A Small Molecule RIG-I Agonist Serves as an Adjuvant to Induce Broad Multifaceted Influenza Virus Vaccine Immunity.

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing noncanonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.

SARS-CoV-2 Serologic Assays in Control and Unknown Populations Demonstrate the Necessity of Virus Neutralization Testing.

BACKGROUND: To determine how serologic antibody testing outcome links with virus neutralization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we evaluated individuals for SARS-CoV-2 antibody level and viral neutralization. METHODS: We compared serum Ig levels across platforms of viral antigens and antibodies with 15 positive and 30 negative SARS-CoV-2 controls followed by viral neutralization assessment. We then applied these platforms to a clinically relevant cohort of 114 individuals with unknown histories of SARS-CoV-2 infection. RESULTS: In controls, the best-performing virus-specific antibody detection platforms were SARS-CoV-2 receptor binding domain (RBD) IgG (sensitivity 87%, specificity 100%, positive predictive value [PPV] 100%, negative predictive value [NPV] 94%), spike IgG3 (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%), and nucleocapsid protein (NP) IgG (sensitivity 93%, specificity 97%, PPV 93%, NPV 97%). Neutralization of positive and negative control sera showed 100% agreement. Twenty individuals with unknown history had detectable SARS-CoV-2 antibodies with 16 demonstrating virus neutralization. Spike IgG3 provided the highest accuracy for predicting serologically positive individuals with virus neutralization activity (misidentified 1/20 unknowns compared to 2/20 for RBD and NP IgG). CONCLUSIONS: The coupling of virus neutralization analysis to a spike IgG3 antibody test is optimal to categorize patients for correlates of SARS-CoV-2 immune protection status.

DHX15 Is a Coreceptor for RLR Signaling That Promotes Antiviral Defense Against RNA Virus Infection.

RNA helicases play an important role in the response to microbial infection. Retinoic acid inducible gene-I (RIG-I) and members of the RIG-I-like receptor (RLR) family of helicases function as cytoplasmic pattern recognition receptors (PRRs) whose actions are essential for recognition of RNA viruses. RIG-I association with pathogen-associated molecular patterns (PAMPs) within viral RNA leads to its activation and signaling via the mitochondrial antiviral signaling (MAVS) adapter protein. This interaction mediates downstream signaling events that drive the innate immune response to virus infection. Here we identify the DEAH-box RNA helicase DHX15 as a RLR binding partner and signaling cofactor. In human cells, DHX15 is required for virus-induced RLR signaling of innate immune gene expression. Knockdown of DHX15 increased susceptibility to infection by RNA viruses of diverse genera, including Paramyxoviridae, Rhabdoviridae, and Picornaviridae. DHX15 associates with RIG-I caspase activation and recruitment domains (CARDs) through its amino terminus, in which the complex is recruited to MAVS on virus infection. Importantly, although DHX15 cannot substitute for RIG-I in innate immune signaling, DHX15 selectively binds PAMP RNA to promote RIG-I ATP hydrolysis and signaling activation in response to viral RNA. Our results define DHX15 as a coreceptor required for RLR innate immune responses to control RNA virus infection.

Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway.

UNLABELLED: The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen-associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor interferon regulatory factor 3 (IRF3). IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines, that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infections caused by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with viruses of the family Flaviviridae, including West Nile virus, dengue virus, and hepatitis C virus, as well as viruses of the families Filoviridae (Ebola virus), Orthomyxoviridae (influenza A virus), Arenaviridae (Lassa virus), and Paramyxoviridae (respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infections caused by RNA viruses of distinct genera. IMPORTANCE: Incidences of emerging and reemerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infections caused by viruses of distinct genera. We identified small molecule compounds that can selectively activate IRF3 for the purpose of identifying drug-like molecules that can be developed for the treatment of viral infections. Here, we report the discovery of a hydroxyquinoline family of small molecules that can activate IRF3 to promote cellular antiviral responses. These molecules can prophylactically or therapeutically control infection in cell culture by pathogenic RNA viruses, including West Nile virus, dengue virus, hepatitis C virus, influenza A virus, respiratory syncytial virus, Nipah virus, Lassa virus, and Ebola virus. Our study thus identifies a class of small molecules with a novel mechanism to enhance host immune responses for antiviral activity against a variety of RNA viruses that pose a significant health care burden and/or that are known to cause infections with high case fatality rates.

Role of the mTOR pathway in LPS-activated monocytes: influence of hypertonic saline.

BACKGROUND: As heightened protein synthesis is the hallmark of many inflammatory syndromes, we hypothesize that the mammalian target of rapamycin (mTOR) pathway, which control the cap-dependent translation initiation phase, was activated by lipopolysaccharide (LPS). In addition, we studied the effect of hypertonic saline solution (HTS) on the mTOR cascade in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: PBMCs were isolated from healthy volunteers and treated with LPS. Cells were pretreated with phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors, or with HTS. Supernatants were harvested 20 h following LPS treatment, and interleukin-10 (IL-10), interleukin-6 (IL-6) and tumor necrosis alpha (TNFα) were analyzed by ELISA. Immunoblot experiments were performed for components of the PI3K/Akt/mTOR pathway at various time points. RNA was extracted after 90 min for real-time RT-PCR quantification. RESULTS: The mTOR pathway is activated in PBMCs within 1 h of LPS stimulation. Pretreatment with rapamycin, a specific inhibitor of mTOR, resulted in a significant decrease of IL-10 and IL-6 translation and expression but did not affect the LPS-induced TNFα production. Both the mTOR pathway and the LPS-induced IL-6 production were down-regulated by HTS pretreatment. CONCLUSIONS: The PI3k/Akt/mTOR cascade modulates LPS-induced cytokines production differentially. IL-10 and IL-6 expression are both up-regulated by activation of the mTOR pathway in response to LPS in PBMCs, while TNFα is not controlled by the mTOR cascade. Meanwhile, pretreatment of PBMCs with a HTS solution suppresses mTOR activity as well as LPS-induced IL-6, suggesting a more central role for mTOR as a regulator of the immuno-inflammatory response.