George S. Deepe

Advances in combating fungal diseases: vaccines on the threshold

The dramatic increase in fungal diseases in recent years can be attributed to the increased aggressiveness of medical therapy and other human activities. Immunosuppressed patients are at risk of contracting fungal diseases in healthcare settings and from natural environments. Increased prescribing of antifungals has led to the emergence of resistant fungi, resulting in treatment challenges. These concerns, together with the elucidation of the mechanisms of protective immunity against fungal diseases, have renewed interest in the development of vaccines against the mycoses. Most research has used murine models of human disease and, as we review in this article, the knowledge gained from these studies has advanced to the point where the development of vaccines targeting human fungal pathogens is now a realistic and achievable goal.

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.

Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum

A protective role for antibodies has not previously been described for host defense against the pathogenic fungus Histoplasma capsulatum (Hc). Mouse mAbs were generated from mice immunized with Hc yeast that binds the cell surface of Hc. Administration of mAbs before Hc infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model. Protection mediated by mAbs was associated with enhanced levels of IL-4, IL-6, and IFN-gamma in the lungs of infected mice. The mAbs increased phagocytosis of yeast by J774.16 cells through a CR3-dependent process. Ingestion of mAb-opsonized Hc by J774.16 macrophage-like cells was associated with yeast cell growth inhibition and killing. The mAbs bound to a 17-kDa antigen expressed on the surface of Hc. The antigen was identified as a histone H2B-like protein. This study establishes that mAbs to a cell surface protein of Hc alter the intracellular fate of the fungus and mediate protection in a murine model of lethal histoplasmosis, and it suggests a new candidate antigen for vaccine development.

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.

Chloroquine induces human macrophage killing of Histoplasma capsulatum by limiting the availability of intracellular iron and is therapeutic in a murine model of histoplasmosis.

We investigated the role of intracellular iron on the capacity of Histoplasma capsulatum (Hc) yeasts to multiply within human macrophages (Mphi). Coculture of Hc-infected Mphi with the iron chelator deferoxamine suppressed the growth of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by iron-free transferrin (apotransferrin). Chloroquine, which prevents release of iron from transferrin by raising endocytic and lysosomal pH, induced human Mphi to kill Hc. The effect of chloroquine was reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. Chloroquine (40-120 mg/kg) given intraperitoneally for 6 d to Hc-infected C57BL/6 mice significantly reduced the growth of Hc in a dose-dependent manner. At 120 mg/kg there was a 17- and 15-fold reduction (P < 0.01) in CFU in spleens and livers, respectively. The therapeutic effect of chloroquine also correlated with the length of treatment. As little as 2 d of chloroquine therapy (120 mg/kg), when started at day 5 after infection, reduced CFU in the spleen by 50%. Treatment with chloroquine for 10 d after a lethal inoculum of Hc protected six of nine mice; all control mice were dead by day 11 (P = 0.009). This study demonstrates that: (a) iron is of critical importance to the survival and multiplication of Hc yeasts in human Mphi; (b) in vitro, chloroquine induces Mphi killing of Hc yeasts by restricting the availability of intracellular iron; and (c) in vivo, chloroquine significantly reduces the number of organisms in the spleens and livers of Hc-infected mice and can protect mice from a lethal inoculum of Hc yeasts. Thus, chloroquine may be effective in the treatment of active histoplasmosis and also may be useful in preventing relapse of histoplasmosis in patients with acquired immunodeficiency syndromes.

Granulocyte Macrophage-Colony Stimulating Factor Induced Zn Sequestration Enhances Macrophage Superoxide and Limits Intracellular Pathogen Survival

Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like zinc (Zn). The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF-activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription-factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H⁺ channel function and triggered reactive oxygen species generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.

Immune Defects in 28-kDa Proteasome Activator γ-Deficient Mice

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-γ-inducible PA28α/β complex in Ag processing. Although the noninducible and predominantly nuclear PA28γ complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28γ-deficient mice and investigated their immune function. PA28γ−/− mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28γ−/− mice, like PA28α−/−/β−/− mice, are deficient in the processing of only specific Ags.

Modulation of Immune Responses in Murine Pulmonary Histoplasmosis

The influence of endogenous interleukin (IL)-12 on the course of pulmonary histoplasmosis was examined in naive and immune mice. All naive animals pretreated with anti-IL-12 monoclonal antibody (MAb) died by day 14. All mice died when anti-IL-12 MAb was initiated as late as postinfection day 3. Unlike those of controls, lungs of naive mice given anti-IL-12 MAb had depressed levels of interferon (IFN)-gamma and increased tumor necrosis factor (TNF)-alpha. The 2 groups had similar IL-4 levels. Administration of anti-IL-4 MAb rescued mice from the inimical effects of anti-IL-12 MAb. Survival of mice given both anti-IL-12 and anti-IL-4 MAb was associated with a blunted TNF-alpha response. In reinfection histoplasmosis, treatment with anti-IL-12 MAb did not alter survival. Fungus burden in lungs, livers, and spleens differed at week 2, but not at week 1, of infection. Thus, endogenous IL-12 is critical for optimal generation of a protective immune response in pulmonary histoplasmosis.

The CCL7-CCL2-CCR2 Axis Regulates IL-4 Production in Lungs and Fungal Immunity

Expression of the chemokine receptor CCR2 can be detrimental or beneficial for infection resolution. Herein, we examined whether CCR2 was requisite for control of infection by the dimorphic fungus Histoplasma capsulatum. H. capsulatum-infected CCR2−/− mice manifested defects in inflammatory cell recruitment, increased IL-4, and progressive infection. Increased IL-4 in CCR2−/− mice primarily contributed to decreased host resistance as demonstrated by the ability of IL-4-neutralized CCR2−/− mice to resolve infection without altering inflammatory cell recruitment. Surprisingly, numerous alveolar macrophages and dendritic cells contributed to IL-4 production in CCR2−/− mice. IL-4-mediated impairment of immunity in CCR2−/− mice was associated with increased arginase-1 and YM1 transcription and increased transferrin receptor expression by phagocytic cells. Immunity in mice lacking the CCR2 ligand CCL2 was not impaired despite decreased inflammatory cell recruitment. Neutralization of the CCR2 ligand CCL7 in CCL2−/− mice, but not wild type, resulted in increased IL-4 and fungal burden. Thus, CCL7 in combination with CCL2 limits IL-4 generation and exerts control of host resistance. Furthermore, increased phagocyte-derived IL-4 in CCR2−/− mice is associated with the presence of alternatively activated phagocytic cells.

PCR Assay for Identification of Histoplasma capsulatum Based on the Nucleotide Sequence of the M Antigen

ABSTRACT The major diagnostic antigens of Histoplasma capsulatum var. capsulatum are the H and M antigens, pluripotent glycoproteins that elicit both humoral and T-cell-mediated immune responses. The gene encoding the M antigen has previously been sequenced, and its sequence has significant overall homology to those of the genes for fungal catalases. Regions of the M-antigen gene with little or no homology were used to design four oligonucleotide sequences for application in the PCR detection and identification of H. capsulatum var. capsulatum. The PCR correctly identified the 31 H. capsulatum var. capsulatum strains isolated from human, animal, and soil specimens and 1 H. capsulatum var. duboisii isolate. PCR products of 111 and 279 bp were amplified with primers Msp1F-Msp1R and Msp2F-Msp2R, respectively. No amplification product was obtained from DNA extracted from an H. capsulatum var. farciminosum isolate. The specificity of the PCR with the M-antigen-derived primers was confirmed by the total absence of amplification products when genomic DNA from Paracoccidioides brasiliensis, Candida spp., Sporothrix schenckii, Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides immitis, Aspergillus niger, and Aspergillus fumigatus were applied in the reaction. This rapid, sensitive, and specific assay provides a way to identify typical and atypical isolates of H. capsulatum var. capsulatum.