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

描述（由申请方提供）：活疫苗在提供有效保护以抵抗随后的感染性攻毒方面具有悠久的历史。然而，在许多情况下，导致保护的机制并没有得到很好的界定。我们的目标是确定革兰氏阴性细菌疫苗株的免疫保护机制，使用新型鼠疫耶尔森菌菌株作为模型系统。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，探索性疫苗具有不同的预防肺炎疾病的能力，这是生物恐怖袭击后预期的形式。我们已经开发了一种新的方法，用于产生有效的疫苗株，以保护免受鼠疫和潜在的其他微生物，基于增强固有的细菌Toll样受体（TLR）-4介导的佐剂活性。与其它革兰氏阴性菌相似，Y。鼠疫杆菌在37 ℃下产生具有低刺激能力的脂多糖（LPS）。TLR 4是LPS通过其脂质A的细胞受体。我们生成了一个新的Y。表达LpxL的鼠疫菌株，E. coli脂质A生物合成酶，并发现该酶在37 ℃下产生有效的LPS。该菌株通过外周接种在小鼠中是无毒的，这是由于通过TLR 4诱导抗菌先天免疫机制，该途径也与强佐剂效应相关。我们的研究结果表明，用Y。鼠疫LpxL菌株诱导针对用强毒细菌的小鼠皮下和鼻内攻击的完全保护，模拟腺鼠疫和肺鼠疫。我们的主要假设是，许多含有具有增加的效力的LPS的活细菌疫苗菌株是有效的疫苗，并且增加的TLR 4信号传导将提供增强的适应性免疫应答。我们建议使用活的和死的Y来确定影响疫苗效力的机制。通过与没有增加的TLR 4刺激的菌株进行比较，测试体外和体内反应，将使用现有的和新的减毒株。依靠原代树突状细胞和T细胞和遗传缺陷小鼠，我们将研究TLR信号通路导致树突状细胞在体内和体外活化，抗原呈递和T细胞亚群活化。我们将分析疫苗对皮下和鼻内感染的效果。这些研究的完成将提供关于疫苗株对革兰氏阴性菌感染可能起作用的机制的信息。将TLR刺激佐剂活性直接掺入免疫逃避病原体中可构成用于减毒和产生疫苗的新方法。公共卫生相关性：该提案将确定针对某些细菌感染的活疫苗的工作机制。我们将使用新型的无毒、免疫刺激性鼠疫杆菌鼠疫耶尔森氏菌菌株作为模型系统。我们的研究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.