Journal: Journal of Translational Medicine

Article Title: Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

doi: 10.1186/s12967-023-04353-7

Figure Lengend Snippet: rMVA- M1-V5 production by cytometry-based RGGSM. A infection/transfection of CEF cells with MVA-77-RED and TP-HAG- M1-V5 DNA. Green fluorescent cells were sorted from the POS-SW, yielding a mixed “green + red” cell population (inset). B An enriched green fluorescent population (inset) were obtained by a second round of Cell-Sorting, from which, after F/T/S, green virus clones were isolated by TD. In this experiment, approximately 5000 events were sorted and 8/48 “green” microcultures were obtained at TD, which corresponds to 0.19 ffu/culture, with a 13:1 ratio of single to multiple infections. C Flow cytometry of CEFs infected with virus isolated from a green clone. Untagged rMVA-infected cells were sorted from a double-negative NEG-SW. Finally, after F/T/S, untagged virus clones were isolated by TD. In this experiment, approximately 5000 events were sorted and 6/48 "green" microculture were obtained at TD, which corresponds to 0.12 ffu/culture, with a 16:1 ratio of single to multiple infections. D M1-specific PCR confirmed the presence of the M1 transgene in both green HAG-tagged (lane 1) and untagged clones (lanes 2 to 4) as in the TP-HAG- M1-V5 construct, which served as a positive control (PC), but unlike in a sample from MVA-77-RED-infected CEFs, which served as a negative control (NC). E HA-specific WB confirmed the presence of the HAG fusion gene in the control green isolate (lane 1) and its absence in untagged clones (lanes 2 to 4). CEFs infected with rMVA-SHAG (see Methods) served as a positive control (PC HA). The V5-specific WB confirms the presence of the M1-V5 protein in all isolates (lanes 1 to 4). CEFs infected with rMVA-Rome- M1-V5 (see Methods) served as a positive control for the presence of the M1-V5 transgene (PC M1-V5). Non-infected CEFs and MVA-77-RED-infected CEFs served as negative controls (NC1 and NC2, respectively)

Article Snippet: Fresh Chick Embryo Fibroblasts (CEFs) were prepared from 11-day old embryonated Specific Pathogen Free chicken eggs (Charles River, Paris, France) and maintained in a serum-free medium containing 10 ng/mL EGF (VP-SFM, GIBCO), supplemented with 2% glutamine and 1% Pen/Strep, as previously described [ , ].

Techniques: Cytometry, Infection, Transfection, FACS, Virus, Clone Assay, Isolation, Flow Cytometry, Construct, Positive Control, Negative Control, Control

Journal: Journal of Translational Medicine

Article Title: Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

doi: 10.1186/s12967-023-04353-7

Figure Lengend Snippet: A membrane bound marker for RGGSM is expressed at the cell surface. A EGFP-expressing rMVA-77-G-M1-V5-infected CEFs show a “cytoplasmic green” signal B rMVA-77-SHAG-infected CEFs show a "stable green" signal displayed also at the cell surface and on actin projections mainly as membrane vesicles. Actin was labeled in red by phalloidin-TRITC. Transgene cassettes schemes below the micrographs are detailed in Fig.

Article Snippet: Fresh Chick Embryo Fibroblasts (CEFs) were prepared from 11-day old embryonated Specific Pathogen Free chicken eggs (Charles River, Paris, France) and maintained in a serum-free medium containing 10 ng/mL EGF (VP-SFM, GIBCO), supplemented with 2% glutamine and 1% Pen/Strep, as previously described [ , ].

Techniques: Membrane, Marker, Expressing, Infection, Labeling

Journal: Journal of Translational Medicine

Article Title: Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

doi: 10.1186/s12967-023-04353-7

Figure Lengend Snippet: Substantial release of virions in supernatants of MVA-infected cells. Supernatants from rMVA-77-SHAG- infected CEFs were harvested at various time points upon infection. Viral titers (ffu/ml, blue curve) and lysis-released cellular dsDNA (ng/ml, red curve) were determined; titer and DNA values are averages ± SD of 3 replicas. Excel log trend and equation for the first part of the viral titer curve are shown. Photographic inserts show cell morphology at 10, 30 and 70 h p.i

Article Snippet: Fresh Chick Embryo Fibroblasts (CEFs) were prepared from 11-day old embryonated Specific Pathogen Free chicken eggs (Charles River, Paris, France) and maintained in a serum-free medium containing 10 ng/mL EGF (VP-SFM, GIBCO), supplemented with 2% glutamine and 1% Pen/Strep, as previously described [ , ].

Techniques: Infection, Lysis

Journal: Journal of Translational Medicine

Article Title: Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

doi: 10.1186/s12967-023-04353-7

Figure Lengend Snippet: Visualization of fluorescent EEVs by Flow Virometry analysis. A Physical, FSC vs. Violet SSC plot (left panel), and fluorescence, B525/40 (green) vs. Violet SSC plot (right panel), characterisation of Megamix beads (900 nm—red, 500 nm—blue, 300 nm—yellow and 100 nm—green dots) by BC CytoFLEX S analysis, setting the instrument for flow virometry. B CEFs were infected with either rMVA-77-SHAG or rMVA-77-G- M1-V5, and supernatants were harvested 30 h p.i. Supernatants were analysed with the settings established using the Megamix beads (B525/40 vs. Violet SSC), revealing signals for entities with the “stable green” surface marker (SHAG-EEVs, left panel), but not so for the “cytoplasmic green” marker (G-EEVs, middle panel), unless these entities were stained in the DNA with the SYTO 13 dye (G-EEVs + SYTO 13, right panel). C and D BC CytoFLEX S analysis with B 525/40 (green) versus R 660/20 (red) settings of SHAG-EEVs. The green fluorescence is progressively lost with sample dilution. E) F/T/S treatment of SHAG EEVs abolishes the green signal, confirming its membrane localization

Article Snippet: Fresh Chick Embryo Fibroblasts (CEFs) were prepared from 11-day old embryonated Specific Pathogen Free chicken eggs (Charles River, Paris, France) and maintained in a serum-free medium containing 10 ng/mL EGF (VP-SFM, GIBCO), supplemented with 2% glutamine and 1% Pen/Strep, as previously described [ , ].

Techniques: Fluorescence, Infection, Marker, Staining, Membrane

Journal: Journal of Translational Medicine

Article Title: Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

doi: 10.1186/s12967-023-04353-7

Figure Lengend Snippet: Flow Virometry sorting of green fluorescent rMVA virions. A Supernatants at 30 h p.i. of rMVA-77-SHAG- or rMVA-77-RED- infected CEFs (SHAG-EEVs and RED-EEVs) were first analysed on BD FACSAria Fusion, based on their physical characteristics (FSC vs. SSC plot, left panel) and a sorting window (rectangle) was chosen such as to exclude larger particles including cells. Next, entities selected from that sorting window were sorted based on green fluorescence (B530/30 vs. SSC plot, middle panel). NEG-SW and POS-SW were then set for further analysis. The POS-SW was defined such that there were no RED-EEVs detectable for that window (right panel). B The two sorted populations from the NEG-SW and POS-SW were re-analyzed by BC CytoFLEX S based on physical characteristics (Violet-SSC) and green fluorescence (B525/40), confirming the enrichment of green fluorescent entities (reflecting the presence of SHAG-EEVs) from the POS-SW, as compared to those from the NEG-SW

Article Snippet: Fresh Chick Embryo Fibroblasts (CEFs) were prepared from 11-day old embryonated Specific Pathogen Free chicken eggs (Charles River, Paris, France) and maintained in a serum-free medium containing 10 ng/mL EGF (VP-SFM, GIBCO), supplemented with 2% glutamine and 1% Pen/Strep, as previously described [ , ].

Techniques: Infection, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting

doi: 10.1186/s12967-023-04353-7

Figure Lengend Snippet: Untagged rMVA-M1-V5 production by virometry-based RGGSM. A Infection/transfection of CEF cells with MVA-77-RED and TP-HAG- M1-V5 DNA. Supernatants at 30 h were directly subjected to Virus-Sorting (BD FACSAria Fusion) based on green fluorescence and physical characteristics (B530/30 vs. SSC). Candidate rMVAs, HAG-M1-V5-EEVs were sorted in bulk from the POS-SW as defined in Fig. A, F/T/S-treated and subjected to TD. In this experiment, approximately 3500 events were sorted and 10/48 “green” microcultures were obtained at TD, which corresponds to 0.18 ffu/culture, with a 7:1 ratio of single to multiple infections. C The supernatant at 30 h of one green fluorescent TD-clone was subjected to Virus-Sorting as in (A). Non fluorescent events (containing the candidate untagged rMVA EEVs) were sorted in bulk from the NEG-SW, as defined in Fig. A, F/T/S-treated and subjected to TD. In this particular experiment, approximately 2000 non-fluorescent events were sorted in bulk and 7/48 non-fluorescent virus infected cultures were obtained at TD, which corresponds to 0.17 ffu/culture, with a 14:1 ratio of single to multiple infections. PCR ( C ) and WB analysis ( D ) of infected cell lysates (as in Fig. D & E) confirmed that all four clones indeed expressed the M1-V5 transgene. Abbreviations: PC (positive control = rMVA-M1-V5-Rome-infected CEFs) (see Methods), NC1 (negative control = non-infected CEFs), NC2 (negative control = MVA-77-RED-infected CEFs)

Article Snippet: Fresh Chick Embryo Fibroblasts (CEFs) were prepared from 11-day old embryonated Specific Pathogen Free chicken eggs (Charles River, Paris, France) and maintained in a serum-free medium containing 10 ng/mL EGF (VP-SFM, GIBCO), supplemented with 2% glutamine and 1% Pen/Strep, as previously described [ , ].

Techniques: Infection, Transfection, Virus, Fluorescence, Clone Assay, Positive Control, Negative Control