L MONOCYTOGENES AS A LIVE VACCINE VEHICLE

我们已经开发了一种表达和分泌外源基因的遗传系统， 细胞内细菌单核细胞增生李斯特菌的抗原。 重组李斯特菌疫苗株表达淋巴细胞 脉络丛脑膜炎病毒（LCMV）核蛋白（NP）或特定的MHC类 I限制性NP表位，能够诱导LCMV特异性CD8+细胞毒性 接种疫苗后小鼠的T淋巴细胞（CTL）应答。 这些菌株 也赋予抗病毒保护，如免疫的 小鼠清除LCMV感染。 表达E1的李斯特菌菌株 棉尾兔乳头瘤病毒（CRPV）蛋白也已被 构建了 用这些重组体免疫兔子， CRPV诱导的乳头状瘤消退， carcinoma. 我们的目标是描述活L. 单核细胞增生疫苗株和免疫系统。 我们将重点关注MHC I类抗原的加工和呈递导致产生 CD8+ T细胞。 使用LCMV模型的研究将确定 对于有效诱导保护性细胞介导的免疫至关重要。 本提案产生的信息将为以下方面提供基础： 未来的努力将使用活的减毒李斯特菌重组体作为新的 用于提供针对细胞内病原体和癌症的保护的工具。 具体目标是： 1.构建减毒L.单核细胞增生疫苗株和确定 其毒性和持久性特征。 L.单核细胞增生 人类病原体 优化免疫原性的减毒方法， 将确定最小毒力。 2.构造L.抗原改变的单核细胞增生疫苗株 表达水平、抗原稳定性和抗原定位。 我们将 确定将抗原递送至MHC I类的最有效方式 加工和呈递途径。 3.确定减毒突变和改变的影响， 抗原递送对诱导抗病毒CTL的影响。 初级CTL 反应，CTL前体频率和对LCMV的保护将是 测定了 CTL存储器的持续时间和特性也将被 测定 这些和其他分析将提供定量评估 重组李斯特菌疫苗诱导的CTL介导的免疫。

We have developed a genetic system for expression and secretion of foreign antigens by the intracellular bacterium Listeria monocytogenes. Recombinant Listeria vaccine strains expressing the lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP), or a specific MHC class I restricted NP epitope, are able to induce LCMV specific CD8+ cytotoxic T lymphocyte (CTL) responses in mice following vaccination. These strains also confer antiviral protection as indicated by the ability of immunized mice to clear LCMV infection. Listeria strains that express the E1 protein of cottontail rabbit papillomavirus (CRPV) have also been constructed. Immunization of rabbits with these recombinants resulted in regression of CRPV induced papillomas and consequent protection from carcinoma. Our objective is to characterize the interaction between live L. monocytogenes vaccine strains and the immune system. We will focus on MHC class I antigen processing and presentation leading to the generation of CD8+ T cells. Studies with the LCMV model will identify parameters that are critical for efficient induction of protective cell mediated immunity. Information resulting from this proposal will provide the foundation for future efforts to use live, attenuated Listeria recombinants as novel tools for providing protection against intracellular pathogens and cancer. The specific aims are to: 1. Construct attenuated L. monocytogenes vaccine strains and determine their virulence and persistence characteristics. L. monocytogenes is human pathogen. Methods for attenuation that optimize immunogenicity and minimize virulence will be identified. 2. Construct L. monocytogenes vaccine strains with alterations in antigen expression level, antigen stability, and antigen localization. We will determine the most effective way to deliver antigen to the MHC class I processing and presentation pathway. 3. Determine the effects of attenuating mutations and alterations in antigen delivery on the induction of antiviral CTL. Primary CTL responses, CTL precursor frequencies and protection against LCMV will be measured. The duration and characteristics of CTL memory will also be determined. These and other assays will provide a quantitative assessment of CTL mediated immunity induced by vaccination with recombinant Listeria.