Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: SPR-experiment testing for the presence of a two state reaction. 500 nM SARS-CoV-2 S1-S2 spike protein was injected over a constant immobilization level of 25 RU hACE2 and normalized to 100 % RU for the time point of injection phase end. Injection times were gradually increased (150 – 600 s).

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection

Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: (A) Multicycle experiment with SARS-CoV-2 RBD. hACE2-fc was immobilized on a protein A/G sensor chip and SARS-CoV-2 RBD was injected in concentration range of 3.9 – 1000 nM. The K D was globally fitted with a 1:1 Langmuir based interaction model. The kinetic parameters were determined with K D of 21.3 nM, k a of 4.25 E+5 +/- 2.2 E+2 [1/Ms] and k d of 9.1 E-3 +/- 4.2E-6 [1/s]. ( B ) Test on secondary state reaction. hACE2-fc was immobilized on a protein A sensor surface and 500 nM of SARS-CoV-2 was injected at a constant concentration for increasing contact intervals. Time points of injection phase end were normalized to 100 % and the dissociation starting point was aligned on the time scale.

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection, Concentration Assay

Journal: bioRxiv

Article Title: Conversion rate to the secondary conformation state in the binding mode of SARS-CoV-2 spike protein to human ACE2 may predict infectivity efficacy of the underlying virus mutant

doi: 10.1101/2021.07.14.452313

Figure Lengend Snippet: (A) hACE2-fc was immobilized on a protein A/G sensor surface and CoV trimer proteins were injected in concentration range of 0.62 – 50 nM. The sensograms were globally fitted with the secondary state reaction model. (SARS-CoV 2002) : K D1 6.7 nM, k a1 6.72 E+5 ± 4.4E+3 [1/Ms], k d1 4.68 E-3 [1/s] ± 6.9E-5, k a2 8.80 E-3 ± 6.9E-5 [1/Ms], k d2 2.07E-4 ± 1.60 E-6 [1/s]; K D-total 160 pM (SARS-CoV-2 wt) : K D1 1.8 nM, k a1 7.82 E+5 ± 1.2E+3 [1/Ms], k d1 1.41 E-3 ± 3.75E-5 [1/s], k a2 1.05E-2 ± 1.8E-4 [1/Ms], k d2 2.44E-4 ± 3.5E-6 [1/s]; K D-total 41 pM. ( SARS-CoV-2 B.1.1.7 ) K D1 192.7 nM, k a1 1.31 E+6 ± 1.5E+4 [1/Ms], k d1 2.5 E-2 ± 1.5E-3 [1/s], k a2 5.3E-2 ± 1.5E-3 [1/Ms], k d2 1.1 E-4 ± 2.6E-6 [1/s]; K D-total 40 pM. For graphic representation of the distribution of primary and secondary state reaction, a component analysis was performed for each of the trimeric spike proteins using the injection concentration of 5.56 nM spike. The sensogram (total) is composed of the primary binding event (red), followed by a secondary transition event (blue) which results in a highly stable secondary complex ( B ) On–off chart for the kinetic values of the SARS-CoV spike trimers in the primary and secondary state.

Article Snippet: The biotinylated hACE2-fc (residues 18-740) was purchased by Acrobiosystems and contains a C-terminal IgG1 Fc-Tag (residues 100-330), followed by a biotinylated Avitag.

Techniques: Injection, Concentration Assay, Binding Assay

Journal: bioRxiv

Article Title: ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

doi: 10.1101/2022.03.22.485248

Figure Lengend Snippet: A: HMDM and THP-1 cells were analysed by qPCR for ACE2 mRNA expression, with each data point showing an independent donor or experiment (n=3). B: HMDM were stimulated with IFNβ (10 ng/ml) for 6 h and protein extracts were analysed by immunoblot, alongside extracts from THP-1 cells (WT, THP-1-ACE2, THP-1-mSc). C: BAL macrophages from 3 donors were adhered overnight and lysed. Expression of ACE2 in BAL macrophages was analysed by immunoblot, relative to a loading control (Calnexin). Lysate from A549-cells overexpressing ACE2 were used as a positive control. D-E: Cells were infected with SARS-CoV-2 at MOI 0.5 or MOI 5. After 1h the virus inoculum was removed, cells were washed and cells or supernatants harvested at the indicated times. Cellular viral mRNA was analysed by qPCR (D-E), and infectious virions released into cell supernatants were measured by plaque assay (F-G). Data show the mean + SEM of 3-5 independent experiments, with data points representing individual experiments. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (two-way ANOVA, Tukey’s multiple comparison test).

Article Snippet: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin ( ) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids ( ).

Techniques: Expressing, Western Blot, Control, Positive Control, Infection, Virus, Plaque Assay, Comparison

Journal: bioRxiv

Article Title: ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

doi: 10.1101/2022.03.22.485248

Figure Lengend Snippet: A: THP-1 cells were infected with SARS-CoV-2 (MOI 5), which was washed away after 1 h, and incubated for a further 72h, after which cell death was analysed by ATPlite assay. Data are presented as cell viability relative to mock, and are mean + SEM of 3 independent experiments. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (two-way ANOVA, Tukey’s multiple comparison test). B: Schematic of TBK1 (BX-795) inhibition. C-F: THP-1-ACE2 or Calu3 cells were stimulated with SARS-CoV-2 at MOI 5. After 1 h, the viral inoculum was removed and BX-795 added. Supernatants were harvested at 72 h and CXCL10 was analysed by ELISA (C) and viral titres were analysed by plaque assay (D,E). Data show mean + SEM of at least 3 independent experiments, with each individual data point representing a different experiment. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (C: one-way ANOVA, Tukey’s multiple comparison test; D,E: ratio-paired t-test).

Article Snippet: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin ( ) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids ( ).

Techniques: Infection, Incubation, Comparison, Inhibition, Enzyme-linked Immunosorbent Assay, Plaque Assay

Journal: EBioMedicine

Article Title: Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

doi: 10.1016/j.ebiom.2022.103818

Figure Lengend Snippet: Binding characteristics and inhibition activities of monoclonal antibodies specific to SARS-CoV-2 S/RBD. (a) Immunoreactivity of hybridoma clones culture supernatants derived from mice immunized with the spike (S) protein of SARS-CoV-2 and with RBD of Spike (b) in ELISA against the pre-fusion stabilized and trimerized S protein ectodomain (“S protein”) or RBD (“RBD”). Asterisks mark non-binders. (c) Blocking of interaction between RBD and ACE2 in a competition assay based on ELISA (RBD-ACE2), inhibition of the S protein internalization by cells expressing ACE2 (S-ACE2), inhibition of cell infection by MLV pseudotyped with SARS-CoV-2 spike protein (Pseudovirus) and plaque reduction neutralization test performed with an authentic SARS-CoV-2 virus isolate Slovakia/SK-BMC5/2020 (PRNT). Experiments were done with hybridoma culture supernatants adjusted with fresh cell culture medium to the same mAb concentration and then diluted for the assays as follows: RBD-ACE2 competitive ELISA 1:6 in PBS-T; S-ACE2 cell assay 1:50 in DMEM; pseudoviral assay 1:25 in DMEM; live virus PRNT 1:50 in EMEM. Positive control: serum of a mouse immunized with the S protein, diluted 1:200; Negative control: irrelevant mAb. All experiments are an average of at least two measurements, pseudoviral tests were all done in tetraplicates. (d) The purified monoclonal antibodies were tested for blocking the RBD-ACE2 interaction in competitive ELISA, the inhibition of ACE2-mediated S protein internalization by HEK293/17-hACE2 cells (e), and plaque reduction neutralization test (f) performed with strain Slovakia/SK-BMC5/2020. The curves are calculated from two replicates using Prism 6 for Windows (GraphPad Software). (g) Summary of assays from d-f. (h, i) Kinetic characteristics of the interactions of RBD with selected neutralizing antibodies. On-rate (k a ), off-rate (k d ) constants and equilibrium dissociation constant (K D ) of individual antibody-RBD complexes (±SD). Green dots mark antibodies AX290 and AX677 selected for further development.

Article Snippet: Human embryonic kidney HEK293T/17-hACE2 cells with stable expression of human angiotensin-converting enzyme ACE2 (AXON Neuroscience SE) were prepared by stable transfection of pDUO2-hACE2-TMPRSS2a (InvivoGen) into HEK293T/17 cells (ATCC Cat# ACS-4500, RRID:CVCL_4V93) with transfection reagent Lipofectamine 3000 (L3000001, Thermo Fisher Scientific), according the to the manufacturer's recommendations.

Techniques: Binding Assay, Inhibition, Clone Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Competitive Binding Assay, Expressing, Infection, Plaque Reduction Neutralization Test, Virus, Cell Culture, Concentration Assay, Competitive ELISA, Positive Control, Negative Control, Purification, Software

Journal: EBioMedicine

Article Title: Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

doi: 10.1016/j.ebiom.2022.103818

Figure Lengend Snippet: Antibody prophylaxis against SARS-CoV-2 infection in a mouse model. (a) Schematic of the in vivo experimental procedures. Wild-type mice were transduced with AAV-hACE2 by forced inhalation. After 11 days, the mice were inoculated subcutaneously (s.c.) with 1·25 mg of the indicated mAbs antibodies one day (-24 h, n=10 animals per group) before being infected intranasally (i.n.) with SARS-CoV-2 (1 × 10 4 pfu). Health characteristics and body weight were monitored daily for the duration of the experiment. 5 animals per group were sacrificed 3 and 7 days after infection and lung tissue analyzed for viral load (on day 3 only), viral RNA and histopathological changes. (b) Body weight was monitored daily for 7 days (n=5 mice per group). Mean with s.d. is shown. (c) Viral burden in the lungs of mice infected with SARS-CoV-2 (n = 5 per group) as measured 3 dpi by plaque assays. Mean with s.d. is shown. Dashed line indicates the limit of detection (2·14 log pfu/g of tissue). All three treated groups were compared to the Isotype Ctrl group, p values were calculated using non-parametric Mann-Whitney test, in all comparisons p=0·0079 (exact p values were calculated). (d) The analysis of viral RNA copy numbers in the lungs shows clear reduction in all animals treated with anti-Spike antibodies compared to isotype control on day 3 (p=0·0079 for AX290, p=0·0159 for AX677 and p=0·0079 for AX290+AX677; evaluated by non-parametric Mann-Whitney test). On day 7 the copy numbers were greatly reduced in all animals, probably due to the elimination of the replicating viruses. The bars represent mean with s.d. Histological analysis of hematoxylin/eosin-stained lung sections collected 7 days after infection showed accumulation of the interstitial inflammatory infiltrates (e) and reduction of normal tissue (f) in animals treated with isotype antibody control. These histopathological changes were reduced by the anti-Spike antibodies AX290 and AX677 applied either alone or in combination. Data were evaluated by Mann-Whitney test (** p=0·0079, * p=0·0159).

Article Snippet: Human embryonic kidney HEK293T/17-hACE2 cells with stable expression of human angiotensin-converting enzyme ACE2 (AXON Neuroscience SE) were prepared by stable transfection of pDUO2-hACE2-TMPRSS2a (InvivoGen) into HEK293T/17 cells (ATCC Cat# ACS-4500, RRID:CVCL_4V93) with transfection reagent Lipofectamine 3000 (L3000001, Thermo Fisher Scientific), according the to the manufacturer's recommendations.

Techniques: Infection, In Vivo, Transduction, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: Common dandelion ( Taraxacum officinale ) leaf extract efficiently inhibits SARS-CoV-2 Omicron infection in vitro

doi: 10.1101/2022.12.22.521558

Figure Lengend Snippet: Inhibition of SARS-CoV-2 Spike B.1.1.529 Omicron variant by T. officinale extract. A and B) Cells were pre-treated with T. officinale (TO) extract for 30 min before transfection with 7500 TU/mL SARS-CoV-2 Spike Omicron variant virus particles for 24 h. The cells were post-incubated in fresh medium (A) containing TO for 60 h or (B) without TO for 48 h. C) Cells were exposed to TO for 30 min, then washed with PBS, and fresh medium containing 7500 TU/mL SARS-CoV-2 Spike Omicron variant virus particles was added for 24h. Cells were treated again for 30 min with TO in fresh medium, washed 1× with PBS, and post-incubated for another 48 h. Luminescence was measured after 15 min. Solvent control (SC): 10% distilled water. Inhibitor positive control: 100 μg/ml anti-hACE2 antibody. Data are mean value + SD. Significance of difference was calculated relative to respective solvent control by oneway ANOVA followed by Bonferroni correction. * p < 0.05, ** p < 0.01.

Article Snippet: Human lung A549 cells, stably expressing hACE2 and TMPRSS2 (A549-hACE2-TMPRSS2), were obtained from InvivoGen SAS (Toulouse Cedex, France).

Techniques: Inhibition, Variant Assay, Transfection, Virus, Incubation, Solvent, Control, Positive Control

Journal: Cell Host & Microbe

Article Title: Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses

doi: 10.1016/j.chom.2022.10.010

Figure Lengend Snippet:

Article Snippet: HEK-Blue hACE2-TMPRSS2 cells , Invivogen , Cat. # hkb-hace2tpsa.

Techniques: Virus, Recombinant, Transfection, Lysis, Cell Culture, Luciferase, Expressing, Irradiation, Amplification, Plasmid Preparation, Software, Fluorescence, Microscopy, Imaging