A Recombinant HAV Expressing a Neutralization Epitope of HEV Induces Immune Response against HAV and HEV in Mice

Figures

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  Figure 1. Schematic representation of hepatitis A virus-HEV neutralization epitope (HAV-HEp148) constructs containing the HEp148 protein in the 2A/2B junction of HAV. The HEp148 gene was cloned into the 2A/2B junction of the HAV genome in the pT7-HAV-A60-MCS (multiple cloning site). This plasmid contains a polylinker coding for restriction sites (SalI, SnaBI, and SpeI) flanked by 3Gly hinges (red color letters), and HAV 3Cpro cleavage sites (scissors) engineered into the 2A/2B junction of pT7-HAV-A60. The HEp148 gene coding for the hepatitis E virus (HEV) neutralization epitope was cloned into the SalI and SpeI sites of pT7-HAV-A60-MCS, and the resulting plasmid was termed pT7-HAV-HEp148.

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  Figure 2. Rescue of the HAV constructs containing the HEp148 gene at the 2A/2B junction. (A) RNA transcribed from pT7-HAV-HEp148 with T7 RNA polymerase. Lane M indicates RNA size markers, lane 1 indicates plus-strand RNA of the HAV-HEp148 virus, and lane 2 indicates plus-strand RNA from the wild-type HAV virus. The sizes of the marker bands are in kb; (B) RT-PCR results show the HEp148 insertion. Viral RNA was extracted and fragments were amplified with RT-PCR using HAV primers specific for HAV nucleotides (nts) 3137 to 3161 and 3544 to 3568, spanning the HEp148 insertion. RT-PCR fragments amplified from RNA extracted from HAV-HEp148 (lane 1) and wild type HAV (lane 2) were analyzed with 1.5% agarose gel electrophoresis. The RT-PCR fragments from HAV-HEp148 and parental HAV are indicated by arrowheads, and their sizes given in base pairs (bp). The size of the DNA molecular weight markers (lane M) is indicated in thousand base pairs (kb).

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  Figure 3. Immunofluorescence (IF) analysis of HAV-HEp148-infected Huh-7 cells. Monolayers of Huh-7 cells were either infected with HAV-HEp148 or mock-infected, fixed with acetone at 7 days post infection (p.i.), and stained with antiserum directed against HAV (green), and antibodies against HEV capsid (ORF2) protein (red). Negative serum was used as a negative control for antibodies in transfected cells. Merged images also show nuclear labeling with DAPI. Micrographs were taken with a Nikon microscope at a 400× magnification.

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  Figure 4. Western blot analysis of the HEp148 antigen expressed by HAV-HEp148 virus in Huh-7 cells. The cell extracts were separated by 12% PAGE gel, proteins were transferred onto nitrocellulose membranes and probed with anti-HEV capsid rabbit polyclonal antibodies. HAV-HEp148: HAV-HEp148 infected samples. Mock: Mock-infected samples. N: samples were not heated. H: samples were boiled for 10 min. Molecular mass markers are shown in kDa on the left margin. HEp148 protein dimer (30 kDa) andβ-actin (43 kDa) are indicated by arrowheads.

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  Figure 5. Profiles of HAV- and HEV-specific IgG production in humoral immune responses following vaccination. (A) Anti-HAV IgG and (B) anti-HEV IgG were detected by indirect ELISA after immunization of the immunized groups, as indicated. Each serum effective dose is the reciprocal value of the maximum positive dilution. The geometric mean titer (GMT) values are presented at the tops of the graphs. Error bars represent the 95% confidence level (Cl). *, IgG cannot be detected at 1:80 serum dilution. ns, no significant difference.