Therapeutic DNA vaccine encoding peptide P10 against experimental paracoccidioidomycosis

Abstract

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Figure 1. Summary of the treatment protocols used.

First protocol: BALB/c mice received 4 weekly injections. Animals were infected and sacrificed 30 or 60 days later. Second protocol: BALB/c and B10.A mice were infected intratracheally. Mice received 4 weekly vaccine doses and animals were sacrificed 1 week after the last injection. Third protocol: B10.A mice were infected i.t. and one month after infection, they were immunized with the DNA vaccine. The animals were sacrificed one month after the last injection, six months after infection.

Figure 2. Immunoprophylactic treatment of PCM.

Gene immunization was initiated 30 days before fungal challenge. CFUs are from lungs of BALB/c mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) and/or IL-12 (pIL-12) DNA insert. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 30 (▪) and 60 (□) days after infection. Each bar represents the average counts and standard deviations of CFUs in lungs from 10 animals in each group. Experiments were carried out in triplicate with similar results. ** p≤0.0001, comparing vector with and without insert; ^## p≤0.0001, .comparing untreated and other groups.

Figure 3. Therapeutic treatment of PCM.

Gene immunization started 30 days after infection. CFUs are from lungs of BALB/c (□) and B10.A(▪) mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) or IL-12 (pIL-12) DNA inserts. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 60 days after infection. Each bar represents the average counts and standard deviations of CFU in lungs from 10 animals in each group. Experiments were performed three times and similar results were achieved. * p≤0.05, ** p≤0.005, comparing vector with and without insert; ^## p≤0.005, comparing untreated and other groups.

Figure 4. Long term therapeutic treatment of experimental PCM.

Gene immunization started 30 days after infection and mice were sacrificed 6 months after infection. CFUs were counted in lungs of B10.A mice infected intratracheally with 3×10⁵ yeast cells and immunized with vectors containing the insert encoding P10 (pP10) or IL-12 (pIL-12). Control mice were inoculated with PBS or with vector without insert. Each bar represents the average counts and standard deviations of CFU in lungs from 5 to 10 animals in each group. Experiments were carried out in triplicate, with similar results. ** p≤0.001, comparing vector with and without insert; ^## p≤0.001, comparing untreated and other groups.

Figure 5. Histopathology of lungs from intratracheally infected B10.A mice.

Animals were infected with P. brasiliensis for one month, treated with or without vectors carrying P10 or IL-12 DNA inserts according to protocol 3, and sacrificed 6 months after the initial infection. Infected mice treated with (A) control pcDNA3, (B) pP10, (C) pIL-12 DNA, and (D) P10 and IL-12 DNA. Gomori staining; original magnification, 40×.