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Rous-Whipple Award Lecture |
From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California
The host-virus interactions that determine the outcome of viral infections have fascinated me ever since medical school. My first opportunity to actively pursue this interest came when I was lucky enough to be part of a team at the National Institutes of Health that transmitted the hepatitis B virus (HBV) to chimpanzees.1 Thus began a longstanding interest in the immunobiology of persistent human viral infections, especially those that infect the liver. I am greatly honored that this work has been recognized by the Rous-Whipple Award this year, giving me this opportunity to describe our current state of understanding of these infections.
The Infection
As indicated in Figure 1
, HBV is a
noncytopathic, enveloped, double-stranded DNA virus that causes acute
and chronic hepatitis and hepatocellular carcinoma (HCC). HBV is
transmitted sexually, parenterally, and from mother to infant at birth,
like human immunodeficiency virus (HIV). Most perinatal HBV infections
become persistent, presumably due to the failure to mount an effective
immune response. In contrast, most adult onset HBV infections resolve,
presumably due to the polyclonal, multispecific humoral and cellular
immune response that the patients produce against the viral
proteins.2
More than 2 billion people alive today have
been infected by HBV, and more than 350 million people are chronically
infected, and these individuals have a 100-fold increased risk of
developing HCC.3
Accordingly, HBV causes approximately 1
million deaths each year worldwide. In the United States alone, nearly
300,000 new infections occur annually, more than 1 million people are
chronically infected, and more than 5000 of them die each year from
cirrhosis and HCC.
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The Virus and Its Life Cycle
The infectious 42-nm virion is an enveloped nucleocapsid
containing the viral polymerase bound to an incomplete, open circular
DNA genome, which consists of a full length 3.2-kb minus strand and an
incomplete plus strand (Figure 2)
. The
mechanism of viral entry is not known, although attachment is thought
to be mediated by the interaction of the preS(1) region of the large
envelope polypeptide with one or more currently undefined hepatocyte
receptor(s).7-12
After entry and presumptive uncoating
(Figure 3)
, viral plus strand DNA
synthesis is completed and the nucleocapsid particle delivers the viral
genome to the nucleus. Recent evidence from our laboratory indicates
that cytoplasmic nucleocapsid particles do not enter the
nucleus.13
This suggests that the capsids are arrested at
the nuclear membrane and release the viral genome into the nucleus
where DNA repair enzymes process and join the viral minus and plus
strands, yielding the 3.2-kb covalently closed circular (ccc) DNA
molecule that serves as the viral transcriptional
template.14
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controlled by 4 independent promoters and a single common
polyadenylation signal, yielding 4 extensively overlapping viral RNAs
(3.5, 2.4, 2.1, and 0.7 kb in length) that are exported into the
cytoplasm where the viral proteins are translated and viral particle
assembly and genome replication occurs. The 3.5-kb transcript produces
the polymerase, core, and precore proteins and serves as the pregenomic
RNA template that is reverse transcribed as the first step in viral
genome replication (see below). The polymerase protein contains a viral
RNA packaging signal and performs multiple enzymatic functions
including reverse transcriptase, DNA polymerase, and RNase H activity
that are essential for viral replication.15
The core
protein, known as hepatitis B core antigen (HBcAg), rapidly forms
homodimers that self-assemble into capsid particles16
in
the cytoplasm and the nucleus of the hepatocyte. The intranuclear
capsid particles appear to be empty, and their role in the viral life
cycle is not understood.17
The cytoplasmic capsids are
true nucleocapsids; ie, they contain the pregenomic viral RNA and the
polymerase protein, and it is within these particles that viral genome
replication occurs. HBV replication involves reverse transcription of the RNA pregenome to produce minus strand DNA. The minus strands then serve as the template for viral plus strand DNA synthesis, resulting in an encapsidated double-stranded open circular DNA genome that either recycles back to the nucleus to amplify the pool of cccDNA or becomes enveloped by the viral envelope proteins, buds into the endoplasmic reticulum (ER), and is secreted via the Golgi apparatus into the blood, where it can spread to other hepatocytes.14,18-25 The nascent precore protein contains the entire core protein plus a leader sequence that directs it to the ER, where it undergoes limited proteolysis and is secreted into the plasma as hepatitis B e antigen (HBeAg). Its role in the viral life cycle is poorly understood, although it may have tolerogenic properties that would favor viral persistence,26,27 and it appears to be able to modulate nucleocapsid stability, and therefore replication, by forming heterodimers with the core protein.28-30
The 2.4- and 2.1-kb transcripts produce the large, middle, and small
envelope proteins. The 3 HBV envelope proteins share common carboxy
termini and display progressive amino terminal extensions. The small
envelope protein contains the hepatitis B surface antigen (HBsAg). The
middle envelope protein contains the entire small envelope protein plus
an amino-terminal extension in which is located the pre-S(2) antigen.
The large envelope protein contains an additional N-terminal extension
that defines the pre-S(1) antigen. The envelope proteins are
cotranslationally inserted into the ER membrane where they aggregate,
bud into the ER lumen, and are secreted by the cell, either as 22-nm
subviral envelope particles or as 42-nm infectious virions if they have
enveloped the viral nucleocapsids before budding. When the large
envelope protein is overexpressed, it forms long branching filamentous
particles that accumulate in the ER and are not secreted, causing the
ER to become hyperplastic. This gives the cell a "ground glass"
appearance histologically31
and makes the cell
hypersensitive to the cytopathic effects of interferon 
(IFN-),32
as will be described below. The 0.7-kb
transcript produces the X protein, which has transcriptional
transactivating potential33
and has been shown to be
required to initiate infection in a woodchuck hepatitis virus
infectivity model.34
It has also been shown that the X
protein can function as a cofactor for the development of
hepatocellular carcinoma when it is overexpressed as a transgene in a
mouse strain that has an unusually high baseline incidence of
HCC.35
The T Cell Response to HBV
As summarized in Figure 4
, my
laboratory has been interested in defining the host-virus interactions
that determine the outcome of HBV infection, especially the role of the
cytotoxic T lymphocyte (CTL) response, because antiviral T cells are
believed to play a major role in control of HBV infection by virtue of
their capacity to identify and kill virus-infected cells. We took two
complementary approaches to this objective. First, to determine whether
the CTL response plays a role in viral clearance and/or disease
pathogenesis, we characterized and compared the quality and magnitude
of the CTL response to HBV in patients who resolve the infection and
patients who become persistently infected. Second, we established and
defined the pathogenetic and antiviral functions of HBV-specific CTLs
that we injected into HBV transgenic mice that express the
corresponding viral proteins in their liver.
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that all of the viral proteins are
targeted by class I restricted CD8-positive CTL in patients with acute
hepatitis who ultimately clear the virus, whereas the CTL response is
relatively weak and more narrowly restricted in persistently infected
patients.2
The association between a strong, multifaceted
T cell response with acute hepatitis and viral clearance suggests a
causal relationship between these events. However, it does not prove
causality, nor does it reveal the mechanisms responsible for viral
clearance or disease pathogenesis during HBV infection. This requires
interventional studies that are not possible in humans but that can be
pursued in appropriate animal models (see below).
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Viral Clearance by Destruction of Infected Cells
A large body of evidence suggests that the vigor of the CTL response to HBV is the principal determinant of viral clearance in infected patients.2 It is widely believed that the CTL response clears viral infections by killing infected cells. Although this is probably true for many viruses, it may not be possible for the CTL response to eradicate infections in which the number of infected cells outnumber the antigen-specific T cells by several orders of magnitude. This appears to be the case for at least some of the hepatitis viruses, especially HBV, which can infect virtually all of the hepatocytes in the liver.
The reasons for this are as follows. For CTL to kill a target cell in vivo, the CTL must be induced by encountering antigen in an immunologically stimulatory microenvironment (eg, lymphoid tissue), enter the circulation and eventually the infected tissue, stop, migrate past any tissue barriers (eg, endothelial cells, basement membranes), reach the infected cells, recognize their cognate antigens, kill the infected cells one at a time, move on to find the next target cell, and kill it before the CTL are triggered to die themselves by the process of activation-induced cell death.40 Thus, viral elimination by CTL-mediated killing is not nearly as efficient in vivo as one might assume from the ease with which CTL can kill target cells in vitro, where the only functions needed are antigen recognition and delivery of one or more death signals, and where the target cells are crowded around the CTL and have been highly selected for their exquisite sensitivity to those signals.
In addition, consider that there are approximately 1011 infectible hepatocytes in the human liver and that all of them can be infected by HBV.41 Consider, also, that there are approximately 1012 lymphocytes in the entire body and that the HBV-specific CTL precursor frequency at the height of the CTL response in a strongly reactive patient with acute hepatitis is rarely greater than 10-4; usually it is much lower.42 Hence, there should be no more than 108 HBV-specific CTL in the entire body at any point in time. Therefore, if every HBV-specific CTL in the entire body were to enter the liver at the same time (highly unlikely), and if most of the hepatocytes were infected (very common), there would be 1 specific CTL in the liver for every 1000 infected hepatocytes. Even considering the temporally extended dynamics of the CTL response, under these conditions it is difficult to imagine that viral clearance can be achieved simply by the destruction of all of the infected cells. Even if only 10% of the hepatocytes were infected, the effector-to-target cell ratio in the liver would be 1:100, well beyond the cytolytic capacity of a single T cell. Even at the 1% infection level the ratio would be 1:10, which would require every CTL to find and kill 10 infected hepatocytes that are widely separated from each other by a 100-fold excess of uninfected cells to clear the infection. Although efficient cytolysis can sometimes be achieved in vitro at this effector-to-target cell ratio, it is very unusual; in vitro, the CTL are surrounded by target cells, all of which express their cognate antigen, so their task is much simpler than it is in vivo. Thus, as the number of infected cells decreases, the challenge of finding them increases, further contributing to the difficulty of clearing an infection by killing alone. Furthermore, if clearance were due entirely to the destruction of infected cells, one might reasonably expect the incidence of fatal acute hepatitis to be much higher than it is during HBV infection. For all of these reasons, we suggest that although the liver disease in viral hepatitis is certainly due to the destructive potential of the CTL response, viral clearance probably requires additional CTL functions besides their ability to kill infected cells.
CTL-Induced Liver Disease in HBV Transgenic Mice
Proof that an MHC-restricted cytolytic immune response to viral
encoded antigens expressed at the surface of the hepatocyte plays an
important role in viral clearance and, in the pathogenesis of
HBV-induced liver disease, required the development of a readily
manipulable small animal model in an immunologically well defined
species. Because HBV does not infect such animals, we decided to
produce HBV transgenic mice to achieve this objective.43
Initially, we showed that mice that express HBV envelope proteins in
their hepatocytes develop acute viral hepatitis after adoptive transfer
of CD8-positive, MHC class I restricted, envelope (HBsAg)-specific CTL
lines and clones.44,45
Furthermore, we showed that the
disease progresses through an orderly series of clearly definable
steps. The first step occurs within 1 hour of CTL administration, with
antigen recognition by the CTL and delivery of a signal that results in
the death of CTL-associated hepatocytes by apoptosis (Figure 6a)
, causing the formation of acidophilic
Councilman bodies (apoptotic hepatocytes) that are characteristic of
acute viral hepatitis in man.46
The second step begins
between 4 and 12 hours after CTL injection, when many host-derived
inflammatory cells are recruited into the vicinity of the CTLs (Figure 6b)
, resulting in the formation of necroinflammatory foci and the
destruction of additional hepatocytes. Importantly, the
effector-to-target cell ratio in vivo in these experiments
is very low (~1/30–1/100), so only a small fraction of the
hepatocytes are killed by the combined effects of the CTLs plus the
ensuing inflammatory response.47-49
However, if many
HBsAg-positive ground glass hepatocytes are present in the liver, a
third process ensues in which the animal may die from fulminant
hepatitis because ground glass cells are exquisitely sensitive to
destruction by IFN-, and this cytokine is actively secreted by CTLs
after antigen recognition.32
The striking similarities
between the immunopathological and histopathological features of this
model and acute viral hepatitis in man suggest that similar events may
contribute to the pathogenesis of the human disease as well.
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Interestingly, the CTLs do not induce a second episode of
hepatitis if they are readministered to the mice less than 4 weeks
after the first CTL injection.50
While studying the basis
for this observation we learned that, in addition to killing
some of the hepatocytes, the CTL also down-regulated the expression and
replication of HBV by all of the hepatocytes in the liver without
killing them.49
In subsequent experiments using transgenic
mice that contain the complete viral genome as recipients of
CTLs,47
we demonstrated that all of the viral RNAs, their
translation products (HBcAg, HBeAg, and HBsAg), nucleocapsids, and
episomal replicative DNA intermediates, are susceptible to this
remarkable antiviral effect (Figure 7)
.
Two lines of evidence suggest that this antiviral process is
noncytopathic and that it is mediated by inflammatory cytokines. First,
it can be blocked by the prior administration of antibodies to IFN-
or tumor necrosis factor 
(TNF-) without reducing the severity of
the liver disease. Second, it can be reproduced by perforin-deficient
CTLs and Fas ligand-deficient CTLs that do not cause hepatitis in these
animals, but it cannot be reproduced by IFN--deficient CTLs, even
though they cause hepatitis (Figure 8)
.47
In recent studies
(McClary H, Koch R, Chisari FV, Guidotti LG: J Virol, in press) we
reproduced the antiviral effect by injecting wild-type CTLs into HBV
transgenic mice whose IFN- and TNF- receptor (p55) had been
knocked out, indicating that it is the IFN- the CTLs produce
themselves that inhibits HBV replication, not the IFN- produced by
the inflammatory cells that the CTLs recruit. In other experiments we
showed that the regulatory effect of the CTLs becomes independent of
IFN- during the first 24 hours after CTL administration, suggesting
that the cytokines activate a regulatory cascade that ultimately
delivers the final inhibitory signal to the hepatocyte. Furthermore,
using HBV transgenic mice whose inducible nitric oxide synthase (iNOS)
allele has been knocked out (Guidotti L, McClary H, Moorhead J, Chisari
FV; J Exp Med, in press), we showed that nitric oxide inhibits
HBV replication and that it is a downstream mediator of the antiviral
effect of IFN-.
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. According to this
scenario, viral clearance depends on the development of a vigorous
intrahepatic immune response, with the severity of the associated liver
disease being determined by the number of infected hepatocytes and the
balance between the cytopathic and antiviral regulatory effects of the
intrahepatic inflammatory cells. If the T cell response is strong and
the number of infected cells is low, viral clearance should occur
rapidly and efficiently, with little evidence of liver disease, simply
by killing the infected cells. Even a strong T cell response may not be
able to clear a massive viral infection, however, unless the curative
limb of the response is called into play. In the absence of this
component, the cytopathic function of the immune response may not be
able to eliminate all of the infected cells, leading to persistent
infection and chronic liver disease. On the other hand, if the T cell
response is quantitatively suboptimal, the virus could persist even if
the appropriate antiviral cytokines are produced, since, at
insufficient levels, they will suppress viral gene expression without
eliminating it, thereby making the virus less visible to the immune
system.
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that are interrupted by the cytokines,
and to identify the cytokine-regulated hepatocellular genes that
inhibit HBV gene expression and replication in this system. At the
moment, we know that the cytokines inhibit viral gene expression by a
posttranscriptional mechanism that destabilizes the viral mRNA in the
nucleus of the cell,51
and we have shown that this is
associated with the cytokine-induced proteolytic cleavage of a cellular
HBV RNA-binding protein that stabilizes the viral RNA under baseline
conditions.52,53
We also know that the cytokines inhibit
viral replication by posttranslationally eliminating viral nucleocapsid
particles, within which replication occurs, from the cytoplasm of the
hepatocyte (Wieland S, Guidotti LG, Chisari FV, J Virol, in
press). We do not know, however, whether the cytokines prevent
nucleocapsid assembly or accelerate nucleocapsid degradation, and
studies are underway to determine which of these mechanisms is
operative.
It is important to note that other intrahepatic stimuli, in addition to
CTLs, can initiate these antiviral events, as long as they trigger the
production of antiviral cytokines in the liver. For example, we showed
that HBsAg-specific class II restricted T cell clones can trigger the
same effects by secreting IFN- in the liver when they recognize
antigen presented by hepatic macrophages.54
We also showed
that systemically administered recombinant interleukin-12 (IL-12) can
inhibit HBV replication by inducing the production of IFN- in the
liver.55
Since IFN- is a powerful macrophage activator,
it is possible that its antiviral effect is mediated by TNF-
produced by activated macrophages. In keeping with this notion, we have
shown that recombinant TNF- also inhibits HBV gene expression in
these mice,56
as does interleukin-2 (IL-2), the regulatory
effects of which are mediated by TNF-57
by
posttranscriptionally accelerating the degradation of HBV
mRNA58
as described above. One might predict from the
foregoing that coinfection or superinfection of the HBV-infected liver
by other pathogens could facilitate the clearance of HBV if the other
pathogens induce the local production of the antiviral cytokines
to which HBV is susceptible. Precisely these events have been shown to
occur in the HBV transgenic mice during lymphocytic choriomeningitis
virus,48
adenovirus, and cytomegalovirus59
infection of the liver, reminiscent of isolated case reports suggesting
that superinfection by hepatitis A virus or hepatitis C virus (HCV) is
sometimes associated with clearance of HBV in chronically infected
patients.60,61
The potential of these antiviral pathways
to be exploited for therapeutic purposes is self-evident.
These results suggest that a strong intrahepatic immune response to HBV during a natural viral infection can suppress viral gene expression and replication and, if the supercoiled viral genome is also eliminated by this process, perhaps even cure infected hepatocytes of the virus without killing them. Importantly, this illustrates that the infected cells can become active participants in the antiviral response by responding to cytokine-induced signals and activating specific intracellular pathways that interrupt the viral life cycle. This is a significant departure from current dogma, which views the infected cell merely as a victim of the viral infection. As a corollary of this hypothesis, the data suggest that a weak immune response or incomplete viral inactivation could contribute to viral persistence and chronic liver disease by reducing the expression of viral antigens sufficiently for the infected cells to escape immune recognition.
Noncytopathic Clearance of HBV during Acute Infection in Chimpanzees
The transgenic mouse studies suffer from two important
limitations. First, the mice are not infected by HBV, so the
observations are limited to biochemical aspects of viral replication
and gene expression, and they fail to examine the effects of the
cytokines in the context of viral entry and spread. Second, for unknown
reasons, the mice do not produce the episomal covalently closed
circular HBV DNA (cccDNA) species that serves as the viral
transcriptional and that must be eliminated for viral clearance to
occur. Accordingly, confirmation of the hypothesis that viral clearance
during HBV infection reflects noncytopathic processes initiated by the
immune response requires analysis of these events in the liver of
HBV-infected animals that produce cccDNA. Chimpanzees are ideal for
these purposes because they are infectible by HBV1
and
they are known to mount a cellular immune response to HBV similar to
that observed in acutely infected humans.62
Therefore, we
infected two healthy young adult HBV-seronegative chimpanzees with HBV
and compared the kinetics of viral clearance and the kinetics of liver
disease by obtaining liver biopsies every week for 24 weeks after
inoculation. Both chimpanzees developed typical cases of acute,
self-limited viral hepatitis. Importantly, the viral DNA (including the
cccDNA) disappeared from the serum and the liver from both animals
several weeks before the peak of the disease (Figure 10)
. Interestingly, the disappearance of
viral DNA from the liver coincided with the induction of IFN-, which
preceded the major influx of T cells. These results demonstrate that
the destruction of the hepatocytes could not be responsible for the
reduction of viral DNA. They also suggest that the early noncytopathic
control of HBV replication in these animals, which was associated with
the induction of IFN, may be due to the influx into the liver of
IFN--producing non-T cells, perhaps natural killer cells,
that can recognize infected cells in the absence of MHC class I
expression. In this scenario, the IFN- produced by the influx of
natural killer cells could perform dual functions by inhibiting HBV
replication, thereby reducing the number of infected hepatocytes, and
by up-regulating MHC class I, thereby permitting the remaining infected
cells to present viral antigens to the antigen-specific CTLs that
complete the process by killing the residual targets and causing the
disease recognized as viral hepatitis. This tissue-sparing,
noncytolytic antiviral process can be viewed as a host survival
strategy to control infections of vital organs that would otherwise be
destroyed if the only way to eliminate the infections were to kill all
of the infected cells. Interestingly, by down-regulating viral antigen
expression, the same process could also function as a viral evasion
strategy and contribute to viral persistence. Indeed, both scenarios
might be correct, and they could even be operative at the same time in
the same individual in view of the recent discovery that traces of HBV
can persist for several decades after complete serological and clinical
recovery from acute viral hepatitis.19
If so, this
noncytolytic process may be strongly favored during evolution and
possibly extend to other pathogens, since it provides a strong survival
advantage for both virus and host.
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is induced in the liver by antigen-nonspecific stimuli. These results
indicate that, like HBV, LCMV is also susceptible to intracellular
inactivation by cytokine-induced antiviral mechanisms that are
operative in the hepatocyte. However, LCMV is not cleared from
intrahepatic nonparenchymal cells or splenocytes, indicating that,
unlike the hepatocyte, these cells do not produce the factors required
to inactivate LCMV. Antiviral mechanisms like these may have evolved to
maintain the functional integrity of vital organs, such as the liver,
in the face of massive infection. Mechanisms of HBV Persistence
For a noncytopathic virus to persist, it must either overwhelm (or
not induce) an effective antiviral immune response, or it must be able
to evade it (Figure 11)
. All of these
scenarios could be operative in patients chronically infected by HBV.
Indeed, neonatal tolerance is probably responsible for both the lack of
an antiviral immune response and viral persistence after mother-infant
transmission, which is the most common antecedent of persistent HBV
infection worldwide.2
The immunological basis for viral
persistence during adult onset infection is not well understood.
Perhaps the simplest explanation is quantitative, based on the kinetics
of infection relative to the induction of a CTL response during the
early days of an infection. For example, viral persistence would be
predicted if the size of the inoculum or the replication rate of an
incoming virus exceeds the kinetics of the immune response, such that
the effector-to-target cell ratio favors the virus even when the CTL
response is fully in place. However, since the CTL response is much
less vigorous in chronically infected patients than it is during acute
infection,64-67
other factors must be involved as well.
Reasonable candidates are the induction of peripheral tolerance or
exhaustion of the T cell response by the high viral load that
characterizes most persistently infected patients. Additionally,
virus-specific CTLs, which might otherwise become activated by antigen
recognition in the immunostimulatory context of secondary lymphoid
organs, might be inactivated if antigen is presented in the absence of
costimulatory signals in the liver.
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Certain viruses (eg, poxviruses, adenoviruses, herpesviruses) have
evolved the ability to inhibit antigen presentation or to suppress or
neutralize antiviral cytokines as survival
strategies.70,71
Thus far, however, there is no evidence
that these processes are operative during HBV infection. As discussed
earlier, however, inflammatory cytokines, especially IFN-, suppress
HBV gene expression and replication, which could contribute to viral
persistence if the effect is incomplete or if the virus infects
an whose immune response to HBV does not produce this cytokine.
Indeed, the cytopathic potential of the CTL in these individuals would
trigger hepatitis, whereas the failure of their CTL to produce the
appropriate cytokines might contribute to viral persistence. Analysis
of cytokine expression in the liver of patients with chronic HBV
infection should clarify this interesting hypothesis.
The role of viral escape mutations as a cause of viral persistence has attracted considerable interest in recent years. Many conditions must be fulfilled, however, for a mutant virus to be selected by CTL-mediated immune pressure.2 Perhaps the most important condition is the occurrence of a strong CTL response that is focused on a single viral epitope. This scenario would favor the outgrowth of variant viruses because they would not otherwise be visible to the immune system. This type of CTL response is unusual, however, during HBV infection, when the CTL response is typically vigorous and multispecific during acute hepatitis and weak or undetectable during chronic hepatitis.42,65,66,72 Accordingly, mutational inactivation of CTL epitopes is extremely uncommon during chronic HBV infection.73
Nonetheless, strong, narrowly focused CTL responses do occur occasionally in these patients,20 and in this setting viral escape mutations can occur.74 Vigorous oligoclonal expansions of T cells have been described in other persistent viral infections, especially HIV75 and Epstein-Barr virus.76 Even in these infections, however, viral mutations that affect recognition of an epitope by some CTL clones do not automatically affect all CTL clones specific for the same epitope, because different T cell clones can bind different residues in the same epitope.77,78 Although CTL escape can confer a strong survival advantage, it is important to emphasize that selection of escape variants in all of these infections occurs in the setting of a pre-existing persistent infection; ie, viral persistence probably leads to selection of escape variants, not the reverse.
The situation may be somewhat different in early chronic HIV infection where the CTL response is characteristically very strong, yet is unable to clear the virus.75,76 The incredibly high rate of HIV production and the exceptionally high mutation rate of this virus may cause so many different viruses to be generated each day that they exceed the capacity of the immune system to respond effectively simply on a numerical basis.78 In this regard, the ostensibly vigorous immune response HIV appears to be unable to compete with the capacity of the virus to generate mutants. Mutational inactivation of CTL epitopes might thus play an earlier and more important role in the establishment of viral persistence for HIV than for HBV.79 It is important to emphasize, however, that the overwhelming rates of viral replication and spread relative to the ability of the immune system to produce enough CTLs to reach and destroy all of the infected cells, plus the immunosuppressive effects of the virus itself, are more important than viral mutation for the development of persistent infection.
The situation may be different again during chronic HCV infection where an extensive quasispecies of viral variants can coexist with a multispecific CTL response80-83 that is intermediate in strength between the response of patients chronically infected by HBV and those infected by HIV. Unlike HBV and HIV, where the viral load is high, the viral titer is very low during chronic HCV infection,84 so viral persistence cannot easily be blamed on an overwhelming infection in this instance. Therefore, escape mutants may play a greater role in the primary establishment of HCV persistence than is likely for HBV. Importantly, CTL escape has been observed in a chronically HCV-infected chimpanzee.85 However, the extent to which the mutation contributed to or was a consequence of persistent HCV infection in this case remains to be determined.
In view of the multispecificity of the CTL response to most persistent viruses, current data favor the notion that negative selection of CTL escape mutants is most likely to occur after a persistent infection is already established. In this setting, viral mutations could solidify the chronic nature of the infection and perhaps even make it irreversible. Whether such mutations can also serve as the primary cause of viral persistence in the context of a multispecific T cell response remains to be proven.
Immune Pathogenesis of Hepatocellular Carcinoma
The mechanisms responsible for malignant transformation in chronic HBV infection are not well defined, and both viral and host factors have been implicated in the process. On the one hand, all cases of HCC occur after many years of chronic hepatitis which could, theoretically, provide the mitogenic and mutagenic environment to precipitate random genetic and chromosomal damage and lead to the development of HCC. On the other hand, most tumors contain clonally integrated HBV DNA and microdeletions in the flanking cellular DNA which could, theoretically, deregulate cellular growth control mechanisms.86 Furthermore, the HBV X gene product has been shown to transactivate cellular genes associated with cellular growth control87-89 and inhibit p53 gene function in vitro90 , suggesting that deregulated X gene expression from integrated fragments of subviral DNA could play a role in hepatocarcinogenesis.91 Similarly, C-terminally truncated viral envelope proteins expressed from integrated subviral DNA may have transactivating activity92,93 and could, potentially, contribute to carcinogenesis in chronic HBV infection. Like retroviruses, however, HBV integration does not occur in resting hepatocytes; so if HBV integration plays a role in hepatocarcinogenesis, antecedent events must occur that trigger hepatocellular turnover.
In an effort to clarify the carcinogenic potential of chronic hepatitis, we previously showed that transgenic mice that produce hepatotoxic quantities of the HBV large envelope polypeptide43,94-96 display hepatocellular injury, regenerative hyperplasia, chronic inflammation, Kupffer cell hyperplasia, oxygen radical production, glutathione depletion, oxidative DNA damage, transcriptional deregulation and aneuploidy that inexorably progresses to HCC.95-100 While those studies demonstrated that HBV can cause hepatocellular carcinoma in the absence of insertional mutagenesis, X gene expression or genotoxic chemicals, they did not prove that chronic immune-mediated hepatitis was a procarcinogenic stimulus in itself. To determine whether hepatocellular carcinoma can be triggered by a chronic, virus-specific immune response, we developed a model of chronic immune-mediated liver disease using transgenic mice that express nontoxic concentrations of the HBV envelope proteins in the hepatocyte. Similar to human chronic HBsAg carriers, these mice are immunologically tolerant to HBsAg and they develop no evidence of liver disease except ground glass hepatocytes during their lifetime. The mice were thymectomized, lethally irradiated and reconstituted with bone marrow and spleen cells either from syngeneic nontransgenic donors that were previously immunized with a recombinant vaccinia virus that expresses HBsAg and displayed HBsAg-specific CTLs and anti-HBs antibodies, or from immunologically tolerant transgenic donors. All results were compared with unmanipulated, age- and sex-matched transgenic mice. Only mice that were reconstituted with immunologically primed nontransgenic immune systems developed acute hepatitis and cleared HBsAg from their serum. Subsequently, all of these mice developed chronic hepatitis and HCC.101
The pathogenetic importance of immune-mediated hepatocellular injury in
hepatocarcinogenesis in this study is strengthened by the fact that
hepatocellular carcinoma occurs in the context of necrosis,
inflammation and regeneration (cirrhosis) in several human liver
diseases other than hepatitis B, including chronic hepatitis
C,102
alcoholism,103
hemochromatosis,104
glycogen storage
disease,105
-1-antitrypsin
deficiency,106,107
and primary biliary
cirrhosis.108
Irrespective of etiology or pathogenesis,
therefore, it would appear that chronic liver cell injury is a
premalignant condition that initiates a cascade of events characterized
by increased rates of cellular DNA synthesis and production of
endogenous mutagens coupled with compromised cellular detoxification
and repair functions. If these processes are sustained for a
sufficiently long period of time, they would be expected to cause the
multiple genetic and chromosomal changes necessary to trigger the
development of hepatocellular carcinoma (Figure 12)
.
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Summary and Conclusions
The diversity of clinical syndromes and disease manifestations associated with HBV infection strongly suggests that the clinical outcome of this infection is determined by the quality and vigor of the antiviral immune response produced by the infected host. Antibodies to antigens expressed at the surface of virus particles can provide protection from initial infection and can prevent viral spread from cell to cell once infection is established. Antibody-mediated immune complex formation can contribute to extrahepatic syndromes in these patients and may even play a role in liver disease if they can bind to the surface of antigen-positive hepatocytes and recruit Fc receptor-positive killer cells, thereby mediating antibody dependent cellular cytotoxicity. CD4-positive helper T cells serve a critically important regulatory function by secreting a variety of cytokines that can facilitate B cell maturation, expansion, and antibody secretion or that foster the development of a strong CTL response. CD8-positive CTLs can kill infected cells by direct contact, triggering them to undergo apoptosis and recruiting antigen-nonspecific inflammatory cells that amplify their cytopathic potential. They also secrete cytokines when they recognize antigen in the infected tissue, some of which have the potential to inhibit the expression and replication of HBV in the hepatocyte. All limbs of the immune response must cooperate productively to terminate a viral infection. Individual differences in the efficiency of viral antigen processing by hepatocytes and professional antigen-presenting cells, or at the level of antigen recognition and responsiveness by B and T lymphocytes, will affect the strength of the antiviral immune response and the extent to which it contributes to viral clearance and liver disease.
Finally, chronic hepatitis appears to be due to a suboptimal cellular immune response that destroys some of the infected hepatocytes and does not purge the virus from the remaining infected hepatocytes, thereby permitting the persisting virus to trigger a chronic indolent necroinflammatory liver disease that sets the stage for development of HCC.
Rous-Whipple Award
The Rous-Whipple Award was established by the American Society for Investigative Pathology to recognize a career of outstanding scientific contribution. The 1999 recipient of the Rous-Whipple Award, Francis V. Chisari, delivered a lecture entitled "Viruses, Immunity, and Cancer: Lessons from Hepatitis B" after accepting the award on Tuesday, April 20, 1999 in Washington, D.C., at the annual meeting of the American Society for Investigative Pathology.
Acknowledgements
I am indebted to my many colleagues and collaborators who contributed greatly to the work described in this paper, especially Drs. Carlo Ferrari, Kazuki Ando, Yasunari Nakamoto and Luca G. Guidotti. I also thank Ms. Andréa Achenbach for assisting with the manuscript preparation.
Footnotes
Address reprint requests to Francis V. Chisari, M.D., Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: fchisari{at}scripps.edu
Supported by grants AI20001, CA40489, CA54560, and M01-RR00833 from the National Institutes of Health. This is manuscript number 12981-MEM from The Scripps Research Institute.
Accepted for publication February 14, 2000.
References
HCC hypothesis. According to this hypothesis, a
vigorous (+++) immune response to HBV leads to viral clearance while an
absent (-) immune response leads to the "healthy" carrier state,
and an intermediate (+) immune response produces chronic hepatitis.
This indolent necroinflammatory liver disease is characterized by
chronic liver cell necrosis which stimulates a sustained regenerative
response. The inflammatory component includes activated macrophages
that are a rich source of free radicals. The collaboration of these
mitogenic and mutagenic stimuli has the potential to cause cellular and
viral DNA damage, chromosomal abnormalities, genetic mutations, etc,
that deregulate cellular growth control in a multistep process that
eventually leads to hepatocellular carcinoma.