Journal: Nature Immunology

Article Title: Children develop robust and sustained cross-reactive spike-specific immune responses to SARS-CoV-2 infection

doi: 10.1038/s41590-021-01089-8

Figure Lengend Snippet: Plasma from SARS-CoV-2 seropositive children ( n = 21) was assessed for binding to the spike protein of the 4 hCoVs or the spike or nucleocapsid regions of SARS-CoV-2. Plasma was either applied neat (control) or after preabsorption with either recombinant spike S1 domain (spike 1 block) or spike S2 domain (spike 2 block). S1 preabsorption markedly reduced binding to SARS-CoV-2 spike with no effect on hCoV, while S2 preabsorption reduced binding to OC43 and HKU-1. One-way repeated measures ANOVA with Holm–Sidak’s multiple comparison test or Friedman test with Dunn’s multiple comparisons test were used as appropriate.

Article Snippet: Plasma samples were prediluted 1:10 with PBS then preabsorbed by adding an equal volume of either recombinant spike S1 domain (catalog no. 10569-CV-100; R&D Systems) or spike S2 domain (catalog no. 10594-CV-100; R&D Systems) at a concentration of 500 μg ml −1 in PBS or PBS alone (mock).

Techniques: Clinical Proteomics, Binding Assay, Control, Recombinant, Blocking Assay, Comparison

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: ( A ) Human monocytic cells THP1-derived macrophages were stimulated with recombinant S1, S2, M, N, and E proteins of SARS-CoV-2 at a concentration of 500 ng/ml. Four hours post-stimulation, the expression of IL6, IL1B, TNFA, CXCL1, CXCL2, CCL2, IFNA, IFNB, and IFNG was measured by real-time RT-PCR. ( B ) THP1 cells were stimulated with S2 protein at various concentrations for 4 hr and measured the indicated cytokines by real-time RT-PCR. ( C ) THP1 cells were stimulated with S2 protein (500 ng/ml). RNA isolated at 2, 4, and 8 hr post-stimulation was measured for IL6, IL1B, TNFA, CXCL1, and CXCL2 by real-time RT-PCR. ( D ) Human peripheral blood mononuclear cells (PBMCs) were incubated with S2 (500 ng/ml) protein for 4 hr. The expression of IL6, IL1B, TNFA, CXCL1, and CXCL2 was measured by real-time RT-PCR. ( E ) THP1 cells were incubated with S1 (500 ng/ml) or S2 (500 ng/ml) in the presence or absence of ACE2 inhibitor MLN-4760 (10 mM). The expression of cytokines was measured at 4 hr by real-time RT-PCR. Data represent mean ± SD (n=3); *p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments described in ( A ) were repeated three times, and ( B–E ) were repeated two times. Data of representative experiments are presented. Figure 1—source data 1. Raw source data for A-E.

Article Snippet: To validate the immune response of S1 and S2 proteins of RayBiotech, we used recombinant S1 (10569-CV-100), S2 (10594-CV-100), and Spike-trimer (10549-CV-100) proteins purchased from R&D Biosystems.

Techniques: Derivative Assay, Recombinant, Concentration Assay, Expressing, Quantitative RT-PCR, Isolation, Incubation

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: ( A, B ) A549 or Calu3 cells were incubated with SARS-CoV-2 S1 or S2 proteins (500 ng/ml) for 12 and 24 hr. The expression of inflammatory cytokines and chemokines was measured by real-time RT-PCR. ( C ) Primary mouse lung epithelial cells were stimulated with S2 (500 ng/ml) for 12 and 24 hr. The expression of inflammatory cytokines and chemokines was measured by real-time RT-PCR. ( D, E ) Calu3 cells or mouse lung primary epithelial cells were stimulated with S2 (500 ng/ml). Culture supernatant collected at 12 and 24 hr were analyzed for IL-6, IL-1β, and TNFα by ELISA. *p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments in ( A, B ) were repeated three times. Other experiments were repeated two times and data of representative experiments are presented. Figure 2—source data 1. Raw source data for A-E.

Article Snippet: To validate the immune response of S1 and S2 proteins of RayBiotech, we used recombinant S1 (10569-CV-100), S2 (10594-CV-100), and Spike-trimer (10549-CV-100) proteins purchased from R&D Biosystems.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: ( A, B ) Bone marrow-derived macrophages (BMDMs) from WT and Myd88 −/ − mice were stimulated with S2 protein (500 ng/ml). ( A ) The activation of the NF-κB pathway was measured by Western blot analysis of P-P65 and P-IκBα. ( B ) The induction of Il6 , Il1b, and Tnfa was measured by real-time RT-PCR. ( C ) BMDMs from WT, Tlr2 −/ − , and Tlr4 −/ − mice were treated with S2 protein (500 ng/ml). Cell lysates collected at different times were analyzed for the activation of the NF-κB pathway by Western blotting of P-P65 and P-IκBα. ( D ) BMDMs from WT and Tlr2 −/ − mice were treated with S1 protein (500 ng/ml), and the activation of P65 and IκBα was measured by Western blotting. ( E, F ) WT, Tlr2 −/ − , and Tlr4 −/ − macrophages were treated with S2 protein (500 ng/ml) or S-tri (500 ng/ml). The expression of cytokines was measured by real-time RT-PCR at 4 hr post-stimulation. Data represent mean ± SD (n=3); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented. ( G ) THP1 cells were stimulated with S2 protein (500 ng/ml), Pam3CSK4 (500 ng/ml), or LPS (100 ng/ml) in the presence or absence of Tlr2 inhibitor C29 (150 mM) for 4 hr. The expression of IL6, IL1B , and TNFA was measured by real-time RT-PCR. ( H ) WT and Tlr2 −/ − mice were administered with S1 and S2 protein (1 μg each/mouse). Blood collected before and 16 hr post S protein administration was measured for IL-6, IL-1β, and TNFα by ELISA. Data represent mean ± SEM (n=5); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. Experiments were repeated two times and data of representative experiments are presented. Figure 5—source data 1. Raw source data for B, E, F, G, H.

Article Snippet: To validate the immune response of S1 and S2 proteins of RayBiotech, we used recombinant S1 (10569-CV-100), S2 (10594-CV-100), and Spike-trimer (10549-CV-100) proteins purchased from R&D Biosystems.

Techniques: Derivative Assay, Activation Assay, Western Blot, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet: ( A, B ) HEK-Blue-Null, HEK-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 were stimulated with S1, S2, or S-tri for 6 hr. FSL1, Pam3CSK4, and LPS were used as ligands for TLR2/1, TLR2/6, and TLR4, respectively. The activation of NF-κB was monitored by the blue color development ( A ), which was measured at 620 nm ( B ). ( C ) HEK-Blue-Null, HEK-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 cells were stimulated with S2 (500 ng/ml) at indicated times. The activation of P-P65 and P-IκBα was measured by Western blot analysis. ( D ) HEK-Blue-Null, HEK2-Blue-TLR2, HEK-Blue-TLR1/2, HEK-Blue-TLR2/6, and HEK-Blue-TLR4 cells were stimulated with S2 (500 ng/ml) for 6 hr. The induction of IL6 and IL1B was measured by real-time RT-PCR. ( E, F ) HEK-Blue-TLR2, HEK-Blue-TLR2/1, and HEK-Blue-TLR2/6 cells were stimulated with S1 or S2 in the presence or absence of TLR2 inhibitor C29 (150 mM) for 6 hr. The NF-κB activity was monitored colorimetrically at 620 nm. ( G, H ) TLR1, TLR2, TLR6, or TLR1/6 were knocked out in Raw264.7 cells with CRISPR/Cas9. Cells were then stimulated with S2 protein (500 ng/ml) for 4 hr. ( G ) The expression of cytokines was measured by real-time RT-PCR. Data represent mean ± SD (n=5); ***p<0.0001, ****p<0.00001 by unpaired Student’s t-test. All experiments were repeated three times and data of representative experiments are presented. Figure 6—source data 1. Raw source data for B, D, F, G.

Article Snippet: To validate the immune response of S1 and S2 proteins of RayBiotech, we used recombinant S1 (10569-CV-100), S2 (10594-CV-100), and Spike-trimer (10549-CV-100) proteins purchased from R&D Biosystems.

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Activity Assay, CRISPR, Expressing

Journal: eLife

Article Title: SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway

doi: 10.7554/eLife.68563

Figure Lengend Snippet:

Article Snippet: To validate the immune response of S1 and S2 proteins of RayBiotech, we used recombinant S1 (10569-CV-100), S2 (10594-CV-100), and Spike-trimer (10549-CV-100) proteins purchased from R&D Biosystems.

Techniques: Derivative Assay, Recombinant, Plasmid Preparation, CRISPR, Sequencing, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software