Novel Bacillus subtilis based spore vaccine to tuberculosis

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)引起的一种慢性传染病。世界卫生组织估计，每年至少有200万人死于结核病，世界上三分之一的活着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，以及艾滋病患者感染结核分枝杆菌，给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。减毒牛分枝杆菌卡介苗长期以来一直被用作结核病疫苗，但其效力因人群而异。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。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发保护性免疫反应。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疫苗替代品，并为那些较早接种卡介苗但没有保护性免疫反应的人提供增强疫苗。然而，这些蛋白质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表达和纯化，这使得它们的生产成本很高。其次，疫苗配方需要在冰箱中储存。最后，疫苗免疫原必须由医务人员肌肉注射，才能达到最佳效果。为了更好地使用这种疫苗，需要采取替代策略。我们计划改造表达Ag85B和ESAT-6的重组枯草杆菌芽胞，将其用作结核病的颗粒疫苗。芽孢杆菌的孢子处于休眠状态，坚固耐用，在极端条件下仍能保持完整性，包括低pH值、干燥、紫外线照射和高达90摄氏度的温度。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，并被作为益生菌消费在许多新型食品中。芽孢杆菌孢子不需要特殊的储存，可以口服和鼻腔给药。我们首先利用质粒载体克隆了枯草杆菌孢子壳蛋白C基因和枯草杆菌rrno基因下游的Ag85B和ESAT-6蛋白编码基因。然后，我们将通过同源重组将这些构建物整合到枯草杆菌的染色体中。携带这种融合结构的菌株将因营养缺乏而产生孢子。然后，我们将测试孢子在小鼠鼻内或口服免疫后消除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抵抗活的结核分枝杆菌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.