Bioengineering protocells for a new class of viral vaccines

描述（由申请人提供）： 应对实地挑战的新战略。副粘病毒亨德拉病毒和尼帕病毒（HeV，NiV）的致命性和传播性使这些病原体受到严重关注。这些也是在生物工程和病毒学之间的界面上开发创新性新疫苗平台的理想模型。靶向副粘病毒融合蛋白（F）的疫苗可以提供针对一组重要的人类病原体的中和免疫。F的瞬时活化状态-仅在受体结合蛋白结合受体时活化后但在融合前暴露-是广泛中和抗体的最佳靶标。我们建议捕获和纯化这种活化的融合中间体，暴露病毒进入所必需的保守F结构域。然后，我们将使用这种捕获的活性状态分子--通常与免疫系统高度隔绝，不能以固定状态适当地呈递--来引发广泛中和抗体。虽然这种激活的构象通常只存在于活细胞的表面，但我们建议使用工程化的原始细胞来捕获和修饰这种过渡状态。人工原始细胞将在仿生膜设置中呈现受体分子以“触发”融合蛋白。 我们实验室最近的三项进展使这种方法变得可行：（1）HeV/NiV与受体部分相互作用时不可逆地失活，诱导F的构象变化，导致融合准备就绪。(2)脂质包被的二氧化硅基原始细胞可以在表面上呈递受体分子，并通过过早触发融合机制不可逆地消灭副粘病毒假型病毒。(3)对应于融合蛋白的七肽重复序列的肽通过仅在构象变化激活至融合就绪后但在融合进展前结合来抑制融合/进入。我们将使用受体分子，以仿生方式呈现在原始细胞表面，激活F的构象变化，然后使用七肽重复序列将F捕获在其激活状态，从而使捕获的中间体与原始细胞结合。然后将该复合物用于诱导中和抗体。1.捕获尼帕病毒融合（F）机制的活化构象：开发携带受体加肽的原始细胞，旨在使F处于过渡状态。捕获机制的确认。我们将检验携带受体加肽的原始细胞能有效地触发和抑制F的中间构象状态的假设。2.使用与处于其活化构象的F复合的原始细胞来引发免疫。评估免疫反应。我们将测试的假设，暴露的瞬时构象的F，提出和捕获的仿生人工表面，elevenly强大的中和抗体，抑制尼帕病毒感染。这些结果将为基于生物工程的疫苗接种建立新的平台。 公共卫生相关性：副粘病毒引起重要的人类疾病，对全球疾病和死亡率有重大影响。作为本申请主题的人畜共患副粘病毒，亨德拉病毒和尼帕病毒，由于其致命和可传播的性质，是公共卫生的迫切关注点。该项目的结果将导致建立一种新的基于生物工程的疫苗接种策略，该策略将病毒学的新基本发现与新技术相结合，以实现一种全新的方法，即诱导和捕获病毒融合蛋白的相关活化构象中间体。这种新的复合物将以一种通常只能被免疫系统短暂看到的方式呈递关键的病毒抗原，非常模拟自然感染。鉴于副粘病毒对人类健康的重要性以及生物工程方法对这些和其他严重病原体疫苗开发的潜在广泛适用性，这些结果将具有高度相关性。

DESCRIPTION (provided by applicant): A new strategy that addresses challenges in the field. The lethal and transmissible nature of the paramyxoviruses Hendra virus and Nipah virus (HeV, NiV) makes these pathogens of serious concern. These are also ideal models for developing an innovative new vaccine platform at the interface between bioengineering and virology. A vaccine that targets the paramyxovirus viral fusion protein (F) could provide neutralizing immunity against an important group of human pathogens. The transient activated state of F - exposed only after activation when the receptor binding protein binds receptor, but before fusion - is an optimal target for broadly neutralizing antibodies. We propose to capture and immobilize this activated fusion intermediate, exposing the conserved F domains that are essential for viral entry. We will then use this captured active-state molecule - which is normally highly sheltered from the immune system and could not be properly presented in an immobilized state - to elicit broadly neutralizing antibodies. While this activated conformation is normally present only at the surface of live cells, we propose to use engineered protocells to capture and immobilize this transitional state. Artificial protocells will present the receptor molecules in a biomimetic membrane setting to "trigger" the fusion protein. Three recent advances in our lab make this approach feasible: (1) HeV/NiV are irreversibly inactivated when they interact with receptor moieties that induce the conformational changes in F that lead to fusion-readiness. (2) Lipid-coated silica-based protocells can present receptor molecules on the surface, and irreversibly inactivate paramyxovirus pseudotyped viruses by prematurely triggering the fusion mechanism. (3) Peptides corresponding to the heptad repeats of the fusion protein inhibit fusion/entry by binding only after activation of the conformational change to fusion-readiness, but before progression of fusion. We will use receptor molecules, presented in a biomimetic fashion on the surface of protocells, to activate the conformational change in F, and then use heptad-repeat peptides to arrest F in its activated state and thus immobilize the captured intermediate with the protocell. This complex will then be used to induce neutralizing antibodies. 1. Capture of the activated conformation of Nipah virus fusion (F) machinery: Development of protocells bearing receptor plus peptide, designed to immobilize F in its transition state. Validation of capture mechanism. We will test the hypothesis that protocells bearing receptor plus peptide can effectively trigger and immobilize the intermediate conformational state of F. 2. Use of protocells complexed with F in its activated conformation to elicit immunity. Assessment of immune response. We will test the hypothesis that the exposed transient conformation of F, presented and captured on a biomimetic artificial surface, elicits powerful neutralizing antibodies that inhibit Nipah virus infection. These results will establish a new platform for bioengineering-based vaccination. PUBLIC HEALTH RELEVANCE: Paramyxoviruses cause important human illnesses that contribute significantly to global disease and mortality. The zoonotic paramyxoviruses that are the subject of this application, Hendra virus and Nipah virus, are an urgent concern for public health due to their lethal and transmissible nature. The results of this project will lead to setting up a new bioengineering-based strategy for vaccination that combines new basic findings from virology with new technology to achieve an entirely new approach that of inducing and capturing the relevant activated conformational intermediate of a viral fusion protein. The novel complex will present the key viral antigen in a way normally only seen transiently by the immune system, closely mimicking natural infection. The results will be highly relevant in light of the importance of paramyxoviruses to human health and the potential broad applicability of the bioengineering approach to vaccine development for these and other serious pathogens.