Towards a vaccine against Fasciola hepatica using FhSAP2 as an antigen

描述(申请人提供)：肝片吸虫寄生虫引起肝吸虫病或肝片吸虫病，从而影响人类以及绵羊、牛和山羊等哺乳动物的健康。肝片吸虫病每年给农业造成约32亿美元的损失。肝片吸虫病也是一种主要的人畜共患疾病，据估计全世界有1700万人感染。杀虫药物三氯苯达唑是控制肝片吸虫病最有效的药物；然而，治疗费用和感染肝片吸虫的绵羊出现抗药性表明有必要制定可持续的策略，如接种疫苗以控制这种疾病。然而，尽管研究由来已久，但尚未开发出针对肝片吸虫的疫苗。我们鉴定了一种新的肝片吸虫抗原FhSAP2，当在弗氏完全佐剂(FCA)中皮下注射(SC)时，能够诱导兔和小鼠感染肝片吸虫囊虫病后的部分免疫(寄生虫负担减少60%，肝脏损伤显著减少)。FhSAP2是一种11.5 kDa的多肽，属于肝片吸虫皂苷样/NK-裂解蛋白家族。我们还证明了FhSAP2-FCA制剂诱导的免疫与高水平的IgG2a抗体和高水平的IFN3有关，这是Th1免疫反应的标志。这些结果使我们假设FhSAP2诱导的保护作用是通过与CD4+Th1细胞相连的机制介导的，这在肝片吸虫病领域是一个新的概念，因为肝片吸虫和大多数蠕虫一样，传统上与Th2免疫反应有关。本研究的目的是在肝片吸虫病小鼠模型上研究这些制剂对T辅助细胞1、T辅助细胞2应答的调节以及FhSAP2对肝片吸虫的保护作用。为了实现这一目标，我们建议在3年资助期内实现两个具体目标。在特定的目标-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。具体地说，我们将确定在MontanideTM ISM 1312和ISA 70M中使用FhSAP2的NAOVE C57BL/6(H-2b)小鼠的Prime-Boost策略，或者在联合使用IL-12或CpG-ODN的ISCOM中采用的Prime-Boost策略是否可以模拟或增强FhSAP2在CFA中获得的保护水平。这将导致疫苗配方的优化，用于未来的研究。在特定的AIM-2中，我们将研究AIM-1中使用的疫苗配方如何调节T辅助细胞1(Th1)-T辅助细胞2(Th2)反应。我们将观察在NAOVE小鼠中诱导的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，这些参数将与特定AIM-1中获得的保护水平相关。这一结果将使我们能够检验我们的假设，即Th1反应在肝片吸虫病中的保护作用。如果所提出的目标实现，我们不仅将有一个优化的疫苗配方在反刍动物身上进行测试，而且我们还将提出如何诱导对肝片吸虫的保护的新概念，这将为进一步研究阐明Th1免疫反应的免疫机制打开大门，使Th1免疫应答对像肝片吸虫这样的多细胞吸虫有效。此外，这些研究可能为将这一概念应用于由人类肝吸虫病原体引起的其他被忽视的疾病的研究奠定基础。PI使用此SC1应用程序的主要目标是过渡到非分数支持机制，并在这样做的过程中确立自己作为独立竞争调查员的地位。 公共卫生相关性：线虫病是反刍动物的一种主要疾病，最近也是人类的一种疾病。我们已经证明，FhSAP2抗原在小鼠和兔的肝片吸虫病模型中具有显著的保护作用。我们建议优化疫苗配方，提高疫苗效力。在未来这样做的过程中，我们将能够在反刍动物身上评估优化后的疫苗。

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.