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

描述（由申请人提供）：活疫苗具有悠久的历史，可以提供有效的保护，防止随后的传染性挑战。然而，在许多情况下，导致保护的机制没有得到很好的界定。我们的目标是确定革兰氏阴性细菌疫苗菌株的免疫保护机制，使用新的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，而探索性疫苗在预防肺炎方面的能力不一，肺炎是生物恐怖袭击后的一种疾病。我们开发了一种新的方法，基于增强固有的细菌toll样受体(TLR)-4介导的佐剂活性，来产生有效的疫苗菌株，以预防鼠疫和潜在的其他微生物。与其他革兰氏阴性菌类似，鼠疫杆菌在37℃时产生低刺激能力的脂多糖（LPS）。TLR4是脂质a对LPS的细胞受体。我们培养了一株表达脂质a生物合成酶LpxL的鼠疫杆菌菌株，并发现该菌株在37℃下可产生强效的LPS。该菌株通过外周接种在小鼠中是无毒的，这是由于TLR4诱导的抗菌先天免疫机制，这一途径也与强佐剂作用有关。我们的研究结果表明，小鼠接种鼠疫杆菌LpxL菌株可诱导小鼠对模拟腺鼠疫和肺鼠疫的毒力细菌的皮下和鼻内攻击产生充分的保护作用。我们的主要假设是，许多含有增强效力的LPS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。我们打算通过与未增加TLR4刺激的菌株进行比较，在体外和体内测试反应，确定影响疫苗效力的机制，使用活的和死的鼠疫杆菌产生强效LPS。现有的和新的减毒菌株都将被使用。依托原代树突状细胞和T细胞以及基因缺陷小鼠，我们将研究导致体内和体外树突状细胞活化、抗原呈递和T细胞亚群活化的TLR信号通路。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针对革兰氏阴性感染的疫苗菌株可能起作用的机制的信息。将刺激TLR的佐剂活性直接结合到免疫逃避病原体中可能是一种衰减和产生疫苗的新方法。

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.