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PubMed2004

Immunogenicity and efficacy of Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan peptide mimotope-protein conjugates in human immunoglobulin transgenic mice.

Peptide mimotopes of capsular polysaccharides have been proposed as antigens for vaccines against encapsulated pathogens. In this study, we determined the antibody response to and efficacy of P13, a peptide mimetic of the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM), in mice that produce human antibodies. P13 was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and administered subcutaneously in Alhydrogel with or without CpG to mice transgenic for human immunoglobulin loci (XenoMouse mice) and expressing either immunoglobulin G2 (IgG2) (G2 mice) or IgG4 (G4 mice). Mice were vaccinated and revaccinated two or three times. The serum antibody responses of the mice to GXM and P13 and antibody idiotype expression were analyzed by an enzyme-linked immunosorbent assay. The results showed that both P13-TT and P13-DT were antigenic, inducing a mimetic response to P13 in both G2 and G4 mice, and immunogenic, inducing a mimotope response including VH3 (idiotype)-positive antibodies to GXM in G2 but not G4 mice. CpG led to higher titers of IgG to P13 and GXM in P13-TT-vaccinated G2 mice. C. neoformans challenge of P13-protein conjugate-vaccinated and control G2 mice induced anamnestic IgG- and VH3-positive responses to GXM and was associated with a significantly decreased risk of death and a prolongation of survival in P13-DT-vaccinated mice compared to phosphate-buffered saline-treated or protein carrier-vaccinated mice. These findings reveal that P13 elicited a human antibody response with VH3 expression in human immunoglobulin transgenic mice that has been observed for human antibodies to GXM and support the concept that peptide mimotope-based vaccines may hold promise for the treatment of C. neoformans infections.

Animals↗
PubMed2004

Glucuronoxylomannan, the major capsular polysaccharide of Cryptococcus neoformans, inhibits the progression of group B streptococcal arthritis.

Glucuronoxylomannan (GXM), the principal constituent of the Cryptococcus neoformans capsule, modulates the inflammatory response of human monocytes in vitro. Here we examine the efficacy of GXM as a novel anti-inflammatory compound for use against experimental septic arthritis. Arthritis was induced in mice by the intravenous injection of 8 x 10(6) CFU of type IV group B streptococcus (GBS). GXM was administered intravenously in different doses (50, 100, or 200 microg/mouse) 1 day before and 1 day after bacterial inoculation. GXM treatment markedly decreased the incidence and severity of articular lesions. Histological findings showed limited periarticular inflammation in the joints of GXM-treated mice, confirming the clinical observations. The amelioration of arthritis was associated with a significant reduction in the local production of interleukin-6 (IL-6), IL-1beta, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-2 and an increase in systemic IL-10 levels. Moreover, peritoneal macrophages derived from GXM-treated mice and stimulated in vitro with heat-inactivated GBS showed a similar pattern of cytokine production. The present study provides evidence for the modulation of the inflammatory response by GXM in vivo and suggests a potential therapeutic use for this compound in pathologies involving inflammatory processes.

Animals↗
PubMed2004

CNLAC1 is required for extrapulmonary dissemination of Cryptococcus neoformans but not pulmonary persistence.

The pathogenic yeast Cryptococcus neoformans produces a laccase enzyme (CNLAC1), which catalyzes the synthesis of melanin in the presence of phenolic compounds. A number of genes have been implicated in the regulation of laccase and melanization, including IPC1, GPA1, MET3, and STE12. Albino mutants derived from random mutagenesis techniques may contain mutations in genes that regulate multiple virulence factors, including CNLAC1. The goal of our study is to investigate the role of CNLAC1 in virulence and evasion of pulmonary host defenses after infection via the respiratory tract. Using a set of congenic laccase-positive (2E-TUC-4) and laccase-deficient (2E-TU-4) strains, we found that both strains are avirulent at a lower dose (10(4) CFU/mouse) in mice. After the infectious dose was increased to 10(6) CFU/mouse, 70% mortality was observed in mice infected with 2E-TUC-4 compared to no mortality in mice infected with 2E-TU-4 at day 30 postinfection. This observation confirms the requirement for CNLAC1 in virulence. Interestingly, we observed no differences between the two strains in pulmonary growth or in elicitation of cellular immune responses in the lung. The only measurable defect of 2E-TU-4 was in dissemination to extrapulmonary sites. To examine the role of CNLAC1 in dissemination, mice were infected intravenously. By week 3 postinfection, equal numbers of strains 2E-TUC-4 and 2E-TU-4 were recovered from the brain and spleen. This observation indicates that CNLAC1 facilitates escape from the lung, but not growth in the lungs or brain, and suggests a novel role for CNLAC1 in virulence during an infection aquired via the respiratory tract.

Animals↗
PubMed2004

Cryptococcus neoformans capsule structure evolution in vitro and during murine infection.

Cryptococcus neoformans capsule structure modifications after prolonged in vitro growth or in vivo passaging have been reported previously. However, nothing is known about the dynamics of these modifications or about their environmental specificities. In this study, capsule structure modifications after mouse passaging and prolonged in vitro culturing were analyzed by flow cytometry using the glucuronoxylomannan-specific monoclonal antibody E1. The capsule structures of strains recovered after 0, 1, 8, and 35 days were compared by using the level of E1-specific epitope expression and its cell-to-cell heterogeneity within a given cell population. In vitro, according to these parameters, the diversity of the strains was higher on day 35 than it was initially, suggesting the absence of selection during in vitro culturing. In contrast, the diversity of the strains recovered from the brain tended to decrease over time, suggesting that selection of more adapted strains had occurred. The strains recovered on day 35 from the spleen and the lungs had different phenotypes than the strains isolated from the brain of the same mouse on the same day, thus strongly suggesting that there is organ specificity for C. neoformans strain selection. Fingerprinting of the strains recovered in vitro and in vivo over time confirmed that genotypes evolved very differently in vitro and in vivo, depending on the environment. Overall, our results suggest that organ-specific selection can occur during cryptococcosis.

Animals↗
PubMed2004

Antibody to Cryptococcus neoformans glucuronoxylomannan inhibits the release of capsular antigen.

Cryptococcus neoformans releases capsular polysaccharide in the supernatant of liquid cultures and in tissues. Significantly less glucuronoxylomannan (GXM) was released by C. neoformans in the presence of capsule-binding monoclonal antibody (MAb). MAb-mediated inhibition of GXM release may be another mechanism by which humoral immunity can mediate protection against this pathogen.

Antibodies, Fungal↗
PubMed2004

Protective and nonprotective human immunoglobulin M monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan manifest different specificities and gene use profiles.

The features of protective murine antibodies to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been rigorously investigated; however, the characteristics of protective human antibodies to GXM have not been defined. We produced monoclonal antibodies (MAbs) from XenoMouse mice (transgenic mice that express human immunoglobulin M [IgM], IgG2, and kappa) which were immunized with a C. neoformans serotype D strain 24067 GXM-diphtheria toxoid conjugate. This study reports the specificity and efficacy of three human IgM MAbs, G14, G15, and G19, generated from these mice. Each MAb was specific for GXM, but G14 and G19 had different specificity based on their binding to serotype A strain H99 and SB4 GXMs, to which G15 did not bind. Nucleic acid sequence analysis revealed that G15 uses V(H)3-64 in the germ line configuration. G14 and G19 use V(H)6-1, which has somatic mutations. All of the MAbs use V kappa DPK22/A27. Studies of MAb efficacy in BALB/c mice showed that administration of 0.1 mg, but not 1 or 0.01 mg, of G15 prolonged survival against lethal C. neoformans strain 24067 challenge, whereas G14 and G19 were not protective at any dose. This panel of MAbs illustrates that serotype D GXM has epitopes that elicit human antibodies that can be either protective or nonprotective. Our findings suggest that V(H) gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that V(H) gene use may have in resistance and susceptibility to cryptococcosis.

Amino Acid Sequence↗
PubMed2004

Age-related resistance of C57BL/6 mice to Cryptococcus neoformans is dependent on maturation of NKT cells.

Conflicting results have been reported regarding the ability of C57BL/6 mice to clear infections due to Cryptococcus neoformans. Examination of the various experimental protocols used suggested that C57BL/6 mice might develop the ability to resist infection as they mature. We analyzed the ability of C57BL/6 mice of different ages to respond to immunization with cryptococcal antigen or to clear a cryptococcal infection. Mice were immunized with a soluble cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund's adjuvant (CneF-CFA). Delayed-type hypersensitivity (DTH) reactions elicited by the immunization were significantly stronger in 15-week-old C57BL/6 mice than in 7-week-old mice. Analysis of cryptococcal CFU 8 weeks following intratracheal infection of 7-week-old mice or 15-week-old mice revealed a relative inability of the younger animals to control the infection. Six-week-old immunized and infected mice cleared cryptococci from brain, spleen, and liver in a manner similar to that of immunized and infected 15-week-old mice. However, the older mice cleared cryptococci much more efficiently from the lungs. The possible role for NKT cells was determined by passive transfer of thymocytes from 10-week-old mice (containing mature NKT cells) or 2-week-old mice (containing immature NKT cells) to 6-week-old mice. The 10-week-old thymocytes significantly enhanced the ability of the mice to develop a DTH response after immunization with CneF-CFA, while animals treated with 2-week-old thymocytes did not improve their DTH response after immunization. The cells in the 10-week-old thymocyte population responsible for improvement of DTH responses were identified as being NK1.1 positive.

Aging↗
PubMed2004

Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo.

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice. MyD88(-/-) mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Opsonic requirements for dendritic cell-mediated responses to Cryptococcus neoformans.

The encapsulated pathogenic yeast Cryptococcus neoformans is poorly recognized by phagocytic cells in the absence of opsonins. Macrophages will bind and internalize complement- or antibody-opsonized C. neoformans; however, less is known about the role of opsonins in dendritic cell (DC)-mediated recognition of the organism. Thus, we studied the opsonic requirements for binding to C. neoformans by cultured human monocyte-derived and murine bone marrow-derived DCs and whether binding leads to antifungal activity and cytokine release. Binding of unopsonized C. neoformans to human and murine DCs was negligible. Opsonization with pooled human serum (PHS) increased binding, while heat treatment of PHS virtually abolished this binding, thus suggesting a role for heat-labile complement components. PHS plus a monoclonal anticapsular antibody, 3C2, had an additive effect on binding for most cryptococcal strains. Human and murine DCs exhibited pronounced anticryptococcal activity in the presence of the antibody at early (2-h) and late (24-h) time points; however, PHS opsonization did not supplement this anticryptococcal activity. Antifungal activity against C. neoformans opsonized in PHS and/or antibody was partially reduced in the presence of inhibitors of the respiratory burst response. Human, but not murine, DCs released modest amounts of tumor necrosis factor alpha when stimulated with C. neoformans opsonized in PHS and/or antibody. However, opsonized C. neoformans failed to stimulate detectable release of interleukin 10 (IL-10) or IL-12p70 from either DC population. Thus, human and murine DCs show maximal binding to and antifungal activity against C. neoformans via a process highly dependent on opsonization.

Animals↗
PubMed2005

Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based approach.

Caenorhabditis elegans can serve as a substitute host for the study of microbial pathogenesis. We found that mutations in genes of the fungal pathogen Cryptococcus neoformans involved in mammalian virulence allow C. elegans to produce greater numbers of progeny than when exposed to wild-type fungus. We used this property to screen a library of C. neoformans mutants for strains that permit larger C. elegans brood sizes. In this screen, we identified a gene homologous to Saccharomyces cerevisiae ROM2. C. neoformans rom2 mutation resulted in a defect in mating and growth defects at elevated temperature or in the presence of cell wall or hyperosmolar stresses. An effect of the C. neoformans rom2 mutation in virulence was confirmed in a murine inhalation infection model. We propose that a screen for progeny-permissive mutants of microorganisms can serve as a high-throughput method for identifying novel loci related to mammalian pathogenesis.

Animals↗
PubMed2005

Receptor-mediated clearance of Cryptococcus neoformans capsular polysaccharide in vivo.

Cryptococcus neoformans capsular glucuronoxylomannan (GXM) is shed during cryptococcosis and taken up by macrophages. The roles of the putative GXM receptors CD14, CD18, Toll-like receptor 2 (TLR2), and TLR4 in GXM clearance from serum and deposition in the liver and spleen in receptor-deficient mice were studied. While alterations in the kinetics of GXM redistribution were seen in the mutant mice, none of the receptors was absolutely required for serum clearance or hepatosplenic accumulation.

Animals↗
PubMed2005

Antibody-mediated protection against Cryptococcus neoformans pulmonary infection is dependent on B cells.

The pathogenesis of pulmonary Cryptococcus neoformans infection and the efficacy of passive immunoglobulin G1 (IgG1) administration were investigated in B-cell-deficient and C57BL/6J mice. C57BL/6J mice lived longer than B-cell-deficient mice after both intratracheal and intravenous infections. Administration of IgG1 prior to infection prolonged the survival of C57BL/6J mice but had no effect on the survival or numbers of CFU in the lungs of B-cell-deficient mice. C. neoformans infection in B-cell-deficient mice resulted in significantly higher levels of gamma interferon (IFN-gamma), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha) than in C57BL/6J mice. IgG1 administration reduced IFN-gamma and MCP-1 levels in C57BL/6J mice but not in B-cell-deficient mice. In addition, compared to its effect in C57BL/6J mice, C. neoformans infection in FcRgammaIII-deficient, athymic, and SCID mice significantly increased IFN-gamma and MCP-1 levels. IgG1 administration was associated with reduced IFN-gamma levels in C57BL/6J mice but not in FcRgammaIII-deficient, athymic, and SCID mice. These observations suggest that IgG1-mediated protection in this system is a consequence of alterations in the inflammatory response that translate into less damage to the host without directly reducing the fungal burden. For hosts with impaired immunities, the ineffectiveness of passive antibody (Ab) may reflect an inability to down-regulate inflammation and avoid self-damage. The results indicate an important role for B cells in host defense against C. neoformans infection and demonstrate a surprising dependence of Ab-mediated protection on B cells in this system.

Animals↗
PubMed2005

Mannoproteins from Cryptococcus neoformans promote dendritic cell maturation and activation.

Our previous data show that mannoproteins (MPs) from Cryptococcus neoformans are able to induce protective responses against both C. neoformans and Candida albicans. Here we provide evidence that MPs foster maturation and activation of human dendritic cells (DCs). Maturation was evaluated by the ability of MPs to facilitate expression of costimulatory molecules such as CD40, CD86, CD83, and major histocompatibility complex classes I and II and to inhibit receptors such as CD14, CD16, and CD32. Activation of DCs was measured by the capacity of MPs to promote interleukin-12 and tumor necrosis factor alpha secretion. DC-induced maturation and interleukin-12 induction are largely mediated by engagement of mannose receptors and presume MP internalization and degradation. DC activation leads to IkappaBalpha phosphorylation, which is necessary for nuclear factor kappaB transmigration into the nucleus. MP-loaded DCs are efficient stimulators of T cells and show a remarkable capacity to promote CD4 and CD8 proliferation. In conclusion, we have evidenced a novel regulatory role of MPs that promotes their candidacy as a vaccine against fungi.

Antigens, Surface↗
PubMed2005

Phenotypic switching of Cryptococcus neoformans can produce variants that elicit increased intracranial pressure in a rat model of cryptococcal meningoencephalitis.

Increased intracranial pressure (ICP) plays an important role in the morbidity and mortality of cryptococcal meningoencephalitis. The microbial and host factors that contribute to the development of increased ICP are poorly understood. We found that phenotypic switch variants of Cryptococcus neoformans (smooth and mucoid) differed in their abilities to promote increased ICP in a rat model of cryptococcal meningitis. Rats infected with the mucoid variant developed increased ICP, whereas rats infected with the smooth parent did not. This trend correlated with a shorter survival time and a higher cerebrospinal fluid (CSF) fungal burden for mucoid variant-infected rats, although brain fungal burdens were comparable between mucoid variant- and smooth parent-infected rats. Magnetic resonance imaging revealed enhanced T2 signal intensity over the surfaces of the brains of mucoid variant-infected rats. In addition, more polysaccharide accumulated in the CSF and brains of mucoid variant-infected rats. The accumulation of glucorunoxylomannan was associated with elevated levels of MCP-1 (CCL2) and, accordingly, a more pronounced but ineffective monocytic inflammatory response in the meninges of mucoid variant-infected rats. In summary, these findings suggest that strain-specific characteristics can influence the development of increased ICP and indicate a manner in which phenotypic switching could influence the outcome of a central nervous system infection.

Animals↗
PubMed2005

The gamma interferon receptor is required for the protective pulmonary inflammatory response to Cryptococcus neoformans.

Mice with a null deletion mutation in the gamma interferon (IFN-gamma) receptor gene were used to study the role of IFN-gamma responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-gamma receptor gene (IFN-gammaR-/-) and into control mice (IFN-gammaR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-gammaR-/- mice are markedly more susceptible to C. neoformans infection than IFN-gammaR+/+ mice. (ii) In the absence of IFN-gamma signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-gamma receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-gammaR-/- mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-gamma signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- gamma responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-gamma is not affected, IFN-gammaR-/- mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-gamma signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.

Animals↗
PubMed2005

Melanization of Cryptococcus neoformans affects lung inflammatory responses during cryptococcal infection.

The production of melanin pigments is associated with virulence for many microbes. Melanin is believed to contribute to microbial virulence by protecting microbial cells from oxidative attack during infection. However, there is also evidence from various systems that melanins have immunomodulatory properties, which conceivably could contribute to virulence by altering immune responses. To investigate the effect of melanin on the immune response, we compared the murine pulmonary responses to infection with melanized and nonmelanized Cryptococcus neoformans cells. Infection with melanized cells resulted in a greater fungal burden during the early stages of infection and was associated with higher levels of interleukin-4 and MCP-1 and greater numbers of infiltrating leukocytes. Infection with laccase-positive (melanotic) C. neoformans cells also elicited higher MCP-1 levels and more infiltrating leukocytes than did infection with laccase-negative cells. Melanization interfered with phagocytosis in vivo for encapsulated C. neoformans but not for nonencapsulated cells. The results provide strong evidence that cryptococcal melanization can influence the immune response to infection and suggest that immunomodulation is an additional mechanism by which the pigment contributes to virulence.

Animals↗