Prophylactic and therapeutic vaccination using dendritic cells primed with peptide 10 derived from the 43-kilodalton glycoprotein of Paracoccidioides brasiliensis

Abstract

Vaccination with peptide 10 (P10), derived from the Paracoccidioides brasiliensis glycoprotein 43 (gp43), induces a Th1 response that protects mice in an intratracheal P. brasiliensis infection model. Combining P10 with complete Freund's adjuvant (CFA) or other adjuvants further increases the peptide's antifungal effect. Since dendritic cells (DCs) are up to 1,000-fold more efficient at activating T cells than CFA, we examined the impact of P10-primed bone-marrow-derived DC vaccination in mice. Splenocytes from mice immunized with P10 were stimulated in vitro with P10 or P10-primed DCs. T cell proliferation was significantly increased in the presence of P10-primed DCs compared to the peptide. The protective efficacy of P10-primed DCs was studied in an intratracheal P. brasiliensis model in BALB/c mice. Administration of P10-primed DCs prior to (via subcutaneous vaccination) or weeks after (via either subcutaneous or intravenous injection) P. brasiliensis infection decreased pulmonary damage and significantly reduced fungal burdens. The protective response mediated by the injection of primed DCs was characterized mainly by an increased production of gamma interferon (IFN-γ) and interleukin 12 (IL-12) and a reduction in IL-10 and IL-4 compared to those of infected mice that received saline or unprimed DCs. Hence, our data demonstrate the potential of P10-primed DCs as a vaccine capable of both the rapid protection against the development of serious paracoccidioidomycosis or the treatment of established P. brasiliensis disease.

Fig 1

Cell proliferation assay. Splenocytes isolated from BALB/c mice previously immunized subcutaneously with 10.2 μM P10 were stimulated in vitro for 72 h with 2.55 μM P10 or with P10-primed DCs or nonprimed DCs. NS, nonstimulated splenocytes; ConA, positive-control activation of splenocytes with the mitogen concanavalin A. Each bar shows the mean of results from 3 experiments carried out on different days. ∗∗∗, significant difference (P < 0.0001; determined by analysis of variance [2-way ANOVA] and the posttest of Tukey-Kramer) relative to the free P10 stimulated system.

Fig 2

Lung CFU: therapeutic protocol. The results are from two independent experiments. Each group from each experiment (n = 5) was infected i.t. with 3 × 10⁵ yeast cells. After 30 days, groups of mice received either nonprimed dendritic cells (DCs) or P10-primed DCs via either an intravenous (IV) or subcutaneous (SC) route. A second identical immunization was administered 7 days later. The control group (C+) was not treated. Mice were sacrificed at day 45 after infection. ∗, significant difference (P < 0.05) compared with the control and unprimed DCs.

Fig 3

Lung CFU: prophylactic protocol. The results are from two independent experiments. The groups of treated mice (n = 5 per experiment) received either unprimed dendritic cells (DC) or P10-primed DCs via either intravenous (IV) or subcutaneous (SC) route 24 h before the mice were infected i.t. with 3 × 10⁵ yeast cells. The control (C+) group received PBS 1 day prior to infection. Mice were sacrificed 30 days after infection. ∗, significant difference (P < 0.001) compared with the control.

Fig 4

Cytokine detection: therapeutic protocol. Cytokines were assayed in the lung tissue from mice 45 days after i.t. infection. Each group (n = 5) was infected i.t. with 3 × 10⁵ yeast cells. After 30 days, groups of mice received either unprimed dendritic cells (DCs) through subcutaneous route or P10-primed DCs via either an intravenous (IV) or subcutaneous (SC) route. A second identical immunization was administered 7 days later. The control group (C+) was not treated, and Sham represents uninfected and untreated mice. ∗, significant difference (P < 0.05) compared with C+ and DC groups.

Fig 5

Cytokine detection: prophylactic protocol. Cytokines were assayed in the lung tissue from mice 45 days after i.t. infection. The treatment groups (n = 5) of mice received either unprimed dendritic cells (DCs) through the subcutaneous (SC) route or P10-primed DCs via either an intravenous (i.v.) or subcutaneous route 24 h before the mice were infected i.t. with 3 × 10⁵ yeast cells. The control (C+) group received PBS 1 day prior to infection, and Sham represents uninfected and untreated mice. ∗, significant difference (P < 0.05) compared with C+ and DC groups.

Fig 6

Representative lung sections: therapeutic protocol. Histopathological sections of murine lungs 45 days after i.t. infection. (A) Representative section of lung from an infected and untreated mouse. For treatment, mice received at day 30 and 38 either unprimed DCs subcutaneously (B) or P10-primed DCs intravenously (C) or subcutaneously (D). Photographs of sections were taken at 400× magnification.

Fig 7

Representative lung sections: prophylactic protocol. Representative histopathological sections of murine lungs 30 days after i.t. infection. Twenty-four hours prior to infection, mice received PBS (A), unprimed DCs subcutaneously (B), P10-primed DCs intravenously (C), or P10-primed DCs subcutaneously (D). Photographs of sections were taken at 400× magnification.