Live Oral Listeria Vaccine Vector

描述（由申请人提供）：本R21申请的目的是探索将噬菌体耐药性作为减毒口服单核细胞增生李斯特菌活疫苗递送平台特征的可行性。我们正在研究一种噬菌体抗性突变，其中Tn917插入中断了一个基因，该基因的产物是生产噬菌体受体所必需的。这种病变也会导致严重的衰减，但保留了单核细胞增生乳杆菌众所周知的免疫刺激特性。我们打算利用申请的资金进行原理验证实验，以确定该菌株的衍生物在表达异源抗原方面的有效性。我们选择表达的抗原是早期分泌抗原靶6 (ESAT-6)，这是一种由结核分枝杆菌产生的分泌蛋白，其作为保护性免疫原的特性已经得到了很好的证实。噬菌体抗性突变体的几个特性揭示了它作为疫苗平台的潜在价值：首先，该突变体在培养的小鼠肠细胞上形成微小斑块，表明它没有减毒到非免疫原性的程度。其次，在口服给药后，突变体在小鼠肠道中发育良好，但在肝脏和脾脏中无法检测到，这说明了突变体的适用性、实用性和安全性。第三，我们采用的亲本菌株通过口服途径对小鼠具有高度传染性，使其成为研究口服（而不是肠外）李斯特免疫的良好模型。该菌株的另一个优点是，它在血清学上与最流行的人类流行病分离株相似。因此，使用该菌株开发疫苗平台将在保护易感人群免受单核增生乳杆菌感染方面具有附加价值，单核增生乳杆菌是一种潜在的生物恐怖剂。在我们的保护研究中，接种疫苗后22天和52天的小鼠在适应性免疫反应中受到10,000倍的刺激-通过接种疫苗的小鼠与接种假疫苗的小鼠在口服毒性亲本（噬菌体敏感）菌株后5天恢复的脾脏集落形成单位来测量。在本申请中，我们提出构建单核增生乳杆菌噬菌体耐药表达平台，并对其安全性和有效性进行试验。具体而言，我们建议：(1)构建表达结核分枝杆菌抗原ESAT-6的基因融合菌株，并记录小鼠口服疫苗接种后对ESAT-6产生的免疫水平和类型。(2)评估口服esat -6表达菌株疫苗小鼠对气溶胶结核分枝杆菌攻击的保护性免疫水平。该平台对结核分枝杆菌的保护性免疫证明将为测试来自其他感染因子（例如病毒）和肿瘤抗原的抗原提供理由——在这些抗原中，该平台可作为治疗辅助手段用于对抗某些形式的癌症——以造福美国的人类健康。公共卫生相关性：本R21申请的目的是探索将噬菌体耐药性作为活的、口服的单核细胞增生李斯特菌疫苗递送平台的一个特征的可行性。我们提出了构建表达平台的方法，并对其进行了小鼠预防结核病的安全性和有效性试验。该平台的保护性免疫证明将为测试来自其他感染因子（例如病毒）的抗原提供理由，并探索该平台作为对抗某些形式癌症的治疗辅助手段的适用性——所有这些都有利于美国的人类健康。

DESCRIPTION (provided by applicant): The purpose of this R21 application is to explore the feasibility of using bacteriophage resistance as a feature of a live, attenuated, oral Listeria monocytogenes vaccine delivery platform. We are studying a phage resistant mutant in which a Tn917 insertion interrupts a gene whose product is required for the production of the phage receptor. This lesion also results in profound attenuation but retention of the immunostimulatory characteristics for which L. monocytogenes is well known. We intend to use the funding requested to conduct proof-of-principle experiments that will determine the usefulness of a derivative of this strain in expressing a heterologous antigen. The antigen we have chosen to express is the early secretory antigenic target 6 (ESAT-6) a secreted protein produced by Mycobacterium tuberculosis whose properties as a protective immunogen are well established. Several properties of the phage resistant mutant revealed its potential value as a vaccine platform: First, the mutant made minute plaques on cultured mouse enterocytes, indicating that it was not attenuated to the point where it would be nonimmunogenic. Second, the mutant prospered in the mouse intestinal tract following oral administration, but could not be detected in the liver and spleen--features that speak to the applicability, practicality, and safety of the mutant. Third, the parent strain we have employed is highly infective for mice via the oral route, making it a good model for studies of oral (as opposed to parenteral) listerial immunization. The strain is additionally advantageous because it is serologically similar to the most prevalent human epidemic isolates. Consequently, the development of a vaccine platform using this strain will have added value in protecting susceptible populations against infection with L. monocytogenes, a potential bioterror agent. In our protection studies, mice challenged at 22 and 52 days post vaccination had a 10,000-fold stimulation in the adaptive immune response--measured as splenic colony forming units recovered from vaccinated versus sham vaccinated mice 5 days post oral challenge with the virulent parental (phage sensitive) strain. In this application, we propose experiments to construct a L. monocytogenes phage resistant expression platform, and conduct tests of its safety and efficacy. Specifically, we propose to: (1) Construct a gene fusion strain expressing the Mycobacterium tuberculosis antigen ESAT-6 and document the level and type of immunity engendered to ESAT-6 following oral vaccination in mice. (2) Assess the level of protective immunity conferred against an aerosol M. tuberculosis challenge in mice orally vaccinated with the ESAT-6-expressing strain. A demonstration of protective immunity to M. tuberculosis by the platform would provide justification for testing antigens from other infectious agents (e.g., viral), and tumor antigens--where the platform could be applied as a therapeutic adjunct to combat certain forms of cancer--to the benefit of human health in the US. PUBLIC HEALTH RELEVANCE: The purpose of this R21 application is to explore the feasibility of using bacteriophage resistance as a feature of a live, oral, Listeria monocytogenes vaccine delivery platform. We propose means to construct the expression platform, and conduct tests of its safety and efficacy in preventing tuberculosis in mice. A demonstration of protective immunity by the platform would provide justification for testing antigens from other infectious agents (e.g., viral) and exploring the platform's applicability as a therapeutic adjunct to combat certain forms of cancer-- all to the benefit of human health in the US.