Abstract
Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the thermodimorphic fungi of the genus Paracoccidioides spp. Paracoccidioides lutzii (PL) is one of the 5 species that constitute the Paracoccidioides genus. PL expresses low amounts of glycoprotein (Gp) 43 (PLGp43) and PLGp43 displays few epitopes in common with the P. brasiliensis (PB) immunodominant antigen PBGp43, which is commonly used for serological diagnosis of PCM. This difference in structure between the glycoproteins markedly reduces the efficiency of serological diagnosis in patients infected with PL. We previously demonstrated that peptide 10 (P10) from the PBGp43 induces protective immune responses in in vitro and in vivo models of PB PCM. Since, P10 has proven to be a promising therapeutic to combat PB, we sought to identify peptides in PL that could similarly be applied for the treatment of PCM. PL yeast cell proteins were isolated from PL: dendritic cell co-cultures and subjected to immunoproteomics. This approach identified 18 PL peptides that demonstrated in silico predictions for immunogenicity. Eight of the most promising peptides were synthesized and applied to lymphocytes obtained from peptide-immunized or PL-infected mice as well as to in vitro cultures with peptides or dendritic cells pulsed the peptides. The peptides LBR5, LBR6 and LBR8 efficiently promoted CD4+ and CD8+ T cell proliferation and dendritic cells pulsed with LBR1, LBR3, LBR7 or LBR8 stimulated CD4+ T cell proliferation. We observed increases of IFN-γ in the supernatants from primed T cells for the conditions with peptides without or with dendritic cells, although IL-2 levels only increased in response to LBR8. These novel immunogenic peptides derived from PL will be employed to develop new peptide vaccine approaches and the proteins from which they are derived can be used to develop new diagnostic assays for PL and possibly other Paracoccidioides spp. These findings identify and characterize new peptides with a promising therapeutic profile for future against this important neglected systemic mycosis.
Highlights
Paracoccidioidomycosis (PCM) is one of the major systemic fungal diseases in Latin America, with the highest incidence occurring in Brazil, Colombia and Venezuela [1,2,3]
We use the FASTA format of P. lutzii (PL) deposited in the Uniprot and MaxQuant software to perform in silico predictions to determine percent rank for peptide binding to MHC-II according to the Immune Epitope Database and Analysis Resource (IEDB) consensus method described above
In addition to synthesizing and testing the peptide with the IEDB less than 10%, we generated two peptides located in the protein Histone H2B, one peptide located in the Hsp70-like protein, two peptides 60S ribosomal protein for which three peptides were characterized, and two other peptides that showed IEDBs of 16 and 22%
Summary
Paracoccidioidomycosis (PCM) is one of the major systemic fungal diseases in Latin America, with the highest incidence occurring in Brazil, Colombia and Venezuela [1,2,3]. PCM due to each of these 5 Paracoccidioides species causes disease manifestations that are indistinguishable from one another. In Brazil, PCM is not a mandatory reportable disease. There are regions with higher rates of PCM, such as in Rondônia were there are 9.4 cases per 100,000 inhabitants, with two cities reporting incidences of ~40 cases per 100,000 inhabitants [8]. The glycoprotein Gp43 produced by PB (PBGp43) is one of the main serological markers used in the diagnosis of PCM [14,15,16,17]. PL expresses low amounts of Gp43 and PLGp43 displays few epitopes in common with the immunodominant PBGp43, which markedly reduces the efficiency of serological diagnosis in patients infected with PL [18, 19]
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