Improved vaccine manufacture to control foot-and-mouth disease: Production of recombinant vaccines by design

我们面临的最大挑战之一将是满足日益增长的粮食需求，尤其是在发展中国家。动物疾病对畜牧业的生产力有重大影响，保障动物福利将是粮食生产最大化的一个主要部分。口蹄疫在世界许多地区流行，影响大量动物，包括野生动物和牲畜（如牛、绵羊、山羊、猪），由于与疫情相关的巨大经济损失，人们非常担心口蹄疫。因此，改善对口蹄疫的控制可以持续改善畜牧业的绩效，并在世界范围内具有巨大的经济和社会价值，特别是在发展中地区，畜牧业被视为使数百万人摆脱贫困的一种手段。在无口蹄疫区域，如英国，口蹄疫的费用主要来自维持无疾病状态的需要。然而，由于口蹄疫的周期性入侵，无口蹄疫国家可能面临巨大损失，2001年英国爆发的估计成本为80亿至100亿英镑。未来对英国的入侵无疑会发生，并有可能造成巨大的经济损失。疫苗接种仍然是控制病毒性疾病的最有效方法，估计每年接种23.5亿剂口蹄疫疫苗。目前，在印度、东南亚和南美洲正在开展以疫苗接种为主导的根除运动。口蹄疫疫苗接种的两个主要制约因素是：(i)生产能力和（ii）需要从田间分离株生产新疫苗。生产能力：口蹄疫疫苗是在细胞培养中生产的化学灭活病毒制剂，口蹄疫的高度传染性要求它们在昂贵的、高度封闭的设施中生产。因此，提高现有生产设施疫苗产量的措施可以通过增加全球可用于口蹄疫控制的疫苗数量产生巨大影响。新疫苗：口蹄疫由口蹄疫病毒（FMDV）引起。口蹄疫控制的一个主要问题是存在七种口蹄疫病毒血清型，每种血清型都由多种不断进化的病毒亚型构成。重要的是，针对一种血清型接种疫苗并不能预防其他血清型。此外，至关重要的是，在一种血清型内，疫苗可提供针对流行暴发毒株的保护。这就需要定期生产新疫苗，特别是针对目前疫苗不太匹配的新出现的毒株。疫苗生产使用已建立的细胞系（如BHK细胞），这些细胞系通常不是口蹄疫病毒的目标。因此，新疫苗的生产严重依赖于田间病毒在细胞培养物中生长的适应性，这对某些田间病毒来说可能是耗时或不可能的。因此，挑战在于生产足够数量的口蹄疫控制疫苗。我们已经证明，口蹄疫病毒的细胞培养适应性和细胞培养生长的改善是通过病毒衣壳中两个不同位点中的任何一个的特定突变来实现的。我们的假设是，具有这种突变的重组FMDV将具有内置的、改进的细胞培养生长，并可用于克服从田间病毒生产新疫苗的延迟，并通过改进的细胞培养生长，提高现有生产能力的疫苗产量。在这里，我们将使用反向遗传学对FMDV进行基因工程，使其包括病毒衣壳中的突变，从而改善病毒生长并开发这些病毒疫苗。我们将重点关注四种最普遍的血清型，包括目前已知疫苗产量低且已被证明难以在细胞培养中生长的病毒。我们的目标是开发可销售的产品，通过减少口蹄疫在全球的流行以及在英国和欧洲进一步爆发口蹄疫的可能性，这些产品可能具有巨大的价值。

One of our biggest challenges will be to meet a growing demand for food especially in the developing world. Animal diseases have a significant impact on the productivity of the livestock industry and safeguarding animal welfare will be a major part of maximising food production. Foot-and-mouth disease (FMD) is endemic in many regions of the world and affects huge numbers of animals, both wild animals and livestock (e.g. cattle, sheep, goats, pigs) and is greatly feared due to the enormous economic losses associated with outbreaks. Thus improved control of FMD could sustainably improve the performance of livestock industries and have enormous economic and social value worldwide especially in developing regions where livestock are seen as a means to raise millions from poverty. In FMD-free regions, such as UK, the costs of FMD primarily result from the need to maintain a disease free status. However, FMD-free countries can face enormous losses as the result of periodic incursions and the estimated cost of the 2001 UK outbreak was £8-10Bn. Future incursions into the UK will undoubtedly occur with the potential to inflict substantial economic losses. Vaccination remains the most effective approach for controlling viral diseases and an estimated 2.35 billion doses of FMD vaccine are administered annually. Currently vaccination-led eradication campaigns are ongoing in India, South-East Asia and South America. Two major constraints on FMD vaccination are (i) production capacity and (ii) the need to produce new vaccines from field isolates. Production capacity: FMD vaccines are chemically-inactivated virus preparations produced in cell-culture and the highly contagious nature of FMD requires they are produced in expensive, high-containment facilities. Thus measure that improve vaccine yield from existing production facilities could have an enormous impact by increasing the amount of vaccine available globally for FMD control.New vaccines: FMD is caused by FMD virus (FMDV). A major problem for FMD control is the existence of seven FMDV serotypes, each formed by multiple and constantly evolving virus subtypes. Importantly, vaccination against one serotype does not protect against the others. Further, it is essential that within a serotype the vaccine offers protection against the circulating outbreak strain. This necessitates the periodic need to produce new vaccines, especially against emerging strains for which the current vaccines are a poor match. Vaccine production uses established cell lines (such as BHK cells) that are not normally targeted by FMDV. Thus, the production of new vaccines is critically dependent upon adaptation of a field virus for growth in cell-culture which can prove time consuming or impossible for some field viruses. Thus the challenge is to produce sufficient quantities of vaccine for FMD control We have shown that cell-culture adaptation of FMDV and improved cell-culture growth are achieved by specific mutations at either of two distinct sites in the viral capsid. It is our hypothesis that recombinant FMDV with such mutations will have in-built, improved cell-culture growth and can be used to overcome the delays in producing new vaccines from field viruses and, through improved cell-culture growth, increase vaccine yield from the existing manufacturing capacity. Here we will use reverse genetics to genetically engineer FMDV to include mutations in the viral capsid that result in improved virus growth and develop these viruses a vaccines. We will focus on four of the most prevalent serotypes and include viruses that are known to currently give a poor vaccine yield and have proven difficult to grow in cell-culture. Our ambition is to develop marketable products that could have enormous value by reducing the prevalence of FMD worldwide and the likelihood of further outbreaks of FMD in the UK and Europe.