Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting
Abstract Background Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subseq...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-07-01
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| Series: | Journal of Translational Medicine |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12967-023-04353-7 |
| _version_ | 1797556863857852416 |
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| author | Daniela Boselli Maddalena Panigada Simona Di Terlizzi Monica Romanò Emanuele Canonico Chiara Villa Claudia Minici Eelco van Anken Elisa Soprana Antonio G. Siccardi |
| author_facet | Daniela Boselli Maddalena Panigada Simona Di Terlizzi Monica Romanò Emanuele Canonico Chiara Villa Claudia Minici Eelco van Anken Elisa Soprana Antonio G. Siccardi |
| author_sort | Daniela Boselli |
| collection | DOAJ |
| description | Abstract Background Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. Methods The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. Results Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. Conclusions Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases. Graphical Abstract |
| first_indexed | 2024-03-10T17:08:00Z |
| format | Article |
| id | doaj.art-8ed26165057b40fcafc217a4d6c62932 |
| institution | Directory Open Access Journal |
| issn | 1479-5876 |
| language | English |
| last_indexed | 2024-03-10T17:08:00Z |
| publishDate | 2023-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Translational Medicine |
| spelling | doaj.art-8ed26165057b40fcafc217a4d6c629322023-11-20T10:44:18ZengBMCJournal of Translational Medicine1479-58762023-07-0121111410.1186/s12967-023-04353-7Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sortingDaniela Boselli0Maddalena Panigada1Simona Di Terlizzi2Monica Romanò3Emanuele Canonico4Chiara Villa5Claudia Minici6Eelco van Anken7Elisa Soprana8Antonio G. Siccardi9FRACTAL - San Raffaele Scientific InstituteDivision of Genetics and Cell Biology, San Raffaele Scientific InstituteFRACTAL - San Raffaele Scientific InstituteFRACTAL - San Raffaele Scientific InstituteFRACTAL - San Raffaele Scientific InstituteFRACTAL - San Raffaele Scientific InstituteDivision of Genetics and Cell Biology, San Raffaele Scientific InstituteUniversità Vita-Salute San RaffaeleDivision of Genetics and Cell Biology, San Raffaele Scientific InstituteDivision of Genetics and Cell Biology, San Raffaele Scientific InstituteAbstract Background Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. Methods The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. Results Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. Conclusions Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases. Graphical Abstracthttps://doi.org/10.1186/s12967-023-04353-7Recombinant MVArMVAFlow VirometryVirus-SortingEEV and IMV virionsHemagglutinin/egfp fusion protein |
| spellingShingle | Daniela Boselli Maddalena Panigada Simona Di Terlizzi Monica Romanò Emanuele Canonico Chiara Villa Claudia Minici Eelco van Anken Elisa Soprana Antonio G. Siccardi Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting Journal of Translational Medicine Recombinant MVA rMVA Flow Virometry Virus-Sorting EEV and IMV virions Hemagglutinin/egfp fusion protein |
| title | Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting |
| title_full | Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting |
| title_fullStr | Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting |
| title_full_unstemmed | Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting |
| title_short | Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting |
| title_sort | time and cost effective production of untagged recombinant mva by flow virometry and direct virus sorting |
| topic | Recombinant MVA rMVA Flow Virometry Virus-Sorting EEV and IMV virions Hemagglutinin/egfp fusion protein |
| url | https://doi.org/10.1186/s12967-023-04353-7 |
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