Rational design of Francisella tularensis attenuated vaccine strains

土拉热弗朗西斯菌（Ft）是已知最具感染性的细菌之一，是A类选择性病原体， 构成了严重的军事和民用生物恐怖主义威胁。目前唯一有效的Ft疫苗是 减毒活疫苗株（LVS），已成功用作保护性疫苗， 俄罗斯的人类和美国与Ft合作的选定团体。然而，LVS尚未获得许可， 在美国使用，在某种程度上，由于衰减的机制是未知的， 人类的特征尚不明确，并且通过某些感染途径在小鼠中保持致死性。这项建议 旨在系统地识别LVS减毒突变，以帮助进一步提高其获得许可的潜力， 能够合理设计新的疫苗株， 也 我们对六种烈性Ft菌株的基因组进行了测序，以与LVS进行比较，并减少偏差 已经实施了合并所有测序的Ft基因组的方法以生成候选基因的列表。 减毒突变这种多基因组方法正在减少需要进行基因组测序的候选人数量。 随后在鼠感染模型中构建和测试，从而显著降低成本， 时间，以及完成项目最初目标所需的动物数量。Ft的突变株将是 使用新开发的Francisella等位基因交换策略构建，以测试每个等位基因的作用。 候选减毒突变对鼠感染模型中弗朗西斯菌致病性的影响，并确定 突变是衰减的。这些数据将补充正在进行的人类对LVS反应的研究 疫苗接种，并有望促进LVS在美国的许可证。迄今为止积累的数据表明 LVS通过新的机制衰减，这可以为合理设计LVS提供见解。 其他病原体的疫苗。在随后的几年里，我们计划利用这些数据，沿着详细的 在现有的最佳动物模型中进行免疫学分析，用于第二阶段的合理设计。 第二代Ft减毒疫苗。

Francisella tularensis (Ft), one of the most infectious bacteria known, is a Category A Select Agent that poses a serious military and civilian bioterrorism threat. The only current effective vaccine against Ft is an attenuated Live Vaccine Strain (LVS) strain, which has been used successfully as a protective vaccine in humans in Russia and select groups who work with Ft in the US. However, LVS has not been licensed for use in the U.S., in part, because the mechanism of attenuation is unknown, the protective response in humans is not well-characterized, and it retains lethality in mice by some routes of infection. This proposal aims to systematically identify the LVS attenuating mutations to help further it's potential for licensure and to enable the rational design of new vaccine strains that protect against tularemia and perhaps other diseases as well. We have sequenced the genome of six virulent Ft strains for comparison to LVS, and a bias-reducing approach incorporating all sequenced Ft genomes has been implemented to generate a list of candidate attenuation mutations. This multi-genome approach is reducing the number of candidates that need to be subsequently constructed and tested in a murine infection model, thereby dramatically reducing the cost, time, and the number of animals needed to complete the initial aim of the project. Mutant strains of Ft will be constructed using newly-developed allelic exchange strategies for Francisella, to test the effect of each candidate attenuation mutation on Francisella pathogenicity in a murine infection model and determine which mutations are attenuating. This data will complement ongoing studies of the human response to LVS vaccination and will hopefully facilitate LVS licensure in the US. The data accumulated thus far suggests LVS is attenuated through novel mechanisms, which may provide insights into the rational design of vaccines for other pathogens. In subsequent years, we plan to leverage this data, along with detailed immunological profiling in the best animal models available, for use in the rational design of second generation Ft attenuated vaccines.