Novel strategies to enhance vaccine immunity against coccidioidomycosis

Coccidioidomycosis is a potentially life-threatening respiratory mycosis endemic to the Americas and caused by inhalation of spores produced by the molds Coccidioides immitis and C. posadasii

Intrinsic role of MyD88 for anti-fungal Tc17 cell responses (P3338)

Abstract Our previous studies showed that vaccine-induced Tc17 cells are necessary for antifungal defenses to control lethal pneumonia due to experimental blastomycosis. Although dendritic cell cytokines such as IL-6, TGF-β and IL-1 are generally required for differentiation of Th17 cells, the role of intrinsic MyD88 signaling and upstream pattern recognition receptors (PRRs) on CD8 T cells for anti-fungal Tc17 responses has not been studied. In work here, we found that MyD88 expression in CD8 T cells was essential for the generation of Tc17 responses following vaccination. As IL-18, IL-1 and PRRs mediate signaling through MyD88, we investigated the requirement of each of these components for anti-fungal Tc17 responses. We found in vivo that IL-18R was dispensable and that IL-1R1 signaling was insufficient to account for the Tc17 defect observed in MyD88-/- CD8 T cells, suggesting a potential role for PRRs. Our in vitro data also suggested that TLR2 signaling is required for Tc17 differentiation. Interestingly, Tc1 responses were relatively spared in MyD88-/- CD8 T cells. Mechanistically, MyD88-/- Tc17 not Tc1 cells failed to proliferate and levels of Bcl2 and Bcl-xL were unaffected, indicating poor expansion but not death of MyD88-/- Tc17 cells as a cause of poor Tc17 responses. Thus, intrinsic MyD88 and PRR signals in CD8 T cells are crucial for the generation of anti-fungal Tc17 responses. These data may have potential applications for designing fungal vaccines.</jats:p

Long-term maintenance of protective memory CD8+ T cells in the absence of CD4+ T cells in mice vaccinated against <i>Blastomyces dermatitidis</i> (45.3)

Abstract CD8+- and CD4+- T cell mediated immunity is major contributing factor for defense against pathogenic fungi. Our previous studies have shown that CD8+ T cells in the absence of CD4+ T cells protected vaccinated mice from lethal challenge with Blastomyces dermatitidis. However, it has also been shown that CD4+ T-cell help is required for maintenance of memory CD8+ T cells after viral and intracellular bacterial infections. To explore the requirements for maintenance of CD8+ T cell memory against pathogenic fungi, we tested whether fungal-specific memory CD8+ T cell persist in the absence of CD4+ T cell help. Here, we show that protective CD8+ T cell memory endures without CD4+ T-cell help for at least six months after fungal vaccination in mice. Additionally, we show that type I cytokines (IFNγ and TNFα), which are requisite for mediating protection, remained intact in the memory CD8+ T cells after a recall response to a lethal pulmonary challenge in vaccinated mice. The phenotypic markers CD27, CD62L, CD122, and CD127 of memory CD8+ T cells were similar to those on CD8+ T-cells induced with helper CD4+ T cells. These findings imply that CD4+ T cell help is not required for the generation, maintenance, and recall responses of protective memory CD8+ T cells after fungal vaccination. Our studies have potential implications for the usage of fungal vaccines in immune-compromised hosts.</jats:p

CCR6+Tc17 cells induced in the absence of CD4 T-cell help upon vaccination with <i>Blastomyces dermatitidis</i> mediate protective immunity (99.8)

Abstract The increasing incidence of fungal infections in immune-compromised patients calls for the development of suitable vaccines to target CD8 T cells. Accumulating studies indicate that Th17 cells play an important role in anti-fungal immunity. However, whether IL-17 producing CD8 T cells (Tc17) can be induced and mediate protective immunity against fungal infection in the absence of CD4 help is not known. Here, using mouse model of vaccination against lethal Blastomyces dermatitidis infection, we show that upon vaccination Tc17 cells are induced and re-called at the site of lethal challenge in the absence of CD4 T-cell help. Vaccine immunity by CD8 T cells was severely impaired in both vaccinated- IL-17A KO and IL-17-receptor KO mice in the absence of CD4 T cells. Neutralization of IL-17A during the re-call response also impaired fungal clearance by effector CD8 T cells. Further, we show that protective immunity by Tc17 cells was not influenced by deficient IL-12 signaling, required for Type I immunity, but neutrophils were essential in Tc17 cell action. Tc17 cells preferentially expressed CCR6 over CXCR3 chemokine receptor (distinct from IFNγ producing cells, which were CXCR3+ve) and antibody blocking of CXCR3 did not affect the re-call response of Tc17 cells to the lung. Collectively, our studies show that protective Tc17 cells are induced in the absence of CD4 T cell help and may aid in designing fungal vaccines for immune-compromised hosts.</jats:p

The Eng1 β-Glucanase Enhances Histoplasma Virulence by Reducing β-Glucan Exposure

The fungal pathogen Histoplasma capsulatum parasitizes host phagocytes. To avoid antimicrobial immune responses, Histoplasma yeasts must minimize their detection by host receptors while simultaneously interacting with the phagocyte. Pathogenic Histoplasma yeast cells, but not avirulent mycelial cells, secrete the Eng1 protein, which is a member of the glycosylhydrolase 81 (GH81) family. We show that Histoplasma Eng1 is a glucanase that hydrolyzes β-(1,3)-glycosyl linkages but is not required for Histoplasma growth in vitro or for cell separation. However, Histoplasma yeasts lacking Eng1 function have attenuated virulence in vivo, particularly during the cell-mediated immunity stage. Histoplasma yeasts deficient for Eng1 show increased exposure of cell wall β-glucans, which results in enhanced binding to the Dectin-1 β-glucan receptor. Consistent with this, Eng1-deficient yeasts trigger increased tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) cytokine production from macrophages and dendritic cells. While not responsible for large-scale cell wall structure and function, the secreted Eng1 reduces levels of exposed β-glucans at the yeast cell wall, thereby diminishing potential recognition by Dectin-1 and proinflammatory cytokine production by phagocytes. In α-glucan-producing Histoplasma strains, Eng1 acts in concert with α-glucan to minimize β-glucan exposure: α-glucan provides a masking function by covering the β-glucan-rich cell wall, while Eng1 removes any remaining exposed β-glucans. Thus, Histoplasma Eng1 has evolved a specialized pathogenesis function to remove exposed β-glucans, thereby enhancing the ability of yeasts to escape detection by host phagocytes

Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity

Full author list omitted for brevity. For the full list of authors, see article.The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge

Characterization of C-type lectins reveals an unexpectedly limited interaction between <i>Cryptococcus neoformans</i> spores and Dectin-1

<div><p>Phagocytosis by innate immune cells is an important process for protection against multiple pathologies and is particularly important for resistance to infection. However, phagocytosis has also been implicated in the progression of some diseases, including the dissemination of the human fungal pathogen, <i>Cryptococcus neoformans</i>. Previously, we identified Dectin-1 as a likely phagocytic receptor for <i>C</i>. <i>neoformans</i> spores through the use of soluble components in receptor-ligand blocking experiments. In this study, we used gain-of-function and loss-of-function assays with intact cells to evaluate the in vivo role of Dectin-1 and other C-type lectins in interactions with <i>C</i>. <i>neoformans</i> spores and discovered stark differences in outcome when compared with previous assays. First, we found that non-phagocytic cells expressing Dectin-1 were unable to bind spores and that highly sensitive reporter cells expressing Dectin-1 were not stimulated by spores. Second, we determined that some phagocytes from Dectin-1^-/- mice interacted with spores differently than wild type (WT) cells, but the effects varied among assays and were modest overall. Third, while we detected small but statistically significant reductions in phagocytosis by primary alveolar macrophages from Dectin-1^-/- mice compared to WT, we found no differences in survival between WT and Dectin-1^-/- mice challenged with spores. Further analyses to assess the roles of other C-type lectins and their adapters revealed very weak stimulation of Dectin-2 reporter cells by spores and modest differences in binding and phagocytosis by Dectin-2^-/- bone marrow-derived phagocytes. There were no discernable defects in binding or phagocytosis by phagocytes lacking Mannose Receptor, Mincle, Card-9, or FcRγ. Taken together, these results lead to the conclusion that Dectin-1 and other C-type lectins do not individually play a major roles in phagocytosis and innate defense by phagocytes against <i>C</i>. <i>neoformans</i> spores and highlight challenges in using soluble receptor/ligand blocking experiments to recapitulate biologically relevant interactions.</p></div

Absence of Dectin-1 or Dectin-2 does not alter association of cryptococcal spores with primary murine phagocytes.

(A) Macrophage-enriched bone marrow-derived phagocytes from WT (black bars), Dectin-1 knockout (dark gray bars) and Dectin-2 knockout (light gray bars) mice were co-incubated with live C. neoformans spores or heat-killed C. albicans yeast, and microscopy was used to assess association of the fungal cells with the macrophages. X-axis shows the fungal cell types being assessed. Y-axis shows the percent of fungal-associated phagocytes relative to WT. Statistical analysis was carried out on 3 individual microscopic fields containing approximately 50 macrophages each. P-values are the result of a Student’s t-test. (B) Representative microscopy fields at 400X of heat-killed C. albicans co-incubated with WT and Dectin-1 knockout macrophage-enriched phagocytes (no difference was observed with C. neoformans). (Black bars = 50 μm) (C) Macrophage-enriched bone marrow-derived phagocytes from WT, Dectin-1-/-, Dectin-2-/-, Card-9-/-, FcRγ-/-, Mincle-/- and MR-/- mice were co-incubated with live C. neoformans spores (black bars) or live C. neoformans yeast (light gray bars) and association (binding + phagocytosis) frequencies determined by CFU analysis. X-axis shows the macrophage genotypes. Y-axis shows the percent of introduced fungal cells that were associated with the phagocytes. Data show the mean and standard deviations from a single experiment carried out in triplicate. An ANOVA with a Dunnett’s post-hoc test revealed that phagocyte association with spores was not statistically different from WT for any of the knock out phagocytes tested (p>0.05); results are representative of those obtained from 2–5 individual experiments for each knockout line compared to WT.</p

Protective antifungal memory CD8+ T cells are maintained in the absence of CD4+ T cell help and cognate antigen in mice

Individuals who are immunocompromised, including AIDS patients with few CD4+ T cells, are at increased risk for opportunistic fungal infections. The incidence of such infections is increasing worldwide, meaning that the need for antifungal vaccines is increasing. Although CD4+ T cells play a dominant role in resistance to many pathogenic fungal infections, we have previously shown that vaccination can induce protective antifungal CD8+ T cell immunity in the absence of CD4+ T cells. However, it has not been determined whether vaccine-induced antifungal CD8+ T cell memory can be maintained in the absence of CD4+ T cell help. Here, we have shown in a mouse model of vaccination against blastomycosis that antifungal memory CD8+ T cells are maintained in the absence of CD4+ T cells without loss of numbers or function for at least 6 months and that the cells protect against infection. Using a system that enabled us to induce and track antigen-specific, antifungal CD8+ T cells, we found that such cells were maintained for at least 5 months upon transfer into naive mice lacking both CD4+ T cells and persistent fungal antigen. Additionally, fungal vaccination induced a profile of transcription factors functionally linked with persistent memory in CD8+ T cells. Thus, unlike bacteria and viruses, fungi elicit long-term CD8+ T cell memory that is maintained without CD4+ T cell help or persistent antigen. This has implications for the development of novel antifungal vaccine strategies effective in immunocompromised patients