Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation
In this study, we invented and construct novel candidate HIV-1 vaccines. Through genetic and protein engineering, we unknowingly constructed an HIV-1-derived transgene with a homopolymeric run of 11 cytidines, which was inserted into an adenovirus vaccine vector. Here, we describe the virus rescue,...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-06-01
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| Series: | Vaccines |
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| Online Access: | https://www.mdpi.com/2076-393X/10/6/960 |
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| author | Zara Hannoun Edmund G. Wee Alison Crook Stefano Colloca Stefania Di Marco Tomáš Hanke |
| author_facet | Zara Hannoun Edmund G. Wee Alison Crook Stefano Colloca Stefania Di Marco Tomáš Hanke |
| author_sort | Zara Hannoun |
| collection | DOAJ |
| description | In this study, we invented and construct novel candidate HIV-1 vaccines. Through genetic and protein engineering, we unknowingly constructed an HIV-1-derived transgene with a homopolymeric run of 11 cytidines, which was inserted into an adenovirus vaccine vector. Here, we describe the virus rescue, three rounds of clonal purification and preparation of good manufacturing practise (GMP) starting material assessed for genetic stability in five additional virus passages. Throughout these steps, quality control assays indicated the presence of the transgene in the virus genome, expression of the correct transgene product and immunogenicity in mice. However, DNA sequencing of the transgene revealed additional cytidines inserted into the original 11-cytidine region, and the GMP manufacture had to be aborted. Subsequent analyses indicated that as little as 1/25th of the virus dose used for confirmation of protein expression (10<sup>6</sup> cells at a multiplicity of infection of 10) and murine immunogenicity (10<sup>8</sup> infectious units per animal) met the quality acceptance criteria. Similar frameshifts in the expressed proteins were reproduced in a one-reaction <i>in vitro</i> transcription/translation employing phage T7 polymerase and <i>E. coli</i> ribosomes. Thus, the most likely mechanism for addition of extra cytidines into the ChAdOx1.tHIVconsv6 genome is that the adenovirus DNA polymerase lost its fidelity on a stretch of 11 cytidines, which informs future adenovirus vaccine designs. |
| first_indexed | 2024-03-09T22:16:23Z |
| format | Article |
| id | doaj.art-895deba437e546f5b8ca79abe46bea1b |
| institution | Directory Open Access Journal |
| issn | 2076-393X |
| language | English |
| last_indexed | 2024-03-09T22:16:23Z |
| publishDate | 2022-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj.art-895deba437e546f5b8ca79abe46bea1b2023-11-23T19:22:05ZengMDPI AGVaccines2076-393X2022-06-0110696010.3390/vaccines10060960Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine PreparationZara Hannoun0Edmund G. Wee1Alison Crook2Stefano Colloca3Stefania Di Marco4Tomáš Hanke5Nuffield Department of Medicine, The Jenner Institute, University of Oxford, Oxford OX3 7DQ, UKNuffield Department of Medicine, The Jenner Institute, University of Oxford, Oxford OX3 7DQ, UKNuffield Department of Medicine, The Jenner Institute, University of Oxford, Oxford OX3 7DQ, UKReiThera S.r.l., Via di Castel Romano, 100, 00128 Rome, ItalyAdvent S.r.l., Via Pontina km 30600, 00071 Pomezia, ItalyNuffield Department of Medicine, The Jenner Institute, University of Oxford, Oxford OX3 7DQ, UKIn this study, we invented and construct novel candidate HIV-1 vaccines. Through genetic and protein engineering, we unknowingly constructed an HIV-1-derived transgene with a homopolymeric run of 11 cytidines, which was inserted into an adenovirus vaccine vector. Here, we describe the virus rescue, three rounds of clonal purification and preparation of good manufacturing practise (GMP) starting material assessed for genetic stability in five additional virus passages. Throughout these steps, quality control assays indicated the presence of the transgene in the virus genome, expression of the correct transgene product and immunogenicity in mice. However, DNA sequencing of the transgene revealed additional cytidines inserted into the original 11-cytidine region, and the GMP manufacture had to be aborted. Subsequent analyses indicated that as little as 1/25th of the virus dose used for confirmation of protein expression (10<sup>6</sup> cells at a multiplicity of infection of 10) and murine immunogenicity (10<sup>8</sup> infectious units per animal) met the quality acceptance criteria. Similar frameshifts in the expressed proteins were reproduced in a one-reaction <i>in vitro</i> transcription/translation employing phage T7 polymerase and <i>E. coli</i> ribosomes. Thus, the most likely mechanism for addition of extra cytidines into the ChAdOx1.tHIVconsv6 genome is that the adenovirus DNA polymerase lost its fidelity on a stretch of 11 cytidines, which informs future adenovirus vaccine designs.https://www.mdpi.com/2076-393X/10/6/960adenovirus DNA polymeraseT7 polymerasepolymerase fidelityprotein engineeringvaccinesHIVconsvX |
| spellingShingle | Zara Hannoun Edmund G. Wee Alison Crook Stefano Colloca Stefania Di Marco Tomáš Hanke Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation Vaccines adenovirus DNA polymerase T7 polymerase polymerase fidelity protein engineering vaccines HIVconsvX |
| title | Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation |
| title_full | Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation |
| title_fullStr | Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation |
| title_full_unstemmed | Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation |
| title_short | Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation |
| title_sort | adenovirus dna polymerase loses fidelity on a stretch of eleven homocytidines during pre gmp vaccine preparation |
| topic | adenovirus DNA polymerase T7 polymerase polymerase fidelity protein engineering vaccines HIVconsvX |
| url | https://www.mdpi.com/2076-393X/10/6/960 |
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