Nanoformulation of Synergistic TLR Ligands to Enhance Amebiasis Vaccine Efficacy

描述(申请人提供)：含有纳米颗粒成分的常规疫苗佐剂，如水包油乳剂或铝盐，不能促进有效的Th1型细胞免疫反应，这对缺乏有效疫苗的复杂疾病，如疟疾、结核病和阿米巴病是至关重要的。后者是由原生动物寄生虫溶组织内阿米巴(一种生物防御B类病原体)引起的一种被忽视的肠道疾病，占重大疾病负担，全世界每年约有5000万人感染，其中发展中国家的负担最大。这项建议描述了含有合成的Toll样受体(TLR)配体组合的纳米脂质体的开发和物理化学特征，以及它们增强阿米巴病疫苗免疫原性和保护效力的能力的评估。减毒活疫苗的效力可能部分归因于它们触发多个病原体识别受体的能力，如TLRs。然而，对于现代重组或灭活疫苗，FDA批准的疫苗(即Cervarx(R))中使用的唯一基于TLR配体的佐剂只有一个TLR配体。然而，适当的TLR配体组合的纳米制剂可能会促进免疫反应的协同激活，包括Th1型细胞免疫。在拟议的工作中，我们将使用高压均质来制备稳定的纳米脂质体，其中包含合成的TLR4配体(GLA)和合成的TLR7/8配体(3M052)。制备的纳米脂质体的直径分别为~65 nm和100 nm，并且具有不同的表面特性(聚乙二醇化长度和浓度)。纳米脂质体在体外表现出可接受的物理化学稳定性以及与重组阿米巴病抗原(LecA)的相容性，接下来将在皮下给药后评估它们在小鼠模型中增强对LecA的Th1型免疫反应的能力。此外，将对免疫方案进行调整，以包括补充的鼻腔给药，以评估诱导 粘膜免疫球蛋白A反应(这也与对阿米巴病的保护相关)，同时保持强大的Th1型细胞反应。最后，展示最佳免疫原性的纳米脂质体佐剂和免疫方案将在阿米巴病小鼠攻击模型中评估其保护效果。该项目的广泛意义包括证明了保护性Th1型免疫可以通过在生物相容和稳定的脂质体载体中适当的TLR配体组合的纳米配方来实现。这种方法可以应用于加速许多其他生物防御病原体的疫苗开发。为阿米巴病或其他需要Th1型免疫反应的适应症开发的成功疫苗，如疟疾或结核病，将带来显著的公共卫生好处。

DESCRIPTION (provided by applicant): Conventional vaccine adjuvants with nanoparticle components such as oil-in-water emulsions or aluminum salts do not promote effective Th1-type cellular immune responses, which are critical for complex diseases lacking effective vaccines such as malaria, tuberculosis, and amebiasis. The latter is a neglected enteric disease caused by the protozoan parasite Entamoeba histolytica (a biodefense category B pathogen) and accounts for significant disease burden with some 50 million annual infections worldwide, with the greatest burden in the developing world. This proposal describes the development and physicochemical characterization of nanoliposomes containing a combination of synthetic Toll-like receptor (TLR) ligands, and the evaluation of their ability to enhance amebiasis vaccine immunogenicity and protective efficacy. The efficacy of live attenuated vaccines may in part be attributed to their capacity to trigger multiple pathogen recognition receptors such as TLRs. Nevertheless, for modern recombinant or inactivated vaccines, the only TLR ligand-based adjuvant used in an FDA-approved vaccine (i.e. Cervarix(R)) consists of a single TLR ligand. However, appropriate nanoformulation of TLR ligand combinations may promote synergistic activation of immune responses, including Th1-type cellular immunity. In the proposed work, we will employ high pressure homogenization to manufacture stable nanoliposomes containing a synthetic TLR4 ligand (GLA) and a synthetic TLR7/8 ligand (3M052). The nanoliposomes will be manufactured at ~65 and 100 nm diameters as well as with different surface characteristics (PEGylation length and concentration). Nanoliposomes demonstrating acceptable in vitro physicochemical stability as well as compatibility with a recombinant amebiasis antigen (LecA) will next be evaluated for their ability to enhance Th1-type immune responses to LecA in a mouse model after subcutaneous administration. Furthermore, the immunization regimen will be adapted to include complementary intranasal administration in order to assess the ability to elicit mucosal IgA responses (which are also correlated with protection against amebiasis) while maintaining strong Th1-type cellular responses. Finally, the nanoliposome adjuvants and immunization regimens demonstrating optimal immunogenicity profiles will be evaluated in an amebiasis mouse challenge model for their protective efficacy. The broad implications of the project include demonstrating that protective Th1-type immunity can be achieved through appropriate nanoformulation of a combination of TLR ligands in a biocompatible and stable liposomal vehicle. This approach could be applied to accelerate vaccine development for many other biodefense pathogens. Successful vaccines developed for amebiasis or other indications requiring Th1-type immune responses such as malaria or tuberculosis would result in significant public health benefits.