Thanks to vaccines, we’ve been able to reduce the global impact of measles, smallpox, and other infectious diseases. The World Health Organization (WHO) declared smallpox eradicated in 1980 after a global vaccination campaign and also estimated that the measles vaccine had saved 17.1 million lives from 2000 to 2015. However, for many infectious diseases, such as hepatitis C, there are still no vaccines available to protect against infection or to help the immune system.

Professor Alexander Ploss, professor in the Department of Molecular Biology at Princeton, studies the hepatitis C virus (HCV) that causes hepatitis C, as well as other pathogens that infect the human liver. In 2015, the WHO estimated that 71 million people worldwide had a chronic HCV infection. In order to better understand how HCV impacts our cells, the Ploss lab has developed mouse models that express human genes or contain transplanted human tissues.

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Graphic by: The Center for Disease Control

Challenges to Studying HCV

HCV is a virus that mainly infects humans and chimpanzees by targeting the liver cells of its host. It is transmitted through contact with blood from an infected person. The Center for Disease Control estimated that 75-85% of people infected with HCV develop a chronic infection, which can lead to liver diseases, such as cirrhosis and liver cancer.

While studying potential vaccines, researchers have run into problems due to the high genetic variation of the virus. It has at least 7 genotypes, or versions of the virus with a different genetic makeup, that a vaccine would have to protect against in order to be effective. Within each genotype, an individual virus can also mutate so that its genetic makeup is slightly different. In a 2016 study, researchers found that mutations occurred in more than 90% of the HCV genome. The virus benefits from these changes in its DNA because mutations can help it adapt to its environment and survive within its host.

“If the immune system mounts an immune response to the hepatitis C virus and the virus changes a little bit, then the antibod[ies] or T-cell[s] may not be able to recognize it. The virus basically falls under the radar until another immune system response against the mutated virus is mounted,” Ploss said. This explains how the virus can persist in someone’s body. In addition, the mutations of the virus could allow it to become resistant to drugs that were intended to treat against it.

The virus and its potential vaccines are difficult to study in traditional animal models, such as mice, because the virus does not infect these animals. The virus is specialized to human and chimpanzee liver cells and binds to specific proteins found on these cells’ surfaces, according to a 2014 review paper. Mice do not have the same proteins on the surface of their liver cells, and as a result, do not get infected by the virus. On the other hand, research on humans and chimpanzees is limited due to ethical concerns. In 2011, a panel of experts at the Institute of Medicine recognized the previous and possible future applications of chimpanzee research but concluded that at this time, “most current biomedical research use of chimpanzees is not necessary.”

Successful Small Animal Models for HCV

The virus’ adaptability and the ethical concerns relating to human and chimpanzee research make it important to find small animal models that can reflect HCV’s infection in humans. One avenue of research has been genetically modifying the virus itself to more efficiently infect mice. Another method has been using viruses, such as rodent hepacivirus, that are genetically related to HCV but naturally infect rodents. These viruses could potentially be used as a proxy to understand how HCV infects humans.

The Ploss lab has tried a different approach to study HCV, using genetically humanized mice. These are mice that have had their genetic code modified in order to express human genes, such as the genes for proteins typically found on human liver cells. In a 2011 study, the lab successfully demonstrated that these modified mice could be infected with HCV.

However, one initial drawback of this method is the mice’s natural immune system. While the modified mice now had human-like liver cells, their immune systems were more effective at fighting off the infection than humans’ immune systems. As a result, this limited the ability to study the long-term effects of the virus in the mouse models.

“In a follow-up study, we modified the animal further by interfering with its innate immune system and we were able to show that you could go through the entire viral life cycle,” Ploss said.

The benefits of this model include a closer application of the results to medical treatment. For example, a specific patient’s liver cells can be engrafted into a mouse in order to study how that patient may respond to certain treatments.

In addition to genetically humanized mice, the Ploss lab has used another approach where the mouse is humanized through xenotransplantation. This is a process where human liver tissues are incorporated into the bodies of the mice, in contrast to just incorporating specific genes. Using a method called “engraftment,” human liver cells are injected into a mouse whose liver has been damaged. As the liver repairs, it incorporates the human liver cells into the organ and the result is a mouse with transplanted human tissue.

Parts of the human immune system can also be engrafted into mice models to show how the liver and immune system work together to respond to a HCV infection. “This is a very powerful system to validate the efficacy of human vaccine candidates as you can look directly at the human immune system,” Ploss said.

The benefits of this model include a closer application of the results to medical treatment. For example, a specific patient’s liver cells can be engrafted into a mouse in order to study how that patient may respond to certain treatments. However, the cost and resources for engraftment are expensive, and the possibility remains that the technique is not successful for some mice.

“On the plus side, there’s clear evidence that such humanized mice, in particular those with human livers, are really incredibly good systems to look at long-term persistence of pathogens…including hepatitis C [virus],” Ploss said.

Researchers are continuing to develop these two humanized models to study HCV and other diseases that also affect the liver, such as hepatitis B and dengue virus. In addition, potential vaccines could be screened for their effectiveness and safety in mice before human trial testing occurs. These vaccines would be essential in populations that have a high occurrence of hepatitis C, such as injection drug users.

“We are now in a crisis situation with the opioid epidemic and the excessive use of injected drugs,” Ploss said. This is because use of these drugs involve blood contact and are linked to the spread of HCV and other blood-borne pathogens. “There’s certainly a need of continuing to work on a vaccine for this particular virus.”

With humanized mouse models, researchers can hone in on the mechanisms of the virus and study potential vaccines to achieve this goal.

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Serena Mon