Novel Bacillus subtilis based spore vaccine to tuberculosis

基于枯草芽孢杆菌的新型结核病孢子疫苗

• 批准号: 8461506
• 负责人: SUBRAMANIAN DHANDAYUTHAPANI
• 金额: $ 5.87万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2013-07-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8461506
• 关键词: 6-kDa early secretory antigenic target; Acquired Immunodeficiency Syndrome; Adult; Antigens; Attenuated; BCG Live; BCG Vaccine; Bacillus (bacterium); Bacillus subtilis; Bacteria; Capsid Proteins; Cavia; Childhood; Chromosomes, Human, 4-5; Chronic; Communicable Diseases; Cytosol; Desiccation; Disease; Dose; Drug Formulations; Drug Resistant Tuberculosis; Drug Targeting; Engineering; Extreme drug resistant tuberculosis; Food; Genes; Genetic Engineering; Genus Mycobacterium; Goals; Health Personnel; Heating; Human; Human Resources; Immune response; Immune system; Immunity; Immunization; Individual; Infection; Intranasal Administration; Life; Multidrug-Resistant Tuberculosis; Mus; Mycobacterium bovis; Mycobacterium tuberculosis; Nose; Nutritional; Oral; Oral Administration; Particulate; Patients; Peptide Vaccines; Peptides; Phagosomes; Plasmid Cloning Vector; Population; Probiotics; Protein C; Proteins; Protocols documentation; Pulmonary Tuberculosis; Recombinants; Reproduction spores; Research; Surface; Temperature; Testing; Tissues; Training; Tuberculosis; Vaccinated; Vaccine Antigen; Vaccines; Validation; Virulent; World Health Organization; bacterial vector; base; booster vaccine; chemotherapy; deprivation; drug development; gene cloning; homologous recombination; immunogenic; killings; macrophage; mucosal vaccination; mucosal vaccine; nonhuman primate; novel; overexpression; pathogen; prophylactic; research study; ultraviolet irradiation

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a chronic infectious disease caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb). The World Health Organization estimates that at least 2 million people die every year because of TB, and that one-third of the living world population is infected with Mtb. The recent emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant TB (XDR-TB) strains, and the infection of AIDS patients by Mtb, present more challenges in the management and treatment of the disease. Identification of new drug targets and development vaccines are the immediate priorities in TB research. Attenuated Mycobacterium bovis BCG has long been used as a vaccine against TB, but its efficacy is variable for different populations. Now, however, several new and highly immunogenic antigenic peptides/proteins have been identified from M. tuberculosis. The secreted antigenic proteins Ag85B and ESAT- 6, when used together, can elicit protective immune responses in mice, guinea pigs and nonhuman primates. This vaccine provides efficacy equivalent to that of BCG, so there is a potential for this to be a prophylactic vaccine replacement for BCG, and a booster vaccine for those who had BCG vaccine earlier but with no protective immune response. However, these protein antigens present several logistical problems. First, the proteins must be overexpressed and purified in large quantities from bacterial vectors, making them expensive to produce. Second, the vaccine formulation requires storage in freezers. Finally, the vaccine immunogens must be injected intramuscularly, by medical personnel, for optimum effect. Alternate strategies are required for better use of this vaccine. We plan to engineer recombinant Bacillus subtilis spores that express Ag85B and ESAT-6, to be used as a particulate vaccine to TB. Bacillus spores are dormant and rugged, and maintain their integrity under extreme conditions, including low pH, desiccation, UV irradiation and temperatures of up to 90oC. Interestingly, Bacillus spores are harmless to humans; in fact, they are sold in grocery stores and are being consumed in a number of novel foods as probiotics. Bacillus spores do not require special storage, and can be orally and nasally administered. Using plasmid vectors, we first will clone the genes encoding the Ag85B and ESAT-6 proteins downstream of the cotC gene, which encodes spore coat protein C, and the rrno gene of B. subtilis. We will then integrate these constructs into the chromosome of B. subtilis through homologous recombination. Strains carrying this fusion construct will be caused to produce spores by nutritional deprivation. We will then test the spores for their ability to eliit anti-Mtb cellular immune responses, after intranasal or oral immunization of the mice. Finally, we will test whether mice with these immunization groups are protected against challenge doses of live M. tuberculosis H37Rv.

描述（申请人提供）：结核病（TB）是由细菌病原体结核分枝杆菌（Mtb）引起的慢性传染病。世界卫生组织估计，每年至少有200万人死于结核病，世界上三分之一的人口感染了结核病。最近出现的耐多药结核病（MDR-TB）和广泛耐药结核病（XDR-TB）菌株，以及艾滋病患者被Mtb感染，对该疾病的管理和治疗提出了更多的挑战。确定新的药物靶点和开发疫苗是结核病研究的当务之急。 减毒牛分枝杆菌BCG长期以来一直被用作结核病疫苗，但其效力对不同人群是可变的。然而，现在已经从M.结核分泌的抗原蛋白Ag 85 B和ESAT- 6，当一起使用时，可以在小鼠，豚鼠和非人灵长类动物中引发保护性免疫应答。这种疫苗提供的效力与BCG相当，因此有可能成为BCG的预防性疫苗替代品，并为那些早期接种BCG疫苗但没有保护性免疫反应的人提供加强疫苗。然而，这些蛋白抗原存在几个后勤问题。首先，这些蛋白质必须从细菌载体中大量过量表达和纯化，这使得它们的生产成本昂贵。第二，疫苗制剂需要储存在冰箱中。最后，疫苗免疫原必须由医务人员肌肉注射，以获得最佳效果。为了更好地使用这种疫苗，需要替代策略。 我们计划设计表达Ag 85 B和ESAT-6的重组枯草芽孢杆菌孢子，用作结核病的颗粒疫苗。芽孢杆菌孢子处于休眠状态，坚固耐用，并在极端条件下保持其完整性，包括低pH值，干燥，紫外线照射和高达90 ° C的温度。有趣的是，芽孢杆菌孢子对人类无害;事实上，它们在杂货店出售，并作为益生菌在许多新型食品中食用。芽孢杆菌孢子不需要特殊的储存，可以口服和鼻腔给药。 使用质粒载体，我们首先克隆编码Ag 85 B和ESAT-6蛋白的基因，其位于编码孢子外壳蛋白C的cotC基因和B的rrno基因的下游。枯草杆菌。然后我们将这些构建体整合到B的染色体中。枯草杆菌通过同源重组。携带这种融合构建体的菌株将通过营养剥夺产生孢子。然后，我们将测试孢子在小鼠鼻内或口服免疫后激发抗Mtb细胞免疫应答的能力。最后，我们将测试具有这些免疫组的小鼠是否对活M.结核病H37 Rv.