Gene Expression in HSV-1 Latency After Corneal Infection

描述(申请人提供)：复发性疱疹间质角膜炎(HSK)仍然是美国传染性致盲疾病的主要原因，唯一有效的治疗方法是角膜移植。HSK是由单纯疱疹病毒1型(HSV-1)从潜伏状态重新激活而触发的，这种潜伏状态是在初次感染期间在感觉神经元中建立的。预防HSK最有可能的方法是阻断三叉神经节神经触发的HSV-1重新激活。曾经被广泛接受的概念，即HSV-1潜伏期在抗原性上是沉默的(因此对免疫系统是不可见的)，现在提供了压倒性证据，证明CD8+T细胞对潜伏感染的神经元进行主动免疫监控，防止完全重新激活导致病毒产生。潜伏状态与持久的细胞免疫渗透有关，该细胞免疫渗透含有病毒特异性CD8+T细胞，这些细胞具有最近接触病毒抗原的特征。它们还具有阻止延迟重新激活的非凡能力。我们的假设是，在潜伏期内将神经节细胞CD8+T细胞渗透到正确的病毒靶点将提供保护，防止再次激活的感染。这项建议的三个具体目标涉及CD8+T细胞渗透的病毒靶点的特征和增强它的手段。我们的研究使用了C57BL6小鼠模型，其中病毒糖蛋白GB编码一个免疫优势靶点。我们的第一个特定目的是测试这一假设，即CD8+T细胞在病毒DNA复制之前对病毒抗原的识别对于以非细胞溶解方式有效地阻止重新激活至关重要。我们已经开发出一种重组HSV-1，它只有在病毒DNA复制开始后才表达gB。我们将确定gB表达的延迟是否会影响淋巴和神经节CD8+T细胞反应的特异性，TG中CD8+T细胞的激活状态，潜伏期的病毒载量，或者gB特异性CD8+T细胞在体外TG培养中阻止HSV-1重新激活的功能能力。这将确定“真正的晚期”病毒抗原在再激活阻断疫苗开发中的重要性。具体目标2将寻求优化策略，以增强TG的CD8+T细胞渗透。在HSV-1缺乏针对CD8+T细胞的Gb抗原的背景下，我们将检测裂解活性启动子和潜伏活性启动子，以表达多聚体形式的免疫优势多肽，并诱导Gb特异的CD8+T细胞在潜伏感染的神经节中渗透。将对最佳策略进行评估，以增强自然的CD8+T细胞反应，并提供更高水平的保护，防止再次激活。第三个目标将检验这样一个假设，即由HSV-1诱导的更广泛的HSV特异性CD8+TCR谱系，其突变破坏了主要的GB表位，将不会改善CD8+T细胞的免疫监控。我们将确定替代反应的再激活阻断能力，以及CD8+T细胞在HSV潜伏感染神经节中渗透的病毒靶点的广度。这可能表明，当免疫压力导致免疫优势表位丢失时，可以在疫苗设计中潜在地利用这些HSV-1蛋白来阻止重新激活。与公众健康相关：本项目中详细介绍的工作旨在加强免疫系统对单纯疱疹病毒感染的保护，特别是角膜的反复感染，感染反复触发反应，使角膜变得不透明，导致疤痕形成，最终导致失明。这项工作对旨在预防触发感染的疫苗设计具有重要意义。

DESCRIPTION (provided by applicant): Recurrent herpes stromal keratitis (HSK) remains the major cause of infectious blinding disease in the US, with the only effective treatment being corneal transplantation. HSK is triggered by reactivation of herpes simplex virus type 1 (HSV-1) from a latent state that is established in sensory neurons during the primary infection. The most likely means of preventing HSK will be to block the triggering HSV-1 reactivation from nerves at the trigeminal ganglia. The once widely-held concept that HSV-1 latency is antigenically silent (and thus invisible to the immune system) is now yielding to overwhelming evidence for active immunosurveillance of latently infected neurons by CD8+T cells, that prevent full reactivation leading to virus production. The latent state is associated with a persisting cellular immune infiltrate that contains virus-specific CD8+T cells which bear hallmarks of recent contact with viral antigen. These also have the remarkable ability to block reactivation from latency. Our overlying hypothesis is that boosting the ganglionic CD8+T cell infiltrate to the correct viral targets during latency will afford protection against reactivated infections. The three specific aims in this proposal address the characteristics of the viral targets of the CD8+T cell infiltrate and the means to augment it. Our studies use the C57Bl6 mouse model, where the viral glycoprotein gB encodes an immunodominant target. Our first specific aim tests the hypothesis that CD8+T cell recognition of viral antigens made before viral DNA replication is critical to effectively block reactivation in a non-cytolytic manner. We have developed a recombinant HSV-1 that expresses gB only after viral DNA replication has initiated. We will determine if the delay of gB expression influences the specificity of the lymph node and ganglionic CD8+ T cell response, the activation state of CD8+ T cells in the TG, the viral load during latency, or the functional capacity of gB-specific CD8+ T cells to block HSV-1 reactivation in ex vivo TG cultures. This will establish the importance of "true late" viral antigens in development of reactivation-blocking vaccines. Specific Aim 2 will seek to optimize strategies that augment the CD8+T cell infiltration of the TG. In the background of HSV-1 lacking the immunodominant gB target for CD8+ T cells, we will test lytic and latency active promoters to express a multimeric form of the immunodominant peptide and induce the infiltration of gB-specific CD8+ T cells in the latently infected ganglia. The optimal strategy will be evaluated for the ability to augment the natural CD8+Tcell response and afford a higher level of protection against reactivation. The third Aim will test the hypothesis that a broader HSV- specific CD8+ TCR repertoire induced by HSV-1 with a mutation that disrupts the dominant gB epitope will not improve CD8+ T cell immunosurveillance. We will determine the reactivation blocking ability of the alternative response, and the breadth of the viral targets of CD8+T cell infiltrate in the HSV latently infected ganglia. This could indicate those HSV-1 proteins that could be potentially exploited in vaccine design to block reactivation when immunological pressure causes loss of immunodominant epitopes. PUBLIC HEALTH RELEVANCE: The work detailed in this project is designed to augment the protection by the immune system against infections by herpes simplex virus, particularly the repeated infection of the cornea, where the infection repeatedly triggers a response that clouds corneal clarity, causes scarring and eventually leads to blindness. The work has important implications in vaccine design aimed to prevent the triggering infections.