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

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.

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.

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.

RG-HAV offers enhanced replication and production yields, supporting its potential in advancing HAV vaccine development.