Journal: Frontiers in Immunology

Article Title: NOD2/RIG-I Activating Inarigivir Adjuvant Enhances the Efficacy of BCG Vaccine Against Tuberculosis in Mice

doi: 10.3389/fimmu.2020.592333

Figure Lengend Snippet: Binding of SB 9000 (Inarigivir) to retinoic acid-inducible gene I (RIG-I). (A) Sandwich ELISA method : Binding of SB 9000 to RIG-I by using SB 9000 derivatized with biotin at the 5′-OH group of the dinucleotide. ELISA microplate coated with anti-DDK antibody at 1:200 dilution in coating buffer (0.1M carbonate, pH 9.6), followed by 1 µg/ml DDK-tagged RIG-I recombinant protein. Biotinylated SB 9000 was then added as indicated and the SB 9000 bound to RIG-I was detected using HRP-conjugated Streptavidin followed by development with TMB substrate solution and absorbance measurement at 450nm (mean ± SD; n = 2). (B) Surface Plasmon Resonance Method : Human RIG-I (hRIG-I) was generated in mammalian cells using Expi293F cells and DDX58 - HaloTag(R) human ORF in pFN21A. The protein was then purified using HaloTag ® Mammalian Protein Purification System. SPR was measured by using various concentrations of biotinylated SB 9000 dissolved in water manually printed onto PlexArray Nanocapture Sensor Chip at 40% humidity. The signal changes after binding and washing (in AU) were recorded as the assay value. Selected protein-grafted regions in the SPR images were analyzed, and the average reflectivity variations of the chosen areas were plotted as a function of time. Real-time binding signals were recorded and analyzed by Data Analysis Module. Kinetic analysis was performed using BIA evaluation 4.1 software. Data are representative of 2 independent experiments, carried out in duplicates and values are expressed as mean ± SD.

Article Snippet: Surface Plasmon Resonance Method: For this assay, full length hRIG-I was generated in mammalian cells using Expi293F cells (ThermoFisher Scientific, A14527) , ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific, A14524) and DDX58 - HaloTag(R) human ORF in pFN21A (Promega).

Techniques: Binding Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Recombinant, SPR Assay, Generated, Purification, Protein Purification, Software

Journal: Frontiers in Immunology

Article Title: NOD2/RIG-I Activating Inarigivir Adjuvant Enhances the Efficacy of BCG Vaccine Against Tuberculosis in Mice

doi: 10.3389/fimmu.2020.592333

Figure Lengend Snippet: Small molecule nucleotide hybrids (SMNHs, aka. SB series) enhance antigen presentation by BCG vaccine infected macrophages and increase Th1 cytokine secretion. C57Bl/6 mouse bone marrow derived MΦs and dendritic cells (DCs) (aka. APCs) were treated with SMNHs (SB series) at indicated doses, followed by infection with Mycobacterium bovis Bacille Calmette Guerin (BCG) for 4 h (MOI = 1). Mycobacterial Antigen-85B (Ag85B) derived p25 peptide specific BB7 CD4 T cells were overlaid and supernatants collected after 18 h were tested for IL-2 using sandwich ELISA. (A) Initial screening of SB series of SMNHs including Inarigivir (NOD2/RIG-I agonist), SB-2 (TLR-7/9 agonist), and SB-3 and SB-4 (dinucleotides related to Inarigivir). *p values vs. BCG alone. (B , C) Comparison of Inarigivir and its resynthesized isomers Rp-Inarigivir and Sp-Inarigivir (10 µM) with the LPS agonist of TLR-4 (1 µg/ml) and related SB compounds during antigen presentation. * p values for groups compared. (D) Structures of Rp-Inarigivir and Sp-Inarigivir. (E) MΦs and DCs were treated or not with Rp-Inarigivir and Sp-Inarigivir (10 µM), followed by BCG infection. Supernatants collected at 18 h tested for Th1 cytokine levels using sandwich ELISA. * p values for treated vs. BCG alone (*<0.01; **< 0.009; p values, 1-way ANOVA with Tukey’s posttest). Data are representative of two independent experiments carried out in duplicates and values are expressed as mean ± SD.

Article Snippet: Surface Plasmon Resonance Method: For this assay, full length hRIG-I was generated in mammalian cells using Expi293F cells (ThermoFisher Scientific, A14527) , ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific, A14524) and DDX58 - HaloTag(R) human ORF in pFN21A (Promega).

Techniques: Infection, Derivative Assay, Sandwich ELISA

Journal: Frontiers in Immunology

Article Title: NOD2/RIG-I Activating Inarigivir Adjuvant Enhances the Efficacy of BCG Vaccine Against Tuberculosis in Mice

doi: 10.3389/fimmu.2020.592333

Figure Lengend Snippet: Inarigivir activates NOD2/RIG-I receptors enhancing Capsase-1 dependent IL-1β secretion and NOD2 dependent antigen presentation. HEK cells were used for assay of receptor activation (A) and wild type C57Bl/6 (B–F) or NOD2 −/− (G–H) mouse derived APCs were left either untreated or treated with Inarigivir, Rp-Inarigivir and Sp-Inarigivir and SB-2 or SB-3 (10 µM), followed by inhibitors as indicated, and where indicated, a 4-h infection with BCG (MOI = 1). Supernatants were tested for cytokines at 18 h or APCs overlaid with BB7 CD4 T cells for antigen presentation assay to measure IL-2. (A) Induction of NF-kB activity by Inarigivir (mix of Rp-Inarigivir and Sp-Inarigivir) through NOD2 and RIG-I activation. HEK-293 cells were transfected with pcDNA, NOD2, RIG-I, and NF-kB-luciferase. The cells were then incubated with Inarigivir (10 µM). Following 12 h incubation, Luciferase activity was measured and presented as mean ± S.D. from three independent experiments. (B) Rp-Inarigivir and Sp-Inarigivir combinations with BCG increase IL-1β secretion which is decreased after Caspase-1 blockade using Z-YVAD-fmk (50 µM). (C–E) IFN-β, TNF-α, and IL-12 cytokines are not affected by the Caspase-I blockade, although Inarigivir, SB2 and SB3 enhance IFN-β levels. (F) Caspase-1 blockade decreases antigen presentation. (G, H) Rp-Inarigivir and Sp-Inarigivir enhance antigen presentation by BCG infected wt-APCs, which is decreased in NOD2 −/− APCs. (* < 0.01 ** < 0.006 p values using 1-way ANOVA with Tukey’s posttest). Data are representative of 2 independent experiments carried out in duplicates and values are expressed as mean ± SD.

Article Snippet: Surface Plasmon Resonance Method: For this assay, full length hRIG-I was generated in mammalian cells using Expi293F cells (ThermoFisher Scientific, A14527) , ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific, A14524) and DDX58 - HaloTag(R) human ORF in pFN21A (Promega).

Techniques: Activation Assay, Derivative Assay, Infection, Activity Assay, Transfection, Luciferase, Incubation