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

活疫苗在提供有效保护以抵抗随后的免疫方面具有悠久的历史。 传染性挑战然而，在许多情况下， 没有很好的定义。我们的目标是确定革兰氏阴性杆菌的免疫保护机制， 细菌疫苗株，使用新型鼠疫耶尔森氏菌菌株作为模型 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，探索性疫苗已经 对肺炎疾病的保护能力是可变的，肺炎疾病是生物恐怖袭击后的预期形式。 攻击我们已经开发出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 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。鼠疫菌产生脂多糖 (LPS)在37 ℃时刺激能力低。TLR 4是LPS的细胞受体， 脂质a我们生成了一个新的Y。表达LpxL的鼠疫菌株，E.大肠杆菌脂质A 生物合成酶，并发现这在37 ° C下产生有效的LPS。该菌株 通过外周接种在小鼠中无毒力，由于诱导了先天性抗菌 免疫机制通过TLR 4，途径也与强佐剂效应。 我们的研究结果表明，用Y。鼠疫LpxL菌株诱导完全 对小鼠皮下和鼻内攻击的保护作用 细菌，模仿淋巴腺鼠疫和肺鼠疫。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， 有效的疫苗，并且增加的TLR 4信号传导将提供增强的 适应性免疫反应我们建议确定影响 使用活的和死的Y.鼠疫菌产生一个强大的LPS，通过比较， 没有增加的TLR 4刺激的菌株，测试体外和体内应答。 将使用现有的和新的减毒株。依靠初级树突状 细胞和遗传缺陷小鼠，我们将研究TLR信号通路， 体内和体外树突状细胞活化、抗原呈递和T细胞活化。我们 将分析疫苗对皮下和鼻内感染的效果。的 这些研究的完成将提供关于疫苗 革兰氏阴性菌感染的菌株可能起作用。TLR刺激的掺入 佐剂活性直接进入免疫逃避病原体可能构成一种新的方法 用于减毒和疫苗的产生。

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 Gramnegative 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 cells and genetically deficent mice, we will study TLR signaling pathways leading to dendritic cell activation in vivo and in vitro, antigen presentation and T cell 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.