OCULAR SHEDDING OF HERPES SIMPLEX VIRUS

本建议涉及两种预防疱疹性角膜炎的策略。 的 首先是阻断单纯疱疹病毒（HSV-1）的建立 通过模拟抗HSV疫苗潜伏期。 牛痘重组病毒将是 构建了表达HSV-1糖蛋白B（gB）的细胞， 牛痘启动子、从细胞膜分泌的gB和gC。 这些 将牛痘重组体与先前测试的重组体进行比较 在晚期启动子下表达gB（评估对HSV-1的保护 实验动物的角膜疾病和HSV-1的建立 神经节和角膜中的潜伏期）。 将确定 抗HSV细胞毒性T淋巴细胞（CTL）是否由这些诱导 疫苗。 有限稀释分析和过继转移研究将 定量和评估这些CTL的保护作用。 对应于预测的gB和gC CTL表位的肽将被 在体外和体内合成并测试。 第二种更加 推测性策略包括在转录本的潜伏期期间进行检测 对应于神经节和角膜中的立即早期HSV-1基因 组织使用敏感和特异性聚合酶链反应（PCR） 测定。 转录本的正义或反义方向将是 通过PCR技术确定。 这些试验还将用于 检测和定量神经节和角膜中的HSV-1 DNA（实验性 动物和进行角膜移植术的患者）。 组织培养 潜伏期的神经元细胞模型将用于确定是否合成 设计为对应于潜伏相关转录物的寡核苷酸 (in有义或反义方向）可以调节HSV-1的再活化， 文化 这种方法将确定是否有一个合理的基础， 潜伏型HSV-1反义治疗的进展。

This proposal concerns two strategies to prevent herpes keratitis. The first involves blocking the establishment of herpes simplex virus (HSV-1) latency by model anti-HSV vaccines. Vaccinia recombinant viruses will be constructed which express HSV-1 glycoprotein B (gB) under an early vaccinia promoter, gB secreted from the cell membrane, and gC. These vaccinia recombinants will be compared to a previously tested recombinant expressing gB under a late promoter (evaluating protection from HSV-1 corneal disease in experimental animals and the establishment of HSV-1 latency, both in ganglia and in the cornea). It will be determined whether anti-HSV cytotoxic T lymphocytes (CTL) are induced by these vaccines. Limiting dilution analysis and adoptive transfer studies will be done to quantitate and evaluate the protective effect of these CTL's. Peptides corresponding to predicted gB and gC CTL epitopes will be synthesized and tested in vitro and in vivo. The second, more speculative strategy involves the detection during latency of transcripts corresponding to immediate early HSV-1 genes in ganglia and corneal tissue using sensitive and specific polymerase chain reaction (PCR) assays. Sense or anti-sense orientation of transcripts will be determined by the PCR technique. These assays will also be used to detect and quantitate HSV-1 DNA in ganglia and in cornea (of experimental animals and in patients undergoing keratoplasty). A tissue culture neuronal cell model of latency will be used to determine if synthetic oligonucleotides designed to correspond to latency associated transcripts (in a sense or anti-sense orientation) can modulate HSV-1 reactivation in culture. This approach will determine if there is a rational basis for the development of anti-sense therapy for latent HSV-1.