RECOMBINANT LISTERIA VACCINES FOR MELANOMA

单核增生李斯特菌是一种革兰氏阳性细菌，能够进入宿主细胞，从内吞囊泡中逃逸，在细胞质内繁殖，并直接在细胞间传播，而不遇到细胞外环境。这种进入宿主细胞质的能力使细菌分泌的蛋白质能够有效地通过MHC I类抗原加工和递呈途径，从而诱导CD8+细胞毒性T细胞（CTL）。我们建立了单核增生乳杆菌表达和分泌外源抗原的遗传系统。表达淋巴细胞性脉络丛脑膜炎病毒（LCMV）核蛋白（NP）或特定MHC I类限制性NP表位的重组疫苗株能够在接种后诱导小鼠LCMV特异性CD8+细胞毒性T淋巴细胞（CTL）反应。通过免疫小鼠有效清除LCMV感染的能力表明，这些菌株具有抗病毒保护作用。此外，还构建了表达E11蛋白的棉尾兔乳头瘤病毒（CRP）李斯特菌。用这些重组体免疫兔可引起CRPV诱导的乳头状瘤消退和对癌的保护。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效用。我们的目标是探索重组李斯特菌菌株作为抗肿瘤疫苗的效用，并确定衰减和抗原递送的最佳策略。具体目标是：1。构建表达肿瘤排斥抗原的单核增生乳杆菌疫苗株。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TRP2 181-188表位的L. monocytogenes菌株。2. 确定预防或治疗性施用重组李斯特菌疫苗株对肿瘤建立、生长和消退的影响。两种小鼠肿瘤模型将用于测试疫苗的效力。3. 构建无性李斯特菌疫苗株，并与野生型和减毒单核增生李斯特菌进行免疫原性比较。innocua是李斯特菌属的非致病性成员。通过转移单核增生乳杆菌的基因，我们将尝试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.