Journal: Cell Reports

Article Title: A two-adjuvant multiantigen candidate vaccine induces superior protective immune responses against SARS-CoV-2 challenge

doi: 10.1016/j.celrep.2021.110112

Figure Lengend Snippet: Neutralization of SARS-CoV-2 pseudovirus in sera and detection of N- and S-specific T cell responses against the B.1351 and B.1.617.2 variants SARS-CoV-2 wild-type (WT), B.1.351 variant, or B.1.617 variant pseudoviruses were incubated with different serum sample dilutions for 1 h at 37°C before the mixtures were added to ACE2-overexpressing 293T cells. Transduction efficiency was quantified by measuring virus-encoded luciferase activity in cell lysates 48 h after transduction and used to calculate the serum dilution factor that resulted in a 50% reduction in pseudovirus particles that were associated with different degrees of S protein-mediated cell entry. (A and B) The 50% pseudovirus neutralization (pVNT 50 ) in serum from mice immunized with the candidate vaccine (A) and mice immunized with the inactivated vaccine (B) against the B.1.351 and B.1.617.2 variants compared with that against the wild-type (WT) virus (n = 4 mice per group). (C–F) Antigen-specific activation of T cells by the N and S proteins of the B.1.351 and B.1.617.2 variants compared with the homologous WT proteins. N-specific (C) and S-specific (D) activation of T cells in splenocytes from mice immunized with the candidate vaccine or the inactivated vaccine at 14 days after the third immunization. N-specific (E) and S-specific (F) activation of T cells in lung tissues from mice immunized with the candidate vaccine or the inactivated vaccine at 14 days after the third immunization (n = 4 mice per group). Significance was determined via one-way ANOVA with a Tukey multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns, no significance.

Article Snippet: B.1.351 variant N protein , SinoBiological , Cat# 40588-V07E9.

Techniques: Neutralization, Variant Assay, Incubation, Transduction, Luciferase, Activity Assay, Activation Assay

Journal: Cell Reports

Article Title: A two-adjuvant multiantigen candidate vaccine induces superior protective immune responses against SARS-CoV-2 challenge

doi: 10.1016/j.celrep.2021.110112

Figure Lengend Snippet:

Article Snippet: B.1.351 variant N protein , SinoBiological , Cat# 40588-V07E9.

Techniques: Blocking Assay, Recombinant, Variant Assay, Enzyme-linked Immunospot, Luciferase, Reporter Assay, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Filtration, Modification

Journal: Frontiers in Immunology

Article Title: Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients

doi: 10.3389/fimmu.2022.968991

Figure Lengend Snippet: Modelling SARS-CoV-2 infection and IFNα stimulation in different human cell types. (A) Representative confocal microscopy images of lung carcinoma epithelial cells Calu-3, colon carcinoma epithelial cells Caco-2, human umbilical vein endothelial cells HUVEC, and peripheral blood mononuclear cells (PBMC) infected with SARS-CoV-2 or mock infected. Cells were stained with anti-SARS-CoV-2 nucleoprotein antibody (red) at 24 hours post infection. Nuclei were stained with draq5 (blue). 20× magnification. (B) Kinetics of SARS-CoV-2 replication in Calu-3, Caco-2, HUVEC, and PBMC. Viral load was measured by TCID50 assay in cell culture supernatant collected at different hours post infection (hpi) with SARS-CoV-2 at MOI 0.1 or 2. Viral titer is represented as mean ± SD of Log TCID50 values obtained from two experiments conducted in triplicate. (C) Expression of the IFN stimulated genes IFIT1 and IFIT2 , the pro-inflammatory cytokine genes IL6 and IL1B , and the ssRNA sensor genes TLR7 and TLR8 in HUVEC, PBMC, Caco-2 and Calu-3 cells at 24 hpi with SARS-CoV-2 at MOI 01 or 2 or treatment with IFN-α2b 1000 U/mL. mRNA expression was measured by real-time RT-PCR and represented as mean ± SD of log2 fold change vs. mock (calculated with the 2 -ΔΔCT method) obtained from two experiments conducted in triplicate. Comparison between groups (infected or treated cells vs. mock) was down by Mann-Whitney U test. *p<0.05.

Article Snippet: Immunofluorescence staining of SARS-CoV-2 Nucleocapsid protein was done with a rabbit monoclonal primary antibody (40143-R019; Sino Biological Inc., Beijing, China) at the dilution of 1:1000 and anti-rabbit IgG Alexa Fluor-546 secondary antibody (goat, 1:2000, Thermo Fisher Scientific).

Techniques: Infection, Confocal Microscopy, Staining, TCID50 Assay, Cell Culture, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients

doi: 10.3389/fimmu.2022.968991

Figure Lengend Snippet: MicroRNA expression following SARS-CoV-2 infection and IFNα stimulation in different human cell types. The miRNAs investigated in vitro were selected among the differentially expressed (9 upregulated and 9 downregulated) serum miRNAs identified by the study in COVID-19 patients vs. healthy controls (HC). (A) Heatmap representing baseline miRNAs expression in lung carcinoma epithelial cells Calu-3, colon carcinoma epithelial cells Caco-2, human umbilical vein endothelial cells HUVEC, and peripheral blood mononuclear cells (PBMC). Data represent -ΔC T values of miRNA normalized to the endogenous control RNU6B in triplicate samples. The color scale bar represents -ΔC T values. (B) Heatmap representing miRNA fold change in cells infected with SARS-CoV-2 at MOI 0.1 or 2 vs. mock infected cells at 6 h and 24 h post infection. mRNA expression was measured by real-time RT-PCR and represented as mean log2 fold change vs. mock (calculated with the 2 -ΔΔCT method) obtained from two experiments conducted in triplicate. The color scale bar represents mean log2 fold change vs. mock. (C) Heatmap representing miRNA fold change in cells treated for 24 h with IFNα 1000 U/mL vs. mock treated cells. mRNA expression was measured by real-time RT-PCR and represented as mean log2 fold change vs. mock (calculated with the 2 -ΔΔCT method) obtained from two experiments conducted in triplicate. The color scale bar represents mean log2 fold change vs. mock.

Article Snippet: Immunofluorescence staining of SARS-CoV-2 Nucleocapsid protein was done with a rabbit monoclonal primary antibody (40143-R019; Sino Biological Inc., Beijing, China) at the dilution of 1:1000 and anti-rabbit IgG Alexa Fluor-546 secondary antibody (goat, 1:2000, Thermo Fisher Scientific).

Techniques: Expressing, Infection, In Vitro, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients

doi: 10.3389/fimmu.2022.968991

Figure Lengend Snippet: MicroRNAs modulated in COVID-19 patients. (A) Illustration of the study design and results, highlighting relevant serum miRNAs that were differentially expressed between COVID-19 patients vs. HC and between severe COVID-19 vs. mild and moderate COVID-19. The figure also shows serum miRNAs significantly associated with the risk of intensive care unit (ICU) hospitalization and death in COVID-19 patients, as well as male sex and age. (B) Illustration of the results of in vitro experiments, summarizing the effects of SARS-CoV-2 infection and IFN-α2b treatment on human lung (Calu-3), colon (Caco-2), endothelial (HUVEC), and peripheral blood mononuclear cells (PBMCs). Upregulated and downregulated miRNAs are represented in red and blue, respectively.

Article Snippet: Immunofluorescence staining of SARS-CoV-2 Nucleocapsid protein was done with a rabbit monoclonal primary antibody (40143-R019; Sino Biological Inc., Beijing, China) at the dilution of 1:1000 and anti-rabbit IgG Alexa Fluor-546 secondary antibody (goat, 1:2000, Thermo Fisher Scientific).

Techniques: In Vitro, Infection

Journal: Analytical Chemistry

Article Title: Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein

doi: 10.1021/acs.analchem.0c02288

Figure Lengend Snippet: Figure 2. Schematic of the workflow used to develop a PRM assay for the detection and quantitation of SARS-CoV-2 spike and nucleoprotein A) PRM assay development was performed using recombinant SARS CoV-2 spike protein and nucleoprotein. Proteotypic target peptides/transitions were selected to generate a spectral library in Skyline. B) The PRM assay was then used to quantitate the SARS CoV-2 protein levels in a mock sample that was created by adding an inactivated virus sample to in-vitro derived mucus.

Article Snippet: SARS-CoV-2 nucleocapsid-His recombinant protein was purchased from Sino Biological (100 g, 40588-V08B,) The protein was difficult to dissolve and required treatment before S-trap preparation below with 40L dimethyl sulfoxide (276855-100mL, Sigma), 126 L 1% TFA, and 100L 1x S-Trap lysis buffer (5% SDS, 50 mM TEAB, pH adjusted to 7.55 using 12% phosphoric acid).

Techniques: Quantitation Assay, Recombinant, Targeted Proteomics, Virus, In Vitro, Derivative Assay