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.

A C57BL/6 Mouse model of SARS-CoV-2 infection recapitulates age- and sex-based differences in human COVID-19 disease and recovery.

We present a comprehensive analysis of SARS-CoV-2 infection and recovery in wild type C57BL/6 mice, demonstrating that this is an ideal model of infection and recovery that accurately phenocopies acute human disease arising from the ancestral SARS-CoV-2. Disease severity and infection kinetics are age- and sex-dependent, as has been reported for humans, with older mice and males in particular exhibiting decreased viral clearance and increased mortality. We identified key parallels with human pathology, including intense virus positivity in bronchial epithelial cells, wide-spread alveolar involvement, recruitment of immune cells to the infected lungs, and acute bronchial epithelial cell death. Moreover, older animals experienced increased virus persistence, delayed dispersal of immune cells into lung parenchyma, and morphologic evidence of tissue damage and inflammation. Parallel analysis of SCID mice revealed that the adaptive immune response was not required for recovery from COVID disease symptoms nor early phase clearance of virus but was required for efficient clearance of virus at later stages of infection. Finally, transcriptional analyses indicated that induction and duration of key innate immune gene programs may explain differences in age-dependent disease severity. Importantly, these data demonstrate that SARS-CoV-2-mediated disease in C57BL/6 mice accurately phenocopies human disease across ages and establishes a platform for future therapeutic and genetic screens for not just SARS-CoV-2 but also novel coronaviruses that have yet to emerge.

A C57BL/6 Mouse Model of SARS-CoV-2 Infection Recapitulates Age- and Sex-Based Differences in Human COVID-19 Disease and Recovery.

We present a comprehensive analysis of SARS-CoV-2 infection and recovery using wild type C57BL/6 mice and a mouse-adapted virus, and we demonstrate that this is an ideal model of infection and recovery that phenocopies acute human disease arising from the ancestral SARS-CoV-2. Disease severity and infection kinetics are age- and sex-dependent, as has been reported for humans, with older mice and males in particular exhibiting decreased viral clearance and increased mortality. We identified key parallels with human pathology, including intense virus positivity in bronchial epithelial cells, wide-spread alveolar involvement, recruitment of immune cells to the infected lungs, and acute bronchial epithelial cell death. Moreover, older animals experienced increased virus persistence, delayed dispersal of immune cells into lung parenchyma, and morphologic evidence of tissue damage and inflammation. Parallel analysis of SCID mice revealed that the adaptive immune response was not required for recovery from COVID disease symptoms nor early phase clearance of virus but was required for efficient clearance of virus at later stages of infection. Finally, transcriptional analyses indicated that induction and duration of key innate immune gene programs may explain differences in age-dependent disease severity. Importantly, these data demonstrate that SARS-CoV-2-mediated disease in C57BL/6 mice phenocopies human disease across ages and establishes a platform for future therapeutic and genetic screens for not just SARS-CoV-2 but also novel coronaviruses that have yet to emerge.

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.

IRF5 regulates unique subset of genes in dendritic cells during West Nile virus infection.

Pathogen recognition receptor (PRR) signaling is critical for triggering innate immune activation and the expression of immune response genes, including genes that impart restriction against virus replication. RIG-I-like receptors and TLRs are PRRs that signal immune activation and drive the expression of antiviral genes and the production of type I IFN leading to induction of IFN-stimulated genes, in part through the interferon regulatory factor (IRF) family of transcription factors. Previous studies with West Nile virus (WNV) showed that IRF3 and IRF7 regulate IFN expression in fibroblasts and neurons, whereas macrophages and dendritic cells (DCs) retained the ability to induce IFN-ß in the absence of IRF3 and IRF7 in a manner implicating IRF5 in PRR signaling actions. Here we assessed the contribution of IRF5 to immune gene induction in response to WNV infection in DCs. We examined IRF5-dependent gene expression and found that loss of IRF5 in mice resulted in modest and subtle changes in the expression of WNV-regulated genes. Anti-IRF5 chromatin immunoprecipitation with next-generation sequencing of genomic DNA coupled with mRNA analysis revealed unique IRF5 binding motifs within the mouse genome that are distinct from the canonical IRF binding motif and that link with IRF5-target gene expression. Using integrative bioinformatics analyses, we identified new IRF5 primary target genes in DCs in response to virus infection. This study provides novel insights into the distinct and unique innate immune and immune gene regulatory program directed by IRF5.

Differential and Overlapping Immune Programs Regulated by IRF3 and IRF5 in Plasmacytoid Dendritic Cells.

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5, an immune-regulatory transcription factor. We show that the protein kinases IKKα, IKKß, IKKε, and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization, thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets, we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs, linking IRF5 with immune regulatory and proinflammatory gene expression. Thus, TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome.

The Role of Lipopolysaccharide Structure in Monocyte Activation and Cytokine Secretion.

BACKGROUND: The lipopolysaccharide (LPS) molecule is composed of a hydrophobic lipid region (Lipid A), an oligosaccharide core, and an O-Antigen chain. Lipid A has been described as the molecular region responsible for inducing activation of immune cells. We hypothesize that the O-Antigen plays a critical role in the activation and responsiveness of mononuclear cell immune function. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with LPS, LPS with attenuated O-Antigen (RF5), or Lipid A (DPL), which lacks an O-Antigen. Selected cells were pretreated with a blocking antibody to CD14. Western blots were performed to determine activation of mitogen-activated protein kinases (MAPK) p38, ERK, and JNK at selected time-points. RNA was extracted for RT-PCR quantification of TNF-α and IL-10 gene transcription. Supernatants were harvested and analyzed by ELISA for tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10). RESULTS: LPS elicited maximal response, including phosphorylation of p38, ERK, and JNK, synthesis of TNF-α and IL-10 mRNA, and secretion of TNF-α and IL-10. Stimulation with RF5 activated the same pathways to a lesser degree. DPL led to increased phosphorylation of p38 and ERK and increased secretion of IL-10. CD14 blockade was associated with a significant decrease in cytokine secretion by LPS, and abolished cytokine secretion in cells stimulated with RF5 or DPL. CONCLUSIONS: Structural variants of LPS activate monocytes differentially. The complete O-Antigen is important for maximal activation of MAPK, cytokine synthesis, and cytokine secretion. LPS with attenuated O-Antigen and Lipid A activate only certain components of these pathways. LPS with a complete O-Antigen stimulates cytokine secretion that is partially independent of CD14, but shortening or removal of the O-Antigen inhibits this secretion.

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.

Eczema in early childhood, sociodemographic factors and lifestyle habits are associated with food allergy: a nested case-control study.

BACKGROUND: Studies suggest an increase in food allergy prevalence over the last decade, but the contributing factors remain unknown. The aim of this study was to evaluate the association between the most common food allergies and atopic history, sociodemographic characteristics and lifestyle habits. METHODS: We conducted a case-control study nested within the SPAACE study (Surveying Prevalence of Food Allergy in All Canadian Environments) ­ a cross-Canada, random telephone survey. Cases consisted of individuals with probable food allergy (self-report of convincing symptoms and/or physician diagnosis) to milk, egg, peanut, tree nut, shellfish, fish, wheat, soy, or sesame. Controls consisted of nonallergic individuals, matched for age. Cases and controls were queried on personal and family history of atopy, sociodemographic characteristics and lifestyle habits. Multivariate logistic regression was used to evaluate the association between atopy, sociodemographic characteristics and lifestyle habits with probable food allergy. RESULTS: Between September 2010 and September 2011, 480 cases and 4,950 controls completed the questionnaire. For all 9 allergens, factors associated with a higher risk of probable allergy were as follows: (1) personal history of eczema (in the first 2 years of life), asthma or hay fever (odds ratio, OR 2.3, 95% CI 1.6-3.5; OR 2.8, 95% CI 2.2-3.6, and OR 2.3, 95% CI 1.8-3.0, respectively), (2) maternal, paternal or sibling's food allergy (OR 3.7, 95% CI 2.5-5.6; OR 3.0, 95% CI 1.8-5.1, and OR 3.1, 95% CI 2.2-4.2), (3) high household income (top 20%; OR 1.5, 95% CI 1.2-2.0). Males and older individuals were less likely to have food allergy (OR 0.7, 95% CI 0.6-0.9, and OR 0.99, 95% CI 0.99-1.00). Eczema in the first 2 years of life was the strongest risk factor for egg, peanut, tree nut and fish allergy. CONCLUSIONS: This is the largest population-based nested case-control study exploring factors associated with food allergies. Our results reveal that, in addition to previously reported factors, eczema in the first 2 years of life is consistently associated with food allergies.

Prevalence and predictors of food allergy in Canada: a focus on vulnerable populations.

BACKGROUND: Studies suggest that individuals of low education and/or income, new Canadians (immigrated <10 years ago), and individuals of Aboriginal identity may have fewer food allergies than the general population. However, given the difficulty in recruiting such populations (hereafter referred to as vulnerable populations), by using conventional survey methodologies, the prevalence of food allergy among these populations in Canada has not been estimated. OBJECTIVES: To estimate the prevalence of food allergy among vulnerable populations in Canada, to compare with the nonvulnerable populations and to identify demographic characteristics predictive of food allergy. METHODS: By using 2006 Canadian Census data, postal codes with high proportions of vulnerable populations were identified and households were randomly selected to participate in a telephone survey. Information on food allergies and demographics was collected. Prevalence estimates were weighted by using Census data to account for the targeted sampling. Multivariable logistic regression was used to identify predictors of food allergy. RESULTS: Of 12,762 eligible households contacted, 5734 households completed the questionnaire (45% response rate). Food allergy was less common among adults without postsecondary education versus those with postsecondary education (6.4% [95% CI, 5.5%-7.3%] vs 8.9% [95% CI, 7.7%-10%]) and new Canadians versus those born in Canada (3.2% [95% CI, 2.2%-4.3%] vs 8.2% [95% CI, 7.4%-9.1%]). There was no difference in prevalence between those of low and of high income or those with and without Aboriginal identity. CONCLUSION: Analysis of our data suggests that individuals of low education and new Canadians self-report fewer allergies, which may be due to genetics, environment, lack of appropriate health care, or lack of awareness of allergies, which reduces self-report.

The use of incentives in vulnerable populations for a telephone survey: a randomized controlled trial.

BACKGROUND: Poor response rates in prevalence surveys can lead to nonresponse bias thereby compromising the validity of prevalence estimates. We conducted a telephone survey of randomly selected households to estimate the prevalence of food allergy in the 10 Canadian provinces between May 2008 and March 2009 (the SCAAALAR study: Surveying Canadians to Assess the Prevalence of Common Food Allergies and Attitudes towards Food LAbeling and Risk). A household response rate of only 34.6% was attained, and those of lower socioeconomic status, lower education and new Canadians were underrepresented. We are now attempting to target these vulnerable populations in the SPAACE study (Surveying the Prevalence of Food Allergy in All Canadian Environments) and are evaluating strategies to increase the response rate. Although the success of incentives to increase response rates has been demonstrated previously, no studies have specifically examined the use of unconditional incentives in these vulnerable populations in a telephone survey. The pilot study will compare response rates between vulnerable Canadian populations receiving and not receiving an incentive. FINDINGS: Randomly selected households were randomly assigned to receive either a $5 incentive or no incentive. The between group differences in response rates and 95% confidence intervals (CIs) were calculated. The response rates for the incentive and non-incentive groups were 36.1% and 28.7% respectively, yielding a between group difference of 7.4% (-0.7%, 15.6%). CONCLUSION: Although the wide CI precludes definitive conclusions, our results suggest that unconditional incentives are effective in vulnerable populations for telephone surveys.

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.

Oxidant alterations in CD16 expression are cytoskeletal induced.

Oxidative stress during reperfusion of ischemia is associated with a phenotypic change in circulating monocytes from CD14++CD16- to a proinflammatory CD14+CD16+ subpopulation resulting in altered immunity and development of organ failure. However, the mechanism responsible remains unknown. We hypothesize that this phenotypic change, modeled by hydrogen peroxide exposure in vitro, is due to oxidative-induced intracellular calcium flux and distinct cytoskeletal and lipid raft changes. Peripheral blood monocytes obtained from healthy volunteers underwent 100 mM H2O2 exposure for 0 to 24 h. Selected cells were pretreated with 2 microM cytochalasin D, 1 microM lactrunculin A, or 30 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid for 30 min. Cells underwent fluorescence-activated cell sorter for CD14, CD16, and cytokine expression. Cellular and lipid raft CD16 expression was determined by immunoblot and confocal microscopy. H2O2 exposed monocytes underwent a rapid time-dependent increase in the surface expression of CD16 from 12.81% +/- 3.53% to 37.12% +/- 7.61% at 24 h (P = 0.001). Total cellular CD16 was not changed by H2O2, but an increase in lipid raft and decrease in intracellular CD16 expression were seen after H2O2 exposure. This increase in CD16 expression was associated with a 27% increase in intracellular TNF-alpha, an alteration in actin polymerization, and the formation of raft macrodomains. These changes induced by H2O2 were inhibited by inhibition of actin polymerization (cytochalasin D and lactrunculin A) and intracellular calcium flux [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. This study provides the first evidence that phenotypic alterations induced by oxidative stress during reperfusion may occur as a result of changes in cytoskeletal architecture due to calcium flux that result in lipid raft alterations rather than solely from demargination and/or production of bone marrow-derived CD16+ monocytes.

The priming effect of C5a on monocytes is predominantly mediated by the p38 MAPK pathway.

The dysregulation of the inflammatory response after trauma leads to significant morbidity and mortality. Monocytes and macrophages play a central role in the orchestration of the inflammatory response after injury. Serum interleukin-6 (IL-6) concentration correlates with poor outcomes after injury. Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that plays a crucial role in the pathogenesis of multiple organ dysfunction syndrome. Furthermore, in the presence of C5a, monocytes and macrophages have potentiated responses, but the mechanisms underlying this response remain largely unknown. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and pretreated with C5a (100 ng/mL) for 1 h before adding lipopolysaccharide (LPS) (10 ng/mL) for up to 20 h. Inhibitors for the mitogen-activated protein kinases (MAPKs) were added 1 h before adding C5a. C5a primes monocytes for LPS-induced IL-6 and TNF-alpha production. Treatment of PBMCs with C5a leads to a rapid activation of the 3 MAPK pathways. SP600125 (inhibitor of c-Jun NH2-terminal kinase MAPK) and PD98059 (inhibitor of extracellular signal-regulated kinase MAPK) did not affect the C5a priming of the LPS-induced IL-6 and TNF-alpha production, whereas SB203580, a specific inhibitor of p38 MAPK, did suppress the C5a priming effect. These results demonstrate that C5a primes adherent PBMCs and modulates LPS-induced IL-6 and TNF-alpha production. Results from extracellular signal-regulated kinase and c-Jun NH2-terminal kinase MAPK blockade suggest that these signaling pathways have minimal or no role in reprogramming LPS-mediated IL-6 and TNF-alpha production. On the contrary, in PBMCs, C5a activates the p38 cascade, and this pathway plays a major role in the C5a enhancement of LPS-induced IL-6 and TNF-alpha production.