Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection.

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.

The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2.

The nuclear long non-coding RNA LUCAT1 has previously been identified as a negative feedback regulator of type I interferon and inflammatory cytokine expression in human myeloid cells. Here, we define the mechanistic basis for the suppression of inflammatory gene expression by LUCAT1. Using comprehensive identification of RNA-binding proteins by mass spectrometry as well as RNA immunoprecipitation, we identified proteins important in processing and alternative splicing of mRNAs as LUCAT1-binding proteins. These included heterogeneous nuclear ribonucleoprotein C, M, and A2B1. Consistent with this finding, cells lacking LUCAT1 have altered splicing of selected immune genes. In particular, upon lipopolysaccharide stimulation, the splicing of the nuclear receptor 4A2 (NR4A2) gene was particularly affected. As a consequence, expression of NR4A2 was reduced and delayed in cells lacking LUCAT1. NR4A2-deficient cells had elevated expression of immune genes. These observations suggest that LUCAT1 is induced to control the splicing and stability of NR4A2, which is in part responsible for the anti-inflammatory effect of LUCAT1. Furthermore, we analyzed a large cohort of patients with inflammatory bowel disease as well as asthma and chronic obstructive pulmonary disease. In these patients, LUCAT1 levels were elevated and in both diseases, positively correlated with disease severity. Collectively, these studies define a key molecular mechanism of LUCAT1-dependent immune regulation through post-transcriptional regulation of mRNAs highlighting its role in the regulation of inflammatory disease.

NRF2 activators inhibit influenza A virus replication by interfering with nucleo-cytoplasmic export of viral RNPs in an NRF2-independent manner.

In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.

Inhibition of exchange proteins directly activated by cAMP as a strategy for broad-spectrum antiviral development.

The recent SARS-CoV-2 and mpox outbreaks have highlighted the need to expand our arsenal of broad-spectrum antiviral agents for future pandemic preparedness. Host-directed antivirals are an important tool to accomplish this as they typically offer protection against a broader range of viruses than direct-acting antivirals and have a lower susceptibility to viral mutations that cause drug resistance. In this study, we investigate the exchange protein activated by cAMP (EPAC) as a target for broad-spectrum antiviral therapy. We find that the EPAC-selective inhibitor, ESI-09, provides robust protection against a variety of viruses, including SARS-CoV-2 and Vaccinia (VACV)-an orthopox virus from the same family as mpox. We show, using a series of immunofluorescence experiments, that ESI-09 remodels the actin cytoskeleton through Rac1/Cdc42 GTPases and the Arp2/3 complex, impairing internalization of viruses that use clathrin-mediated endocytosis (e.g. VSV) or micropinocytosis (e.g. VACV). Additionally, we find that ESI-09 disrupts syncytia formation and inhibits cell-to-cell transmission of viruses such as measles and VACV. When administered to immune-deficient mice in an intranasal challenge model, ESI-09 protects mice from lethal doses of VACV and prevents formation of pox lesions. Altogether, our finding shows that EPAC antagonists such as ESI-09 are promising candidates for broad-spectrum antiviral therapy that can aid in the fight against ongoing and future viral outbreaks.

Breaking the barrier: membrane fusion triggers innate antiviral immunity.

The sensing of viral infection by the innate immune system is dominated by the recognition of nucleic acids. New data now demonstrate that the fusion of viral and target-cell membranes leads to the activation of an immune response dependent on the adaptor STING.

Identification of FDA-approved bifonazole as a SARS-CoV-2 blocking agent following a bioreporter drug screen.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Harnessing Autophagy to Overcome Antigen-Specific T-Cell Dysfunction: Implication for People Living with HIV-1.

Like other chronic viral infections, HIV-1 persistence inhibits the development of antigen-specific memory T-cells, resulting in the exhaustion of the immune response and chronic inflammation. Autophagy is a major lysosome-dependent mechanism of intracellular large-target degradation such as lipid and protein aggregates, damaged organelles, and intracellular pathogens. Although it is known that autophagy may target HIV-1 for elimination, knowledge of its function as a metabolic contributor in such viral infection is only in its infancy. Recent data show that elite controllers (EC), who are HIV-1-infected subjects with natural and long-term antigen (Ag)-specific T-cell protection against the virus, are characterized by distinct metabolic autophagy-dependent features in their T-cells compared to other people living with HIV-1 (PLWH). Despite durable viral control with antiretroviral therapy (ART), HIV-1-specific immune dysfunction does not normalize in non-controller PLWH. Therefore, the hypothesis of inducing autophagy to strengthen their Ag-specific T-cell immunity against HIV-1 starts to be an enticing concept. The aim of this review is to critically analyze promises and potential limitations of pharmacological and dietary interventions to activate autophagy in an attempt to rescue Ag-specific T-cell protection among PLWH.

Postpartum Disseminated Herpes Simplex Virus Type 1 Infection With Hemophagocytic Lymphohistiocytosis and Fulminant Neonatal Herpes Infection.

The present study describes a 19-year-old woman with systemic herpes simplex virus (HSV)-1 infection and hemophagocytic lymphohistiocytosis (HLH) postpartum, and a fatal course of neonatal herpesvirus infection. Functional investigation of cells from the mother demonstrated significantly impaired induction of antiviral interferons and cytokines in the context of normal activation of the transcription factors NF-κB and IRF3. Whole-exome sequencing did not reveal any functionally validated genetic variants. We suggest that the functionally impaired antiviral responses, potentially caused by a variant in CASP8 or other variants in noncoding regions of the genome, contributed to the unusually severe disease course observed in two generations.

The C-type lectin receptors dectin-1, MR, and SIGNR3 contribute both positively and negatively to the macrophage response to Leishmania infantum.

Macrophages act as the primary effector cells during Leishmania infection through production of reactive oxygen species (ROS) and interleukin-1ß (IL-1ß). However, how macrophage-killing mechanisms are activated during Leishmania-macrophage interactions is poorly understood. Here, we report that the macrophage response against Leishmania infantum in vivo is characterized by an M2b-like phenotype and C-type lectin receptors (CLRs) signature composed of Dectin-1, mannose receptor (MR), and the DC-SIGN homolog SIGNR3 expression. Dectin-1 and MR were crucial for the microbicidal response as indicated by the fact that they activated Syk-p47phox and arachidonic acid (AA)-NADPH oxidase signaling pathways, respectively, needed for ROS production and also triggered Syk-coupled signaling for caspase-1-induced IL-1ß secretion. In contrast, SIGNR3 has divergent functions during Leishmania infantum pathogenesis; this CLR favored parasite resilience through inhibition of the LTB4-IL-1ß axis. These pathways also operated during infection of primary human macrophages. Therefore, our study promotes CLRs as potential targets for treatment, diagnosis, and prevention of visceral leishmaniasis.

SARS-CoV-2 evades immune detection in alveolar macrophages.

Respiratory infections, like the current COVID-19 pandemic, target epithelial cells in the respiratory tract. Alveolar macrophages (AMs) are tissue-resident macrophages located within the lung. They play a key role in the early phases of an immune response to respiratory viruses. AMs are likely the first immune cells to encounter SARS-CoV-2 during an infection, and their reaction to the virus will have a profound impact on the outcome of the infection. Interferons (IFNs) are antiviral cytokines and among the first cytokines produced upon viral infection. In this study, AMs from non-infectious donors are challenged with SARS-CoV-2. We demonstrate that challenged AMs are incapable of sensing SARS-CoV-2 and of producing an IFN response in contrast to other respiratory viruses, like influenza A virus and Sendai virus, which trigger a robust IFN response. The absence of IFN production in AMs upon challenge with SARS-CoV-2 could explain the initial asymptotic phase observed during COVID-19 and argues against AMs being the sources of pro-inflammatory cytokines later during infection.

Dengue Virus Targets Nrf2 for NS2B3-Mediated Degradation Leading to Enhanced Oxidative Stress and Viral Replication.

Dengue virus (DENV) is a mosquito-borne virus that infects upward of 300 million people annually and has the potential to cause fatal hemorrhagic fever and shock. While the parameters contributing to dengue immunopathogenesis remain unclear, the collapse of redox homeostasis and the damage induced by oxidative stress have been correlated with the development of inflammation and progression toward the more severe forms of disease. In the present study, we demonstrate that the accumulation of reactive oxygen species (ROS) late after DENV infection (>24 hpi) resulted from a disruption in the balance between oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant response. The DENV NS2B3 protease complex strategically targeted Nrf2 for degradation in a proteolysis-independent manner; NS2B3 licensed Nrf2 for lysosomal degradation. Impairment of the Nrf2 regulator by the NS2B3 complex inhibited the antioxidant gene network and contributed to the progressive increase in ROS levels, along with increased virus replication and inflammatory or apoptotic gene expression. By 24 hpi, when increased levels of ROS and antiviral proteins were observed, it appeared that the proviral effect of ROS overcame the antiviral effects of the interferon (IFN) response. Overall, these studies demonstrate that DENV infection disrupts the regulatory interplay between DENV-induced stress responses, Nrf2 antioxidant signaling, and the host antiviral immune response, thus exacerbating oxidative stress and inflammation in DENV infection.IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen that threatens 2.5 billion people in more than 100 countries annually. Dengue infection induces a spectrum of clinical symptoms, ranging from classical dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome; however, the complexities of DENV immunopathogenesis remain controversial. Previous studies have reported the importance of the transcription factor Nrf2 in the control of redox homeostasis and antiviral/inflammatory or death responses to DENV. Importantly, the production of reactive oxygen species and the subsequent stress response have been linked to the development of inflammation and progression toward the more severe forms of the disease. Here, we demonstrate that DENV uses the NS2B3 protease complex to strategically target Nrf2 for degradation, leading to a progressive increase in oxidative stress, inflammation, and cell death in infected cells. This study underlines the pivotal role of the Nrf2 regulatory network in the context of DENV infection.

Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling.

The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.

SIRT1 Modulates the Sensitivity of Prostate Cancer Cells to Vesicular Stomatitis Virus Oncolysis.

Oncolytic virotherapy represents a promising experimental anticancer strategy, based on the use of genetically modified viruses to selectively infect and kill cancer cells. Vesicular stomatitis virus (VSV) is a prototypic oncolytic virus (OV) that induces cancer cell death through activation of the apoptotic pathway, although intrinsic resistance to oncolysis is found in some cell lines and many primary tumors, as a consequence of residual innate immunity to the virus. In the effort to improve OV therapeutic efficacy, we previously demonstrated that different agents, including histone deacetylase inhibitors (HDIs), functioned as reversible chemical switches to dampen the innate antiviral response and improve the susceptibility of resistant cancer cells to VSV infection. In the present study, we demonstrated that the NAD+-dependent histone deacetylase SIRT1 (silent mating type information regulation 2 homolog 1) plays a key role in the permissivity of prostate cancer PC-3 cells to VSVΔM51 replication and oncolysis. HDI-mediated enhancement of VSVΔM51 infection and cancer cell killing directly correlated with a decrease of SIRT1 expression. Furthermore, pharmacological inhibition as well as silencing of SIRT1 by small interfering RNA (siRNA) was sufficient to sensitize PC-3 cells to VSVΔM51 infection, resulting in augmentation of virus replication and spread. Mechanistically, HDIs such as suberoylanilide hydroxamic acid (SAHA; Vorinostat) and resminostat upregulated the microRNA miR-34a that regulated the level of SIRT1. Taken together, our findings identify SIRT1 as a viral restriction factor that limits VSVΔM51 infection and oncolysis in prostate cancer cells.IMPORTANCE The use of nonpathogenic viruses to target and kill cancer cells is a promising strategy in cancer therapy. However, many types of human cancer are resistant to the oncolytic (cancer-killing) effects of virotherapy. In this study, we identify a host cellular protein, SIRT1, that contributes to the sensitivity of prostate cancer cells to infection by a prototypical oncolytic virus. Knockout of SIRT1 activity increases the sensitivity of prostate cancer cells to virus-mediated killing. At the molecular level, SIRT1 is controlled by a small microRNA termed miR-34a. Altogether, SIRT1 and/or miR-34a levels may serve as predictors of response to oncolytic-virus therapy.

Crosstalk between Cytoplasmic RIG-I and STING Sensing Pathways.

Detection of evolutionarily conserved molecules on microbial pathogens by host immune sensors represents the initial trigger of the immune response against infection. Cytosolic receptors sense viral and intracellular bacterial genomes, as well as nucleic acids produced during replication. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Although distinct classes of receptors are responsible for the RNA and DNA sensing, the downstream signaling components are physically and functionally interconnected. This review highlights the importance of the crosstalk between retinoic acid inducible gene-I (RIG-I)-mitochondrial antiviral-signaling protein (MAVS) RNA sensing and the cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) DNA sensing pathways in potentiating efficient antiviral responses. The potential of cGAS-STING manipulation as a component of cancer immunotherapy is also reviewed.

An optimized retinoic acid-inducible gene I agonist M8 induces immunogenic cell death markers in human cancer cells and dendritic cell activation.

RIG-I is a cytosolic RNA sensor that recognizes short 5' triphosphate RNA, commonly generated during virus infection. Upon activation, RIG-I initiates antiviral immunity, and in some circumstances, induces cell death. Because of this dual capacity, RIG-I has emerged as a promising target for cancer immunotherapy. Previously, a sequence-optimized RIG-I agonist (termed M8) was generated and shown to stimulate a robust immune response capable of blocking viral infection and to function as an adjuvant in vaccination strategies. Here, we investigated the potential of M8 as an anti-cancer agent by analyzing its ability to induce cell death and activate the immune response. In multiple cancer cell lines, M8 treatment strongly activated caspase 3-dependent apoptosis, that relied on an intrinsic NOXA and PUMA-driven pathway that was dependent on IFN-I signaling. Additionally, cell death induced by M8 was characterized by the expression of markers of immunogenic cell death-related damage-associated molecular patterns (ICD-DAMP)-calreticulin, HMGB1 and ATP-and high levels of ICD-related cytokines CXCL10, IFNß, CCL2 and CXCL1. Moreover, M8 increased the levels of HLA-ABC expression on the tumor cell surface, as well as up-regulation of genes involved in antigen processing and presentation. M8 induction of the RIG-I pathway in cancer cells favored dendritic cell phagocytosis and induction of co-stimulatory molecules CD80 and CD86, together with increased expression of IL12 and CXCL10. Altogether, these results highlight the potential of M8 in cancer immunotherapy, with the capacity to induce ICD-DAMP on tumor cells and activate immunostimulatory signals that synergize with current therapies.

Activation of Nrf2 Signaling Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity.

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.

RIG-I-Mediated STING Upregulation Restricts Herpes Simplex Virus 1 Infection.

UNLABELLED: STING has emerged in recent years as a key player in orchestrating innate immune responses to cytosolic DNA and RNA derived from pathogens. However, the regulation of STING still remains poorly defined. In the present study, we investigated the mechanism of the regulation of STING expression in relation to the RIG-I pathway. Our data show that signaling through RIG-I induces STING expression at both the transcriptional and protein levels in various cell types. STING induction by the RIG-I agonist 5'triphosphorylated RNA (5'pppRNA) was recognized to be a delayed event resulting from an autocrine/paracrine mechanism. Indeed, cotreatment with tumor necrosis factor alpha and type I/II interferon was found to have a synergistic effect on the regulation of STING expression and could be potently decreased by impairing NF-κB and/or STAT1/2 signaling. STING induction significantly contributed to sustainment of the immune signaling cascade following 5'pppRNA treatment. Physiologically, this cross talk between the RNA- and DNA-sensing pathways allowed 5'pppRNA to efficiently block infection by herpes simplex virus 1 (HSV-1) both in vitro and in vivo in a STING-dependent fashion. These observations demonstrate that STING induction by RIG-I signaling through the NF-κB and STAT1/2 cascades is essential for RIG-I agonist-mediated HSV-1 restriction. IMPORTANCE: The innate immune system represents the first line of defense against invading pathogens. The dysregulation of this system can result in failure to combat pathogens, inflammation, and autoimmune diseases. Thus, precise regulation at each level of the innate immune system is crucial. Recently, a number of studies have established STING to be a central molecule in the innate immune response to cytosolic DNA and RNA derived from pathogens. Here, we describe the regulation of STING via RIG-I-mediated innate immune sensing. We found that STING is synergistically induced via proinflammatory and antiviral cytokine cascades. In addition, we show that in vivo protection against herpes simplex virus 1 (HSV-1) by a RIG-I agonist required STING. Our study provides new insights into the cross talk between DNA and RNA pathogen-sensing systems via the control of STING.

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

UNLABELLED: The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. IMPORTANCE: The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.