Development of a Rationally Attenuated Live Vaccine for Francisella tularensis

描述（由申请方提供）：土拉热弗朗西丝菌（Ft）是一种革兰氏阴性细胞内（嗜巨噬细胞）细菌，是土拉菌病的病原体。Ft是机密 作为A类病原体，因为它可以通过呼吸道途径在低接种量下感染，如果不治疗，小鼠和人都会迅速发病和死亡。Ft活疫苗株（LVS），一种来源于Ft亚种的B型毒株。holarctica对人是减毒的，但当鼻内或腹膜内给药时在小鼠中引起兔热病样疾病。尽管Ft LVS显示可降低实验室获得性呼吸道土拉菌病的发生率，但由于其减毒的分子基础尚未完全阐明，菌株表现出表型不一致性，并且Ft LVS免疫接种未能提供针对Ft某些亚种的完全保护，因此在美国未获得许可。因此，必须开发额外的预防性产品和/或减毒疫苗株以用于对抗可能的生物恐怖主义威胁。我们最近发表的结果表明，插入失活的 lpxF基因在F. novicida（Fn）产生具有改变的脂多糖（LPS）结构的减毒菌株，其保护免疫的小鼠免受野生型（WT）Fn的> 25，000 LD 50攻击。我们现在表明，Ft LVS中的类似突变导致脂质A的相同结构修饰，并且在皮下感染C57 BL/6和BALB/c小鼠后在>106 LD 50下是无毒的。 该修订的R21提案的目标是基于Ft LVS（B型）和Ft Schu S4（A型）菌株中lpxF的未标记缺失，开发活的、合理减毒的Ft疫苗。在这两种菌株中工程化未标记的lpxF缺失后，我们将通过质谱法确认来自每个突变体的脂质A的结构，并确认每个突变体确实在小鼠中减毒。此外，我们将测试它们保护WT、IFN-r-/-和TLR 2-/-小鼠免受同源和异源攻击的能力。最后，我们将测试每种突变株诱导与“经典活化”（杀微生物）与“替代活化”（先前显示促进胞质内复制）巨噬细胞相关的巨噬细胞基因表达的能力，以及体外细胞内复制的能力。在本研究完成时，我们预期已开发出一种合理减毒的候选疫苗，可预防多种Ft A型和B型弗朗西斯菌菌株。

DESCRIPTION (provided by applicant): Francisella tularensis (Ft) is a Gram negative, intracellular (macrophage-tropic) bacterium, and the etiologic agent of tularemia. Ft is classified as a Category A agent because it may can be contracted with low inocula by the respiratory route and causes rapid morbidity and mortality if untreated both mice and humans. Ft Live Vaccine Strain (LVS), a type B strain derived from Ft subsp. holarctica, is attenuated for man, but causes a tularemia-like disease in mice when administered intranasally or intraperitoneally. Although Ft LVS was shown to reduce the incidence of laboratory-acquired respiratory tularemia, it is not licensed in the USA because the molecular basis for its attenuation has not been fully elucidated, the strain exhibits phenotypic inconsistencies, and Ft LVS immunization fails to provide complete protection against some subspecies of Ft. Therefore, additional prophylactic products and/or attenuated vaccine strains must be developed for use against possible bioterrorism threats. Our recently published results show that insertional inactivation of the lpxF gene in F. novicida (Fn) resulted in an attenuated strain with an altered lipopolysaccharide (LPS) structure that protected immunized mice against >25,000 LD50 challenge with wild-type (WT) Fn. We now show that a similar mutation in Ft LVS resulted in the same structural modification to the lipid A and was avirulent at >106 LD50 after subcutaneous infection of C57BL/6 and BALB/c mice. The goal of this revised R21 proposal is to develop live, rationally attenuated Ft vaccines based on unmarked deletion of lpxF in both Ft LVS (Type B) and Ft Schu S4 (Type A) strains. After engineering an unmarked lpxF deletion in these two strains, we will confirm the structure of the lipid A from each mutant by mass spectroscopy, and confirm that each mutant is, indeed, attenuated in mice. Additionally, we will test their capacity to protect WT, IFN-r-/-, and TLR2-/- mice against homologous and heterologous challenge. Finally, we will test for the capacity of each mutant strain to induce macrophage gene expression associated with "classically activated" (microbicidal) vs. "alternatively activated" (shown previously to facilitate intracytosolic replication) macrophages and for the ability to replicate intracellularly in vitro. At the completion of this study, we expect to have developed a rationally attenuated vaccine candidate that will protect against multiple Ft Type A and Type B Francisella strains.