Additive effect of rPb27 immunization and chemotherapy in experimental paracoccidioidomycosis

Abstract

Paracoccidioidomycosis, PCM, the major systemic mycosis in Latin America, is caused by the termally dimorphic fungus Paracoccidioides brasiliensis and requires extended periods of chemotherapy with a significant frequency of relapsing disease. The search for new alternatives of treatment is necessary. rPb27 is an antigenic protein from P. brasiliensis that already showed a significant protective activity as a vaccine for PCM in experimental models. The cDNA of rPb27 was subcloned into a pET-DEST 42 plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. Immunization with this recombinant protein and chemotherapy were used together in an attempt to improve treatment of PCM. For this, BALB/c mice were challenged with pathogenic P. brasiliensis strain and after immunized with rPb27, in the presence of Corynebacterium parvum and Al(OH)(3), some groups were also treated with fluconazole. After 40 days of treatment, the combined drug/rPb27 administration controlled PCM in the liver and spleen, with long lasting protection, and largely preserved tissues structures of these organs. Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions. At the same time, the levels of TNF-α were higher than infected-only mice. Moreover, significant levels of anti-rPb27 specific IgG1, IgG2a and IgG2b isotypes were detected in the sera of mice immunized with rPb27 fluconazole treated or not. These results showed an additive protective effect of rPb27 immunization and chemotherapy, suggesting that an rPb27-based vaccine can be used to enhance PCM antifungal treatment.

Figure 1. Purified rPb27 profile.

A. SDS-PAGE analysis of rPb27 after purification on HiTrap™ Chelating HP (Amersham Biosciences, Uppsala Sweden). Aliquot of 20 µl of purified rPb27 was separated on 10% polyacrylamide gels, under reducing conditions, followed by Comassie-blue staining. B. Western blotting analysis of purified rPb27 using mouse IgG anti-his-tag (Amersham Biosciences, Uppsala Sweden). Purified rPb27 was subjected to 10% SDS-PAGE, under reducing conditions, followed by eletrophoretic transfer to nitrocellulose paper. The membrane was incubated with mouse IgG anti-his-tag (1∶100) and revealed with goat anti-mouse IgG conjugated with peroxidase (1: 10000). 1, molecular marker. 2, purified rPb27. The molecular weight (kDa) of molecular marker proteins is showed on the left.

Figure 2. Fungal recovery in lung, spleen and liver of infected mice.

The CFUs were estimated 40 (A) and 90 days (B) post treatment in organs from mice infected intratracheally with 3×10⁵ P. brasiliensis yeast cells and subjected to fluconazole treatment combined or not with rPb27 immunization. Control mice were only infected with P. brasiliensis (Infected), adjuvant mice were inoculated with C. parvum-Al(OH)₃ with fluconazole treatment (Adjuvant/T) or not (Adjuvant), and rPb27 mice were immunized with recombinant protein combined to fluconazole treatment (rPb27/T) or not (rPb27). All groups of mice were infected with the same number of yeast cells. Bars represent the Log₁₀(UFC/g) means and standard deviations from organs of 3 to 5 animals in each group. * significant (p<0,05) difference in relation to the group of mice only infected.

Figure 3. Representative histopathology of lungs, livers and spleens from infected mice, after 40 days of treatment.

BALB/c mice were euthanized 40 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with P. brasiliensis. Infected/T, same as Infected, but treated with fluconazole. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. In each lung photos, the scale bar represents 427.3 µm, while in each liver and spleen photos, the scale bar represents 56.9 µm.

Figure 4. Representative histopathology of lungs, livers and spleens from infected mice, after 90 days of treatment.

BALB/c mice were euthanized 90 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with P. brasiliensis. Infected/T, same as Infected, but treated with fluconazole. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. In each lung photos, the scale bar represents 416.6 µm, while in each liver and spleen photos, the scale bar represents 55.6 µm.

Figure 5. Representative granulome histopathology of lungs from infected mice after 40 and 90 days of infection.

BALB/c mice were euthanized 40 and 90 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with P. brasiliensis. Infected/T, same as Infected, but treated with fluconazole. rPb27/T, group infected and posteriorly immunized with rPb27 and treated with fluconazole. In each photo, the scale bar represents 25.9 µm.

Figure 6. IgG isotypes production against rPb27 by infected and immunized mice.

Antibody response against rPb27 in mice infected and immunized with this recombinant protein associated or not with fluconazole chemotherapy was determined by ELISA assay after 40 (A) and 90 (B) days of treatment. Control, mice without any intervention. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. Bars represent the means and standard deviations of optical density (O.D.) at 1∶400 serum dilution in each experimental group (n = 3). * significant (p<0,05) difference in relation to the control group. # significant (p<0,05) difference in relation to the rPb27 group.