Novel Woodchuck Hepatitis Virus (WHV) transgene mouse models show sex-dependent WHV replicative activity and development of spontaneous immune responses to WHV proteins

Abstract

The woodchuck model is an informative model for studies on hepadnaviral infection. In this study, woodchuck hepatitis virus (WHV) transgenic (Tg) mouse models based on C57BL/6 mice were established to study the pathogenesis associated with hepadnaviral infection. Two lineages of WHV Tg mice, harboring the WHV wild-type genome (lineage 1217) and a mutated WHV genome lacking surface antigen (lineage 1281), were generated. WHV replication intermediates were detected by Southern blotting. DNA vaccines against WHV proteins were applied by intramuscular injection. WHV-specific immune responses were analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The presence of WHV transgenes resulted in liver-specific but sex- and age-dependent WHV replication in Tg mice. Pathological changes in the liver, including hepatocellular dysplasia, were observed in aged Tg mice, suggesting that the presence of WHV transgenes may lead to liver diseases. Interestingly, Tg mice of lineage 1281 spontaneously developed T- and B-cell responses to WHV core protein (WHcAg). DNA vaccination induced specific immune responses to WHV proteins in WHV Tg mice, indicating a tolerance break. The magnitude of the induced WHcAg-specific immune responses was dependent on the effectiveness of different DNA vaccines and was associated with a decrease in WHV loads in mice. In conclusion, sex- and age-dependent viral replication, development of autoimmune responses to viral antigens, pathological changes in the liver in WHV Tg mice, and the possibility of breaking immune tolerance to WHV transgenes will allow future studies on pathogenesis related to hepadnaviral infection and therapeutic vaccines.

FIG 1

Generation and identification of WHV Tg mice. (A) Structure of plasmid puc119WHV-1.3. Shown is a schematic representation of 1.3-fold-overlength WHV constructs for the generation of WHV Tg mice. En, enhancer; Poly A, polyadenylation signal. The black ovals indicate the promoter sequences of WHV ORFs. The three stop codons introduced into the S ORF are underlined. (B) Transgenic copy number and number of integration sites of WHV transgenes. A total of 10 μg of mouse genomic DNA was digested with EcoRI and then subjected to Southern blot analysis with linear full-length WHV copy standards.

FIG 2

Organ-, age-, and sex-dependent WHV gene expression and replication in Tg mice. (A to C) WHV replicative intermediates in the liver, spleen, and kidney of mice of lineages 1281 (A to C) and 1217 (A) were analyzed by Southern blotting. (D) WHV viral loads in the serum of mice of lineage 1217 were determined by real-time PCR. (E) Intrahepatic WHV mRNAs in mice of lineage 1281 were determined by real-time RT-PCR. PWHs, primary woodchuck hepatocytes; M, male; F, female; L, liver; K, kidney; S, spleen; ET, exposure time; GE, genome equivalents.

FIG 3

Histological investigation of liver tissues. Liver tissue samples were fixed in formalin and embedded in paraffin. Sections were cut and processed for histological and microscopic examination according to standard methods. Representative photographs are shown for the histological presentation of normal and dysplastic changes of liver sections. The characteristically histological presentation of HCC found in an aged mouse of lineage 1217 is also shown. The arrows show inflammatory cell infiltrates in the hematoxylin- and eosin-stained section.

FIG 4

WHcAg-specific humoral and cellular immune responses in WHV Tg mice. (A to D) Anti-WHc antibodies in the serum of WHV Tg mice of lineages 1281 (A and B) and 1217 (C and D) were detected by ELISA. (E) WHcAg-specific CTLs in the splenocytes of Tg mice of lineage 1281 were analyzed by flow cytometry. S/N, sample/negative control ratio. Mice T88 and T164 of lineage 1281 are two representative examples positive and negative for WHcAg-specific CTL responses, respectively.

FIG 5

Immunization-induced humoral and cellular responses to WHcAg in WHV Tg mice. WHV Tg mice of lineage 1217 (wild-type WHV) and normal C57BL/6 mice were immunized three times with pWHcIm (A to D) or pCGWHc (E to H). Serum anti-WHc antibodies (A and E), WHcAg-specific CTLs after in vitro expansion in the presence of specific peptides for 7 days (B and C) or ex vivo stimulation for 4 h (F and G), and serum WHV DNA loads (D and H) were determined by ELISA, flow cytometry, and real-time PCR, respectively. PBS, phosphate-buffered saline; n.s., not significant.