Protective effect of rPb40 as an adjuvant for chemotherapy in experimental paracoccidioidomycosis.

The conventional treatment for the most prevalent mycosis in Latin America, paracoccidioidomycosis (PCM), involves long periods of therapy that results in side effects and a high frequency of relapses. The search for a new, alternative treatment is necessary. Pb40 is an antigenic protein from P. brasiliensis fraction F0. This fraction has already been shown to have significant protective activity when used as a PCM vaccine in experimental models. The complete cDNA sequence corresponding to Pb40 was cloned into a pET-21a plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. The predicted protein sequence exhibited nearly 100% homology to a fragment of the hypothetical EF-hand domain containing protein of P. brasiliensis. Immunization with this recombinant protein was used together with chemotherapy in an attempt to improve PCM treatment. The combined drug/rPb40 treatment exhibited long-lasting control of PCM in the liver and spleen and largely preserved the tissue structures of these organs. Despite the lack of a reduction in CFUs in the group that received the combined treatment, there was a significant reduction in the size of the lesions in the lungs after 70 days of infection. At the same time, the IL-10 levels were higher in the treated mice than in the infected-only mice. Moreover, significant levels of rPb40-specific IgG antibodies were detected in the sera of immunized mice. Thus, the treatment protocol consisting of rPb40 immunization in addition to fluconazole chemotherapy showed an additive protective effect after intratracheal challenge, preventing fungal dissemination to other sites of infection and preventing relapses. These results provide new prospects for PCM immunotherapy.

Protective immunity induced by rPb27 of Paracoccidioides brasiliensis.

A cDNA coding for an antigenic protein (rPb27) from the pathogenic fungus Paracoccidioides brasiliensis was cloned and its protective activity was determined against paracoccidioidomycosis (PCM). The cDNA sequence contained an open reading frame (ORF) of 660 base pairs encoding a protein of 219 amino acids with a predicted molecular weight of 25kDa. The deduced amino acid sequence exhibited 100% identity to the 27kDa P. brasiliensis hypothetic protein (access number AA49615). The complete coding cDNA was cloned into a pGEX 4T-2 plasmid and expressed in Escherichia coli as a glutathione-S-transferase-tagged (GST) recombinant protein. Mice immunized with purified rPb27 were able to develop high levels of IgG2b, moderate levels of IgG1 and low levels of IgG2a. At the same time the levels of TGF-beta and IFN-gamma were high while a very low production of IL-10 was verified. Using confocal microscopy with anti-rPb27 mouse serum against P. brasiliensis yeast forms, surface and cytosolic staining pattern were observed. Moreover, immunization of mice with this antigen induced a significant degree of protection in the lungs (93%), liver (93%) and spleen (100%) at 60 days after challenge with infection. Thus, the granulomatous lesions revealed a greater degree of compaction and organization, with few lesions in the lungs and no dissemination of the fungus to other organs. These results showed that a recombinant protein of P. brasiliensis (rPb27) promoted acquired protection against infection with P. brasiliensis yeast forms, suggesting the use of this protein for future development as a prophylactic vaccine for PCM.