Resumo

INTRODUCTION: Fungal infections have emerged as serious public health problem since the majority of therapeutic strategies are ineffective. Therefore, it makes necessary the development of new and efficient strategies for the treatment of mycosis. Passive immunization using monoclonal antibodies is a powerful alternative to eliminate fungal infections. In order to develop a broader reagent against mycosis, we have used lectins, dectin-1 and WGA, with recognized affinity to common fungal structures such b-1,3-glucan and chitin, and fused to effector Fc part of IgG2a. These lectin-Fc fusion proteins were evaluated regarding their antifungal activity against several pathogenic fungi. Hereby, we propose the development and characterization of new chimeras lectin-Fc as a potential therapeutic strategy against mycosis, due to the recognition of universal targets on fungal cell wall. MATERIALS AND METHODS: The RNA of mouse dendritic cells (dectin-1), Triticum aestivum (WGA) and IgG2a hybridomas (Fc CH2-CH3) were extracted by the RiboPure-Yeast Kit. cDNAs were produced using the First Strand cDNA Synthesis Kit and the oligo-dT random primer. The cDNAs were amplified using specific 5’ and 3’ primers for each fragment by PCR. For the construction of the chimeras, genes were fused by overlapping PCR. These products were purified and cloned into a pSecTag plasmid and used to transfect CHO-K1 cells with Lipofectamine. Transfected cells were selected by zeocin resistance and expression of respective fusion proteins by indirect ELISA. Inhibition ELISA was performed to calculate dissociation constants (Kd). Immunofluorescences were conducted to confirm the binding of the chimeras to the cell wall of all fungi tested. RESULTS AND DISCUSSION: We were able to highly express the chimeras dectin-Fc and WGA-Fc in CHO cells as determined by indirect ELISAs against b-glucan and chitin. Inhibition ELISAs were performed to calculate the Kd of both chimeras, which displayed similar values to their respective native proteins. Immunofluorescesces conducted with both chimeras displayed co-localized binding to Uvitex labeled cell wall for WGA-Fc fusion proteins and an outer ring for the dectin-Fc proteins. CONCLUSION: Initial analyses have demonstrated that chimeras were able to bind to their respective purified or native glycans present on the cell wall of the most common pathogenic fungi. Currently we are evaluating these fusion proteins regarding their antifungal and protection properties in vitro and in vivo against these fungal pathogens.