Skip to content
2000
Volume 21, Issue 17
  • ISSN: 1570-1808
  • E-ISSN: 1875-628X

Abstract

Background

The adenovirus vector has been widely studied for vaccines and gene therapies. During the production of the adenovirus vector, a high virus titer is desired to obtain enough virus. The adenovirus vector has been widely studied for the vaccinations and gene therapies, where a high virus titer is desired to obtain sufficient quantities of the virus. For an adenovirus vector-based vaccine, suppression of antigen expression during production would improve the virus titer during production.

Objective

This study aimed to construct an adenovirus vector with lacO-regulated antigen expression using the SARS-CoV-2 spike as a transgene model, which would improve the adenovirus titer during production.

Methods

The lacO expression cassette was designed and prepared as a synthetic gene in pUC57. The lacO expression cassette was then subcloned into pShuttle-CMV. The SARS-CoV-2 spike gene was then inserted into the pShuttle-CMV harboring lacO to generate pShuttle-lacO_S and pShuttle-lacO-intron_S. Recombinant pShuttle was then used to generate a recombinant adenovirus genome using BJ5183 pAdeasy-1. Transfection of the I-linearized adenovirus genome into AD293 and HEK293 cells was used to generate adenovirus primary stock for 14 days of incubation.

Results

Recombinant adenovirus genomes, pAdeasy-lacO_S and pAdeasy-lacO-intron_S, were successfully generated and characterized using I restriction and PCR. In the production of adenovirus primary stocks, the adenovirus titer produced in AD293 cells was higher than in HEK293 cells. The primary stock titer of AdV_lacO-intron-S was higher than AdV_lacO-S and AdV_S titers.

Conclusion

Production of adenovirus with lacO and spike gene, either with or without intron, was successful with a higher titer as compared to AdV_S titer.

© 2024 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/lddd/10.2174/0115701808293712240516105131
2024-05-21
2025-07-24

  • From This Site

    /content/journals/lddd/10.2174/0115701808293712240516105131
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. LeeC.S. BishopE.S. ZhangR. YuX. FarinaE.M. YanS. ZhaoC. ZengZ. ShuY. WuX. LeiJ. LiY. ZhangW. YangC. WuK. WuY. HoS. AthivirahamA. LeeM.J. WolfJ.M. ReidR.R. HeT.C. Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine.Genes Dis.201742436310.1016/j.gendis.2017.04.001 28944281
     [Google Scholar]
  2. TatsisN. ErtlH.C.J. Adenoviruses as vaccine vectors.Mol. Ther.200410461662910.1016/j.ymthe.2004.07.013 15451446
     [Google Scholar]
  3. CharmanM. HerrmannC. WeitzmanM.D. Viral and cellular interactions during adenovirus DNA replication.FEBS Lett.2019593243531355010.1002/1873‑3468.13695 31764999
     [Google Scholar]
  4. CrenshawB.J. JonesL.B. BellC.R. KumarS. MatthewsQ.L. Perspective on adenoviruses: Epidemiology, pathogenicity, and gene therapy.Biomedicines2019736110.3390/biomedicines7030061 31430920
     [Google Scholar]
  5. ChavdaV. BezbaruahR. ValuD. PatelB. KumarA. PrasadS. KakotiB. KaushikA. JesawadawalaM. Adenoviral vector-based vaccine platform for COVID-19: Current status.Vaccines 202311243210.3390/vaccines11020432 36851309
     [Google Scholar]
  6. BellamkondaN. LambeU.P. SawantS. NandiS.S. ChakrabortyC. ShuklaD. Immune response to SARS-CoV-2 Vaccines.Biomedicines2022107146410.3390/biomedicines10071464 35884770
     [Google Scholar]
  7. CoughlanL. KremerE.J. ShayakhmetovD.M. Adenovirus-based vaccines—a platform for pandemic preparedness against emerging viral pathogens.Mol. Ther.20223051822184910.1016/j.ymthe.2022.01.034 35092844
     [Google Scholar]
  8. KyriakidisN.C. López-CortésA. GonzálezE.V. GrimaldosA.B. PradoE.O. SARS-CoV-2 vaccines strategies: A comprehensive review of phase 3 candidates.NPJ Vaccines2021612810.1038/s41541‑021‑00292‑w 33619260
     [Google Scholar]
  9. ArtariniA. HadiantiT. Giri-RachmanE.A. TanM.I. SafitriI.A. HidayatN.A. RetnoningrumD.S. NataliaD. Development of adenovirus-based covid-19 vaccine candidate in indonesia.Mol. Biotechnol.202466222223210.1007/s12033‑023‑00749‑4 37076664
     [Google Scholar]
  10. GallJ.G.D. LizonovaA. EttyReddy, D.; McVey, D.; Zuber, M.; Kovesdi, I.; Aughtman, B.; King, C.R.; Brough, D.E. Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes.Mol. Biotechnol.200735326327310.1007/BF02686012 17652790
     [Google Scholar]
  11. IngusciS. VerlengiaG. SoukupovaM. ZucchiniS. SimonatoM. Gene therapy tools for brain diseases.Front. Pharmacol.20191072410.3389/fphar.2019.00724 31312139
     [Google Scholar]
  12. ChaurasiyaS. HittM.M. Adenoviral vector construction I: Mammalian systems. In: In Adenoviral Vectors for Gene Therapy: Second Edition; 85-112.Elsevier Inc.2016
     [Google Scholar]
  13. ZhaoC. CrewsC.J. DerdeynC.A. BlackwellJ.L. Lac-regulated system for generating adenovirus 5 vaccine vectors expressing cytolytic human immunodeficiency virus 1 genes.J. Virol. Methods20091601-210111010.1016/j.jviromet.2009.04.028 19409930
     [Google Scholar]
  14. MatthewsD.A. CummingsD. EveleghC. GrahamF.L. PrevecL. Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.J. Gen. Virol.199980234535310.1099/0022‑1317‑80‑2‑345 10073694
     [Google Scholar]
  15. BerenjianS. AkusjärviG. Binary AdEasy vector systems designed for Tet-ON or Tet-OFF regulated control of transgene expression.Virus Res.20061151162310.1016/j.virusres.2005.07.001 16102868
     [Google Scholar]
  16. HuS.X. JiW. ZhouY. LogothetisC. XuH.J. Development of an adenovirus vector with tetracycline-regulatable human tumor necrosis factor alpha gene expression.Cancer Res.1997571633393343 9269991
     [Google Scholar]
  17. RubinchikS. DingR. QiuA.J. ZhangF. DongJ. Adenoviral vector which delivers FasL–GFP fusion protein regulated by the tet-inducible expression system.Gene Ther.200071087588510.1038/sj.gt.3301172 10845726
     [Google Scholar]
  18. HuangH. LiuY. LiaoW. CaoY. LiuQ. GuoY. LuY. XieZ. Oncolytic adenovirus programmed by synthetic gene circuit for cancer immunotherapy.Nat. Commun.2019101480110.1038/s41467‑019‑12794‑2 31641136
     [Google Scholar]
/content/journals/lddd/10.2174/0115701808293712240516105131
Loading
Construction of an Adenovirus Vector with Laco-regulated Antigen Expression Using the SARS-Cov-2 Spike as a Transgene Model
Letters in Drug Design & Discovery 21, 3723 (2024); https://doi.org/10.2174/0115701808293712240516105131
/content/journals/lddd/10.2174/0115701808293712240516105131
/content/journals/lddd/10.2174/0115701808293712240516105131
Loading

Data & Media loading...


  • Article Type:     Research Article
Keyword(s): Adenovirus; COVID-19; intron; LacO; primary stock; spike

Most Cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test