Worldwide, human hepatitis B virus (HBV) infection causes liver-related death in more than 600 thousand people annually (www.who.int/mediacentre/factsheets/fs204/en/). Approximately 400 million people are persistently infected with HBV with dramatically increased risk of developing liver cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Thus, over half of the 700 thousand annual liver cancer cases are caused by HBV. However, current therapy of chronic HBV is suboptimal and expensive and, in most treated patients, does not lead to a cure. Treatment options include nucleos(t)ide analogs tenofovir and entecavir, which are highly effective in lowering viremia level, but only rarely lead to sustained clearance or long-term suppression of viral load. Another option is treatment with pegylated interferon-alpha (PEG-IFN-α), which, in a small number of cases, has been associated with late viral clearance, thus suggesting that induction of relevant immune responses might lead to a cure for persistent HBV. Taken together, there is a great need for studies of alternative and less-expensive treatment options, and it would be highly relevant to have a readily available immunocompetent animal model for human HBV.
Studies in nonhuman primates have been of major importance for experimental studies of human hepatitis viruses, including analyses of hepatitis A virus in New World monkeys (tamarins and owl monkeys), HBV, hepatitis D virus, and hepatitis C virus (HCV) in hominoids (chimpanzees), and hepatitis E virus in Old World monkeys (cynomolgus and rhesus macaques).[2-5] Chimpanzees are the only animal model for studies of human HBV and HCV infections and related innate and adaptive host immune responses. Chimpanzees have been available primarily for research in the United States, where several animal facilities can perform studies in a suitable environment. However, a report from the Institute of Medicine (released December 15, 2011), evaluating the role of this model in biomedical research, has limited or eliminated most experimental research in chimpanzees funded by the National Institutes of Health, a major funding agency for such research. The use of chimpanzees for persistent HBV was already rather limited because the chronicity rate in experimental infections is low, and only a small number of animals have been available. Cost has been another limiting factor. However, a recent study by Lanford et al. showed how the chimpanzee model could be used to determine the effect of molecules affecting pathways of the immune system; it was demonstrated that a Toll-like receptor 7 agonist could effectively lower HBV viral load partly by inducing antigen-specific T- and natural killer cell responses.
New World monkeys (e.g., tamarins, marmosets, and owl and spider monkeys commonly used in biomedical research) do not appear to be susceptible to human HBV. An HBV variant was identified in Woolly monkeys (endangered species) and could lead to acute, but not persistent, experimental infection in Spider monkeys.[9, 10] Among Old World monkeys, there is evidence of occult human HBV infection of subgenotype A2 in baboons with detection of the HBV DNA genome at low titers in serum, but not the HBV surface antigen (HBsAg). However, HBV could be transmitted to naïve baboons and HBV DNA could be detected for at least 6 months. It remains to be determined whether this will be a relevant model for studying chronic HBV infection. Cynomolgus and rhesus macaques are frequently used in biomedical research, but it has been unclear whether human HBV could be transmitted to these animals. The possibility of using rhesus monkeys for experimental human HBV infection was examined early after the discovery of HBV. Thus, in 1972, London et al. reported that HBV could be transmitted to rhesus monkeys (Macaca mulatta) and serially passaged to naïve monkeys, but this could not be confirmed subsequently. In 2002, Gheit et al. reported that Barbary apes (M. sylvanus) could be acutely infected with detection of HBV DNA and HBsAg after intrahepatic transfection with cloned human HBV. Overall, however, Old World monkeys have, so far, not been relevant as animal models in experimental HBV research, and, with the exception of chimpanzees, animal models with robust persistent infection remain unavailable.
Human HBV or HBV variants closely related to human HBV have been isolated from greater (orangutan, gorilla, and chimpanzees) and lesser apes (gibbons), as reviewed previously. In this issue of HEPATOLOGY, an interesting study by Dupinay et al. describes the discovery of human HBV in cynomolgus monkeys of the species M. fascicularis on Mauritius Island. This species of Old World monkeys, also called crab-eating or long-tailed macaque, was originally brought from Java to Mauritius Island, located in the Indian Ocean east of Madagascar, off the South East coast of Africa (Fig. 1). Amazingly, the investigators found that approximately 25% of the tested monkeys were positive for HBV DNA in serum; HBsAg expression was detected in hepatocytes. However, many of the animals had what appeared to be occult low-titer HBV infections. More important, persistence of HBV DNA in serum was demonstrated in 6 animals followed for up to 8 months, thus providing evidence of persistent HBV infection. The HBV DNA titers in these particular animals were similar to titers reported in HBV chronically infected chimpanzees. Furthermore, the virus was transmissible to naïve monkeys (M. sylvanus), with the appearance of HBV DNA and HBsAg markers and evidence of acute hepatitis; transmission to M. fascicularis monkeys was apparently not attempted. Finally, sequence analysis of HBV genomes of viruses recovered from M. fascicularis revealed that animals were infected with human HBV genotype D; genotype D is a common HBV genotype found worldwide. The recovered viruses did have potentially important mutations, compared with HBV currently circulating in humans, which could perhaps explain permissiveness in Old World monkeys.
The major obstacle for translating this novel finding of persistent human HBV infection in macaques into an available animal model is the establishment of persistent experimental infections. This could involve neonatal infections and/or immunosuppression. However, such approaches did not result in persistent experimental infections of Woolly monkey HBV in spider monkeys. It would be relevant to test different Old World monkey species for susceptibility to this human HBV variant. This research could involve the generation of molecular clones representing the exact HBV variant identified in cynomolgus monkeys on Mauritius Island, or cloned human HBV with the insertion of specific identified mutations, such as a unique substitution identified in the pre-S1 domain of the L glycoprotein involved with receptor binding. This research might define the genetic elements responsible for susceptibility of this human HBV to Old World monkeys and might contribute to our understanding of HBV evolution. Here, it is of interest that an HBV receptor called NTCP (sodium taurocholate cotransporting polypeptide) with interaction with the pre-S1 domain has recently been identified, and human and cynomolgus monkey NTCP have differences that could require the adaptation of human HBV to be able to infect monkeys.
Although natural HBV infection in adult humans is cleared in most cases, associated with vigorous host T-cell responses and liver inflammation, it has not yet been possible to develop treatment strategies that efficiently eliminate HBV infection. The current approaches with nucleos(t)ide analogs tenofovir and entecavir and/or PEG-IFN-α do not directly target the nonreplicating and stable form of nuclear HBV DNA, covalently closed circular DNA (cccDNA). HBV cccDNA is the template for viral RNA transcription, and to cure HBV, this form of HBV DNA must eventually be eliminated. Therefore, efforts are ongoing to develop drugs that prevent RNA transcription, for example, by applying HBV cccDNA-targeted enzymes, such as zinc-finger nucleases, inducing double-strand DNA breaks. Because these DNA breaks are not efficiently repaired, the thought is that this will lead to inactivation of viral genes and prevent HBV replication.[18, 19] Preliminary investigations could be performed with animal variants of HBV. However, the study of these novel HBV drugs will eventually require animal models of human hepatitis B for testing of safety and efficacy.
In conclusion, given the lack of suitable and readily available animal models for persistent human HBV infection, it would be a major breakthrough if chronic HBV infection in Old World monkeys can be developed, based on the unique adapted HBV strain identified by Dupinay et al. in M. fascicularis. Old World monkeys are immunologically closely related to humans, and HBV-infected monkeys could thus be used to examine drugs designed to stimulate the immunity of chronically infected individuals or to directly target the cccDNA template in attempts to cure chronic HBV. However, it remains to be determined whether the generation of persistent experimental infections can be achieved in Old World monkeys. Thus, additional studies will be required to confirm the utility of this model in experimental studies of chronic human HBV infection.
Jens Bukh, M.D.1,2
Robert E. Lanford, Ph.D.3
Robert H. Purcell, M.D.4
1Copenhagen Hepatitis C Program (CO-HEP)
Department of Infectious Diseases and Clinical Research Centre
Copenhagen University Hospital, Hvidovre
2Department of International Health, Immunology and Microbiology
Faculty of Health and Medical Sciences
University of Copenhagen
3Department of Virology and Immunology
Texas Biomedical Research Institute
Southwest National Primate Research Center
San Antonio, TX
4Laboratory of Infectious Diseases
National Institute of Allergy and Infectious Diseases
National Institutes of Health