An immunoproteomic approach revealing peptides from Sporothrix brasiliensis that induce a cellular immune response in subcutaneous sporotrichosis

Sporothrix brasiliensis is the most virulent fungus of the Sporothrix complex and is the main species recovered in the sporotrichosis zoonotic hyperendemic area in Rio de Janeiro. A vaccine against S. brasiliensis could improve the current sporotrichosis situation. Here, we show 3 peptides from S. brasiliensis immunogenic proteins that have a higher likelihood for engaging MHC-class II molecules. We investigated the efficiency of the peptides as vaccines for preventing subcutaneous sporotrichosis. In this study, we observed a decrease in lesion diameters in peptide-immunized mice, showing that the peptides could induce a protective immune response against subcutaneous sporotrichosis. ZR8 peptide is from the GP70 protein, the main antigen of the Sporothrix complex, and was the best potential vaccine candidate by increasing CD4(+) T cells and higher levels of IFN-gamma, IL-17A and IL-1 beta characterizing a strong cellular immune response. This immune environment induced a higher number of neutrophils in lesions that are associated with fungus clearance. These results indicated that the ZR8 peptide induces a protective immune response against subcutaneous sporotrichosis and is a vaccine candidate against S. brasiliensis infection.FAPESPUniv Sao Paulo, Fac Pharmaceut Sci, Dept Clin & Toxicol Anal, Sao Paulo, BrazilUniv Sao Paulo, Inst Chem, Dept Biochem, Sao Paulo, BrazilUniv Sao Paulo, Inst Biomed Sci, Dept Immunol, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Dept Biol Sci, Diadema, BrazilUniv Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Dept Biol Sci, Diadema, BrazilFAPESP: 2016/04729-3Web of Scienc

ScFv from antibody that mimics gp43 modulates the cellular and humoral immune responses during Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells

TLR3 Is a Negative Regulator of Immune Responses Against Paracoccidioides brasiliensis

Toll-like receptors (TLRs) comprise the best-characterized pattern-recognition receptor (PRR) family able to activate distinct immune responses depending on the receptor/adaptor set assembled. TLRs, such as TLR2, TLR4 and TLR9, and their signaling were shown to be important in Paracoccidioides brasiliensis infections. However, the role of the endosomal TLR3 in experimental paracoccidioidomycosys remains obscure. In vitro assays, macrophages of the bone marrow of WT or TLR3−/− mice were differentiated for evaluation of their microbicidal activity. In vivo assays, WT or TLR3−/− mice were infected intratracheally with Paracoccidioides brasiliensis yeasts for investigation of the lung response type induced. The cytotoxic activity of CD8+ T cells was assessed by cytotoxicity assay. To confirm the importance of CD8+ T cells in the control of infection in the absence of tlr3, a depletion assay of these cells was performed. Here, we show for the first time that TLR3 modulate the infection against Paracoccidioides brasiliensis by dampening pro-inflammatory response, NO production, IFN+CD8+T, and IL-17+CD8+T cell activation and cytotoxic function, associated with granzyme B and perforin down regulation. As conclusion, we suggest that TLR3 could be used as an escape mechanism of the fungus in an experimental paracoccidioidomycosis

TLR3 influence in experimental PCM model.

Os receptores do tipo Toll compreendem a família de receptores de reconhecimento de padrões melhor caracterizados, que podem ativar diferentes respostas imunes, dependendo de quais receptores e conjuntos de adaptadores são utilizados. Os TLRs, como TLR2, TLR4 e TLR9, e sua sinalização foram implicados no reconhecimento de P. brasiliensis e na regulação da resposta imune, no entanto, o papel do TLR3 ainda não está claro. Assim, a compreensão da função endossomal do TLR3 na PCM experimental é crucial. Utilizamos modelos in vitro e in vivo de infecção por P. brasiliensis, camundongos C57Bl/6 e TLR3-/-, para avaliar a contribuição da TLR3 no desenvolvimento da infecção. Mostramos que ausência de TLR3 leva o aumento de óxido nítrico e a capacidade fagocítica por macrófagos nas primeiras 4 horas de interação com leveduras P. brasiliensis. Mostramos ainda que os camundongos TLR3-/- desempenham papel protetor após 30 dias de infecção intratraqueal com P. brasiliensis, mostrando diminuição do aumento de CFU, perfil de resposta Th1 e Th17, bem como aumento de células citotóxicas T CD8+ produtoras de IFN-γ e IL-17. As células citotóxicas T CD8+ mostraram ser essenciais para o controle da infecção nos camundongos TLR3-/-, uma vez que a depleção dessas células levou a progressão da doença. Em estágios iniciais, 3 e 5 dias de infecção, observamos aumento do recrutamento de neutrófilos para o pulmão. Estudos recentes indicam que o TLR3 é um receptor importante para a resposta imune na micose e sua ausência favorece a infecção por fungos. Em contraste, nossos resultados mostram que, no caso do PCM, o TLR3 é prejudicial ao hospedeiro, sugerindo que a ativação do TLR3 pode ser um possível mecanismo de escape de P. brasiliensis.Toll-like receptors comprise the best-characterized pattern-recognition receptor family that can activate different immune responses, depending on which receptor and adaptor set are utilized. TLRs, such as TLR2, TLR4 and TLR9, and their signaling have been implicated in the recognition of P. brasiliensis and regulation of the immune response, however, the role of TLR3 remains unclear. Thus, understanding the endosomal function of TLR3 in experimental PCM is crucial. We used in vitro and in vivo models of infection by P. brasiliensis, C57Bl/6 and TLR3-/- mice, to assess the contribution of TLR3 on development of infection. We show that absence of TLR3 leads to increased nitric oxide and phagocytic capacity by macrophages in the first 4 hours of interaction with yeasts P. brasiliensis. We also showed that TLR3-/- mice play a protective role after 30 days of intratracheal infection with P. brasiliensis, showing a decrease in the CFU increase, Th1 and Th17 response profile, as well as an increase in cytotoxic CD8+ cells producing IFN-γ and IL-17. The cytotoxic T CD8+ cells were shown to be essential for the control of infection in TLR3-/- mice, since the depletion of these cells led to the progression of the disease. In the initial stages, 3 and 5 days of infection, we observed increased recruitment of neutrophils to the lung. Recent studies indicate that TLR3 is an important receptor for the immune response in mycosis and its absence favors fungal infection. In contrast, our results show that in the case of PCM, TLR3 is detrimental to the host, suggesting that TLR3 activation may be a possible escape mechanism of P. brasiliensis

Phenotypic characterization of dendritic cells transfected with scFv derived from monoclonal anti-β-idiotypic Ab2 which mimics the antigen gp43 of Paracoccidioides brasiliensis

A paracoccidioidomicose (PCM) é um a micose profunda de natureza granulomatosa, causada pelo fungo Paracoccidioides brasiliensis (P. brasiliensis) e que compromete preferencialmente o tecido pulmonar. O P. brasiliensis sintetiza várias substâncias, dentre estas, destaca-se a glicoproteína de 43 kDa (gp43), a qual é considerada o principal componente antigênico do fungo. Dada a importância da gp43, anticorpos monoclonais (Mabs) contra esta glicoproteína foram obtidos e posteriormente caracterizados. Baseado na hipótese da rede idiotípica, Jerne (1974) propôs que cada anticorpo, quando produzido em resposta a um antígeno, induz a formação de outros anticorpos dirigidos contra sua região variável, na qual é única. Anticorpos anti-idiotípicos, que reconhecem o paratopo, contendo a imagem interna do antígeno, são denominados de Ab2-β e seu potencial terapêutico tem sido explorado em diferentes sistemas. Apesar da molécula de anticorpo ser complexa estruturalmente, sabemos que a região Fab, é a responsável pelo mimetismo do antígeno. Então, nosso grupo de pesquisa construiu uma nova molécula de anticorpo à partir do Mab anti-idiotípico Ab2-β, que mimetiza o antígeno gp43 de P. brasiliensis, denominada de fragmento variável de cadeia única (scFv). A transfecção dessa molécula em células dendríticas (DCs) mostrou ser promissora na PCM experimental, visto que estes transfectomas foram eficientes em apresentar a proteína scFv às células dos linfonodos, induzindo linfoproliferação, além de diminuírem a carga fúngica pulmonar. Visto que a molécula de scFv que possui a região de reconhecimento e ativação de linfócitos T mostrou eficiência no modelo de terapia na PCM experimental, o principal objetivo deste trabalho foi analisar os mecanismos pelos quais os transfectomas de DCs ativam a resposta imune em camundongos infectados com o fungo, para que possamos entender melhor a capacidade destas células em modular a resposta imune na PCM experimental. Dessa maneira, avaliamos a capacidade das DCs trasfectadas com pMAC/PS-scFv em migrar para os linfonodos regionais e induzirem uma resposta protetora com produção de IgG, bem como, analisamos o fenótipo destas células e produção de citocinas. Nosso modelo de terapia com pMAC/PS-scFv mostrou-se eficiente, uma vez que observamos diminuição de células T regulatórias nos linfonodos regionais, bem como aumento da produção de citocinas como IFN-γ e IL-12. Ainda, observamos aumento do isotipo IgG2b, sugerindo a modulação de resposta imune para o tipo Th1, importante na proteção da PCM.The paracoccidioidomycosis (PCM) is the nature of granulomatous mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis) and preferentially compromises lung tissue. The P. brasiliensis synthesizes various substances, among these, there is a 43 kDa glycoprotein (gp43), which is considered a major antigenic component of the fungus. Given the importance of gp43 monoclonal antibodies (Mabs) against this glycoprotein were obtained and further characterized. Based on the idiotypic network hypothesis, Jerne (1974) proposed that each antibody, when produced in response to an antigen, induces the several of other antibodies production against its variable region, which is unique. Anti-idiotypic antibodies that recognize the paratope containing the internal image of the antigen are called Ab2 β and their therapeutic potential has been explored in different systems. Although the antibody molecule is structurally complex, we know that the Fab region, is responsible for antigen mimicking. Therefore, our research group has engineered a new molecule from the antibody Mab-anti-idiotypic Ab2 β that mimics the antigen gp43 of P. brasiliensis, known as single chain variable fragment (scFv). Transfection of this molecule on dendritic cells (DCs) has shown promise in experimental PCM, since these transfection were effective scFv protein present in the cells of the lymph nodes, inducing lymphoproliferation, in addition to reducing fungal burden in the lung. Since the scFv molecule that has the region recognition and activation of T lymphocytes showed efficiency in therapy model in experimental PCM, the main objective of this study was to analyze the mechanisms by which transfected DCs activate the immune response in infected mice with the fungus, so that we can better understand the ability of these cells to modulate the immune response in experimental PCM. Thus, the capacity of transfected DCs with pMAC / PS-scFv to migrate to regional lymph nodes and induce a protective response with IgG production, as well as analyze the phenotype of these cells and cytokine production. Our therapy model with pMAC / PS-scFv was efficient, since we observed decrease of regulatory T cells in regional lymph nodes, as well as increased production of cytokines such as IFN-γ and IL-12. Furthermore, we observed increased IgG2b isotype, suggesting modulation of the immune response to a Th1 response, the protect model of experimental PCM

The Role of Dimorphism Regulating Histidine Kinase (Drk1) in the Pathogenic Fungus <i>Paracoccidioides brasiliensis</i> Cell Wall

Dimorphic fungi of the Paracoccidioides genus are the causative agents of paracoccidioidomycosis (PCM), an endemic disease in Latin America with a high incidence in Brazil. This pathogen presents as infective mycelium at 25 °C in the soil, reverting to its pathogenic form when inhaled by the mammalian host (37 °C). Among these dimorphic fungal species, dimorphism regulating histidine kinase (Drk1) plays an essential role in the morphological transition. These kinases are present in bacteria and fungi but absent in mammalian cells and are important virulence and cellular survival regulators. Hence, the purpose of this study was to investigate the role of PbDrk1 in the cell wall modulation of P. brasiliensis. We observed that PbDrk1 participates in fungal resistance to different cell wall-disturbing agents by reducing viability after treatment with iDrk1. To verify the role of PbDRK1 in cell wall morphogenesis, qPCR results showed that samples previously exposed to iDrk1 presented higher expression levels of several genes related to cell wall modulation. One of them was FKS1, a β-glucan synthase that showed a 3.6-fold increase. Furthermore, confocal microscopy analysis and flow cytometry showed higher β-glucan exposure on the cell surface of P. brasiliensis after incubation with iDrk1. Accordingly, through phagocytosis assays, a significantly higher phagocytic index was observed in yeasts treated with iDrk1 than the control group, demonstrating the role of PbDrk1 in cell wall modulation, which then becomes a relevant target to be investigated. In parallel, the immune response profile showed increased levels of proinflammatory cytokines. Finally, our data strongly suggest that PbDrk1 modulates cell wall component expression, among which we can identify β-glucan. Understanding this signalling pathway may be of great value for identifying targets of antifungal molecular activity since HKs are not present in mammals

Cytokines.

<p>BALB/c mice (seven/group) were infected with 1x10⁶ of <i>P</i>. <i>brasiliensis</i> yeast. On 7 and 14 days, the animals were treateds with pMAC/PS-scFv. As control, the BALB/c mice was treateds with PBS (50μL) or empty vector. After seven days of last burst, the lung (A) and lynph nodes (B) cells were cultivated in vitro for 24 hours and the IFN-γ, IL-12 and IL-4 were measured by ELISA. *p<0.05 e **p<0,001. Results are representative of three independent experiments.</p

DCs molecule expression.

<p>DCs were transfected with pMAC/PS-scFv. After 48 hours the total DCs were stained with anti-MHCII/CD11c, anti-CD86, anti-CD40 and anti-CD80. The data were analyzed by flow cytometer. Results are representative of three independent experiments.</p

Humoral response.

<p>BALB/c mice (seven/group) were infected previously with <i>P</i>. <i>Brasiliensis</i> yeast (1x10⁶) by intratracheal pathway. The animals were treateds twice with pMAC/PS-scFv and total IgG was measured (A). The ratio between IgG2a/IgG1 was analyse (B) *p<0.05. Results are representative of three independent experiments.</p

Phenotype of lung cells.

<p>BALB/c mice (seven/group) were infected with <i>P</i>. <i>brasiliensis yeast</i> (1x10⁶). After that, the animals received twice treatments with PBS (50μL), DC (1x10⁶), DC-pMAC/PS (1x10⁶ cells plus 20ng/mL DNA) and DC-pMAC/PS-scFv (1x10⁶ cells plus 20ng/mL DNA). After seven days from the last treatment, the lung cells were stained with anti-CD3e/CD4 (A), anti-CD3e/CD8 (B) and anti-CD40/CCR7 (C). As control of the infection, BALB/c mice received only PBS (50μL) by intratracheal pathway. The data was analyzed by flow cytometer. Flow cytometry graphs: one representative experiment is shown.</p