Journal: bioRxiv

Article Title: MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

doi: 10.1101/2020.12.30.424878

Figure Lengend Snippet: Expression of modified S proteins. ( A, B ) Western blots. HeLa cells were mock infected or infected with 5 PFU per cell of indicated rMVA for 18 h and total lysates were analyzed by SDS polyacrylamide gel electrophoresis. After membrane transfer, the proteins were detected with antibody to RBD or FLAG. The positions and masses in kDa of marker proteins are indicated on left and the positions of S, S1, S2 and RBD on right. ( C, D ) Flow cytometry. HeLa cells were infected in triplicate and permeabilized or stained directly with anti-SARS-CoV-2 Spike RBD mAb followed by APC-conjugated goat anti-mouse IgG. Infected cells were identified by GFP fluorescence and the percent that express S was determined by antibody staining. Bars represent the geometric mean. ( E ) Mean fluorescent intensities. HeLa cells were infected in duplicate and incubated with soluble hACE2 followed by Alexa Fluor 647-conjugated anti-hACE2 antibody. Cells that express S were identified as in the previous panels and the intensity of anti-hACE2 antibody determined. A representative of two experiments is shown.

Article Snippet: The binding of hACE2 to surface expressed S protein on infected HeLa cells was detected by incubating with 100 ng/10 6 cells of biotinylated human ACE2 protein (Cat# 10108-H08H-B, Sino Biological) followed by Alexa Fluor 647-conjugated anti-hACE2 antibody (Cat# FAB9332R, R&D Systems).

Techniques: Expressing, Modification, Western Blot, Infection, Polyacrylamide Gel Electrophoresis, Membrane, Marker, Flow Cytometry, Staining, Fluorescence, Incubation

Journal: bioRxiv

Article Title: MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

doi: 10.1101/2020.12.30.424878

Figure Lengend Snippet: Challenge of transgenic mice following prime and boost vaccinations. ( A ) Protocol consisted of vaccinating five groups of six K18 hACE2 mice (female, 7 weeks old) IM in each hind leg with 10 7 PFU of MVA or rMVA on days 0 (prime) and 21 (boost) and challenging unvaccinated (naive) and vaccinated mice with 10 5 TCID50 of CoV-2 IN on day 35. A second IN challenge of surviving mice and two added naive mice was performed 2 weeks after the first challenge. Mice were weighed daily and observed for signs of morbidity. Mice were bled before vaccination (pre-bleed) and before the boost and 2 weeks after the boost. After challenge, 2 mice from each group were sacrificed on days 2 (*) and 5 (**) to determine the amounts of CoV-2 virus and subgenomic RNA. ( B ) Binding antibody was determined by ELISA on serum from each of the six mice of each vaccinated group and plotted as 1/end-point dilution. Dotted line indicates limit of detection. Abbreviations: X1 refers to sera collected 3 weeks after prime; X2 refers to sera collected 2 weeks after homologous boost; /indicates heterologous boost with RBD protein. ( C ) Neutralizing antibody was determined by a pseudovirus assay on serum from each of the six mice in each group and plotted as NT50. ( D ) Weights of surviving mice were determined daily and plotted as per cent of starting weight. Asterisks indicate number of mice that died or euthanized on a specific day. Data for one of the naive mice was obtained in a preliminary experiment. ( E) Weights were determined following the second challenge of surviving mice and two naive mice. ( F ) Virus titers in lung homogenates obtained on days 2 and 5 were determined by end point dilution and plotted as TCID50 per gram of tissue. The lower two data points for naive mice on day 2 were determined in a preliminary experiment. ( G ) Virus titers in nasal turbinate homogenates obtained on days 2 and 5 were determined as in panel F and plotted as TCID50 per sample. ( H ) RNA was isolated from lung homogenates on days 2 and 5. CoV-2 N and S subgenomic RNAs were determined by ddPCR and plotted as copies per 10 8 copies of 18s rRNA in the same sample. ( I ) RNA was isolated from nasal turbinate homogenates as described in panel H.

Article Snippet: The binding of hACE2 to surface expressed S protein on infected HeLa cells was detected by incubating with 100 ng/10 6 cells of biotinylated human ACE2 protein (Cat# 10108-H08H-B, Sino Biological) followed by Alexa Fluor 647-conjugated anti-hACE2 antibody (Cat# FAB9332R, R&D Systems).

Techniques: Transgenic Assay, Virus, Binding Assay, Enzyme-linked Immunosorbent Assay, Isolation

Journal: bioRxiv

Article Title: MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

doi: 10.1101/2020.12.30.424878

Figure Lengend Snippet: Challenge of transgenic mice after a single vaccination and after passive transfer of immune sera. ( A-F ) K18-hACE2 mice (female, 7 weeks old) were vaccinated IM on each hind leg with 10 7 PFU of MVA (n=5) or rMVA Tri (n=8) and 5 mice of each group were challenged 3 weeks later with 10 5 TCID50 of CoV-2 IN. ( A ) S-binding antibody prior to challenge. ( B ) Neutralizing antibody prior to challenge. ( C ) Weights of mice after challenge. ( D ) Lung virus titers. ( E ) Nasal turbinate virus titers. ( F ) N and S subgenomic RNA copies per 10 8 copies of 18s RNA in lung and nasal turbinates. (G) Passive serum transfer. K18-hACE2 mice were injected IP with 0.4 ml of pooled serum from mice vaccinated with parental MVA (n=3) or with MVA-S (n=4) and challenged with 10 5 TCID50 of CoV-2 IN. Mice were weighed on the indicated days and values plotted as percent of starting weight of each mouse. Asterisks indicate number of mice that died or were sacrificed due to morbidity on day 7.

Article Snippet: The binding of hACE2 to surface expressed S protein on infected HeLa cells was detected by incubating with 100 ng/10 6 cells of biotinylated human ACE2 protein (Cat# 10108-H08H-B, Sino Biological) followed by Alexa Fluor 647-conjugated anti-hACE2 antibody (Cat# FAB9332R, R&D Systems).

Techniques: Transgenic Assay, Binding Assay, Virus, Injection

Journal: bioRxiv

Article Title: Bromodomain and Extraterminal Inhibition Blocks Inflammation-Induced Cardiac Dysfunction and SARS-CoV-2 Infection (Pre-Clinical)

doi: 10.1101/2020.08.23.258574

Figure Lengend Snippet: A) Schematic of the experiment. B) Lungs of SARS-CoV-2 infected K18-hACE2 mice 5 d.p.i. In comparison to the control there is sloughing of bronchial epithelium, and white arrowheads show (A) collapse of alveolar spaces and (B) bronchiolar lumen. C) Qualification of mapped reads in RNA-seq reveal severe lung infection with no/negligible heart infection. n = 5 mice per group. D) Volcano plot of lung RNA-seq reveals a robust upregulation (logFC > 0.5) of 419 genes and downregulation (logFC < −0.5) of 98 genes, both FDR < 0.05. n = 5 mice per group. E) Activation of viral responses in lungs revealed using KEGG pathway analysis of upregulated genes. Size represents number of genes regulated and the pathways of the coloured circles are highlighted by the text. F) Analysis of the transcriptional response predicts Stat1 and Ep300 as key mediators of infection in the lungs. G) Hearts of SARS-CoV-2 infected K18-hACE2 mice 5 d.p.i. The hearts appeared relatively normal without significant necrosis, fibrosis (Masson’s Tri-chrome not shown) or immune infiltrates. H) Volcano plot of heart RNA-seq reveals a robust upregulation (logFC > 0.5) of 249 and downregulation (logFC < −0.5) of 159 genes, both FDR < 0.05. n = 5 mice per group. I) Activation of viral responses in hearts revealed using KEGG pathway analysis of upregulated genes. Repression of ECM in hearts revealed using KEGG pathway analysis of downregulated genes. Size represents number of genes regulated and the pathways of the coloured circles are highlighted by the text. J) Analysis of the transcriptional response predicts Stat1 and Ep300 as key mediators of the response in the heart. K) PCA plot of heart RNA-seq samples. n = 4-5. L) Volcano plot of heart RNA-seq reveals a robust upregulation (logFC > 0.5) of 11 genes and downregulation (logFC < −0.5) of 91 genes, both FDR < 0.05 in SARS-CoV-2 infected K18-hACE2 mice treated with INCB054329. n = 4-5 mice per group. M) Repression of viral responses in hearts revealed using KEGG pathway analysis of down-regulated genes. Size represents number of genes regulated and the pathways of the coloured circles are highlighted by the text. N) Analysis of the transcriptional response predicts Ep300 as the key mediator of INCB054329 effects in the heart. O) Cross-analysis of the transcriptional responses in hCO with hearts of SARS-CoV-2 infected K18-hACE2 mice. P) Co-regulated genes in panel (O) reveals a consistent activation of viral responses in both models using KEGG pathway analysis of upregulated genes. Size represents number of genes regulated and the pathways of the coloured circles are highlighted by the text. Q) Heat-map of genes induced by both CS in hCO and SARS-CoV-2 infected K18-hACE2 mouse hearts that are also repressed with INCB054329 treatment. R) Severe weight loss by 4-5 d.p.i in SARS-CoV-2 infected K-18-hACE2 mice is due to severe lung infection and also brain infection and euthanasia is required. d.p.i. – days post infection. Data presented as mean ± standard error of the mean. ** p < 0.01, using Mann-Whitney *** p < 0.001 and **** p < 0.0001 using two-way ANOVA with Sidak’s post hoc test compared to CTRL. For D, H, L – red dots are regulated as per the described cut-offs and grey dots are not. For F,J and N - Values presented are adjusted P values, number of genes regulated by the transcription factor/number of genes regulated, and % of genes regulated over the total. The size of the coloured slices represent the fraction of genes regulated (180° = 100%), and overlaps for each transcription factor are also depicted.

Article Snippet: For ACE2 assays the following was used for control, 1:200 Goat IgG Alexa Fluor 647-conjugated antibody, and assay 1:200 anti-human ACE2 AlexFluor 647 conjugated antibody and 1:200 anti-human CD90 (all RnD Systems) and were incubated for 60 min at 4°C in Binding Buffer.

Techniques: Infection, Comparison, Control, RNA Sequencing, Activation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Bromodomain and Extraterminal Inhibition Blocks Inflammation-Induced Cardiac Dysfunction and SARS-CoV-2 Infection (Pre-Clinical)

doi: 10.1101/2020.08.23.258574

Figure Lengend Snippet: A) All BETi (except ABBV-744) previously used in clinical trials prevent CS-induced diastolic dysfunction. n = 12-19 hCOs per condition from 3 experiments. hPSC cardiac cells- HES3 (no endothelial cells). B) BETi specific for the BD2 domain (RVX-2157) or selective for the BD2 domain (apabetalone) prevent CS-induced diastolic dysfunction. n = 12-18 hCOs per condition from 3 experiments. hPSC cardiac cells- HES3 (no endothelial cells). C) Apabetalone decreases serum LGALS3BP in the phase IIb ASSURE clinical trial. Data are presented as a change from baseline. n = 47 placebo and 47 apabetalone. D) BETi specific for the BD2 domain (RVX-2157) or selective for the BD2 domain (apabetalone) decrease ACE2 expression after 3 days. hPSC cardiac cells – HES3. E) Pre-treatment with BETi specific for the BD2 domain (RVX-2157) or selective for the BD2 domain (apabetalone) for 3 days block SARS-CoV-2 infection. E-gene expression in 2D cultured hPSC-CM 3 days after infection. n = 6-8 from 2 experiments (pooled from hPSC cardiac cells- HES3 and AA). F) Dose-response curve for apabetalone 3 day pre-treatment to block SARS-CoV-2 infection. E-gene expression in 2D cultured hPSC-CM 3 days after infection. n = 6 from 1 experiment. hPSC cardiac cells- HES3. G) Apabetalone or JQ-1 3 day pre-treatment reduces SARS-CoV-2 titre in hPSC-CM 3 days after infection. n = 6 from 1 experiment. hPSC cardiac cells- HES3. TCID50 - Fifty-percent tissue culture infective dose. Data presented as mean ± standard error of the mean for all plots except for C which is median ± interquartile range. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001, using one-way ANOVA with Dunnett’s multiple comparisons test (A,B – CS, E,F,G - DMSO) or Mann-Whitney (C).

Article Snippet: For ACE2 assays the following was used for control, 1:200 Goat IgG Alexa Fluor 647-conjugated antibody, and assay 1:200 anti-human ACE2 AlexFluor 647 conjugated antibody and 1:200 anti-human CD90 (all RnD Systems) and were incubated for 60 min at 4°C in Binding Buffer.

Techniques: Clinical Proteomics, Expressing, Blocking Assay, Infection, Gene Expression, Cell Culture, MANN-WHITNEY