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review article
mechanisms of disease
Hepatitis B Virus Infection — Natural History
and Clinical Consequences
Don Ganem, M.D., and Alfred M. Prince, M.D.
virologic features
classification and structure
Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae (hepatotropic
DNA virus) family. Hepadnaviruses have a strong preference for infecting liver cells, but
small amounts of hepadnaviral DNA can be found in kidney, pancreas, and mononu-
clear cells. However, infection at these sites is not linked to extrahepatic disease.10-13
HBV virions are double-shelled particles, 40 to 42 nm in diameter (Fig. 1A),14 with
an outer lipoprotein envelope that contains three related envelope glycoproteins (or
surface antigens).15 Within the envelope is the viral nucleocapsid, or core.16 The core
contains the viral genome, a relaxed-circular, partially duplex DNA of 3.2 kb, and a po-
lymerase that is responsible for the synthesis of viral DNA in infected cells.17 DNA se-
quencing of many isolates of HBV has confirmed the existence of multiple viral geno-
types, each with a characteristic geographic distribution.18
In addition to virions, HBV-infected cells produce two distinct subviral lipopro-
tein particles: 20-nm spheres (Fig. 1B) and filamentous forms of similar diameter
HBV
Recycling
Vesicular transport
to cell membrane
Budding into
endoplasmic
reticulum
Core particle
Translation
Core assembly and
RNA packaging
Cytoplasm
Transcription
cccDNA
Repair
Nucleus
identity of which remains unknown. After mem- All viral RNA is transported to the cytoplasm,
brane fusion, cores are presented to the cytosol and where its translation yields the viral envelope, core,
transported to the nucleus. There, their DNA ge- and polymerase proteins, as well as the X and preC
nomes are converted to a covalently closed circular polypeptides. Next, nucleocapsids are assembled
(ccc) form,26 which serves as the transcriptional in the cytosol, and during this process a single mol-
template for host RNA polymerase II. This enzyme ecule of genomic RNA is incorporated into the as-
generates a series of genomic and subgenomic sembling viral core.28 Once the viral RNA is en-
transcripts.27 capsidated, reverse transcription begins.28 The
synthesis of the two viral DNA strands is sequential. pecially the responses of cytotoxic T lymphocytes,
The first DNA strand is made from the encapsi- play a central role in viral clearance. Figure 3 sum-
dated RNA template; during or after the synthesis marizes the major types of cellular immune re-
of this strand, the RNA template is degraded and sponse to HBV.
the synthesis of the second DNA strand proceeds, The mechanisms by which cytotoxic T lympho-
with the use of the newly made first DNA strand as a cytes kill liver cells and cause viral clearance have
template.25,27,29 Some cores bearing the mature been incisively investigated in transgenic mice that
genome are transported back to the nucleus, where express viral antigens or contain replication-com-
their newly minted DNA genomes can be convert- petent viral genomes in the liver.32,33 Because these
ed to cccDNA to maintain a stable intranuclear pool mice harbor HBV genes in their germ-line DNA, they
of transcriptional templates.26 Most cores, how- are largely tolerant to HBV proteins, and according-
ever, bud into regions of intracellular membranes ly, clinically significant liver injury does not devel-
bearing the viral envelope proteins. In so doing, they op. However, if antiviral cytotoxic T lymphocytes of
acquire lipoprotein envelopes containing the viral syngeneic animals are transferred into such mice,
L, M, and S surface antigens and are then exported acute liver injury with many of the features of clini-
from the cell. cal hepatitis B develops.34 It is striking that, in this
model, the number of hepatocytes killed by direct
pathogenesis of hepatitis b engagement between cytotoxic T lymphocytes and
The HBV replication cycle is not directly cytotoxic their targets is very small and clearly insufficient
to cells. This fact accords well with the observation to account for most of the liver damage. This sug-
that many HBV carriers are asymptomatic and have gests that much of the injury is due to secondary
minimal liver injury, despite extensive and ongo- antigen-nonspecific inflammatory responses that
ing intrahepatic replication of the virus.30 It is now are set in motion by the response of the cytotoxic
thought that host immune responses to viral anti- T lymphocytes. Presumably, much of the damage
gens displayed on infected hepatocytes are the prin- occurring in this context is due to cytotoxic by-prod-
cipal determinants of hepatocellular injury. This no- ucts of the inflammatory response, such as tumor
tion is consistent with the clinical observation that necrosis factor (TNF), free radicals, and proteas-
patients with immune defects who are infected with es. Other immune-cell populations, notably natu-
HBV often have mild acute liver injury but high rates ral killer T cells,35 probably also contribute to liver
of chronic carriage.31 injury.
The immune responses to HBV and their role in Recent experiments suggest that some of the
the pathogenesis of hepatitis B are incompletely inflammatory by-products, notably interferon-g
understood. Correlative clinical studies show that (IFN-g) and TNF-a, can have antiviral effects that do
in acute, self-limited hepatitis B, strong T-cell re- not involve killing the target cells. When cytotoxic
sponses to many HBV antigens are readily demon- T lymphocytes are transferred to mice that bear rep-
strable in the peripheral blood.32 These responses licating HBV, viral DNA and RNA throughout the
involve both major-histocompatibility-complex liver rapidly disappear, even from viable, uninjured
(MHC) class II–restricted, CD4+ helper T cells and hepatocytes — an effect that can be blocked by the
MHC class I–restricted, CD8+ cytotoxic T lympho- administration of antibodies to TNF-a and IFN-g.34
cytes. The antiviral cytotoxic T-lymphocyte response Such noncytocidal antiviral effects may be impor-
is directed against multiple epitopes within the HBV tant for viral clearance in natural infection. In fact,
core, polymerase, and envelope proteins; strong cytokine release triggered by unrelated hepatic in-
helper T-cell responses to C and P proteins have also fections in HBV-transgenic mice can have the same
been demonstrated in acute infection. By contrast, effect.36 This phenomenon may explain the sup-
in chronic carriers of HBV, such virus-specific T-cell pression and occasional clearance of chronic HBV
responses are greatly attenuated, at least as assayed infection in patients with superimposed acute hep-
in cells from the peripheral blood. However, anti- atitis caused by unrelated viruses.
body responses are vigorous and sustained in both
situations (although free antibodies against HBsAg natural history
[anti-HBs antibodies] are not detectable in carriers Primary HBV infection in susceptible (nonimmune)
because of the excess of circulating HBsAg). This hosts can be either symptomatic or asymptomatic.
pattern strongly suggests that T-cell responses, es- The latter is more common than the former, espe-
Antigen-
presenting cell HBV
MHC
class II
HBV
antigens
CD4+
T cell
HBV
peptides
HBV DNA
MHC Down-
CD8+ Infected
class I regulation
T cell hepatocyte HBV RNA
of viral
replication
TNF-a
Interferon-g HBV
peptides
CD8+
T cell
HBV cores
HBsAg
MHC
class I
cially in young children. Most primary infections in ic hepatitis B. The resulting hepatic insufficiency
adults, whether symptomatic or not, are self-lim- and portal hypertension make this process one of
ited, with clearance of virus from blood and liver the most feared consequences of chronic HBV in-
and the development of lasting immunity to rein- fection.
fection.37,38 However, some primary infections in
healthy adults (generally less than 5 percent) do not Primary Infection
resolve but develop into persistent infections. In In primary infection, HBsAg becomes detectable
such cases, viral replication continues in the liver in the blood after an incubation period of 4 to 10
and there is continual viremia, although the titers weeks, followed shortly by antibodies against the
of virus in the liver and blood are variable. Persis- HBV core antigen (anti-HBc antibodies), which ear-
tent HBV infection may be symptomatic or asymp- ly in infection are mainly of the IgM isotype.38 Vire-
tomatic. People with subclinical persistent infec- mia is well established by the time HBsAg is detect-
tion, normal serum aminotransferase levels, and ed, and titers of virus in acute infection are very high
normal or nearly normal findings on liver biopsy — frequently 109 to 1010 virions per milliliter.39 Cir-
are termed asymptomatic chronic HBV carriers; culating HBeAg becomes detectable in most cases,
those with abnormal liver function and histologic and studies of chimpanzees and other animals with
features are classified as having chronic hepatitis B. primary hepadnaviral infection show that 75 to 100
Cirrhosis, a condition in which regenerative nod- percent of hepatocytes are infected when this anti-
ules and fibrosis coexist with severe liver injury, gen is evident.40 Thus, it is not surprising that epi-
develops in about 20 percent of people with chron- demiologic studies consistently show high rates of
both vertical and horizontal transmissibility during aminotransferase levels, known as a flare, which
acute HBV infection.41 suggests that the process reflects immune-mediat-
When liver injury does occur in primary infec- ed destruction of infected hepatocytes. Reductions
tion, alanine aminotransferase levels do not increase in the level of viremia as great as five orders of mag-
until after viral infection is well established, reflect- nitude may accompany seroconversion to anti-HBe
ing the time required to generate the T-cell–mediat- antibodies.44 Thus, the natural history of HBV per-
ed immune response that triggers liver injury. Once sistence suggests that there is an ongoing immune
this response is under way, titers of virus in blood attack on infected cells in the liver — an attack that
and liver begin to drop. The fact that infection can is usually inadequate to eradicate infection alto-
be cleared from virtually all hepatocytes without gether, but that does reduce the number of infected
massive hepatic destruction (in most cases) is a tes- cells and thereby lowers the circulating viral load.
tament to the extraordinary power of the noncy- Figure 4 shows typical patterns of serologic and
tolytic clearance mechanisms described above. With molecular markers in both acute self-limited and
clearance of the infection, the viral antigens HBsAg chronic HBV infection.
and HBeAg disappear from the circulation, and free The widely held view that circulating viral DNA
anti-HBs antibodies become detectable. disappears when anti-HBe antibodies appear is in-
Surprisingly, in self-limited infection, as defined correct; this idea reflects the fact that, for many
by the disappearance of the viral antigens and the years, HBV DNA was measured by relatively insen-
appearance of anti-HBs antibodies, low levels of sitive hybridization methods with a detection limit
HBV DNA in the blood may persist for many years, of 105 to 106 virions per milliliter. Thanks to the ad-
if not for life.42 It is not known whether this DNA vent of the polymerase-chain-reaction (PCR) meth-
contains the entire HBV genome, or even whether od, we now know that at least 70 to 85 percent of
it is contained in virions. However, inoculation of people with anti-HBe antibodies have detectable
serum from three subjects with persistent HBV viral DNA in the circulation, typically in the range
DNA into chimpanzees has not led to documented of 103 to 105 molecules per milliliter, and some-
infectivity.42 times higher.44 Although these levels of HBV DNA
are relatively low, they are hardly negligible. (For
Persistent Infection reference, they are similar to levels of human im-
In persistent HBV infection, the early events unfold munodeficiency virus [HIV] and HCV DNA in many
as in self-limited infection, but HBsAg remains in patients with symptomatic acquired immunodefi-
the blood and virus production continues, often for ciency syndrome or hepatitis C.) Given the short
life. However, levels of viremia in chronic infection half-life of HBV virions (approximately one day),39
are generally substantially lower than during pri- such levels can be sustained only by ongoing viral
mary infection, although they can vary consider- replication; therefore, the claim that HBV enters a
ably from person to person. High titers of HBV in so-called nonreplicative phase later in its course is
the blood are often indicated by the continued pres- not correct. For this reason, anyone who has a pos-
ence of HBeAg. Typically, there are 107 to 109 virions itive test for HBsAg should be presumed to have
per milliliter in the blood43 in such cases, which are some level of ongoing viremia. For example, when
highly infectious. But most people with persistent a decision must be made about immunoprophy-
infection, especially those with anti-HBe antibod- laxis after a needle stick involving blood from an
ies, have somewhat lower levels of viremia. HBsAg-positive patient, prophylaxis should be of-
One feature of chronic HBV infection that is not fered irrespective of that patient’s HBeAg status.
widely appreciated is its dynamic natural history. HBeAg-negative carriers are a heterogeneous
Even though, in most cases, HBsAg remains detect- group. Most such carriers have low levels of viral
able for life, titers of viral DNA tend to decline over DNA, relatively normal levels of alanine amino-
time. With the passage of time, there is also a ten- transferase, and a good prognosis.30 However, par-
dency for HBeAg to disappear from the blood, along ticularly in southern Europe and in Asia, at least 15
with seroconversion to positivity for anti-HBe anti- to 20 percent of such carriers have elevated levels
bodies — a progression that occurs at a rate of of alanine aminotransferase and viral DNA in the
5 to 10 percent per year in persistently infected peo- blood.45 The virus in many such carriers harbors
ple.39 Often, the disappearance of HBeAg is pre- mutations in the preC region that prevent the pro-
ceded or accompanied by a transient rise in alanine duction of HBeAg.46 It has been suggested that per-
0 5 10 15 20 48 2 4 6 8 >10
Weeks since Exposure Years since Exposure
HBV DNA
HBeAg
Anti-HBs
HBsAg
Anti-HBc
Anti-HBe
ALT
0 5 10 15 20 48 2 4 6 8 >10
Weeks since Exposure Years since Exposure
sistently abnormal levels of alanine aminotransfer- begin. Furthermore, screening is imperfect — alpha
ase and elevated levels of viral DNA may denote a fetoprotein screening, for example, has an excel-
subgroup of HBeAg-negative carriers who should lent negative predictive value, but its positive pre-
receive active antiviral therapy.47 dictive value ranges from 9 to 30 percent.47
values tend to have a relatively stable course, with patients. Moreover, in many patients a flare of liver
low rates of clinical or pathological progression.30 injury occurs during administration of interferon
At present, therapy is usually not offered to such alfa, often just before or during clearance of HBeAg.
persons. As noted above, some HBeAg-negative pa- This phenomenon may reflect the immunomodu-
tients have liver dysfunction and substantial vire- latory activity of interferon alfa, which, in addition
mia (>105 molecules per milliliter). Discussions to impairing HBV replication, can also cause up-reg-
of the treatment of such patients are rare in the pub- ulation of MHC class I antigens on hepatocytes and
lished literature, but results of a recent trial suggest thereby augment the recognition of infected cells
that many of these patients would also benefit from by cytotoxic T lymphocytes. Although sometimes
effective antiviral therapy.56 A recent clinical prac- disquieting to patients and physicians alike, these
tice guideline47 includes this group in its discus- flares are intrinsic to the therapy and, as markers of
sion of treatment; clearly, future clinical investiga- enhanced antiviral immune responsiveness, often
tions should focus more attention on this group. presage a successful outcome. However, treatment
The usual markers of successful therapy are the with interferon alfa is generally contraindicated in
loss of HBeAg, seroconversion to anti-HBe anti- very advanced liver disease, since in such cases the
bodies, and reduction of the circulating viral load. flares may precipitate overt liver failure. Moreover,
These are useful indicators, since patients with sta- patients with advanced cirrhosis and splenomeg-
ble seroconversion to anti-HBe–positive status typ- aly usually have base-line leukopenia and throm-
ically have improved histologic findings in the liv- bocytopenia, which can be exacerbated by the drug.
er, and this improvement tends to be maintained
over the long term.57 True cure of infection (loss of antiviral drugs
HBsAg and complete disappearance of viremia, as Lamivudine
measured by stringent PCR assays) is achieved only In the past decade, therapy for HBV has been revo-
infrequently (in 1 to 5 percent of patients) with cur- lutionized by the advent of drugs that directly block
rent regimens, although the increasing numbers of replication of the HBV genome. All these drugs (to
active antiviral drugs might lead to an upward revi- date) are nucleoside or nucleotide analogues that
sion of this figure in the future. In the case of pa- selectively target the viral reverse transcriptase. The
tients with HBeAg-negative chronic hepatitis, there first successful drug, lamivudine, emerged from
is no information about which markers best mea- screening for inhibitors of the HIV reverse trans-
sure the response to therapy. Quantitation of vire- criptase and was introduced into clinical practice
mia by PCR assays would seem a logical starting for the management of HIV infection. Carriers of
place, but there have been no systematic studies to HIV who are also infected with HBV had substan-
guide the clinical interpretation of results. tial declines in HBV viremia when treated with la-
mivudine,60 and such declines were also observed
interferon in patients with chronic hepatitis B who did not
For many years, administration of interferon alfa have HIV infection.61 In general, treatment with la-
(5 million to 10 million units subcutaneously three mivudine results in a reduction of 3 to 4 log in circu-
times per week, for at least three months) was the lating levels of HBV DNA in the first three months
mainstay of therapy. About 30 percent of patients of therapy; this decline is associated with more rap-
who tolerated this regimen had a successful re- id loss of HBeAg, seroconversion to anti-HBe–pos-
sponse, defined as a loss of HBeAg, the develop- itive status, and improvement in serum aminotrans-
ment of anti-HBe antibodies, and a decline in se- ferase levels. The drug is usually well tolerated, a
rum alanine aminotransferase levels.58 With HBe factor that has led to the rapid displacement of in-
seroconversion and normalization of alanine ami- terferon alfa from the roster of first-line therapies
notransferase levels, improvement is usually sus- for HBV. Lamivudine is not immunomodulatory and
tained well after therapy has been discontinued.58 can be used in patients with decompensated cir-
Interferon alfa treatment of chronic HBV infections rhosis.62 Even polyarteritis nodosa associated with
in patients with cirrhosis has even been reported to HBV has been shown to respond dramatically to
reduce the risk of hepatocellular carcinoma.59 treatment with lamivudine plus plasma exchange.63
However, the side effects of therapy with inter- Although lamivudine is not an immunomodu-
feron alfa (fever, myalgias, thrombocytopenia, and lator, there is strong evidence that successful treat-
depression) make it a difficult treatment for many ment with lamivudine relies to some extent on an
adequate host immune response. This evidence but no evidence of overt clinical failure, especially
emerged from a retrospective examination of sub- since transient exacerbations of liver injury devel-
groups of patients with optimal responses to ther- op in some patients when antiviral therapy is with-
apy, which revealed a strong correlation between drawn.67 (Such postwithdrawal flares are not lim-
HBeAg clearance and elevated pretreatment val- ited to lamivudine but have also been observed with
ues for alanine aminotransferase.64 HBeAg sero- other anti-HBV regimens.56,68) Now that newer
conversion occurred in 65 percent of cases in which anti-HBV drugs are available, additional options
pretreatment values for alanine aminotransferase exist for patients with resistant strains of HBV.
were more than five times the upper limit of the
normal range, as compared with only 26 percent Other Nucleotide Analogues
in patients with elevations in alanine aminotrans- Recently, the Food and Drug Administration (FDA)
ferase values that were two to five times the upper approved a second antiviral drug, adefovir, to treat
limit of the normal range. Only 5 percent of pa- HBV infection. Adefovir, a nucleotide (adenosine
tients with pretreatment alanine aminotransferase monophosphate) analogue, is a prodrug that un-
values that were less than twice the upper limit of dergoes two intracellular phosphorylations to yield
the normal range had clearance of HBeAg — a the active drug, an inhibitor of the viral polymer-
rate similar to that for the spontaneous loss of this ase. Initially developed as an inhibitor of HIV re-
marker. This finding suggests that by reducing the verse transcriptase, it proved nephrotoxic in the dos-
viral load, lamivudine allows the immune and in- es that were required for effective inhibition of HIV
flammatory responses to deal more effectively with replication. However, in lower doses (10 mg per day)
the remaining infected hepatocytes in the host. it has shown little nephrotoxicity and retains good
The principal limitation of lamivudine mono- efficacy against HBV in HBeAg-positive patients,
therapy is the development of drug resistance, with a reduction of 3 to 4 log in viremia; the fre-
which is mediated largely by point mutations at quency of HBeAg seroconversion is enhanced, and
the YMDD motif at the catalytic center of the viral there is histologic improvement in the liver.68 Simi-
reverse transcriptase. The resulting mutants are lar efficacy was documented in HBeAg-negative pa-
slightly less fit than wild-type HBV in the absence tients with abnormal liver function and elevated lev-
of the drug, but they are strongly selected for in its els of viral DNA.56 Moreover, the drug effectively
presence.65 Viral resistance emerges much more inhibits the replication of lamivudine-resistant HBV
slowly in HBV infection than in HIV infection, for mutants, both in vitro and in vivo.69,70 Clear evi-
complex reasons beyond the scope of this review. dence of the emergence of adefovir-resistant HBV
By the end of one year of therapy, 15 to 20 percent mutants has not been found in the clinical trials
of patients have resistant variants in the circula- performed to date,56,68,71 although this issue re-
tion; the figure rises to 40 percent by two years, and mains open now that large numbers of patients
to 67 percent by the fourth year.66 will be using the drug.
The clinical significance of the development of Tenofovir, another adenine nucleotide analogue
resistance is still being debated. Clearly, in many that was approved by the FDA for the treatment of
patients, resistance presages a return of higher- HIV, also has activity against the HBV polymerase.
level viremia, and in some of these patients further In recent trials in HIV carriers who were positive for
liver injury develops. However, although the level HBeAg, treatment with standard doses of tenofovir
of viremia rises, in many patients it may still remain led to a reduction of 4 log in circulating HBV DNA
below pretreatment levels — perhaps as a result levels, even in patients who had evidence of lamiv-
of the reduced fitness of the variants. In addition, udine-resistant virus.72,73 However, the FDA has
some patients continue to undergo conversion from not yet approved tenofovir for use in patients with
HBeAg-positive status to HBeAg-negative status, HBV infection.
even after the appearance of lamivudine-resistant Several investigational drugs are now in ad-
mutants in the circulation; by the end of four years vanced stages of clinical trials. Entecavir is a guan-
of therapy, 40 to 50 percent of patients treated with osine analogue that, unlike the drugs discussed
lamivudine have undergone such conversion. There- above, is highly selective for the HBV polymerase
fore, some experts favor the continuation of lamiv- and has no activity against HIV. It is extremely po-
udine therapy in patients with resistant variants tent on a molar basis, and doses as low as 0.1 mg per
day can cause reductions of 4 log in levels of HBV ously or after therapeutic vaccination with HBV
DNA; a dose of 0.5 mg per day caused a reduction antigens?
of nearly 5 log in 24 weeks of monotherapy.74 The
drug is also active against lamivudine-resistant HBV. liver transplantation
l-deoxythymidine is a thymidine analogue that also Liver transplantation for HBV disease is attended
selectively inhibits the HBV polymerase and is ad- by recurrent viral infection in more than 80 percent
vancing through clinical trials; it, too, appears to be of patients if no interventions are undertaken to pre-
active against lamivudine-resistant virus.75 Two vent reinfection.77 Recurrent HBV infection in the
other nucleoside analogues (emtricitabine and cle- context of the level of immunosuppression needed
vudine) have also shown promise in vitro and in vivo, to prevent graft rejection is typically associated with
although neither is reliably active against all lamiv- high viral loads and a poor outcome, especially
udine-resistant variants.76 for patients with chronic HBeAg-positive hepati-
Thus, the past few years have witnessed the de- tis. Hepatitis B immune globulin, administered over
velopment of a plethora of new drugs for the treat- long periods, cuts the reinfection rate in half and
ment of HBV infection. It seems likely that combi- extends the two-year survival rate from 50 percent
nation therapy will become the wave of the future to approximately 80 percent.77,78 Use of lamivu-
in HBV therapy, but many questions remain to be dine after transplantation has resulted in similar
resolved by clinical investigation. Is combination improvement,79 but without question, the best re-
therapy truly superior to monotherapy, clinically as sults have been obtained with post-transplantation
well as virologically? If, as most expect, it proves to prophylaxis consisting of both hepatitis B immune
be superior, which combination of drugs should be globulin and lamivudine. This regimen reduces
used for initial treatment? Will there be an evolution the rate of reinfection to about 10 percent and has
of cross-resistance among drugs that are effective boosted the five-year rate of HBV-free survival to
against lamivudine-resistant viruses? What should approximately 80 percent.80 However, many prob-
be the duration of therapy? (One recent practice lems remain. Hepatitis B immune globulin is very
guideline recommends stopping antiviral therapy expensive and contributes substantially to the cost
at one year in HBeAg-positive patients who have of transplantation. It seems likely that, in the near
seroconversion to anti-HBe antibodies but continu- future, a more limited course of prophylaxis with
ing therapy in those who do not have seroconver- hepatitis B immune globulin (6 to 12 months), in
sion in the first year or in whom breakthrough vi- concert with long-term drug therapy, perhaps with
remia develops.47) More speculatively, could more more than one agent, will become popular. Wheth-
potent combination therapy reduce HBV levels to er the use of hepatitis B immune globulin can be
a point where the remaining virus could be cleared avoided altogether by the use of multidrug regi-
by the host’s own immune system, either spontane- mens will require careful study.
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