A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines
<b>Background/Objectives</b>: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of m...
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2025-05-01
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| author | Maria Rubtsova Yuliana Mokrushina Dmitry Andreev Maria Poteshnova Nikita Shepelev Mariya Koryagina Ekaterina Moiseeva Diana Malabuiok Yury Prokopenko Stanislav Terekhov Aleksander Chernov Elena Vodovozova Ivan Smirnov Olga Dontsova Alexander Gabibov Yury Rubtsov |
| author_facet | Maria Rubtsova Yuliana Mokrushina Dmitry Andreev Maria Poteshnova Nikita Shepelev Mariya Koryagina Ekaterina Moiseeva Diana Malabuiok Yury Prokopenko Stanislav Terekhov Aleksander Chernov Elena Vodovozova Ivan Smirnov Olga Dontsova Alexander Gabibov Yury Rubtsov |
| author_sort | Maria Rubtsova |
| collection | DOAJ |
| description | <b>Background/Objectives</b>: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation <i>in vivo</i>. The composition of 5′ and 3′ UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for <i>in vivo</i> selection of translationally active UTRs. <b>Methods</b>: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. <b>Results</b>: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. <b>Conclusions</b>: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines. |
| format | Article |
| id | doaj-art-390975625e8c445f8f7379cf4fa47efb |
| institution | OA Journals |
| issn | 2076-393X |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj-art-390975625e8c445f8f7379cf4fa47efb2025-08-20T02:33:58ZengMDPI AGVaccines2076-393X2025-05-0113553010.3390/vaccines13050530A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA VaccinesMaria Rubtsova0Yuliana Mokrushina1Dmitry Andreev2Maria Poteshnova3Nikita Shepelev4Mariya Koryagina5Ekaterina Moiseeva6Diana Malabuiok7Yury Prokopenko8Stanislav Terekhov9Aleksander Chernov10Elena Vodovozova11Ivan Smirnov12Olga Dontsova13Alexander Gabibov14Yury Rubtsov15Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, RussiaDepartment of Chemistry, Lomonosov Moscow State University, 119991 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, Russia<b>Background/Objectives</b>: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation <i>in vivo</i>. The composition of 5′ and 3′ UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for <i>in vivo</i> selection of translationally active UTRs. <b>Methods</b>: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. <b>Results</b>: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. <b>Conclusions</b>: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines.https://www.mdpi.com/2076-393X/13/5/530mRNA vaccinetranslationUTRssecreted nanoluciferaselipid nanoparticlesimmunization |
| spellingShingle | Maria Rubtsova Yuliana Mokrushina Dmitry Andreev Maria Poteshnova Nikita Shepelev Mariya Koryagina Ekaterina Moiseeva Diana Malabuiok Yury Prokopenko Stanislav Terekhov Aleksander Chernov Elena Vodovozova Ivan Smirnov Olga Dontsova Alexander Gabibov Yury Rubtsov A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines Vaccines mRNA vaccine translation UTRs secreted nanoluciferase lipid nanoparticles immunization |
| title | A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines |
| title_full | A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines |
| title_fullStr | A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines |
| title_full_unstemmed | A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines |
| title_short | A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines |
| title_sort | luciferase based approach for functional screening of 5 and 3 untranslated regions of the mrna component for mrna vaccines |
| topic | mRNA vaccine translation UTRs secreted nanoluciferase lipid nanoparticles immunization |
| url | https://www.mdpi.com/2076-393X/13/5/530 |
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