Characterization of response immune of human cells to proteins recombinant LACK and KMP-11 of leishmania amazonensis. / Caracterização da resposta imune de células humanas às proteínas recombinantes LACK e KMP-11 de leishmania amazonensis

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Vaccination represents a reasonable perspective for leishmaniasis control besides being a cost-effective approach to infectious disease control in general. This study was performed to evaluate the immune response of human cells to the Leishmania amazonensis proteins KMP-11 and LACK, both of them considered as candidate antigens for a leishmaniasis vaccine. We studied three groups of subjects: 35 patients with active cutaneous leishmaniasis, 35 individuals immunized with Leishvacin and 13 healthy individuals from non-endemic areas (naive). The following antigens were used for the in vitro stimulation of peripheral mononuclear blood cells (PBMC) from the study subjects: total extract of killed Leishmania amazonensis promastigotes (La) and the recombinant proteins KMP-11 and LACK of L. amazonensis. The production of the cytokines IFN-γ, IL-10, IL-5 e TNF-a by PBMC was evaluated by ELISA. ELISPOT was used to determine the frequencies of IFN-γ- and IL-4-producing cells. We also characterized the PBMC phenotypes in order to analyze the expression of the surface markers CD56, CD4, CD8, CD25, CD69 and CD45RO. The assessment of cytokine levels in PBMC cultures by ELISA in the patients group in response to La, showed higher IFN-g levels than those seen among the vaccinees and the naive individuals, and higher TNF-α levels in comparison to those from the vaccinees; the IFN-g levels exhibited a positive correlation with the Montenegro skin test diameters and a inverse correlation with the time of disease activity. There was a positive correlation between the levels of the IFN-g and IL-10 induced by La, and a positive correlation between IFN-γ levels stimulated by La and the total extract composed of killed promastigotes of L. braziliensis (Lb), with IFN-γ levels induced by Lb higher than those induced by La. In the vaccinated group, LACK induced a higher IFN-g production and lower production of IL-10 and TNF-α in comparison to the naive group. Correlations were found in this group between the results obtained with the three antigens with respect to the IFN-g, IL-10 and TNF-α production. When we compared the stimuli in the patients group, La induced the highest IFN-g levels and IL-10 levels lower than those induced by LACK. Among the vaccinees, LACK induced higher IFN-g levels than those obtained after La and KMP-11 stimulation. In the naive group, LACK induced higher IL-10 levels than those stimulated by La or by KMP-11, and higher TNF-α levels than those stimulated by La. ELISPOT with La-stimulated cells from the patients showed a higher frequency of IFN-γ-producing cells than that observed in the naive group, in agreement with the IFN-g-ELISA results. The frequencies of IFN-γ- and IL-4-producing cells in response to LACK among the patients and vaccinees were higher than those found for the naive individuals. The vaccinated group exhibited higher proportion of CD8+ memory cells and CD8+ cells expressing CD25 as compared to the naive group, in response to all antigens used. For La stimulation we found a higher proportion of CD8+ cells among the vaccinees as compared to the patients, a higher proportion of CD8+ cells expressing the activation markers CD25 and CD69 for the vaccinees in comparison to the patients and the naive individuals, and a higher proportion of CD8+ memory cells in the vaccinated group in comparison to the patients and the naive subjects. Also, in response to La, there was a higher proportion of CD4+ memory cells among the vaccinees than in the naive group. KMP-11 induced higher proportions of CD4+ and CD8+ memory cells, CD8+CD25+ and CD8+CD69+ cells in the vaccinated group as compared to the other groups (patients and naive). LACK induced higher proportions of CD4+ and CD8+ memory cells and CD8+CD25+ cells in the vaccinated group in comparison to those found among patients and naive individuals, as well as higher proportions of CD8+CD69+ cells than those from the naive group. The comparison among the three antigens revealed in the patients group a lower proportion of CD8+ cells under La stimulation than after KMP-11 and LACK stimulation. In the vaccinated group, La stimulated a higher proportion of CD8+ cells than did KMP-11. Taken together, these results indicate significant differences between the responses to Leishmania antigens (crude and defined) induced by natural infection versus immunization, which could be exploited for the identification of Leishmania molecules with immunoprotective potential for humans.

ASSUNTO(S)

leishmania braziliensis vacina leishmania/citologia doencas infecciosas e parasitarias vaccine leishmania braziliensis leishmania/cytology recombinant proteins proteínas recombinantes

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