Therapeutic Vaccination with Cationic Liposomes Formulated with Dioctadecyldimethylammonium and Trehalose Dibehenate (CAF01) and Peptide P10 Is Protective in Mice Infected with Paracoccidioides brasiliensis

Abstract

The peptide P10 is a vaccine candidate for Paracoccidioidomycosis, a systemic mycosis caused by fungal species of the genus Paracoccidioides spp. We have previously shown that peptide P10 vaccination, in the presence of several different adjuvants, induced a protective cellular immune response mediated by CD4⁺ Th₁ lymphocytes that was associated with the increased production of IFN-γ in mice challenged with a virulent isolate of Paracoccidoides brasiliensis. Cationic liposomes formulated with dioctadecyldimethylammonium and trehalose dibehenate (DDA/TDB, termed also CAF01-cationic adjuvant formulation) have been developed for safe administration in humans and CAF01 liposomes are utilized as an adjuvant for modulating a robust Th₁/Th₁₇ cellular response. We evaluated the efficacy of the adsorption of peptide P10 to CAF01 cationic liposomes and used the generated liposomes to vaccinate C57Bl/6 mice infected with P. brasiliensis. Our results showed that P10 was efficiently adsorbed onto CAF01 liposomes. The vaccination of infected mice with cationic liposomes formulated with DDA/TDB 250/50 µg/mL and 20 µg of P10 induced an effective cellular immune response with increased levels of Th₁₇ cytokines, which correlated with significant decreases in the fungal burdens in lungs and protective granulomatous tissue responses. Hence, cationic liposomes of DDA/TDB 250/50 µg/mL with 20 µg of P10 are a promising therapeutic for safely and effectively improving the treatment of paracoccidioidomycosis.

Keywords: CAF01; DDA/TDB; P. brasiliensis; adjuvant; cationic liposome; paracoccidioidomycosis; peptide vaccine.

Figure 1

TEM of cationic liposomes. The liposomes were prepared by the film hydration method and fixed with Uranyl for morphological analyses. The micrographs show unilamellar liposomes of DDA/TDB alone (A); DDA/TDB plus P10 (B).

Figure 2

Vaccination with DDA/TDB/P10 decreases the fungal load. The fungal burden was measured in the lungs of mice infected with P. brasiliensis via Colony Forming Units (CFU) assay and the results were expressed as CFU/g of lung tissue. Infected animals either received PBS (1), DDA/TDB alone (2), or different formulations of DDA/TBD with P10 (3, 4, and 5). The data represent the mean and SD of results from 3 experiments using 6 mice per group. An asterisk (*) represents a statistically significant difference, *** p < 0.001 and * p < 0.05.

Figure 3

Evaluation of immune responses induced by P10 peptide adsorbed on DDA/TDB liposomes. Th₁, Th₂, and Th₁₇-associated cytokines were measured in pulmonary homogenates 75 days after infection by capture enzyme-linked immunosorbent assay (ELISA). Infected mice with P. brasiliensis received three doses of either PBS as a control (1), DDA/TDB alone (2), or different concentrations of DDA/TDB with P10 (3–5). The data are shown are the mean and SD of results from three independent experiments using 6 mice per group. The asterisk represents a statistically significant difference * p < 0.05 and ** p < 0.01.

Figure 4

Photomicrographs of pulmonary tissues from C57BL/6 mice with or without P. brasiliensis infection. The lung sections were stained with Hematoxylin and Eosin to analyze the presence of inflammation. Uninfected lung tissue (A) is presented for comparison with P. brasiliensis infected lungs from mice that received PBS only (B), treated with DDA/TDB alone (C), or vaccinated with DDA: DDA/TDB 250/50 μg/P10 20 μg (D) DDA/TDB 312.5/62.5 μg/P10 20 μg (E) or DDA/TDB 500/100 μg/P10 20 μg (F). (10× magnification).