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制造预防多种疾病的疫苗(多价疫苗)。这项技术允许我们通过RNA基因组的DNA拷贝(CDNA)来突变RNA病毒基因组。然后，新的基因组cdna可以用来获得变异形式的病毒。在这些研究中，我们将使用现有的小反刍动物病毒(PPRV)疫苗作为载体，传递来自其他重要经济病毒的抗原。猪繁殖与呼吸综合征病毒对小型反刍动物造成毁灭性的瘟疫，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。在以前使用这项技术对相关病毒牛瘟病毒(RPV)进行的研究中，我们能够在感染细胞中高效表达外源蛋白，生产有效的RPV标记疫苗，并确定决定病毒株之间毒力差异的一些分子因素。最近有研究表明，麻疹病毒(MV)的基因组可以被人工分割，并且这些片段的cDNA可以用类似于全长非片段cDNA的方式来拯救活病毒。猪伪狂犬病病毒的分离和拯救将为运送其他小型反刍动物病毒病原体的免疫原提供新的途径。与其他非分段负链(NNS)病毒的研究表明，在病毒活性下降之前，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基因组的分割可以有效地克服这一限制，证明了分割后的MV能够高效地编码多达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，同时预防几种重要的反刍动物疾病，并提高它们的成本效益。虽然由于过去20年的协调一致的疫苗接种运动，RPV在全球几乎被消灭，但PPRV是一种在世界新地区出现的疾病，目前正在许多发展中世界以及欧盟边界造成巨大的经济损失。目前为猪繁殖与呼吸综合征病毒开发的减毒活疫苗是安全和高效的，因此是理想的疫苗载体，可以标记以区分感染动物和接种疫苗的动物。我们希望探索使用PPRV作为媒介传递来自其他具有重要经济意义的病毒的多种抗原的分段方法，如蓝舌病毒(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，后者也能够感染人类。BTV和RVFV曾被认为是外来疾病，尽管最近BTV已经进入欧盟，对农业造成了毁灭性的影响。RVFV也有可能进入欧洲，因为携带BTV的昆虫媒介可能也能感染RVFV。目前使用的PPRV疫苗产生一种灭菌免疫，对病毒提供终身保护，对于RVFV，被认为在接种疫苗后产生类似的反应。然而，对于BTV来说，存在一些不同的遗传变异，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地区传播的病毒的疫苗。RVFV和BTV在亚洲和非洲的大部分地区都是地方病，有效的疫苗接种战略对它们的控制是不可或缺的。本提案中针对的三种病毒性疾病符合BBSRC抗击发展中国家疾病的战略以及DFID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.