The role of cGAMP via the STING pathway in modulating germinal center responses and CD4 T cell differentiation.

Germinal center (GC) responses are essential for establishing protective, long-lasting immunity through the differentiation of GC B cells (BGC) and plasma cells (BPC), along with the generation of antigen-specific antibodies. Among the various pathways influencing immune responses, the STING (Stimulator of Interferon Genes) pathway has emerged as significant, especially in innate immunity, and extends its influence to adaptive responses. In this study, we examined how the STING ligand cGAMP can modulate these key elements of the adaptive immune response, particularly in enhancing GC reactions and the differentiation of BGC, BPC, and follicular helper T cells (TFH). Employing in vivo models, we evaluated various antigens and the administration of cGAMP in Alum adjuvant, investigating the differentiation of BGC, BPC, and TFH cells, along with the production of antigen-specific antibodies. cGAMP enhances the differentiation of BGC and BPC, leading to increased antigen-specific antibody production. This effect is shown to be type I Interferon-dependent, with a substantial reduction in BPC frequency upon interferon (IFN)-ß blockade. Additionally, cGAMP's influence on TFH differentiation varies over time, which may be critical for refining vaccine strategies. The findings elucidate a complex, antigen-specific influence of cGAMP on T and B cell responses, providing insights that could optimize vaccine efficacy.

Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

Cyclic nucleotide-induced helical structure activates a TIR immune effector.

Cyclic nucleotide signalling is a key component of antiviral defence in all domains of life. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway1, which originated in bacteria2. These defence systems require a sensor domain to bind the cyclic nucleotide and are often coupled with an effector domain that, when activated, causes cell death by destroying essential biomolecules3. One example is the Toll/interleukin-1 receptor (TIR) domain, which degrades the essential cofactor NAD+ when activated in response to infection in plants and bacteria2,4,5 or during programmed nerve cell death6. Here we show that a bacterial antiviral defence system generates a cyclic tri-adenylate that binds to a TIR-SAVED effector, acting as the 'glue' to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organize and complete a composite active site, allowing NAD+ degradation. Activation requires extended filament formation, both in vitro and in vivo. Our study highlights an example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

African Swine Fever Virus EP364R and C129R Target Cyclic GMP-AMP To Inhibit the cGAS-STING Signaling Pathway.

African swine fever virus (ASFV) is a highly pathogenic swine DNA virus with high mortality that causes African swine fever (ASF) in domestic pigs and wild boars. For efficient viral infection, ASFV has developed complex strategies to evade key components of antiviral innate immune responses. However, the immune escape mechanism of ASFV remains unclear. Upon ASFV infection, cyclic GMP-AMP (2',3'-cGAMP) synthase (cGAS), a cytosolic DNA sensor, recognizes ASFV DNA and synthesizes the second messenger 2',3'-cGAMP, which triggers interferon (IFN) production to interfere with viral replication. In this study, we demonstrated a novel immune evasion mechanism of ASFV EP364R and C129R, which blocks cellular cyclic 2',3'-cGAMP-mediated antiviral responses. ASFV EP364R and C129R with nuclease homology inhibit IFN-mediated responses by specifically interacting with 2',3'-cGAMP and exerting their phosphodiesterase (PDE) activity to cleave 2',3'-cGAMP. Particularly notable is that ASFV EP364R had a region of homology with the stimulator of interferon genes (STING) protein containing a 2',3'-cGAMP-binding motif and point mutations in the Y76S and N78A amino acids of EP364R that impaired interaction with 2',3'-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2',3'-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2',3'-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines.

A photoaffinity labeling strategy identified EF1A1 as a binding protein of cyclic dinucleotide 2'3'-cGAMP.

2'3'-cyclic GMP-AMP (2'3'-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2'3'-cGAMP, it is important to find out whether 2'3'-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2'3'-cGAMP-based photoaffinity probes that capture and isolate 2'3'-cGAMP-binding proteins. These probes enable the identification of some potential 2'3'-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2'3'-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2'3'-cGAMP interactome, discover unknown functions of 2'3'-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.

A two-adjuvant multiantigen candidate vaccine induces superior protective immune responses against SARS-CoV-2 challenge.

An ideal vaccine against SARS-CoV-2 is expected to elicit broad immunity to prevent viral infection and disease, with efficient viral clearance in the upper respiratory tract (URT). Here, the N protein and prefusion-full S protein (SFLmut) are combined with flagellin (KF) and cyclic GMP-AMP (cGAMP) to generate a candidate vaccine, and this vaccine elicits stronger systemic and mucosal humoral immunity than vaccines containing other forms of the S protein. Furthermore, the candidate vaccine administered via intranasal route can enhance local immune responses in the respiratory tract. Importantly, human ACE2 transgenic mice given the candidate vaccine are protected against lethal SARS-CoV-2 challenge, with superior protection in the URT compared with that in mice immunized with an inactivated vaccine. In summary, the developed vaccine can elicit a multifaceted immune response and induce robust viral clearance in the URT, which makes it a potential vaccine for preventing disease and infection of SARS-CoV-2.

The cyclic dinucleotide 2'3'-cGAMP induces a broad antibacterial and antiviral response in the sea anemone Nematostella vectensis.

In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis Using RNA sequencing, we found that 2'3'-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis Many of the antiviral genes induced by 2'3'-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2'3'-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2'3'-cGAMP signaling in animals.

STING Ligand-Mediated Priming of Functional CD8+ T Cells Specific for HIV-1-Protective Epitopes from Naive T Cells.

Functional HIV-1-specific CD8+ T cells primed from naive T cells are expected to act as effector T cells in a "shock-and-kill" therapeutic strategy for an HIV-1 cure since less functional HIV-1-specific CD8+ T cells are elicited from memory T cells in HIV-1-infected individuals on combined antiretroviral therapy (cART). CD8+ T cells specific for HIV-1 conserved and protective epitopes are candidates for such T cells. We investigated the priming with STING ligand of CD8+ T cells specific for HLA-B*52:01 or HLA-C*12:02-restricted protective epitopes from naive T cells. STING ligand 3'3'-cGAMP effectively primed CD8+ T cells specific for 3 of 4 HLA-B*52:01-restricted epitopes but failed to prime those specific for all 3 HLA-C*12:02-restricted epitopes from the naive T cells of HIV-1-uninfected individuals having an HLA-B*52:01-C*12:02 protective haplotype. These HLA-B*52:01-restricted CD8+ T cells had a strong ability to suppress HIV-1 replication and expressed a high level of cytolytic effector molecules. The viral suppression ability of these T cells was significantly correlated with the expression level of perforin and showed a trend for a positive correlation with the expression level of CD107a. The present study highlighted the priming with STING ligand of functional CD8+ T cells specific for protective epitopes, which T cells would contribute as effector T cells to a shock-and-kill therapy. IMPORTANCE The current "shock-and-kill" therapeutic strategy for HIV cure has been directed toward eliminating latent viral reservoirs by reactivation of latent reservoirs with latency-reversing agents followed by eradication of these cells by immune-mediated responses. Although HIV-1-specific T cells are expected to eradicate viral reservoirs, the function of these T cells is reduced in HIV-1-infected individuals with long-term cART. Therefore, priming of HIV-1-specific T cells with high function from naive T cells is to be expected in these individuals. In this study, we demonstrated the priming with STING ligand 3'3'-cGAMP of CD8+ T cells specific for HIV-1-protective epitopes from naive T cells. cGAMP primed CD8+ T cells specific for 3 HLA-B*52:01-restricted protective epitopes, which cells expressed a high level of cytolytic effector molecules and effectively suppressed HIV-1 replication. The present study suggested that the priming with STING ligand of functional CD8+ T cells specific for protective epitopes would be useful in a therapy for an HIV-1 cure.

Cytosolic d-type CpG-oligonucleotides induce a type I interferon response by activating the cGAS-STING signaling pathway.

Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d-type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self-nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.

Regulation of Anion Channel LRRC8 Volume-Regulated Anion Channels in Transport of 2'3'-Cyclic GMP-AMP and Cisplatin under Steady State and Inflammation.

The recently identified anion channel LRRC8 volume-regulated anion channels (VRACs) are heteromeric hexamers constituted with the obligate LRRC8A subunit paired with at least one of the accessory LRRC8B to LRRC8E subunits. In addition to transport chloride, taurine, and glutamate, LRRC8 VRACs also transport the anticancer agent cisplatin and STING agonists 2'3'-cyclic GMP-AMP (cGAMP) and cyclic dinucleotides; hence, they are implicated in a variety of physiological and pathological processes, such as cell swelling, stroke, cancer, and viral infection. Although the subunit composition largely determines VRAC substrate specificity, the opening of various VRAC pores under physiological and pathological settings remains enigmatic. In this study, we demonstrated that VRACs comprising LRRC8A and LRRC8E (LRRC8A/E-containing VRACs), specialized in cGAMP transport, can be opened by a protein component present in serum under resting condition. Serum depletion ablated the tonic activity of LRRC8A/E-containing VRACs, decreasing cGAMP transport in various human and murine cells. Also, heating or proteinase K treatment abolished the ability of serum to activate VRAC. Genetic analyses revealed a crucial role for cGAMP synthase (cGAS) in serum/TNF-promoted VRAC activation. Notably, the presence of cGAS on the plasma membrane, rather than its DNA-binding or enzymatic activity, enabled VRAC activation. Moreover, phospholipid PIP2 seemed to be instrumental in the membrane localization of cGAS and its association with VRACs. Corroborating a role for LRRC8A/D-containing VRACs in cisplatin transport, serum and TNF markedly potentiated cisplatin uptake and killing of cancer cells derived from human or mouse. Together, these observations provide new insights into the complex regulation of VRAC activation and suggest a novel approach to enhance the efficacy of cGAMP and cisplatin in treating infection and cancer.

The cGAS-STING pathway as a therapeutic target in inflammatory diseases.

The cGAS-STING signalling pathway has emerged as a key mediator of inflammation in the settings of infection, cellular stress and tissue damage. Underlying this broad involvement of the cGAS-STING pathway is its capacity to sense and regulate the cellular response towards microbial and host-derived DNAs, which serve as ubiquitous danger-associated molecules. Insights into the structural and molecular biology of the cGAS-STING pathway have enabled the development of selective small-molecule inhibitors with the potential to target the cGAS-STING axis in a number of inflammatory diseases in humans. Here, we outline the principal elements of the cGAS-STING signalling cascade and discuss the general mechanisms underlying the association of cGAS-STING activity with various autoinflammatory, autoimmune and degenerative diseases. Finally, we outline the chemical nature of recently developed cGAS and STING antagonists and summarize their potential clinical applications.

STING Agonist Mitigates Experimental Autoimmune Encephalomyelitis by Stimulating Type I IFN-Dependent and -Independent Immune-Regulatory Pathways.

The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of IFN genes (STING) pathway induces a powerful type I IFN (IFN-I) response and is a prime candidate for augmenting immunity in cancer immunotherapy and vaccines. IFN-I also has immune-regulatory functions manifested in several autoimmune diseases and is a first-line therapy for relapsing-remitting multiple sclerosis. However, it is only moderately effective and can induce adverse effects and neutralizing Abs in recipients. Targeting cGAMP in autoimmunity is unexplored and represents a challenge because of the intracellular location of its receptor, STING. We used microparticle (MP)-encapsulated cGAMP to increase cellular delivery, achieve dose sparing, and reduce potential toxicity. In the C57BL/6 experimental allergic encephalomyelitis (EAE) model, cGAMP encapsulated in MPs (cGAMP MPs) administered therapeutically protected mice from EAE in a STING-dependent fashion, whereas soluble cGAMP was ineffective. Protection was also observed in a relapsing-remitting model. Importantly, cGAMP MPs protected against EAE at the peak of disease and were more effective than rIFN-ß. Mechanistically, cGAMP MPs showed both IFN-I-dependent and -independent immunosuppressive effects. Furthermore, it induced the immunosuppressive cytokine IL-27 without requiring IFN-I. This augmented IL-10 expression through activated ERK and CREB. IL-27 and subsequent IL-10 were the most important cytokines to mitigate autoreactivity. Critically, cGAMP MPs promoted IFN-I as well as the immunoregulatory cytokines IL-27 and IL-10 in PBMCs from relapsing-remitting multiple sclerosis patients. Collectively, this study reveals a previously unappreciated immune-regulatory effect of cGAMP that can be harnessed to restrain T cell autoreactivity.

Tuning the Innate Immune Response to Cyclic Dinucleotides by Using Atomic Mutagenesis.

Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.

Development of novel highly sensitive methods to detect endogenous cGAMP in cells and tissue.

Intracellular DNA triggers interferon release during the innate immune response. Cyclic GMP-AMP synthase (cGAS) senses intracellular double-stranded DNA not only in response to viral infection but also under autoimmune conditions. Measuring the levels of cyclic GMP-AMP (cGAMP) as a second messenger of cGAS activation is important to elucidate the physiological and pathological roles of cGAS. Therefore, we generated monoclonal antibodies against cGAMP using hybridoma technology to test antibody specificity and establish methods to detect intracellular cGAMP. The resulting cGAMP-specific antibody enabled the development of a time-resolved fluorescence energy transfer assay with a quantifiable range of 0.1 nM to 100 nM cGAMP. Using this assay, we detected cellular and tissue cGAMP. We confirmed that the cGAMP antibody successfully targeted intracellular cGAMP through immunocytochemical analyses. These results demonstrated that the cGAMP antibody is a powerful tool that allows determining cGAS involvement in autoimmunity and disease pathology at the cell and tissue levels.

STING activation in cancer immunotherapy.

Cancer immunotherapy modulates and leverages the host immune system to treat cancer. The past decade has witnessed historical advancement of cancer immunotherapy. A myriad of approaches have been explored to elicit or augment anticancer innate immunity and/or adaptive immunity. Recently, activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor residing in the endoplasmic reticulum, has shown great potential to enhance antitumor immunity through the induction of a variety of pro-inflammatory cytokines and chemokines, including type I IFNs. A number of natural and synthetic STING agonists have been discovered or developed, and tested in preclinical models and in the clinic for the immunotherapy of diseases such as cancer and infectious diseases. Cyclic dinucleotides (CDNs), such as cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic GMP-AMP (cGAMP), are a class of STING agonists that can elicit immune responses. However, natural CDNs are hydrophilic small molecules with negative charges and are susceptible to enzymatic degradation, leading to low bioavailability in target tissues yet unwanted toxicities and narrow therapeutic windows. Drug delivery systems, coupled with nucleic acid chemistry, have been exploited to address these challenges. Here, we will discuss the underlying immunological mechanisms and approaches to STING activation, with a focus on the delivery of STING agonists, for cancer immunotherapy.

Cyclic GMP-AMP Triggers Asthma in an IL-33-Dependent Manner That Is Blocked by Amlexanox, a TBK1 Inhibitor.

Extracellular host-derived DNA, as one of damage associated molecular patterns (DAMPs), is associated with allergic type 2 immune responses. Immune recognition of such DNA generates the second messenger cyclic GMP-AMP (cGAMP) and induces type-2 immune responses; however, its role in allergic diseases, such as asthma, has not been fully elucidated. This study aimed to determine whether cGAMP could induce asthma when used as an adjuvant. We intranasally sensitized mice with cGAMP together with house dust mite antigen (HDM), followed by airway challenge with HDM. We then assessed the levels of eosinophils in the broncho-alveolar lavage fluid (BALF) and serum HDM-specific antibodies. cGAMP promoted HDM specific allergic asthma, characterized by significantly increased HDM specific IgG1 and total IgE in the serum and infiltration of eosinophils in the BALF. cGAMP stimulated lung fibroblast cells to produce IL-33 in vitro, and mice deficient for IL-33 or IL-33 receptor (ST2) failed to develop asthma enhancement by cGAMP. Not only Il-33-/- mice, but also Sting-/-, Tbk1-/-, and Irf3-/-Irf7-/- mice which lack the cGAMP-mediated innate immune activation failed to increase eosinophils in the BALF than that from wild type mice. Consistently, intranasal and oral administration of amlexanox, a TBK1 inhibitor, decreased cGAMP-induced lung allergic inflammation. Thus, cGAMP functions as a type 2 adjuvant in the lung and can promote allergic asthma in manners that dependent on the intracellular STING/TBK1/IRF3/7 signaling pathway and the resultant intercellular signaling pathway via IL-33 and ST2 might be a novel therapeutic target for allergic asthma.

Enhancing Immune Response and Heterosubtypic Protection Ability of Inactivated H7N9 Vaccine by Using STING Agonist as a Mucosal Adjuvant.

Influenza vaccines for H7N9 subtype have shown low immunogenicity in human clinical trials. Using novel adjuvants might represent the optimal available option in vaccine development. In this study, we demonstrated that the using of the STING agonist cGAMP as a mucosal adjuvant is effective in enhancing humoral, cellular and mucosal immune responses of whole virus, inactivated H7N9 vaccine in mice. A single dose of immunization was able to completely protect mice against a high lethal doses of homologous virus challenge with an significant dose-sparing effect. We also found that intranasal co-administration of H7N9 vaccine with cGAMP could provide effective cross protection against H1N1, H3N2, and H9N2 influenza virus. Furthermore, cGAMP induced significantly higher nucleoprotein specific CD4+ and CD8+ T cells responses in immunized mice, as well as upregulated the IFN-γ and Granzyme B expression in the lung tissue of mice in the early stages post a heterosubtypic virus challenge. These results indicated that STING agonist cGAMP was expected to be an effective mucosal immune adjuvant for pre-pandemic vaccines such as H7N9 vaccines, and the cGAMP combined nasal inactivated influenza vaccine will also be a promising strategy for development of broad-spectrum influenza vaccines.

Cyclic GMP-AMP signalling protects bacteria against viral infection.

The cyclic GMP-AMP synthase (cGAS)-STING pathway is a central component of the cell-autonomous innate immune system in animals1,2. The cGAS protein is a sensor of cytosolic viral DNA and, upon sensing DNA, it produces a cyclic GMP-AMP (cGAMP) signalling molecule that binds to the STING protein and activates the immune response3-5. The production of cGAMP has also been detected in bacteria6, and has been shown, in Vibrio cholerae, to activate a phospholipase that degrades the inner bacterial membrane7. However, the biological role of cGAMP signalling in bacteria remains unknown. Here we show that cGAMP signalling is part of an antiphage defence system that is common in bacteria. This system is composed of a four-gene operon that encodes the bacterial cGAS and the associated phospholipase, as well as two enzymes with the eukaryotic-like domains E1, E2 and JAB. We show that this operon confers resistance against a wide variety of phages. Phage infection triggers the production of cGAMP, which-in turn-activates the phospholipase, leading to a loss of membrane integrity and to cell death before completion of phage reproduction. Diverged versions of this system appear in more than 10% of prokaryotic genomes, and we show that variants with effectors other than phospholipase also protect against phage infection. Our results suggest that the eukaryotic cGAS-STING antiviral pathway has ancient evolutionary roots that stem from microbial defences against phages.