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

迄今为止的证据表明，在慢性感染F。在慢性感染的牛[1]、绵羊[2，3]和大鼠[4]中，Th 2应答占主导地位，IgG 1是主要的抗体同种型。相比之下，在对感染产生抗性的动物中，已经观察到高水平的IgG 2和IFN-δ [5]。这表明虽然Th 2应答与感染易感性相关，但Th 1应答可能与抗性相关。寄生虫抗原的鉴定是在感染过程中诱导Th 1应答的关键，用于开发针对寄生虫的疫苗。我们最近报道了片形吸虫saposin-like /NK-lysin蛋白家族的新成员，称为FhSAP-2，其在小鼠和兔中具有高度免疫原性。它是一种片形吸虫/血吸虫交叉反应性抗原，在F.肝脏感染此外，FhSAP-2对人红细胞和外周血单核细胞表现出有效的裂解活性。详细的结构 对FhSAP-2的分析表明，它含有排列在5个两亲性α-螺旋结构域内的6个保守的半胱氨酸残基和在严格保守位置的7个疏水残基。此外，应用于FhSAP-2的一级结构的计算机算法已经预测了几个MHC II类复合物结合区域以及可能形成T细胞表位的区域的存在[ 16，17]。由于FhSAP-2是F. hepatica / S. mansoni交叉反应性抗原，显然这些T细胞表位中的一些可能是F. hepatica / S. mansoni交叉反应表位。该提议的第一个长期目标是鉴定和表征来自FhSAP-2的限于Th 1或Th 2表型的表位，然后鉴定那些是片形吸虫/血吸虫交叉反应性表位。为此， 来自FhSAP-2的整个序列的文库将在最后10个氨基酸中重叠合成。在细胞增殖试验中，用单一肽估计用FhSAP-2致敏并用肽库加强的BALB/c（H-2d）和C57 BL/6（H-2b）小鼠的脾和腹股沟淋巴结淋巴细胞。将根据抗体同种型和Th细胞因子谱研究通过接种含有T细胞表位的肽在动物中产生的免疫应答的幅度。本提案的第二个长期目标是评估作为实验模型的小鼠和兔中由应答肽池和两种线性MAP构建体赋予的保护水平，并建立疫苗诱导的免疫应答与接种动物中攻击后寄生虫负荷之间的相关性。为此，小鼠和兔将用应答肽或多肽的池进行初免。 肝片吸虫攻击感染前的MAP。通过减少寄生虫负荷、减少肝损伤和/或减少寄生虫卵排泄来测量保护作用。这些结果将有助于鉴定FhSAP-2表位，这些表位可以形成针对片形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.