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

基本信息

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
项目类别：
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

项目摘要

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结核菌。近年来，耐多药结核病（MDR-TB）和广泛耐药结核病（XDR-TB）菌株的出现，以及艾滋病患者结核分枝杆菌的感染，给该病的管理和治疗带来了更多挑战。确定新药物靶点和开发疫苗是结核病研究的当务之急。减毒牛分枝杆菌卡介苗长期以来一直被用作结核病疫苗，但其功效对于不同人群而言存在差异。然而，现在已经从结核分枝杆菌中鉴定出几种新的、高免疫原性的抗原肽/蛋白质。分泌的抗原蛋白 Ag85B 和 ESAT-6 一起使用时，可以在小鼠、豚鼠和非人类灵长类动物中引发保护性免疫反应。该疫苗的功效与卡介苗相当，因此有可能成为卡介苗的预防性疫苗替代品，以及为那些较早接种卡介苗但没有保护性免疫反应的人提供的加强疫苗。然而，这些蛋白质抗原存在一些后勤问题。首先，这些蛋白质必须从细菌载体中大量表达和纯化，这使得它们的生产成本昂贵。其次，疫苗配方需要储存在冰箱中。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，需要采取替代策略。我们计划设计表达 Ag85B 和 ESAT-6 的重组枯草芽孢杆菌孢子，用作结核病颗粒疫苗。芽孢杆菌孢子处于休眠状态且坚固，在极端条件下（包括低 pH、干燥、紫外线照射和高达 90oC 的温度）仍能保持其完整性。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奇食品中食用。芽孢杆菌不需要特殊储存，可以口服和鼻腔给药。使用质粒载体，我们首先将编码 Ag85B 和 ESAT-6 蛋白的基因克隆到编码孢子外壳蛋白 C 的 cotC 基因下游，以及枯草芽孢杆菌的 rrno 基因。然后，我们将通过同源重组将这些构建体整合到枯草芽孢杆菌的染色体中。携带这种融合构建体的菌株将因营养缺乏而产生孢子。然后，在对小鼠进行鼻内或口服免疫后，我们将测试孢子引发抗结核分枝杆菌细胞免疫反应的能力。最后，我们将测试这些免疫组的小鼠是否能够免受攻击剂量的活结核分枝杆菌 H37Rv 的影响。