Journal: Microbiology

Article Title: The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups

doi: 10.1099/mic.0.035642-0

Figure Lengend Snippet: Strains used in this study

Article Snippet: PRB94, the glycerol dehydratase (Δ pduC ) mutant of ATCC PTA-6475, was used as a negative control.

Techniques: Isolation, Mutagenesis

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