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

蓝舌病病毒（BTV）可感染所有反刍动物，包括牛、绵羊和山羊，在大多数情况下不会引起疾病。然而，当这种病毒感染绵羊，特别是欧洲品种的绵羊时，它会引起严重的疾病，导致高达70%的受感染动物死亡。该病毒通过叮咬蠓（库蠓种）传播，特别是那些喜欢温暖气候的蠓。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的蠓正在向北移动。这使得欧洲比以前面临更大的疾病风险。事实上，自1998年以来，六种不同的BTV毒株在七次不同的情况下侵入地中海区域，影响到所有南欧国家，并共同造成有史以来最大的疾病爆发（造成150多万头动物死亡）。目前可用的抗蓝舌病疫苗是蓝舌病病毒的活株，在细胞培养物中经过许多代的生长而被削弱。这些疫苗应该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侵害。然而，有证据表明，这些疫苗毒株也很危险，它们会引起疾病，并与野生型病毒一样，由同样的叮咬蚊传播。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）仍需在实地进行测试。蓝舌病病毒颗粒有一个由两种蛋白质组成的外蛋白层，VP2和VP5，以及一个由VP7组成的核心，这些蛋白质被感染动物的免疫系统识别，产生一种可以预防疾病的反应。该项目将制作病毒基因的DNA副本，并用它们在细菌或昆虫细胞中合成病毒蛋白质。然后，这些蛋白质将被纯化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颗粒的外表面蛋白质的结构，以了解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，或者将其复制到另一种病毒（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结合到微小的合成小珠中，这些小珠会随着时间的推移以受控的方式分解。这将延长动物接触疫苗的时间，使它们有更多的时间产生更高水平的病毒保护，从而更好地预防这种疾病。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，以确定哪种最有效，并设计出更好（和更安全）的疫苗。由于这些新型疫苗显示出的希望，我们将利用这个机会更详细地研究它们是如何起作用的。我们想要弄清楚蛋白质在小球上或小球内的位置是否会影响免疫系统对它们的反应。与此同时，我们想研究哪一种反应最好：是在一个单珠系统上使用每种成分，还是在含有不同蛋白质和/或dna的珠上使用。我们的最终目标是研制出单次注射后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.