A genetically engineered viral and receptor system for enabling Hepatitis B virus infection in marmoset models

Background

Hepatitis B virus (HBV) infection remains a significant global health concern, leading to chronic liver disease, cirrhosis, and hepatocellular carcinoma in millions of individuals worldwide. Research into HBV pathogenesis, immune responses, and therapeutic interventions has been hampered by the lack of suitable animal models that faithfully recapitulate human infection. While chimpanzees are susceptible to HBV and have historically served as the gold standard for in vivo studies, ethical concerns, high costs, and limited availability have severely restricted their use. As a result, there is a pressing need for alternative small non-human primate models that are genetically and physiologically similar to humans, enabling robust and reproducible studies of HBV biology and the development of new treatments.

Current approaches to modeling HBV infection in animals are limited by species-specific barriers at the level of viral entry, primarily due to differences in the sodium taurocholate co-transporting polypeptide (NTCP) receptor, which mediates HBV attachment and internalization. Most non-human primates, including Old World and New World monkeys, possess NTCP orthologues that are structurally incompatible with HBV, preventing efficient viral binding and productive infection. Even when some degree of viral attachment is observed, subsequent steps such as internalization and replication are often blocked, resulting in non-permissive systems that fail to support the full HBV life cycle. This lack of a genetically tractable, immunocompetent, and physiologically relevant small primate model has impeded progress in understanding HBV host specificity, immune evasion, and the evaluation of antiviral strategies, underscoring the urgent need for innovative solutions to overcome these species barriers.

Technology

This technology enables robust Hepatitis B virus (HBV) infection in the common marmoset, a small non-human primate, by overcoming species-specific barriers at the level of the sodium taurocholate co-transporting polypeptide (NTCP) receptor. Through a combination of phylogenetic, structural, and mutagenesis analyses, researchers identified key amino acid residues in NTCP that determine HBV binding and internalization, pinpointing the marmoset as an ideal model due to its NTCP’s structural similarity to the human version. To further enhance infectivity, a simian-tropic HBV was engineered by replacing the first 48 amino acids of the HBV preS1 region with the corresponding sequence from woolly monkey HBV. This chimeric virus demonstrated significantly improved binding, entry, and replication in both primary and induced pluripotent stem cell-derived marmoset hepatocytes, establishing a functional in vitro system for HBV research.

The differentiation of this solution lies in its precise molecular dissection of host-virus interactions and its strategic engineering of both the viral genome and host receptor. By identifying and modifying specific NTCP residues that control viral entry, and by adapting the viral preS1 region to better match the marmoset’s receptor, the approach overcomes a long-standing species barrier that has limited HBV research to humans and chimpanzees. The use of both primary and stem cell-derived marmoset hepatocytes provides a scalable and genetically tractable platform, while the small size and reproductive advantages of marmosets make them practical for laboratory studies. This technology thus establishes the first robust, immunocompetent small primate model for HBV, enabling advanced studies of viral pathogenesis and the preclinical evaluation of antiviral therapies.

Benefits

• Overcomes species-specific barriers to HBV infection by engineering a simian-tropic HBV adapted to marmoset NTCP receptor.
• Enables robust HBV infection and replication in primary and induced pluripotent stem cell-derived marmoset hepatocytes.
• Provides a genetically tractable, small non-human primate model for studying HBV pathogenesis and testing antiviral therapies.
• Utilizes targeted viral adaptation to enhance infectivity without compromising viral replication competence.
• Facilitates mechanistic insights into HBV entry and host specificity through structural and mutagenesis analyses of NTCP.
• Supports development of immunocompetent animal models for chronic HBV infection research.
• Combines advanced cell culture techniques and stem cell differentiation to create physiologically relevant in vitro infection systems.

Applications

• Preclinical HBV drug testing
• HBV vaccine efficacy studies
• HBV pathogenesis research
• Gene therapy safety evaluation

Additional info

This work details a method to overcome Hepatitis B virus (HBV) species barriers by identifying critical NTCP receptor residues (84-87, 157-165) governing viral entry. A simian-tropic HBV was engineered by replacing its preS1 region (first 48 amino acids) with the corresponding woolly monkey HBV sequence. This modified virus robustly infects primary and iPSC-derived marmoset hepatocytes, establishing a functional in vitro system for HBV studies.