Avalia??o da dose-resposta e da associa??o de adjuvantes na pot?ncia de vacinas quim?ricas contra leishmaniose visceral : estudo de fase I em camundongos BALB/c.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.No Brasil, a leishmaniose visceral canina (LVC) encontra-se em expans?o sendo um s?rio problema de sa?de p?blica. A alta preval?ncia de c?es infectados refor?a a necessidade de uma vacina para ser empregada em campanhas de vacina??o de forma profil?tica. Nesse trabalho foram avaliadas duas quimeras desenhadas a partir do mapeamento de ep?topos de c?lulas T pertencentes a prote?nas de Leishmania infantum descritas na literatura como candidatas vacinais. O objetivo do estudo foi avaliar a dose-resposta e o efeito da associa??o de adjuvantes na efic?cia de vacinas quim?ricas contra a leishmaniose visceral em camundongos BALB/c. Os animais foram divididos em 14 grupos Salina, Saponina e Monofosforil lip?dio A (SM) e quimeras A e B, nas doses de 5, 10 e 20 ?g, associados ou n?o aos adjuvantes. Esses foram imunizados com 3 doses, com intervalo de 15 dias entre as doses, e desafiados com 1x107 promastigotas de L. infantum. Foi avaliada a atividade proliferativa de linf?citos T totais (CD3+ ) e suas subpopula??es (CD3+CD4+ e CD3+CD8+ ) e a produ??o de citocinas intracitoplasm?ticas (IL-2, IFN-?, TNF-?, IL-4 e IL-10) ap?s est?mulo com ant?geno sol?vel de L. infantum (ASLi) por citometria de fluxo. Al?m disso, foi determinada a produ??o de ?xido n?trico (NO) em sobrenadante de cultura de esplen?citos estimulados com ASLi e produ??o de imunoglobulinas murinas anti-Leishmania (IgG total, IgG1 e IgG2) no soro dos camundongos por ELISA. A quantifica??o da carga parasit?ria foi feita no ba?o por PCR em tempo real (qPCR). Ambas quimeras nas tr?s doses avaliadas, apresentaram aumento da prolifera??o de linf?citos T e suas subpopula??es quando comparadas ao grupo Salina. Quando associadas aos adjuvantes, o mesmo comportamento foi observado sendo o aumento em compara??o aos grupos Salina e SM. Observou-se aumento de c?lulas T CD4+ e CD8+ produtoras de IFN-?, TNF-? e IL-2, enquanto houve redu??o de IL-4 e IL-10, tanto em A quanto em B nas tr?s doses. Cabe ressaltar, que em B na dose de 5 ?g associada a SM houve aumento na produ??o de IFN-? e IL-2 e redu??o de IL-4 e IL-10 pelos linf?citos TCD4+ em compara??o ao grupo Salina. Os resultados da reatividade anti-Leishmania, mostraram que as prote?nas quim?ricas A e B aumentaram a produ??o de IgG total e IgG2 nas tr?s doses avaliadas. Quando associadas a SM, apenas a prote?na quim?rica A, mostrou-se reativa. Em rela??o ao subclasse IgG1, houve aumento nas doses de 5 ?g e 10 ?g. E para IgG2 houve diferen?a significativa das doses de 5 e 10 ?g com a dose de 20 ?g. Quando quantificamos a carga parasit?ria foi observado que as prote?nas quim?ricas sozinhas n?o foram capazes de reduzir a carga no ba?o dos animais. Em contrapartida, quando foram associadas ao sistema SM, houve redu??o da carga parasit?ria no ba?o em todas as doses avaliadas para ambas quimeras. Observou-se aumento na produ??o de NO em todos grupos que foram imunizados com as quimeras associadas ao sistema SM. Os resultados obtidos foram promissores, demonstrando que mesmo em doses reduzidas as quimeras foram capazes de desencadear uma resposta imunol?gica satisfat?ria. Os resultados mostram que as duas quimeras sozinhas e ou associadas ao sistema de adjuvantes foram imunog?nicas mesmo em doses reduzidas. Al?m disso, esse estudo contribuir? para o desenvolvimento de novas vacinas contra a LV.In Brazil, canine visceral leishmaniasis (CVL) is expanding and is a serious public health problem. The high prevalence of infected dogs reinforces the need for a vaccine for use in prophylactic vaccination campaigns. In this study, two chimeras designed from the mapping of T cell epitopes belonging to Leishmania infantum proteins, described in the literature as vaccine candidates were evaluated. The aim of the study was to evaluate the immunogenicity and vaccination of two chimeric proteins at different doses against visceral leishmaniasis in BALB/c mice. The animals were divided into 14 groups Saline, Saponin and Monophosphoryl lipid A (SM) and chimeras A and B, at doses of 5, 10 and 20 ?g, associated or not with adjuvants. These were immunized with 3 doses, 15 days apart, and challenged with 1x107 L. infantum promastigotes. The proliferative activity of total T lymphocytes (CD3+ ) and their subpopulations (CD3+CD4+ and CD3+CD8+ ) and the production of intracytoplasmic cytokines (IL-2, IFN-?, TNF-?, IL-4 and IL-10) after stimulation with soluble L. infantum antigen (ASLi) were assessed by flow cytometry. Besides, the production of supernatant nitric oxide (NO) from ASLi-stimulated splenocyte culture and production of anti-Leishmania murine immunoglobulins (total IgG, IgG1 and IgG2) in serum was determined by ELISA. The quantification of parasite load was performed by real time PCR (qPCR). Both chimeras at the three doses evaluated increased lymphocyte proliferation and its subpopulations when compared to the Saline group. When associated with adjuvants, the same behavior was observed as the increase compared to groups Salina and SM. Increased CD4+ and CD8+ T cells IFN-?, TNF-? and IL-2 products were observed over the three doses, while there was a reduction in IL-4 and IL-10. It is noteworthy that in B at a dose of 5 ?g associated with SM there was an increase in IFN-? and IL-2 production and a reduction of IL4 and IL-10 by T CD4+ lymphocytes compared to the Saline group. Anti-Leishmania reactivity results, which show chimeric proteins A and B increased total IgG and IgG2 production at the three available doses. When associated with SM, only chimeric protein A was reactive. Regarding the IgG1 subclass, there was an increase in doses of 5 ?g and 10 ?g. And for IgG2 there was a significant difference in the 5 ?g and 10 ?g doses with a 20 ?g dose. When we quantified the parasite load it was observed that the chimeric proteins alone were not able to reduce the load in the animals. In contrast, when they were associated with the SM system, there was a reduction in parasite load in the spleen at all doses available for chimeras. An increase in NO production was observed in all groups that were immunized with chimeras associated with the SM system. The data obtained were promising, demonstrating that even in reduced doses the chimeras were able to trigger a satisfactory immune response. Results show that two chimeras alone and or associated with the adjuvant system were immunogenic even at reduced doses. Also, this study contributed to the development of new vaccines against a VL