Cells and Vaccine Immunity to the Endemic Mycosis of North America

Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors exert divergent contributions to the development of antifungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, Histoplasma capsulatum, and Coccidioides posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking Ag-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival, and migration. Whereas Card9 signaling is essential, C-type lectin receptors offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells.

Tc17 Cells Mediate Vaccine Immunity against Lethal Fungal Pneumonia in Immune Deficient Hosts Lacking CD4+ T Cells

Vaccines may help reduce the growing incidence of fungal infections in immune-suppressed patients. We have found that, even in the absence of CD4+ T-cell help, vaccine-induced CD8+ T cells persist and confer resistance against Blastomyces dermatitidis and Histoplasma capsulatum. Type 1 cytokines contribute to that resistance, but they also are dispensable. Although the role of T helper 17 cells in immunity to fungi is debated, IL-17 producing CD8+ T cells (Tc17 cells) have not been investigated. Here, we show that Tc17 cells are indispensable in antifungal vaccine immunity in hosts lacking CD4+ T cells. Tc17 cells are induced upon vaccination, recruited to the lung on pulmonary infection, and act non-redundantly in mediating protection in a manner that requires neutrophils. Tc17 cells did not influence type I immunity, nor did the lack of IL-12 signaling augment Tc17 cells, indicating a distinct lineage and function. IL-6 was required for Tc17 differentiation and immunity, but IL-1R1 and Dectin-1 signaling was unexpectedly dispensable. Tc17 cells expressed surface CXCR3 and CCR6, but only the latter was essential in recruitment to the lung. Although IL-17 producing T cells are believed to be short-lived, effector Tc17 cells expressed low levels of KLRG1 and high levels of the transcription factor TCF-1, predicting their long-term survival and stem-cell like behavior. Our work has implications for designing vaccines against fungal infections in immune suppressed patients.

The WI-1 Adhesin Blocks Phagocyte TNF-α Production, Imparting Pathogenicity on Blastomyces dermatitidis

The WI-1 adhesin is indispensable for pathogenicity of Blastomyces dermatitidis and is thought to promote pulmonary infection by fixing yeast to lung tissue and cells. Recent findings suggest that WI-1 confers pathogenicity by mechanisms in addition to adherence. Here, we investigated whether WI-1 modulates host immunity by altering production of pro-inflammatory cytokines. Production of TNF-α in lung alveolar fluids of mice infected with B. dermatitidis was severalfold higher for WI-1 knockout yeast compared with wild-type yeast, and in vitro coculture of unseparated lung cells with these isogenic yeast disclosed similar differences. Upon coculture with purified macrophages and neutrophils, wild-type yeast blocked TNF-α production, yet WI-1 knockout yeast stimulated production. Coating knockout yeast with purified WI-1 converted them from stimulating TNF-α production to inhibiting production. Addition of purified WI-1 into stimulated phagocyte cultures led to concentration-dependent inhibition of TNF-α production. Neutralization of TNF-α in vivo exacerbated experimental pulmonary infection, particularly for the nonpathogenic WI-1 knockout yeast. Inducing increased TNF-α levels in the lung by adenovirus-vectored gene therapy controlled infection with wild-type yeast. Thus, the WI-1 adhesin on yeast modulates host immunity through blocking TNF-α production by phagocytes, which fosters progression of pulmonary infection.

BAD1, an Essential Virulence Factor of Blastomyces dermatitidis, Suppresses Host TNF-α Production Through TGF-β-Dependent and -Independent Mechanisms

We investigated how BAD1, an adhesin and virulence factor of Blastomyces dermatitidis, suppresses phagocyte proinflammatory responses. Wild-type yeast cocultured with murine neutrophils or macrophages prompted release of a soluble factor into conditioned supernatant that abolished TNF-α production in response to the fungus; isogenic, attenuated BAD1 knockout yeast did not have this effect. Phagocytes released 4- to 5-fold more TGF-β in vitro in response to wild-type yeast vs BAD1 knockout yeast. Treatment of inhibitory, conditioned supernatant with anti-TGF-β mAb neutralized detectable TGF-β and restored phagocyte TNF-α production. Similarly, addition of anti-TGF-β mAb into cultures of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-α production. Conversely, TGF-β treatment of phagocytes cultured with knockout yeast suppressed TNF-α production. Hence, TGF-β mediates BAD1 suppression of TNF-α by wild-type B. dermatitidis cultured in vitro with phagocytes. In contrast to these findings, neutralization of elevated TGF-β levels during experimental pulmonary blastomycosis did not restore BAD1-suppressed TNF-α levels in the lung or ameliorate disease. Soluble BAD1 was found to accumulate in the alveoli of infected mice at levels that suppressed TNF-α production by phagocytes. However, in contrast to yeast cell surface BAD1, which induced TGF-β, soluble BAD1 failed to do so and TNF-α suppression mediated by soluble BAD1 was unaffected by neutralization of TGF-β. Thus, BAD1 of B. dermatitidis induces suppression of TNF-α and progressive infection by both TGF-β-dependent and -independent mechanisms.