Development of a Novel Nipah Virus Pre-Fusion Protein Recombinant Vaccine.

尼帕病毒（Nipah virus, NiV）是亨尼帕病毒属（副粘病毒科）的一员，是东南亚呼吸道和脑病散发暴发的病原体。在马来西亚和新加坡最初爆发NiV时，大多数人类NiV病例是由与猪的密切接触引起的。在2001年至2012年孟加拉国和印度随后爆发的疫情中，饮用被果蝠粪便、尿液和唾液污染的新鲜椰枣汁以及与感染者密切接触被发现是NiV感染的主要来源。然而，猪对NiV非常敏感，通常作为病毒的扩增宿主。由于疾病的致死性和潜在风险，不建议将减毒活疫苗逆转为野生型。因此，许多新冠病毒候选疫苗都是“重组”疫苗，即用插入另一种更安全的病毒（称为载体）的基因生产新冠病毒蛋白。通常选择NiV的融合(F)和(G)蛋白，因为它们在体内提供有效的免疫反应。G蛋白允许病毒附着在细胞上，F蛋白使病毒包膜与细胞膜融合，这样病毒核酸就能进入细胞。最近发现，在其他几种副粘病毒中，成熟病毒中的F蛋白形式（融合后形式）在诱导免疫反应方面不如病毒进入细胞时发生的F蛋白形式（融合前形式）好。融合前的F蛋白是不稳定的，但我们能够通过稍微改变序列来稳定它。我们将使用副粘病毒科的另一个“安全”成员仙台病毒（SeV，一种啮齿动物病毒）作为载体。我们将比较单独产生两种F蛋白中的一种或每一种与G蛋白结合产生的SeV疫苗的免疫反应。越来越多的证据表明，SeV具有作为疫苗载体的巨大潜力，部分原因是该病毒使用常见的唾液酸受体进入细胞，这有助于感染来自不同物种（包括猪和人类）的多种细胞类型。虽然SeV的生物安全风险很小，但我们还将开发该系统，以产生不能复制的SeV NiV疫苗，这种疫苗可以感染宿主细胞并产生NiV F和G蛋白，但可以消除活的SeV载体在动物或人类之间传播的任何残留的生物安全问题。我们假设，基于表达预融合NiV F蛋白的复制能力和不复制能力的SeV载体的新型疫苗方法将为生产高度成功的NiV疫苗提供重大突破。我们进一步假设，基于表达融合前NiV F的rSeV的NiV疫苗将比融合后F疫苗诱导更强大和更持久的保护性免疫。作为猪疫苗，这些系统将大大减少猪对人类的传播。此外，生产非复制性七价病毒疫苗解决了生物安全问题。由于Sev病毒也感染人类细胞，这些疫苗随后可用于人体试验。

Abstract Nipah virus (NiV) is a member of the Henipavirus genus (family Paramyxoviridae) and is the causative agent of sporadic outbreaks of respiratory and encephalitic disease in Southeast Asia. During the initial NiV outbreaks in Malaysia and Singapore, most human NiV cases were caused by close contact with pigs. In subsequent outbreaks during 2001 to 2012 in Bangladesh and India, drinking fresh date palm sap, which was contaminated by fruit bat’s droppings, urine and saliva, and close contact with infected humans were found to be the major source of NiV infection. However, pigs are highly susceptible to NiV and generally serve as an amplifying host of the virus. Due to the lethal nature of disease and potential risk of reversion of a live attenuated vaccine to wild-type is not advisable. Therefore, many candidate vaccine for NiV are 'recombinant', where NiV proteins are produced from inserted genes in another safer virus (referred to as the vector). The fusion (F) and (G) proteins of NiV are commonly chosen as they provide an effective immune response in the body. The G protein allows the virus to attach to the cell and the F protein causes fusion of the virus envelope with the cell membrane so that the virus nucelic acid can enter the cell. Recently it has been discovered for several other paramyxovirus that the form of the F protein in the mature virus (post fusion form) is not as good at inducing an immune response as the form of F which occurs when the virus is entering cells (pre-fusion form). The pre-fusion F protein is unstable but we are able to stabilise this by making slight sequence changes. We will use another ‘safe’ member of the Paramyxovirdae, Sendai virus (SeV, a rodent virus) as the vector. We will compare the immune response of the SeV vaccines which produce one of the 2 types of F protein alone or each in combination with the G protein. Increasing evidence indicates that SeV has substantial potential as a vaccine vector in part because the virus uses common sialic acid receptors for cell entry, which facilitates infection of a wide range of cell types from different species including pigs and humans. Although SeV biosafety risks are minimal, we will also develop the system to generate replication-incompetent SeV NiV vaccines that infect host cells and produce the NiV F and G proteins but removes any residual biosafety concerns of a live SeV vector spreading between animals or humans. We hypothesise that novel vaccine approach based on replication-competent and incompetent SeV vectors expressing pre-fusion NiV F protein will provide a significant breakthrough in the generation of highly successful NiV vaccines. We further hypothesise that NiV vaccines based on rSeV expressing pre-fusion NiV F will induce more robust and longer lasting protective immunity than post fusion F vaccines. Used as a pig vaccine these systems will greatly reduce porcine to human transmission. Futhermore, production of non-replicating SeV vaccines addresses biosafety. As Sev also infects human cells, these vaccines could be subsequently trialled for human use.