Infection phenotypes of CpG-recoded RNA viruses in animal models with immature immunity

拟议研究计划的长期目标是更好地了解增加病毒基因组中CpG（胞嘧啶-磷酸-鸟嘌呤）二核苷酸的数量如何影响感染和宿主免疫反应，并利用这些知识来改善兽医领域的疫苗接种。我们特别感兴趣的疫苗技术，将是安全的，适用于怀孕期间和新生儿和年幼的动物与不成熟的免疫力。** 增加RNA病毒基因组中的CpG二核苷酸数量，同时保留蛋白质的天然氨基酸组成和抗原谱，从而减弱感染。然而，减弱的感染会导致强烈的免疫反应。已经证明，宿主免疫比野生型病毒更有效地靶向CpG编码的突变体。尽管取得了重大进展，但对CpG组合物影响毒力的机制还没有完全了解。除了令人兴奋的病毒-宿主相互作用的基本问题外，CpG重编码还可能为更安全的活疫苗提供尖端技术，这将导致减弱的感染，没有病理学，但有强大的免疫反应。到目前为止，一种CpG候选疫苗已经在成年小鼠中进行了表征，显示出有希望的结果。然而，CpG重新编码技术的安全范围一般来说，特别是在具有不成熟免疫系统的动物中，没有定义。* 我们选择Zika病毒（ZIKV）作为模型，因为它会影响胎儿和新生儿。我们的研究应用了一种新的反向遗传学系统来操纵病毒RNA基因组中的CpG频率，新生小鼠模型和世界上第一个ZIKV感染的胎猪模型。为了更好地理解RNA病毒基因组中CpG二核苷酸的增加如何影响感染，并确定CpG二核苷酸的增加是否会导致新生儿和胎儿中感染表型的减弱，我们将解决四个目标：* 首先，我们将评估RNA病毒基因组中CpG二核苷酸数量与不同细胞系中感染表型之间的机制关系。其次，我们将比较感染野生型和CpG编码的ZIKV的新生动物的感染结果（临床体征/死亡率）和免疫应答。最后，我们将探讨胎儿在发育的早期和中期对野生病毒和CpG编码病毒的子宫内感染动力学、病理学和免疫反应。这些实验将提高我们对病毒基因组中的CpG编码如何影响感染和宿主免疫以及CpG编码病毒在免疫力不成熟的动物中的安全范围的理解。这些基础知识将影响CpG疫苗技术的理论和应用，特别是在兽医领域，使加拿大人和全球社会受益。重要的是，该计划还将为年轻研究人员提供优秀的多学科培训。

The long-term goal of the proposed research program is to understand better how increasing the number of CpG (cytosine-phosphate-guanine) dinucleotides in viral genomes affects infection and host immune responses and harness this knowledge to improve vaccination in the veterinary field. We are particularly interested in vaccine technology that will be safe to apply during pregnancy and in newborn and young animals with immature immunity. ******Increasing CpG dinucleotide numbers in RNA viral genomes, while preserving natural amino acid composition of proteins and antigenic profile, attenuates infection. Attenuated infection, however, leads to robust immune responses. It has been demonstrated that host immunity more efficiently targets CpG-recoded mutants than wild-type viruses. Despite significant progress, there is no full understanding of the mechanism(s) through which CpG composition influences virulence. In addition to exciting fundamental questions of virus-host interactions, CpG recoding potentially provides a cutting-edge technology for safer live vaccines, which will cause attenuated infection, no pathology, but robust immune responses. So far, one CpG vaccine candidate has been characterized in adult mice, showing promising results. However, safety margins of the CpG recoding technology in general, and particularly in animals with an immature immune system, are not defined. ******We chose to work with Zika virus (ZIKV) as a model because it affects fetuses and neonates. Our research applies a novel reverse genetics system to manipulate CpG frequencies in viral RNA genomes, a neonatal mouse model, and the first fetal pig model for ZIKV infection in the world. To better understand how increasing of CpG dinucleotides in an RNA viral genome affects infection and to determine if increasing of CpG dinucleotides leads to attenuated infection phenotypes in neonates and fetuses, we will address four objectives:******First, we will evaluate a mechanistic relationship between the number of CpG dinucleotides in an RNA viral genome and infection phenotypes in different cell lines. Second, we will compare infection outcomes (clinical signs/mortality) and immune responses in newborn animals infected with wild and CpG-recoded ZIKV. Finally, we will address in utero infection kinetics, pathology, and immune responses to wild and CpG-recoded viruses in fetuses at the early stage and mid-stage of development.******These experiments will improve our understanding of how CpG recoding in viral genomes affects infection and host immunity, and safety margins of the CpG-recoded viruses in animals with immature immunity. This basic knowledge will influence theories and application of the CpG vaccine technology particularly in the veterinary field benefiting Canadians and global community. Significantly, the program will also provide outstanding multidisciplinary training for young researchers.