VACCINE: Development of Novel BRSV Pre-Fusion Protein Recombinant Bovine Vaccine.

牛呼吸道合胞病毒（BRSV）是一种与呼吸道病毒相关的牛呼吸道疾病复合体，是养牛业面临的主要问题。它在全世界范围内导致牛，特别是小牛的大量发病和死亡。目前疫苗的效力是有限的，并且存在对更有效的疫苗的主要需求。BRSV与人RSV（HRSV）高度相似，后者是导致婴幼儿住院的最常见原因，并在老年人中引起严重疾病。由于疗效差和安全性问题，迄今为止还没有HRSV疫苗可用。由于疗效差和安全性问题，迄今为止还没有HRSV疫苗可用。在牛中，当前疫苗的免疫系统的应答由于存在中和疫苗的母体抗体而受到损害。目前的提案将针对开发新技术的组合来生产新型BRSV疫苗。具体而言，我们将汇集副粘病毒反向遗传学、基于结构的疫苗抗原设计和疫苗开发方面的世界知名专业知识，以产生新一代BRSV疫苗，我们相信该疫苗将克服当前疫苗的许多局限性。这将包括使用仙台病毒（SeV，一种小鼠病毒）和5型副流感病毒（PIV 5，一种犬病毒）作为载体（载体），将BRSV基因递送到细胞中并从中产生蛋白质（抗原）。由于这两种病毒均未在牛中发现，它们感染牛细胞，但不会在牛中引起疾病，并且与牛抗体没有已知的交叉反应性，因此可以避免与母源抗体存在相关的问题。SeV和PIV 5的使用将促进初免/加强疫苗方案的开发，该方案将避免第一次疫苗接种诱导的病毒载体特异性免疫应答，因为两种病毒的抗原性不同。这样的方案可能使对BRSV抗原的免疫应答最大化。我们将为SeV和PIV 5开发复制能力强和复制能力弱的病毒。后者是通过产生病毒感染性颗粒而产生的，这些颗粒具有用于进一步复制的基因之一。然而，这些颗粒可以进入细胞并产生BRSV蛋白。我们的累积数据（已发表和未发表）证实，两种复制型病毒在动物模型中均为人RSV疫苗抗原的独立高效载体。无复制能力的SeV和PIV 5病毒载体的开发将解决与重组病毒的环境传播相关的一些安全性问题，并将涉及两种病毒的新型拯救技术（从病毒基因的拷贝生产病毒）的产生。BRSV和HRSV含有一种称为融合（F）蛋白的蛋白质，该蛋白质允许病毒进入细胞，并且与与细胞相互作用时（融合后）相比，在病毒体表面（融合前）的形式不同。F蛋白诱导免疫系统的应答，并且最近已经显示融合前形式产生比融合后形式高几倍的免疫应答（在实验HRSV疫苗中）。我们将利用这一发现，设计和生产最高免疫原性的BRSV疫苗，基于使用融合前稳定的F蛋白。对于基于人RSV融合前F的实验疫苗证明了这一点的优先性，作为疫苗抗原，其显示出比天然F高上级许多倍。最后，我们将把所有这些互补但不同的元素结合在一起，以开发新型BRSV疫苗和疫苗方案，并测试它们在使用这些疫苗的最终宿主靶点中的效力，即，小牛这些疫苗及其研究结果也将作为设计有效的HRSV人用疫苗的基础。

Bovine respiratory syncytial virus (BRSV) is a respiratory virus associated bovine respiratory disease complex, which is a major problem for the cattle industry. It results in considerable morbidity and mortality in cattle, and particularly young calves, worldwide. Efficacy of current vaccines is limited and there is a major need for a much more effective vaccine. BRSV is highly similar to Human RSV (HRSV) which is the most common cause of hospitalization in young infants and causes severe disease in elderly. No HRSV vaccines are available to date due to poor efficacy and safety issues. No HRSV vaccines are available to date due to poor efficacy and safety issues. In cattle the response of the immune system of current vaccines is compromised by the presence of maternal antibodies which neutralise the vaccine. This current proposal will target the exploitation of a combination of novel technologies to generate novel BRSV vaccines. Specifically, we will bring together world-renowned expertise in paramyxovirus reverse genetics, structure-based vaccine antigen design, and vaccine development to generate a new generation of BRSV vaccines that we are confident will circumvent many of the limitations of current vaccines. This will include the use of Sendai virus (SeV, a mouse virus) and parainfluenza virus type 5 (PIV5, a dog virus) as vehicles (vectors) to deliver a BRSV gene into the cell and produce the protein (antigen) from this. As neither of these viruses is found in cattle, they infect bovine cells but do not cause disease in cattle and there is no known cross-reactivity with bovine antibodies, problems associated with the presence of maternal antibodies will be circumvented. The use of SeV and PIV5 will facilitate development of a prime/boost vaccine regimen, which will circumvent a virus vector-specific immune response induced by the 1st vaccine administration, as both viruses are antigenically distinct. Such a regimen is likely to maximize immune responses to the BRSV antigen. We will develop both replication competent and incompetent viruses for both SeV and PIV5. The latter are produced by producing virus infectious particles that have one of the genes for further replication removed. However, these particles can enter cells and produce BRSV proteins. Our cumulative data, both published and unpublished, confirms that both replication competent viruses are independently highly efficient vectors for human RSV vaccine antigens in animal models. The development of replication-incompetent SeV and PIV5 viral vectors will address some safety concerns related to environmental dissemination of recombinant viruses, and will involve the generation of novel rescue technologies (production of virus from copies of the virus genes) for both viruses. BRSV and HRSV contain a protein called the fusion (F) protein which allows the virus to get into cells and is in a different form on the virion surface (pre-fusion) compared to when it interacts with a cell (post-fusion). The F protein induces a response by the immune system and the pre-fusion form has recently been shown to produce a several fold higher immune response than the post fusion form (in an experimental HRSV vaccine. We will exploit this finding to design and generate the most highly immunogenic BRSV vaccine, based on the use of a pre-fusion stable F protein. Precedence for this was demonstrated for human RSV pre-fusion F-based experimental vaccine, which was shown to be many fold superior to native F as a vaccine antigen. Finally, we will bring all of these complementary but disparate elements together to develop novel BRSV vaccines and vaccine regimens and test their efficacy in the final host target for the use of these vaccines, i.e., calves. These vaccines and the results from their study will also serve as the basis for design of effective human vaccines for HRSV.