Selection Versus Mutation: Reducing the Risk of Vaccine Reversion

针对众多地方性病原体的疫苗接种是家禽业的重要组成部分。如果没有这些疫苗，鸡将在早期死于感染，使该行业的生产率远远低于可持续水平。IBV是一种地方性病毒，在全世界的鸡中引起严重的疾病暴发。目前已有针对IBV的有效且经济可行的疫苗，主要由致病病毒株通过在鸡蛋中传递约100次而生产。在这些传代过程中，病毒从原始致病序列积累了多个序列变异。这最终导致病毒的衰减和减毒活疫苗的生产。这些疫苗已经失去了致病的能力，但仍能在鸡体内引起保护性免疫反应，从而保护鸡免受未来的感染。然而，由于这些是活病毒，考虑到野生毒株和疫苗毒株之间很少的序列变化，恢复到致病性形式的可能性相当大。尽管这些疫苗对家禽业很重要，而且有病毒逆转的风险，但在鸡蛋传代过程中发生的过程和驱动病毒衰减的选择性力量是未知的。重要的是，与病毒种群中已经存在的次要变异的选择相比，病毒序列突变的差异贡献尚未确定。了解这些基本过程对于开发未来疫苗以减少疾病复发的威胁至关重要。这项研究将使用以与疫苗相同的方式生产的传代致病性IBV菌株。与此同时，我们将使用一种独特的系统，使我们能够传递单个病毒克隆，而不是混合病毒群。利用现代深度测序技术，我们将以精细分辨率研究衰减过程中发生的分子变化。这将首次揭示在疫苗生产过程中混合病毒群是如何变化的，以及单个病毒可能发生突变的程度。然后，这些结果将为操纵驱动病毒变化的力量的一系列研究提供信息。第一种方法将使用IBV毒株，其中含有一种来自另一种毒株的蛋白质，这种蛋白质会影响鸡的免疫反应，第二种方法将使用比野生型病毒突变快得多的病毒。通过与野生型病毒相同的方式对这些病毒进行传代和深度测序，我们将了解不同的力量如何驱动病毒序列突变。然后将对这些重组传代病毒进行测试，以确定这一过程是否导致病毒衰减，以及它们是否仍有可能感染接触过接种疫苗禽类的其他鸡。最终，这项研究将揭示IBV是如何通过鸡蛋传世而减弱的，并确定基因组中防止病毒致病但不损害其作为疫苗潜力的关键区域。此外，我们将进一步发展我们对不同压力如何影响衰减过程的理解，并可能通过工程减毒病毒来确定改进疫苗设计过程的方法。通过了解和操纵控制病毒衰减和疫苗生产的过程，我们的目标是确定减少疫苗毒株恢复和造成破坏性疾病暴发的危险的方法。

Vaccination against numerous endemic pathogens is an essential component of the poultry industry. Without these vaccines chickens would succumb to infection at an early age reducing the productivity of the industry well below sustainable levels. IBV is an endemic virus that causes severe disease outbreaks in chickens worldwide. Effective and economically viable vaccines against IBV are available and mainly produced from pathogenic virus strains by passing in eggs approximately one hundred times. During these passages the virus accumulates multiple sequence variations from the original pathogenic sequence. This ultimately leads to attenuation of the virus and the production of a live attenuated vaccine. These vaccines have lost their ability to cause disease but still elicit a protective immune response in the chicken, thus protecting the bird from future infections. However, as these are live viruses the potential for to revert back to a pathogenic form is considerable considering the few sequence changes between wild and vaccine strains.Despite the importance of these vaccines to the poultry industry and the risk of reversion, the processes that occur and the selective forces that drive virus attenuation during egg passage are unknown. Importantly, the differential contribution of virus sequence mutation compared to the selection of minor variants already present in the virus population has not been determined. Understanding these basic processes is essential to the development of future vaccines to reduce the threat of reversion.This study will use passaged pathogenic IBV strains produced in the same way as vaccines. In parallel we will use a unique system that allows us to passage a single virus clone rather than a mixed virus population. Using contemporary deep sequencing technology we will study the molecular changes that occur at fine resolution during the attenuation process. This will for the first time reveal how a mixed population of virus changes during vaccine manufacture and the extent to which individual viruses can mutate. These results will then inform a series of studies that manipulates the forces that drive virus change. The first will use IBV strains that contain a protein from another strain that influences the immune response in the chicken, and the second will use viruses that mutate much faster than wild type viruses. By passaging and deep sequencing these viruses in the same way as the wild type viruses, we will understand how different forces drive virus sequence mutation. These recombinant passaged viruses will then be tested to determine if this process has led to attenuation and also if they maintain the potential to infect other chickens that are exposed to vaccinated birds. Ultimately this research will reveal how IBV is attenuated by egg passage and identify key regions of the genome that prevent the virus from causing disease but do not impair its potential as a vaccine. Moreover, we will further develop our understanding of how different pressures influence the attenuation process and potentially identify ways to improve the process of vaccine design by engineering attenuated viruses. By understanding and manipulating the processes that govern virus attenuation and vaccine production we aim to identify ways of reducing the danger of vaccine strains reverting and causing damaging disease outbreaks.