HBV-infected Chimpanzees Research Articles

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

THU-213 - Treatment of Chronically HBV-Infected Chimpanzees with RNA Interference Therapeutic ARC-520 LED to Potent Reduction of Viral MRNA, DNA and Proteins without Observed Drug Resistance

THU-213 - Treatment of Chronically HBV-Infected Chimpanzees with RNA Interference Therapeutic ARC-520 LED to Potent Reduction of Viral MRNA, DNA and Proteins without Observed Drug Resistance

From the Editor’s desk...: June 2015

Selection of the month Targeting Toll-like receptor 7 in chronic HBV infection Toll-like receptor (TLR) signaling has been implicated to play a significant role in the context of cytokine-induced ‘curing' of HBV-infected cells. In previous studies, the oral small molecule TLR7 agonist GS-9620 caused a marked HBsAg reduction in chronically HBV-infected chimpanzees. The present study by Menne et al. evaluated GS-9620 in the HBV Woodchuck animal model demonstrating that short duration treatment with the TLR7 agonist was able to induce a sustained antiviral response which is likely due to the induction of local interferon-alpha and natural killer cell and CD8 + T cell responses as well as activation of B cells. A striking reduction in the incidence of hepatocellular carcinoma was also observed in the treated animals. The safety and efficacy of GS-9620 (attaining a functional cure?) will be further developed in a phase II multi-center study in virally-suppressed subjects with chronic hepatitis B (ClinicalTrials.gov NCT02166047).

MicroRNA-122 Abundance in Hepatocellular Carcinoma and Non-Tumor Liver Tissue from Japanese Patients with Persistent HCV versus HBV Infection

Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify an important role for HCV replication late in the progression to malignancy.

OL-023 Modeling dynamics of five acutely HBV infected chimpanzees

OL-023 Modeling dynamics of five acutely HBV infected chimpanzees

CD8+ T Cell Control of Hepatitis B Virus Replication: Direct Comparison between Cytolytic and Noncytolytic Functions

Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell-mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8(+) T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8(+) T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8(+) T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8(+) T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8(+) T cells produce an array of cytokines, among which IFN-gamma and TNF-alpha are responsible for HBV inactivation in the target cells. Blockade of IFN-gamma and TNF-alpha abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-gamma and rTNF-alpha resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8(+) T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN-gamma and TNF-alpha.

A novel hepatitis B virus surface antigen immunoassay as sensitive as hepatitis B virus nucleic acid testing in detecting early infection

The aim was to considerably enhance the sensitivity of hepatitis B virus (HBV) surface antigen (HBsAg) detection and investigate whether the window period for HBV detection could be reduced. A high-sensitivity chemiluminescent enzyme immunoassay (CLEIA) was developed for quantitative HBsAg detection by a combination of monoclonal antibodies, each one for a specific epitope of HBsAg, and by improving the conjugation technique. The sensitivity of the assay was compared with that of the existing chemiluminescent immunoassay (CLIA). Commercially available seroconversion panels and samples of HBV-infected chimpanzees were tested with the developed prototype to assess whether the window period for HBsAg detection could be reduced to that for DNA detection. Compared to the existing CLIA, the CLEIA prototype detected HBsAg with approximately 230-fold higher sensitivity and showed a reduced window period. HBsAg detection by the CLEIA prototype and HBV DNA detection by polymerase chain reaction (PCR) occurred simultaneously. The mean time for the CLEIA prototype to first detect HBsAg was approximately 17.4 days less than that for the existing systems. Further, CLEIA prototype enabled HBsAg detection even in anti-HBs-positive seroconversion samples. In the inoculated chimpanzees the HBsAg and HBV DNA became detectable simultaneously and concentrations increased in parallel, whereas HBsAg remained detectable longer than HBV DNA in the declining phase of viremia. The CLEIA prototype yielded results comparable with those of HBV DNA PCR. This novel high-sensitivity assay may be useful for early detection of HBV infection and monitoring patients with a history of infection.

Complete-genome analysis of hepatitis B virus from wild-born chimpanzees in central Africa demonstrates a strain-specific geographical cluster

In order to determine whether geographical or species clustering accounts for the distribution of hepatitis B virus (HBV) in subspecies of chimpanzees in Africa, four complete chimpanzee HBV (ChHBV) genome sequences were obtained from eight hepatitis B surface antigen-positive wild-born chimpanzees from Cameroon, Republic of Congo and Gabon. The serological profiles of these chimpanzees corresponded to the acute or chronic highly replicative phase of HBV infection, as confirmed by high plasma HBV loads. Analysis of the sequence alignment of 256 aa (S region) from the eight HBV-infected chimpanzees allowed us to determine the HBV amino acid patterns specific to each chimpanzee subspecies and to their geographical origin. Phylogenetic analysis of both the S region and the complete genome confirmed this distinctive clustering of eight novel ChHBV strains within Pan troglodytes. The strong phylogenetic associations of ChHBV sequences with both chimpanzee subspecies and their geographical origin were therefore confirmed.

Hepatitis viruses in non‐human primates

Previous epidemiological studies of rural human populations in Gabon reveal a high prevalence of human hepatitis A, B, C and D viruses. In order to investigate the prevalence of the blood-born hepatitis viruses in apes and monkeys living in the same area, we performed an epidemiological survey of HBV, HCV and HDV in wild-born non-human primates. We tested 441 wild-born non-human primates from Gabon and Congo and 132 imported monkeys for the presence of serological markers of HBV, HCV and HDV infections. None of Cercopithecidae monkeys were reactive against HBV/HDV and HCV. In contrast, 29.2% of wild-born great apes (154 chimpanzees and 14 gorillas) were positive for HBV serological markers. Nine chimpanzees were in the replicative phase of HBV infection. None of these HBV infected chimpanzees exhibited symptoms or significant changes in serum clinical chemistry related to HBV infection. The negativity to HCV-related viruses and the negativity of the Cercopithecidae species tested against HBV/HDV do not allow us to definitively rule out the presence of an animal counterpart of human hepatitis viruses in non-human primates.

Genomic analysis of the host response to hepatitis B virus infection.

Previous studies in hepatitis B virus (HBV)-infected humans and chimpanzees suggest that control of HBV infection involves the cells, effector functions, and molecular mediators of the immune response. The objective of the current study was to identify, in the liver of acutely HBV-infected chimpanzees, the spectrum of virus-induced and immune response-related genes that regulate the infection. The results demonstrate that HBV does not induce any genes during entry and expansion, suggesting it is a stealth virus early in the infection. In contrast, a large number of T cell-derived IFN-gamma-regulated genes are induced in the liver during viral clearance, reflecting the impact of an adaptive T cell response that inhibits viral replication and kills infected cells, thereby terminating the infection.

Titration of the infectivity of hepatitis D virus in chimpanzees.

The infectivity of hepatitis D virus (HDV) was evaluated by intravenous inoculation of chimpanzees. HDV was present in the inoculum at a titer of 10(11) chimpanzee infectious doses (CID). In contrast, the titer of hepatitis B virus (HBV) in the same inoculum was 10(6) CID. All HBV-infected chimpanzees inoculated with less than or equal to 10(-11) dilutions of the HDV-positive plasma were superinfected; an animal receiving a 10(-12) dilution did not develop markers of HDV replication in serum or liver. All HDV-infected chimpanzees had marked elevations of serum alanine aminotransferase activities. The incubation period from exposure to development of hepatitis was inversely related to the dose of HDV inoculum, although the severity and duration of hepatitis were independent of it. All animals recovered and rapidly developed antibody to hepatitis D antigen.

Unrelatedness of factor VIII-derived non-A/non-B hepatitis and hepatitis B virus.

A DNA hybridization assay was used to detect hepatitis B virus (HBV)-specific DNA sequences in extracted sera obtained from chimpanzees infected with HBV, hepatitis A virus (HAV), and a factor VIII-derived non-A/non-B (NANB) agent. The results did not reveal any HBV-DNA homology with sera obtained from animals infected with HAV or factor VIII-derived NANB. Sera obtained from two HBV-infected chimpanzees demonstrated that HBV-specific DNA could be detected during the acute phase of the disease. In addition, an HBV-specific DNA-dependent DNA polymerase assay did not demonstrate any statistically significant activity in 12 of 12 NANB acute-phase specimens or in 6 of 6 NANB chronic-phase specimens. These results suggest that the factor VIII-derived NANB agent is unrelated to HBV.