Characterization of T and B Cell Epitopes for a Fasciola/Schistosoma Vaccine

到目前为止的证据表明，在慢性感染肝片吸虫的动物中，Th2反应占主导地位，在慢性感染的牛[1]、羊[2，3]和大鼠[4]中，IgG1是主要的抗体亚型。相比之下，在对感染产生抵抗力的动物中，观察到了高水平的IgG2和干扰素-β[5]。这表明，虽然Th2反应与感染的易感性有关，但Th1反应可能与耐药性有关。寄生虫抗原在感染过程中参与诱导Th1反应，对寄生虫疫苗的研制至关重要。我们最近报道了片状吸虫皂苷样蛋白/NK-裂解蛋白家族的一个新成员，命名为FhSAP-2，它在小鼠和兔身上具有高度的免疫原性。它是一种肝片吸虫/血吸虫交叉反应抗原，在肝片吸虫感染的早期阶段表达。此外，FhSAP-2对人红细胞和外周血单核细胞具有很强的裂解活性。一个详细的结构 对FhSAP-2的分析表明，它含有6个保守的半胱氨酸残基，分布在5个两亲性的α-螺旋结构域中，7个疏水残基位于严格保守的位置。此外，应用于FhSAP-2一级结构的计算机算法预测了几个MHC II类复杂结合区的存在以及可能形成T细胞表位的区域[16，17]。由于FhSAP-2是肝片吸虫/曼氏血吸虫交叉反应抗原，很明显，其中一些T细胞表位可能是肝片吸虫/曼氏血吸虫交叉反应表位。这项建议的第一个长期目标是鉴定FhSAP-2中仅限于Th1或Th2表型的表位，然后鉴定那些片状吸虫/血吸虫交叉反应表位。对于这个，一种多肽 从FhSAP-2的整个序列中合成的Libray将在最后10个氨基酸中重叠。用FhSAP-2对BALB/c(H-2d)和C57BL/6(H-2b)小鼠的脾和腹股沟淋巴结淋巴细胞进行细胞增殖试验。通过接种含有T细胞表位的多肽在动物体内产生的免疫反应的大小将根据抗体的同种类型和Th细胞因子谱进行研究。这项建议的第二个长期目标是评估作为实验模型的小鼠和兔的应答多肽池和两个线性MAP结构所提供的保护水平，并建立疫苗诱导的免疫反应与免疫后寄生虫负担之间的相关性。为此，小鼠和兔子将用反应物多肽池或 肝片吸虫挑战感染前的地图。将通过减少寄生虫负担、减少肝脏损害和/或减少寄生虫卵排泄来衡量保护措施。这些结果将有助于识别FhSAP-2表位，这些表位可能构成针对肝片吸虫病或血吸虫病的多组分疫苗的一部分。

Evidence to date suggests that in animals chronically infected with F. hepatica a Th2 response is dominant with IgG1 being the dominant antibody isotype in chronically infected cattle [1], sheep [2, 3], and rats [4]. By contrast, in animals that develop resistance against infection, high levels of IgG2 and IFN-delta have been observed [5]. This indicates that while Th2 response is associated to susceptibility of infection, the Th1-response could be associated to resistance. The identification of parasite antigens which are involved in inducing the Th1 response during infection is crucial for the development of a vaccine against parasite. We have recently reported a novel member of the Fasciola saposin-like / NK-lysin protein family, termed FhSAP-2, which is highly immunogenic in mice and rabbits. It is a Fasciola / Schistosoma cross-reactive antigen that is expressed at an early stage of F. hepatica infection. In addition, FhSAP-2 exhibit a potent lytic activity on human erythrocytes and peripheral blood mononuclear cells. A detailed structural analysis of FhSAP-2 has shown that it contains 6 conserved cysteine residues arranged within 5 amphipathic alpha-helix domains and seven hydrophobic residues in strictly conserved positions. Additionally, computer algorithms applied to primary structure of FhSAP-2 have predicted the existence of several MHC class II complex-binding regions as well as of regions that might form T-cell epitope [ 16, 17]. Since FhSAP-2 is a F. hepatica / S. mansoni cross-reactive antigen it is obvious that some of these T-cell epitopes could be F. hepatica / S. mansoni cross-reactive epitopes. The first long term objective of this proposal is to identify and characterize epitopes from FhSAP-2 which are restricted to the Th1 or Th2 phenotype and then to identify those that are Fasciola / Schistosoma cross-reactive epitopes. For this, a peptide libray from the entire sequence of FhSAP-2 will be synthetized with overlapping in the last ten amino acids. Spleen and inguinal lymph node lymphocytes from BALB/c (H-2d) and C57BL/6 (H-2b) mice primed with FhSAP-2 and boosted with pools of peptides will be estimulated with single peptides in cell-proliferation assays. The magnitude of the immune response generated in the animals by vaccination with peptides containing T-cell epitopes will be studied in terms of isotype of antibodies and Th cytokine profile. The second long term objective of this proposal is to asses the level of protection conferred by pools of responders peptides and two lineal MAP constructs in mouse and rabbit as experimental model and to establish the correlation between vaccine-induced immune responses and post-challenge parasite burden in the animals vaccinated. For this, mice and rabbits will be prime with pools of responders peptides or MAPs before a Fasciola hepatica challenge infection. Protection will be measured by reduction of parasite burden, reduction of liver damage, and/or reduction of parasite egg excretion. The results will be useful to identify FhSAP-2 epitopes that could form part of a multi-component vaccine against fascioliasis or schistosomiasis.