Blocking TGFβ prevents acute CHIKV-induced disease in O mice.

<p>A and O B6 mice were inoculated and treated with 100ug of anti-TGFβ antibody or isotype control and swelling was measured daily as described in Methods. Data are mean ± SEM (n = 8 per group). Statistical significance was determined using mixed model, repeated measures analyses of variance (ANOVA) as detailed in Statistics. Red stars indicate reduction of swelling in O mice from αIgG1 to αTGFβ treated; black stars indicate reduction of swelling in A mice from αIgG1 to αTGFβ treated.</p

Age increases acute CHIKV-induced joint swelling.

<p>(A) A (12 weeks) and O (18–20 months) B6 mice were inoculated via f.p. and swelling was measured daily as described in Methods. Data are mean ±SEM (<i>n</i> = 10–16 per group). Statistical significance was determined using mixed model, repeated measures analyses of variance (ANOVA) as detailed in Statistics. ***<i>P</i>< 0.001.</p

Prolonged CHIKV viremia with age and evidence of persistent infection.

<p>(A) Serum and (B) CHIKV-inoculated feet were harvested on indicated days post-infection and assayed for viral titer by plaque assay. (A) Serum viral titers on days 1–4 post-infection and (B) viral titer of CHIKV feet on days 3 and 9 post-infection (<i>n</i> = 7–8 per group). (C) Genome copies of virus in CHIKV feet on day 60 post-infection (<i>n</i> = 16–18 per group). (D) CHIKV-inoculated feet were harvested at day 90 p.i. and assayed for the presence of fluorescent infectious virus by co-culture on C6/36 insect cells (n = 8–10 per group). Dashed line indicates limit of detection for all assays. Horizontal lines indicate the median. Statistical significance determined on log-transformed data by unpaired student’s <i>t</i>-test. *<i>P</i>< 0.05; **<i>P</i>< 0.01; ***<i>P</i>< 0.001.</p

Age-related impairment of adaptive immune response to CHIKV.

<p>(A) Popliteal LNs collected and quantified from either naïve or CHIKV-infected A and O mice at day 3, 7, or 9 post-infection. The LN draining from the CHIKV-inoculated foot is indicated as dLN and from the non-inoculated foot as ndLN. Table under graph indicates the average fold-increase from naïve for each age in either the dLN or ndLN (<i>n</i> = 6–8 per group). Horizontal lines indicate the median. Statistical significance determined by student’s <i>t</i>-test. (B-C) Lymphocytes from popliteal LNs on d7 post-infection were stimulated with CHIKV peptides in the presence of protein transport inhibitor. Total number of IFNγ⁺ CD4 T cells (B) and frequency (C) for each age. Data are mean ± SEM (<i>n</i> = 10 per group). Statistical significance determined by unpaired Student’s <i>t</i>-test. (D) CHIKV-specific IgM and (E) IgG2c in serum determined by ELISA at the indicated day post-infection. Data are mean (<i>n</i> = 4–24 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (F) Plaque reduction neutralizing test on serum from days 9 and 60 post-infection. Data are mean + SEM (<i>n</i> = 12 per group). Statistical significance was determined by two-way ANOVA with Bonferroni post-test. (G) Serum samples collected from patients experiencing acute CHIKV-disease were evaluated by plaque reduction neutralizing test. Data are mean + SEM (<i>n</i> = 24 young and 15 aged). Statistical significance was evaluated by unpaired student’s <i>t</i>-test. In all panels black indicates A, red indicates O; * <i>P</i>< 0.05; ** <i>P</i>< 0.01; *** <i>P</i>< 0.001.</p

Dysregulated cytokine production with age.

<p>Serum was collected from A and O mice and assayed by ELISA for (A) CXCL9 or (B) TGFβ concentration at days 2 or 9 and 30, respectively. Data are mean + SEM (<i>n</i> = 3 naïve and 7–8 infected per age). (C) Human samples from IgM-positive CHIKV patients or age and sex-matched controls were assayed for Free-active TGFβ cytokine by ELISA. Data are mean + SEM (<i>n</i> = 39 each group). Statistical significance was evaluated by unpaired student’s <i>t</i>-test. * <i>P</i>< 0.05; ** <i>P</i>< 0.01; *** <i>P</i>< 0.001.</p

Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses

<div><p>Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005324#ppat.1005324.ref001" target="_blank">1</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005324#ppat.1005324.ref004" target="_blank">4</a>]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.</p></div

IRF3 is Required for G10-Dependent Transcription and Anti-Alphaviral Activity.

<p>(A) Immunoblot showing IRF3, STAT1, and GAPDH in THF-ISRE stably transduced with Cas9 and CRISPR gRNA directed against either STAT1 (THF-ISRE-ΔSTAT1) or IRF3 (THF-ISRE-ΔIRF3) as indicated. (B) Induction of IRF3/IFN-dependent LUC in THF lacking STAT1 following 7h exposure to 100μM G10, UV-inactivated CMV, or 1000U/mL IFNβ. Values displayed are average fold changes ±SD of quadruplicate measurements relative to cells exposed only to 1% DMSO. (C) Induction of IRF3/IFN-dependent LUC in THF lacking IRF3 following 7h exposure to 100μM G10, UV-inactivated CMV, or 1000U/mL IFNβ. Values displayed are as in B. (D) Immunoblot of lysates from THF-ISRE following 6h exposure to DMSO, UV-CMV, SeV or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, and GAPDH. (E) Average media titers +SD of CHIKV, VEEV, and SINV at 24 (VEEV) or 48hpi (CHIKV, SINV) obtained from THF-ISRE-ΔIRF3 cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p

G10 Elicits IRF3 phosphorylation and Anti-Alphaviral Activity in Cells Lacking IPS1.

<p>(A) Immunoblot of lysates from THF-ISRE-ΔIPS1 following 6h exposure to DMSO, UV-CMV, SeV or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, IPS1, STING, and GAPDH. (B) Average media titers +SD of CHIKV, VEEV, and SINV at 24h (VEEV) or 48h (CHIKV, SINV) post infection obtained from THF-ISRE-ΔIPS1 cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p

Comparative Kinetics and Dose-Dependence of Innate Immune Activation by G10 and 2’3’-cGAMP.

<p>(A) Immunoblot of lysates from THF-ISRE cells following exposure to G10 (100μM) or transfected 2’3’-cGAMP (42.3μM) for indicated time showing phosphorylation status of IRF3 S386, total IRF3, and GAPDH. (B) mRNA synthesis of indicated genes in THF following 8h exposure to indicated concentration of G10 (blue) or 2’3’-cGAMP (red). Indicated values represent average mRNA fold change ±SD from duplicate experiments relative to cells exposed to 1% DMSO.</p

G10 Elicits Antiviral Activity.

<p>Average titers ±SD of VACV, WNV, CHIKV, and VEEV grown on THF cells in the presence of indicated G10 concentration (DMSO concentration normalized to 1%). Infections were performed in triplicate and virus harvested at 48h post infection (CHIKV, WNV, CHIKV) or 24h post infection (VACV, VEEV).</p