Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3’ UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 × 10⁶ L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4⁺, TCD8⁺ and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-6. In parallel groups of mice, a challenge consisting on 1 × 10⁶ virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis.

婴儿利什曼原虫感染可能会引起内脏利什曼病（VL），这是一种致命性疾病，分布在世界各地，可能是无症状的或无症状的。后者表明免疫力在某些个体中自然产生，因此，针对VL的疫苗是可能的。基因表达的分子机制在利什曼原虫中被理解，这些知识可能对疫苗开发有用。这项研究的目的是通过以阶段特异性方式调节有毒蛋白质的表达来开发减毒菌株。为了这个目的，选择了amastin基因的3'UTR，以其在a蛇毒阶段中的表达增加而闻名，用于指导外源蛋白的表达。该构建体（PFL-AMA），首先，被证明有效的mCherry的具体在的细胞内形式的表达婴儿利什曼，如通过荧光显微镜证实，流式细胞术和Western印迹。然后，在pFL-AMA质粒中，mCherry编码序列被卵亲和素或活性形式的牛胰蛋白酶所取代。在鞭毛前体的无菌培养和体外评估了寄生虫的存活率。巨噬细胞的体外感染。两条转染的寄生虫都显示出在巨噬细胞内繁殖的能力有限。将BALB / c小鼠腹膜内（ip）接种单剂量，该剂量由转染了带有毒性基因的质粒的2×10 ⁶ L. babyum前鞭毛体组成。接种后10周后，通过在肝脏或脾脏中进行有限的稀释，没有发现任何寄生虫，但是检测到了特异性的免疫反应。用转染的寄生虫免疫可诱导细胞和体液免疫反应，并激活TCD4 ⁺，TCD8 ⁺B细胞，具有TH1型应答，并具有促炎性细胞因子（如IFN-γ，TNF-α和IL-6）水平升高。在平行组的小鼠中，进行了皮下注射（l ）婴儿乳杆菌（腹膜内接种）或亚马逊乳杆菌的1×10 ^6个毒性寄生虫组成的攻击。接种了婴儿乳杆菌的小鼠，其肝，脾和骨髓中的寄生虫负担比携带野生乳杆菌的小鼠（未接种疫苗）要低。同样，接种疫苗的小鼠脚掌发炎比对照组小。这些数据支持该策略作为旨在开发针对不同形式利什曼病的疫苗的有效免疫系统。