Maria Jesus Gomez

Generation of a Recombinant 65-Kilodalton Mannoprotein, a Major Antigen Target of Cell-Mediated Immune Response to Candida albicans

ABSTRACT A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogenCandida albicans. An expression library of C. albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa. This serum recognized the CaMp65 from a cell wall extract of C. albicans. After cloning and sequencing of the relevant C. albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified. Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65. A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65. A putative adhesin RGD sequence was also present in the C-terminal region of the molecule. This triplet was absent in the ScMp65. The relevant gene (designatedCaMP65) was localized to chromosome R of C. albicans as determined by pulse-field gel electrophoresis. Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus. A recombinant, His6-tagged protein (rCaMp65) was expressed inEscherichia coli under an inducible promoter. After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies. Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors. While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors. In addition, a number of CD4+ T-cell clones were generated using a C. albicans mannoprotein fraction as an antigenic stimulant. Several of these clones specifically responded to both the native and the recombinant C. albicans Mp65 but not to ScMp65. Thus, the recombinant Mp65 of C. albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

Proliferative and cytotoxic responses to mannoproteins of Candida albicans by peripheral blood lymphocytes of HIV-infected subjects.

Mucosal candidiasis is one of the first opportunistic diseases in HIV‐infected subjects. In order to understand the relationship between this disease and immunodeficiency to chemically defined, immunodominant Candida antigens, a mannoprotein fraction from C. albicans cell wall (GMP) was used to analyse proliferative and non‐MHC‐restricted cytotoxic responses of peripheral blood mononuclear cells (PBMC) from normal and HIV‐infected subjects. In the former, GMP induced extensive blastogenesis, generation of powerful cytotoxicity against a tumour cell line (K562), and production of substantial amounts of interferon‐gamma (IFN‐γ). Cultured PBMC from HIV‐infected subjects manifested an early decreased ability for proliferative as well as differentia live cytotoxic responses to the candidal mannoproteins. This inability became clearly evident in subjects with stage III (CDC) of the disease, was total in CDC stage IV and occurred even in some subjects with a normal number of CD4+ cells. Low or absent response to GMP correlated with lack of response to tetanus toxoid. In contrast, both lymphoproliferative and cytotoxic responses to exogeneous IL‐2 was highly preserved at all stages of infection. The production of IFN‐γ in GMP‐stimulated PBMC cultures critically fell to negligible values in most of the subjects in CDC stages II and III. Thus, the lowered or absent cell‐mediated immune responses to candidal mannoprotein may be one factor to explain the early, elevated susceptibility of HIV‐infected subjects to mucosal candidiasis. This study also shows that our mannoprotein preparation may be used as a probe to detect the overall efficiency of T cell responses in the above subjects.

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

ABSTRACT 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 α/β 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 ofC. 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.

Mannoprotein from Cryptococcus neoformans Promotes T-Helper Type 1 Anticandidal Responses in Mice

ABSTRACT We previously demonstrated that mannoprotein (MP) from Cryptococcus neoformans (CnMP) stimulates interleukin-12 production by human monocytes, thus fostering a T-helper type 1 (Th1) protective anticryptococcal response. In this paper we show that CnMP was also able to induce a Candida albicans-directed protective Th1 response. This was demonstrated for mice immunized with CnMP by induction of a delayed-type hypersensitivity (DTH) reaction to C. albicans MP (CaMP) as well as induction of gamma interferon production by CD4+ and CD8+ splenic T cells stimulated in vitro with CaMP. CnMP-immunized mice were also partially protected from lethal systemic challenge with C. albicans, as shown by prolonged median survival times and decreased fungal burden in the kidney. Much evidence supports the validity of these cross-reactive and functional Th1 responses: (i) a non-cross-reactive C. albicans antigen, such as enolase, did not produce a DTH response to CaMP; (ii) passive adoptive transfer of T cells primed with CnMP induced a DTH reaction; (iii) C. neoformans extract elicited a DTH response to CaMP; and (iv) a monoclonal antibody (7H6) directed against a major and immunodominant T-cell-stimulatory 65-kDa MP (MP65) of C. albicans also recognized discrete 100-kDa constituents of C. neoformans extracts, as well as secretory constituents of the fungus. These results suggest the presence of common Th1 antigenic determinants in the mannoproteic material of C. neoformans and C. albicans epitopes, which should be considered in devising common strategies for immunoprophylactic or immunotherapeutic control of the fungi.

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.

Biochemical and Immunological Characterization of MP65, a Major Mannoprotein Antigen of the Opportunistic Human Pathogen Candida albicans

ABSTRACT In the search of the antigenic determinants of a 65-kDa mannoprotein (MP65) of Candida albicans, tryptic fragments of immunoaffinity-purified MP65 preparations were tested for their ability to induce lymphoproliferation of human peripheral blood mononuclear cells (PBMC). Five major peptides (T1 to T5) were shown to induce a vigorous proliferation of PBMC from the majority of the eight healthy human subjects tested. With the use of synthetic peptides, critical amino acid sequences of the two most immunoactive (T1 and T2) peptides were determined. Similar to what was found for the MP65 molecule, no PBMC multiplication was induced by the antigenic peptides in cultures of naive cord blood cells. The amino acid sequence analysis of tryptic and chymotryptic peptides of MP65 demonstrated a substantial homology with the deduced sequences of two cell wall proteins ofSaccharomyces cerevisiae, encoded by the genesYRM305C and YGR279C. However, the antigenic peptides were those showing the least similarity with the corresponding regions of the above proteins. In particular, the lymphoproliferation-inducing sequence of the T1 peptide scored only 20% identity with the homologous regions of S. cerevisiaeproteins. Besides disclosing the amino acid sequence of MP65, this study provides an initial characterization of some of its antigenic determinants, as well as of synthetic peptides of potential use to detect specific immune responses against MP65, a major target of anticandidal cell-mediated immunity in humans.

Biochemical and antigenic characterization of mannoprotein constituents released from yeast and mycelial forms of Candida albicans

Yeast or mycelial cultures of Candida albicans released comparable amounts of Concanavalin A-reactive mannoprotein material after 24-h of growth, and in both cases this material showed a qualitatively similar SDS-PAGE pattern, with predominantly polydisperse constituents of high molecular mass. The two secretion mixtures also showed similar reactivity by ELISA with serum from a subject with high titre anti-Candida antibodies, as well as with an anti-Candida hyperimmune antiserum raised in rabbits. Both secreted extracts were separated by ion-exchange chromatography into two major fractions (designated F1 and F2), each containing mannoprotein antigens recognized by rabbit and human sera, although the immunoreactivity of the two fractions from the two growth forms was not uniform. The mannoproteins released from mycelial cultures, in particular those present in the F1 fraction, were poorly reactive or not reactive at all in ELISA with a monoclonal antibody (mAbAF1) which strongly recognized the material released from yeast cultures. Immunoblots of the more acidic, more antigenic F2 fractions with mAbAF1 and polyclonal anti-Candida antisera demonstrated that the monoclonal antibody did not recognize several mannoprotein molecules which were recognized by the polyclonal antibodies, in particular a 45-47 kDa component present only in the secreted extract from mycelium. A quantitative ELISA-inhibition method showed that the rate of release of mannoprotein antigen during growth in the yeast form was either constant (as assayed with polyclonal antibodies) or fluctuated without any definite trend (as seen with mAbAF1). On the other hand, cultures of mycelial cells exhibited an early (90 min) peak of antigen release, followed by either a decrease to a rate corresponding to that of yeast cells (with polyclonal antibodies) or a total lack of secretion (with mAbAF1). This modulation in the secretion of mAbAF1 reactive molecules was temporarily associated with germ tube emergence-elongation, and was not observed in an agerminative mutant of C. albicans grown under germination permissive conditions. These results highlight the dynamic aspects of the secretion of specific mannoprotein epitopes released from C. albicans during hyphal growth, and the direct relationship between this release and the dynamic expression of the same epitopes on the cell surface demonstrated previously.

Type 1 T Helper Cells Specific for Candida albicans Antigens in Peripheral Blood and Vaginal Mucosa of Women with Recurrent Vaginal Candidiasis

The cytokine profile of circulating and vaginal T cells specific for immunodominant mannoprotein antigens of Candida albicans was analyzed in patients with recurrent vaginal candidiasis (RVC). Peripheral blood mononuclear cells (PBMC) from patients with RVC proliferated more than those from healthy subjects and expressed higher type 1:type 2 T helper cell cytokine ratios in response to C. albicans stimulation. A higher number of C. albicans-specific T cells was generated in PBMC from patients with RVC than in PBMC from healthy donors. C. albicans-specific T cell clones from patients with RVC produced higher levels of interferon (IFN)-gamma and lower levels of interleukin (IL)-4 than clones from control women. More important, a higher proportion of C. albicans-specific T cell clones was generated from lesional mucosa of patients with RVC than from normal mucosa, all of which produced IFN-gamma but not IL-4. These findings provide direct evidence that RVC is characterized by a highly polarized local and circulating type 1 T helper cell-like response against C. albicans antigens.

Generation of a highly immunogenic recombinant enolase of the human opportunistic pathogen Candida albicans

Enolase, a 46 kDa glycolytic enzyme, is an immunodominant antigen of Candida albicans, an important human opportunistic pathogen. The full‐length coding sequence of C. albicans enolase gene was subcloned into the prokaryotic expression vector pDS56/RBSII, His6/E− under the control of an inducible promoter to produce a His6‐tagged enolase. The recombinant protein was purified to homogeneity by one‐step nickel‐chelate affinity chromatography. It was recognized by a monoclonal antibody specific for C. albicans enolase, as well as by anti‐enolase antibodies present in human sera (IgG). The recombinant protein promptly elicited antibody in mice and detected immune responses in normal human subjects that were comparable to those generated by the native C. albicans enolase. Thus this new recombinant enolase constitutes a valuable reagent for studying the possible role of this protein in anti‐Candida immune response.

Cell Wall Constituents of Candida Albicans as Biological Response Modifiers

Candida albicans is an opportunistic fungal agent which has become a highly prevalent and incident cause of disease, especially life-threatening in neutropenic, bone-marrow transplanted subjects with underlying malignant hemopathy1,2. An extensive experimental evidence demonstrates that this fungus, and materials extracted from it, can also be used to influence or modify multiple biologic functions3,4. A particular point of interest in the “Biological Response Modifier” (BRM)-activities of Candida, that make this microorganism quite particular in comparison to other more popular microbial immunomodulators (for instance, BCG), is that Candida is a human commensal. Thus, almost every normal subject is primed for immune response to candidal antigens, as shown by the presence in its serum of measurable, sometimes elevated, levels of specific antibodies, and positive cell-mediated response to Candida antigens 5,6. While generating restrictions to the use of certain fungal materials as immunomodulators (mostly concerning undesired hypersensitivity reactions) the human commensalism also tells us that Candida-induced immunomodulation may take place under natural conditions and can be easily amplified. A rather dramatic example of this amplification is the generation of LAK- like effectors following Candida vaccination of normal mice7 or exposure of peripheral blood mononuclear cells (PBMC) from normal human donors to a mannoprotein extract of C. albicans 8. Moreover, certain fungal products are potent recall antigens to probe the efficiency of the immune system, both in normal and pathological conditions9,10 Most of the BRM-effects of C. albicans are mediated by the glucan and the mannoprotein constituents of the fungal cell wall4. In this note, we will mostly address the latter components (hereafter referred to as MP), and will attempt to summarize some of our recent results on the immunogenic and immunomodulatory effects of a purified and chemically-characterized mannoprotein fraction (F2).