VZV vaccine attenuation and the DNA damage response

VZV 疫苗减毒和 DNA 损伤反应

• 批准号: 10657725
• 负责人: Paul R. Kinchington
• 金额: $ 63.5万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657725
• 关键词: Adult; Affect; Alleles; Area; Attenuated; Attenuated Vaccines; Automobile Driving; Binding; Biological Assay; Chickenpox; Chickenpox Vaccine; Clinical; Complement; Country; Cytokeratin; DNA Damage; DNA Repair; Data; Defect; Development; Disease; Epithelium; Exanthema; Experimental Models; Filament; Foundations; Ganglia; Gene Expression; Gene Frequency; Genes; Genetic Transcription; Genotype; Growth; Herpes zoster disease; Herpesviridae; Herpesvirus Type 3; Human; Immunity; Immunize; Incidence; Infection; Infectious Skin Diseases; Life Cycle Stages; Link; Modeling; Mutation; Neurons; Pain; Parents; Pathway interactions; Phenotype; Proteins; Public Health; Recombinants; Reporter; Risk; Simplexvirus; Single Nucleotide Polymorphism; Skin; Stimulus; System; Target Populations; Testing; Trans-Activators; Up-Regulation; VZV vaccine; Vaccines; Viral Genes; Virus; Virus Diseases; Virus Latency; Virus Replication; Work; Zoster Vaccine; attenuation; cell type; chromatin immunoprecipitation; chronic pain; epidermal stem cell; experience; genetic regulatory protein; human embryonic stem cell; improved; insight; keratinocyte; keratinocyte differentiation; neuronal growth; novel; overexpression; prevent; promoter; reactivation from latency; recombinant virus; response; stem cells; uptake; vaccine candidate; varicella-zoster virus immediate early protein 62

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.

抽象的 由人类疱疹病毒带状疱疹病毒（VZV）引起的疾病广泛而令人衰弱，但 可以通过使用活衰减的VZV疫苗来限制。水痘疫苗在 我们，但是许多国家并没有广泛使用它，有些则根本不使用。同一疫苗病毒的高滴度版本是 然后发展以使成年人免疫以增强VZV免疫力并降低疱疹带状疱疹（Hz）的发生率， 神经元潜伏期的VZV重新激活的结果。 Hz令人衰弱且复杂，通常是由慢性 疼痛很难治疗。 HZ仍然是公共健康问题，因为大多数成年人都有野生型（WT） VZV在其神经节中，有HZ风险，Hz疫苗远非目标范围 人群。活疫苗病毒Voka需要改进。它可能导致皮疹，潜在并引起罕见 Hz病例。它在遗传上是异质的，具有数百个单核苷酸多态性（SNP） 发生在不同的父/疫苗等位基因频率上。病毒衰减的基础尚不清楚。 5个SNP是 完全或几乎完全固定在疫苗等位基因上，并被怀疑直接衰减。有趣的是，四个躺在 编码IE62的VZV基因，一种调节所有VZV基因表达的关键蛋白。令人兴奋的是，我们的数据 表明WT VZV通过其IE62打开了干细胞表皮标记KRT15的表达 角质形成细胞和皮肤，而疫苗病毒及其IE62则没有。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水平影响 角质形成细胞DNA损伤反应（DDR）。综上所述，数据支持一个全球假设，即IE62 上调KRT15以控制DDR的促病毒方面。 Voka在皮肤中被衰减，因为它的IE62确实 不能触发KRT15的上调以调节DDR促病毒途径。为了检验这一假设，AIM 1将 寻求在IE62中建立疫苗SNP，这是皮肤模型中生长衰减的基础。首先，我们将使用 互补测定法描绘了那些防止IE62增强复制的疫苗SNP 角质形成细胞中的Voka疫苗病毒。其次，我们将开发含有ORF62的WT VZV重组者 带有疫苗SNP的基因，然后在包括人皮外植体在内的皮肤模型中量化其复制。在 AIM 2，我们将表征新颖的IE62-KRT15-DDR攻击病毒途径的步骤，该途径受差异调节 KRT15级别和IE62。这包括研究IE62疫苗基因型如何影响KRT15 转录； KRT15水平如何影响DDR和VZV复制；以及DDR的哪些组成部分 人类上皮分化模型中VZV的前病毒。 AIM 3将寻求确定IE62是否具体 SNP是使用培养的人神经元的神经元潜伏期中VOKA的不良重新激活表型的基础 成功建模了VZV潜伏期和实验重新激活的模型。这些研究将在一起 定义管理VZV衰减的机制，并为生成定义的基础建立基础 均质的活疫苗候选者在皮肤中衰减，无法从潜在状态重新激活。