Adeno-Associated Virus-Mediated Gene Transfer Leads to Persistent Hepatitis B Virus Replication in Mice Expressing HLA-A2 and HLA-DR1 Molecules

Abstract

H epatitis B virus (HBV) infection is a major health problem.There are more than 350 million chronic carriers worldwide, and they are at high risk of developing liver cirrhosis and hepatocellular carcinoma (1). Chronic HBV infection is the result of impaired HBV-specific immune responses such that the infected hepatocytes cannot be eliminated or cured efficiently, but many of the associated issues remain unclear (2, 3).Due to the paucity of in vitro and in vivo models for HBV infection, HBV-transgenic mice are the most widely used model. These mice have the viral genome integrated into the chromosome and produce infectious HBV particles or viral antigens in the liver; however, the main limitation of HBV-transgenic mouse models is that they are immunologically tolerant to viral antigens (4, 5). Various routes have been exploited to introduce the HBV genome into the hepatocytes of adult mice. One is to introduce a replication-competent HBV genome into the mouse liver by hydrodynamic injection (HDI) through the tail vein (6); although HBV replicates in the mouse liver, the virus is rapidly cleared by immune responses against HBV proteins (7). Recently, Huang and colleagues used HDI to create a nontransgenic model of persistent HBV replication (8). The virus persisted in 40% of mice or was eliminated according to the genetic background. These mice rapidly develop anti-hepatitis B virus core (HBc) antibody, which is the first serological marker of acute HBV infection in humans. An alternative method uses adenoviral vectors to transfer 1.3 copies of the HBV genome into immunocompetent mice (9, 10), and acute or chronic HBV infection was obtained depending on the dose of adenoviral vector injected.Here, we describe an alternative murine model for the study of HBV persistence based on the liver-targeted transduction of adeno-associated virus serotype 2/8 (AAV2/8). We produced an AAV2/8 construct carrying a replication-competent HBV DNA genome and by intravenous injection established a model of HBV persistence in humanized HLA-A2/DR1 immunocompetent mice. Hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), and HBV DNA persisted for at least 1 year in sera of all AAV2/8-injected mice, and viral replication intermediates and transcripts were detected in their livers. HBcAg was expressed in 60% of hepatocytes without significant inflammation in the liver. The persistence of infection was associated with the presence of regulatory T cells (Tregs) in the liver. This mouse model of HBV persistence recapitulates viral and histological characteristics of human chronic HBV infection in the immunetolerant stage of the disease (11,12).In HLA-A2/DR1 mice, cellular immune responses were completely restricted to HLA molecules. Antibody, T-helper, and cytotoxic-T-lymphocyte responses to vaccination with recombinant HBsAg or HBsAg-expressing DNA were similar to those in vaccinated humans (13,14) or in HBV-infected individuals (15). Therefore, this AAV2/8-HBV-transduced HLA-A2/DR1 murine model may be useful fo...

Adenoviral Delivery of Recombinant Hepatitis B Virus Expressing Foreign Antigenic Epitopes for Immunotherapy of Persistent Viral Infection

“…It is noteworthy that DNA vaccination with pCMV-S2S, a plasmid encoding the middle and small envelope proteins of HBV (34, 35), did not affect HBV persistence (Fig. 7B), indicating a prominent immune tolerance developed in the mouse liver (36).…”

“…We used a recently developed mouse model of rAAV8-HBV persistence in which prominent immune tolerance was established by a defect in the HBV-specific functional T-cell response and a high number of regulatory T cells in the liver (36). This tolerance was fundamentally different from that of HBV transgenic mice, which produce HBV proteins as self-antigens in the liver from birth.…”

Mouse Models of Hepatitis B Virus Pathogenesis

“…However, low levels of cccDNA have been found in the livers of these animals after crossing them with hepatocyte nuclear factor 1 a-null mice, suggesting that the impairment of cccDNA synthesis in mouse hepatocytes is not absolute (Raney et al 2001). Alternative methods to deliver replication-competent HBV genomes to mouse hepatocytes, such as hydrodynamic injection of naked plasmid DNA and adenovirus-or adeno-associated virus (AAV)-mediated transfer, did not lead to detectable cccDNA formation, confirming the existence of a species restriction on the production of cccDNA (Sprinzl et al 2001;Yang et al 2002Yang et al , 2013Huang et al 2011;Dion et al 2013).…”

“…On a final note, immuno-competent mouse models based on the use of recombinant AAV carrying replication-competent HBV genomes have been recently reported to display varying levels of hepatic HBV gene expression and replication, which are capable of persisting for many months (Huang et al 2011;Dion et al 2013;Yang et al 2013). In one study, the persistence of hepatic HBVexpression and replication did not cause liver inflammation and liver pathology because of the induction of regulatory T cells or other mechanisms of peripheral tolerance (Dion et al 2013).…”

“…In one study, the persistence of hepatic HBVexpression and replication did not cause liver inflammation and liver pathology because of the induction of regulatory T cells or other mechanisms of peripheral tolerance (Dion et al 2013). In another study, the authors reported that AAV-infected animals develop HCC by 12 -16 months of age (Huang et al 2011), but it remains to be determined whether such a phenotype is linked to an HBV-specific adaptive immune response.…”