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APMIS 117: 427–439
r 2009 The Authors
Journal Compilation r 2009 APMIS
DOI 10.1111/j.1600-0463.2009.02454.x
The hepatitis C virus enigma
HELGE MYRMEL,1 ELLING ULVESTAD1,2 and BIRGITTA ÅSJØ1,2
1
Department of Microbiology and Immunology, Haukeland University Hospital; and 2Section for Microbiology
and Immunology, The Gade Institute, University of Bergen, Bergen, Norway
Myrmel H, Ulvestad E, Åsj B. The hepatitis C virus enigma. APMIS 2009; 117: 427–39.
Hepatitis C virus (HCV) has a high propensity to establish chronic infection with end-stage liver disease. The high turnover of virus particles and high transcription error rates due to lack of proof-reading
function of the viral polymerase imply that HCV exists as quasispecies, thus enabling the virus to evade
the host immune response. Clearance of the virus is characterized by a multispecific, vigorous and
persistent T-cell response, whereas T-cell responses are weak, narrow and transient in patients who
develop chronic infection. At present, standard treatment is a combination of pegylated interferon-a
and ribavirin, with a sustained viral response rate of 40–80%, depending on genotype. The mechanisms
for the observed synergistic effects of the two drugs are still not known in detail, but in addition to
direct antiviral mechanisms, the immunomodulatory effects of both drugs seem to be important, with a
shift from Th2- to Th1-cytokine profiles in successfully treated patients. This article describes virus–
host relations in the natural course of HCV infection and during treatment.
Key words: Hepatitis; virus; variability; immunology.
Helge Myrmel, Department of Microbiology and Immunology, Haukeland University Hospital,
N-5021 Bergen, Norway. e-mail: [email protected]
Since its identification in 1989 (1), hepatitis C
virus (HCV) infection has been recognized as a
major health problem worldwide. Approximately 170 million people are infected globally
(2), and HCV infection is at present the most
common cause of chronic hepatitis and cirrhosis
in the United States and other western countries
(3). The prevalence rate in Scandinavia is
0.2–0.5% (2, 4).
Contrary to the situation in hepatitis B virus
(HBV) infection, the majority of hepatitis C
cases develop chronic infection, and possibly
around 50–80% of infected individuals fail to
clear the virus and develop chronic infection (5).
Once chronicity is established, there is no spontaneous resolution of the viremia.
HCV infection is characterized by a wide range
of clinical manifestations, including asymptomatic chronic carriage, acute and chronic hepatitis, cirrhosis, hepatocellular carcinoma and
Invited review
extrahepatic manifestations. The latter are commonly observed and may represent the first sign of
the disease. Although the major target for HCV is
the hepatocyte, the virus also displays lymphotropism, and HCV genome sequences have been
detected in circulating lymphocytes (T and B cells)
(6,7) and antigen-presenting cells (8), as well as
in other compartments such as the brain. Its
wide cellular tropism may be explained by the
ubiquitous expression of its putative cell receptor,
CD81 (9).
THE VIRUS
HCV is a small, enveloped, positive singlestranded RNA virus that belongs to the genus
Hepacivirus within the family of Flaviviridae
(Fig. 1A). The genome has approximately 9600
nucleotides and contains one large open reading
frame flanked by non-translated regions at the
5 0 and 3 0 ends (Fig. 1B). The polyprotein is
cleaved by host-cell proteases and virus-encoded
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MYRMEL et al.
A
Env 1
Env 2
RNA genome
Env lipid
Capsid proteins
HCV RNA
B
polyprotein precursor encoding region
5’ NTR
3’ NTR
Structural proteins
p22
gp35
gp70
C
E1
E2
envelope
glycoproteins
nucleocapsid
p7
p23
NS1 NS2
Nontructural proteins
p70
NS3
p8
p27
p56/58
p68
NS4A
NS4B
NS5A
NS5B
metalloprotease
serine protease
RNA helicase
transmembrane protein
IFN-resistance
protein
RNA
polymerase
cofactors
Fig. 1. Schematic model of HCV (A) with envelope (env) proteins 1 and 2, capsid proteins, RNA genome and the
various proteins after cleavage of the precursor protein (B).
proteases into structural (core, envelope 1 and 2)
and non-structural proteins (NS2, NS3, NS4A,
NS4B, NS5A and NS5B). NS5B encodes the
viral RNA-dependent RNA polymerase that
lacks a proof-reading function. Replication of
the positive-stranded HCV genome is thus characterized by ongoing error rates of 104 and
105 nucleotide substitutions per base pair copied. Together with the high production of 1012
virions per day, with an average of 2.7 h half-life
and a turn-over rate close to 99% (10), the calculated mutation rate is in the order of
1.5–2.0103 nucleotide substitutions per site
and per year, and every possible mutation in every single position of the genome can theoretically be generated every day. This implies that
HCV, like other RNA viruses, typically exists as
quasispecies, which is a collection of related but
not identical genomes. Based on phylogenetic
analysis, HCV has been classified into six major
genotypes. The genotypes differ from each other
by 31–33% on the nucleotide level, and these
genotypes can be further divided into multiple
epidemiologically distinct subtypes that differ by
20–25% (11).
The origin of HCV has not yet been properly
identified, but bovine viral diarrhoea virus has
been suggested as a possible ancestral virus (12).
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However, the divergence of HCV genotypes,
geographical distribution and presence of single
types with numerous subtypes is compatible
with a pattern that suggests long periods of endemic infection and existence for 500–2000
years. It has been suggested that various regions
of the African continent are home for genotypes
1, 2, 4 and 5, whereas South East Asia is an endemic region for genotype 6. Of particular interest is the finding of more than one virus
genotype, but each of them represented by only
a few subtypes. This could indicate recent and
limited introduction from endemic areas, exemplified by types 1a, 1b, 2a, 2b and 3a in
Northern Europe and North America. For example, types 1a and 3a are found in young
adults, often with intravenous drug use as a risk
factor. The recent dissemination of these viruses
is supported by more limited genetic diversity
within these subtypes (13), a pattern also seen
for spreading of human immunodeficiency virus
type 1 (HIV-1), with restricted genetic variability
in risk groups with rapid dissemination (14). In
contrast, subtypes 2a, 2b and 2c from some
African countries show extreme diversity in line
with a longstanding infection. Epidemics linked
to medical practice are the spreading of subtype
1b by contaminated anti-D immunoglobulin in
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the 1970s in Ireland and former East Germany.
The very high prevalence of subtype 4a in Egypt
has been linked to cross-infection by mass treatment with anti-schistosomal agents. In earlier
times, virus transmission may have been caused
by variolation as a means of protection against
smallpox, use of unsterilized needles and syringes after injection of arsphenamine for treatment of syphilis and association with
scarification as part of culture-specific rites (13).
At present, the standard treatment of HCVinfection is a combination therapy with pegylated interferon-a (PEG-IFN) and ribavirin (15).
Although their precise mode of action is not
known in detail, both drugs have substantial
antiviral and immunomodulatory effects. The
response rate depends on the infecting genotype,
which suggests that sequence differences between genotypes influence the susceptibility to
the drugs.
The understanding of the HCV viral life cycle,
its mode of pathogenesis and elaboration of antiviral drugs and vaccines, have all been hampered by the lack of a robust cell culture system
for HCV replication and a small animal model.
The chimpanzee is the only non-human species
susceptible to HCV infection. Recently, however, mouse models with reconstituted human liver grafts have shown promising results (16, 17).
A break-through in HCV research came in
2005, when cell-culture systems were established
that allowed production of infectious HCV particles from a cloned viral genome transfected
into human hepatocellular carcinoma Huh-7derived cells (18, 19). Also, a human hepatocyte
cell line immortalized with human papilloma
virus 18/E6E7 was susceptible to HCV infection
(20). However, these cell lines were not susceptible to infection with primary HCV isolates
from patients, and recent improvements have
allowed the infection of normal hepatocytes by
naturally occurring HCV genotypes. Primary
hepatocytes were isolated and cultures were established from organ donors. After 5 days these
cells were infected by addition of serum from
chronically HCV-infected patients (21).
HCV infection and virus entry of a susceptible
cell is a complex multistep process. The initial
host-cell attachment may involve glycosaminoglycans and the low-density lipoprotein receptor. Then the particle interacts sequentially
with the surface molecules SR-B1 (scavenger rer 2009 The Authors Journal Compilation r 2009 APMIS
ceptor), the tetraspannin CD81 and the tightjunction protein claudin-1 (CLDN1). The particle is then internalized by clathrin-mediated endocytosis and fusion, most likely occurring in
early endosomes. However, more entry factors
probably play a role in the infectious process,
because some human cell lines expressing CD81,
CLDN1 and SR-B1 still remain resistant to
HCV (22).
HOST DEFENCE
Based on evolutionary and functional considerations, the molecular and cellular entities
that make up the host response to HCV infection can be divided into innate and adaptive subcomponents. The innate components, including
phagocytes and antigen-presenting cells, serve as
a first line of defence. Upon infection, they recognize pathogen-associated molecular patterns
through a variety of pattern-recognition receptors, including the Toll-like receptors (TLR).
Recognition of microbial patterns or danger in
turn triggers the whole cascade of effector mechanisms of the innate and adaptive immune
systems (23). Acting through a restricted portal
of four intracellular adapter molecules, a limited
number of TLR are able to induce the transcriptional activation of hundreds of genes that
code for systemic and localized defence factors.
These include genes for proinflammatory
cytokines, molecules that drive dendritic cell
migration to the lymph nodes and MHC and costimulatory molecules.
The dispersed signals that emanate from the
innate immune response trigger the components
of the adaptive immune system within the secondary lymphoid organs, including the spleen
and lymph nodes, where antigen-specific naı̈ve T
and B cells interact with antigen and antigen
presenting cells (24). At this point, the extremely
complex characteristics of the infectious agents
are being met by a similarly complex response
performed by the adaptive immune system. A
major event at this early stage is the polarization
of the T helper (Th) response into Th1 and Th2
cells (25). Whereas Th1 cells produce interleukin
(IL) 2 and IFN-g and activate macrophages and
cytotoxic CD81 T cells, Th2 cells produce IL-4,
IL-5, IL-10 and IL-13, the secretion of which
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MYRMEL et al.
leads to the activation and differentiation of B
cells into antibody-producing plasma cells.
The diversification of the Th repertoire depends upon the microenvironment that is being
generated at the contact surface between the T
cell and the dendritic cell, within which signalling molecules of both cells become congregated.
While immature dendritic cells energetically
sample their external antigenic environment, the
processed molecules are not expressed on their
surface MHC molecules. However, upon activation the cells stop internalizing antigen and
instead focus on generating complexes between
peptides and MHC class II molecules for T-cell
activation. Recognition of the MHC-antigen
complex activates the T cell, which in turn further activates the dendritic cell to provide more
stimulating signals (26).
But also the site in the body where infection or
damage occurs is important for the ensuing polarization of the immune reaction. Different tissues mount different immune responses to the
same antigenic stimulus. This is partly owing to
the complex pattern of tissue-specific distribution of dendritic cells, but also because epithelial
and stromal cells at the site of infection generate
TLR-mediated signals that contribute to the activation of T cells (27). The liver has a unique
capacity to induce antigen-specific tolerance,
and it has been speculated as to whether this
may be due to the tolerogenic potential of resident dendritic cells (28).
The adaptive immune response is influenced
by properties of both the organism and the antigen. Organismal properties that may regulate
the output include genetic makeup, previous encounters with similar infectious agents, and
route of infection, while important properties of
the antigen include its concentration and the
timing and duration of antigen encounter. These
considerations may be visualized as a lens metaphor (Fig. 2) in which both the evolutionary and
the developmental aspects are taken into consideration. In this representation, evolutionary
derived experiences (convex lens) serve to focus
signals towards a species-specific norm of responses, while developmentally shaped experiences (concave lens) lead to diverging organismspecific responses (29). Accordingly, the various
phenotypes that the immune response could
take should emanate from the way the evolutionary selected genetic makeup of an organism
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Signal
Decision
Skin
Response
Malfunctional
(autoimmunity)
I
n
n
a
t
e
Mucosa
Central nervous system
Functional
Malfunctional
(allergy)
Feed back mechanism
Fig. 2. The lens metaphor. The combined use of innate and adaptive immune components provides the
organism with rapid response kinetics and ability to
mount a powerful and appropriate response to a diversity of infectious agents. The immune system receives signals from the microbial world and the
peripheral tissues by receptors of the innate immune
system. Upon stimulation the receptors trigger secondary events, including release of inflammatory
mediators. These secondary signals are made prominent within the lymphoid tissues, and thus constitute
the context in which decisions are being made. The
focusing role of the innate immune system as well as
the contextual parameters sets the lead for the activation of the adaptive immune system. The adaptive
immune system generates an immunodominant response towards specific antigens, which may be fit or
unfit.
intersects and maps onto its developmentally
shaped adaptive systems.
THE BALANCE
Both pathogenesis and the eventual outcome of
any viral infection are significantly influenced by
the host immune response. As one of few RNA
viruses able to cause persistent infection in man,
HCV has evolved through the eons in an intimate relationship with its host. This adaptation has led to the evolution of several methods
of evading or counteracting the host’s innate
and adaptive antiviral defences. The mutability
and replicative capacity of the virus also enables
it to respond rapidly to new selection pressures,
e.g. differences in B- and T-cell epitopes encountered in different individuals.
The genetic variability of HCV can be perceived at different levels. The main genotypes of
the virus represent an obvious and substantial
divergence often showing geographical or demographic (risk group) ranges. On another level
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is the variability observed between strains or individual variants. Finally, there is the diversification of viruses within an infected individual
over time (quasispecies). Taken together, this
genetic variability linked with a large and rapidly replicating virus population facilitates the
adaptation of HCV to new selection pressures
such as immune responses and antiviral therapy.
HCV has a profound impact on the immune
system of the host, not only through evasion and
modification of the immune response, but also
through a direct tropism for immune cells such
as B lymphocytes. The virus is thought to both
downregulate the type I IFN-a/b receptor and
block type I IFN signalling pathways as well as
to impair NK cell effector functions by interaction of the E2 protein with CD81 on the cell
surface, thus counteracting viral clearance by
the innate system (30–32).
Infection results in antibody production
against various viral proteins, but the humoral
immune response does not correlate with a favourable outcome of infection (33). Virus-specific antibodies can usually be detected within
50–60 days after HCV infection (34). To what
extent antibodies neutralize HCV infectivity is
not clear. HCV infectivity in chimpanzees has
been neutralized by in vitro treatment of HCV
pseudoparticles with antibodies (35), and polyclonal IgG from a chronically infected patient
has recently been shown to convey sterilizing
immunity towards the ancestral HCV strain in
vivo (36). In addition, sequence changes in the
hypervariable region 1 (HVR1) of the E2 protein, a major target for the immune response,
has been shown to predict infection outcome in
humans. These sequence changes coincide with
seroconversion and probably represent escape
mutations, implying that HCV adapts to immune selection pressure exerted by antibodies
(37). In contrast to the ability of anti-HBs antibodies to prevent HBV reinfection, anti-HCV
antibodies do not seem to prevent HCV reinfection in immune humans, and resolution of HCV
infection has been reported to occur even in the
absence of seroconversion (38). Taken together,
these findings indicate that neutralizing antibodies (nAbs) are isolate specific, and raise concerns for the development of a broadly reactive
vaccine against HCV.
There is strong evidence that virus-specific
CD41 and CD81 T cells play a crucial role in
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both viral control and liver injury. Thus, the pathogenesis of liver damage associated with HCV
infection is thought to be largely immunomediated, although a possible direct cytopathic effect is suggested by histopathological
features such as steatosis (39).
In both humans and chimpanzees, the clearance of acute infection is accompanied by a
strong and multispecific CD41 and CD81 T-cell
response (40, 41). CD41 T cells exert a regulatory function by modulating antigen-specific
B-cell activity (Th2 profile) through the recently
described CXCR51 T follicular helper cell subset (42), and the CD81 T-cell responses (Th1
profile). Th1 cytokines, e.g. IL-2, IFN-g and tumour necrosis factor (TNF-) a, activate several
antiviral mechanisms, such as enhancing immune recognition by increasing the expression of
HLA-molecules on infected cells or activating
CD81 T cells and NK cells. Several studies have
shown a correlation between a multispecific and
sustained CD41 T-cell response directed against
non-structural HCV proteins, often targeting
immunodominant epitopes within the NS3-region, and viral clearance in acute HCV infection
(43, 44). It seems that patients with acute HCV
infection displaying a Th1 profile (IFN-g and
IL-2) when stimulated with HCV antigens are
more likely to clear the virus than patients developing a Th2 phenotype response (IL-4 and
IL-10) (45, 46). Indeed, even in chronically infected patients, a strong Th1 response seems to
be associated with a more favourable and less
inflammatory course of disease (47).
An interesting aspect of chronic HCV infection is the development of weak CD41 and
CD81 T-cell responses. This phenomenon,
which may be owing to the tolerogenic potential
of the liver, may either be caused by a primary
T-cell failure in which the dendritic cells fail to
stimulate the antigen specific T cells, or by T-cell
exhaustion in which virus-specific T cells become
depleted owing to a continuous high viral load
(48) (Fig. 3).
THE BATTLE
In the encounter between HCV and its human
host there seems to be a ‘moment of truth’ during the first 12 weeks of infection (49). At this
point, about 20–25% of patients with acute
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MYRMEL et al.
A
B
priming
antigen
inexperienced
T cells
expansion
accumulation
in the liver
antigen
presenting cell
antigen
recognition
effector
functions
cytotoxic
non-cytotoxic
Fig. 3. Successful T-cell response to HCV, with priming of antigen inexperienced T cells, expansion and accumulation in the liver of virus-specific T cells. Upon recognition of their antigen, the T cells exert both cytotoxic
and non-cytotoxic functions. Lack of HCV-specific T cells can be caused by primary T cell failure (A) due to
impaired antigen presentation of antigen presenting cells, or by T cell exhaustion (B) following the initial priming
[modified from (48)].
hepatitis C will have cleared the infection,
whereas the majority becomes chronic carriers.
However, if both HCV RNA-positive and -negative cases at the time of anti-HCV seroconversion are included, estimate of clearance has been
found to be around 40%, indicating that the
clearance rate may be underestimated (50). Indeed, several studies have shown that up to 53%
of patients with acute HCV-infection show a selflimiting course of disease (49, 51, 52). This is in
line with previous findings of presumed seroreversion cases among healthy blood donors (53).
Accordingly, it may well be that the commonly
assumed frequency of 80% chronic carriers is an
overestimate, and that more patients actually
clear the virus during the acute stage.
The characteristics of the immune response in
cases of clearance have been studied extensively
[for review, see (50)]. Several factors are associated with viral clearance, among them symptomatic hepatitis with icterus, female gender and
the strength and pattern of HCV-specific CD41
T-cell responses. In a systematic review of 31
longitudinal studies, Micallef et al. (50) found
that female gender and a clinically acute hepatitis were independent predictors of spontaneous
clearance. The reason for a better prognosis in
females is currently not known, but it is a phenomenon also found in IFN response studies
(54). One hypothesis is that clearance may be
facilitated by oestrogen hormone.
A high rate of viral clearance among acute
symptomatic hepatitis C patients, paralleling
what is also observed in HBV infection with viral clearance, is consistent with the hypothesis of
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the importance of a vigorous broad initial immune response. Distinct patterns of host–virus
interactions related to viral clearance or persistence have been identified. As previously mentioned, the antiviral T-cell response seems to be
of paramount importance, and a lack of Th1
effector cells during the first few weeks of HCVinfection has been found to predict viral persistence (55). However, such immunologic testing
has hitherto not been a part of the standard diagnostic procedures of acute hepatitis C. And
while there is strong evidence to support the hypothesis of T-cell exhaustion accompanied by
narrow specificity as determining factors for
viral persistence, no testable hypothesis has so
far been launched as to what factors will influence this. On the other hand, patients clearing
the virus spontaneously can most often be identified by monitoring viral load during the first
few weeks of infection. Accordingly, the strategy
for treatment of acute hepatitis C should be to
wait for 3 months before treating the persistently
viremic patients (51).
SHIFTING THE BALANCE – EFFECT OF
TREATMENT
Effect of IFN-a
IFN-a, which belongs to a large group of closely
related cytokines, is invariably produced in
mammalian cells following virus infections.
While plasmacytoid dendritic cells seem to be
the main producers of IFN, just about any cell
can produce IFN. The production is induced by
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virally derived pathogen-associated molecular
patterns, including double- and single-stranded
RNA, that bind to and activate corresponding
molecules in host cells. Upon activation of such
host cell pattern-recognition receptors, which
include the TLR, intracellular signalling cascades become activated, leading to transcription
of IFN genes and enhanced production and secretion of IFN. Once secreted, the IFN binds to
IFN-receptors on the producer as well as neighbouring cells and initiates an intracellular signalling cascade, which leads to the upregulation
of hundreds of IFN-responsive genes, many of
which have antiviral effects.
The antiviral effects of IFN have led to its use
in treatment of select viral diseases. A modified
IFN molecule termed PEG-IFN has become the
cornerstone for treatment of chronic HCV infection. In comparison with native IFN,
PEG-IFN exhibits sustained exposure with
once-weekly dosing, a better adverse effects
profile and superior clinical efficacy. Recent data
obtained from patients with chronic HCV infection indicate that the effects of PEG-IFN on
viral clearance depend upon the baseline level of
IFN stimulation in each patient (56). Paired liver
biopsies obtained from patients before and 4
hours after administration of the first dose of
PEG-IFN demonstrated that patients responding poorly to therapy expressed high levels of
IFN-stimulated genes before therapy and had
little additional effect on the same genes after
PEG-IFN. In contrast, patients who responded
well to therapy showed a strong upregulation of
the IFN-stimulated genes following PEG-IFN
administration. It is not known how IFN-stimulated gene preactivation is connected to
treatment failure, but it may be due to some intricacies involved in the host–parasite interaction; like many other viruses HCV has evolved a
great diversity of molecular mechanisms to
evade the effects of IFN (57).
IFN is regarded as an important player in the
innate immune response and therefore crucial in
limiting the early replication and spread of infectious agents. But IFN is also important for
indirect activation of the B and T lymphocytes of
the adaptive immune system. This activity is
mediated by the effects of IFN on dendritic cells,
which are both producers and consumers of
IFN; upon infection, dendritic cells are induced
to produce large amounts of IFN, while producr 2009 The Authors Journal Compilation r 2009 APMIS
tion of IFN by infected cells can lead to the maturation of dendritic cells (58). The observation
that IFN induces maturation of dendritic cells
suggests that IFN should have adjuvant effects
on the T- and B-cell response. Although such
immunostimulatory activity has been demonstrated in mice, affecting both the humoral and
cellular immune responses (59), the same effects
have not been thoroughly demonstrated in humans. While it appears evident that IFN treatment increases the levels of polyclonal IgG and
decreases the concentration of lymphocytes (60),
the immunostimulatory effect of IFN on the antigen-specific immune response has not been reproduced in humans vaccinated against HBV
(61). Results from the study even suggested that
administration of IFN leads to a reduction of the
antibody response to the stimulating antigen.
Effect of ribavirin
Ribavirin, a guanosine analogue first synthesized in the 1970s, exhibits a broad antiviral effect against several RNA and DNA viruses both
in vivo and in vitro. Several antiviral mechanisms
have been suggested, such as inhibition of the
HCV polymerase and early chain termination.
Higher mutation rates in the presence of ribavirin have also been favoured as a key antiviral
mechanism. However, the exact antiviral mechanisms of ribavirin in treatment of HCV infection have not yet been established.
Ribavirin monotherapy has a minimal impact
on HCV viremia, despite reducing the level of
alanine transaminase (ALT) in a number of patients as well as improving the histological grade
of inflammation (62, 63). Therefore, other mechanisms of action have been explored. In addition to its specific antiviral mechanisms, ribavirin
exerts a modulating effect on the host response.
Induction of an antiviral state is achieved by enhancement of antiviral Th1 cytokines and suppression of the anti-inflammatory Th2 cytokine
expression in the T cells (64, 65). There is
evidence that ribavirin may act differentially on
specific Th1- and Th2-like responses (66). Thus,
altering the T-cell balance may well be the major
therapeutic mechanism of ribavirin, rather
than its strictly virostatic abilities, although the
direct antiviral efficacy of the drug may be underestimated. The reason for this is that measuring the viral RNA in plasma with reverse
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MYRMEL et al.
transcription polymerase chain reaction (RTPCR) does not distinguish between infectious
and defective genomes. RNA rendered noninfectious by the mutagenic effect of the compound could be packaged and released in serum,
and expression of defective antigens accompanied by reduced immune reaction may lead to
reduced cellular damage and the observed fall in
ALT levels (67). On the other hand, ribavirin has
been shown to totally inhibit an exclusively Th2mediated HBe-antigen specific immune response
in transgenic mice, and such a suppressive effect
may also partly explain the observed normalization of ALT-levels in HCV-infected patients
in the absence of a decrease in viral load.
Ribavirin seems to affect humoral immune
responses during chronic HCV infection, with
transient decrease in both anti-HCV-core antibodies and anti-NS3 specific IgG (66). In any
case, the simultaneous lack of effect on serum
levels of HCV RNA suggests that the decrease in
antibodies occurs independently of the virus
load, at least as measured by RT-PCR.
The overall effect of ribavirin in vitro seems to
be immunosuppressive. However, it does not have
a generally immunosuppressive effect on B cells,
but rather seems to act selectively. This is evidenced by its effect on shift in IgG subclasses in
transgenic mice, where a dose-dependent decrease
of IgG1 (negatively regulated by Th1-derived cytokines) and an increase in IgG2a (positively
regulated by Th1-derived cytokines) has been
shown (68). It would seem, therefore, that ribavirin influences antibody levels or subclass distribution through its effects on other immune
cells, such as regulatory T cells. A shift towards a
Th1-like immune response does not necessarily
imply a reduction of humoral responses in a
mixed Th1-/Th2-like polyclonal immune response, as both Th1- and Th2-like cytokines promote humoral responses. Nevertheless, the shift
towards a Th1-response fits well with the observed
responses in patients who clear the virus.
In conclusion, ribavirin may be viewed as an
immunomodulating rather than a strictly antiviral compound in the treatment of chronic
HCV-infection.
IFN/ribavirin synergy
When combining IFN and ribavirin, synergistic
effects in clinical efficacy appear, exceeding that
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of the individual monotherapies (69, 70).
Whereas PEG-IFN monotherapy is associated
with an overall sustained viral response (SVR)
rate (i.e. no detectable virus 6 months after cessation of treatment) in 30–39% of treated patients (71, 72), and ribavirin alone has virtually
no impact on viral clearance, the combination
therapy results in SVR rates of 42–80% (73, 74).
This may be due to the effect of ribavirin in determining the adaptive immune response bias
towards a Th1 cytokine profile, and this effect
acting synergistically with the effect of IFN-a. It
may also partly be explained by the multistep
antiviral mechanisms of ribavirin, especially the
mutagenic effect. Ribavirin may slow down the
HCV replication and impair its infectivity, while
the accompanying IFN-a antiviral effects may
be needed for clearance of the virus. Whatever
the mechanisms, adding ribavirin with PEGIFN is the key to success in the treatment of
chronic HCV.
FUTURE PERSPECTIVES
New antiviral drug designs
With an SVR rate of 40–80% for the susceptible
genotypes, there is an urgent need for other
treatment strategies. The access to culture systems for primary HCV isolates (21) and chimeric
mice with ‘human livers’ (17) for evaluation of
drug metabolism (human cytochrome P450),
pharmacokinetics and toxicity has dramatically improved the design of new specific
anti-HCV drugs (75, 76). Several companies are
currently developing specific, small-molecular
antiviral HCV drugs, such as inhibitors of HCV
protease, polymerase and NS5A. One example is
the protease inhibitor boceprevir that has advanced into clinical phase II trials (77). However,
the efficiency of monotherapeutic strategies is
questionable. Lessons learned from the antiviral
therapy of HIV-1 infection are that combinations of non-cross-resistant classes of drugs are
key to successful antiviral therapy (78).
Perspectives for development of a vaccine
There is evidence that chimpanzees that have
cleared a primary infection can be protected
against re-exposure to the same virus, and that
protection is associated with a rapid, multi-antigen
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memory T-cell response (79). Alternatively, a new
infection leads to rapid control (80). Moreover,
chimpanzees that recovered from a genotype 1 infection were protected from challenges with mixtures of four genotypes and complex inocula
exhibiting up to 30% amino acid divergence (81).
The rapid clearance of the challenge virus was associated with demonstrable increases in intrahepatic interferon stimulating gene transcripts
in liver biopsies. However, another study showed
that the protection may not resist repeated challenges. After five homologous (genotype 1a) and
six heterologous (genotypes 1b or 2a) rechallenges
the chimpanzee finally became persistently infected by the original virus (82).
With respect to HCV infections in injecting
drug users, there are contradicting reports. Some
studies find that drug users who had cleared initial HCV infection remained uninfected despite
ongoing exposure, or if reinfected the magnitude
of viremia and frequency of viral persistence was
significantly lower than in controls (83). Other
studies show a high incidence of HCV reinfection in injecting drug users, which would indicate no increased immunity against further
infections (84).
Major hurdles in vaccine studies have been the
lack of small animal models and the inability to
grow HCV in vitro. These difficulties have partly
been overcome by use of mouse models reconstituted with human liver cells (16, 85), tissue
culture systems allowing replication of primary
HCV isolates (21) and testing for neutralization.
Remaining difficulties are associated with the
great genetic variability and mutation rate of
HCV. Many linear and conformational epitopes
targeted by nAbs have been identified in the E1
and E2 glycoproteins near the HVR1 (86). In a
very elegant study, the impact of nAbs on the
HVR1 was shown in serum samples collected
from one infected individual over 26 years. It
turns out that nAb responses always lag behind
the rapidly evolving quasispecies population
(87). In spite of this, there is a growing number
of vaccine candidates under development [reviewed in (88, 89)]. There are three approaches
for HCV vaccine design: (1) to prevent initial
infection (sterilizing immunity), (2) to prevent
viral persistence, and (3) to improve virologic
response rates in chronic infection (therapeutic
vaccines). Of particular interest is the use of
nAbs as therapeutic vaccines and antiviral drugs
r 2009 The Authors Journal Compilation r 2009 APMIS
for potential treatment of liver transplant patients to prevent HCV from reinfecting the
transplant (90–92). One of these broadly reacting nAbs competes directly with binding to the
cellular receptor CD81 (93).
CONCLUSION
The partnership between HCV and its human
host has evolved over millennia, whereas we
have only known the virus for a couple of decades. The virus displays numerous mechanisms
to evade the host defence, and there is still much
to be learned about the regulatory and effector
mechanisms of the host immune response. An
efficient, multispecific and vigorous T-cell response at the early stage of infection is critical to
clear the virus. The vaccine issue is complex and
at present the outlook is not very heartening
owing to the extreme adaptability of the virus.
Nevertheless, rapidly increasing knowledge of
viral mechanisms and strategies and accompanying progress in therapeutic measures hold
promise that man in the foreseeable future will
equalize the millenial lead of the virus.
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