The role of LPS-TLR4 signaling in live vaccine-induced protective responses

描述(由申请人提供)：活疫苗在提供有效保护以抵御随后的感染挑战方面有很长的历史。然而，在许多情况下，导致保护的机制没有得到很好的界定。我们的目标是以新型鼠疫耶尔森氏菌株为模型系统，确定革兰氏阴性菌疫苗株的免疫保护机制。革兰氏阴性菌鼠疫耶尔森氏菌是鼠疫的病原体。目前还没有获得许可的鼠疫疫苗，而且探索性疫苗对肺炎的预防能力各不相同，预计在生物恐怖袭击后会出现这种形式。我们开发了一种新的方法来产生有效的疫苗株，用于预防鼠疫和潜在的其他微生物，基于固有的细菌Toll样受体(TLR)-4介导的佐剂活性的增强。与其他革兰氏阴性菌相似，鼠疫耶尔森氏菌在37℃时产生一种刺激能力较低的脂多糖(LPS)。TLR4是通过其脂类A对脂多糖的细胞受体。我们产生了一株新的表达LpxL的鼠疫杆菌菌株，LpxL是一种大肠杆菌脂类A生物合成酶，并发现它在37℃时能产生有效的内毒素。该菌株在小鼠周围接种是无毒的，因为通过TLR4诱导了抗菌天然免疫机制，这一途径也与强佐剂作用有关。我们的结果表明，用鼠疫耶尔森氏菌LpxL株免疫小鼠，可以完全抵抗皮下和鼻腔内感染强毒细菌的攻击，类似于腺鼠疫和肺鼠疫。我们的主要假设是，许多含有增强效力的内毒素的活细菌疫苗株是有效的疫苗，并且增加的TLR4信号将提供增强的适应性免疫反应。我们建议通过与没有增加TLR4刺激的菌株进行比较，测试体外和体内反应，确定使用产生有效内毒素的活的和灭活的鼠疫杆菌来确定影响疫苗效力的机制。现有的和新的减毒株都将使用。依托原代树突状细胞和T细胞以及基因缺陷小鼠，我们将研究体内和体外导致树突状细胞激活的TLR信号通路、抗原提呈和T细胞亚群激活。我们将分析疫苗对皮下和鼻腔感染的效果。这些研究的完成将提供有关革兰氏阴性感染疫苗株可能发挥作用的机制的信息。将刺激TLR的佐剂活性直接结合到免疫逃避病原体中可能构成一种新的减毒和疫苗生成方法。与公共卫生相关：该提案将确定针对某些细菌感染的活疫苗发挥作用的机制。我们将使用新型无毒、免疫刺激的鼠疫杆菌菌株作为模型系统。我们的结果可能有助于设计未来针对鼠疫和其他传染病的疫苗。

DESCRIPTION (provided by applicant): Live vaccines have a long history for providing efficient protection against subsequent infectious challenge. However, the mechanisms leading to protection are in many cases not well defined. Our goal is to define mechanisms for immune protection by Gram-negative bacterial vaccine strains, using novel Yersinia pestis strains as model systems. The gram-negative bacterium Yersinia pestis is the causative agent of plague. Currently there is no available licensed plague vaccine, and exploratory vaccines have variable ability to protect against pneumonic disease, the form expected after a bioterror attack. We have developed a new method for the generation of efficient vaccine strains for protection against plague and potentially other microorganisms, based upon enhancement of inherent bacterial Toll-like receptor (TLR)-4 mediated adjuvant activity. Similar to various other gram-negative bacteria, Y. pestis produces a lipopolysaccharide (LPS) with low stimulatory ability at 37:C. TLR4 is the cellular receptor for LPS via its lipid A. We generated a new Y. pestis strain expressing LpxL, an E. coli lipid A biosynthesis enzyme, and found this to produce a potent LPS at 37:C. This strain is avirulent in mice by peripheral inoculation, due to induction of antibacterial innate immune mechanisms via TLR4, a pathway also associated with strong adjuvant effects. Our results indicate that vaccination of mice with the Y. pestis LpxL strain induces full protection against both subcutaneous and intranasal challenge of mice with virulent bacteria, mimicking bubonic and pneumonic plague. Our main hypotheses are that many live bacterial vaccine strains containing LPS with increased potency are efficient vaccines, and that the increased TLR4 signaling will provide enhanced adaptive immune responses. We propose to determine mechanisms influencing the vaccine efficacy using live and killed Y. pestis producing a potent LPS, by comparing to strains without increased TLR4 stimulation, testing both in vitro and in vivo responses. Both existing and novel attenuated strains will be used. Relying on primary dendritic and T cells and genetically deficient mice, we will study TLR signaling pathways leading to dendritic cell activation in vivo and in vitro, antigen presentation and T cell subset activation. We will analyze vaccine effects against both subcutaneous and intranasal infection. The completion of these studies will provide information on the mechanism by which vaccine strains towards Gram-negative infections may act. Incorporation of TLR- stimulating adjuvant activity directly into immune-evading pathogens may constitute a novel method for attenuation and generation of vaccines. PUBLIC HEALTH RELEVANCE: This proposal will identify mechanisms by which live vaccines against some bacterial infections work. We will use novel avirulent, immunostimulatory strains of the plague bacillus Yersinia pestis as a model system. Our results may help in the design of future vaccines against plague and other infectious diseases.