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

Fig. 1. Generation of an HAV vaccine strain using reverse genetics in Vero cells. (A) Schematic of generating an HAV vaccine strain using reverse genetics. (B) pUC57S-HAV was linearized, and HAV cDNA was isolated after electrophoresis (M: DNA size marker, P: pUC57-HAV, L: linearized HAV cDNA [arrow]). (C) In vitro transcription product (HAV RNA) isolated after electrophoresis (M: RNA size marker, R: HAV RNA generated in vitro). (D) Ten days after transfection of HAV RNA in Vero cells, the generated HAV was assessed using quantitative RT-PCR (cell control: no HAV RNA transfected sample). HAV, hepatitis A virus; RT-PCR, reverse transcription polymerase chain reaction; GE, genomic equivalent.

Fig. 2. Adaptation of the HAV in Vero cells. (A) RG-HAV was re-infected in Vero cells serially. The samples collected 12 days after re-infection were analyzed using quantitative RT-PCR. (B) Transmission electron microscopy images of RG-HAV. The image on the right is a magnified view of the red box in the left image. ELISA results for RG-HAV (C) and rotavirus (D). HAV, hepatitis A virus; RG-HAV, hepatitis A virus vaccine generated via reverse genetics; RT-PCR, reverse transcription polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; GE, genomic equivalent; O.D., optical density.

Fig. 3. Comparison of the growth kinetics of RG-HAV and HM-175 in MRC-5 and Vero cells. Following infection with 10 MOI of either RG-HAV or HM-175 in MRC-5 or Vero cells, the growth kinetics were evaluated by subjecting the culture supernatants to RT-PCR (A, B) and ELISA (C, D). RG-HAV, hepatitis A virus vaccine generated via reverse genetics; MOI, multiplicity of infection; RT-PCR, reverse transcription polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; GE, genomic equivalent.

Fig. 4. Analysis of the nucleotide and amino acid sequences of RG-HAV. (A) Structure of the HAV genome. (B) Variations in the nucleotide and amino acid sequences between RG-HAV and HM-175. (C) Location of the altered amino acids in RG-HAV (Red box: epitope within the antigenic region; Yellow box: epitope with neutralizing antigenic properties; Green box: C-terminal domain; Yellow highlights indicate the altered amino acids). RG-HAV, hepatitis A virus vaccine generated via reverse genetics; HAV, hepatitis A virus; UTR, untranslated region.

Fig. 5. Comparison of the IRES between HM-175 and RG-HAV. (A) Comparative analysis of the nucleotide sequences of the IRES between HM-175 and RG-HAV. The predicted secondary structures of the IRES in HM-175 (B) and RG-HAV (C) are shown. (D) Relative codon deoptimization index of the capsid of HM-175 and RG-HAV. IRES, internal ribosomal entry site; RG-HAV, hepatitis A virus vaccine generated via reverse genetics; RCDI, relative codon deoptimization index.

Fig. 6. Immunogenicity assessment of RG-HAV. (A) Six-week-old BALB/c mice were intramuscularly immunized with RG-HAV (2×104 GE) with AH or PBS twice at a 2-week interval. (B) Sera were collected two weeks after primary and booster immunization, and the HAV-specific Ab titers were analyzed using ELISA. RG-HAV, hepatitis A virus vaccine generated via reverse genetics; GE, genomic equivalent; AH, alum hydroxide; PBS, phosphate-buffered saline; HAV, hepatitis A virus; ELISA, enzyme-linked immunosorbent assay; N.D., not detected. ***p<0.001.