About a Virus

By Jason Socrates Bardi

"The first [precept] was never to accept anything for true which I did not clearly know to be such; that is to say, carefully to avoid precipitancy and prejudice, and to comprise nothing more in my judgement than what was presented to my mind so clearly and distinctly as to exclude all ground of doubt."

———Rene Descartes, Discourse on the Method of Rightly Conducting the Reason, and Seeking Truth in the Sciences, 1638

It is the early 1970s and a nurse named Johnny accidentally sticks himself with a needle while doing a routine blood draw, exposing himself to the hepatitis B virus (HBV). There are at this time no screens for the virus, and aside from admonishing himself for his clumsiness and washing the tiny wound with antibiotic soap, Johnny thinks nothing of it.

A few weeks later, while the virus is replicating prolifically in his liver, he transmits hepatitis B to his wife during intercourse. Unbeknownst to either of them, she is already pregnant. And when their baby is born, he is also infected.

The odds, in this fictionalized case, are that Johnny and his wife both fully recover from their infection. Their immune systems mount a strong defense against the virus and it is eliminated from their systems. The odds do not favor their son, however. His immature immune system will most likely not be able to fight off the hepatitis B virus, and it will establish a chronic, lifelong infection in his liver. Like all other chronically infected people, he will have potentially lethal health problems and an enormously increased risk of developing liver cancer.

In the United States, there are 1,250,000 people living with chronic hepatitis B, according to the Centers for Disease Control and Prevention (CDC). The average American’s lifetime chances of being infected by HBV are about five percent, and about five percent of those infections will become chronic.

Globally, the situation is more dire. Nearly half the world’s population lives in areas where more than eight percent of people are chronically infected by hepatitis B. Anyone living in those areas has a greater than 60 percent chance of being infected by hepatitis B virus at some point in their lifetime. Hepatitis B is the leading cause of liver cancer in the world.

“There are 350 million people in the world who are chronically infected with hepatitis B,” says physician Frank Chisari, who is professor in The Scripps Research Institute (TSRI) Department of Molecular and Experimental Medicine.

“My lifelong dream is to contribute to the termination of hepatitis B infection in all those chronically infected people—that has been driving my research throughout my career.”

Cells Can Cure Themselves

Hearing of goals, rather than of accomplishments, is strange from someone who has recently received more than one lifetime achievement award.

A few weeks ago, Chisari and TSRI Chemistry Professor Chi-Huey Wong were elected to the National Academy of Sciences, becoming two of the now 14 investigators at TSRI who have been admitted to this august body. And just days ago, he was elected to the American Academy of Microbiology, the highest honor the American Society of Microbiology bestows upon its members. Both honors recognize the work Chisari has done on hepatitis since coming to TSRI in 1973.

In the last three decades, by studying infections in patients and in a closely related species, and by developing transgenic models to study the HBV immunobiology and pathogenesis, Chisari and his collaborators have characterized the course of HBV infection in the liver, the immune system’s response to the virus, and the mechanisms whereby a chronic HBV infection can lead to liver cancer. In recent years, he and his collaborators completed a comprehensive analysis of the virological and immunological features of HBV infection using liver biopsies and blood samples they obtained from infected subjects every week for six months after inoculation, describing the course of infection with a level of detail that had never before been attempted.

The insights they gained from these studies and their earlier human and transgenic model experiments have revolutionized the way we think the immune system can control a viral infection. Furthermore, they also demonstrated that there’s a dark side to the antiviral immune response, which can produce progressive tissue damage and even trigger the development of cancer when it goes awry.

Hepatitis is caused by one of several evolutionarily distinct viruses (called A, B, C, D and E) that all target hepatocytes, the parenchymal cells of the liver. Hepatocytes are the tiny chemical factories in the liver that produce most of the proteins present in blood, nurturing all the other organs of the body. They also produce bile, a fluid used for digestion of fat in the diet and for the elimination of waste.

Hepatitis B virus is a circular, double-stranded DNA virus just over 3,000 base pairs long belonging to the Hepadnaviridae family. The infectious particle, or virion, contains this tiny genome and a viral polymerase enzyme in a protein capsid shell surrounded by a lipid coat. HBV infection starts when these virions are introduced into the bloodstream through routes that are similar to those used by the human immunodeficiency virus (HIV)—unprotected sex, contaminated needles, and mother-infant transmission.

Once inside the bloodstream, the virions eventually pass through the liver, and the process of disease starts when HBV infects one or more liver hepatocytes. The initial number of cells infected may be small—just a few—but within six to eight weeks, the virus rapidly replicates and can infect every hepatocyte cell in the liver.

Upon reaching this widespread infection, there is a rapid spike of viral DNA in the bloodstream and the initiation of an immune response, which is evident by the appearance of T cells in the liver and a reduction by several orders of magnitude in the amount of virus in the blood.

Most adults who are infected with hepatitis B suffer an acute infection. After the HBV activity peaks, the body mounts an immune response and the virus disappears. The immune response is so effective that the liver goes from having all its hepatocytes infected to having none of them infected.

Over a decade ago, Chisari suspected that the immune system must be using an unexpected way of clearing the virus from infected liver cells during an HBV infection, because in many cases it was clearing the virus without killing off all the infected cells.

For years, scientists had recognized that one of the principal ways that the immune system deals with a viral infection like HBV is to unleash cytotoxic T lymphocytes (CTL), also called killer T cells, which carry a receptor on their surface that specifically recognizes tell-tale viral markers on the surface of infected cells that indicate these “target” cells should be eliminated. CTLs then kill these infected cells by inducing them to undergo apoptosis, the cellular equivalent of suicide. Until recently, however, this destructive process was thought to be the only antiviral mechanism that CTLs had at their disposal.

But Chisari realized that this couldn’t be the primary mechanism for clearing HBV because killing requires direct contact between a CTL and an infected hepatocyte, and there are simply not enough killer T cells to kill off every hepatocyte in the liver. And even if there were, killing your liver is the last thing your body would want to do, because it is impossible to live without this vital organ.

So, in the early 1990s he started looking into the possibility that CTLs might be able to coax infected cells into curing themselves without being destroyed. The mechanism of this clearance occupied nearly a decade of Chisari’s time, and a few years ago he and his colleague Luca Guidotti, an Associate Professor at TSRI, demonstrated that the immune system can indeed help cure infected cells and how this “intracellular effector” function may actually be the primary way that the immune system controls HBV infection—something that took most people by surprise.

This unprecedented concept established a new paradigm in our understanding of the host-virus relationship, and like many revolutionary ideas, was initially met with surprise and skepticism. In the past several years, however, it was independently confirmed for HBV and it has been extended to a number of other infections as well.

The Immune System Helps Cells that Help Themselves

Basically, in addition to the direct killing of infected hepatocytes, the activated killer T cell will start to produce and secrete chemicals, called cytokines, that bind to surrounding cells that are also infected and that carry specific markers to which the cytokines bind.

Once these cytokines bind to an infected cell, that binding event activates genes within the infected cell that produce proteins that intercept the lifecycle of the pathogen, leading to an internal elimination of the virus without destroying the cell. In hepatitis, the primary cytokine that drives this process—which also occurs in other cells that are infected with other pathogens—is called interferon-gamma (IFN-g).

In hepatitis, Chisari and Staff Scientist Stefan Wieland in his group demonstrated that the first defense mechanism of INF-g involves interrupting the assembly of the viral RNA and associated proteins into infectious capsids.

“Assembly of [capsid] is very rapidly abolished by signals that are delivered by IFN-g,” says Chisari. In recent years, he has worked to categorize the molecules that are produced by HBV-infected liver cells after they are activated by IFN-g.

One candidate class he and his postdoctoral fellow Michael Robek have found to be upregulated in response to the cytokines are proteins of the proteasome, the cell organelle responsible for degrading protein in the cell’s cytoplasm. Chisari has demonstrated that treating HBV-infected cells with inhibitors of these proteasome proteins blocks the antiviral activity of IFN-g.

Clearance is not limited to this one mechanism. A second, slower mechanism that HBV-infected cells engage after they are turned on by IFN-g is to remove all the viral RNA from the cell by destroying a cellular protein that protects the viral RNA. Without the protection of this cellular protein, the viral RNA is susceptible to ribonuclease enzymes in the cytosol, which destroy it.

Nor is the production of cytokines during such an immune response limited to one type of immune cell. Multiple cells of the immune system, including cytotoxic T lymphocytes, helper T cells, natural killer cells, macrophages, and dendritic cells all release such cytokines. And when these cells are activated to produce IFN-g in the liver, the infection will be cleared.

“We think this is what happens in most of the acute infections in adult patients,” says Chisari. “[Cytokine-induced viral clearance] is the dominant effector force for the control of HBV infection.”

Not every intracellular event involved in this clearance is known, and a large portion of Chisari’s laboratory is busy mapping all the details. Nevertheless, the usefulness of purging the infection while preserving the integrity of the cells is obvious when one compares acutely infected patients to the more serious, chronic cases: acute infections are rapidly controlled by the immune system and chronic infections are not.

Chronic Infections

Chronic hepatitis is a “smoldering” infection, in which the body fails to clear the virus from all the infected cells. What the body does do, however, is to unleash its killer T cells in an attempt to clear the infection. However, for reasons that are not entirely clear, the T cell response isn’t vigorous enough to eliminate the infection. Indeed, the number of HBV-specific T cells produced by these patients is 100 to 1000 fold lower than in patients who clear the infection. This leads to a slow, progressive process in which the outnumbered T cells are able to kill some of the infected hepatocytes, but not enough of them to terminate the infection of a large organ like the liver.

However, what they can do is continue killing... and that sets the stage for the rest of the story.

The antiviral effect may be essentially unrecognizable, but the cumulative destruction can lead to a serious condition known as cirrhosis. Cirrhosis is caused by progressive destruction and regeneration of hepatocytes, inflammation, and scarring.

“This terribly compromises the function of the liver and shortens the life of the patient if the disease is severe, and it can progress to cancer,” says Chisari.

The scar tissue impedes blood flow in the liver. The decreased blood flow can then cause a number of other complications to the body, including jaundice, which can be seen as a yellow coloring of the eyes and skin because of the release of bile into the bloodstream. Cirrhosis itself often kills chronically infected patients, and even when it does not it can lead to cancer of the liver.

Cancer is caused by the exposure of the liver to mutagens released by the inflammatory cells and to an increased probability of random point mutations due to the active regeneration spurred on by the continual CTL activity.

"[People with chronic hepatitis] have a 100- to 200-fold increased risk for developing liver cancer," says Chisari. "By comparison, heavy smokers have a 10-fold higher risk of developing lung cancer."

In all, 15 to 25 percent of people who are chronically infected with HBV die from liver disease. In the United States, liver diseases related to HBV infections claim about 5,000 lives a year. Worldwide, this number is 1 million per year.

The situation is particularly dire for children. Nine out of ten infants who are infected with HBV will suffer a chronic infection, whereas only two to five percent of individuals who are infected as adults will become chronically infected. In fact, the CDC estimates that 20 to 30 percent of the 1.25 million Americans who are chronically infected with HBV were infected as children.

A Less Vigorous Defense

HBV infections are more serious in chronically infected patients because their immune systems mount a quantitatively inadequate defense. That’s why infants are at a dramatically increased risk of acquiring a chronic infection if they are infected by their mothers through neonatal transmission when their immune systems are immature. The virus establishes itself in this immunologically immature population and tolerizes them so that they will not make an adequate immune response.

When the viral infection spreads, so does the amount of viral antigen in the blood. The immune system recognizes specific antigens, or epitopes and uses this recognition as the basis of a targeted attack. Chisari’s group first discovered in the late 1980s that people who clear the infection make a polyclonal, vigorous response to many different epitopes from all the viral proteins.

"It’s a profound and effective immune response directed at so many elements that mutational escape [is not possible]," says Chisari.

In chronically infected patients, on the other hand, the response is rather weak. Several years ago, Chisari looked at the immune response in infected humans by comparing virus-specific cytotoxic T cells with characteristics of the disease. When he looked at the blood of chronically infected patients, Chisari saw few cytotoxic T lymphocytes and the ones that were there were specific for very few epitopes. This profound difference, suggests Chisari, is the basis for chronic infection.

"Chronically infected patients develop an ineffective immune response," he says. "If we can find some way to boost this immune response that they are, in fact, capable of making but are not, maybe they would then be cured."

Current treatment for chronic HBV involves taking antivirals, which control but do not eliminate the infection. As soon as the course of medicine is stopped, the HBV rebounds. Chisari and his collaborators are now looking for ways to couple antiviral therapy with immune stimulation.

The General Clinical Research Center

Significantly, Chisari carries out a number of his studies at the General Clinical Research Center (GCRC), which he also directs.

"If there is one thing you can do in this article," Chisari says to me, "bring the GCRC to the attention of the TSRI faculty and postdoctoral fellows."

The GCRC is a TSRI-managed clinical research facility located in the Green Hospital. The center is open to any TSRI-affiliated investigator or postdoctoral fellow who is interested in clinical studies involving humans, and it provides substantial financial assistance for these studies by providing for the care, monitoring, and testing of patients.

"[Investigators] don’t need to seek additional funding to pay for the patient-related costs," says Chisari.

In fact, the GCRC enables TSRI investigators to determine definitively the bearing of their discoveries on human biology. It brings together basic scientists with physicians and nurses who are trained to take care of patients and collect valuable samples. The center has a laboratory, directed by TSRI Associate Professor Daniel Salomon, that processes samples to stabilize them for further study. The center also has a large database to track samples and draws upon the talents of TSRI Professor James Koziol, a biostatistician.

"It has been used very effectively by a number of TSRI investigators and also by a large number of clinical investigators," says Chisari, who is the GCRC director. Associate Professor Bruce Zuraw is associate director. Professor Ernest Beutler, Chair of the Department of Molecular and Experimental Medicine, is the principal investigator on the grant from the National Institutes of Health, which provides the majority of the GCRC’s funding and provides strict guidelines designed to protect the rights and safety of patients in any human trial.

Any researcher who wishes to conduct a study in the GCRC must submit a protocol to the TSRI Human Subjects Committee, which is independent of the GCRC. This committee reviews the safety, ethical, and human-protection aspects of the study. If the protocol passes, it is then reviewed by the GCRC Scientific Advisory Committee, which meets every month or so to evaluate proposed studies for scientific merit.

Chisari notes that this procedure is supportive of scientists while rigorously enforcing National Institutes of Health guidelines. “Investigators who are using the GCRC will be alerted to any risks and provided with education and guidance on how they can be avoided,” he says.

TSRI researchers who are interested in using the facilities establish a collaboration with a clinician who has admitting privileges to the hospital, and the studies are carried out by this licensed physician.

Investigators use the GCRC for a number of purposes, the simplest of all being to safely obtain blood for their investigations. The center tracks blood donors and screens all blood for HIV and hepatitis B and C viruses.

A slightly more involved study might find a TSRI investigator correlating some marker in blood or other bodily fluid with the manifestation of a disease. The investigator might, for instance, ask the doctors and nurses at the GCRC to conduct clinical exams of a study group to monitor patients' progress, at the same time as collecting samples. Clinical exams can range from routine interviews and X-rays to magnetic resonance imaging and spinal taps.

At the highest level, the GCRC provides a way to bring together patients with diseases and conditions for which there is no known cure with investigators who have potential therapies. And during such clinical investigations, the center can monitor the procedures for beneficial or adverse effects, drug levels in the blood, pharmacokinetics, and toxicology.

"It could be an important outlet for chemists who make small molecules they think could be important in the life of a cell or the life of an organism," says Chisari. "A treatment can be administered to the patient in the setting of the GCRC once approval is obtained by [TSRI’s Institutional Review Board] and by the U.S. Food and Drug Administration."




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Professor Francis Chisari hopes to find an answer to chronic hepatitis B infections—a condition affecting millions worldwide. (Photo by Alan McPhee.)















These two views of a section of a transgenic liver replicating the hepatitis B virus dramatically demonstrate how cytotoxic T lymphocytes (CTLs) control infection. The cells are stained for the viral core protein, and the abundant red splotches in the top section are evidence of widespread viral replication before CTLs are added. The bottom panel shows the same section after CTLs have been added. The dramatic disappearance of virus is due to the antiviral effect of interferon gamma produced by the CTLs after they recognize the virus in the liver.