The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide

Abstract

Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4(+)-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund's adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM.

Keywords: FliC flagellin; P10; Paracoccidioides brasiliensis; adjuvants; aluminum hydroxide; complete Freund’s adjuvant; dioctadecyl-dimethylammonium bromide; paracoccidioidomycosis.

FIGURE 1

Colony forming units (CFU) from lungs of BALB/c mice infected intratracheally with 3 × 10⁵ yeast cells of Pb18 and immunized at 30, 37, and 44 days after infection with the different adjuvants with or without P10. Animals were sacrificed after 60 days of infection. Control animals were infected by not immunized (IFN). The adjuvants used were: aluminum hydroxide alone (ALU) or with P10 (AP10), FliC flagellin alone (FLA) or with P10 (FP10), complete Freund’s adjuvant alone (CFA) or with P10 (CF10), and cationic lipid alone (CLI) or with P10 (CP10). Significant difference *p < 0.05, **p < 0.01.

FIGURE 2

Cytokine detection was assayed in the lung tissue from BALB/c mice 52 days after infection. Analyzed cytokines were: (A) IFN-γ, (B) TNF-α, (C) IL-4, and (D) IL-10. Each group was infected i.t. with 3 × 10⁵ yeast cells and immunized at 30, 37, and 44 days after infection with different adjutants with or without P10.The groups of mice included unimmunized, infected control mice (INF); animals infected and immunized with aluminum hydroxide alone (ALU) or with P10 (AP10), FliC flagellin alone (FLA) or with P10 (FP10), and cationic lipid alone (CLI) or with P10 (CP10). *p < 0.05: significance p < 0.05 compared to control mice (only infected). **p < 0.01: compared to control mice (only infected).

FIGURE 3

Histological sections of murine lungs from i.t. infected BALB/c mice submitted to immunization with P10 associated to cationic lipid. Groups of mice included (A) uninfected controls, (B) unimmunized infected controls, (C) infected and immunized only with the cationic lipid, (D) infected and immunized with cationic lipid plus P10, (E) infected and immunized only with FliC flagellin, (F) infected and immunized with FliC flagellin plus P10, (G) infected and immunized only with aluminum hydroxide, (H) infected and immunized with aluminum hydroxide plus P10 after 52 days of infection. H&E staining, ×10 magnification.

FIGURE 4

Histological sections of murine lungs from i.t. infected BALB/c mice immunized after 30 days of infection with P10 associated with FliC flagellin, aluminum hydroxide, or cationic lipid. (A) infected, unimmunized control mice, (B) infected and immunized with FliC flagellin plus P10, (C) infected and immunized with aluminum hydroxide plus P10, and (D) infected and immunized with cationic lipid plus P10 after 52 days of infection. Masson’s trichrome staining, ×40 magnification. Blue staining and arrows indicate type I collagen fibers.