Identification of new antigens for a plague vaccine

描述（由申请人提供）：从历史上看，鼠疫是人类已知的最具破坏性的流行病之一（仅次于天花），在三次有记录的鼠疫大流行中，总共造成2亿多人死亡。由于鼠疫菌有可能引起大规模爆发，世界卫生组织将鼠疫归类为再出现的传染病，并且由于全球变暖，人们担心可能会出现第四次大流行，导致鼠疫在啮齿动物宿主中的流行率增加。目前鼠疫杆菌作为一种生物武器的相关性是由于其高毒力和多种抗生素耐药菌株的发展。虽然用鼠疫疫苗对人类进行免疫将阻止使用鼠疫杆菌作为生物武器，但目前还没有针对鼠疫的疫苗。在目前的资助期间，我们发现了一种新的鼠疫杆菌抗原（博朗脂蛋白），这种抗原促进了腺鼠疫和肺鼠疫的发展。我们的研究表明，用lpp和色素沉着位点（pgm）基因缺失的鼠疫菌突变株免疫小鼠，可预防由高毒力鼠疫菌CO92菌株引起的肺鼠疫。我们现在已经描述了Lpp引发的导致宿主损伤的信号通路。最重要的是，我们的数据表明lpp突变体不能在巨噬细胞内存活，这与该突变体中应激反应基因（htrA）的下调有关。我们从这些数据推断，其他与应激相关的基因（例如，外核糖核酸酶）也可能参与唇酶介导的细菌毒性减弱。事实上，缺失编码多核苷酸磷酸化酶（PNPase）的基因也能在小鼠全身感染模型中减弱鼠疫杆菌，并提供对鼠疫的保护。我们为这笔赠款提出了三个具体目标。目的1是产生鼠疫杆菌CO92的双突变体，其中编码纤溶酶原激活蛋白酶（pla）、pnp和其他两种显性外核糖核酸酶（如rnb [RNase II]和rnr [RNase R]）的基因将从lpp基因缺失的鼠疫杆菌CO92背景菌株中删除。这些突变体将在腺鼠疫和肺鼠疫模型（小鼠和大鼠）中进行衰减测试。在目标2中，我们将在动物模型中描述最高度减毒突变体的保护性免疫反应，以及这种突变体对亲本CO92菌株的攻击所提供的保护。我们已经在WT CO92菌株中发现了几种免疫原性蛋白，它们与感染CO92菌株的大鼠的免疫血清发生反应。这些抗原可能是重组鼠疫疫苗中添加的优秀候选抗原。因此，在目标3中，我们将首先从WT细菌中删除这些基因，以证明它们对细菌毒力的影响。其次，我们将纯化这些免疫原性蛋白，并在小鼠和大鼠免疫并随后感染毒力鼠疫菌后评估其保护作用。总的来说，我们的研究重点是鉴定新的鼠疫杆菌减毒活疫苗株，并表征新的免疫保护性CO92抗原，这可能对重组鼠疫疫苗很重要。

DESCRIPTION (provided by applicant): Historically, plague is one of the most devastating epidemic diseases known to mankind (second only to smallpox), resulting overall in more than 200 million deaths related to three recorded plague pandemics. Since Y. pestis has the potential to cause large-scale outbreaks, the WHO has categorized plague as a re-emerging infectious disease, and there is a concern for a possible fourth pandemic because of global warming, resulting in an increased prevalence of plague in rodent hosts. The current relevance of Y. pestis as a bioweapon is due to its high virulence and the development of multi-antibiotic-resistant strains. Although immunization of humans with plague vaccine will discourage the use of Y. pestis as a bioweapon, currently there is no vaccine against plague. During the current funding of the grant, we identified a new antigen (Braun lipoprotein) of Y. pestis that contributed to the development of bubonic and pneumonic plague. Our studies indicated that mice immunized with the mutant strain of Y. pestis deleted for the lpp and pigmentation locus (pgm) genes were protected against developing pneumonic plague caused by the highly virulent Y. pestis CO92 strain. We have now delineated the signaling pathways initiated by Lpp to cause host damage. Most importantly, our data indicated that the lpp mutant was unable to survive within macrophages, which was linked to the down-regulation of a stress response gene (htrA) in this mutant. We inferred from these data that other stress-associated genes (e.g., exoribonucleases) could also be involved in lpp-mediated, attenuated virulence of the bacterium. Indeed, deletion of the gene encoding polynucleotide phosphorylase (PNPase) also attenuated Y. pestis in a mouse model of systemic infection and provided protection against plague. We have proposed 3 specific aims for this grant. Aim 1 is to generate double mutants of Y. pestis CO92 in which genes encoding plasminogen-activating protease (pla), pnp, and two other predominant exoribonucleases (e.g., rnb [RNase II] and rnr [RNAse R]) will be deleted from the lpp gene minus background strain of Y. pestis CO92. These mutants will be tested for their attenuation in bubonic and pneumonic plague models (mice and rats). In aim 2, we will characterize protective immune responses of the most highly attenuated mutant in an animal model and the protection afforded by such a mutant against challenge with the parental CO92 strain. We have identified several immunogenic proteins in the WT CO92 strain that reacted with the immune sera of rats infected with CO92 strain. These antigens may represent excellent candidates for addition in the recombinant plague vaccine. Therefore in aim 3, we will first delete these genes from the WT bacterium to demonstrate their effects on bacterial virulence. Second, we will purify such immunogenic proteins and evaluate their protective effects after immunization of mice and rats followed by subsequent infection with the virulent Y. pestis. Overall, our studies are focused on identifying new live-attenuated vaccine strains of Y. pestis and to characterize the new immuno-protective CO92 antigens that could be important for the recombinant plague vaccine. PUBLIC HEALTH RELEVANCE: Y. pestis is a category A select agent and its potential to be used as a biothreat agent has caused significant concerns. In addition, plague represents a re-emerging infectious disease because of an increased number of cases worldwide. Currently, there is no vaccine available against this deadly disease, and hence our efforts are to develop new and novel countermeasures against plague, as well as to study new mechanisms of pathogenesis in Y. pestis.