Genetically engineered mice could boost fight against aggressive hepatitis

Article provided by the Department of Molecular Biology

Hepatitis delta virus (HDV) causes the most aggressive form of viral hepatitis in humans, putting at least 20 million people worldwide at risk of developing liver fibrosis, cirrhosis, and liver cancer. Efforts to develop effective treatments against HDV have been hampered by the fact that laboratory mice are not susceptible to the virus. But, in a study published June 27, 2018, in the journal Science Translational Medicine, Alexander Ploss, assistant professor of molecular biology at Princeton University and colleagues describe a genetically engineered mouse that can be persistently infected with HDV.

HDV is a small, RNA-based “satellite” virus that produces just a single protein of its own and therefore requires additional proteins provided by another liver virus, hepatitis B virus (HBV). HDV can infect patients already carrying HBV, or both viruses can infect patients simultaneously. Though infections can be prevented with an anti-HBV vaccine, there are no antiviral therapies available to cure existing HDV infections.

HDV and HBV infect the liver by binding to a protein called NTCP that is present on the surface of liver cells. But the viruses only recognize the version of NTCP present in humans and a few other primates, and therefore can’t infect mice or other small mammals that produce their own versions of NTCP. This has made it difficult to study HBV and HDV infections in the laboratory. Researchers have tried transplanting human liver cells into immunocompromised mice before infecting them with virus, but this approach has produced inconsistent results and is both expensive and time-consuming.

Ploss and colleagues, led by graduate student Benjamin Winer, took a different approach. They generated mice that express the human NTCP protein in their liver cells, allowing these cells to be infected by HBV and HDV.

In these mice, HBV failed to replicate after entering mouse liver cells but HDV was able to establish persistent infection when provided with the HBV proteins it needs to propagate. For example, mice genetically engineered to produce both human NTCP and the entire HBV genome could be infected with HDV for up to 14 days. “To our knowledge, this is the first time the entire HDV life cycle has been recapitulated in a mouse model with inheritable susceptibility to HDV,” Ploss said.

The mice were able to rid themselves of HDV before they developed any liver damage, apparently by mounting an immune response involving antiviral interferon proteins and various white blood cell types, including Natural Killer (NK) cells and T cells. Accordingly, mice expressing human NTCP and the HBV genome, but lacking functional B, T, and NK cells could be infected with HDV for two months or more.

These immunocompromised animals allowed Ploss and colleagues to test the effectiveness of two drugs that are currently being developed as treatments for HDV infection. Both drugs—either alone or in combination—suppressed the levels of HDV in immunocompromised mice after viral infection. But the drugs were not able to completely clear the mice of HDV; viral levels rose again within weeks of stopping treatment.

“This is largely in line with recently reported data from clinical trials, showing the utility of our model for preclinical antiviral drug testing,” Winer said.

“Our model is amenable to genetic manipulations, robust, and can be adopted as a method to rapidly screen for potential treatments,” Ploss added.

Timothy M. Block, president of the Hepatitis B Foundation and its Baruch S. Blumberg Institute who was not involved in the study, said “These systems should be able to provide practical, and presumably economical  tools. Their work is urgently needed, and a desperate community welcomes it. I emphasize that it is often the new methods in science that revolutionize a field such as drug discovery, almost as much as the new drugs themselves.”

The research team included collaborators from Princeton University; Weill Medical College of Cornell University; The Jackson Laboratory; University Medical Center Hamburg-Eppendorf, Hamburg; New York University Medical Center; and North Carolina State University College of Veterinary Medicine.

This study was supported by grants from the National Institutes of Health (R01 AI079031, R01 AI107301, R21AI117213 to Alexander Ploss), a Research Scholar Award from the American Cancer Society (RSG-15-048-01-MPC to Alexander Ploss), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (to Alexander Ploss) and a Graduate fellowship from the Health Grand Challenge from the Global Health Fund of Princeton University (to Benjamin Y. Winer). The NYU Experimental Pathology Immunohistochemistry Core Laboratory is supported in part by the Laura and Isaac Perlmutter Cancer Center Support Grant; NIH/NCI P30CA016087 and the National Institutes of Health S10 Grants NIH/ORIP S10OD01058 and S10OD018338. Benjamin Y. Winer is a recipient of F31 NIH/NRSA Ruth L. Kirschstein Predoctoral awarded from the NIAID. Julie Sellau and Elham Shirvani-Dastgerdi are both recipients of postdoctoral fellowships from the German Research Foundation. Michael V. Wiles was funded by The Jackson Laboratory.

Benjamin Y. Winer, Elham Shirvani-Dastgerdi, Yaron Bram, Julie Sellau, Benjamin E. Low, Heath Johnson, Tiffany Huang, Gabriela Hrebikova, Brigitte Heller, Yael Sharon, Katja Giersch, Sherif Gerges, Kathleen Seneca, Mihai-Alexandru Pais, Angela S. Frankel, Luis Chiriboga, John Cullen, Ronald G. Nahass, Marc Lutgehetmann, Jared Toettcher, Michael V. Wiles, Robert E. Schwartz, and Alexander Ploss. Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for persistent hepatitis delta virus infection. Science Translational Medicine. 2018. DOI: 10.1126/scitranslmed.aap9328

Princeton researchers report new system to study chronic hepatitis B

A co-culture of human hepatocytes
A co-culture of human hepatocytes and non-parenchymal stromal cells self-assembles into liver-like structures that can be infected for extended periods with the hepatitis B virus. Image courtesy of Benjamin Winer and Alexander Ploss, Princeton University Department of Molecular Biology.

By the Department of Molecular Biology

Scientists from Princeton University‘s Department of Molecular Biology have successfully tested a cell-culture system that will allow researchers to perform laboratory-based studies of long-term hepatitis B virus (HBV) infections. The technique, which is described in a paper published July 25 in the journal Nature Communications, will aid the study of viral persistence and accelerate the development of antiviral drugs to cure chronic hepatitis B, a condition that affects over 250 million people worldwide and can cause severe liver disease, including liver cancer.

HBV specifically infects the liver by binding to a protein called sodium-taurocholate co-transporting polypeptide (NTCP) that is only present on the surface of liver cells. Once inside the cell, HBV hijacks its host’s cellular machinery to convert the virus’s DNA into a stable “mini-chromosome.” This allows the virus to establish persistent, long-term infections that can ultimately cause liver fibrosis, cirrhosis and hepatocellular carcinoma. The World Health Organization estimates that 600,000 people die every year as a result of HBV infection.

Researchers have so far failed to develop drugs that can cure chronic HBV infections, partly because they have not been able to study the long-term infection of liver cells grown in the laboratory. Liver cells—also known as hepatocytes—lose their function within days of being isolated from donor livers, preventing researchers from studying anything other than the acute stage of HBV infection. Hepatocytes can be maintained for longer when they are co-cultured with other, supportive cells.

“In previous studies using hepatocytes and cells known as fibroblasts grown on micro-patterned surfaces, HBV infections worked with only a few donors, and infection lasted for no longer than 14-19 days and required the suppression of antiviral cell signaling pathways, which poses problems for studying host-cell responses to HBV and for antiviral drug testing,” said Alexander Ploss, an assistant professor of molecular biology at Princeton University.

Dr. Ploss and colleagues at Princeton and the Hurel Corporation, led by graduate student Benjamin Winer, tested a different system, in which primary human hepatocytes are co-cultured with non-parenchymal stromal cells, which are cells that support the function of the parenchymal hepatocytes in the liver. When plated in collagen-coated labware, the co-cultures self-assemble into liver-like structures. These self-assembling liver-like cultures could be persistently infected with HBV for over 30 days, without the aid of antiviral signaling inhibitors. Moreover, the system worked with hepatocytes grown from a variety of donors and with viruses isolated from chronically-infected patients, which are harder to work with than lab-grown strains of HBV.

“The establishment of a co-culturing system of human primary hepatocytes and non-parenchymal stromal cells for extended HBV infection is a valuable addition to the armamentarium of cell culture model systems for the study of HBV biology and therapeutic development, which has been hampered by a relative lack of efficient infectious cell culture systems,” said T. Jake Liang, a senior investigator at the National Institute of Diabetes and Digestive and Kidney Diseases, who was not involved in the research.

Ploss and colleagues were able to scale down their co-culture infections to volumes as small as a few hundred microliters. This will be important for future high-throughput screens for anti-HBV drug candidates. As a proof-of-principle for these screens, the researchers found that they could block HBV infections in their co-culture system using drugs that either prevent the virus’ entry into hepatocytes or inhibit a viral enzyme that is essential for the virus’ replication. “The platform presented here may aid the identification and testing of novel therapeutic regimens,” Ploss said.

This study is supported in part by grants from the National Institutes of Health (R21AI117213 to Alexander Ploss and R37GM086868 to Tom W. Muir), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (to Alexander Ploss) and funds from Princeton University (to Alexander Ploss). Benjamin Y. Winer is a recipient of F31 NIH/NRSA Ruth L. Kirschstein Predoctoral awarded from the National Institute of Allergy and Infectious Diseases. Felix Wojcik is supported by a German Research Foundation (DFG) postdoctoral fellowship.

The study, “Long-term hepatitis B infection in a scalable hepatic co-culture system,” by Benjamin Y. Winer, Tiffany S. Huang, Eitan Pludwinski, Brigitte Heller, Felix Wojcik, Gabriel E. Lipkowitz, Amit Parekh, Cheul Cho, Anil Shrirao, Tom W. Muir, Eric Novik, Alexander Ploss, was published in Nature Communications on July 25, 2017. DOI: 10.1038/s41467-017-00200-8.

Read more in this commentary in Nature Microbiology.

New mouse model for hepatitis C (Nature)

By Catherine Zandonella, Office of the Dean for Research

Hepatitis C affects about three million people in the U.S. and is a leading cause of chronic liver disease, so creating a vaccine and new treatments is an important public health goal. Most research to date has been done in chimpanzees because they are one of a handful of species that become infected and spread the virus.

Now researchers led by Alexander Ploss of Princeton University and Charles Rice of the Rockefeller University have generated a mouse that can become infected with hepatitis C virus (HCV).  They reported the advance in the Sept 12 issue of the journal Nature. “The entire life cycle of the virus — from infection of liver cells to viral replication, assembly of new particles, and release from the infected cell — occurs in these mice,” said Ploss, who joined the Princeton faculty in July 2013 as assistant professor of molecular biology.

Ploss and his colleagues have been working for some time on the challenge of creating a small animal model for studying the disease. Four years ago, while at the Rockefeller University in New York, Ploss and Rice identified two human proteins, known as CD81 and occludin, that enable mouse cells to become infected with HCV (Nature 2009). In a follow up study Ploss and colleagues showed that a mouse engineered to express these human proteins could become infected with HCV, although the animals could not spread the virus (Nature 2011).

In the present study, which included colleagues at Osaka University and the Scripps Research Institute, the researchers bred the human-protein-containing mice with another strain that had a defective immune system – one that could not easily rid the body of viruses. The resulting mice not only become infected, but could potentially pass the virus to other susceptible mice.

The availability of this new way to study HCV could help researchers discover new vaccines and treatments, although Ploss cautioned that more work needs to be done to refine the model.

The study was supported in part by award number RC1DK087193 from the National Institute of Diabetes and Digestive and Kidney Diseases; R01AI072613, R01AI099284, and R01AI079031 from the National Institute for Allergy and Infectious Disease; R01CA057973 from the National Cancer Institute; and several foundations and contributors, as well as the Infectious Disease Society of America and the American Liver Foundation.

Read the abstract

Marcus Dorner, Joshua A. Horwitz, Bridget M. Donovan, Rachael N. Labitt, William C. Budell, Tamar Friling, Alexander Vogt, Maria Teresa Catanese, Takashi Satoh, Taro Kawai, Shizuo Akira, Mansun Law, Charles Rice & Alexander Ploss. 2013. Completion of the entire hepatitis C virus life cycle in genetically humanized mice. Nature 501, 237–241 (First published online on 31 July 2013)  doi:10.1038/nature12427.