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

基本信息

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
项目类别：
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

项目摘要

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）：土拉弗朗西斯菌（Ft）是一种革兰氏阴性、细胞内（嗜巨噬细胞）细菌，也是土拉菌病的病原体。 Ft 已分类之所以被列为 A 类病原体，是因为它可能通过呼吸道途径感染少量接种物，如果不治疗小鼠和人类，会导致快速发病和死亡。 Ft 活疫苗菌株 (LVS)，一种源自 Ft 亚种的 B 型菌株。 holarctica 对人类有减毒作用，但当鼻内或腹膜内给药时，会在小鼠中引起兔热病样疾病。尽管 Ft LVS 被证明可以降低实验室获得性呼吸道兔热病的发病率，但它在美国并未获得许可，因为其减毒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 免疫无法提供完整的疫苗接种。针对 Ft 某些亚种的保护。因此，必须开发额外的预防产品和/或减毒疫苗株以应对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 F. novicida (Fn) 中的 lpxF 基因产生了脂多糖 (LPS) 结构改变的减毒菌株，可保护免疫小鼠免受野生型 (WT) Fn >25,000 LD50 的攻击。我们现在表明，Ft LVS 中的类似突变会导致脂质 A 发生相同的结构修饰，并且在皮下感染 C57BL/6 和 BALB/c 小鼠后，LD50 > 106 时无毒。修订后的 R21 提案的目标是基于 Ft LVS（B 型）和 Ft Schu S4（A 型）毒株中 lpxF 的无标记删除，开发合理减毒的 Ft 活疫苗。在这两个菌株中工程化未标记的 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，并确认每个突变体确实在小鼠中减毒。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攻击的能力。最后，我们将测试每种突变菌株诱导与“经典激活”（杀菌）与“替代激活”（之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。这项研究完成后，我们预计将开发出一种合理减毒的候选疫苗，能够预防多种 Ft A 型和 B 型弗朗西斯菌菌株。