RECOMBINANT LISTERIA VACCINES FOR MELANOMA

单核细胞增生性李斯特菌是一种革兰氏阳性细菌，它能够进入宿主细胞，逃离胞内小泡，在细胞质内繁殖，并在不接触细胞外环境的情况下直接在细胞间传播。进入宿主细胞胞浆的能力使细菌分泌的蛋白质能够有效地通过MHC-I类抗原处理和递呈途径诱导CD8+细胞毒性T细胞(CTL)。我们建立了单核细胞增多性李斯特氏菌表达和分泌外源抗原的遗传系统。表达淋巴脉络膜脑膜炎病毒(LCMV)核蛋白(NP)或特定MHC-I类限制性NP表位的重组疫苗株在免疫小鼠后能诱导LCMV特异性CD8+细胞毒性T淋巴细胞(CTL)反应。免疫小鼠有效清除LCMV感染的能力表明，这些毒株具有抗病毒保护作用。还构建了表达E11蛋白棉尾兔乳头瘤病毒(CRP)的李斯特菌株。用这些重组子免疫兔，可使CRPV诱导的乳头状瘤消退，并对肿瘤具有保护作用。这些和其他结果证明了李斯特菌疫苗株在诱导抗病毒和抗肿瘤免疫方面的有效性。我们的目标是探索重组李斯特菌菌株作为抗肿瘤疫苗的用途，并确定最佳的减毒和抗原递送策略。其具体目的是：1.构建表达肿瘤排斥抗原的单核细胞增多性乳杆菌疫苗株。将构建表达黑色素瘤相关肿瘤排斥抗原gp100、MART1、Trp2或H-2K/b限制性Trp2181-188表位的单核细胞增多性乳杆菌。2.确定预防性或治疗性应用重组李斯特氏菌疫苗株对肿瘤的建立、生长和消退的影响。两个小鼠肿瘤模型将被用来测试疫苗的有效性。3.构建无菌李斯特菌疫苗株，比较其与野生型和减毒单核细胞增多性李斯特氏菌的免疫原性。诺氏乳杆菌是李斯特菌属的非致病成员。通过转移单核细胞增多性乳杆菌的基因，我们将试图构建具有免疫原性但无毒的嵌合菌株。

Listeria monocytogenes is a gram positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm and spread directly from cell-to-cell without encountering the extracellular milieu. The ability to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently the MHC class I antigen processing and presentation pathway leading to the induction of CD8+ cytotoxic T cells (CTL). We developed a genetic system for expression and secretion of foreign antigens by L. monocytogenes. Recombinant 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 confer antiviral protection as indicated by the ability of immunized mice to efficiently clear LCMV infection. Listeria strains that express the E11 protein cottontail rabbit papillomavirus (CRP) have also been constructed. Immunization of rabbits with these recombinants causes regression of CRPV induced papillomas and protection from carcinoma. These and other results demonstrate the utility of Listeria vaccine strains for inducing antiviral and anti-tumor immunity. Our objectives are to explore the utility of recombinant Listeria strains as anti-tumor vaccines and determine optimal strategies for attenuation and antigen delivery. The specific aims are to: 1. Construct L. monocytogenes vaccine strains expressing tumor rejection antigens. L. monocytogenes strains expressing the melanoma associated tumor rejection antigens gp100, MART1, TRP2, or an H-2K/b restricted TRP2 181-188 epitope will be constructed. 2. Determine the effects of prophylactic or therapeutic administration of recombinant Listeria vaccine strains on tumor establishment, growth and regression. Two murine tumor models will be used to test vaccine efficacy. 3. Construct Listeria innocua vaccine strains and compare their immunogenicity with wild type and attenuated L. monocytogenes. L. innocua is a non-pathogenic member of the Listeria genus. By transferring genes from L. monocytogenes, we will attempt to construct chimeric strains that are immunogenic yet avirulent.