Immunotherapy in PCM: evaluation of combined therapy with monoclonal antibodies targeting CTLA-4 and antifungals drugs.

Abstract

In paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America, previous studies have revealed that host immunity is strongly regulated by various suppressive mechanisms mediated by plasmacytoid dendritic cells, the enzyme indoleamine 2,3-dioxygenase (IDO1), regulatory T cells, as well as negative co-stimulatory molecules such as CTLA-4 and PD-1. Immunotherapy with monoclonal antibodies has emerged as an effective therapeutic approach in the treatment of cancer and immune disorders. In addition to their use in these areas, monoclonal antibodies have also shown potential in combating infectious diseases, enhancing the effectiveness of existing antimicrobial drugs. In the context of fungal infections, monoclonal antibodies have been explored as a promising strategy. Studies have demonstrated that immune checkpoint inhibitors, such as those targeting to the PD-1 or CTLA-4, can modify the course of these diseases by promoting a more effective immune response and reducing the severity of the infection. A previous study conducted by our research group demonstrated that blockade of CTLA-4 and PD-1 through treatment with anti-PD-1 or anti-CTLA-4 reduced the fungal burden in affected organs, improved lung lesions, and increased the survival of treated animals by strengthening protective immune responses. Furthermore, anti-CTLA-4 treatment was more effective in disease control compared to anti-PD-1 treatment. Therefore, this work aims to establish an immunotherapeutic approach for PCM involving the inhibition of CTLA-4 molecules in combination or not with three different antifungal agents with the purpose of reverse the typical immunosuppression of this chronic infection and promote host recovery. For this purpose, C57BL/6 mice will be inoculated with 1x106 P. brasiliensis yeast cells, after 6 weeks, the animals will be treated with anti-CTLA-4 alone or in combination with amphotericin B, fluconazole, or itraconazole. After two weeks of treatment, mice will be evaluated for disease progression through colony-forming units (CFU), histopathology, and morbidity curves. Additionally, ELISA and flow cytometry will be performed to assess the immune response resulting from the use of this novel immunotherapeutic approach. In an additional protocol, the animals will also be evaluated three weeks after the completion of treatments. In this way, we intend to develop a protocol aimed at interrupting the course of the disease by controlling fungal growth associated with the restoration of the immune response, which together may result in significant improvement or even cure of this chronic infection.