Understanding the immune mechanism of host disease resistance and development of marker vaccines and DIVA tests for Peste des Petits Ruminants (PPR)

小反刍兽疫（PPR），也称为“山羊鼠疫”，是绵羊和山羊的一种高度传染性病毒性疾病。据估计，仅在印度，这种疾病每年造成的直接经济损失就高达18亿印度卢比（3900万美元），而在全球范围内，有60%的小反刍动物被认为处于危险之中。然而，对PPRV的发病机制知之甚少。重要的是，病毒复制的主要位点尚未被正确定义，而宿主对小反刍兽疫的特异性抗性的机制尚不清楚。有效的减毒活疫苗可用于控制小反刍猪瘟，IVRI和Pirbright开发了c-ELISA试剂盒，用于监测针对H蛋白的抗体反应。然而，这些疫苗和相关的c-ELISA试剂盒的关键缺点是它们无法区分接种动物的感染（DIVA），从而减缓了通过疫苗接种控制小反瘟的实施。我们假设PPRV感染呼吸道粘膜的免疫细胞，然后这些细胞迁移到局部淋巴器官富含t细胞的区域，从那里病毒进入全身循环。我们的目标是开发一种重组DIVA减毒活疫苗，该疫苗将雾化到主要病毒复制位点，并能够产生快速和适当的免疫反应，包括粘膜免疫。通过进一步描述绵羊、山羊和大型反刍动物对PPRV感染的不同易感性背后的免疫机制，我们的目标是加强我们对疾病抗性机制的理解，从而能够开发出更好的疫苗来控制PPRV。目的和目标：为了提高控制和根除小反刍兽疫的能力，需要三个互补的研究目标：1)了解小反刍兽疫病毒在病毒前阶段的发病机制，以确定病毒复制的主要部位作为疫苗接种的靶点；2)进一步表征小型反刍动物和大型反刍动物对小反刍兽疫的免疫反应，以了解不同的抗病能力；3)开发适当的小反刍兽疫标记疫苗，并进行相关的DIVA测试，以提供有效和有针对性的小反刍兽疫控制，最终从印度根除。研究方法：与我们的Nigeria75/1 GFP疫苗拯救病毒类似，构建并拯救含有GFP基因的强毒重组小反刍病毒。这种病毒将被雾化感染山羊。从呼吸道黏膜和相关淋巴结收集组织，通过组织中绿色荧光蛋白的自身荧光和免疫组化来定位病毒的主要复制位点。如果我们在病毒血症前阶段确定PPRV的主要复制位点，则后续疫苗接种将使用鼻内途径。利用反向遗传学技术，我们将开发两种针对印度PPRV的阴性标记疫苗（Sungri 96-山羊源和Arasur 1987-绵羊源）。H蛋白中对单克隆抗体结合至关重要的一个区域的表位缺失将允许使用当前的cH-ELISA在接种疫苗和自然感染的动物之间进行血清学区分。为了简单快速的现场诊断，我们打算开发环介导等温扩增法（LAMP）和横向流动装置。印度实时RT-PCR检测和生物传感器检测的开发和验证将有助于小反刍兽疫控制规划。将使用无偏倚的转录组学方法，对小型和大型反刍动物物种/品种对PPRV的不同易感性背后的免疫机制进行调查。增加我们对PPRV早期发病机制的了解，使用标记疫苗产生适当的免疫反应，以及区分感染与疫苗接种的能力，再加上对免疫功能的更深入了解，将使我们能够控制PPRV。该项目将通过汇集印度和英国小反刍兽疫研究的主要参与者，帮助在印度和其他地方控制小反刍兽疫。

Peste des petits ruminants (PPR), also known as 'goat plague', is a highly contagious viral disease of sheep and goats. The direct economic losses of this disease have been estimated to be INR 1800 million (US$ 39 million) annually in India alone, whilst globally, >60% of the small ruminant population is considered at risk. However little is known about the pathogenesis of PPRV. Importantly, the primary site of viral replication has yet to be properly defined and the mechanism of the contrasting host specific disease resistance to PPR is unknown. Effective live attenuated vaccines are available to control PPR and a c-ELISA kit, to monitor the antibody response against the H protein, has been developed at IVRI and Pirbright. However, the critical drawback of these vaccines and associated c-ELISA kits is their inability to differentiate infection in vaccinated animals (DIVA) that slows down the implementation of PPR control through vaccination. We hypothesise that PPRV infects immune cells of the respiratory mucosa, which then migrate to T-cell-rich areas of local lymphoid organs, from which virus enters the general circulation. We aim to develop a recombinant live attenuated DIVA vaccine that will be aerosolised to the primary viral replication site and is capable of generating a rapid and appropriate immune response including mucosal immunity. By further characterising the immune mechanisms underlying the differential susceptibility of sheep, goats and large ruminants to PPRV infection we aim to enhance our understanding of disease resistance mechanisms that will enable the development of better vaccines for the control of PPRV.Aims and objectives: Three complementary research objectives are necessary to advance the ability to control and eradicate PPR: 1) understand PPRV pathogenesis in the pre-viraemic stage to identify the primary site of virus replication as a target for vaccination; 2) further characterise the immune response to PPR in small and large ruminants to understand differential disease resistance; and 3) develop appropriate PPR marker vaccines with associated DIVA tests to provide effective and targeted PPR control, ultimately leading to eradication from India. Research approaches: Similar to our Nigeria75/1 GFP vaccine virus rescue, a virulent recombinant PPR virus containing the GFP gene will be constructed and rescued. This virus will be aerosolised to infect goats. Tissues will be collected from respiratory mucosa and associated lymph nodes and the primary viral replication site will be located by auto fluorescence of GFP and by IHC in tissues. Subsequent vaccinations will use the intranasal route if we define the primary replication site of PPRV during the pre-viremic stage. Using reverse genetics techniques we will develop two negative marker vaccines against Indian PPRV (Sungri 96- goat origin and Arasur 1987- sheep origin). Epitope deletion of a region of the H protein critical for monoclonal antibody binding will allow serological differentiation between vaccinated and naturally infected animals using the current cH-ELISA. For simple and rapid field diagnosis, we intend to develop loop mediated isothermal amplification assay (LAMP) and a lateral flow device. Real-time RT-PCR assay and biosensor assay development and validation in India will help the PPR control programme. Interrogation of the immune mechanisms that underlie the differential susceptibility of species / breeds of small and large ruminants to PPRV will be carried forward using an unbiased transcriptomics approach. Increasing our knowledge on early pathogenesis, using a marker vaccine to generate an appropriate immune response and the ability to differentiate infection from vaccination, in combination with a deeper understanding of immune function will enable the control of PPRV. This project will help in delivering PPR control in India and elsewhere by bringing together major players in PPR research in India and the UK.