Development of a Safe and Potent Vaccine Against Melioidosis using the LVS dcapB Vector Platform

项目总结/摘要 类鼻疽伯克霍尔德菌（Burkholderia pseudomallei，Bp）是一种能够感染人和动物的环境细菌 是类鼻疽病的病原体，这是一种在许多热带地区流行的高度致命的疾病。此外 由于其在流行地区的重大公共卫生负担，每年造成约89，000人死亡，Bp被归类为 作为生物恐怖主义的一级选择剂，在很大程度上是因为吸入低剂量的BP可以迅速引起 致命的肺炎由于自然获得性类鼻疽通常很难诊断， 接触后预防并不是一项切实可行的公共卫生政策， 肺炎类鼻疽从生物恐怖袭击，一个安全有效的暴露前疫苗将是高度 在这两种情况下都有好处。目前，没有针对Bp的许可疫苗。因此，疫苗 对生活在流行地区的人们、旅行者和军事人员来说， 它还将减少在生物恐怖袭击中故意释放的风险。 本申请的目的是开发一种安全、高效的抗Bp疫苗， 生物防御和公共卫生目的，基于易于扩展的单载体平台疫苗。 具体而言，我们将使用一种新型、安全、有效的减毒活载体LVS ΔcapB。LVS ΔcapB载体， 在这个实验室开发的，是一种进一步减毒的版本（在小鼠中毒性降低10，000倍）的多- 缺失型土拉弗朗西斯菌疫苗（F.土拉热亚种北极熊活疫苗株; LVS）已经 在人体中进行了广泛的测试;因此，预计LVS ΔcapB非常安全。此外，重组 包含该载体的疫苗已经显示出诱导有效的体液和细胞介导的（包括两者）免疫应答 CD 4+和CD 8+）免疫应答，所有这些对于针对Bp的持久有效免疫保护都可能是重要的。 在初步研究中，用表达免疫保护性Bp的rLVS ΔcapB疫苗进行同源加强免疫， 抗原已经证明了对呼吸道攻击的惊人的保护作用， 敏感的BALB/c小鼠;保护作用上级于未经许可的减毒Bp 82疫苗，后者不太可能 由于担心毒力返强和可能导致慢性疾病， 为了实现我们的目标，开发一个安全和高效的疫苗对BP，我们将a）构建 表达多种免疫保护性Bp抗原作为融合蛋白的优化的rLVS ΔcapB疫苗; B） 在体外系统地评估疫苗的蛋白质表达、生长动力学和稳定性; c）评估 通过不同途径施用的疫苗在免疫和非免疫接种中的安全性、传播、清除和稳定性 d）评估针对高毒力Bp攻击的保护效力 通过呼吸（IN）途径，这是生物恐怖袭击中最受关注的途径， 途径，主要负责自然感染的途径;以及e）探索保护性免疫的相关性 使用效力不断提高的疫苗。

PROJECT SUMMARY/ABSTRACT Burkholderia pseudomallei (Bp), an environmental bacterium capable of infecting humans and animal hosts, is the causative agent of melioidosis, a highly fatal disease endemic in many tropical areas. In addition to its significant public health burden in endemic regions, causing ~89,000 deaths annually, Bp is categorized as a Tier 1 Select Agent of bioterrorism, in large part because inhalation of low doses of Bp can cause rapidly fatal pneumonia. As naturally acquired melioidosis is often difficult to diagnose and requires very lengthy treatment, and as post-exposure prophylaxis is not a practical public health policy for countering an outbreak of pneumonic melioidosis from a bioterrorist attack, a safe and effective pre-exposure vaccine would be highly beneficial in both circumstances. Currently, there are no licensed vaccines against Bp. Hence, a vaccine against Bp would be of great benefit to people living in endemic regions, travelers, and military personnel stationed in these areas, and it would also reduce the risk from an intentional release in a bioterrorist attack. The goal of this application is to develop a safe and highly potent vaccine against Bp, suitable for both biodefense and public health purposes, based on a readily expandable single vector platform vaccine. Specifically, we shall use a novel, safe, yet potent live attenuated vector, LVS ΔcapB. The LVS ΔcapB vector, developed in this laboratory, is a further attenuated version (>10,000 fold less virulent in mice) of a multi- deletional Francisella tularensis vaccine (F. tularensis subsp. holarctica Live Vaccine Strain; LVS) already extensively tested in humans; hence, LVS ΔcapB is anticipated to be exceedingly safe. Moreover, recombinant vaccines comprising this vector have been shown to induce potent humoral and cell-mediated (including both CD4+ and CD8+) immune responses, all likely important to long-lasting potent immunoprotection against Bp. In preliminary studies, homologous boosting with rLVS ΔcapB vaccines expressing immunoprotective Bp antigens has already demonstrated impressive protective efficacy against respiratory challenge with virulent Bp in sensitive BALB/c mice; protection was superior to the unlicensed attenuated Bp82 vaccine, which is unlikely to be approved because of concerns about reversion to virulence and the possibility of causing chronic disease. To accomplish our goal of developing a safe and highly potent vaccine against Bp, we shall a) construct optimized rLVS ΔcapB vaccines expressing multiple immunoprotective Bp antigens as fusion proteins; b) evaluate the vaccines systematically in vitro for protein expression, growth kinetics, and stability; c) evaluate the vaccines administered by different routes for safety, dissemination, clearance, and stability in both immune competent and immune deficient mice; d) evaluate protective efficacy against challenge with highly virulent Bp by the respiratory (IN) route, the route of greatest concern in a bioterrorist attack, and by the subcutaneous route, the route principally responsible for natural infection; and e) explore correlates of protective immunity using vaccines of escalating efficacy.