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）引起的疾病是广泛的和使人衰弱的， 可以通过使用减毒活VZV疫苗来限制。水痘疫苗在美国取得了巨大的成功， 美国，但许多国家没有广泛使用它，有些根本没有。同一种疫苗病毒的高滴度版本， 然后开发用于免疫成人以增强VZV免疫力并降低带状疱疹（HZ）的发病率， VZV从神经元潜伏期再激活的结果。HZ是衰弱和复杂的，最常见的是慢性 HZ仍然是一个公共卫生问题，因为大多数成年人携带野生型（WT） VZV在他们的神经节中存在，并且有HZ的风险，HZ疫苗在目标中的覆盖率远未达到最佳 人口。活疫苗病毒vOka需要改进。它可以导致皮疹，去潜伏和导致罕见的 例HZ。它具有遗传异质性，有数百个单核苷酸多态性（SNP） 发生在不同的亲本/疫苗等位基因频率。病毒减毒的基础尚不清楚。5 SNPs 对于疫苗等位基因完全或几乎完全固定，并且怀疑直接减毒。有趣的是，四个谎言 编码IE62的VZV基因，IE62是调节所有VZV基因表达的关键蛋白。令人兴奋的是，我们的数据 显示WT VZV通过其IE62开启干细胞表皮标志物KRT 15在细胞中的表达， 角质形成细胞和皮肤，而疫苗病毒及其IE62不。然后，我们发现KRT 15在我们的细胞中的表达， VZV复制需要上皮分化模型。此外，KRT 15水平影响 角质形成细胞DNA损伤反应（DDR）。综上所述，这些数据支持一个全球假设，即IE62 上调KRT 15以控制DDR的前病毒方面。vOka在皮肤中衰减，因为其IE62 不触发KRT 15的上调以调节DDR前病毒途径。为了验证这一假设，目标1将 试图确定IE62中疫苗SNP是皮肤模型中生长衰减的基础。首先，我们将使用 互补测定来描绘那些阻止IE62促进免疫缺陷病毒复制的疫苗SNP。 角质形成细胞中的vOka疫苗病毒。其次，我们将开发含有ORF 62的WT VZV重组体， 基因与疫苗SNPs，然后量化他们的复制模型的皮肤，包括人类皮肤外植体。在 目的2，我们将表征新的IE62-KRT 15-DDR前病毒途径的步骤，其被差异调节 KRT 15和IE62的水平。这包括研究IE62疫苗基因型如何影响KRT 15 转录; KRT 15水平如何影响DDR和VZV复制;以及DDR的组成部分是什么。 在人上皮分化模型中VZV的前病毒。目标3将寻求确定IE62特定 使用培养的人神经元，SNP是来自神经元潜伏期的vOka的不良再激活表型的基础 成功模拟VZV潜伏期和实验再激活的模型。这些研究将 定义控制VZV衰减的机制，并为生成定义的 在皮肤中减毒且不能从潜伏状态再活化的均质活疫苗候选物。

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.