Development of novel multivalent vaccines

该项目的目标是利用反向遗传学开发更好的方法来生产预防多种疾病的疫苗（多价疫苗）。这项技术使我们能够通过RNA基因组的DNA拷贝（cDNA）使RNA病毒基因组发生突变。然后，新的基因组cDNA可以用来获得病毒的突变形式。在这些研究中，我们将使用现有的小反刍兽疫病毒（PPRV）疫苗作为载体，传递来自其他经济上重要病毒的抗原。PPRV在小反刍动物中造成毁灭性的瘟疫，并对非洲和亚洲许多国家的动物福利和经济产生严重影响。在先前的研究中，我们利用该技术对相关的牛瘟病毒（RPV）进行了研究，我们能够在受感染的细胞中有效地表达外源蛋白，并生产出有效的RPV标记疫苗，并确定了一些决定病毒株之间毒力差异的分子因素。最近的研究表明，麻疹病毒（MV）是一种与麻疹病毒密切相关的病毒，其基因组可以被人工分割，并且这些片段的cDNA可以以类似于全长非片段cDNA的方式来拯救活病毒。PPRV的分离和拯救将为其他小反刍动物病毒病原体的免疫原传递提供新的途径。对其他非分节负链病毒的研究表明，在观察到病毒活力降低之前，可以添加到NNS病毒基因组中的额外遗传物质的数量是有限的。NNS基因组的片段化可以有效地克服这一限制，片段化的MV能够有效地编码多达6个外源蛋白。如果这适用于相关病毒，那么它将增加它们的编码能力，使我们能够生产多价疫苗，同时预防几种经济上重要的反刍动物疾病，并提高其成本效益。虽然由于过去20年协调一致的疫苗接种运动，RPV实际上已经从全球消灭，但PPRV是世界上新出现的一种疾病，目前正在许多发展中国家以及欧洲联盟边界造成巨大的经济损失。目前针对PPRV开发的减毒活疫苗是安全且高效的，因此是用作疫苗载体的理想候选疫苗，可以标记以区分感染动物和接种动物。我们希望利用PPRV作为多种抗原的载体传递系统来探索分段方法，这些抗原来自其他经济上重要的病毒，如蓝舌病病毒（BTV）和裂谷热病毒（RVFV），这些昆虫传播的病原体可以感染牛和羊，后者也可以感染人类。BTV和裂谷热病毒曾被认为是外来疾病，尽管最近BTV进入了欧盟，对农业产生了毁灭性的影响。裂谷热病毒也有可能进入欧洲，因为携带BTV的昆虫媒介也可能感染裂谷热病毒。目前使用的PPRV疫苗会产生一种绝育免疫，对病毒提供终身保护，而对于裂谷热病毒，人们认为在接种疫苗后也会产生类似的反应。然而，对于BTV来说，存在许多不同的遗传变异，尽管不同，但它们聚集在不同的地理区域。我们希望开发针对在特定区域内传播的病毒的疫苗。裂谷热病毒和BTV在亚洲和非洲大部分地区都是地方性的，有效的疫苗接种战略是控制这两种病毒的必要条件。该提案针对的三种病毒性疾病符合BBSRC的发展中国家防治疾病战略，也符合英国国际发展部对改善发展中国家农业可持续性的长期承诺。

The objective of this project is to use reverse genetics to develop better ways of making vaccines that protect against more than one disease (multivalent vaccines). This technology allows us to mutate RNA virus genomes through DNA copies (cDNA) of the RNA genome. The new genome cDNA can then be used to obtain the mutated form of the virus. In these studies we will use an existing vaccine for peste des petits ruminants virus (PPRV), as a vector to deliver antigens from other economically important viruses. PPRV causes a devastating plague in small ruminants and has a severe impact on animal welfare and the economies of many countries in Africa and Asia. In previous studies using this technology with a related virus, rinderpest virus (RPV), we were able to express foreign proteins efficiently in infected cells and to produce effective marker vaccines for RPV as well as identify some of the molecular factors which determine differences in virulence between virus strains. Recently it has been shown that the genome of measles virus (MV), a closely related virus, can be artificially segmented and that cDNAs of these segments can be used in a similar way to the full-length nonsegmented cDNA to rescue viable virus. The segmentation and rescue of PPRV will provide a new way to deliver immunogens from other small ruminant viral pathogens. Work with other nonsegmented negative strand (NNS) viruses has shown that there is a limit to the amount of extra genetic material that can be added to NNS virus genomes before a reduction in virus viability is seen. Segmentation of NNS genome can effectively overcome this limit, as evidenced by the ability of the segmented MV to encode at up to six foreign proteins efficiently. If this is applicable to related viruses then it would increase their coding capacity and enable us to produce multivalent vaccines to simultaneously protect against several economically important diseases of ruminants and increase their cost-effectiveness. Whilst RPV has been virtually eliminated from the globe as a result of a concerted vaccination campaign over the past 20 years, PPRV is a disease emerging in new regions of the world and is now causing great economic losses across much of the developing world as well as on the borders of the European Union. The current live-attenuated vaccines developed for PPRV are safe and highly effective and are, therefore, ideal candidates for use as vaccine vectors that can be tagged to allow differentiation between infected and vaccinated animals. We wish to explore the segmented approach using PPRV as a vector delivery system for multiple antigens from other economically significant viruses such as bluetongue virus (BTV) and Rift Valley Fever virus (RVFV), insect borne pathogens which can infect cattle and sheep, the latter also being able to infect humans. BTV and RVFV were once considered exotic diseases although recently BTV has entered the European Union, having a devastating effect on agriculture. RVFV has the potential to also enter Europe as insect vectors that carry BTV may also competent for RVFV infection. Current use of the PPRV vaccine generates a sterilising immunity that gives lifelong protection against the virus and, for RVFV, a similar response is thought to be generated post vaccination. However, for BTV a number of distinct genetic variants exist which, although diverse, cluster across distinct geographical regions. We wish to develop vaccines that target viruses circulating within a specific areas. Both RVFV and BTV are endemic across much of Asia and Africa and effective vaccination strategies are integral to their control. The three viral diseases targeted in this proposal are in line with the BBSRC's combating diseases of the developing world strategy as well as DFID's long term commitment to improving the sustainability of agriculture in developing countries.