[Preparation of Mycobacterium tuberculosis EsxV lipid nanoparticles subunit vaccine and its immunological characteristics].

To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.

Long non-coding RNA ENST00000469812 promotes Enterovirus type 71 replication via targeting the miR-4443/NUPR1 axis in rhabdomyosarcoma cells.

Hand, foot, and mouth disease (HFMD) caused by Enterovirus type 71 (EV71) is a serious threat to children's health. However, the pathogenic mechanism of EV71 is still unclear. Long non-coding RNAs (lncRNAs), some of which bind to miRNA as competitive endogenous RNAs (ceRNA) and weaken the silencing effect on the mRNA of downstream target genes, play a key role in regulating the viral infection process. In this study, through experimental verification, we found miR-4443 to be downregulated in cells infected with EV71. Next, by predicting lncRNAs that potentially regulate miR-4443, we found that EV71 infection induced upregulation of lncRNA ENST00000469812 and then further downregulated miR-4443 expression by direct interaction. We also demonstrated that nuclear protein 1 (NUPR1) is one of the target genes of miR-4443 and is involved in the ENST00000469812/miR-4443/NUPR1 regulatory axis. Finally, the ENST00000469812/miR-4443/NUPR1 regulatory axis exhibited a positive effect on EV71 replication. Here, we lay a foundation for exploring the pathogenic mechanism of EV71 and identify potential targets for HFMD treatment.

Cyclic di-AMP as endogenous adjuvant enhanced BCG-induced trained immunity and protection against Mycobacterium tuberculosis in mice.

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.

[Immune responses induced by subunit vaccine of Ag85B-ESAT-6 delivered by mucosal route to Mycobacterium tuberculosis].

Objective To identify the immune responses induced by subunit vaccine of Ag85B-ESAT-6 (AE) fusion protein by mucosal route and the protection against Mycobacterium tuberculosis (MTB) infection in mice. Methods AE and AE with c-di-AMP as adjuvant were inoculated intranasally in mice. The generation of specific IgG, cytokines secreted by Th1 cells (IFN-γ, IL-2) and Th2 cells (IL-10) were detected by ELISA. The transcriptional levels of IFN-γ, IL-2, IL-10, and TNF-α were determined using real-time quantitative PCR. After MTB infection by vein, the antibodies level in mice sera and cytokines secretion of splenocytes were detected by ELISA. Histopathological changes in mice lung was illustrated by HE staining, and bacteria burdens of spleen and lung were counted by colony-forming units (CFUs) on plate. Results AE and AE combined with c-di-AMP via nasal mucosal immunization could induce high level specific antibodies in sera, promote splenocyte proliferation, and lead to increased Th1/Th2 cytokines and TNF-α transcription in spleen and lung, and secret more Th1/Th2 cytokines in spleen. After MTB infection, compared with the control group, the specific antibody levels of AE and AE combined with c-di-AMP immunized mice still increased, with enhanced the Th1/Th2 cellular immune responses, inflammatory response in the lung tissues, and reduced bacteria loads in spleen and lung, especially in mice immunized with AE combined with c-di-AMP. Conclusion Intranasal mucosal vaccination of AE subunit vaccine can induce humoral and cellular immune responses, and provide protection against MTB infection in mice, c-di-AMP as an adjuvant can improve the immunogenicity of AE to a certain extent.

Cyclic-di-AMP Phosphodiesterase Elicits Protective Immune Responses Against Mycobacterium tuberculosis H37Ra Infection in Mice.

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-ß 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.

c-di-AMP Accumulation Regulates Growth, Metabolism, and Immunogenicity of Mycobacterium smegmatis.

Cyclic dimeric adenosine monophosphate (c-di-AMP) is a ubiquitous second messenger of bacteria involved in diverse physiological processes as well as host immune responses. MSMEG_2630 is a c-di-AMP phosphodiesterase (cnpB) of Mycobacterium smegmatis, which is homologous to Mycobacterium tuberculosis Rv2837c. In this study, cnpB-deleted (ΔcnpB), -complemented (ΔcnpB::C), and -overexpressed (ΔcnpB::O) strains of M. smegmatis were constructed to investigate the role of c-di-AMP in regulating mycobacterial physiology and immunogenicity. This study provides more precise evidence that elevated c-di-AMP level resulted in smaller colonies, shorter bacteria length, impaired growth, and inhibition of potassium transporter in M. smegmatis. This is the first study to report that elevated c-di-AMP level could inhibit biofilm formation and induce porphyrin accumulation in M. smegmatis by regulating associated gene expressions, which may have effects on drug resistance and virulence of mycobacterium. Moreover, the cnpB-deleted strain with an elevated c-di-AMP level could induce enhanced Th1 immune responses after M. tuberculosis infection. Further, the pathological changes and the bacteria burden in ΔcnpB group were comparable with the wild-type M. smegmatis group against M. tuberculosis venous infection in the mouse model. Our findings enhanced the understanding of the physiological role of c-di-AMP in mycobacterium, and M. smegmatis cnpB-deleted strain with elevated c-di-AMP level showed the potential for a vaccine against tuberculosis.

Integrative analysis of lncRNA-miRNA-mRNA-associated competing endogenous RNA regulatory network involved in EV71 infection.

The competing endogenous RNA (ceRNA) axis has been shown to play a critical role in the pathogenesis of various viral infections. Generally, the ceRNA network involves long non-coding RNAs (lncRNAs) that act as sponges for miRNA to regulate mRNA expression. However, no information is available regarding the involvement of ceRNA networks in Enterovirus type 71 (EV71) infections. In the present study, data obtained from Gene Expression Omnibus (GEO) database was analyzed using various bioinformatics tools. EV71 infection in rhabdomyosarcoma (RD) cells was associated with differential expression of six lncRNAs, 28 miRNAs, and 349 mRNAs. Gene function enrichment analysis suggested induction of cytoplasmic vesicle process upon EV71 infection. The ceRNA networks were constructed, in which 20 hub genes were predicted by protein-protein interaction. To confirm the MALAT1/miR-194-5p/DUSP1 ceRNA regulatory axis in EV71 infection, real-time quantitative polymerase chain reaction (qRT-PCR) and luciferase reporter assay were performed. The results of the study also revealed the involvement of the MALAT1/miR-194-5p axis in apoptosis induced by EV71 infection, while no association with autophagy was observed. Thus, the present study provided novel insights into the pathogenic mechanism of EV71 infection.

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

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.

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

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-ß, IL-1ß 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.

Immunological characteristics of Mycobacterium tuberculosis subunit vaccines immunized through different routes.

Tuberculosis is chronic infectious disease caused by Mycobacterium tuberculosis (M.tb) that is prevalent worldwide. Several specific antigens, such as Antigen 85B (Ag85B) and 6 kDa early secretory antigenic target (ESAT-6) protein of M.tb, are listed as some of the candidate subunit vaccines against M.tb. ESAT-6, as a virulent factor and differential gene in M.tb, shows insufficient immunogenicity in animal model. In order to investigate the ways to improve the immunogenicity of ESAT-6, we immunized ESAT-6 by subcutaneous and intramuscular routes with different adjuvants. We found that ESAT-6 immunized alone did not induce significant humoral immunity in both immunization routes. However, subcutaneous immunization of ESAT-6 plus incomplete Freund's adjuvant can induce a significant humoral immune response, enhanced proliferation and elevated secretion of IFN-γ from splenocytes. Intramuscular immunization of ESAT-6 plus adjuvant aluminum salt or poly(I:C) did not enhance humoral and cellular immune responses. Therefore, it is concluded that immunization of ESAT-6 subcutaneously plus incomplete Freund's adjuvant induces stronger humoral and cellular immune responses, which can be considered of ESAT-6 as a subunit vaccine in further research against tuberculosis.

Neuroprotective effects of the SCR1-3 functional domain of CR1 on acute cerebral ischemia and reperfusion injury in rats.

OBJECTIVE: Complement receptor type 1 (CR1), one of the most potent inhibitors in complement activation, shows a protective effect on cerebral ischemia/reperfusion (CI/R) injury due to its ability to bind C3b and C4b and to inactivate C3/C5 convertases. So far, no study assessed the effect of the first three short consensus repeats (SCR1-3) with low molecular weight, one of the most active functional domains of CR1, binding C4b with a powerful decay-acceleration effect on classical and alternative C3/C5 convertases pathways. Therefore, we aim to assess this effect on CI/R injury in the present study. METHODS: Seventy-five adult male Sprague-Dawley rats were randomly divided into three groups: sham operation group (n = 15), CI/R group (n = 30), and CI/R group treated with CR1-SCR1-3 protein (n = 30). After middle cerebral artery occlusion (MCAO) for 1 hour and reperfusion for 24 hours, neurological motor deficits, cerebral infarct size, and biochemical parameters including myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) were assessed. Meanwhile, tissues in cerebral cortex were collected and processed for western blotting, immunohistochemistry, and HE staining. RESULTS: CR1-SCR1-3 could improve neurological functions in brain with a 26.8% decrease in neurological motor deficit score and could lead to a 63.8% reduction in cerebral infarct size. Besides, pretreatment using CR1-SCR1-3 could prevent neutrophil infiltration and alleviate inflammation severity and subsequent tissue damage. Decreased C4b expression and action, as well as improved morphological changes, were also observed in cerebral tissues of CI/R+CR1-SCR1-3 rats. CONCLUSION: CR1-SCR1-3 protein could possess a neuroprotective effect on acute CI/R injury.