Abstract
The available cell-adapted hepatitis A virus (HAV) strains show a very slow replication phenotype hampering the affordable production of antigen. A fast-growing strain characterized by the occurrence of mutations in the internal ribosome entry site (IRES), combined with changes in the codon composition has been selected in our laboratory. A characterization of the IRES activity of this fast-growing strain (HM175-HP; HP) vs. its parental strain (HM175; L0) was assessed in two cell substrates used in vaccine production (MRC-5 and Vero cells) compared with the FRhK-4 cell line in which its selection was performed. The HP-derived IRES was significantly more active than the L0-derived IRES in all cells tested and both IRES were more active in the FRhK-4 cells. The translation efficiency of the HP-derived IRES was also much higher than the L0-derived IRES, particularly, in genes with a HP codon usage background. These results correlated with a higher virus production in a shorter time for the HP strain compared to the L0 strain in any of the three cell lines tested, and of both strains in the FRhK-4 cells compared to Vero and MRC-5 cells. The addition of wortmannin resulted in the increase of infectious viruses and antigen in the supernatant of FRhK-4 infected cells, independently of the strain. Finally, the replication of both strains in a clone of FRhK-4 cells adapted to grow with synthetic sera was optimal and again the HP strain showed higher yields.
Highlights
Hepatitis A outbreaks are on the rise
It has been described that the replication rate of hepatitis A virus (HAV), in some cell lines, is controlled to a significant degree by the efficiency of translation, by the Internal Ribosome Entry Site (IRES) activity (Funkhouser et al, 1999)
The most expensive reagent to produce viruses in cell culture is fetal calf serum (FBS), when using substrates such as the FRhK-4 cell line which require a supplement of 15% fetal bovine serum (FBS)
Summary
Hepatitis A outbreaks are on the rise. Recently, a huge outbreak has hit the men-having-sex-withmen (MSM) group across Europe with over 4,000 cases (Beebeejaun et al, 2017; Freidl et al, 2017; Werber et al, 2017; Ecdc., 2018). The control of these outbreaks has been hampered by low vaccination coverage and by vaccine shortages, which have sometimes been overcome by restricting the number of vaccine doses and their content. While these measures attempt to avoid as much as possible the spread of the outbreaks and to protect the patients’ health, they may prompt the selection of vaccine-escape virus variants (Pintó and Bosch, 2019; Sabrià et al, 2019). Production of large amounts of antigens, required for inactivated vaccines, is still a critical step, for viruses with poor growth in cell culture such as the hepatitis A virus (HAV) (Lemon et al, 1992). Several strains of HAV have been adapted to replicate in vitro, including the HM175, CR-326, GBM, TZ84, Lv-8, YN5, and RG-SB strains (Lemon et al, 2017), but production of industrial batches require large scale-ups resulting in very high costs
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