Abstract
Bacillus Calmette-Guerin (BCG) is a live attenuated vaccine against tuberculosis (TB) and remains the most commonly used vaccine worldwide. However, BCG has varied protective efficiency in adults and has safety concerns in immunocompromised population. Thus, effective vaccines are necessary for preventing the prevalence of TB. Cyclic di-AMP (c-di-AMP) is a bacterial second messenger which regulates various cellular processes and host immune response. Previous work found that c-di-AMP regulates bacterial physiological function, pathogenicity and host type I IFN response. In this study, we constructed a recombinant BCG (rBCG) by overexpressing DisA, the diadenylate cyclase of Mycobacterium tuberculosis (Mtb), and observed the physiological changes of rBCG-DisA. The immunological characteristics of rBCG-DisA were investigated on humoral and cellar immune responses in a mice infection model. Our study demonstrated that overexpression of DisA in BCG does not affect the growth but reduces the length of BCG. rBCG-DisA-immunized mice show similar humoral and cellar immune responses in BCG-immunized mice. After Mtb infection, the splenic lymphocytes from both BCG and rBCG-DisA-immunized mice produced more IFN-γ, IL-2, and IL-10 than the un-immunized (UN) mice, while the cytokine levels of the rBCG-DisA group increased significantly than those of the BCG group. The transcription of IFN-β, IL-1β and autophagy related genes (Atgs) were up-regulated in macrophages after treated with c-di-AMP or bacterial infection. The productions of IL-6 were increased after Mtb challenge, especially in the rBCG-DisA-immunized mice. Strikingly, H3K4me3, the epigenetic marker of innate immune memory, was found in both two immunized groups, and the rBCG-DisA group showed stronger expression of H3K4me3 than that of BCG. In addition, the pathological changes of rBCG-DisA immunized mice were similar to that of BCG-immunized mice. The bacterial burdens in the lungs and spleens of BCG- and rBCG-DisA-immunized mice were significantly decreased, but there was no significant difference between the two immunized groups. Together, these results suggested that compared to BCG, rBCG-DisA vaccination, induces stronger immune responses but did not provided additional protection against Mtb infection in this study, which may be related to the innate immunity memory. Hence, c-di-AMP is a promising immunomodulator for a further developed BCG as a better vaccine.
Highlights
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most deadly killers of infectious diseases worldwide [1]
DNA integrity scanning protein (DisA) expressions were very low in Mtb and Bacillus Calmette-Guerin (BCG) detected by anti-DisA sera [36], which were consisted with the low level of c-di-AMP in bacteria [15, 25]
Our study showed that recombinant BCG (rBCG)-DisA-infected RAW264.7 macrophages induced higher IFN-β transcription than un-stimulated cells, but not BCG-infected cells (Figure 6A). rBCG-DisA activated autophagy related genes (Atgs) increase as Beclin1 and Atg7 transcription augmented (Figure 6B), but the overall autophagy did not increase significantly (Figure S4), which was inconsistent with a previous c-di-AMP Induces Elevated Immune Responses paper reporting that Mtb with overexpressed DisA induced potent autophagy [25]
Summary
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most deadly killers of infectious diseases worldwide [1]. The only vaccine currently available for TB is Mycobacterium bovis Bacillus Calmette-Guerin (BCG), which can effectively prevent severe TB in children but is less successful in controlling pulmonary TB in adults, and may cause disseminated infection in immunocompromised populations when it is administered as a live attenuated vaccine. Vaccines that can replace traditional BCG have not yet been obtained. In addition to BCG’s effects on TB, it has been shown that BCG induces heterologous protections against non-mycobacterial reinfection, allergic diseases, and certain malignancies [2]. More attempts should be done to improve the safety and protection efficiency of BCG against TB
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