Gene Expression in HSV-1 Latency After Corneal Infection

描述（由申请方提供）：复发性疱疹性基质角膜炎（HSK）仍然是美国感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1型单纯疱疹病毒（HSV-1）从原发感染期间在感觉神经元中建立的潜伏状态重新激活而触发的。预防HSK最可能的方法是阻断三叉神经节神经对HSV-1的激活。HSV-1潜伏期是抗原沉默的（因此对免疫系统不可见）这一曾经被广泛持有的概念现在被压倒性的证据所取代，即CD 8 +T细胞对潜伏感染的神经元进行主动免疫监视，防止完全重新激活导致病毒产生。潜伏状态与持续的细胞免疫浸润有关，细胞免疫浸润含有病毒特异性CD 8 +T细胞，这些细胞具有最近与病毒抗原接触的标志。这些也具有阻止潜伏期再激活的显着能力。我们的假设是，在潜伏期期间促进神经节CD 8 +T细胞浸润到正确的病毒靶点将提供针对再活化感染的保护。在这个建议中的三个具体目标解决的CD 8 +T细胞浸润的病毒目标的特性和手段，以增强it. We的研究使用C57 B16小鼠模型，其中病毒糖蛋白gB编码的免疫显性目标。我们的第一个具体目标测试的假设，即CD 8 +T细胞识别病毒DNA复制前的病毒抗原是至关重要的，以有效地阻止再活化的非细胞溶解的方式。我们已经开发了一种重组HSV-1，仅在病毒DNA复制开始后表达gB。我们将确定gB表达的延迟是否影响淋巴结和神经节CD 8 + T细胞应答的特异性、TG中CD 8 + T细胞的活化状态、潜伏期期间的病毒载量或gB特异性CD 8 + T细胞在离体TG培养物中阻断HSV-1再活化的功能能力。这将确立“真正晚期”病毒抗原在再激活阻断疫苗开发中的重要性。具体目标2将寻求优化增加TG的CD 8 +T细胞浸润的策略。在HSV-1缺乏针对CD 8 + T细胞的免疫显性gB靶标的背景下，我们将测试裂解和潜伏活性启动子以表达免疫显性肽的多聚体形式并诱导gB特异性CD 8 + T细胞浸润潜伏感染的神经节。将评估最佳策略增强天然CD 8 + T细胞应答并提供更高水平的保护以防止再活化的能力。第三个目的将检验以下假设：由具有破坏显性gB表位的突变的HSV-1诱导的更广泛的HSV特异性CD 8 + TCR库将不会改善CD 8 + T细胞免疫监视。我们将确定替代反应的再激活阻断能力，以及HSV潜伏感染神经节中CD 8 +T细胞浸润的病毒靶点的宽度。这可能表明，当免疫压力导致免疫显性表位丢失时，这些HSV-1蛋白可能在疫苗设计中被利用来阻断再活化。公共卫生相关性：该项目中详细介绍的工作旨在增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，感染反复引发反应，使角膜清晰度模糊，导致疤痕，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.