VZV vaccine attenuation and the DNA damage response

摘要 由人类疱疹病毒水痘带状疱疹病毒(VZV)引起的疾病广泛存在，并使人虚弱，但 可以通过使用VZV减毒活疫苗来限制。水痘疫苗在中国取得了巨大的成功。 美国，但许多国家并不广泛使用它，有些国家根本不使用。同一疫苗病毒的高滴度版本是 后来发展成为成年人接种疫苗，以提高VZV免疫力，降低带状疱疹(HZ)的发病率， VZV从神经元潜伏期重新激活的结果。赫兹使人虚弱和复杂，最常见的是慢性 难以治愈的疼痛。赫兹仍然是一个公共卫生问题，因为大多数成年人都有野生型(WT)。 VZV在他们的神经节中存在，并有感染HZ的风险，而HZ疫苗在目标区域远未达到最佳覆盖率 人口。活疫苗病毒Voka需要改进。它可以引起皮疹，潜伏，并导致罕见 HZ 3例。它在遗传上是异质性的，有数百个单核苷酸多态(SNPs) 发生在不同的父母/疫苗等位基因频率上。病毒衰减的基础尚不清楚。5个SNPs是 完全或几乎完全固定的疫苗等位基因，并被怀疑直接衰减。耐人寻味的是，有四个 VZV基因编码IE62，这是一种调节所有VZV基因表达的关键蛋白质。令人兴奋的是，我们的数据 结果表明，WT VZV通过其IE62，启动干细胞表皮标记KRT15在 角质形成细胞和皮肤，而疫苗病毒及其IE62不能。然后我们发现KRT15在我们的 VZV复制需要上皮分化模型。此外，KRT15水平会影响 角质形成细胞DNA损伤反应(DDR)。总而言之，这些数据支持一个全球性的假设，即IE62 上调KRT15以控制DDR的亲病毒方面。Voka在皮肤上是衰减的，因为它的IE62 不触发KRT15的上调来调节DDR亲病毒通路。为了检验这一假设，目标1将 寻求确定IE62中的疫苗SNPs是皮肤模型生长减弱的基础。首先，我们将使用 应用互补分析技术筛选可阻止IE62基因启动复制的疫苗SNPs 角质形成细胞中的沃卡疫苗病毒。其次，我们将开发含有ORF62的WT VZV重组体 用疫苗SNPs检测基因，然后量化它们在皮肤模型中的复制，包括人类皮肤外植体。在……里面 目的2，我们将表征新的IE62-KRT15-DDR亲病毒途径的差异调控步骤 通过KRT15级别和IE62。这包括研究IE62疫苗的基因型如何影响KRT15 转录；KRT15水平如何影响DDR和VZV复制；DDR的哪些组成部分 人上皮细胞分化模型中VZV的前病毒。目标3将寻求确定IE62是否特定于 利用培养的人类神经元，SNPs是Voka从神经元潜伏期重新激活表型差的基础 已成功模拟VZV潜伏期和实验性重新激活的模型。总而言之，这些研究将 定义管理VZV衰减的机制，并为生成定义的 均质活疫苗候选疫苗，在皮肤中减毒，无法从潜伏状态重新激活。

ABSTRACT Diseases caused by the human herpesvirus Varicella Zoster Virus (VZV) are widespread and debilitating but can be limited by using live attenuated VZV vaccines. The varicella vaccine has been hugely successful in the US, but many countries do not use it widely, some not at all. A high titer version of the same vaccine virus was then developed to immunize adults to boost VZV immunity and reduce the incidence of Herpes Zoster (HZ), the result of VZV reactivation from neuronal latency. HZ is debilitating and complicated, most often by chronic pain that is difficult to treat. HZ remains a public health concern, because most adults harbor wild-type (WT) VZV in their ganglia and are at risk for HZ, and the HZ vaccines have far from optimal coverage in the target populations. The live vaccine virus, vOka, needs improvement. It can cause rashes, go latent and cause rare cases of HZ. It is genetically heterogeneous, with hundreds of single nucleotide polymorphisms (SNPs) occurring at different parent/vaccine allele frequencies. The basis of virus attenuation is not known. 5 SNPs are fully or nearly fully fixed for the vaccine allele and are suspected to direct attenuation. Intriguingly, four lie in the VZV gene encoding IE62, a critical protein that regulates expression of all VZV genes. Excitingly, our data shows that WT VZV, through its IE62, turns on expression of the stem cell epidermal marker KRT15 in keratinocytes and skin, while vaccine virus and its IE62 do not. We then found that KRT15 expression in our epithelial differentiation model is required for VZV replication. Furthermore, KRT15 levels influence the keratinocyte DNA Damage Response (DDR). Taken together, the data support a global hypothesis that IE62 upregulates KRT15 to control pro-viral aspects of the DDR. vOka is attenuated in skin because its IE62 does not trigger the upregulation of KRT15 to regulate DDR pro-viral pathways. To test this hypothesis, Aim 1 will seek to establish that vaccine SNPs in IE62 underlie growth attenuation in models of skin. First, we will use a complementation assay to delineate those vaccine SNPs that prevent IE62 from boosting the replication of vOka vaccine virus in keratinocytes. Second, we will develop WT VZV recombinants that contain ORF62 genes with vaccine SNPs, then quantify their replication in models of skin, including human skin explants. In Aim 2, we will characterize steps of the novel IE62-KRT15-DDR pro-viral pathway that is differentially regulated by KRT15 levels and IE62. This includes studying how the IE62 vaccine genotype influences KRT15 transcription; how KRT15 levels affect the DDR and VZV replication; and what components of the DDR are proviral for VZV in the human epithelial differentiation model. Aim 3 will seek to determine if IE62 specific SNPs underlie the poor reactivation phenotype of vOka from neuronal latency, using cultured human neuron models that have successfully modeled VZV latency and experimental reactivation. Together, these studies will define mechanisms governing VZV attenuation and establish foundations for generating a defined homogeneous live vaccine candidate that is attenuated in skin and unable to reactivate from the latent state.