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