Novel Bacillus subtilis based spore vaccine to tuberculosis

描述（由申请人提供）：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杆菌。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，以及艾滋病患者被结核分枝杆菌感染，给该疾病的管理和治疗带来了更多挑战。确定新的药物靶点和开发疫苗是结核病研究的当务之急。长期以来，减毒牛分枝杆菌卡介苗一直被用作结核病疫苗，但其效力在不同人群中是不同的。然而，现在已经从结核分枝杆菌中鉴定出一些新的和高度免疫原性的抗原肽/蛋白。当分泌的抗原蛋白Ag85B和ESAT- 6一起使用时，可以在小鼠、豚鼠和非人灵长类动物中引起保护性免疫反应。这种疫苗的效力与卡介苗相当，因此有可能成为卡介苗的预防性替代疫苗，并为以前接种过卡介苗但没有保护性免疫反应的人提供加强疫苗。然而，这些蛋白抗原存在一些后勤问题。首先，这些蛋白质必须从细菌载体中大量过度表达和纯化，这使得它们的生产成本很高。第二，疫苗制剂需要冷藏。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。为了更好地利用这种疫苗，需要采取其他策略。我们计划设计表达Ag85B和ESAT-6的重组枯草芽孢杆菌孢子，用于结核病颗粒疫苗。芽孢杆菌孢子处于休眠状态，坚固耐用，在极端条件下保持完整性，包括低pH值，干燥，紫外线照射和高达90℃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，并作为益生菌被用于许多新型食品中。芽孢杆菌孢子不需要特殊储存，可以口服和鼻腔给药。我们首先利用质粒载体克隆枯草芽孢杆菌编码孢子外壳蛋白C的cotC基因下游的Ag85B和ESAT-6蛋白基因以及rno基因。然后通过同源重组将这些结构整合到枯草芽孢杆菌的染色体中。携带这种融合结构的菌株会因营养剥夺而产生孢子。然后，我们将在小鼠鼻内或口服免疫后，测试孢子对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抵抗活结核分枝杆菌H37Rv的攻击剂量。

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.