Recombinant BCG With Bacterial Signaling Molecule Cyclic di-AMP as Endogenous Adjuvant Induces Elevated Immune Responses After Mycobacterium tuberculosis Infection

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.

Subunit Vaccine ESAT-6:c-di-AMP Delivered by Intranasal Route Elicits Immune Responses and Protects Against Mycobacterium tuberculosis Infection

“…For inflammatory cytokines, ESAT-6:c-di-AMP inoculation resulted in elevated IL-18 ( P < 0.05) and TNF-α ( P < 0.01) mRNA levels in lung than those in Naïve mice, but not significantly different from the group vaccinated with ESAT-6 alone ( Figures 3E–I ) . IFN-β levels were induced mainly by c-di-AMP in ESAT-6:c-di-AMP immunization group ( P < 0.01), consistent with studies of c-di-AMP on IFN-β response in macrophages ( Dey et al., 2015 ; Rueckert et al., 2017 ; Ning et al., 2019 ). Taken together, inoculation ESAT-6:c-di-AMP elicited Th17 and inflammatory cytokine responses in lung.…”

“…Mouse spleen was aseptically removed and homogenized with 40 μm cell strainers. Single cell suspension was prepared as our previous work ( Ning et al., 2019 ). Splenocytes were seeded in 96-well microplates at 2 × 10 5 cells/well and stimulated with 5 μg/ml ESAT-6 at the indicated timepoints.…”

“…In our previous work we found that rBCG with elevated c-di-AMP induced more cytokines related to trained immunity such as IL-1b, IL-6, and TNF-a in splenocytes (Ning et al, 2019). Both IL-1 and IL-18 belong to the interleukin-1 family of cytokines and were secreted following inflammasome activation, which involved in innate and adaptive immune system (Mantovani et al, 2019).…”

“…Pathogen specific antigens with c-di-AMP as a mucosal adjuvant conferred protection against influenza virus ( Sanchez et al., 2014 ) or Trypanosoma cruzi ( Matos et al., 2017 ). Our previous work demonstrated that c-di-AMP as endogenous adjuvant of recombinant BCG (rBCG) induced stronger immune responses in mice after Mtb infection, which was related to trained immunity ( Ning et al., 2019 ). Another report of this rBCG enhanced the protective efficacy against TB in a guinea pig model ( Dey et al., 2020 ).…”

Lessons from Bacillus Calmette-Guérin: Harnessing Trained Immunity for Vaccine Development

“…Enhancing and utilizing BCG's trained immunity effects in future vaccines against Mtb should be a priority and utilizing recombinant technologies to enhance BCG immunogenicity or reduce Mtb pathogenicity presents a unique opportunity to enhance anti-Mtb candidates. A recombinant BCG with overexpression of Mtb di-adenylate cyclase, which produces bacterial secondary messenger cyclic di-AMP, demonstrated comparable protection to BCG-immunized mice, while cellular analyses demonstrated increased IL-6 production following Mtb challenge and higher expression of H3K4 trimethylation than BCG [61]. Live-attenuated M. tuberculosis Vaccine Candidate (MTBVAC), the first genetically modified, live attenuated vaccine based on Mtb which has demonstrated safety and efficacy in initial clinical trials, induces trained immunity effects in vitro through shifts in metabolism and epigenetic changes at proinflammatory promoters, and can protect subcutaneously vaccinated mice from lethal intranasal doses of Streptococcus pneumoniae [62].…”