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Additive Effect of rPb27 Immunization and Chemotherapy in Experimental Paracoccidioidomycosis

Fernandes VC, Martins EM, Boeloni JN, Coitinho JB, Serakides R, Goes AM - PLoS ONE (2011)

Bottom Line: Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions.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.

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. cfernandes.viviane@gmail.com

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.

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.
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pone-0017885-g002: 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.

Mentions: To explore the combined effect of rPb27 immunization and fluconazole treatment in BALB/c mice, animals immunization and chemotherapy started 30 days after infection. Analysis of organ CFU was done after 40 and 90 days of treatment. A significant reduction of fungi recovered from lung, spleen and liver of animals (CFU) was obtained in mice immunized with rPb27 and treated with fluconazole at the first time point. In the lung of these animals it was determined, 40 days post treatment, a 60% reduction in the CFUs in relation to infected-only group. Besides, in the liver and spleen of these animals there wasn't recovered any fungi colonie at the two time points analyzed. The rPb27 immunization alone failed to reduce fungi recovered from these organs. And the treatment with the antifungal drug alone also failed to reduce the number of CFU in the lung and liver at two time points, and in the spleen after 90 days of treatment despite a reduction after 40 days (Fig. 2 A, B).

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Additive Effect of rPb27 Immunization and Chemotherapy in Experimental Paracoccidioidomycosis

Fernandes VC, Martins EM, Boeloni JN, Coitinho JB, Serakides R, Goes AM - PLoS ONE (2011)

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.
© Copyright Policy
pone-0017885-g002: 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.
Mentions: To explore the combined effect of rPb27 immunization and fluconazole treatment in BALB/c mice, animals immunization and chemotherapy started 30 days after infection. Analysis of organ CFU was done after 40 and 90 days of treatment. A significant reduction of fungi recovered from lung, spleen and liver of animals (CFU) was obtained in mice immunized with rPb27 and treated with fluconazole at the first time point. In the lung of these animals it was determined, 40 days post treatment, a 60% reduction in the CFUs in relation to infected-only group. Besides, in the liver and spleen of these animals there wasn't recovered any fungi colonie at the two time points analyzed. The rPb27 immunization alone failed to reduce fungi recovered from these organs. And the treatment with the antifungal drug alone also failed to reduce the number of CFU in the lung and liver at two time points, and in the spleen after 90 days of treatment despite a reduction after 40 days (Fig. 2 A, B).

Bottom Line: Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions.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.

Affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. cfernandes.viviane@gmail.com

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.

View Similar Images In: Results  - Collection
View Article: PubMed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=3053394&rFormat=json&query=null&req=5