Immunity to Pneumonic Tularemia

摘要：图拉氏方济氏菌是图拉热病的病原体，可通过多种途径感染人类，包括通过媒介传播。然而，吸入细菌和由此导致的肺炎图拉热症是最危险的疾病形式。这是由于未经治疗的患者的潜伏期短(3-5天)、非特异性症状以及高死亡率(大于80%)。此外，美国和前苏联都已将其武器化，使其成为用作生物武器的可行候选者。尽管世界各地对图拉氏杆菌进行了80多年的研究，但人们对这种细菌与宿主的动态相互作用知之甚少，特别是在气溶胶感染之后。 在过去的几年里，我的实验室提供了大量的证据，证明图拉氏杆菌成功感染宿主并在宿主体内复制的主要机制之一是通过主动抑制宿主在肺部的免疫反应。我们建立了一种可重现的小鼠模型，在该模型中，小鼠暴露于低剂量的全身气雾剂(30-50CFU)中，以研究感染的动态变化和进展。该模型揭示了有关肺炎性图拉热症的几个重要问题。最重要的观察之一是，与更弱的菌株不同，毒力图拉氏杆菌在感染的头几天积极抑制宿主免疫反应，包括肺树突状细胞。虽然我们还没有确定抑制的主要机制，但似乎有几个宿主分子参与其中，包括转化生长因子-β(TGF-β)、前列腺素E2(PGE2)和血管内皮生长因子(VEGF)。图拉氏丝孢子菌调节这些分子表达的具体作用和机制目前正在实验室进行研究。 抗击肺炎图拉热症的一个难以实现的目标是开发有效的非抗生素疗法，这种疗法可以在感染前或感染后不久提供保护。以前的报告表明，适当的免疫原可能能够克服方济各氏菌引起的免疫抑制，使宿主能够有效地根除细菌。例如，在致命的图拉氏丝虫活疫苗株(LVS)挑战前3天注射的减毒图拉氏杆菌活疫苗株(LVS)中提纯的内毒素可以保护所有受感染的动物。在注射CpG核酸基序或阳离子脂质DNA复合体(CLDC)后也观察到了类似的现象。虽然先前已经表明，LVS内毒素不能预防完全毒力的图拉氏F菌的感染，但尚不清楚CpG或CLDC是否能对类似菌株产生保护性免疫。与Juvaris BioTreateutics合作，我们目前正在测试CLDC对图拉氏杆菌毒力气雾剂攻击的保护效果，目的是开发一种易于管理的治疗方法，可以在自然爆发的恐怖事件后迅速分发。 除了了解图拉氏丝虫操纵宿主先天免疫反应的方式外，我们还在研究保护性适应反应发展所需的宿主成分。到目前为止，唯一可用的疫苗(尽管没有在美国获得许可)是一种被称为活疫苗株或LVS的图拉氏F菌B型减毒株。然而，这种疫苗的使用存在一些问题，包括其内毒素的不可预测的相变，使该细菌对肺炎图拉热症完全无效。与科罗拉多州立大学的John Belisle博士合作，我们正在测试来自LVS的无细胞疫苗。使用粗制的亚细胞组分，我们已经能够产生对低剂量气雾剂的保护，几乎与在接种LVS疫苗的动物中观察到的保护相同。此外，我们目前正在使用这些疫苗结合佐剂来确定免疫与肺炎图拉热症存活率的相关性，这些佐剂旨在以两极分化的方式扭曲免疫反应。这种方法将使我们能够确定宿主的特定需求，而不会出现使用转基因小鼠的复杂情况。

Summary: Francisella tularensis, the causative agent for tularemia, can infect humans by a number of routes, including vector-borne transmission. However, it is inhalation of the bacterium, and the resulting pneumonic tularemia, that represents the most dangerous form of disease. This is due to the short incubation time (3-5 days), non-specific symptoms, and a high mortality rate (greater than 80%) in untreated individuals. Furthermore, F. tularensis has been weaponized by both the United States and the former Soviet Union making it a viable candidate for use as a biological weapon. Despite over 80 years of research on F. tularensis around the world, very little is understood about the dynamic interaction of this bacterium with the host, especially following aerosol infection. In the last several years my laboratory has provided abundant evidence that one of the primary mechanisms by which F. tularensis successfully infects and replicates in the host is via active suppression of the host immune response in the lungs. We have a developed a reproducible murine model in which mice are exposed to a low dose, whole body, aerosol (30-50 CFU) in which to study the dynamic changes and progress of infection. This model has revealed several important points concerning pneumonic tularemia. One of the most important observations is that, unlike more attenuated strains, virulent F. tularensis actively suppresses the host immune response, including pulmonary dendritic cells, during the first few days of infection. Although we have not identified the primary mechanism of suppression there are several host molecules that appear to be involved, including Transforming Growth Factor-beta (TGF-beta), Prostaglandin E2 (PGE2) and Vascular Endothelial Growth Factor (VEGF). The specific role and the mechanism by which F. tularensis modulates expression of these molecules is currently under investigation in the laboratory. One elusive goal in combatting pneumonic tularemia is the development of effective non-antibiotic based therapeutics that can provide protection shortly before or after infection. Previous reports have suggested that appropriate immunogens may be able to overcome the immunosuppression invoked by Francisella and enable the host to effectively eradicate the bacterium. For example, LPS purified from the attenuated F. tularensis Live Vaccine Strain (LVS) injected 3 days prior to a lethal LVS challenge protects all infected animals. A similar phenomenon has been observed following injections of CpG nucleic acid motifs or cationic-lipid DNA complexes (CLDC). While it has been previously shown that LVS LPS does not protect against infections with fully virulent F. tularensis, it is not known if CpG or CLDC can engender protective immunity against similar strains. In collaboration with Juvaris Biotherapeutics, we are currently examining the protective efficacy of CLDC against aerosol challenge with virulent F. tularensis with the goal of developing an easily administrated therapeutic that could be quickly distributed following a natural outbreak of terror event. In addition to understanding the way in which F. tularensis manipulates the host innate immune response we are investigating host components required for development of a protective adaptive response. To date, the only vaccine available (although not licensed in the United States) is an attenuated, Type B strain of F. tularensis known as Live Vaccine Strain or LVS. However, there are a number of problems associated in the use of this vaccine including an unpredictable phase shift in its LPS which renders the bacterium completely ineffective against pneumonic tularemia. In collaboration with Dr. John Belisle (Colorado State University) we are testing acellular vaccines derived from LVS. Using crude sub-cellular fractions we have been able to generate protection against low dose aerosols nearly equivalent as that observed in animals vaccinated with LVS. Furthermore, we are currently identifying correlates of immunity for survival of pneumonic tularemia using these vaccines combined in adjuvants designed to skew the immune response in a polarized fashion. This approach will allow us to identify specific requirements in the host without the complications of using genetically modified mice.