Towards a vaccine against Fasciola hepatica using FhSAP2 as an antigen

描述（由申请人提供）：寄生虫肝片吸虫病引起肝吸虫病或片吸虫病，从而影响人类、绵羊、牛和山羊以及其他哺乳动物的健康。片形吸虫病每年在农业上造成的损失估计为100亿至32亿美元。片吸虫病也是一种主要的人畜共患疾病，据估计全世界有1700万人受到感染。杀氟剂三氯咪唑是控制片形吸虫病最有效的药物；然而，治疗费用和感染肝f.s ca的绵羊出现耐药性表明，需要制定可持续的战略，例如为控制这种疾病而接种疫苗。然而，尽管进行了长期的研究，一种针对肝片形吸虫的疫苗尚未开发出来。我们已经鉴定出一种新的被称为FhSAP2的肝炎F. c .抗原，当在弗氏完全佐剂（FCA）中皮下递送（SC）时，能够诱导部分免疫（减少60%的寄生虫负担和显著减少肝损伤）。FhSAP2是一种11.5kDa的多肽，属于肝猪肝皂苷样蛋白/ nk -溶素蛋白家族。我们还证明，FhSAP2-FCA制剂诱导的免疫与高水平的IgG2a抗体和高水平的IFN3相关，这是Th1免疫应答的标志。这些结果使我们假设FhSAP2诱导的保护作用是通过与CD4+ Th1细胞相关的机制介导的，这在片形吸虫病领域是一个新的概念，因为肝梭菌和大多数蠕虫一样，传统上与Th2免疫应答有关。本研究的目的是研究这些制剂在片形吸虫病小鼠模型中对T-辅助性1、T-辅助性2反应的调节及其对FhSAP2诱导的肝f的保护作用。为实现这一目标，我们建议在三年资助期内实现两个具体目标。在特异性的aim-1中，我们将研究FhSAP2在含有免疫刺激序列的佐剂中递送SC时的保护作用。具体来说，我们将确定携带FhSAP2的幼年C57BL/6 （H-2b）小鼠在MontanideTM ISM 1312和ISA 70M中或与IL-12或CpG-ODN共同给药的ISCOMs中是否可以模拟或增强FhSAP2在CFA中获得的保护水平。这将为今后的研究优化疫苗配方。在具体的aim-2中，我们将研究aim-1中使用的疫苗制剂如何调节T辅助性1(Th1)-T辅助性2 （Th2）反应。我们将观察Th1/Th2细胞因子谱、抗体亚类水平以及B细胞和t细胞、巨噬细胞（MX）和树突状细胞（dc）的激活状态，这些参数将与特异性aim-1获得的保护水平相关。该结果将使我们能够验证我们关于Th1反应在片形吸虫病中的保护作用的假设。如果上述目标得以实现，我们不仅将获得一种可用于反刍动物试验的优化疫苗配方，而且我们还将提出如何诱导对肝F.虫的保护的新概念，这将为进一步研究阐明使Th1免疫反应成为可能的免疫机制打开大门，从而有效地对抗肝F.虫等多细胞吸虫。此外，这些研究可能为将这一概念应用于人类肝吸虫病原体引起的其他被忽视疾病的研究奠定基础。PI与SC1应用程序的主要目标是过渡到非score支持机制，并以此建立自己作为一个独立的有竞争力的调查员。

DESCRIPTION (provided by applicant): The helminth parasite Fasciola hepatica causes liver fluke disease or fascioliasis, thereby affecting the health of humans, as well as sheep, cattle and goats, among others mammalians. Fascioliasis causes losses in agriculture estimated at >US3.2billion per year. Fascioliasis is also a major zoonotic disease and it is estimated that 17 million people worldwide are infected. The flukicide triclabendazole is the most effective drug to control fascioliasis; however, the cost of treatment and the emergence of drug resistance in sheep infected with F. hepatica suggest a need to develop sustainable strategies, such as vaccination for the control of this disease. However, despite the long-standing research, a vaccine against Fasciola hepatica has not yet been developed. We have identified a novel F. hepatica antigen termed FhSAP2 that when delivered subcutaneously (SC) in Freund's complete adjuvant (FCA) is able to induce partial immunity (>60% reduction in parasite burden and significant reduction of liver damage) in rabbits and mice challenged with F. hepatica metacercariae. FhSAP2 is an 11.5kDa polypeptide belonging to the F. hepatica saposin-like / NK-lysin protein family. We also demonstrated that the immunity induced by the FhSAP2-FCA formulation is associated with high levels of IgG2a antibodies and high levels of IFN3, which are signatures of a Th1 immune response. These results led us to hypothesize that the protection induced by FhSAP2 is mediated by a mechanism linked to CD4+ Th1 cells, which is a new concept in the field of fascioliasis since F. hepatica, like most helminthes, is traditionally associated to Th2 immune responses. The goal of the present study is to study the modulation of T-helper 1, T- helper 2 responses and the protective effect against F. hepatica induced by FhSAP2 with these formulations in the mouse model of fascioliasis. To address this goal we propose to fulfill two specific aims in the 3-year funding period. In specific aim-1 we will study the protective effect of FhSAP2 when delivered SC in adjuvants containing immunostimulatory sequences. Specifically, we will ascertain whether the prime-boost strategy of naove C57BL/6 (H-2b) mice with FhSAP2 in MontanideTM ISM 1312 and ISA 70M or trapped into ISCOMs co- administered with IL-12 or CpG-ODN could mimic or enhance the protection levels obtained with FhSAP2 in CFA. This will lead to an optimization of the vaccine formulation for future studies. In the specific aim-2 we will study how the vaccine formulations used in the aim-1 modulate the T helper 1(Th1)-T helper 2 (Th2) responses. We will look at the Th1/Th2 cytokine profile, the levels of antibody subclasses as well as the activation status of B- and T-cells, macrophages (MX) and dendritic cells (DCs) elicited in naove mice and these parameters will be correlated with the levels of protection obtained in specific aim-1. The outcome will allow us to test our hypothesis of the protective role of Th1 responses in fascioliasis. If the proposed aims are achieved, we not only would have in hand an optimized vaccine formulation to be assayed in ruminants but we will also put forward a new concept of how to induce protection against F. hepatica, which will open the door to further studies to elucidate the immune mechanisms that make possible the Th1 immune responses which are effective against a multicellular fluke like F. hepatica. Moreover, these studies might serve as the basis for the application of this concept in the study of other neglected diseases caused by human liver fluke pathogens. The main objective of PI with this SC1 application is to make a transition to a non-SCORE support mechanism and in doing so establish herself as an independent competitive investigator. PUBLIC HEALTH RELEVANCE: Fasioliasis is a major disease of ruminants and more recently humans. We have demonstrated that an FhSAP2 antigen induces significant protection in a mouse and rabbit model of fascioliasis. We propose to optimize the vaccine formulation and improve the efficacy of the vaccine. In doing so in the future we will be able to evaluate the optimized vaccine in ruminants.