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.

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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) infection, remains the most common cause of death from a single infectious disease. More safe and effective vaccines are necessary for preventing the prevalence of TB. In this study, a subunit vaccine of ESAT-6 formulated with c-di-AMP (ESAT-6:c-di-AMP) promoted mucosal and systemic immune responses in spleen and lung. ESAT-6:c-di-AMP inhibited the differentiations of CD8+ T cells as well as macrophages, but promoted the differentiations of ILCs in lung. The co-stimulation also enhanced inflammatory cytokines production in MH-S cells. It was first revealed that ESAT-6 and c-di-AMP regulated autophagy of macrophages in different stages, which together resulted in the inhibition of Mtb growth in macrophages during early infection. After Mtb infection, the level of ESAT-6-specific immune responses induced by ESAT-6:c-di-AMP dropped sharply. Finally, inoculation of ESAT-6:c-di-AMP led to significant reduction of bacterial burdens in lungs and spleens of immunized mice. Our results demonstrated that subunit vaccine ESAT-6:c-di-AMP could elicit innate and adaptive immune responses which provided protection against Mtb challenge, and c-di-AMP as a mucosal adjuvant could enhance immunogenicity of antigen, especially for innate immunity, which might be used for new mucosal vaccine against TB.

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Bacillus Calmette-Guérin (BCG) is a licensed prophylactic vaccine against tuberculosis (TB). Current TB vaccine efforts focus on improving BCG effects through recombination or genetic attenuation and/or boost with different vaccines. Recent years, it was revealed that BCG could elicit non-specific heterogeneous protection against other pathogens such as viruses through a process termed trained immunity. Previously, we constructed a recombinant BCG (rBCG-DisA) with elevated c-di-AMP as endogenous adjuvant by overexpressing di-adenylate cyclase of Mycobacterium tuberculosis DisA, and found that rBCG-DisA induced enhanced immune responses by subcutaneous route in mice after M. tuberculosis infection. In this study, splenocytes from rBCG-DisA immunized mice by intravenous route (i.v) elicited greater proinflammatory cytokine responses to homologous and heterologous re-stimulations than BCG. After M. tuberculosis infection, rBCG-DisA immunized mice showed hallmark responses of trained immunity including potent proinflammatory cytokine responses, enhanced epigenetic changes, altered lncRNA expressions and metabolic rewiring in bone marrow cells and other tissues. Moreover, rBCG-DisA immunization induced higher levels of antibodies and T cells responses in the lung and spleen of mice after M. tuberculosis infection. It was found that rBCG-DisA resided longer than BCG in the lung of M. tuberculosis infected mice implying prolonged duration of vaccine efficacy. Then, we found that rBCG-DisA boosting could prolong survival of BCG-primed mice over 90 weeks against M. tuberculosis infection. Our findings provided in vivo experimental evidence that rBCG-DisA with c-di-AMP as endogenous adjuvant induced enhanced trained immunity and adaptive immunity. What’s more, rBCG-DisA showed promising potential in prime-boost strategy against M. tuberculosis infection in adults.

CR1-SCR1-3 protein could possess a neuroprotective effect on acute CI/R injury.

Many antigens from Mycobacterium tuberculosis (M. tuberculosis) have been demonstrated as strong immunogens and proved to have application potential as vaccine candidate antigens. Cyclic di-AMP (c-di-AMP) as a bacterial second messenger regulates various bacterial processes as well as the host immune responses. Rv2837c, the c-di-AMP phosphodiesterase (CnpB), was found to be relative to virulence of M. tuberculosis and interference with host innate immune response. In this study, recombinant CnpB was administered subcutaneously to mice. We found that CnpB had strong immunogenicity and induced high levels of humoral response and lung mucosal immunity after M. tuberculosis intranasally infection. CnpB immunization stimulated splenocyte proliferation and the increasing number of activated NK cells but had little effects on Th1/Th2 cellular immune responses in spleens. However, CnpB induced significant Th1/Th2 cellular immune responses with a decreased number of T and B cells in the lungs, and significantly recruits of CD4+ and CD8+ T cells after M. tuberculosis attenuated strain H37Ra infection. Besides, we first reported that CnpB could stimulate IFN-β expression transitorily and inhibit the autophagy of macrophages in vitro. In mice intranasally infection model, CnpB immunization alleviated pathological changes and reduced M. tuberculosis H37Ra loads in the lungs. Thus, our results suggested that CnpB interferes with host innate and adaptive immune responses and confers protection against M. tuberculosis respiratory infection, which should be considered in vaccine development as well as a drug target.

Background: Coagulation function indicators are often used to evaluate patients’ coagulation function.They are rarely used in disease diagnosis;their use is limited to somecoagulopathies. We described the pattern of coagulation function indicators in patients with pleural effusions and determined the diagnostic value of different abnormal patterns of these indicators for distinguishing tuberculous pleural effusions (TPEs) from malignant pleural effusions associated with lung adenocarcinoma (AD-MPEs).Methods: Patients with AD-MPEs or TPEs participated in this retrospective study.Coagulation functionindicators, including activated partial thromboplastin time, prothrombin time (PT), fibrinogen (FIB), thrombin time, fibrinogen degradation products (FDP), and D-dimer(D-D), were measured; their levels and abnormal patterns were analyzed separately and in combination,to determine the diagnostic performance of individual and combined values and the optimal diagnostic model.Results: One hundred patients with AD-MPEs and 84 patients with TPEs participated.PT, FIB, FDP, and D-Dlevels in patients with TPE were significantly higher than those in patients with AD-MPE (all P<0.01).Receiver operating characteristiccurves revealed that PT, FIB, FDP, and D-Dvalues, particularly FDP and D-D, could distinguish betweenAD-MPE and TPE. The diagnostic performance was better for the combination of the four indicators than for any single indicator (Youden’s index: 0.513; area under the curve: 0.810).The proportion of patients with abnormal patterns of all four indicators was significantly higher in those with TPE than in those with AD-MPE (P<0.001).Conclusions: Thecombined values of PT, FIB, FDP, and D-Dhave diagnostic value for distinguishing TPE from AD-MPE.