Martins Vivian Tamietti, Chávez-Fumagalli Miguel Angel, Lage Daniela Pagliara, Duarte Mariana Costa, Garde Esther, Costa Lourena Emanuele, da Silva Viviane Grazielle, Oliveira Jamil Silvano, Magalhães-Soares Danielle Ferreira de, Teixeira Santuza Maria Ribeiro, Fernandes Ana Paula, Soto Manuel, Tavares Carlos Alberto Pereira, Coelho Eduardo Antonio Ferraz
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Programa de Pós-Graduação em Ciências da Saúde: Infectologia e Medicina Tropical, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
PLoS One. 2015 Sep 14;10(9):e0137683. doi: 10.1371/journal.pone.0137683. eCollection 2015.
In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.
在本研究中，将无鞭毛体阶段存在的两种婴儿利什曼原虫假定蛋白LiHyp1和LiHyp6与前鞭毛体蛋白IgE依赖性组胺释放因子（HRF）结合，组成一种多蛋白疫苗，用于评估其对婴儿利什曼原虫感染的效果。此外，分析了这三种蛋白的抗原性，并评估了它们在犬内脏利什曼病（CVL）血清诊断中的应用。将LiHyp1、LiHyp6和HRF的DNA编码序列克隆到原核表达载体中，并对重组蛋白进行纯化。在ELISA检测中，所有蛋白均能被内脏利什曼病（VL）犬的血清识别，且与接种巴西商业疫苗的犬血清、克氏锥虫感染或犬埃立克体感染动物的血清均无交叉反应。此外，来自利什曼病流行或非流行地区的未感染动物的抗体不能识别这些抗原。在VL小鼠模型（感染婴儿利什曼原虫的BALB/c小鼠）中评估了单独或联合（组成多蛋白疫苗）使用皂苷时这三种蛋白的免疫原性和保护效果。接种单个蛋白或多蛋白疫苗加皂苷的小鼠的脾细胞在体外刺激后显示出蛋白特异性的γ干扰素、白细胞介素-12和粒细胞-巨噬细胞集落刺激因子的产生，感染后仍保持这种状态。与接种生理盐水或皂苷的小鼠相比，这些动物在不同评估器官中的寄生虫负荷显著降低。寄生虫负荷的降低与针对寄生虫总提取物的γ干扰素的白细胞介素-12依赖性产生有关（主要由CD4+T细胞产生），这与寄生虫蛋白驱动的一氧化氮产生的诱导相关。接种基于重组蛋白疫苗的小鼠也显示出高水平的寄生虫特异性IgG2a抗体。与单个疫苗相比，多蛋白疫苗接种在攻击感染前后诱导了更明显的Th1反应，这与对寄生虫向内部器官传播的更高控制相关。
