Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer.

In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.

CpG-C ODN M362 as an immunoadjuvant for HBV therapeutic vaccine reverses the systemic tolerance against HBV.

Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both in vivo and in vitro, overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8+ and CD4+ T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5+ CD8+ T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.

Unique Roles for Streptococcus pneumoniae Phosphodiesterase 2 in Cyclic di-AMP Catabolism and Macrophage Responses.

Cyclic di-AMP (c-di-AMP) is an important signaling molecule for pneumococci, and as a uniquely prokaryotic product it can be recognized by mammalian cells as a danger signal that triggers innate immunity. Roles of c-di-AMP in directing host responses during pneumococcal infection are only beginning to be defined. We hypothesized that pneumococci with defective c-di-AMP catabolism due to phosphodiesterase deletions could illuminate roles of c-di-AMP in mediating host responses to pneumococcal infection. Pneumococci deficient in phosphodiesterase 2 (Pde2) stimulated a rapid induction of interferon ß (IFNß) expression that was exaggerated in comparison to that induced by wild type (WT) bacteria or bacteria deficient in phosphodiesterase 1. This IFNß burst was elicited in mouse and human macrophage-like cell lines as well as in primary alveolar macrophages collected from mice with pneumococcal pneumonia. Macrophage hyperactivation by Pde2-deficient pneumococci led to rapid cell death. STING and cGAS were essential for the excessive IFNß induction, which also required phagocytosis of bacteria and triggered the phosphorylation of IRF3 and IRF7 transcription factors. The select effects of Pde2 deletion were products of a unique role of this enzyme in c-di-AMP catabolism when pneumococci were grown on solid substrate conditions designed to enhance virulence. Because pneumococci with elevated c-di-AMP drive aberrant innate immune responses from macrophages involving hyperactivation of STING, excessive IFNß expression, and rapid cytotoxicity, we surmise that c-di-AMP is pivotal for directing innate immunity and host-pathogen interactions during pneumococcal pneumonia.

The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.

Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8+ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.

Signal Transduction of Streptococci by Cyclic Dinucleotide Second Messengers.

Since the discovery of cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP) in 1987, the role of cyclic dinucleotides in signal pathways has been extensively studied. Many receptors and effectors of cyclic dinucleotides have been identified which play important roles in cellular processes. Example of such effectors include cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP)-binding proteins and endoplasmic reticulum membrane adaptor. Accumulating evidence indicate that cyclic dinucleotides act as second messengers that not only regulate the bacterial physiological processes but also affect host immune responses during infections. Streptococci species, which produce cyclic dinucleotides, are responsible for many human diseases. Numerous studies suggest that the cyclic dinucleotides are vital in signal transduction pathways as second messengers and influence the progression of infectious diseases. Here, we provide an overview of the molecular principles of cyclic dinucleotides synthesis and degradation and discuss recent progress on streptococcal signal transduction pathways by cyclic dinucleotide second messengers and their role in regulating host immune reaction. This review will provide a better understanding of the molecular mechanisms of streptococcal cyclic dinucleotide second messengers thereby revealing novel targets for preventing infections.

Immunomodulatory asthma therapy in the equine animal model: A dose-response study and evaluation of a long-term effect.

INTRODUCTION: Equine asthma represents a naturally occurring animal model for human allergic neutrophilic asthma. Inhalative nanoparticle-bound cytosine-phosphate-guanosine (CpG-GNP) immunotherapy, independent of specific allergens, has already shown promising clinical and immunological results in previous studies and offers the possibility to treat the underlying cause of the disease. This study analyses the relationship between dose and response, and evaluates a possible long-term effect. METHODS: In the prospective, randomised, double-blind clinical field study, 29 horses suffering from equine asthma received 10 inhalation treatments with either 187.5 µg CpG-GNP (CpG single dose [CpGsd]; n = 11), 375 µg CpG-GNP double dose (CpG double dose [CpGdd]; n = 9) (q48h for 20 days) or 1600 µg beclomethasone (n = 9) (q24h for 10 days). Each horse was examined three times: before the treatment (I), immediately after the 10 inhalations (II), and 8 weeks after the final inhalation (III). The three groups were compared according to clinical and laboratory parameters. The study examined the sustainability of the long-term effect of the treatment after 8 weeks, as well as the tolerability of the formula as a double dose. RESULTS: The CpGsd resulted in a significant improvement in 82% of the parameters, the CpGdd in 72%. In the long-term evaluation, the CpGsd showed a significant improvement in 100% of the parameters in comparison to the initial values, the CpGdd in 67%. On the immunological level, the bronchoalveolar lavage revealed a significant reduction of IL-4, IL-8, and interferon-γ. CONCLUSION: Both CpG groups displayed significant improvements in clinical and laboratory parameters, especially regarding the long-term effect of CpGsd. Doubling the CpG dose did not result in any improvement in comparison to the original single dose. On the immunological level, an anti-inflammatory, as well as an immunomodulatory effect, apart from a Th2-dominated immune response, could be observed. This immunomodulatory inhalation treatment could indicate a new possibility for human allergic asthma therapy.

A Visible Codelivery Nanovaccine of Antigen and Adjuvant with Self-Carrier for Cancer Immunotherapy.

Codelivery nanovaccines of antigens and adjuvants have achieved positive therapy for cancer immunotherapy. The insufficient immunogenicity of these vaccines leads to the difficulty of eliciting robust immune effects for immune clearance due to the inadequate loading efficiency, complex preparation processes, low safety concerns, and weak immune responses. Herein, a visible codelivery nanovaccine of an antigen and adjuvant based on self-cross-linked antigen nanoparticles (ovalbumin nanoparticles (ONPs)) combined with the adjuvant (CpG) for cancer immunotherapy was prepared using antigens themselves as carriers. ONPs not only provide sufficient antigens for continuous simulation of the immune response with high antigen loading efficiency but also serve as natural carriers of CpG. In vitro and in vivo experiments proved that ONPs-CpG can elicit a robust immune response including DC maturity, T cell activation, and IFN-γ production. ONPs-CpG induced strong tumor-specific immunity and exhibited remarkable antitumor immunotherapy effects in vivo using mouse models of lymphoma. Furthermore, to perform the precise vaccine delivery, the dual fluorescent codelivery nanovaccine was monitored in real time in vivo by the visible imaging method. With regard to migration tracking, fluorescence imaging allowed for both high resolution and sensitivity of visible detection based on the fluorescence of ONPs and CpG. The multifunctional nanovaccine could function as a robust platform for cancer immunotherapy and a visible system for antigen-adjuvant tracking.

The ER membrane adaptor ERAdP senses the bacterial second messenger c-di-AMP and initiates anti-bacterial immunity.

Cyclic diadenylate monophosphate (c-di-AMP) is secreted by bacteria as a secondary messenger. How immune cells detect c-di-AMP and initiate anti-bacterial immunity remains unknown. We found that the endoplasmic reticulum (ER) membrane adaptor ERAdP acts as a direct sensor for c-di-AMP. ERAdP-deficient mice were highly susceptible to Listeria monocytogenes infection and exhibited reduced pro-inflammatory cytokines. Mechanistically, c-di-AMP bound to the C-terminal domain of ERAdP, which in turn led to dimerization of ERAdP, resulting in association with and activation of the kinase TAK1. TAK1 activation consequently initiated activation of the transcription factor NF-κB to induce the production of pro-inflammatory cytokines in innate immune cells. Moreover, double-knockout of ERAdP and TAK1 resulted in heightened susceptibility to L. monocytogenes infection. Thus, ERAdP-mediated production of pro-inflammatory cytokines is critical for controlling bacterial infection.

Synthesis of All Possible Canonical (3'-5'-Linked) Cyclic Dinucleotides and Evaluation of Riboswitch Interactions and Immune-Stimulatory Effects.

The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.

Ag-presenting CpG-activated pDCs prime Th17 cells that induce tumor regression.

Plasmacytoid dendritic cells (pDC) rapidly and massively produce type I IFN and other inflammatory cytokines in response to foreign nucleic acids, thereby indirectly influencing T-cell responses. Moreover, antigen (Ag)-presenting pDCs directly regulate T-cell differentiation. Depending on the immune environment, pDCs exhibit either tolerogenic or immunogenic properties. Here, we show that CpG-activated pDCs promote efficient Th17 differentiation. Indeed, Th17 responses are defective in mice selectively lacking MHCII on pDCs upon antigenic challenge. Importantly, in those mice, the frequency of Th17 cells infiltrating solid tumors is impaired. As a result, the recruitment of infiltrating leukocytes in tumors, including tumor-specific cytotoxic T lymphocytes (CTL), is altered and results in increased tumor growth. Importantly, following immunization with tumor Ag and CpG-B, MHCII-restricted Ag presentation by pDCs promotes the differentiation of antitumor Th17 cells that induce intratumor CTL recruitment and subsequent regression of established tumors. Our results highlight a new role for Ag presenting activated pDCs in promoting the development of Th17 cells and impacting on antitumor immunity.

Variable induction of PRDM1 and differentiation in chronic lymphocytic leukemia is associated with anergy.

Despite antigen engagement and intact B-cell-receptor (BCR) signaling, chronic lymphocytic leukemia (CLL) cells fail to undergo terminal differentiation. We hypothesized that such failure may be due to anergy, as CLL cells exhibit variable levels of nonresponsiveness to surface IgM stimulation that is reversible in vitro. Moreover, anergy is associated with reduced differentiation capacity in normal B cells. We investigated responses of CLL cells to two potent differentiation-promoting agents, IL-21 and cytosine guanine dinucleotide-enriched oligo-deoxynucleotides. The induction of PR domain-containing protein 1 (PRDM1; also known as Blimp-1), a critical regulator of plasmacytic differentiation, by these agents was closely correlated but varied between individual cases, despite functionally intact IL-21 receptor- and Toll-like receptor 9-mediated signal transducer and activator of transcription 3, and nuclear factor-κB pathways. PRDM1 induction was inversely correlated with the extent of anergy as measured by the ability to mobilize intracellular Ca(2+) following BCR crosslinking. PRDM1 responsiveness was associated with other markers of differentiation and proliferation but not with differences in apoptosis. The ability to induce PRDM1 did correlate with differential transcriptional and epigenetic regulation of the PRDM1 gene. These studies extend our understanding of CLL pathobiology, demonstrating that reduced differentiation capacity may be a consequence of anergy. Epigenetic drugs may offer possibilities to reactivate PRDM1 expression as part of novel differentiation therapy approaches.

STING-dependent type I IFN production inhibits cell-mediated immunity to Listeria monocytogenes.

Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-ß and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon.

CD40L--a costimulatory molecule involved in the maturation of antigen presenting cells in Atlantic salmon (Salmo salar).

The CD40L/CD40 signalling pathway is critically involved in the final stage of the maturation of DCs. This paper reports the identification and functional characterization of CD40L and CD40 from Atlantic salmon (Salmo salar). Salmon CD40L is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, while CD40 is a type I membrane-bound receptor with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The salmon CD40L and CD40 were widely expressed, particularly in immune tissues, and while CD40L expression was induced by in vitro stimulation of HKLs with PHA and ConA, CpG increased CD40 expression. A CD40L construct was overexpressed in the CHSE-214 cell line and co-cultivation of the CD40L-CHSE transfectants with HKL induced a rapid and long-lasting upregulation of important costimulatory molecules like CD40, CD83, B7-H1 and the cytokines IL-12p40, IL-10, IL-1ß and IFNs, which all are involved in T-helper cell responses. Furthermore, the CD40L transfected cells increased the percentage of HKLs expressing surface MHCIIß but unlike other APC maturation stimuli, like CpG, they did not reduce the capacity to internalise antigen. Our results provide the first evidence for the existence of a functional CD40L mediated costimulatory pathway in Atlantic salmon.

Collective action of hematopoietic cell subsets mediates anti-IL10R1 and CpG tumor immunity.

Based on the specificity of antigen recognition and the ability to generate long-lived memory responses, cancer immunotherapies primarily target tumor-associated T cells. Systemic administration of anti-IL-10R1 antibody in combination with local CpG administration has been shown to induce tumor regression in a T-cell-dependent manner. Here, we confirmed the anti-tumor efficacy of anti-IL-10R1 and CpG therapy in the highly aggressive B16F10 melanoma model. However, T cells were not required for tumor growth inhibition. Through cellular depletions and genetic models of leukocyte deficiency, we demonstrated that T, B, and NK cells, and neutrophils are not essential for anti-tumor efficacy. Nevertheless, hematopoietic cells as a whole are required for anti-IL-10R1- and CpG-induced tumor growth inhibition, suggesting that the collective action of multiple subsets of hematopoietic-derived cells is required for anti-tumor efficacy.

Adjuvant effect of liposome-encapsulated natural phosphodiester CpG-DNA.

Immunostimulatory CpG-DNA targeting TLR9 is one of the most extensively evaluated vaccine adjuvants. Previously, we found that a particular form of natural phosphodiester bond CpG-DNA (PO-ODN) encapsulated in a phosphatidyl-Β-oleoyl- γ-palmitoyl ethanolamine (DOPE) : cholesterol hemisuccinate (CHEMS) (1 : 1 ratio) complex (Lipoplex(O)) is a potent adjuvant. Complexes containing peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Here, we showed that IL-12 production was increased in bone marrow derived dendritic cells in a CpG sequence-dependent manner when PO-ODN was encapsulated in Lipoplex(O), DOTAP or lipofectamine. However, the effects of Lipoplex(O) surpassed those of PO-ODN encapsulated in DOTAP or lipofectamine and also other various forms of liposome-encapsulated CpG-DNA in terms of potency for protein antigen-specific IgG production and Th1- associated IgG2a production. Therefore, Lipoplex(O) may have a unique potent immunoadjuvant activity which can be useful for various applications involving protein antigens as well as peptides.

Bis-(3',5')-cyclic dimeric adenosine monophosphate: strong Th1/Th2/Th17 promoting mucosal adjuvant.

New effective adjuvants are required to improve the performance of subunit vaccines. Here, we showed that bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP), a second messenger molecule in bacteria and archaea, exerts strong adjuvant activities when delivered by mucosal route. In vitro studies showed that c-di-AMP was able to both stimulate pre-activated murine macrophages and promote the activation and maturation of dendritic cells of murine and human origin. Co-administration of c-di-AMP with ß-galactosidase (ß-Gal) by intranasal route to BALB/c mice resulted in the elicitation of significantly higher serum antigen-specific IgG titres than in controls. The induction of local immune responses was shown by the production of antigen-specific secretory IgA in different mucosal territories. In addition, strong cellular immune responses were observed against both the ß-Gal protein and a peptide encompassing its MHC class I-restricted epitope. The ratio of ß-Gal-specific antibodies and the secreted cytokine profiles by in vitro re-stimulated splenocytes suggested that a balanced Th1/Th2/Th17 response pattern is promoted by c-di-AMP. When C57BL/6 mice were immunized with OVA and c-di-AMP, vigorous in vivo CTL responses were also observed. These results indicated that c-di-AMP exhibits a high potential as adjuvant for the development of mucosal vaccines, in particular when cellular immunity is needed.

Novel immunostimulatory phosphodiester oligodeoxynucleotides with CpT sequences instead of CpG motifs.

The innate immune system recognizes bacterial DNA as a nonself to induce rapid immune activation. TLR9 recognizes synthetic oligodeoxynucleotides (ODNs) and bacterial DNA containing unmethylated CpG dinucleotides in the context of specific base sequences (CpG-DNA). Here, we demonstrate that phosphorothioate backbone CT-ODN (PS-CT-ODN), a derivative of phosphorothioate backbone CpG-DNA (PS-ODN) with CT sequences substituted for the CG sequences, stimulates IL-8 promoter activation and gene expression. Furthermore, we identified an immunostimulatory phosphodiester bond CT-ODN (PO-CT-ODN) from Staphylococcus aureus chromosomal DNA and found that the PO-CT-ODN induces cytokine production in a TLR9-dependent manner when encapsulated with a proper liposome. Our experimental analyses also demonstrate that the immunostimulatory PO-CT-ODN can act as an adjuvant for the induction of Ag-driven IgG production. Further investigation of the functional role of PO-CT-ODN may support the future application of PO-CT-ODN in immunotherapeutics.

Resolution of infection promotes a state of dormancy and long survival of CD4 memory T cells.

Memory T cells survive throughout the lifetime of an individual and are protective upon recall. It is not clear how memory T cells can live so long. Here, we demonstrate that at the resolution of a viral infection, low levels of antigen are captured by B cells and presented to specific CD4(+) memory T cells to render a state of unresponsiveness. We demonstrate in two systems that this process occurs naturally during the fall of antigen and is associated with a global gene expression program initiated with the clearance of antigen. Our study suggests that in the absence of antigen, a state of dormancy associated with low-energy utilization and proliferation can help memory CD4(+) T cells to survive nearly throughout the lifetime of mice. The dormant CD4(+) memory T cells become activated by stimulatory signals generated by a subsequent infection. We propose that quiescence might be a mechanism necessary to regulate long-term survival of CD4 memory T cells and to prevent cross-reactivity to self, hence autoimmunity.

Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice.

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.