Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
PLoS Negl Trop Dis. 2013;7(3):e2148. doi: 10.1371/journal.pntd.0002148. Epub 2013 Mar 28.
The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL.
METHODOLOGY/PRINCIPAL FINDINGS: The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed.
CONCLUSIONS/SIGNIFICANCE: The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.
本研究旨在评估一种假设的利什曼原虫无鞭毛体特异性蛋白（LiHyp1），该蛋白先前通过在利什曼原虫婴儿期进行的免疫蛋白质组学方法鉴定，其与超氧化物酶基因家族具有同源性，试图选择一种新的候选抗原用于特异性血清诊断，并组成一种针对内脏利什曼病（VL）的疫苗。
方法/主要发现：克隆了 LiHyp1 DNA 序列；纯化了重组蛋白（rLiHyp1），并评估其抗原性和免疫原性。rLiHyp1 蛋白被无症状和有症状的犬内脏利什曼病（CVL）动物血清中的抗体识别，但与用巴西商业疫苗 Leish-Tec 接种的狗、恰加斯病或健康动物的血清无交叉反应。此外，还评估了 rLiHyp1 加皂苷在皮下接种强毒利什曼原虫前鞭毛体的 BALB/c 小鼠中的免疫原性和保护效力。rLiHyp1 加皂苷接种的小鼠在体外刺激重组蛋白后，表现出高水平和特异性的 IFN-γ、IL-12 和 GM-CSF 产生。与对照组（生理盐水和皂苷）相比，免疫和感染的小鼠在肝脏、脾脏、骨髓和爪子引流淋巴结中发现的寄生虫数量显著减少。保护与依赖于 IL-12 的 IFN-γ产生有关，主要由 CD4 T 细胞产生。在这些小鼠中，还可以观察到寄生虫介导的 IL-4 和 IL-10 反应的减少。
结论/意义：本研究表明，这种利什曼虫氧化酶无鞭毛体特异性蛋白可用于更敏感和特异性的无症状和有症状 CVL 的血清诊断，并且当与 Th1 型佐剂结合使用时，也可作为候选抗原用于开发针对 VL 的疫苗。
