A novel c-di-AMP-based recombinant BCG vaccine

摘要 结核病(TB)仍然是一种全球流行病，目前世界人口的四分之一受到感染和 每年约有1000万新的活跃病例。然而，我们对致病因子的了解， 结核分枝杆菌(Mtb)仍然有限，唯一获得许可的结核病疫苗(BCG)不足以 控制疫情。更好地理解结核分枝杆菌和卡介苗的生物学差异是至关重要的。 开发一种更有效的结核病疫苗。我们这个项目的长期目标是开发一种新的 重组卡介苗更好地控制结核病。近年来，环二AMP(c-di-AMP)已被公认为 一种新的细菌信号分子和一种有效的疫苗佐剂。我们已经证明了Mtb Rv3586 Rv2837c(CnpB)编码c-二-AMP磷酸二酯酶。 与MTB野生型(WT)相比，∆cnpB分泌的c-di-AMP明显更多，并刺激 在感染的巨噬细胞中有更强的I型干扰素(干扰素-I)反应。我们还透露，卡介苗 ∆cnpB产生但不分泌c-diamp；c-diamp的分泌和mtb区的差异1(Rd1) 是c-di-AMP诱导的干扰素-I反应所必需的。有趣的是，众所周知，卡介苗在 诱导干扰素-I，加入干扰素-I可增强卡介苗的免疫原性。此外，还利用了c-di-amp 作为一种潜在的疫苗佐剂，可引起强烈的体液和细胞免疫反应。C-的生产过剩 在卡介苗中表达DISA的DI-AMP对感染动物也有更好的保护作用。然而， 改善的可能性是中等的，因为重组卡介苗仍然不能分泌c-diamp和 诱导大量干扰素-I。因此，我们假设操纵c-diamp的动态平衡和分泌 卡介苗的研究将使卡介苗能够更好地预防结核病。此应用程序的目标是 构建分泌c-diAMP并诱导干扰素-I应答的重组卡介苗 接种疫苗。我们提出了两个具体的目标：(1)构建分泌c-diAMP的重组卡介苗菌株，并 (2)评价重组卡介苗对干扰素-I的诱导和对结核分枝杆菌感染的保护作用。这些 重组卡介苗很有可能在疫苗效果上优于目前的卡介苗。此外，我们的 这些发现还将为其他细菌病原体的疫苗策略提供基本的见解。因此，这一点 这项提议对公众健康有广泛的影响。

SUMMARY Tuberculosis (TB) remains a global epidemic, with one-fourth of the current world population infected and approximately 10 million new active cases annually. However, our knowledge about the causative agent, Mycobacterium tuberculosis (Mtb), is still limited, and the only licensed TB vaccine (BCG) is inadequate to control the epidemic. It is critical to better understand the biological difference between Mtb and BCG in order to develop a more effective TB vaccine. Our long-term goal of this project is to develop a novel recombinant BCG vaccine to better control TB. Recently, cyclic di-AMP (c-di-AMP) has been recognized as a new bacterial signaling molecule and a potent vaccine adjuvant. We have demonstrated that Mtb Rv3586 (disA) encodes a diadenylate cyclase and Rv2837c (cnpB) encodes a c-di-AMP phosphodiesterase. Compared to the Mtb wild-type (WT), ∆cnpB secretes a significantly larger amount of c-di-AMP and stimulates a stronger type I interferon (IFN-I) response in the infected macrophages. We have also revealed that BCG ∆cnpB produces but does not secrete c-di-AMP; both c-di-AMP secretion and Mtb region of difference 1 (RD1) are required for the c-di-AMP-induced IFN-I response. Interestingly, it is well known that BCG is defective in inducing IFN-I, and addition of IFN-I enhances BCG's immunogenicity. Moreover, c-di-AMP has been utilized as a potential vaccine adjuvant that elicits strong humoral and cellular immune response. Overproduction of c- di-AMP in BCG by expressing disA also results in better protection in infected animals. However, the improvement is moderate likely because that the recombinant BCG is still unable to secrete c-di-AMP and induce substantial IFN-I. Therefore, we hypothesize that manipulation of c-di-AMP homeostasis and secretion in BCG will enable BCG to provide a better protection against TB. The objective of this application is to construct a recombinant BCG that secretes c-di-AMP and induces optimal levels of IFN-I response during vaccination. We propose two specific aims: (1) to construct c-di-AMP-secreting recombinant BCG strains, and (2) to evaluate the recombinant BCG strains in induction of IFN-I and protection against Mtb infection. These recombinant BCG strains will be very likely superior to the current BCG in vaccine efficacy. Furthermore, our findings will also provide fundamental insights into vaccine strategies for other bacterial pathogens. Thus, this proposal has a broad impact on public health.