A novel hyper-immunogenic low virulent BCG vaccine against tuberculosis

总结 由细菌病原体分枝杆菌引起的结核病（TB）导致的人类发病率和死亡率 结核病（Mtb）仍然是全世界重大的健康问题。卡介苗 (BCG)仍然是唯一被批准的结核病疫苗。尽管BCG被认为是安全的， 有效对抗肺外儿童结核病，其保护儿童和成人肺结核病的能力 仍然值得怀疑。此外，人们担心BCG不会诱导长期持久的免疫应答， 免疫个体。然而，我们对结核分枝杆菌和卡介苗衍生的重组疫苗的研究结果强烈地表明， 这表明BCG可以被改进以更有效地对抗结核病。 该提案的目标是通过合理删除基因来改进BCG。我们计划依次删除 BCG的三个基因即sapM、zmp 1和nuoG，通过同源重组， 敲除BCG（BCG-TKO）菌株。这些基因编码的蛋白质使卡介苗能够逃避宿主免疫 通过阻止吞噬体-溶酶体融合、自噬、细胞凋亡和其他相关过程， 抗原呈递细胞（APC），如树突细胞和巨噬细胞。我们假设， 这些基因将允许BCG-TKO菌株被APC有效地加工，这将导致增加的 抗原呈递给免疫细胞并增强体内免疫原性和功效。另外我们 预期这些基因的缺失将降低BCG的毒力，从而使BCG-TKO成为 艾滋病毒安全。我们计划通过三个目标来实现我们的目标：1）通过同源重组构建BCG-TKO菌株， 2）在SCID小鼠模型中分析BCG-TKO菌株的免疫原性和安全性，和 3)研究BCG-TKO在有和没有HIV的常规和人源化小鼠模型中的功效 感染总的来说，我们希望这项建议将产生一种高效的第三代卡介苗 抗TB，其将适合于甚至在HIV感染的婴儿中施用。

SUMMARY Human morbidity and mortality due to tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), continue to be of significant health concern throughout the world. Bacille Calmette-Guerin (BCG) still remains the only approved vaccine against TB. Although BCG is considered safe and partially effective against extra-pulmonary childhood TB, its ability to protect against childhood and adult pulmonary TB is still questionable. In addition, there is a concern that BCG does not induce long lasting immune responses in the immunized individuals. However, our findings with Mtb- and BCG- derived recombinant vaccines strongly suggest that BCG can be improved to be more efficacious against TB. The goal of this proposal is to improve BCG by rationally deleting genes. We plan to sequentially delete three genes of BCG namely sapM, zmp1 and nuoG, through homologous recombination, to result in a triple knockout BCG (BCG-TKO) strain. These genes encode proteins that enable BCG to evade host immune response by preventing phagosome-lysosomal fusion, autophagy, apoptosis and other related processes in the antigen presenting cells (APCs) such as dendritic cells and macrophages. We hypothesize that deletion of these genes will allow the BCG-TKO strain to be efficiently processed by APCs, which will lead to increased antigen presentation to the immune cells and enhanced in vivo immunogenicity and efficacy. Additionally, we anticipate that deletion of these genes will reduce the virulence of the BCG, thus making the BCG-TKO to be HIV safe. We plan to accomplish our goal with three aims: 1) construct a BCG-TKO strain through homologous recombination, 2) analyze the immunogenicity and safety of the BCG-TKO strain in a SCID mouse model, and 3) investigate the efficacy of the BCG-TKO in the regular and humanized mouse models with and without HIV infection. Overall, we expect that this proposal will produce a highly efficacious, third generation BCG vaccine against TB that will be suitable for administration even in HIV infected infants.