Paracoccidioides brasiliensis vaccine formulations based on the gp43-derived P10 sequence and the Salmonella enterica FliC flagellin

Abstract

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4(+) T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

FIG. 1.

Characterization of the specific serum IgG subclass response elicited in mice i.n. immunized with gp43 or gp43 admixed with FliC. (A) Anti-gp43-specific IgG subclass detected 1 week after the last immunization dose. (B) Anti-gp43-specific IgG subclass detected in vaccinated mice 2 months after i.t. challenge with the Pb18 strain. gp43, mice immunized with 3 doses of purified gp43; gp43 + FliCd, mice immunized with purified gp43 admixed with FliC. For the other groups (FliCd, FliCd-P10, FliCd-P10L, and P10 + FliCd), a gp43-specific antibody response was not detected. Values are means of endpoint titers plus standard deviations for serum pools (n = 8) prepared from each mouse group. Data are representative of two independent experiments with similar results. The IgG1/IgG2a ratio of each immunization group is indicated at the top of the figure. Asterisks indicate a statistically significant difference observed between mice immunized with gp43 and mice immunized with gp43 admixed with FliC (P ≤ 0.01).

FIG. 2.

Generation of recombinant hybrid flagellins genetically fused to the CD4⁺ T-cell-specific gp43-derived P10 epitope. (A) Schematic representation of the recombinant flagellins after P10 in-frame insertion at FliC central hypervariable domain. The two recombinant flagellins carried the P10 peptide (FliCd-P10) or the P10 peptide with lysine residues on each side of the fusion site (FliCd-P10L). (B) Coomassie blue-stained 12% polyacrylamide gel loaded with flagellins extracted from different Salmonella strains. Lane 1, molecular mass markers (Fermentas); lane 2, FliCd flagellin extracted from S. Dublin strain SL5930 (with no insert); lane 3, FliCd-P10 flagellin extracted from S. Dublin strain SLP10; lane 4, FliCd-P10L flagellin extracted from S. Dublin strain SLP10L; lane 5, purified gp43. Each well was loaded with approximately 2 μg of purified flagellins or gp43.

FIG. 3.

Cytokine relative ratios measured in lungs of mice after challenge with the Pb18 strain. All cytokines were measured in whole extracts of lung cells. Immunization groups and specific cytokine values used in the cytokine ratio determination were as depicted in Table 2.

FIG. 4.

Detection of viable fungal cells in lung tissues of vaccinated mice after i.t. challenge with P. brasiliensis Pb18. (A) Detection of viable fungi in mice i.n. immunized with purified gp43 or gp43 admixed with FliC. Immunization groups are as described in the legend to Fig. 2. (B) Detection of viable fungal cells in mice i.n. immunized with P10 epitope genetically fused with flagellin (FliCd-P10 or FliCd-P10L) or synthetic P10 peptide admixed with FliC flagellin. PBS, mice immunized only with PBS; FliCd, mice immunized only with FliC; FliCd-P10 and FliCd-P10L, mice immunized with purified recombinant FliCd-P10 and FliCd-P10L, respectively; P10+FliCd, mice immunized with P10 admixed with FliCd. All mice groups were i.t. challenged with the Pb18 strain and sacrificed 2 months later for determination of CFU in homogenized lung tissue. The same experiments were repeated three times. Each bar represents the medium number plus standard deviation in organs collected from 8 to 10 animals in each group. Asterisks indicate statistically significant differences between results detected in mice immunized with gp43 and P10 and those in mice immunized only with FliC (*, P ≤ 0.05; **, P ≤ 0.01).

FIG. 5.

Representative histopathology of lung lesions caused by P. brasiliensis strain Pb18 in mice immunized with different vaccine formulations. Tissue samples were collected 2 months after i.t. challenge with strain Pb18. (A) Lung section from PBS-immunized mouse with granuloma containing multiple viable fungal cells. (B) Lung section from a mouse immunized with gp43 admixed with FliC. Observe the extensive granulomatous lesions with intense cellular infiltration and large number of multiplying fungal cells. (C) Lung section from mouse immunized with the hybrid FliCd-P10L. (D) Lung section from mouse immunized with P10 admixed with FliC showing preserved alveolar structure and absence of granulomatous lesions and fungal cells. All sections were amplified 40-fold and stained with hematoxylin-eosin.