Vaccine development against Theileria parasites using liposome as adjuvant

细胞介导的免疫（CMI）被认为是一种有效的控制感染的细胞内病原体，如原虫。为研制能诱导CMI的多肽疫苗，构建了牛白血病病毒囊膜糖蛋白gp 51的20肽甘露聚糖包被脂质体。脂质体诱导小鼠特异性迟发型超敏反应、淋巴细胞增殖反应和细胞毒性淋巴细胞反应，免疫小鼠脾细胞经肽刺激后产生大量IFN-γ和IL-2，但不产生IL-4和IL-10。结果表明，合成肽脂质体对小鼠Th-1型免疫有增强作用。由瑟氏锥虫引起的牛梨形虫病是日本放牧牛经济损失的主要原因。日本的寄生虫种群和分离株由遗传混合种群组成。疟原虫主要表面蛋白p32的氨基酸序列中含有Lys-Glu-Lys（KEK）基序，是疟原虫侵入红细胞所必需的三肽基序之一。我们合成了含有KEK序列的合成肽，并将其包裹在甘露聚糖包被的脂质体中作为佐剂，因为肽可以诱导CMI。含有KEK基序的合成肽与脂质体的免疫导致低寄生虫血症，并减少临床症状相比，对照未接种疫苗的小牛。有趣的是，具有对应于用作免疫原的p32等位基因形式的寄生虫被抑制。因此，在日本，需要含有源自C型和I型寄生虫的KEK肽的鸡尾酒疫苗来控制泰勒虫寄生虫感染。

Cell-mediated immunity (CMI) is considered to be an effective in controlling infection with intracellular pathogenes, such as protozoa. To develope apeptide-based vaccine capable inducing CMI,mannan-coated liposome encapsulating 20-mer synthetic peptide of bovine leukemia virus envelope glycoprotein, gp51 was constructed. The liposome induced specific delayd-type hypersensitivity, lymphocyte proliferative responses and a cytotoxic lymphocyte response in mice.By stimnlation with the peptide, the spleen cells from the immunized mice produced a large amount of IFN-gamma and IL2, whereas they released neither IL4 or IL10. These results indicate the augmentation of Th-1 type immunity in mice by the synthetic peptide-liposome. Bovine piroplasmosis caused by T.sergenti is a major cause of economical loss in grazing cattle in Japan. Parasite stocks and isolates in Japan consist of genetically mixed population. Amino acid sequence of major piroplasm surface protein, p32 contains Lys-Glu-Lys (KEK) motif which is one of tripeptide motif necessary for Malaria parasite to invade erythrocytes. We produced synthetic peptides containing KEK sequences and encapsulated in mannan-coated liposome as adjuvant bacause of the induction of CMI by peptide. Immunization of synthetic peptides containing KEK motifs with liposome resulted in low parasitemia and reduced the clinical symptoms compared to that of control non-vaccinated calves. Interestingly, parasites with a p32 allelic form corresponding to one used as immunogen were suppressed.Therefore, a cocktail vaccine containing KEK peptides derived from C and I type parasites is desired for control Theileria parasite infection in Japan.