Development of a Rationally Attenuated Live Vaccine for Francisella tularensis

土拉弗朗西斯菌合理减毒活疫苗的研制

• 批准号: 8650788
• 负责人: Robert K Ernst
• 金额: $ 23.03万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-10 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8650788
• 关键词: Address; Aerosols; Anabolism; Animals; Antibodies; Asia; Attenuated; Attenuated Live Virus Vaccine; Attenuated Vaccines; Bacteria; Baltimore; Bioterrorism; Categories; Centers for Disease Control and Prevention (U.S.); Clinic; Complement; Contracts; Core Facility; Data; Development; Disease; Engineering; Enzymes; Europe; Exhibits; Francisella; Francisella tularensis; Funding; Gene Expression; Genes; Goals; Human; Immune response; Immunity; Immunization; In Vitro; Inbred BALB C Mice; Incidence; Infection; Interferons; Kanamycin Resistance; Knowledge; Laboratories; Lead; Lethal Dose 50; Licensing; Life; Lipid A; Lipopolysaccharides; Location; Maryland; Mass Spectrum Analysis; Mediating; Membrane; Modification; Molecular; Molecular Profiling; Morbidity - disease rate; Mus; Mutation; North America; O Antigens; Pathogenesis; Pharmacy facility; Preclinical Testing; Publications; Publishing; Relative (related person); Research; Research Personnel; Research Project Grants; Risk; Route; Structure; TLR2 gene; Testing; Therapeutic; Tularemia; Vaccine Design; Vaccines; Virulence; Work; attenuation; base; design; in vivo; insight; macrophage; man; meetings; microbial; microbicide; mortality; mutant; novel; pathogen; prophylactic; public health relevance; research clinical testing; respiratory; subcutaneous; vaccine candidate; vaccine development

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.

描述（由申请人提供）：Francisella tuarlarensis（ft）是革兰氏阴性，细胞内（巨噬细胞 - 冰球）细菌，是toraremia的病因学药物。 ft被分类 作为一种类别，药剂可能会因呼吸道途径而与接种量低接种，如果未经治疗的小鼠和人类都会引起快速发病和死亡率。 FT活疫苗应变（LVS），一种源自FT亚种的B型菌株。 Holarctica是针对人减弱的，但是当鼻内或腹膜内施用时，在小鼠中引起类似小鼠的疾病。尽管FT LV被证明可以降低实验室获得的呼吸道血症的发生率，但在美国未获得许可，因为其衰减的分子基础尚未完全阐明，该菌株表现出表型不一致，并且FT LVS免疫无法提供完全的保护，以防止某些亚基ft ft ft ft。因此，必须开发其他预防性产品和/或衰减的疫苗菌株，以抗可能的生物恐怖威胁。我们最近发表的结果表明，插入失活 novicida（FN）中的LPXF基因导致脂肪糖酸（LPS）结构改变的菌株，该结构改变了野生型（WT）FN的25,000 LD50挑战，从而保护了免疫小鼠> 25,000 LD50挑战。现在，我们表明，FT LV中的类似突变导致与脂质A进行相同的结构修饰，并且在皮下感染C57BL/6和BALB/C小鼠后，在> 106 ld50中均在> 106 ld50中进行了反复修饰。 这项修订后的R21提案的目的是基于FT LVS（B型）和FT Schu S4（A型）菌株中未标记的LPXF缺失，开发现场，合理地减弱的FT疫苗。在这两种菌株中设计了未标记的LPXF缺失后，我们将通过质谱确认每个突变体的脂质A的结构，并确认每个突变体的确在小鼠中已减弱。此外，我们将测试它们保护WT，IFN-R - / - 和TLR2 - / - 小鼠免受同源和异源挑战的能力。最后，我们将测试每个突变菌株诱导与“经典活化”（杀生）与“替代激活”相关的巨噬细胞基因表达的能力（以前显示的是促进to骨内复制）巨噬细胞的，并具有在体外重复内细胞内复制的能力。这项研究完成后，我们预计将开发出合理减弱的疫苗候选者，该疫苗将防止多个FT A型和B型Francisella菌株。