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

蓝舌病毒基因和蛋白质的表达和配制，用于制定有效的疫苗接种策略

• 批准号: BB/D014204/1
• 负责人: Peter Mertens
• 金额: $ 35.53万
• 依托单位: The Pirbright Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD014204%2F1
• 关键词: Expression; formulation; Bluetongue; virus; genes

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.

蓝舌病病毒（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，特别是欧洲品种时，它会导致严重的疾病，杀死多达70%的受感染动物。该病毒通过蚊虫（库蠓属）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的蠓正在向北移动。这使欧洲面临比以前更大的疾病风险。事实上，自1998年以来，六种不同的BTV毒株已经入侵地中海地区，总共发生了七次不同的事件，影响了南欧的所有国家，并共同造成了有史以来最大的疾病爆发（超过150万只动物死亡）。目前可用于对抗蓝舌病的疫苗是蓝舌病病毒的活毒株，其已通过在细胞培养物中生长许多代而减弱（减毒）。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，这些疫苗株也是危险的，会引起疾病，并通过与野生型病毒相同的叮咬蠓传播。最近已经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。蓝舌病病毒颗粒具有由两种蛋白质VP 2和VP 5组成的外部蛋白质层，以及具有由VP 7组成的表面的核心，这些蛋白质被感染动物的免疫系统识别，产生可以保护免受疾病的反应。该项目将制作病毒基因的DNA拷贝，并利用它们在细菌或昆虫细胞中合成病毒蛋白。然后将这些蛋白质纯化并用于研究它们与动物免疫系统的相互作用，特别是保护性反应的产生，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，以了解哪些氨基酸序列与免疫系统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，在接种疫苗的动物中合成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质、DNA或MVA掺入显微合成珠粒中，这些珠粒以受控的方式随时间分解。这将延长动物接触疫苗的时间，从而有更多的时间产生更高水平的抗病毒保护，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，以确定哪种组合最有效，并设计出更好（更安全）的疫苗。由于这些新型疫苗所显示的前景，我们将利用这个机会更详细地研究它们是如何工作的。我们想知道珠子上或珠子中蛋白质的位置是否会影响免疫系统对它们的反应。与此同时，我们希望研究哪种方法能给出最佳响应：在单个珠系统上使用每种组分，还是使用含有不同蛋白质和/或DNA的珠。我们的最终目标是一种疫苗，在单剂量后100%有效。

项目成果

Biolistic transfection of human embryonic kidney (HEK) 293 cells.

人胚肾 (HEK) 293 细胞的基因枪转染。

• DOI: 10.1007/978-1-62703-110-3_10
• 发表时间: 2013
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Li X

The immunogenicity of recombinant vaccines based on modified Vaccinia Ankara (MVA) viruses expressing African horse sickness virus VP2 antigens depends on the levels of expressed VP2 protein delivered to the host.

• DOI: 10.1016/j.antiviral.2018.04.015
• 发表时间: 2018-06
• 期刊: Antiviral research
• 影响因子: 7.6
• 作者: Calvo-Pinilla E;Gubbins S;Mertens P;Ortego J;Castillo-Olivares J
• 通讯作者: Castillo-Olivares J

Protection of IFNAR (-/-) mice against bluetongue virus serotype 8, by heterologous (DNA/rMVA) and homologous (rMVA/rMVA) vaccination, expressing outer-capsid protein VP2.

• DOI: 10.1371/journal.pone.0060574
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Jabbar TK;Calvo-Pinilla E;Mateos F;Gubbins S;Bin-Tarif A;Bachanek-Bankowska K;Alpar O;Ortego J;Takamatsu HH;Mertens PP;Castillo-Olivares J
• 通讯作者: Castillo-Olivares J