Expression and formulation of Bluetongue virus genes and proteins for development of effective vaccination strategies

蓝舌病毒(BTV)可以感染所有反刍动物，包括牛、绵羊和山羊，在大多数情况下不会引起疾病。然而，当病毒感染绵羊，特别是欧洲品种的绵羊时，它可以导致严重的疾病，高达70%的受感染动物死亡。该病毒通过叮咬的蚊子(库蚊物种)传播，特别是那些喜欢温暖气候的物种。由于最近欧洲气候的变化被认为是由全球变暖引起的，可以传播BTV的蚊子正在向北移动。这使欧洲面临比以前更大的疾病风险。事实上，自1998年以来，6种不同的BTV菌株在总共7次不同的情况下入侵地中海区域，影响到南欧所有国家，共同造成了有史以来最大规模的疾病暴发(有150多万头动物死亡)。目前可用于对抗蓝舌病的疫苗是蓝舌病病毒的活毒株，这种病毒已在细胞培养中生长了许多代而减弱(减毒)。这些药物被认为只会产生轻微的症状，但可以保护动物免受更危险的病毒株的伤害。然而，有证据表明，这些疫苗毒株也是危险的，会导致疾病，并通过与野生型病毒相同的叮咬蚊子传播。最近出现了基于部分纯化和化学处理的病毒的替代灭活疫苗，尽管对这些疫苗的反应性质(保护性或非保护性)仍需在现场进行测试。蓝舌病毒颗粒的外层由两种蛋白质VP2和VP5组成，核心由VP7组成，VP7是受感染动物的免疫系统识别的蛋白质，产生的反应可以预防疾病。该项目将制作病毒基因的DNA副本，并使用它们在细菌或昆虫细胞中合成病毒蛋白质。然后，这些蛋白质将被提纯，并用于研究它们与动物免疫系统的相互作用，特别是产生保护性反应，例如可以中和病毒的抗体。该项目还将检查BTV颗粒外表面蛋白质的结构，看看哪些氨基酸序列与免疫系统发生反应，并导致中和反应。该项目将直接使用病毒基因的DNA拷贝，或复制到另一种病毒(牛痘病毒-MVA)中，在接种疫苗的动物体内合成BTV蛋白，从而激活它们的免疫系统。通过将合成和纯化的病毒蛋白、DNA或MVA结合到微观合成珠子中，这些珠子会随着时间的推移以受控的方式分解。这应该会延长动物接触疫苗的时间，让更多的时间产生对病毒的更高水平的保护，从而更好地预防疾病。这些新的疫苗接种策略被认为是完全安全的，因为它们不使用活的BTV来诱导免疫/保护。因此，它们将以不同的组合使用，以观察哪种疫苗最有效，并设计出更好(更安全)的疫苗。由于这些新型疫苗显示出的前景，我们将利用这个机会更详细地研究它们是如何发挥作用的。我们想找出蛋白质在珠子上或珠子中的位置是否会影响免疫系统对它们的反应方式。同时，我们想要研究哪一种反应最好：在单个珠子系统中使用每种成分，还是在含有不同蛋白质和/或DNA的珠子上使用。我们的最终目标是一种在单剂接种后100%有效的疫苗。

Bluetongue virus (BTV) can infect all ruminants, including cows, sheep and goats, in most cases without causing disease. However, when the virus infects sheep, particularly the European breeds, it can cause severe disease, killing up to 70% of the infected animals. The virus is transmitted by biting midges (Culicoides species), particularly those species which prefer warmer climates. As a result of recent changes in the European climate that are thought to have been caused by global warming, midges that can transmit BTV are moving northwards. This places Europe at much greater risk from the disease than before. Indeed, since 1998, six different strains of BTV have invaded the Mediterranean region, on a total of seven different occassions, affecting all of the countries of Southern Europe and collectively causing the largest outbreak of the disease ever recorded (with over 1.5 million dead animals). The vaccines that are currently available to fight bluetongue are live strains of the bluetongue virus that have been weakened (attenuated) by growth for many generations in cell cultures. These are supposed to produce only mild symptoms but protect the animal from more dangerous virus strains. However, there is evidence that these vaccine strains are also dangerous, causing disease and being transmitted by the same biting midges as the wild type viruses. Alternative inactivated vaccines have recently become available, based on partially purified and chemically treated virus, although the nature of the response to these vaccines (protective or not) still has to be tested in the field. The bluetongue virus particle has an outer protein layer composed of two proteins, VP2 and VP5, and a core, with a surface composed of VP7, proteins which are recognised by the immune system of infected animals, generating a reponse that can protect against the disease. The project will make DNA copies of viral genes and use them to synthesise viral proteins in bacteria, or in insect cells. The proteins will then be purified and used to study their interactions with the animal's immune system, particularly the production of a protective response e.g. antibodies that can neutralise the virus. The project will also examine the structure of the outer surface proteins of BTV particles, to see which aminoacid sequences react with the immune system and lead to a neutralising response. The project will use DNA copies of the viral genes directly, or copied into another virus (vaccinia virus - MVA), to synthesise the BTV proteins in vaccinated animals and thus ativating their immune system. By incorporating the synthesised and purified viral proteins, DNAs or MVA into microscopic synthetic beads that breakdown over time in a controlled way. This should prolong the exposure of the animals to the vaccine, allowing more time to generate a higher level of protection against the virus, and therefore better protection against the disease. These new vaccination strategies are considered to be entirely safe, since they do not use live BTV to induce immunity/protection. They will therefore be used in different combinations to see which is most effective, and to design a better (and safer) vaccine. Because of the promise shown by these new types of vaccines, we will use this opportunity to study in more detail how they work. We want to find out if the position of the proteins on or in the beads affects the way the immune system responds to them. At the same time, we want to study which gives the best response : the use of each component on a single bead system, or beads containg different proteins and/or DNAs. Our ultimate goal being a vaccine that is 100% efficient after a single dose.