Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii.

BACKGROUND: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2'OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. METHODS: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. CONCLUSION: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.

Morphological changes and phagocytic activity during the interaction of human neutrophils with Sporothrix schenckii: An in vitro model.

Histopathological studies of human sporotrichosis lesions show pyogenic and granulomatous processes in which polymorphonuclear neutrophils (PMNs) play a central role. Few studies regarding the events associated with the interaction of human PMNs with Sporothrix schenckii have been made despite their importance in the clinical manifestations of the disease. In this study, human PMNs were co-cultured with conidia or yeast cells of S. schenckii to compare the phagocytic activity and morphological changes that could provide a clearer insight into the role of these phagocytes in the initial phase of sporotrichosis. PMNs showed increased cell size and separation of the nuclear lobes after phagocytosis. Through Scanning Electron Microscopy (SEM) analysis, an increase in cells with flattened filaments and vesicles on their surface was observed. Phagocytosed conidia showed a significant increase in width and size. The phagocytic activity was greater against yeasts than with conidia, but the viability of both S. schenckii cellular morphotypes was not drastically affected even after 2 h of co-culture. In conclusion, morphological changes in PMNs suggest that S. schenckii induces processes that may favor proinflammatory events. These phagocytes show a high ability to bind or ingest S. schenckii cells without affecting their viability. Morphological changes recorded in ingested conidia, suggest that this fungus could make the dimorphic switching in PMNs.

The importance of Toll-like receptor 4 during experimental Sporothrix brasiliensis infection.

Here we investigated the importance of Toll-like receptor 4 (TLR-4) in innate immune response to Sporothrix brasiliensis, a virulent fungus of Sporothrix spp. In vitro assays, using C57Bl/6 (wild type [WT]) bone marrow-derived macrophages (BMDMs), and TLR-4 knockout (TLR-4-/-) showed that the absence of TLR-4 resulted in impaired phagocytosis and lower levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and nitric oxide. In vivo assays were also performed, and the mice (WT and TLR-4-/-) were intraperitoneally infected with S. brasiliensis yeast ATCC MyA-4831 and euthanized on days 7, 14, and 28 postinfection, with the following parameters evaluated: fungal burden in liver, spleen, kidney, and brain, and the production of cytokines interferon γ (IFN-γ), TNF-α, IL-2, IL-4, IL-6, and IL-10. The results demonstrate the macrophages dependency on TLR-4 for inflammatory activation and in the absence of TLR-4 during experimental S. brasiliensis infection enhanced dissemination occurred after 14 and 28 days. These data show that TLR-4 signals are important for the recognition of S. brasiliensis by macrophages, and their absence promotes the persistence of the infection.

Natural killer cells are pivotal for in vivo protection following systemic infection by Sporothrix schenckii.

Natural killer (NK) cells are one of the first cell types to enter inflammation sites and have been historically known as key effector cells against tumours and viruses; now, accumulating evidence shows that NK cells are also capable of direct in vitro activity and play a protective role against clinically important fungi in vivo. However, our understanding of NK cell development, maturation and activation in the setting of fungal infections is preliminary at best. Sporotrichosis is an emerging worldwide-distributed subcutaneous mycosis endemic in many countries, affecting humans and other animals and caused by various related thermodimorphic Sporothrix species, whose prototypical member is Sporothrix schenckii. We show that following systemic infection of BALB/c mice with S. schenckii sensu stricto, NK cells displayed a more mature phenotype as early as 5 days post-infection as judged by CD11b/CD27 expression. At 10 days post-infection, NK cells had increased expression of CD62 ligand (CD62L) and killer cell lectin-like receptor subfamily G member 1 (KLRG1), but not of CD25 or CD69. Depletion of NK cells with anti-asialo GM1 drastically impaired fungal clearance, leading to a more than eightfold increase in splenic fungal load accompanied by heightened systemic inflammation, as shown by augmented production of the pro-inflammatory cytokines tumour necrosis factor-α, interferon-γ and interleukin-6, but not interleukin-17A, in the spleen and serum. Our study is, to the best of our knowledge, the first to demonstrate that a fungal infection can drive NK cell maturation in vivo and that such cells are pivotal for in vivo protection against S. schenckii.

Sporotrichosis between 1898 and 2017: The evolution of knowledge on a changeable disease and on emerging etiological agents.

The description of cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. Sporothrix schenckii causes a benign chronic subcutaneous mycosis, Sporothrix brasiliensis is highly virulent, and Sporothrix globosa mainly causes fixed cutaneous lesions. Furthermore, S. brasiliensis is the prevalent species related to cat-transmitted sporotrichosis. Sources of infection, transmission, and distribution patterns also differ between species, and variability differs between species because of different degrees of clonality. The present review article will cover several aspects of the biology of clinically relevant agents of sporotrichosis, including epidemiological aspects of emerging species. Genomic information of Sporothrix spp. is also discussed. The cell wall is an essential structure for cell viability, interaction with the environment, and the host immune cells and contains several macromolecules involved in virulence. Due to its importance, aspects of glycosylation and cell wall polysaccharides are reviewed. Recent genome data and bioinformatics analyses helped to identify specific enzymes of the biosynthetic glycosylation routes, with no homologs in mammalian cells, which can be putative targets for development of antifungal drugs. A diversity of molecular techniques is available for the recognition of the clinically relevant species of Sporothrix. Furthermore, antigens identified as diagnostic markers and putative vaccine candidates are described. Cell-mediated immunity plays a key role in controlling infection, but Sporothrix species differ in their interaction with the host. The adaptive branch of the immune response is essential for appropriate control of infection.

Experimental sporotrichosis in a cyclophosphamide-induced immunosuppressed mice model.

This report describes a model of host resistance for Sporothrix schenckii, an opportunistic fungi in immunosuppressed mice with cyclophosphamide (CY) to be used in studies of immunotoxicology and immunopharmacology. Two doses of CY were administered intraperitoneally: 200 mg/kg and a booster of 150 mg/kg at 9-day intervals. Three days after the first dose of CY the animals were infected subcutaneously with 1.8 × 108 yeast/ml (S. schenckii ATCC 16345). At 7 and 14 days post-infection, the animals were euthanized and analyzed the fungal load by unit forming colony count in the spleen and popliteal lymph nodes. The relative weight of thymus and spleen, splenic index, the frequency of T and B cells in spleen by flow cytometry, the hind paw inflammation index and cytokine (interleukin [IL]-17, IL-10, and interferon [IFN]-γ) profile were measured. Histopathological studies of the spleen and the hind paw were also assessed. The immunosuppression status was confirmed at the evaluated days by reduction of relative weight of thymus, reduction of the splenic white pulp, the population of B and T lymphocytes, and the cytokine profile in the treated mice with CY in comparison with nontreated groups, associated to higher fungal load in hind paw and spleen in the infected mice. The described model reveals an increasing in susceptibility to infection and severity when associated with immunosuppression. This model can serve as a reference for studies of S. schenckii host resistance in pharmaceutical and toxicological studies.

The NLRP3 inflammasome contributes to host protection during Sporothrix schenckii infection.

Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen-associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated with fungal recognition is the nucleotide-binding oligomerization domain-like receptor (NLR). After PAMP recognition, NLR family pyrin domain-containing 3 (NLRP3) binds to apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 to form the NLRP3 inflammasome. When activated, this complex promotes the maturation of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 and cell death through pyroptosis. In this study, we aimed to evaluate the importance of the NLRP3 inflammasome in the outcome of S. schenckii infection using the following three different knockout (KO) mice: NLRP3-/- , ASC-/- and caspase-1-/- . All KO mice were more susceptible to infection than the wild-type, suggesting that NLRP3-triggered responses contribute to host protection during S. schenckii infection. Furthermore, the NLRP3 inflammasome appeared to be critical for the ex vivo release of IL-1ß, IL-18 and IL-17 but not interferon-γ. Additionally, a role for the inflammasome in shaping the adaptive immune response was suggested by the lower frequencies of type 17 helper T (Th17) cells and Th1/Th17 but not Th1 cells in S. schenckii-infected KO mice. Overall, our results indicate that the NLRP3 inflammasome links the innate recognition of S. schenckii to the adaptive immune response, so contributing to protection against this infection.

Molecular Components of the Sporothrix schenckii Complex that Induce Immune Response.

Sporotrichosis is a fungal disease caused by the Sporothrix schenckii complex that includes species such as S. brasiliensis, S. schenckii sensu stricto, S. globosa, S. luriei, S. mexicana, and S. pallida, which exhibit different potentially antigenic molecular components. The immune response of susceptible hosts to control infection and disease caused by these fungi has been little studied. Besides, the fungus-host interaction induces the activation of different types of immune response. This mini-review analyzes and discusses existing reports on the identification and functional characterization of molecules from species of the S. schenckii complex with clinical relevance, and the mechanisms that mediate the type and magnitude of the immune response in experimental models in vivo and in vitro. This knowledge is expected to contribute to the development of protective and therapeutic strategies against sporotrichosis and other mycoses.

Response of Cytokines and Hydrogen Peroxide to Sporothrix schenckii Exoantigen in Systemic Experimental Infection.

The response of hydrogen peroxide (H2O2) and cytokines during an experimental sporotrichosis in male Swiss mice was assessed over a period of 10 weeks by monitoring macrophage activation challenged with exoantigen (ExoAg) from the fungus Sporothrix schenckii. The studied endpoints were: H2O2 production, fungal burden at spleen, apoptosis in peritoneal macrophages, and IL-1ß, IL-6, IL-2, IL-10 production. During the two first weeks of infection was observed low burden of yeast in spleen and high response of H2O2, IL-2, and IL-1ß. The weeks of highest fungal burden (fourth-sixth) coincided with major apoptosis in peritoneal macrophages, normal production of IL-6 and lower production of H2O2, IL-2, and IL-1ß, suggesting a role for these three last in the early control of infection. On the other hand, IL-1ß (but not IL-6) was recovered since the sixth week, suggesting a possible role in the late phase of infection, contributing to the fungal clearance in conjunction with the specific mechanisms. The IL-10 was elevated until the sixth, principally in the second week. These results evidences that ExoAg is involved in the host immune modulation, influencing the S. Schenckii virulence, and its role is related with the time of the infection in the model used.

Therapeutic vaccine using a monoclonal antibody against a 70-kDa glycoprotein in mice infected with highly virulent Sporothrix schenckii and Sporothrix brasiliensis.

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungi that comprise the Sporothrix complex. The latter are widely distributed in nature, developing a saprophytic mycelial form on plant debris and soil. Formerly, the S. schenckii species was thought to be the only species capable of causing sporotrichosis. However, in recent years, the existence of a group of highly genotypically and phenotypically variable species has been reported as etiologic agents of this mycosis. Recently, it has become important to study aspects such as virulence and the immune response against key members of the Sporothrix complex and to observe the presence of glycoprotein (gp) 70 and efficacy of the P6E7 monoclonal antibody against more virulent strains. The data presented here demonstrate that the strain isolated from a case of feline sporotrichosis, that is, strain 5110 (American Type Culture Collection MYA-4823) is the most virulent and the only one able to secrete gp70. This glycoprotein is apparently an important factor in the virulence of Sporothrix spp. because treatment with MAb P6E7 resulted in the reduction of fungal burden in the analyzed organs. Additional studies of the role of gp70 in modulating the immune response of the host are needed to understand the pathology of sporotrichosis.

New posology of potassium iodide for the treatment of cutaneous sporotrichosis: study of efficacy and safety in 102 patients.

BACKGROUND: The first therapeutic choice for the treatment of cutaneous sporotrichosis is oral itraconazole; however, the increase in cases of zoonotic transmission outbreak necessitates a search for effective and safe treatment alternatives. OBJECTIVE: To evaluate a new potassium iodide (KI) posology as an alternative for the treatment of limited cutaneous forms of sporotrichosis. METHODS: One hundred and two patients with sporotrichosis diagnosed by isolation of Sporothrix sp. were included and were divided into 2 groups that received different doses of KI: group A received the conventional dose, and group B received the reduced dose. The cure criteria were based on clinical and serological data. RESULTS: Seventy-nine patients (77.4%) reached clinical cure: 70.6% and 84.3% of groups A and B respectively. Sixteen patients (15.6%) were lost during follow-up, and seven changed drug therapy: five in group A and two in group B. The incidence of adverse events was similar for both groups (64.7%): predominantly metallic taste (44%), followed by mild gastrointestinal intolerance and acneiform eruption (10.7% each). No serious adverse events occurred, and there were no recurrences. Analysis of the results showed no statistically significant difference between groups (P = 0.9255). The improvement in serologic titres was significant in both treatment groups. CONCLUSION: Through statistical analysis, the usual posology was not shown to be superior to the one proposed in this study. Serology for sporotrichosis may be used as a valuable tool in the clinical monitoring of these patients.

Involvement of major components from Sporothrix schenckii cell wall in the caspase-1 activation, nitric oxide and cytokines production during experimental sporotrichosis.

Sporotrichosis is a chronic infection caused by the dimorphic fungus Sporothrix schenckii, involving all layers of skin and the subcutaneous tissue. The role of innate immune toll-like receptors 2 and 4 in the defense against this fungus has been reported, but so far, there were no studies on the effect of cell wall major components over the cytosolic oligo-merization domain (NOD)-like receptors, important regulators of inflammation and responsible for the maturation of IL-1ß and IL-18, whose functions are dependents of the caspase-1 activation, that can participate of inflammasome. It was evaluated the percentage of activation of caspase-1, the production of IL-1ß, IL-18, IL-17, IFN-γ and nitric oxide in a Balb/c model of S. schenckii infection. It was observed a decreased activity of caspase-1 during the fourth and sixth weeks of infection accompanied by reduced secretion of the cytokines IL-1ß, IL-18 and IL-17 and high production of nitric oxide. IFN-γ levels were elevated during the entire time course of infection. This temporal reduction in caspase-1 activity coincides exactly with the reported period of fungal burden associated with a transitory immunosuppression induced by this fungus and detected in similar infection models. These results indicate the importance of interaction between caspase-1, cytokines IL-1ß and IL-18 in the host defense against S. schenckii infection, suggesting a participation the inflammasome in this response.

[Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium]. / Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

Sporothrix schenckii yeasts induce ERK pathway activation and secretion of IL-6 and TNF-α in rat mast cells, but no degranulation.

Sporothrix schenckii is a dimorphic fungus that causes sporotrichosis, a subcutaneous mycosis found throughout the world in humans and other mammals. After contact with conidia, transition to the yeast stage is required for establishment of infection. Mast cells are one of the first components of the immune system to make contact with invading pathogens. They release potent mediators that are decisive in initiating and directing the course of immune and inflammatory responses in the host. It remains unknown whether or not yeast cells of S. schenckii activate mast cells. Our aim in this study was to evaluate the in vitro response of mast cells to S. schenckii yeasts cells. Mast cells became activated after interaction with the yeasts, although exocytosis of preformed mediators was not stimulated. Sporothrix schenckii yeasts induced the release of early response cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 and activation of the extracellular signal-regulated kinase (ERK) signaling pathway in mast cells. As TNF-α and IL-6 are considered crucial mediators in the defense of the host against fungal disease, the release of both mediators from mast cells may contribute to the overall response of the host immune system during S. schenckii infection.

Influence of TLR-2 in the immune response in the infection induced by fungus Sporothrix schenckii.

Toll-like receptors (TLRs) play an important role in immunity, since they bind to pathogen surface antigens and initiate the immune response. However, little is known about the role of TLR-2 in the recognition of S. schenckii and in the subsequent immune response. Therefore, the aim of this study was to evaluate the involvement of TLR-2 in the immune response induced by S. schenckii. C57BL/6 mice (WT) and C57BL/6 TLR-2 knockout (TLR-2-/-) were used to evaluate, over a period of 10 weeks of sporotrichotic infection, the influence of TLR-2 over macrophages production of IL-1ß, IL-12 and TNF-α, their stimulation level by NO release and the production of IFN -γ, IL-6, IL-17 and TGF-ß by spleen cells. The results showed that the production of pro-inflammatory mediators and NO, TLR-2 interference is striking, since its absence completely inhibited it. IL-17 production was independent of TLR-2. The absence of Th1 response in TLR2-/- animals was concomitant with IL-17 production. Therefore, it can be suggested that TLR-2 absence interferes with the course of the infection induced by the fungus S. schenckii.

Role of TLR-2 and fungal surface antigens on innate immune response against Sporothrix schenckii.

Sporotrichosis is an infection caused by the dimorphic fungus Sporothrix schenckii. Toll-like receptors (TLRs) play an important role in immunity, since they bind to pathogen surface antigens and initiate the immune response. However, little is known about the role of TLR-2 and fungal surface antigens in the recognition of S. schenckii and in the subsequent immune response. This study aimed to evaluate the involvement of TLR-2 and fungal surface soluble (SolAg) and lipidic (LipAg) antigens in phagocytosis of S. schenckii and production of immune mediators by macrophages obtained from WT and TLR-2(-/-) animals. The results showed that TLR-2(-/-) animals had had statistical lower percentage of macrophages with internalized yeasts compared to WT. SolAg and LipAg impaired phagocytosis and immunological mediator production for both WT and TLR-2(-/-). The absence of TLR-2 led to lower production of the cytokines TNF-α, IL-1ß, IL-12 and IL-10 compared to WT animals. These results suggest a new insight in relation to how the immune system, through TLR-2, recognizes and induces the production of mediators in response to the fungus S. schenckii.

The predominance of alternatively activated macrophages following challenge with cell wall peptide-polysaccharide after prior infection with Sporothrix schenckii.

Sporotrichosis is a subcutaneous mycosis that is caused by the dimorphic fungus Sporothrix schenckii. This disease generally occurs within the skin and subcutaneous tissues, causing lesions that can spread through adjacent lymphatic vessels and sometimes leading to systemic diseases in immunocompromised patients. Macrophages are crucial for proper immune responses against a variety of pathogens. Furthermore, macrophages can play different roles in response to different microorganisms and forms of activation, and they can be divided into "classic" or "alternatively" activated populations, as also known as M1 and M2 macrophages. M1 cells can lead to tissue injury and contribute to pathogenesis, whereas M2 cells promote angiogenesis, tissue remodeling, and repair. The aim of this study was to investigate the roles of M1 and M2 macrophages in a sporotrichosis model. Toward this end, we performed phenotyping of peritoneal exudate cells and evaluated the concomitant production of several immunomediators, including IL-12, IL-10, TGF-ß, nitric oxide, and arginase-I activity, which were stimulated ex vivo with cell wall peptide-polysaccharide. Our results showed the predominance of the M2 macrophage population, indicated by peaks of arginase-I activity as well as IL-10 and TGF-ß production during the 6th and 8th weeks after infection. These results were consistent with cellular phenotyping that revealed increases in CD206-positive cells over this period. This is the first report of the participation of M2 macrophages in sporotrichosis infections.

Mast cell activation by conidia of Sporothrix schenckii: role in the severity of infection.

Mast cells are abundant in the skin and other peripheral tissues, where they are one of the first immune cells to make contact with invading pathogens. As a result of pathogen recognition, mast cells can be activated and release different preformed and de novo-synthesized mediators. Sporothrix schenckii is the fungus that causes sporotrichosis, a worldwide-distributed subcutaneous mycosis considered as an important emerging health problem. It remains unknown whether or not mast cells are activated by S. schenckii. Here, we investigated the in vitro response of mast cells to conidia of S. schenckii and their in vivo involvement in sporotrichosis. Mast cells became activated after interaction with conidia, releasing early response cytokines as TNF-α and IL-6. Although histamine release was not significantly stimulated by S. schenckii, we determined that conidia potentiate histamine secretion induced by compound 48/80. Furthermore, functional depletion of peritoneal mast cells before S. schenckii infection significantly reduced the severity of cutaneous lesions of the sporotrichosis. These data demonstrate that mast cells are important contributors in the host response to S. schenckii infection, suggesting a role of these cells in the progress of clinical manifestations in sporotrichosis.

Antibodies Against Sporothrix schenckii Enhance TNF-α Production and Killing by Macrophages.

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti-gp70. Additionally, we show an increase in the levels of pro-inflammatory cytokines such as TNF-α and IL-1ß. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF-α production and fungus killing by macrophages in experimental sporotrichosis.

Cell-free antigens of Sporothrix brasiliensis: antigenic diversity and application in an immunoblot assay.

Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis.