Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD
Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection an...
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| Format: | Article |
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MDPI AG
2022-04-01
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| Series: | Biomedicines |
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| Online Access: | https://www.mdpi.com/2227-9059/10/5/990 |
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| author | Cathy Ventura Dalinda Eusébio Ana M. Gonçalves Jorge Barroca-Ferreira Diana Costa Zhengrong Cui Luís A. Passarinha Ângela Sousa |
| author_facet | Cathy Ventura Dalinda Eusébio Ana M. Gonçalves Jorge Barroca-Ferreira Diana Costa Zhengrong Cui Luís A. Passarinha Ângela Sousa |
| author_sort | Cathy Ventura |
| collection | DOAJ |
| description | Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (<i>p</i>-value < 0.05) and presented a non-significant lack of fit (<i>p</i>-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO<sub>2</sub> in the fermentation step during 5 h and 30% pO<sub>2</sub> in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry. |
| first_indexed | 2024-03-10T03:17:34Z |
| format | Article |
| id | doaj.art-83e76953dab24fe580126a64b805b387 |
| institution | Directory Open Access Journal |
| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-10T03:17:34Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomedicines |
| spelling | doaj.art-83e76953dab24fe580126a64b805b3872023-11-23T10:09:28ZengMDPI AGBiomedicines2227-90592022-04-0110599010.3390/biomedicines10050990Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBDCathy Ventura0Dalinda Eusébio1Ana M. Gonçalves2Jorge Barroca-Ferreira3Diana Costa4Zhengrong Cui5Luís A. Passarinha6Ângela Sousa7CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, PortugalCICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, PortugalCICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, PortugalCICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, PortugalCICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, PortugalDivision of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USACICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, PortugalCICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, PortugalNucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (<i>p</i>-value < 0.05) and presented a non-significant lack of fit (<i>p</i>-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO<sub>2</sub> in the fermentation step during 5 h and 30% pO<sub>2</sub> in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.https://www.mdpi.com/2227-9059/10/5/990bioreactorCOVID-19design of experimentsminicircle DNA vaccine |
| spellingShingle | Cathy Ventura Dalinda Eusébio Ana M. Gonçalves Jorge Barroca-Ferreira Diana Costa Zhengrong Cui Luís A. Passarinha Ângela Sousa Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD Biomedicines bioreactor COVID-19 design of experiments minicircle DNA vaccine |
| title | Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD |
| title_full | Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD |
| title_fullStr | Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD |
| title_full_unstemmed | Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD |
| title_short | Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD |
| title_sort | maximization of the minicircle dna vaccine production expressing sars cov 2 rbd |
| topic | bioreactor COVID-19 design of experiments minicircle DNA vaccine |
| url | https://www.mdpi.com/2227-9059/10/5/990 |
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