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

项目摘要/摘要 假鼻疽伯克霍尔德氏菌(BP)，一种能感染人和动物的环境细菌 类鼻疽病是一种在许多热带地区流行的高度致命的地方病。此外 由于BP在流行地区造成了重大的公共卫生负担，每年导致约89,000人死亡，BP被归类为 作为生物恐怖主义的一级选择剂，很大程度上是因为吸入低剂量的BP会迅速导致 致命性肺炎。由于自然获得的类鼻疽病通常很难诊断，而且需要非常长的时间。 治疗，以及暴露后预防并不是一项实际的公共卫生政策，以对抗暴发的 来自生物恐怖袭击的肺炎类鼻炎，一种安全有效的暴露前疫苗将高度 在两种情况下都是有益的。目前，还没有获得许可的针对BP的疫苗。因此，一种疫苗 对英国石油公司的打击将对生活在疫区的人们、旅行者和军事人员有很大的好处 驻扎在这些地区，这也将减少在生物恐怖袭击中故意释放的风险。 这项应用的目标是开发一种安全和高效的BP疫苗，适用于两者 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。 具体地说，我们将使用一种新颖、安全但有效的减毒活载体，LVSΔCAPB。LVSΔCAPB载体， 在本实验室开发的，是一种进一步减毒版本(对小鼠的毒性降低10,000倍)的多- 图拉氏弗氏杆菌缺失型疫苗(图拉氏亚种)Holarctica活疫苗株；LVS)已经 在人体中进行了广泛的测试；因此，预计LVSΔCAPB是非常安全的。此外，重组的 含有该载体的疫苗已被证明能诱导出强大的体液和细胞介导性(包括两者 CD4+和CD8+)免疫反应，所有这些都可能对BP的持久有效免疫保护很重要。 在初步研究中，用表达免疫保护性BP的RLVΔCAPB疫苗进行同源增强 抗原已经显示出令人印象深刻的保护效果，以对抗强毒BP对呼吸道的挑战 敏感的BALB/c小鼠；保护效果优于未经许可的Bp82减毒疫苗，后者不太可能 由于担心恢复毒力和可能导致慢性病，因此获得批准。 为了实现我们开发安全和高效的BP疫苗的目标，我们将a)构建 优化的表达多种免疫保护性BP抗原融合蛋白的RLVΔCAPB疫苗； 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 通过不同途径接种的疫苗在两种免疫中都具有安全性、传播性、清除性和稳定性 有能力和免疫缺陷的小鼠；d)评估对高毒力BP攻击的保护效果 通过呼吸(IN)路线，这是生物恐怖袭击中最令人担忧的路线，以及皮下 途径，自然感染的主要责任途径；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.