Attenuation of a highly virulent oncogenic herpesvirus by computer-aided virus reengineering

疫苗是对抗传染病，特别是病毒感染的最有价值和最具成本效益的工具。“千切致死”是一种新的革命性战略，能够以一种可控的方式，以前所未有的效率和速度将病毒衰减到所需的水平。这种策略也被称为合成减毒病毒工程（SAVE），其基础是在编码序列中引入数百个核苷酸突变，而不改变编码蛋白的氨基酸组成。因此，突变的病毒，一种改良的活病毒疫苗（MLV），在抗原性上与亲本致病病毒相同，但其复制和致病能力受损。开放阅读框（orf）中氨基酸的实际编码是有偏倚的，一些同义密码子的使用频率高于其他密码子（密码子偏倚）。因此，使用稀有密码子会导致次优翻译。类似地，但独立于密码子偏倚，orf中的密码子对组合也不是随机的。一些同义密码子对在orf中发现的频率明显高于或低于根据形成密码子对的两个密码子的总体频率（密码子对偏差）所预期的频率。密码子对的使用比密码子偏好对翻译效率的影响更大，因此，不利的密码子对通常很少被使用。利用天然代表性不足的密码子对为有效的蛋白质翻译创造了不利的条件，这导致病毒的衰减。SAVE策略已成功地用于抑制两种不相关的RNA病毒（脊髓灰质炎病毒和流感病毒）和一种致病性细菌（肺炎链球菌），但尚未对大型双链DNA病毒（如阿斯法病毒、痘病毒或疱疹病毒）进行试验。在将这项技术用于生产病毒疫苗之前，必须对其进行全面的研究和测试。拟议的项目将首次探索该技术在大型DNA病毒上的应用前景。拟议项目的总体目标是确定如何通过SAVE技术最有效地减毒疱疹病毒。为了实现这一目标，我们将使用一种独特的小动物模型，该模型涉及高毒力和致癌性马立克氏病疱疹病毒（MDV）和鸡。本项目的中心假设是，MDV基因的密码子对失优将产生完全减毒的疱疹病毒，减毒后的病毒将成为高效的MLV疫苗。突变病毒将首先在体外构建和测试，然后通过体内实验评估其诱导疫苗保护而自身没有毒性的能力。我们预计，所提出的建议将对病毒学和疫苗学产生重大和积极的影响，对SAVE技术的全面了解将最终导致更好的动物和人类疫苗的开发。

Vaccines are the most valuable and cost-effective tools to fight infectious diseases, particularly virus infections. The ´death by thousand cut´ is a new and revolutionary strategy that has enabled attenuation of viruses to a desired level, in a controlled manner, and with unprecedented efficiency and speed. This strategy, also known as synthetic attenuated virus engineering (SAVE), is based on introduction of several hundred nucleotide mutations into coding sequences without changing the amino acid composition of encoded proteins. Thus, the mutated virus, a modified live viral vaccine (MLV), is antigenically identical with the parental pathogenic virus, but its replication and pathogenic capacity is impaired. The actual encoding of amino acid in open reading frames (ORFs) is biased and some synonymous codons are used more often than others (codon bias). Consequently, utilization of rare codons results in suboptimal translation. Similarly, but independently of codon bias, codon pair combinations in ORFs are also not random. Some synonymous codon pairs are found in ORFs significantly more or less frequently than it would be expected based on the overall frequencies of two codons that form a codon pair (codon pair bias). Codon pair usage affects translational efficiency more than codon bias, and thus, unfavorable codon pairs are normally rarely used. Utilization of naturally underrepresented codon pairs creates unfavorable conditions for efficient protein translation, and this causes attenuation of a virus. The SAVE strategy has been successfully used in attenuation of two unrelated RNA viruses (poliovirus and influenza virus) and a pathogenic bacterium (Streptococcus pneumoniae), but it has not been tested on large double-stranded DNA viruses, such as asfarviruses, poxviruses or herpesviruses. Before this technology can be applied for production of viral vaccines, it must be comprehensively studied and tested.The proposed project will explore promising applications this technology for the first time on a large DNA virus. The overall goal of the proposed project is to determine how a herpesvirus can be attenuated most efficiently by the SAVE technology. To address this objective we will use a unique small animal model, which involves the highly virulent and oncogenic Marek´s disease herpesvirus (MDV), and the chicken. The central hypothesis for this project is that codon pair deoptimization of MDV genes will result in fully attenuated herpesviruses and that the attenuated viruses will be highly efficacious MLV vaccines. The mutant viruses will be first constructed and tested in vitro, and then evaluated for their ability to induce vaccinal protection without being virulent themselves by in vivo experiments.We expect that the proposed will have significant and positive impact on virology and vaccinology and that comprehensive understanding of the SAVE technology will ultimately lead the development of superior animal and human vaccines.