The infectivity and pathogenicity of hepatitis A virus live-attenuated vaccine strain H2 in type I interferon receptor-deficient mice

Abstract

Hepatitis A virus (HAV) live-attenuated vaccine H2 strain has been approved for clinical use for decades with ideal safety profiles in nonhuman primate models and humans. Recently, type I interferon (IFN) receptor-deficient mice were shown to be susceptible to HAV infection. Herein, we sought to determine the infection and replication dynamics of the H2 in Ifnar^-/- mice that lack type I IFN receptor. Following intravenous injection, the H2 failed to cause obvious clinical symptoms in Ifnar^-/- mice, and no significant upregulation in serum alanine aminotransferase (ALT) levels was observed. Notably, the histopathological examination showed that there were significant focal infiltrations of lymphocytes and neutrophils in the portal area, but no focal necrosis was observed in liver tissues. Viral RNAs sustained in the liver, and the infectious virus could be recovered from the liver tissue until 42 days post-infection. More importantly, H2 infection induced obvious viremia and persistent viral shedding in feces. In addition, robust HAV-specific humoral immune responses were induced in Ifnar^-/- mice. Overall, our study revealed the safety profile of H2 in Ifnar^-/- mice, which not only helps understand the attenuation mechanism of H2, but also expands the application of the Ifnar^-/- mouse model for HAV studies.

Keywords: Hepatitis A; Hepatitis A virus (HAV); Live-attenuated vaccine; Mouse model.

Fig. 1

H2 strain infection in Ifnar^−/− mice. A Schematic diagram of inoculation and sampling in Ifnar^−/− mice. B Serum HAV RNA in Ifnar^−/− mice infected with 10⁷ RNA copies of the H2 strain was quantified by real-time RT-qPCR. C Fecal HAV RNA in infected Ifnar^−/− mice was quantified by real-time RT-qPCR. Dotted lines indicate the limits of detection.

Fig. 2

Hepatic HAV RNA and ALT level in Ifnar^−/−mice infected by H2 strain. A HAV RNA in the liver of Ifnar^−/− mice at day 42 post-inoculation. Briefly, the liver was homogenized in DMEM followed by centrifugation at 8000×g for 10 ​min and detected by real-time RT-qPCR. Dotted lines indicate the limits of detection. B Serum ALT activity was measured using the alanine transaminase activity assay kit with Color Endpoint (Abcam, USA). Ifnar^−/− mice were infected with 10⁷ RNA copies of H2 strain and mice serum were collected at different time.

Fig. 3

H2 strain infection in the liver of Ifnar^−/−mice. RNA ISH: ISH assay for HAV RNA detection in liver tissues. Red arrows show brown positive cells. IHC: Immunohistochemical staining of liver tissues with HAV-specific antibody. HAV protein is indicated in brown. Red arrows show brown positive cells. H&E: Histopathological assays of the mice liver. The red arrow shows inflammatory cell infiltration. Scale bars, 20 ​μm.

Fig. 4

Isolation of the virus from the liver of Ifnar^−/− mice infected with the H2 stain. 2BS cells were inoculated with liver extracts from H2 strain-infected mice, and HAV RNA was detected in cell cultures at different time points. Dotted lines indicate the limits of detection.

Fig. 5

Humoral immune responses in Ifnar^−/− mice infected with the H2 strain. A HAV-specific IgG antibody titers of infected Ifnar^−/− mice at different time points were determined by ELISA at different time points. Dotted lines indicate the limits of detection. B Virus neutralization assay. Wild type HAV (HM175/18f strain) was incubated with heat-inactivated serum of H2 infected Ifnar^−/− mice or uninfected mock group or PBS, respectively, on day 42 post-inoculation (H2 or mock group) or PBS (negative control, NC group), and the mixture was used to infect BS-C-1 ​cells in 12-well plates to detect anti-HAV neutralizing antibodies. Data are shown as the mean ​± ​SEM. Significance was calculated using nonparametric statistical analysis (∗P ​< ​0.05).