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.