Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Anti-beta-glucan antibodies in healthy human subjects.

Previous data by our group demonstrated the antifungal efficacy of a vaccine consisting of laminarin (beta-(1,3)-glucan), conjugated with diphtheria toxoid, which generated protective anti-laminarin antibodies in mice. In this paper, we sought for the presence, isotype and subclass composition of natural anti-laminarin antibodies in an unselected population of human healthy subjects, in a comparison with antibodies directed against beta-(1,6)-glucan (pustulan) and branched beta-(1,3/1,6)-glucan (Pool 1) and mannan from Candida albicans. Almost all subjects showed detectable levels of anti-beta-glucan antibodies, with IgG largely prevailing on IgM, little, if any, IgA and no IgE. However, the titer of anti-beta-glucan antibodies was overall about 1log lower than that of anti-mannan antibodies of the corresponding isotype. In particular, the level of anti-laminarin IgG was the lowest one, its geometrical mean titer (95% confidence interval, CI) being 1838 (1245-2714) as compared to 8157 (6067-10,931) and 3940 (2911-5332) for pustulan and Pool 1 fungal glucan, respectively. Analysis of IgG subclass composition showed that IgG2 was the prevalent subclass against any antigen, and again the concentration of anti-laminarin IgG2 was the lowest one, its geometrical mean concentration being 0.13 (0.07-0.24)microg/ml as compared to anti-pustulan and anti-Pool 1 glucan and mannan IgG2 levels, which were 0.33 (0.2-0.5), 1.35 (0.9-2.0), and 36.1 (25.2-51.3)microg/ml, respectively. These data show that anti-laminarin antibodies are present at low levels in humans as compared to other anti-beta-glucan and, mostly, anti-mannan antibodies, and suggest that a protective antifungal vaccination in humans should attempt to tip the balance of antifungal antibodies in favour of the anti-laminarin ones.

The interaction of human dendritic cells with yeast and germ-tube forms of Candida albicans leads to efficient fungal processing, dendritic cell maturation, and acquisition of a Th1 response-promoting function.

T helper cell type 1 (Th1) cell-mediated immunity plays a critical role in protection against the opportunistic pathogen Candida albicans. Virulence of the fungus is closely associated with its ability to form germ-tubes (GT), the early phase of the dimorphic transition from the commensal yeast (Y) to the more invasive hyphal (H) form. In this study, we examined the functional outcome of the interaction of Y or GT forms with human dendritic cells (DCs), professional antigen-presenting cells, which are pivotal for initiation and modulation of T cell responses. DCs phagocytosed and killed Y and GT cells with a comparable efficiency, becoming able to trigger strong proliferative responses by Candida-specific, autologous T cell clones. Both fungal forms induced DC maturation, as indicated by up-regulation of CD83, CD80, CD86, CD40, and major histocompatibility complex classes I and II surface antigens. Chemokine receptors were also modulated in Candida-DCs, which showed increased CCR7/CXCR4 and decreased CCR5 expression. Y- and GT-activated DCs differed in the pattern of cytokine expression. In particular, GT cells, in common with fully differentiated H cells, induced significantly more elevated levels of interleukin (IL)-10 than Y cells. Nevertheless, Y-, GT-, or H-pulsed DCs secreted comparable amounts of IL-12p70. In addition, irrespective of the fungal form triggering DC activation, Candida-DCs acquired the ability to prime naive T lymphocytes with a defined Th1 phenotype. Overall, our findings highlight the induction of substantially similar functional patterns in human DCs encountering the different forms of growth of C. albicans, both seemingly activating the Th1-type immunity which is characteristic of the healthy human subjects, naturally immunized and protected against the fungus.

Antigenic properties and processing requirements of 65-kilodalton mannoprotein, a major antigen target of anti-Candida human T-cell response, as disclosed by specific human T-cell clones.

T-cell-mediated immunity is known to play a central role in the host response to Candida albicans. T-cell clones are useful tools for the exact identification of fungal T-cell epitopes and the processing requirements of C. albicans antigens. We isolated human T-cell clones from an HLA-DRB1*1101 healthy donor by using an antigenic extract (MP-F2) of the fungus. Specific clones were T-cell receptor alpha/beta and CD4(+)/CD8(-) and showed a T-helper type 1 cytokine profile (production of gamma interferon and not interleukin-4). The large majority of these clones recognized both the natural (highly glycosylated) and the recombinant (nonglycosylated) 65-kDa mannoprotein (MP65), an MP-F2 minor constituent that was confirmed to be an immunodominant antigen of the human T-cell response. Surprisingly, most of the clones recognized two synthetic peptides of different MP65 regions. However, the peptides shared the amino acid motif IXSXIXXL, which may be envisaged as a motif sequence representing the minimal epitope recognized by these clones. Three clones recognized natural and pronase-treated MP65 but did not detect nonglycosylated, recombinant MP65 or the peptides, suggesting a possible role for polysaccharides in T-cell recognition of C. albicans. Finally, lymphoblastoid B-cell lines were efficient antigen-presenting cells (APC) for recombinant MP65 and peptides but failed to present natural, glycosylated antigens, suggesting that nonprofessional APC might be defective in processing highly glycosylated yeast proteins. In conclusion, this study provides the first characterization of C. albicans-specific human T-cell clones and provides new clues for the definition of the cellular immune response against C. albicans.

Differential chemokine response of human monocytes to yeast and hyphal forms of Candida albicans and its relation to the beta-1,6 glucan of the fungal cell wall.

Hyphae formation from yeast cells is a virulence trait enabling the human opportunistic pathogen Candida albicans to invade host tissues. Hyphal cells proved to be much less efficient than yeast cells in stimulating production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, interleukin-8 (IL-8), and particularly, monocyte chemotactic protein-1 (MCP-1) by human monocyte. This different stimulation did not depend on the monocyte inability to ingest the hyphae nor did it imply hyphal resistance to the extracellular killing by the monocytes. Purified hyphal and yeast cell walls reproduced the differences shown by the intact cells, and chemical-enzymatic dissection of cell wall components suggested that cell wall beta-1,6 rather than beta-1,3 glucan was the main chemokine inducer. Coherently, immunofluorescence studies with an anti beta-1,6 glucan serum showed that the surface expression of this polysaccharide was much lower on hyphae than on yeast cells. By minimizing chemokine induction, the formation of hyphal filaments might facilitate C. albicans escaping from host immunity.

Defective induction of interleukin-12 in human monocytes by germ-tube forms of Candida albicans.

Yeast (Y) to germ-tube (GT) transition of Candida albicans is considered a putative virulence trait. On the other hand, interleukin-12 (IL-12) is a key promoter of T-helper type 1 protective immunity against this human opportunistic pathogen. We studied IL-12 production by human monocytes cocultured in vitro with Y or GT forms of C. albicans. Following stimulation by Y cells, monocytes produced appreciable levels of IL-12, which, upon addition of gamma interferon (IFN-gamma), compared to those achievable by lipopolysaccharide (100 ng/ml) stimulation (140 +/- 65 and 185 +/- 80 pg/ml, respectively [mean +/- standard deviation in four independent experiments]). In contrast, IL-12 production by GT cell-stimulated monocytes was much lower or absent (<5 pg/ml) and could not be brought to the level induced by Y cells by the addition of IFN-gamma (30 +/- 10 pg/ml in the four independent experiments above). Besides being observed as actual cytokine production, this lower response was also observed as specific IL-12 p40 mRNA transcript and was not associated with hyperproduction of the IL-12-competing cytokine IL-10. Phagocytosis and killing experiments in the presence of cytochalasin D showed that IL-12 production by Y cell-stimulated monocytes was phagocytosis dependent and that GT cells of C. albicans were not phagocytized by the human monocytes. Importantly, however, Y and GT cells were equally killed by the monocytes. Thus, the virulence trait attributed to the Y-GT transition of C. albicans might also be related to the lack of induction by GT cells of a protective anticandidal immunity through defective IL-12 production.

In vitro and in vivo anticandidal activity of human immunodeficiency virus protease inhibitors.

Highly active antiretroviral therapy that includes human immunodeficiency virus (HIV) aspartyl protease inhibitors (PIs) causes a decline in the incidence of some opportunistic infections in AIDS, and this decline is currently attributed to the restoration of specific immunity. The effect of two PIs (indinavir and ritonavir) on the enzymatic activity of a secretory aspartyl protease (Sap) of Candida albicans (a major agent of mucosal disease in HIV-infected subjects) and on growth and experimental pathogenicity of this fungus was evaluated. Both PIs strongly (>/=90%) and dose dependently (0.1-10 microM) inhibited Sap activity and production. They also significantly reduced Candida growth in a nitrogen-limited, Sap expression-dependent growth medium and exerted a therapeutic effect in an experimental model of vaginal candidiasis, with an efficacy comparable to that of fluconazole. Thus, besides the expected immunorestoration, patients receiving PI therapy may benefit from a direct anticandidal activity of these drugs.

Role of protease inhibitors in preventing recurrent oral candidosis in patients with HIV infection: a prospective case-control study.

This study was conducted to evaluate the efficacy of highly active anti-retroviral therapy (HAART) in preventing recurrence of oral candidosis (OC) associated with HIV. A prospective case-controlled observational study was performed in an inner-city university-hospital HIV/AIDS clinic. Ninety-three HIV-positive study subjects with a history of recurrent OC were divided into two groups: protease inhibitors (PI)-treated patients (group 1, n = 30) and non-PI-treated patients (group 2, n = 63). Study subjects were matched for sex, age, stage of HIV infection, and peripheral CD4+ T-cell counts. The non-PI-treated group was further subdivided into the following three subgroups: HIV-positive study subjects treated with reverse transcriptase inhibitors (RTI; groups 2a and 2c) and HIV-positive study subjects not treated with RTIs (group 2b). Group 2c met the same inclusion criteria as group 2a had but was matched 6 months after the beginning of the study. We also assessed in vitro peripheral blood mononuclear cells (PBMC) and their lymphoproliferative response, as well as cutaneous delayed-type hypersensitivity (DTH) response to Candida-associated antigens in a randomly selected sample of study subjects divided into those treated with PIs and those who were not. During a 1-year follow-up, OC was diagnosed in 2 (7%) PI-treated and 23 (36%) non-PI-treated patients (p<.001). In addition to comparing findings in group 1 with those in group 2c, OC was detected in 14 (50%) non-PI-treated patients compared with no HAART-treated study subjects (p<.001). Only 41% of PI-treated study subjects had positive lymphoproliferative response in PBMCs and none was positive in terms of DTH to Candida antigens (p = not significant versus non-PI-treated study subjects). While objectively demonstrating a beneficial effect of HAART in preventing recurrence of OC infections, our findings suggest this effect cannot be not fully accounted for by reconstitution of anti-Candida cell-mediated immunity, given that other mechanisms, even of a nonimmune nature, could have some effect.

Fusarium infections in patients with severe aplastic anemia: review and implications for management.

BACKGROUND AND OBJECTIVE: The prognosis of severe fungal infections, such as fusarium infections, in patients with aplastic anemia is directly related to the recovery of bone marrow functions. In this study, in vitro anti-Fusarium activity of granulocytes was investigated, the case of disseminated infection in a child with very severe aplastic anemia is reported, and implications for management of such infective complications are discussed. DESIGN AND METHODS: The in vitro efficiency of PMNL from three untreated, normal blood donors and from two G-CSF-treated WBC donors in contrasting the growth of the Fusarium sp strain isolated from the patient we present was measured by a 3H-glucose uptake inhibition assay and confirmed by microscopic examination. RESULTS: Basic growth inhibitory activity of unstimulated PMNL on Fusarium cells was significantly enhanced in the presence of GM-CSF in all three blood donors tested. In one of the two G-CSF-treated donors, in vitro efficiency of PMNL in contrasting the growth of the fungus increased notably after G-CSF treatment. We report the case of a 3-year-old girl with very severe aplastic anemia unresponsive to conventional immunosuppressant therapy who developed a disseminated fusarium infection. The child initially responded to liposomal amphotericin B and granulocyte transfusions from G-CSF stimulated donors. Subsequently she was given a cord blood stem cell transplantation but died of disseminated infection. INTERPRETATION AND CONCLUSIONS: Including the present case, there are only ten reports of invasive infections caused by the genus Fusarium in aplastic anemia patients and only two of the patients survived. In vitro data seem to suggest that in vivo treatment with rh-G-CSF could have a stimulatory effect on the anti-Fusarium activity of neutrophils. Despite the efficacy of granulocyte transfusions by G-CSF-stimulated donors in the temporary control of fusarium infection, treatment of the underlying hematologic disease is required to cure the infection in patients with severe aplastic anemia. Granulocyte transfusions by G-CSF-stimulated donors while awaiting bone marrow recovery following the blood stem cell transplant should be considered.

Interaction between human interleukin-2-activated natural killer cells and heat-killed germ tube forms of Candida albicans.

Human interleukin-2-activated natural killer (LAK) cells are able to recognize and to bind to both live and heat-killed germ tube forms of Candida albicans, establishing a wide and intimate contact as revealed by electron microscopic observations. Following the interaction, LAK cells are activated: an increased expression of some cytokine mRNA (in particular, TNF-alpha, GM-CSF, and IFN-gamma) has been revealed by RT-PCR and perforin secretion has been suggested by immunofluorescence microscopy. Nonetheless, neither morphological damage or growth inhibition of fungal target cells have been detected. Instead, evident signs of cell damage could be noticed in interacting LAK cells. Moreover, the observation by transmission electron microscopy of LAK cell-germ tube conjugates revealed the presence of apoptotic cells. The analysis of LAK cell cytotoxic activity against DAUDI cells showed that the lymphocytic effector underwent a significant reduction in its lytic capability after the interaction with C. albicans. The results obtained in this in vitro study seem to indicate that in such an interaction LAK cells cannot directly inhibit or kill the fungal pathogen by using their lytic machinery but they secrete those cytokines which have stimulatory effects on phagocytic cells. The ultimate results are the programmed death of LAK cells and the enhancement of the fungicidal activity exerted by competent cells.

Possible participation of polymorphonuclear cells stimulated by microbial immunomodulators in the dysregulated cytokine patterns of AIDS patients.

Macrophages and polymorphonuclear cells (PMN) play a major role as cells primarily responsive to microbial biological response modifiers (BRM). Although much attention has been given to macrophages, PMN have been relatively underinvestigated. We have recently studied the responses of PMN from HIV- and HIV+ subjects after stimulation with a powerful immunomodulatory fraction from the cell wall of Candida albicans (MP-F2) and compared this to bacterial lipopolysaccharide (LPS). Both cytokine patterns and PMN anticandidal activity were investigated. MP-F2, like LPS, was an active inducer of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), IL-6, and IL-1 beta production by PMN and monocytes from all subjects. IL-12 was also produced by MP-F2-stimulated PMN in the presence of interferon-gamma (IFN-gamma). PMN from HIV+ subjects showed increased in vitro expression of TNF-alpha and IL-6 genes as determined by semiquantitative reverse transcriptase-polymerase chain reaction. In all subjects, cytokine gene expression was strongly stimulated by MP-F2 or LPS and inhibited by IL-10. Production of IL-6 and TNF-alpha protein (measured by ELISA) was higher in PMN from HIV+ subjects in at least one of the conditions tested (unstimulated or stimulated by LPS or MP-F2). However, the amount of the C-X-C chemokine IL-8 was equal in PMN from HIV- and HIV+ subjects. PMN from HIV+ subjects were at least as active in inhibiting candide growth as PMN from HIV- controls. In both groups PMN were equally stimulated by MP-F2 and LPS. Only in severely neutropenic subjects was there some reduction in the anticandidal activity but not in cytokine responses. When appropriately stimulated by microbial BRM, PMN are active producers of pro-inflammatory and immunomodulatory cytokines. This production is not only totally preserved in HIV+ subjects but may be higher than in PMN from HIV- subjects and may be coupled with an efficient anticandida activity. We suggest that during common bacterial or fungal infections PMN may contribute to the dysregulated production of inflammatory cytokines in AIDS patients.

Responsiveness of human polymorphonuclear cells (PMNL) to stimulation by a mannoprotein fraction (MP-F2) of Candida albicans; enhanced production of IL-6 and tumour necrosis factor-alpha (TNF-alpha) by MP-F2-stimulated PMNL from HIV-infected subjects.

IL-1beta, IL-6, IL-8 and TNF-alpha production by PMNL from 21 HIV-infected (HIV+), including 11 full-blown AIDS, and 20 HIV-uninfected (HIV-) subjects (matched for age and sex to HIV+ ones) was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA. PMNL from both categories of subjects were strongly stimulated in their actual cytokine production by a mannoprotein fraction (MP-F2) of Candida albicans, as well as by the bacterial lipopolysaccharide (LPS). These stimulatory effects were apparently due to increased cytokine gene expression and were substantially reversed by the physiological inhibitor IL-10. However, PMNL from HIV+ subjects showed increased IL-6 and TNF-alpha gene expression and produced more IL-6 and TNF-alpha than PMNL from HIV- controls, under similar stimulation conditions. This difference could not be attributed to a given stage of HIV infection, any associated medication, or to a generalized increase of gene expression, as quantitatively similar beta-actin and IL-1beta transcripts were detected. Moreover, no significant difference in IL-8 production by the PMNL from HIV+ and HIV- subjects was observed. Our studies suggest that PMNL from HIV+ subjects might add to other cellular sources of IL-6 and TNF-alpha (e.g. monocytes-macrophages) in contributing to the cytokine-dysregulated pattern typical of the HIV+ patient.

Purification and biochemical characterization of a 65-kilodalton mannoprotein (MP65), a main target of anti-Candida cell-mediated immune responses in humans.

A 65 kDa-constituent (MP65) of a whole-cell mannoprotein (MP) fraction of Candida albicans was purified by immunoaffinity chromatography with monoclonal antibodies directed against periodate-insensitive, protease-sensitive MP epitopes, putatively polypeptide in nature. These antibodies were obtained by immunization of mice with concanavalin A bead-coupled, low-glycosylated MP from hyphal cells of C. albicans grown in the presence of a subinhibitory dose of tunicamycin. The immunoaffinity-purified MP65 molecule had a pI of 4.1 and a protein/polysaccharide ratio of 1.8:1. It was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, or N-glycoffanases but still reactive with concanavalin A. The polysaccharide moiety of MP65 was composed exclusively of mannose and glucose at a ratio of 12.7 to 1. The protein moiety showed numerous potential O-glycosidic linkage sites as suggested by the high proportion of serine and threonine (together accounting for more than 20% of the total amino acid composition) and susceptibility to diluted alkali. This treatment and digestion with alpha-mannosidase caused a reduction in the MP65 molecular mass to around 54 kDa. The N-terminal sequence of MP65 protein moiety was rich in alanine and valine (7 of 13 amino acids) and did not show any significant homology with deposited sequences in data banks. Purified MP65, at doses of a few nanograms, induced extensive T-cell proliferation of human peripheral blood mononuclear cells. This proliferation was specifically inhibited, in a dose-response fashion, by the antigen-binding fragment of the monoclonal antibody used for immunoaffinity purification. Overall, these results highlight biochemical and molecular details of MP65, a main target of human T-cell response to C.albicans.

Mannoproteins from Candida albicans elicit a Th-type-1 cytokine profile in human Candida specific long-term T cell cultures.

Since T lymphocytes are active producers of regulatory cytokines, long-term T cell cultures (LTTC) specific for a major mannoproteic antigen (MP) of Candida albicans from peripheral blood mononuclear cells (PBMC) of healthy donors were generated and their cytokine profile studied. LTTC consisted of an expanded CD3CD4 T-helper (Th) populations, with a high proportion of CD45R0 T memory cells. Stimulation of LTTC by MP induced a Th-1 type cytokine profile, as indicated by the presence of IFN-gamma and the absence of IL-5 (both as mRNA and protein) in cell cultures and supernatants. These results suggest a predominant Th1 response elicited by C. albicans mannoprotein antigen in human PBMC.

Noninhibitory binding of human interleukin-2-activated natural killer cells to the germ tube forms of Candida albicans.

During incubation in vitro with yeast or germ tube forms of Candida albicans, only 2 to 6% of freshly isolated human natural killer (NK) cells (> 85% CD16+, CD56+, CD3-; < 15% CD3+; cytolytic for the NK-susceptible target K562 but not for the NK-resistant target DAUDI), were seen to interact with the fungal cells. As seen under the electron microscope, the contact area had a limited extent and was narrow, and neither the surface nor the intracytoplasmic organization of the NK cell was altered. In contrast, more than 30% of interleukin-2-activated NK (LAK) cells (> 96% CD16+, CD56+, CD3-; 1.5% CD3+; cytolytic for both K562 and DAUDI targets) interacted closely with the fungus. This interaction was particularly extensive with the surface of the fungal germ tube that was intimately enveloped by villous protrusions from the lymphocyte surface. The fungus-interacting LAK cell also showed a remarkable redistribution of surface microvilli and polarization of cytoplasmic organelles, such as the Golgi apparatus, centrioles, and granules, toward the area of fungal contact. Together with the elevated cytolytic potential against the K562 and DAUDI targets, all the morphological data suggested the presence of a potentially active lytic machinery in the fungus-interacting LAK cell. Nonetheless, two independent assays for anticandidal activity did not show consistent killing or fungal growth inhibition by either fresh NK or LAK cells. While offering direct evidence of the strong interaction between human LAK cells and the germ tubes, precursors of tissue-invasive hyphal forms of C. albicans, our observations also suggest that this interaction may not be sufficient to kill the fungus or arrest its growth.

Differential release of an immunodominant 65 kDa mannoprotein antigen from yeast and mycelial forms of Candida albicans.

The release of mannoprotein (MP) antigen from Candida albicans grown at 28 degrees C (yeast form) or 37 degrees C (mycelial form), and the ability of each released material to stimulate a cell-mediated immune (CMI) response by human lymphocytes in vitro, were studied. Overall, the mycelial cells released more MP per unit of dry mass increase and the released material was relatively enriched with MP constituents of lower molecular mass with respect to the material released from yeast cells. Moreover, the mycelial MP contained a 65 kDa component (MP65) which was the largely predominant MP recognized by a rabbit anti-mycelium antiserum. When peripheral blood mononuclear cells from normal human subjects were stimulated in vitro with graded amounts of yeast or mycelial MP, the latter was about one order of magnitude more potent than the former in inducing lymphocyte proliferation. Following MP separation by gel permeation chromatography, an appreciable CMI response was stimulated only by the MP65-containing MP fractions, and to a degree apparently related to the amount of MP65 itself. Altogether, these data confirm our previous findings about the MP65 antigen as a major target of CMI response to C. albicans, and demonstrate that this antigen is released predominantly by the mycelial cells of the fungus in vitro.

A mannoprotein constituent of Candida albicans that elicits different levels of delayed-type hypersensitivity, cytokine production, and anticandidal protection in mice.

To identify major immunogenic constituents of Candida albicans, the effect of a mannoprotein fraction (MP-F2) on the elicitation of a delayed-type hypersensitivity (DTH) reaction, cytokine production, and protection from a virulent Candida challenge in a mouse candidiasis model was studied. In mice immunized with whole cells of a low-virulence strain of C. albicans and thus protected against a challenge with a highly virulent strain of this fungus, MP-F2 was able to elicit a strong DTH response that was accompanied by splenocyte proliferation in vitro in the presence of Candida antigen. The supernatants of MP-F2-stimulated splenocyte cultures contained gamma interferon (IFN-gamma, a typical CD4+ T helper-1 (Th1) cytokine, but no interleukin-4, (IL-4), a typical CD4+ Th2 cytokine. IFN-gamma was produced by CD4+ cells, and its level could be greatly increased by the addition of anti-IL-4 or, mostly, anti-IL-10 antibodies to the CD4+ cell cultures. Upon a suitable schedule of immunization, MP-F2 was also able to induce a vigorous DTH response in Candida-uninfected mice, a response that could be efficiently transferred into naive recipients by CD4+ cells from the spleens of MP-F2-immunized mice. The immunization described above also conferred to mice a low degree of protection against a virulent Candida challenge, both in terms of median survival time and in the number of Candida cells in the kidney. However, while DTH induction by MP-F2 was as strong as that induced by whole cells, MP-F2-induced protection was significantly weaker than that conferred by Candida whole-cell immunization. Mice immunized with either MP-F2 or Candida whole cells had an inverted ratio between the number of CD4+ splenocytes producing IFN-gamma and that of cells producing IL-4, compared with nonimmunized animals. However, the number of IL-4-producing CD4+ cells was significantly higher in MP-F2-vaccinated, weakly protected mice than in Candida whole-cell-vaccinated, highly protected animals. Overall, our data suggest that the MP-F2 fraction contains one or more major immunogens of C. albicans which are capable of interfering with the balance of CD4+ Th1 and Th2 responses that is so critical in the outcome of host-Candida relationship and are thus potentially relevant in the mechanisms of Candida-specific DTH regulation and protection.

Mannoprotein-induced anti-U937 cell cytotoxicity in peripheral blood mononuclear cells from uninfected or HIV-infected subjects: role of interferon-gamma and tumor necrosis factor-alpha.

A mannoprotein fraction (MP-F2: mannan, > 90%; protein, 4.5%) from the human commensal microorganism Candida albicans was as efficient as interleukin-2 (IL-2) in generating cytotoxicity against the uninfected or human immunodeficiency virus type-1 (HIV-1) persistently infected monocytoid U937 cell line in cultured peripheral blood mononuclear cells (PBMC) from healthy human subjects. MP-F2-activated killing of U937 cells (U937-MAK) decreased progressively with advancing stages of HIV-1 infection to virtually no killing effect in PBMC from advanced AIDS subjects (AIDS PBMC). This decrease paralleled a lowered susceptibility of U937 cells to natural killer cell activity. In contrast, IL-2-activated killing of U937 cells (U937-LAK) was not affected by the progression of HIV infection and persisted at high levels in AIDS PBMC. To shed light on the mechanisms of U937-MAK and its decrease during HIV infection, IL-1 beta, IL-6, TNF-alpha, GM-CSF, and IFN-gamma production was analyzed. Decreases in TNF-alpha, GM-CSF, and IFN-gamma, but not IL-1 beta or IL-6, levels were observed in MP-F2-stimulated PBMC from HIV-infected subjects, compared to healthy controls. Interestingly, these cytokine levels fell before the onset of AIDS. The greatest relative drop was that of IFN-gamma, from 4600 (+/- 600) to 290 (+/- 160) and 217 (+/- 110) mean pg/ml (+/- SE) in PBMC from healthy donors (11 subjects), CDC stages II + III (14 subjects), and CDC stage IV (10 subjects), respectively. The following observations suggest that decreased IFN-gamma production plays a role in the abrogation of U937-MAK activity: (i) addition of neutralizing anti-IFN-gamma antibodies abolished both IFN-gamma and U937-MAK activity in PBMC from healthy subjects; (ii) substantial levels of IFN-gamma were detected in supernatants of PBMC cultures stimulated by IL-2, in line with preserved U937-LAK activity. Interestingly, anti-IFN-gamma antibodies also abolished TNF-alpha production, and the anti-TNF-alpha antiserum effect was comparable to that of anti-IFN-gamma in U937-MAK inhibition. In contrast, anti-TNF-alpha antibodies abrogated TNF-alpha activity, but only partially reduced IFN-gamma production. Thus, in human PBMC, U937-MAK activity progressively decreases with advancing stages of HIV infection, whereas U937-LAK activity is sustained. Furthermore, the present results indicate a pivotal role for IFN-gamma in U937 MAK activity, possibly through activation of TNF-alpha production.

Identification of a 65-kDa mannoprotein as a main target of human cell-mediated immune response to Candida albicans.

To identify molecular targets of anticandidal cell-mediated immunity (CMI) in humans, a highly immunogenic mannoprotein fraction (MP-F2) of Candida albicans was studied. SDS-PAGE and gel-permeation chromatography separated MP-F2 into polydisperse mannoproteins of > 200-31.5 kDa. However, only a 65-kDa constituent specifically induced proliferation of human peripheral blood mononuclear cells (PBMC). Lymphoproliferation was accompanied by production of interleukin (IL)-1 beta, interferon-gamma, and IL-6 but not IL-4. MP-F2- and MP-65-induced PBMC proliferation was inhibited by an antagonist anti-T cell receptor antibody. Neither the purified protein derivative of Mycobacterium tuberculosis nor MP-65 activated naive lymphocytes from umbilical cord blood, although these cells proliferated extensively in response to both phytohemagglutinin and IL-2. These data strongly suggest that MP-65 is an immunodominant mannoprotein antigen that is ordinarily expressed as a target of anti-Candida CMI in healthy humans.