Comparative efficacy and toxicity of two vaccine candidates against Sporothrix schenckii using either Montanide™ Pet Gel A or aluminum hydroxide adjuvants in mice.

Sporotrichosis is an important zoonosis in Brazil and the most frequent subcutaneous mycosis in Latin America, caused by different Sporothrix species. Currently, there is no effective vaccine available to prevent this disease. In this study, the efficacy and toxicity of the adjuvant Montanide™ Pet Gel A (PGA) formulated with S. schenckii cell wall proteins (ssCWP) was evaluated and compared with that of aluminum hydroxide (AH). Balb/c mice received two subcutaneous doses (1st and 14th days) of either the unadjuvanted or adjuvanted vaccine candidates. On the 21st day, anti-ssCWP antibody levels (ELISA), the phagocytic index, as well as the ex vivo release of IFN-γ, IL-4, and IL-17 by splenocytes and IL-12 by peritoneal macrophages were assessed. Cytotoxicity of the vaccine formulations was evaluated in vitro and by histopathological analysis of the inoculation site. Both adjuvanted vaccine formulations increased anti-ssCWP IgG, IgG1, IgG2a, and IgG3 levels, although IgG2a levels were higher in response to PGA+CWP100, probably contributing to the increase in S. schenckii yeast phagocytosis by macrophages in the opsonophagocytosis assay when using serum from PGA+CWP100-immunized mice. Immunization with AH+CWP100 led to a mixed Th1/Th2/Th17 ex vivo cytokine release profile, while PGA+CWP100 stimulated a preferential Th1/Th2 profile. Moreover, PGA+CWP100 was less cytotoxic in vitro, caused less local toxicity and led to a similar reduction in fungal load in the liver and spleen of S. schenckii- or S. brasiliensis-challenged mice as compared with AH+CWP100. These results suggest that PGA may be an effective and safe adjuvant for a future sporotrichosis vaccine.

Gamma radiation effects on Sporothrix schenckii yeast cells.

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii. Zoonotic transmission to man can occur after scratches or bites of animals, mainly cats. In this study, the gamma radiation effects on yeast of S. schenckii were analyzed with a view of developing a radioattenuated vaccine for veterinary use. The cultures were irradiated at doses ranging from 1.0 to 9.0 kGy. The reproductive capacity was measured by the ability of cells to form colonies. No colonies could be recovered above 8.0 kGy, using inocula up to 10(7) cells. Nevertheless, yeast cells irradiated with 7.0 kGy already were unable to produce infection in immunosuppressed mice. Evaluation by the FungaLight™ Kit (Invitrogen) indicated that yeast cells remained viable up to 9.0 kGy. At 7.0 kGy, protein synthesis, estimated by the incorporation of [L-(35)S] methionine, continues at levels slightly lower than the controls, but a significant decrease was observed at 9.0 kGy. The DNA of 7.0 kGy irradiated cells, analyzed by electrophoresis in agarose gel, was degraded. Cytoplasmic vacuolation was the main change verified in these cells by transmission electron microscopy. The dose of 7.0 kGy was considered satisfactory for yeast attenuation since irradiated cells were unable to produce infection but retained viability, metabolic activity, and morphology.

Attempts at a peptide vaccine against paracoccidioidomycosis, adjuvant to chemotherapy.

Chemotherapy is the basis of treatment of paracoccidioidomycosis in its various forms. Depending on the Paracoccidioides brasiliensis virulence, the status of host immunity, the degree of tissue involvement and fungal dissemination, treatment can be extended for long periods with an alarming frequency of relapses. Association of chemotherapy with a vaccine to boost the cellular immune response seemed a relevant project not only to reduce the time of treatment but also to prevent relapses and improve the prognosis of anergic cases. The candidate immunogen is the gp43 major diagnostic antigen of P. brasiliensis and more specifically its derived peptide P10, carrying the CD4+ T-cell epitope. Both gp43 and P10 protected Balb/c mice against intratracheal infections with virulent P. brasiliensis strain. P10 as single peptide or in a multiple-antigen-peptide (MAP) tetravalent construction was protective without adjuvant either by preimmunization and intratracheal challenge or as a therapeutic agent in mice with installed infection. P10 showed additive protective effects in drug-treated mice stimulating a Th-1 type immune response with high IFN-gamma and IL-12. P10 and few other peptides in the gp43 were selected by Tepitope algorithm and actually shown to promiscuously bind several prominent HLA-DR molecules suggesting that a peptide vaccine could be devised for a genetically heterogenous population. P10 was protective in animals turned anergic, was effective in a DNA minigene vaccine, and increased the protection by monoclonal antibodies in Balb/c mice. DNA vaccines and peptide vaccines are promising therapeutic tools to be explored in the control of systemic mycoses.