Enhanced replication of a hepatitis A virus vaccine strain via adaptation in Vero cells.

Sang Hwan Seo, Jung-Ah Choi, Mi Sun Kim, Eunji Yang, Sumin Choi, Dong Won Seo, Manki Song
Author Information
  1. Sang Hwan Seo: Science Unit, International Vaccine Institute, Seoul, Korea. ORCID
  2. Jung-Ah Choi: Science Unit, International Vaccine Institute, Seoul, Korea. ORCID
  3. Mi Sun Kim: Science Unit, International Vaccine Institute, Seoul, Korea. ORCID
  4. Eunji Yang: Science Unit, International Vaccine Institute, Seoul, Korea. ORCID
  5. Sumin Choi: Gyeongbuk Institute for Bio Industry, Andong, Korea. ORCID
  6. Dong Won Seo: Gyeongbuk Institute for Bio Industry, Andong, Korea. ORCID
  7. Manki Song: Science Unit, International Vaccine Institute, Seoul, Korea. ORCID

Abstract

Purpose: Hepatitis A virus (HAV) production has been limited by its slow replication rate and reliance on diploid cell lines like MRC-5, which present challenges in scalability, passage limitations, and serum-free culture conditions. This study aimed to develop an HAV vaccine strain with enhanced replication capacity.
Materials and Methods: We generated a reverse genetically modified HAV vaccine strain (RG-HAV) and adapted it to Vero cells through sequential culturing. Replication rates of RG-HAV and a commercially used strain, HM-175, were compared in Vero and MRC-5 cells. Nucleotide sequences, including coding and non-coding regions like the internal ribosomal entry site (IRES), were analyzed. Structural assessments included 3-dimensional modeling of IRES and relative codon deoptimization analysis of the capsid. Immunogenicity was evaluated by measuring HAV-specific antibody responses in mice.
Results: Vero-adapted RG-HAV achieved a 30-fold increase in production yield compared to initial transfection. In Vero cells, RG-HAV peaked at 15 days post-infection, compared to 20 days for HM-175. In MRC-5 cells, RG-HAV and HM-175 reached peak production at 10 and 15 days, respectively. RG-HAV produced over 5-fold more HAV in Vero cells and 8-fold more in MRC-5 cells than HM-175. Sequence analysis revealed nine amino acid differences in RG-HAV structural proteins and five nucleotide changes in the type III IRES region, potentially enhancing IRES functionality. Immunization with inactivated RG-HAV with alum hydroxide induced HAV-specific antibody responses in mice.
Conclusion: RG-HAV offers enhanced replication and production yields, supporting its potential in advancing HAV vaccine development.

Keywords

References

  1. Public Health. 2019 Mar;168:150-156 [PMID: 30442468]
  2. Euro Surveill. 2015 Jan 29;20(4): [PMID: 25655054]
  3. J Med Virol. 2006 Nov;78(11):1398-405 [PMID: 16998883]
  4. J Virol. 1994 Aug;68(8):5253-63 [PMID: 8035522]
  5. World J Gastroenterol. 2004 Sep 1;10(17):2571-3 [PMID: 15300909]
  6. Lancet Infect Dis. 2014 Oct;14(10):976-81 [PMID: 25195178]
  7. N Engl J Med. 1992 Aug 13;327(7):453-7 [PMID: 1320740]
  8. Front Microbiol. 2021 Feb 18;12:642267 [PMID: 33679679]
  9. Biotechnol Adv. 2020 Nov 15;44:107608 [PMID: 32768520]
  10. PLoS Pathog. 2010 Mar 05;6(3):e1000797 [PMID: 20221432]
  11. Arch Intern Med. 2010 Nov 8;170(20):1811-8 [PMID: 21059974]
  12. Zhonghua Yi Xue Za Zhi. 2002 May 25;82(10):678-81 [PMID: 12133465]
  13. J Virol. 2002 Feb;76(3):1171-80 [PMID: 11773393]
  14. Proc Soc Exp Biol Med. 1984 Jan;175(1):10-5 [PMID: 6364147]
  15. Appl Environ Microbiol. 1987 Dec;53(12):2967-71 [PMID: 2829721]
  16. Cold Spring Harb Perspect Med. 2018 Oct 1;8(10): [PMID: 29530949]
  17. Cold Spring Harb Perspect Med. 2018 Dec 3;8(12): [PMID: 29610147]
  18. Am J Health Syst Pharm. 2017 Nov 15;74(22):1879-1886 [PMID: 28970246]
  19. J Clin Microbiol. 1984 Jul;20(1):28-33 [PMID: 6086708]
  20. Lancet Infect Dis. 2018 Jan;18(1):33-34 [PMID: 29303741]
  21. Can J Infect Dis Med Microbiol. 2023 Jan 4;2023:4263309 [PMID: 36644336]
  22. Hepatol Int. 2021 Oct;15(5):1068-1082 [PMID: 34345993]
  23. J Infect Dis. 1991 Apr;163(4):735-9 [PMID: 1849160]
  24. J Gen Virol. 1989 Aug;70 ( Pt 8):2051-62 [PMID: 2549182]
  25. J Virol. 1994 Feb;68(2):1066-74 [PMID: 8289336]
  26. Euro Surveill. 2021 May;26(20): [PMID: 34018484]
  27. J Med Virol. 1987 May;22(1):35-44 [PMID: 3035078]
  28. Virology. 1993 Jun;194(2):475-80 [PMID: 8389072]
  29. Virulence. 2021 Dec;12(1):1174-1185 [PMID: 33843464]
  30. J Virol. 1996 Feb;70(2):1041-9 [PMID: 8551562]
  31. Nature. 2015 Jan 1;517(7532):85-88 [PMID: 25327248]
  32. Cold Spring Harb Perspect Med. 2019 Feb 1;9(2): [PMID: 29661808]
  33. Proc Soc Exp Biol Med. 1981 Jun;167(2):201-6 [PMID: 6262835]
  34. Euro Surveill. 2015 Jul 23;20(29):21192 [PMID: 26227370]
  35. Sci Rep. 2016 Nov 03;6:35962 [PMID: 27808108]
  36. J Virol. 2003 Jun;77(11):6574-9 [PMID: 12743317]

Word Cloud

Created with Highcharts 10.0.0RG-HAVcellsVeroHAVproductionreplicationMRC-5vaccinestrainHM-175IRESviruscompareddaysHepatitiscelllikeenhancedribosomalentrysiteanalysisHAV-specificantibodyresponsesmice15Purpose:limitedslowratereliancediploidlinespresentchallengesscalabilitypassagelimitationsserum-freecultureconditionsstudyaimeddevelopcapacityMaterialsMethods:generatedreversegeneticallymodifiedadaptedsequentialculturingReplicationratescommerciallyusedNucleotidesequencesincludingcodingnon-codingregionsinternalanalyzedStructuralassessmentsincluded3-dimensionalmodelingrelativecodondeoptimizationcapsidImmunogenicityevaluatedmeasuringResults:Vero-adaptedachieved30-foldincreaseyieldinitialtransfectionpeakedpost-infection20reachedpeak10respectivelyproduced5-fold8-foldSequencerevealednineaminoaciddifferencesstructuralproteinsfivenucleotidechangestypeIIIregionpotentiallyenhancingfunctionalityImmunizationinactivatedalumhydroxideinducedConclusion:offersyieldssupportingpotentialadvancingdevelopmentEnhancedhepatitisviaadaptationCodonInternalReversegenetics

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