Scientists have created the first transgenic nonhuman primate model susceptible to hepatitis B virus (HBV), potentially enabling preclinical testing of curative therapies. The study used PiggyBac transposon technology to insert a liver-specific human sodium-taurocholate cotransporting polypeptide (hNTCP) expression cassette into rhesus macaque embryos.
Two macaque offspring were born from surrogate mothers after zygote modification. Both express hNTCP in the liver. Hepatocytes isolated from the macaques were shown to be susceptible to HBV infection, confirming functional expression of the viral receptor.
HBV is limited by strict species specificity, which has slowed the development of immunocompetent animal models. Prior work showed that vector-mediated expression of hNTCP in macaques permits infection. This study expands on that approach by producing a transgenic model capable of persistent liver-specific expression.
The findings offer a new platform for testing curative therapies for HBV and studying host-virus interactions in a preclinical setting.
In an email Q&A, Ben Burwitz, PhD, associate professor at Oregon Health & Science University’s Oregon National Primate Research Center, discussed the significance of the new model, the advantages of germline hNTCP expression over vector-based methods, and the technical process behind creating the transgenic macaques. He also outlined how the model could support antiviral development while acknowledging its current limitations as a developing research tool.
Contagion: Can you explain the significance of creating a transgenic rhesus macaque model for HBV research, and how does it address the challenges of studying HBV in nonhuman primates?
Burwitz: “One of the major barriers to the study of curative HBV therapies is the lack of a translational, immunocompetent model of HBV infection. HBV has a narrow host range, infecting only humans and chimpanzees. Chimpanzee research was halted in 2012 by the NIH, and this left a huge gap in the field. Very early on we showed that rhesus macaque hepatocytes could be infected with HBV in vitro if you artificially expressed the HBV receptor on their surface. This was the first data showing that the only block to HBV infection in rhesus macaques was the viral entry step. From there, we created viral vectors (e.g., Ad5 and AAV) that expressed the HBV receptor and could be given to rhesus macaques in vivo. These animals became susceptible to HBV infection, which drove our desire to make transgenic rhesus macaques expressing the HBV receptor naturally from the germline.”
Contagion: What specific advantages does the liver-specific expression of the human NTCP receptor provide for studying HBV infection in this model, and how does it replicate the natural susceptibility of human hepatocytes?
Burwitz: “Rhesus macaques expressing human NTCP (HBV receptor) from the germline have several advantages over creating HBV susceptible rhesus macaques through viral vectors:
1) Viral vectors are immunogenic
2) Not all hepatocytes are transduced by viral vectors, limiting HBV spreading
3) The viral vector genomes and hNTCP expression can be lost during cell division. Therefore, an optimal rhesus macaque HBV model would have transgenic, hepatocyte-specific hNTCP expression to overcome these challenges.”
Burwitz: “In terms of replicating the HBV susceptibility of human hepatocytes, all measures of HBV replication that we have looked at thus far in vitro in rhesus macaque hepatocytes expressing human NTCP look identical to human infection.”
Contagion: Could you expand on the methodology used in this study, particularly the use of PiggyBac transposon technology to create the transgenic macaques? What are the key steps involved in generating these animals?
Burwitz: “PiggyBac is a transposon. Transposons, also known as ‘jumping genes’, were first discovered by Barbara McClintock in the 1940s while studying maize. There are two components of the system. First, the transposon is the genetic element that can be moved from one genomic location to another (in our case, moving human NTCP from a circular DNA template to the rhesus macaque genome). Second, there is an enzyme called a transposase that facilitates the moving of the transposon. Therefore, we needed to inject single-cell rhesus macaque embryos (called zygotes) with both our transposase and our transposon.”
What You Need To Know
The transgenic rhesus macaque model enables more accurate study of HBV infection and therapy development by overcoming the limitations of previous animal models.
Germline expression of the human NTCP receptor in macaques provides better liver-specific HBV susceptibility, reducing immunogenicity and allowing for more consistent infection.
The model offers a new platform for studying HBV transmission, chronicity, and testing antiviral therapies, though it still requires further validation for human-like HBV responses.
Burwitz: “To be honest, the genetic engineering components of the project were far more straightforward than the Assisted Reproductive Technologies components. The Assisted Reproductive Technologies core at the Oregon National Primate Research Center capitalizes on decades of rhesus macaque reproductive and developmental science (the repro division was the first and only division upon the opening of the Oregon National Primate Research Center) to finally bring to bear our ability to make transgenic rhesus macaques. This core is second to none at in vitro fertilization, microinjection of single cell embryos, and biopsies and cryopreservation of transgenic embryos. My lab does none of this, we were just along for the amazing ride and had a good idea on how to use the technology.”
Contagion: What implications do these transgenic macaques have for the development of HBV therapies, and how do you envision this model being used in future drug development and therapeutic testing?
Burwitz: “There has been a successful HBV vaccine since the early 1980s, yet many parts of the world continue to have high rates of HBV transmission. The vast majority of people living with chronic HBV infection (i.e., an infection that their body was unable to clear naturally) were infected at the time of birth. Therefore, the most relevant way we can use our model is to study the critical aspects of vertical transmission (mother-to-child at birth) that lead to chronicity, as well as to study new drugs that can eliminate HBV from the liver at different times in life. Humans living with chronic HBV live for decades, and therefore we have a lot of time to study these models if we can show that they also maintain chronic HBV infection indefinitely.”
Contagion: Are there any challenges or limitations in using this model, and how might researchers overcome them as they move forward with HBV-related studies?
Burwitz: “In the end, no matter what manipulations we make to rhesus macaques, they are still not natural carriers of HBV infection. Therefore, there may still be barriers to the use of this model that we cannot anticipate. For instance, there may be immune responses to HBV in rhesus macaques that are not present in humans, simply because HBV has co-evolved with humans for over 10,000 years. We are excited about our new model, but there is still (and always seems to be) a lot of work left ahead of us.”
This new transgenic rhesus macaque model is the first immunocompetent nonhuman primate system naturally susceptible to hepatitis B virus. While it still requires further validation, it provides a useful tool for studying chronic infection, vertical transmission, and testing potential curative therapies in a biologically relevant setting.
Reference
Rust LN, Wettengel JM, Biswas S, Ryu J, Piekarski N, Yusova S, Lutz SS, Naldiga S, Hinrichs BJ, Sullivan MN, Lo JO, Protzer U, Smedley JV, Sacha JB, Hanna CB, Bimber BN, Hennebold JD, Burwitz BJ. Liver-specific transgenic expression of human NTCP in rhesus macaques confers HBV susceptibility on primary hepatocytes. Proc Natl Acad Sci U S A. 2025 Feb 18;122(7):e2413771122. doi: 10.1073/pnas.2413771122. Epub 2025 Feb 12. PMID: 39937851; PMCID: PMC11848295
