A hepatitis B virus transgenic mouse model with a conditional, recombinant, episomal genome

Abstract

Background & aims: Development of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell.

Methods: To solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase.

Results: We demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response.

Conclusions: The HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression.

Lay summary: Hepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.

Keywords: AAV, adeno-associated virus; Ad, adenovirus; Cre/LoxP; HBx, HBV X-protein; Hepatitis B virus; ORF, open reading frame; PFUs, plaque-forming units; Transgenic mouse; cccDNA; cccDNA, covalently closed circular DNA; pgRNA, pregenomic RNA; rcccDNA, recombinant cccDNA.

Graphical abstract

Fig. 1

A novel transgenic HBV mouse was designed with LoxP sites to facilitate excision of HBV genome. (A) A LoxP-flanked HBV genome was cloned with downstream HBV-overlength sequences maintaining open reading frames and supplying native polyadenylation for HBV protein expression. The LoxP-HBV cassette is excised by Cre recombinase to form a recombinant HBV cccDNA molecule. (B) The HBV1.1X cassette allows for complete expression of the pgRNA transcript and complete HBx protein to be expressed during integration. (C) The amino-acid sequence for the inserted LoxP site (red) and SacII site (underlined) at the HBx/overlength junction is depicted. cccDNA, covalently closed circular DNA; HBx, HBV X-protein; pgRNA, pregenomic RNA.

Fig. 2

HBV1.1X mice exhibit HBc expression in a small number of hepatocytes. (A) Hydrodynamic injection of pCMV-NLS Cre into heterozygous HBV1.1X mice, demonstrated no difference in HBsAg levels before and after Cre plasmid injection (n = 4). (B) Immunofluorescent staining in male mice showed a small percentage of HBc-positive hepatocytes, the frequency did not increase with Cre injection. (C) HBc expression could be detected in the renal tubule cells in the kidneys of mice, consistent with previous HBV transgenic mice. (D) Homozygous HBV1.1X mice exhibited high HBsAg expression levels at baseline (n = 4). (E) Male and female homozygous mice, aged 3 months, were assessed for the presence of HBsAg IgG antibodies, with both below background detection limit by ELISA assay. Bars represent mean and SEM; ∗significant, p <0.05 using parametric, 2-tailed t tests.

Fig. 3

Southern blot demonstrates rcccDNA formation from integrated HBV1.1X genome after Cre recombinase introduction. A representative diagram of HBV1.1X genome digestion for Southern blot is provided (A), where integrated HBV can be released with MfeI digestion, or alternatively released after introduction of Ad-Cre. The probe is localised over the PreS region of the HBV genome. (B) Southern blot revealed a small band released with the introduction of Cre, while the control remains unstained. Upon MfeI digestion, the integrated HBV DNA is released, while the rcccDNA form is now shifted upward owing to linearisation (~3.2 kb). Ad, adenovirus; rcccDNA, recombinant covalently closed circular DNA.

Fig. 4

HBsAg expression can be eliminated in HBV1.1X mice using transgenic Cre recombinase tools. HBV1.1X mice were crossed with Alb-Cre transgenic mice to induce liver-specific excision of the HBV genome. (A) Male HBV1.1X/Alb-Cre heterozygous for both alleles had undetectable serum HBsAg compared to control mice without Alb-Cre (n = 3). (B) Immunofluorescent staining for HBc protein in the livers of both mice showed less HBc staining of HBV1.1X/Alb-Cre mice. (C) HBV1.1X mice were crossed with ROSA26-CreERT2 mice to create ROSA26-CreERT2/HBV1.1X mice. Tamoxifen induction of CreERT2 resulted in complete loss of HBsAg expression 1 week after induction cycle ended (n = 8). (D) HBc staining in the liver of mice untreated and treated with tamoxifen revealed continued presence of HBc positive cells 3 months after tamoxifen induction period. Bars represent mean and SEM; ∗significant, p <0.05 using parametric, 2-tailed t tests. Alb, albumin.

Fig. 5

Both HBsAg and HBc expression can be eliminated from the liver by administering Ad-CMV-Cre into mice. Heterozygous HBV1.1X male mice were obtained and injected with Ad-CMV-Cre (n = 4) or Ad-CMV-GFP (n = 3), or not injected (n = 3) with virus as a control. (A) Serum HBsAg levels were followed post-injection in the mice. All mice continued to express HBsAg over the first week post injection, but by Day 21, Ad-Cre mice had lost expression. Each point represents mean and SEM; ∗significant, p <0.05 using parametric, 2-tailed t tests. (B–D) Staining for HBc antigen by immunofluorescence at Day 34 post injection, observed the continued presence of HBc-positive cells in non-injected and Ad-CMV-GFP injected mice, while Ad-CMV-Cre mice lacked observed HBc staining, beyond rare hepatocytes (1 positive cell in a lobe). Ad, adenovirus.

Fig. 6

Mechanism of gene expression regulation in HBV1.1X transgenic mice with Cre recombinase. A model is presented of how HBV gene expression may be regulated by Cre recombinase in HBV1.1X transgenic mice. The model predicts at baseline, the ROSA26 locus helps drive expression of the HBV1.1X genome in a small fraction of hepatocytes, with the rest of the genomes transcriptionally silent. After Cre recombinase is introduced, the HBV genome is excised and converted into rcccDNA, which can continue expression, but expression levels are reduced without genomic context. The cellular environment may compensate by helping to activate gene expression of rcccDNA, such as seen after adenoviral introduction. Overall, HBsAg levels fall near the detection limit given the low level of HBV-expressing hepatocytes with 1 or 2 genomes expressing HBV antigens, much lower than the amount of copies delivered by hydrodynamic delivery of plasmids or viral vector approaches. rcccDNA, recombinant covalently closed circular DNA.