Journal: ACS nano

Article Title: Re-Engineered Pseudoviruses for Precise and Robust 3D Mapping of Viral Infection.

doi: 10.1021/acsnano.3c07767

Figure Lengend Snippet: Figure 1. Development of clickVSVs bearing the SARS-CoV-2 spike (S) Glycoprotein. (a) Schematic representation of the SARS- CoV-2 virus, the vesicular stomatitis virus (VSV), and the clickable pseudovirus (VSVΔG-eGFP-SARS-CoV-2-Sclick), generated using Genetic Code Expansion via amber suppression using orthogonal tRNA/tRNA-synthetase pairs (aaRS/tRNAaa). (b) Widefield fluorescence images of GFP (green) and Hoechst (white) of infected Vero E6 cells after 24 h of infection with VSVΔG-eGFP- SARS-CoV-2-S variants. (c) Influence of click-labeling on infectivity of clickVSVs: without (gray) or with prior click-labeling with Tetrazine-Cy5 (Tet-Cy5) (magenta). (d) Neutralization of wildtype VSVΔG-eGFP-SARS-CoV-2-S and clickable VSVΔG- eGFP-SARS-CoV-2-SCy5 with a neutralizing antibody (+nAB) or additional treatment with soluble ACE2 ectodomain (+sACE2).

Article Snippet: Vero E6 (CLS, 305008) and HEK293T (DSMZ, ACC635) cells were cultivated at 37 °C and 5% CO2 in T25-culture flasks (Thermo Scientific, #156340) with DMEM (Dulbeccos’s Modified Eagle’s Medium, Sigma-Aldrich, #D5796) supplemented with 10% FCS (Sigma-Aldrich, #F7524), 1% L-glutamine, and 1% Pen-Strep (Sigma-Aldrich, #P4333).

Techniques: Virus, Generated, Fluorescence, Infection, Labeling, Neutralization

Journal: BMC Medicine

Article Title: Humoral immune response after different SARS-CoV-2 vaccination regimens

doi: 10.1186/s12916-021-02231-x

Figure Lengend Snippet: Development of SARS-CoV-2 neutralising antibodies (VNA) after first (empty circles) and second (filled circles) immunisation with the vector vaccine AZD1222 or the messenger ribonucleic acid (mRNA)-based vaccines BNT162b2 or mRNA-1273. A surrogate neutralisation assay ( A ) and a Vero-cell-based virus-neutralisation test (cVNT) using the SARS-CoV-2 variant of concern B.1.1.7 (alpha) strain ( B ) were applied to measure the VNAs. The assay cut-offs are indicated by dashed lines. The median and the 95% confidence interval were calculated for each group in A and B . Ns non-significant; * p < 0.05; *** p < 0.001; **** p < 0.0001 (Kruskal-Wallis test)

Article Snippet: In brief, 2.5 × 10 4 Vero cells (order no. 605372, CLS Cell Lines Service GmbH, Eppelheim, Germany) were seeded per well and incubated at 37 °C under standard conditions.

Techniques: Plasmid Preparation, Vaccines, Virus, Variant Assay

Journal: BMC Medicine

Article Title: Humoral immune response after different SARS-CoV-2 vaccination regimens

doi: 10.1186/s12916-021-02231-x

Figure Lengend Snippet: Anti-SARS-CoV-2 immunoglobulin G (IgG) response in Binding Antibody Units (BAU) per millilitre (ml) after first (open circles) and second (filled circles) immunisation with the vector vaccine AZD1222 (green), the messenger ribonucleic acid (mRNA)-based vaccine BNT162b2 (blue) and after a heterologous vaccination scheme, starting with AZD1222, followed by an mRNA-based vaccine boost (BNT162b2 or mRNA-1273; red) with regard to the detection of virus-neutralising antibodies (VNA). The latter were measured in a Vero-cell-based neutralisation test (cVNT) using the SARS-CoV-2 variant of concern B.1.1.7 (alpha). Cut-off values for positivity of the anti-trimeric spike (S) IgG assay ( A ), anti-S IgG assay ( B ) and anti-receptor binding domain (RBD) IgG assay ( C ), respectively, and the cVNT cut-off value for the presence of VNA are indicated by black dashed lines. The Spearman correlation coefficients of log(reciprocal titre) were calculated with 0.86, 0.86 and 0.88, respectively. The probability of detecting VNA at a given BAU/ml in the anti-SARS-CoV-2 IgG assays was calculated by logistic regression ( D – F ): VNA were present in 95% of samples when IgG concentrations of 886 BAU/ml (anti-trimeric S IgG), 323 BAU/ml (anti-S IgG) and 448 BAU/ml (anti-RBD IgG), respectively, were measured (green dashed lines; 95% confidence intervals (CI) 59.4 to 99.6%). Vertical black dashed lines represent the threshold values set by the manufacturers of the antibody assay; red dashed lines represent the BAU/ml concentrations (anti-trimeric S IgG: 350 BAU/ml; anti-S IgG: 119 BAU/ml; anti-RBD IgG: 174 BAU/ml) with a 50% probability of VNA detection. The distribution of the cVNT titres, the medians, and the 95% CIs between the three plotted thresholds (dashed black, red and green lines in A – F ) are shown ( G – I )

Article Snippet: In brief, 2.5 × 10 4 Vero cells (order no. 605372, CLS Cell Lines Service GmbH, Eppelheim, Germany) were seeded per well and incubated at 37 °C under standard conditions.

Techniques: Binding Assay, Plasmid Preparation, Virus, Variant Assay

Journal: BMC Medicine

Article Title: Humoral immune response after different SARS-CoV-2 vaccination regimens

doi: 10.1186/s12916-021-02231-x

Figure Lengend Snippet: Correlation of the surrogate neutralisation test (sVNT) results with results obtained by the laboratory-developed Vero-cell-based virus-neutralisation test (cVNT) using a B.1.1.7 strain as antigen ( A ). The Spearman correlation coefficient of log(reciprocal titre) was calculated with 0.88; empty circles : first vaccination; filled circles: second vaccination; red: heterologous vaccination with AZD1222/mRNA; green: homologous vaccination with AZD1222; blue: homologous vaccination with BNT162b2. Probability of detecting virus-neutralising antibodies (VNA) with the cVNT at a given percentage inhibition of sVNT calculated by logistic regression (B) ; e.g. at 20% inhibition (black dashed line), 63% inhibition (red dashed line), and at 87% inhibition of sVNT (green dashed line), the probabilities of detecting VNA with cVNT are 4% (95% confidence interval (CI) 1–16%), 50 % (95% CI 34–66 %) and 85% (95% CI 73–92%), respectively. The distribution of the cVNT titres, their medians, and their 95% CIs between the three plotted thresholds (dashed black, red and green lines in A , B ) are shown ( C )

Article Snippet: In brief, 2.5 × 10 4 Vero cells (order no. 605372, CLS Cell Lines Service GmbH, Eppelheim, Germany) were seeded per well and incubated at 37 °C under standard conditions.

Techniques: Virus, Inhibition

Journal: BMC Medicine

Article Title: Humoral immune response after different SARS-CoV-2 vaccination regimens

doi: 10.1186/s12916-021-02231-x

Figure Lengend Snippet: Presence of virus-neutralising antibodies (VNA) against the SARS-CoV-2 variants of concern B.1.1.7 (alpha, filled circles) and B.1.617.2 (delta, empty circles) after the second immunisation. Sera from 26 age- and gender-matched individuals who received a heterologous (AZD1222/BNT162b2, n = 9) or a homologous vaccination scheme (AZD1222/AZD1222, n = 9; BNT162b2/BNT162b2, n = 8) were tested (see Table ). An individual VNA titre > 1:10 was defined as neutralising in our Vero-cell-based virus-neutralisation test (cVNT). †The significance of the median VNA titre differences against B.1.1.7 and B.1.617.2 was calculated using the Wilcoxon test (** p < 0.01). ‡Comparison of the median VNA titre differences achieved with different immunisation schemes against B.1.617.2 (Kruskal-Wallis test; ns not significant; ** p < 0.01; **** p < 0.0001)

Article Snippet: In brief, 2.5 × 10 4 Vero cells (order no. 605372, CLS Cell Lines Service GmbH, Eppelheim, Germany) were seeded per well and incubated at 37 °C under standard conditions.

Techniques: Virus, Comparison

Journal: Microorganisms

Article Title: Emergence and Characterization of Three Pseudorabies Variants with Moderate Pathogenicity in Growing Pigs

doi: 10.3390/microorganisms13040851

Figure Lengend Snippet: Isolation, identification, and biological characteristics of the three isolated field PRV strains. ( A ) Vero cells were inoculated with SD1501, SD1701, and SD1801, and the cytopathic effects and fluorescence were observed. ( B ) Identification of SD1501, SD1701, and SD1801 using Western blot analysis. ( C ) Plaque purification of SD1501, SD1701, and SD1801. ( D , E ) Growth curves of SD1501, SD1701, and SD1801 using qPCR ( D ) and TCID50 determination ( E ).

Article Snippet: Vero cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Solarbio, Shanghai, China), supplemented with 10% fetal bovine serum (FBS, NULEN BIOTECH, Shanghai, China), and maintained at 37 °C in 5% CO 2 .

Techniques: Isolation, Fluorescence, Western Blot, Purification

Journal: The Journal of Experimental Medicine

Article Title: Inherited human OX40 deficiency underlying classic Kaposi sarcoma of childhood

doi: 10.1084/jem.20130592

Figure Lengend Snippet: R65C is a loss-of-function mutation. (A) PHA-activated T cell blasts from healthy controls (C), R65C heterozygous family members (Het), or the patient (P) were incubated with biotinylated recombinant soluble OX40L. Unbound OX40L molecules were washed out, and the levels of cell-bound OX40L were measured by flow cytometry with allophycocyanin-labeled streptavidin (SA-APC). Representative histograms and MFIs (mean fluorescence intensities) of CD3 + CD4 + cells (CD4 + T cells) and CD3 + CD8 + cells (CD8 + T cells) in three independent experiments are shown. (B) PHA-activated T cell blasts from the patient were transduced with bicistronic lentiviral vectors encoding luciferase (Luc), OX40-WT, or OX40-R65C, together with IRES-RFP. Cell surface OX40 levels measured with ACT35 and binding to OX40L measured with biotinylated recombinant soluble OX40L are shown for CD3 + CD4 + RFP + cells. One result representative of two independent experiments is shown. (C) OX40L binding to Jurkat or HEK-293 cells transduced with bicistronic retroviruses with an empty vector (Mock) or encoding OX40-WT or OX40-R65C, together with IRES-GFP, was assessed as in A. The MFIs of OX40L binding for GFP + cells, normalized with respect to those for isotype controls, are shown. The mean of three independent experiments is shown. Error bars indicate the SEM. *, P < 0.05. (D) CFSE-labeled PBMCs from healthy controls (C1–C8) and the patient (P) were incubated for 3 d with 1 ng/ml of plate-bound anti-CD3 antibody and Vero cells infected with retroviruses either with an empty vector (Vero-Mock) or encoding OX40L (Vero-OX40L). Percentages of CFSE-diluted cells in the CD3 + CD4 + population are plotted. One result representative of two independent experiments is shown. (E) CFSE-labeled PBMCs from healthy controls (C1–C8) and the patient (P) were incubated for 3 d with the indicated concentrations of plate-bound anti-CD3 antibody or PHA. Percentages of CFSE-diluted cells in the CD3 + CD4 + population are plotted. One result representative of two independent experiments is shown.

Article Snippet: The final concentrations and the source of recall antigens were as follows: 5 μg/ml tuberculin PPD, 7 Lf/ml TT (Statens Serum Institut), uninfected normal human dermal fibroblast (NHDF) extract, CMV-infected NHDF extract, VZV-infected NHDF extract, uninfected Vero cell extract, HSV-1–infected Vero cell extract, and 2.5–5 μg/ml EBV-infected human B cell extract (EastCoast Bio).

Techniques: Mutagenesis, Incubation, Recombinant, Flow Cytometry, Labeling, Fluorescence, Transduction, Luciferase, Binding Assay, Plasmid Preparation, Infection

Journal: The Journal of Experimental Medicine

Article Title: Inherited human OX40 deficiency underlying classic Kaposi sarcoma of childhood

doi: 10.1084/jem.20130592

Figure Lengend Snippet: Impaired CD4 + T cell recall antigen response. (A and C) PBMCs from eight healthy controls (C1–C8), an R65C heterozygous family member (I.1), and the patient (P) were stimulated with PPD or PHA for 5 d. IFN-γ (A) or IL-10 (C) levels in the supernatant were measured by ELISA. The dotted lines indicate the limit of detection. One result representative of three independent experiments is shown. BCG+, BCG vaccinated; BCG−, no prior BCG vaccination; NS, nonstimulated. (B and D) IFN-γ (B) or IL-10 (D) production in response to various recall antigens was assessed as in A and C. Except for TT, which was provided as purified protein, the recall antigens were provided as virus-infected crude cell lysate. A lysate of uninfected cells tested in the same experiment did not trigger IFN-γ production (not depicted). The dotted lines indicate the limit of detection. One result representative of three independent experiments is shown. (E) CFSE-labeled PBMCs were incubated with the indicated recall antigens for 6 d. T cell proliferation was assessed by determining the proportion of cells with CFSE levels lower than the undivided peak. Results are shown for CD4 + T cells (CD3 + CD4 + ). One result representative of three independent experiments is shown. (F) PBMCs from two healthy controls were incubated with 1 ng/ml of plate-bound anti-CD3 and Vero cells infected with retroviruses with an empty vector (Vero-Mock) or encoding OX40L (Vero-OX40L) for 3 d, or PPD or TT for 6 d. PBS, isotype antibody, or anti-OX40L antibody was added every other day to a concentration of 1 µg/ml. IFN-γ levels in the culture supernatant were measured by ELISA. Means and SEM from two experiments for one of the healthy controls are shown. **, P < 0.01; ns, not significant.

Article Snippet: The final concentrations and the source of recall antigens were as follows: 5 μg/ml tuberculin PPD, 7 Lf/ml TT (Statens Serum Institut), uninfected normal human dermal fibroblast (NHDF) extract, CMV-infected NHDF extract, VZV-infected NHDF extract, uninfected Vero cell extract, HSV-1–infected Vero cell extract, and 2.5–5 μg/ml EBV-infected human B cell extract (EastCoast Bio).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Virus, Infection, Labeling, Incubation, Plasmid Preparation, Concentration Assay

Journal: Advanced Science

Article Title: Host‐Directed Antiviral Activity of SB2960 Through Selective Induction and Remodeling of Stress Granules

doi: 10.1002/advs.202512972

Figure Lengend Snippet: Structure‐activity relationship (SAR) analysis of benzopyranylpyrazole‐based stress granule (SG) inducers. A) SAR strategy highlighting modification at R 1 , R 2 , and R 3 positions on the benzopyranylpyrazole scaffold. B,C) Quantification of SG formation in Vero cells treated with each analog (20 µ m ) or sodium arsenite (NaAs, 250 µ m ) as a positive control. SGs were visualized by G3BP1 immunofluorescence, and SG number per cell was quantified. Data represent mean ± SD (n ≥ 3).

Article Snippet: Vero kidney epithelial cells derived from the African green monkey were obtained from the Korean Cell Line Bank [KCLB] (KCLB No.10081).

Techniques: Activity Assay, Modification, Positive Control, Immunofluorescence

Journal: Advanced Science

Article Title: Host‐Directed Antiviral Activity of SB2960 Through Selective Induction and Remodeling of Stress Granules

doi: 10.1002/advs.202512972

Figure Lengend Snippet: SB2960 induces stress granules (SGs), modulates innate immunity, and exhibits low cytotoxicity. A) Dose‐dependent SG induction in Vero cells by SB2960, visualized by G3BP1 staining. SB2910 served as an inactive control and did not induce SG formation even at 100 µ m . B) qRT‐PCR analysis of antiviral genes in poly(I:C)‐stimulated G3BP1‐GFP U‐2 OS cells treated with SB2960 (20 µ m ). Data represent mean ± SD (n = 3–4). Statistical values were calculated using one‐way ANOVA. ** P < 0.01; *** P < 0.001; **** P < 0.0001. C) Proteomic profiling comparing poly(I:C) versus poly(I:C) + SB2960 in G3BP1‐GFP U‐2 OS cells. The line graph (top) shows the average expression of proteins upregulated by poly(I:C) and downregulated by SB2960 (n = 3). The bar graph (bottom) presents the top seven Gene Ontology (GO) biological processes enriched in this protein cluster, ranked by p‐ value. D,E) Annexin V/PI flow cytometry and LDH release assay in Vero cells showing minimal cytotoxicity up to 100 µ m SB2960, in contrast to sodium arsenite (NaAs) (n = 3). F) Co‐immunofluorescence staining for G3BP1 and SARS‐CoV‐2 nucleocapsid (N) protein in infected Vero cells treated with SB2960. Scale bar is 30 µm.

Article Snippet: Vero kidney epithelial cells derived from the African green monkey were obtained from the Korean Cell Line Bank [KCLB] (KCLB No.10081).

Techniques: Staining, Control, Quantitative RT-PCR, Expressing, Flow Cytometry, Lactate Dehydrogenase Assay, Immunofluorescence, Infection

Journal: Advanced Science

Article Title: Host‐Directed Antiviral Activity of SB2960 Through Selective Induction and Remodeling of Stress Granules

doi: 10.1002/advs.202512972

Figure Lengend Snippet: Identification of RACK1 as a mechanistic mediator of SB2960. A) Chemical structure of SB2991, a molecular glue degrader probe derived from SB2960 for chemoproteomic target identification. B) 2D differential gel electrophoresis (2D‐DIGE)‐based identification of proteins selectively degraded by SB2991. Green spots indicate SB2991‐depleted proteins and were analyzed by LC‐MS. C,D) Western blot showing dose‐dependent RACK1 degradation by SB2991 and rescue by SB2960 treatment. Numbers below each blot represent mean values from independent experiments, normalized to the DMSO control. E) Cellular thermal shift assay (CETSA) showing thermal stabilization of RACK1 upon SB2960 treatment in G3BP1‐GFP U‐2 OS cells; α‐tubulin served as control. Data represent mean ± SD (n = 2). F) G3BP1 immunofluorescence analysis in Vero cells with or without RACK1 knockdown and SB2960 treatment. RACK1 knockdown enhanced SB2960‐induced SG formation. Scale bar is 30 µm. Data represent mean ± SD (n = 3).

Article Snippet: Vero kidney epithelial cells derived from the African green monkey were obtained from the Korean Cell Line Bank [KCLB] (KCLB No.10081).

Techniques: Derivative Assay, Drug discovery, Nucleic Acid Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control, Thermal Shift Assay, Immunofluorescence, Knockdown