FAILURE OF IMMUNITY AGAINST HEPATITIS C VIRUS:
IMPLICATIONS OF DEFECTS IN EARLY IMMUNITY
Job Fermie, August 2012
Supervisor: Dr. D. van Baarle
Masterthesis, Infection & Immunity
About the cover
A 3D interpretation of the Hepatitis C Virus virion, the causative agent of hepatitis C. Visible in orange are the
glycoproteins required for viral adhesion and cell entry. Shown in blue and purple are the viral envelope and
nucleocapsid structure, respectively. The viral genome is contained within the nucleocapsid. Image courtesy of
the scientific illustrator Russel Kightley.
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ABSTRACT
The hepatitis C virus (HCV) is an upcoming human pathogen. Although HCV infection is mostly
asymptomatic, persistent infection may lead to liver failure or liver cancer. Research into HCV has shown
that the immune response against HCV differs from more 'classic' immune responses observed against other
viral pathogens, as human immunity often fails to clear HCV infection. One of the striking observations is
that T cell induction, a crucial process in anti-HCV immunity, is delayed by 8-10 weeks in comparison to T cell
immunity against other viruses. Research suggests that this delayed and aberrant immune response is
mostly the result of events during the initiation of the antiviral response, but the total picture on this regard
remains mostly unclear. This thesis will focus on the processes involved in early immunity against HCV, and
how failure of these processes leads to the delayed and disturbed immune response that is seen in patients.
Job Fermie, August 2012
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TABLE OF CONTENTS
Abstract .................................................................................................................................................................. 3
Introduction: ........................................................................................................................................................... 5
Historical perspective ......................................................................................................................................... 5
Molecular biology of HCV ................................................................................................................................... 5
HCV pathology .................................................................................................................................................... 5
Unsuccessful clearing of HCV: failure of human immunity .................................................................................... 6
Failure of T cell immunity ................................................................................................................................... 6
Innate immunity: ................................................................................................................................................ 8
Pattern recognition ........................................................................................................................................ 8
Natural killer cells .......................................................................................................................................... 9
Dendritic cells............................................................................................................................................... 10
Challenges in clearing HCV ................................................................................................................................... 11
Reactivation of anti-HCV immunity .................................................................................................................. 11
Prevention of HCV infection ............................................................................................................................. 11
Discussion ............................................................................................................................................................. 12
References ............................................................................................................................................................ 14
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INTRODUCTION:
HISTORICAL PERSPECTIVE
HCV was first identified in 1989, as hepatitis not caused by HAV or HBV1. Currently the disease is most often
transmitted through injection drug misuse, and less frequently through perinatal (mother to child)
transmission or sexual contact, especially during same-sex contact. Previously, HCV was often transmitted
through blood transfusions, although this route of transmission was eliminated with the introduction of
specific screening tools after the discovery of HCV2. Despite best efforts in treatment and vaccine
development, 170 million people over the world are infected with HCV, making it one of the most prevalent
causative agents for hepatitis. Several major genotypes of HCV exist, which are ordered by a number.3 Of these
genotypes, genotype 1 is most common in the western world, causing a large majority of all infections. There is
no real difference in virulence or infectivity between the different genotypes, but they do differ greatly in their
responsiveness to treatment. The variability between strains poses challenges for vaccination, since an
effective vaccine will have to protect against the majority of all strains.
MOLECULAR BIOLOGY OF HCV
HCV is a small (55-65nm) enveloped positive stranded RNA virus, and is a member of the Hepacivirus genus in
the Flaviviridae family.3,4 It has a 10kbp positive single stranded RNA genome encoding a 3010 amino acid long
polyprotein in one open reading frame. So far, one alternative open reading frame has been identified,
although the function of its gene product(s) remains unknown. Viral entry into cells requires interactions
between HCV envelope proteins E1 and E2 and the tetraspanin CD81, a scavenger receptor, as well as
scavenger receptor class B type 1(SR-BI) and the tight junction proteins claudin and occludin5,6. After entry, the
nucleocapsid falls apart and the HCV RNA is released into the cytoplasm, where it is translated. Translation
initiation occurs through an intraribosomal entry site (IRES) and results in a single large polyprotein, which is
processed by both cellular and viral proteases, resulting in 9 separate proteins: HCV core protein, which
functions as the nucleocapsid, the envelope spike proteins HCV E1 and E2, a small protein p7, and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B which aid in viral replication 7,8 (figure 1). After
processing, a replication complex is formed in the cytosol, using NS5B as the viral RNA polymerase, producing
negative stranded copies of the HCV genome, which then serve as templates for new positive-stranded
progeny. The virus particles are then assembled, and bud into internal membranes, giving them their
envelope. Viral secretion from hepatocytes is suspected to use the lipoprotein release pathways, as the virus is
typically associated with VLDL, LDL and HDL particles in the blood of infected patients9,10.
HCV PATHOLOGY
Hepatitis C is usually diagnosed when serum levels of alanine aminotransferase (ALT) rise above normal, 8-12
weeks after initial infection. Around the same time, HCV specific T cells and antibodies start to emerge, and
HCV titers begin to drop. However, in many cases HCV infection is not cleared at this point, and approximately
70-80% of patients go on to develop chronic hepatitis C, which puts these patients at risk for other liver
diseases like liver fibrosis, steatosis and hepatocellular carcinoma.
Cellular immunity is essential in clearing HCV, yet many patients do not establish a robust immune response
against the pathogen. Many suggestions have been made, but it seems that a combination of factors is causing
this deficiency. A striking observation in this matter is the fact that T-cell development seems delayed. HCV
specific T-cells appear in the liver 8-12 weeks after initial infection coinciding with a rise of serum IFN-γ levels,
whereas T cells usually become detectable in the blood within 4-5 days after a viral infection8. It is currently
uncertain why T cells are so heavily delayed, and it is also unknown why T cells are unable to clear the
infection after their late induction. Humoral immunity seems less important for HCV clearance, as neutralizing
antibodies are not crucial for resolving HCV infection. HCV-specific antibodies do appear in infected
individuals, and appear to apply selective pressure on HCV, as escape mutations in sequences targeted by αHCV antibodies do appear. However, HCV can be cleared by patients with antibody deficiencies, suggesting
that cellular immunity is the key in resolving HCV infection.
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FIGURE 1 GENOME ORGANIZATION OF HCV AND THE RESULTING GENE PRODUCTS. THE HCV RNA IS TRANSLATED AS ONE POLYPROTEIN,
WHICH IS THEN CLEAVED BY BOTH CELLULAR AND VIRAL PROTEASES INTO HCV PROTEINS. HCV P7 HAS BEEN SUGGESTED TO BE AN ION
CHANNEL AND IS IMMUNOGENIC. FIGURE ADAPTED FROM REHERMANN ET AL.7
UNSUCCESSFUL CLEARING OF HCV: FAILURE OF HUMAN IMMUNITY
FAILURE OF T CELL IMMUNITY
HCV specific CD8+ T cells are essential for clearing HCV infection. They are the main effector cell type for the
clearance of intracellular pathogen. Through recognition of non-self peptides presented by MHC class I
proteins they are able to detect and kill infected cells. Despite the fact that T cells are essential for clearing
HCV infections, they fail to do so in the majority of infected individuals. In HCV infections, T cell immunity is
very delayed compared to many other viral infections, with HCV specific T cells emerging in the periphery
several weeks after infection instead of several days. It takes 8-12 weeks for virus-specific T cells to emerge,
which coincides with an increase in serum ALT levels and higher levels of IFN-γ, as well as a decrease of viral
load7. It is not understood why T cell induction is so heavily delayed, and how this delayed induction relates to
the development of chronic infection. Several mechanisms have been suggested as to why T cells are so
heavily delayed and why they are not effective in clearing HCV.
A striking observation is that HCV specific T cells undergo massive apoptosis shortly after HCV infection. T cells
in the blood display high levels of the receptor programmed death 1 (PD-1) and are highly susceptible to
apoptosis11. These cells display markedly reduced functional capability in a process described as stunning,
during which T cells exhibit defective proliferation, reduced cytotoxicity, and reduced cytokine production 12.
This decline in T cell functionality seems to occur independent of disease outcome13, but the recovery of T cell
function only occurs in individuals that eventually clear HCV infection 14. These stunned T cells may be the
result of the high antigen loads early in infection. This state must be overcome, after which the infection can
be cleared. Although this hypothesis has been regarded as a solid explanation to the late induction of HCVspecific T cells, the results supporting this hypothesis have been obtained from patient material, which has
been cultured prior to experimentation15. As culturing of activated antigen-specific T cells is a difficult and
tedious process with high losses of cells, it seems likely that results based on these culturing assays are not
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always fully representative of the in vivo situation. On the other hand, cells obtained from chronically infected
patients show very similar deficiencies compared to the ‘stunned’ T-cells16. These T cells are characterized as
exhausted T-cells, signified through their inability to produce effector cytokines like TNF-α and IFN-γ, defective
proliferation and loss of cytolytic function17. These cells can be characterized by surface expression of the
inhibitory receptors PD-1 and Tim-3, both of which are present on T cells during chronic HCV infection 16,17,18.
These observations would suggest that the stunning seen during acute infection might be the onset of the T
cell exhaustion seen later during chronic infection. In patients with resolving infection, PD-1- and Tim-3- CD8+ T
cells greatly outnumber PD-1+ and Tim-3+ CD8+ T cells, reaffirming that these T cells are pivotal in clearing HCV
infection14,19. Therefore, the level of PD-1 and Tim-3 expressing T cells may serve as a reliable risk indicator to
determine if a patient is at risk for developing chronic infection. PD-1 and Tim-3 may also serve as therapeutic
targets, as blockade of these receptors partially reverses T cell exhaustion. 18
Although stunning and exhaustion of T cells is regarded as one of the keystones of T cell failure in HCV
infection, other factors have also been suggested. The priming of T cells may be important in this regard, as
this process is essential in determining the specificity and intensity of an immune response. Experimental
evidence shows that HCV core protein is able to impair in vitro priming of T cells by both hepatocytes and
dendritic cells, which even leads to increased IL-10 production in the primed cells, showing a phenotype similar
to that of regulatory T cells20,21. Combined with a relatively tolerogenic hepatic environment, it is likely that
HCV induces and exploits priming defects in order to increase its survival in the liver 22. Defects in priming will
also be examined in more detail below.
It was also suspected that HCV escapes immune surveillance by defects in T cell recruitment to the infected
tissue. However, research in chimpanzees suggests that recruitment of T cells is not defective, as levels of
chemokines CXCL10, CXCL11, CCL4 and CCL5 all increased within one month of infection 15. T cell recruitment
was not defective, as T cells entered the liver immediately after their emergence in the blood. Interesting
enough, experimental infection in chimpanzees shows remarkable similarity to infection in humans: T cell
induction was also delayed in these animals, and similar to human infection, a serum ALT peak could be
detected between 10 and 12 weeks after infection.15 This reinforces the hypothesis that T cell failure can be
attributed to a defect in T cell induction, and not to factors influencing T cell recruitment.
A crucial part of functional T cell immunity is the correct presentation of parts of viral proteins (epitopes) on
MHC class I complexes on infected cells. The proteasome of a cell will digest proteins in a cell to peptides of 810 amino acids long. These peptides are transported to the ER, and loaded onto MHC I, and presented on the
cell surface. Antigen-specific T cells can recognize these complexes, and kill infected cells if non-self peptide
sequences are detected on cells. Defects in antigen presentation on MHC I will reduce the chance of correct
recognition of infected cells, and allow the virus to persist in its host23. Due to failure of T cell immunity in HCV
patients, this process may be key to explaining this failure. Many viruses such as the Epstein-Barr virus (EBV)
and herpesviruses express genes that target parts of the MHC machinery in order to stay invisible to immunity,
including MHC loading, processing and trafficking of MHC to the cell surface23. Until now, no such factors have
been determined in HCV. However, HCV’s genome structure allows a different strategy to avoid being
detected through MHC. HCV’s genome is RNA based, which is inherently less stable than a DNA genome.
Combined with a high replication rate and an error-prone RNA-replicase HCV NS5B, the virus has a high
mutation rate24,25. This allows HCV to outpace T cell immunity, which can mainly be attributed to the
development of a viral quasispecies in each infected individual. This poses the risk that a T cell response
develops too narrowly and fails to target all HCV variations. A second problem arising from the high mutation
rate of HCV is the speed at which escape mutations may develop: even after a successful T cell response
against the quasispecies present in an individual, mutated copies of the HCV genome may still escape from
immune surveillance26. This mechanism gains part of its potency from the locations where mutations develop:
reports suggest that HCV mutations occur in the ‘anchor’ regions of an epitope, the amino acids that are
essential for successful loading of peptides24. Mutations of HCV proteins in areas involved in proteasome
processing have also been suggested, which will lead to further degradation of HCV peptides, instead of
loading onto MHC class I. Thus, to successfully clear HCV, a T cell response needs to be broadly directed
against multiple epitopes in the HCV genome, which will ensure that HCV does not escape adaptive immunity.
Even though it is clear that a defective T cell response is a hallmark of failure to clear an HCV infection, less is
known about the underlying factors of the defective T cell response. It is uncertain why T cell development is
so heavily delayed, and why HCV infection becomes chronic in such a large part of infected individuals.
Although part of the problem can be attributed to defects in T cells themselves, it is likely that the aberrant T
cell behaviour can be attributed to earlier immune processes, such as the priming of T cells by DCs in the
lymphatic tissue and the liver, or the ability of innate immunity to manage the viral load in the liver. Failure of
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any of these processes is likely to affect the process of adaptive immunity, and may in the end lead to chronic
HCV infection.
INNATE IMMUNITY:
PATTERN RECOGNITION
The first step in innate immunity is recognition of a pathogen through pattern recognition receptors (PRR).
These receptors are capable of sensing structurally conserved molecules such as double-stranded RNA
(dsRNA), lipopolysaccharide (LPS) and other molecules not found in the healthy host, and are present on many
different cell types of the body. Recognition of these molecules activates these receptors, which leads to the
initiation of the innate immune response. For HCV, the most important PRRs are Toll-like receptors (TLR) 3, 7
and 8 and retinoic acid-inducible gene I (RIG-I)27,28. These receptors recognize viral single stranded (ss)RNA and
dsRNA, a prerequisite in the replication of the HCV genome. Activation of PRRs leads to the translocation of
NF-kB and interferon regulatory factor 3 (IRF3) to the nucleus, where they induce the transcription of
interferon (IFN)-β. Secreted IFN-β is a potent inducer of an antiviral state in neighbouring hepatocytes.
Through the JAK-STAT signalling pathway, the IFN-stimulated gene factor 3 (ISGF3) complex is formed, which
translocates to the nucleus, where it induces the transcription of IFN stimulated genes (ISGs).8 Transcription of
these genes upregulates IFN-α and IFN-β production as well as the expression of major histocompatibility
complexes (MHC) I and II, thereby increasing antigen presentation and the chance of killing infected cells by
cytotoxic T cells and NK cells. Another essential function of IFN-β is the inhibition of viral replication. Among
the ISGs are the OAS1/RNAse L system, ADAR1, p56, and PKR.27,29 Their gene products are capable of
destabilizing and degrading RNA structures and phosphorylating eukaryotic translation initiation factors, which
limit viral replication. Induction of the IFN response also further increases IFN production, thereby amplifying
the entire response via a positive feedback loop.
However, HCV has developed resistance against the IFN response, by interfering with the processes on several
key points. Part of this process is mediated by the HCV NS3/4A serine protease, which is able to block Toll-IL-1
receptor domain-containing adaptor inducing IFN-β (TRIF), an important adaptor protein in the signalling
through TLR3. TRIF cleavages blocks the translocation of both NF-kB and IRF3, thereby preventing IFN-β
production30,31. HCV also interferes with the response to IFN-β: HCV core protein is able to inhibit JAK-STAT
signalling as well as reducing the transcriptional activity of ISGF332. Other gene products of the ISGs are also
targeted by HCV: HCV NS5A inhibits 2’-5’ oligoadenylate synthetase (2’-5’ OAS), thereby blocking RNA
degradation33. Both HCV E2 and NS5A target protein kinase R (PKR)34. E2 acts as a decoy target for PKR35,
whereas NS5A heterodimerizes with PKR33. Both of these interactions block the ability of PKR to phosphorylate
initiation factors, thus reducing the translation inhibition normally caused by PKR activity. The IRES of the HCV
genome also makes the HCV genome less sensitive to phosphorylation of translation initiation factors,
meaning that HCV is partially resistant to the effects of the IFN signalling 36. The attenuation of IFN production
not only means that HCV is able to reproduce without interference from the antiviral state in the liver, it also
means that cytokine production in the liver is disturbed. Since disturbance of signalling through IFN receptors
results in reduced production of pro-inflammatory cytokines, the correct inflammatory environment required
for HCV clearance may not be achieved, resulting in the mismanagement of disease.
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FIGURE 2 HCV ATTENUATES THE IFN-Β-INDUCED ANTIVIRAL STATE THROUGH SEVERAL DISTINCT MECHANISMS. FIRST, HCV GENE
PRODUCTS WILL ATTENUATE IFN RECEPTOR SIGNALING. HCV PROTEINS ALSO INHIBIT GENE PRODUCTS OF THE IFN RESPONSE. IMAGE
ADAPTED FROM [8].
NATURAL KILLER CELLS
A cell type thought to be of importance in the defence against viral pathogens is the natural killer (NK) cell. As
part of the innate immune system, these cells serve as early guardians against infection. Contrary to CD8+ T
cells, NK cells display nonspecific cytotoxicity against tumour cells as well as virally infected cells. NK cell
cytotoxicity is mediated through mechanisms similar to those seen in CTLs. NK cells are able to induce FasL-Fas
mediated cell death, and carry large granules containing perforin and granzyme, but unlike in CTLs, prior
activation is not necessary to produce these granules. This ability allows them to control viral replication
during the time needed for T cells to develop and emerge from lymphatic tissue, a period normally taking
about 7 days. NK cells are regulated through activating and inhibitory receptors. Activation may occur by
ligands on host cells, and by proinflammatory cytokines, which include IFN-α, IFN-β and IL-1237. Inhibition is
mainly achieved through the presence of MHC molecules on host cells, which are recognized by killer-cell
immunoglobulin-like receptors (KIR) and provide a powerful inhibitory signal to prevent killing of cells. Since
absence of MHC on nucleated cells is usually an indicator of viral infection, regulation of NK activity through
MHC provides an easy, regulated mechanism for killing by NK cells. Due to these properties, it seems likely that
NK cells are an important factor in determining HCV outcome.
A first problem for NK cell functionality during HCV infection may be the reduced cytokine production of
infected hepatocytes, such as the inhibition of IFN-α and IFN-β production in infected hepatocytes. Normally,
NK cells are activated by cytokines produced by infected cells or DCs present at the site of infection. Since HCV
is able to manipulate the cytokine balance in the liver by interfering with the interferon pathway in
hepatocytes, it seems likely that NK cells receive less stimulation from the infected tissue, thus reducing NK
cell activity.38,39 Subsequently, reduced NK cell activity may induce defects downstream in the development of
adaptive immunity, since NK cells are partially responsible for the induction of a proper inflammatory
environment in the liver.
HCV gene products may also directly manipulate NK cell function. Experimental evidence shows that at high
levels, recombinant HCV E2 protein is able to crosslink the tetraspanin CD8140, thereby inhibiting cytotoxicity
and cytokine production in NK cells. However, this observation could not be reproduced using HCV virus
particles, which leaves the importance of this mechanism unclear in vivo. Not all mechanisms examined
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require NK cell infection: under experimental conditions, contact with infected hepatoma cells seems enough
to alter NK cell functionality, with NK cells showing diminished ability to lyse infected cells and displayed a
downregulation of activating receptors on their cell surface.41 Crosslinking of CD81 has been suggested as the
mechanism behind this downregulation, despite contradictory observations. However, expression of viral
glycoproteins may have a different effect than the presence of E1 and E2 assembled in viral particles. In
addition to the reduction of cytolytic effects, cytokine production is also manipulated: during HCV infection, NK
cells are able to produce IL-10, which has been suggested to be an effect of HCV NS5A.38
NK cells also seem more directly involved with the development of adaptive immunity. Recent research shows
intricate interactions between NK cells and adaptive immunity, attributing a regulatory role to NK cells in this
process42. Experimental evidence suggests that NK cells are able to suppress T cell immunity in order to
prevent damage to the host. Since inflammatory and immune responses are frequently processes that can
damage healthy host tissue, tight regulation is essential for preventing excessive host damage. Indeed, NK celldeficient mice are at risk to die from excessive IFN-γ production and immune pathology after a non-lethal viral
infection, suggesting the importance of this regulatory mechanism. 43 However, NK cell-mediated immune
suppression can also be counterproductive in infection: in experiments using lymphocytic choriomeningitis
virus (LCMV) in mice, NK cell depletion through neutralizing antibodies results in an enhanced T cell immune
response, and the animals clear the infection earlier than animals with normal NK cell levels 44. Interestingly, NK
cell depletion also prevents chronic LCMV infection44. Although it may be hard to translate these results to
HCV infections, it is tempting to suggest that NK cells are in part responsible for the later induction of T cells
and possible chronic infection. Several mechanisms of regulation have been suggested, the most remarkable
being the killing of CD8+ T cells through a perforin-mediated mechanism. Interestingly, blockade of this
mechanism may be beneficial for clearance of HCV or other pathogens, as perforin-deficient mice showed
higher T cell activity and cleared LCMV earlier than their wildtype counterparts 44. Other inhibitory mechanisms
may include the production of anti-inflammatory cytokines, as it is known that NK cells are able to produce IL10, an important cytokine in regulating immunity. This observation is supported by the fact that NK cells from
chronically infected individuals produce IL-10, which indicates that NK cells may suppress immunity in an
attempt to prevent immunopathology42 in HCV infection although the production of IL-10 might also be the
result of manipulation by HCV gene products38,42,45. Thus, NK cells serve an immunoregulatory role in adaptive
immunity, with both beneficial and detrimental effects on the course of HCV infection. Regulation of T cell
activity prevents immunopathology after HCV infection, but may also lead to impaired T cell development and
the development of chronic HCV infection.
DENDRITIC CELLS
Together with NK cells, dendritic cells (DCs) are essential for linking innate and adaptive immunity. Immature
DCs constantly sample their environment to survey for possible pathogens. Upon antigen detection, DCs will
mature, drastically improving their ability to present the captured antigen and cytokine production. The
antigens are presented to cells of the adaptive immune system, which helps the adaptive immune system to
adequately respond to pathogens. Dendritic cells can be separated into two main classes, myeloid and
plasmacytoid DCs46. Myeloid DCs (mDCs) are mainly characterized by their ability to produce IL-12 upon
stimulation, while plasmacytoid (pDCs) are known for their ability to produce large amounts of IFN-α, an
important cytokine in the development of a Th1-type adaptive immune reaction47,48. The link to adaptive
immunity provided by DCs is an interesting target for HCV. Disrupted DC signalling will result in a delayed
adaptive immune response, and will most likely be a factor in establishing chronic HCV infection. During HCV
infection, DC functionality seems to be altered. pDCs are enriched in the liver of individuals infected with HCV,
but these cells appear to be defective in IFN-α production, which disturbs their function in immunity49. The
same is seen in MDCs: the functionality of these cells appears to be lower than normal 46. It seems likely that
DCs are targeted by HCV in an attempt to disturb the bridge between innate and adaptive immunity. Several
mechanisms have already been observed. For example, the HCV gene products NS3 and core protein induce
the production of TNF-α through TLR2 stimulation, which inhibits IFN-α production, and induces apoptosis in
these cells8,20,50. Interesting enough, infection does not seem to be necessary for this effect, suggesting
another mechanism to trigger this effect. This is further supported by the observation that DCs do not express
claudin-1, and that HCV cannot replicate in these cells in vitro.51 Other mechanisms in DCs also appear to be
defective or inhibited as a result of HCV infection: in experimental setups, DCs carrying plasmids encoding HCV
gene products like HCV core, E1 or E2 produce less IL-2 and present less antigen through MHC class II, making
these cells less effective or defective at priming and activating CD4+ T cells 46. Another striking observation is
that after incubation with the serum of HCV positive individuals, dendritic cells will become unresponsive to
CCL21, the chemokine recruiting DCs to lymphatic tissue for T cell priming 40. Impaired recruitment of DCs will
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result in less T cell priming and activation, leading to a lower chance of clearing HCV infection. Important to
note is that most of these observations are in vitro, so it may be hard to translate these observations to an in
vivo situation. Combined with the fact that many experiments using DCs from patients are from patients who
have developed chronic hepatitis C infection, the importance of DC dysfunction in the development of chronic
HCV infection remains under investigation.
CHALLENGES IN CLEARING HCV
REACTIVATION OF ANTI-HCV IMMUNITY
With the high rate of patients unable to clear HCV by themselves, the development of an easy and affordable
treatment plan has a high priority. Currently PEGylated IFN-α is used as the standard treatment in patients
with chronic HCV infection, which is mainly used to ‘reactivate’ the immune system, causing partial reversal of
exhaustion in T cells52,53, and a boost of NK cell functionality 54. However, many patients (about 50%) do not
respond to treatment when chronically infected, and the treatment has many side effects. Despite efforts to
analyse the mechanisms stimulated by IFN-α treatment, it remains unclear why the administration of
exogenous IFN-α induces clearance of HCV, whereas endogenous IFN-α production appears to have little to no
effect on HCV clearance. The exact mechanisms behind IFN-α treatment are uncertain, but work in the
genetics field has given suggestions to how this mechanism would work. Response to IFN-α is largely
determined by a single nucleotide polymorphism (SNP) in IL-2855, which heavily correlates with treatment
outcome. Currently, the information of IL-28 polymorphisms is combined with other genetic factors, which
have mostly been associated with the ability of NK cells to manage and clear HCV infections. Several
combinations of polymorphisms have been implicated in the outcome of HCV infection: these compound
genotypes will have impact on the ability of the individual to clear an infection. For example, KIR2DL3 (killercell immunoglobulin-like receptor 2DL3), KIR2DS3 and several group 1 HLA-C alleles have been correlated with
the chance of clearing HCV infections44,56,57. The functional implications of these compound genotypes are not
fully understood, but it is suggested that favourable compound genotypes reduce the activation threshold of
NK cells55,57–59. Following this hypothesis, NK cells with an unfavourable compound genotype will have a higher
activation threshold and a worse reaction to treatment. Although the information obtained from these SNPs is
far from conclusive, they at least provide some explanation to the question of how exogenous IFN-α restores
immunity. Unfortunately, it does not provide any information as to why the body’s own IFN-α production is
insufficient to induce a successful antiviral response.
Improvement could also be made on earlier HCV detection and treatment, although this poses a challenge for
appropriate treatment: in the past, IFN-α treatment during acute infection has had an adverse effect on
patients, causing chronic infection in these patients instead of clearance. 60 This is not necessarily a problem,
although it means new treatment strategies will have to use a different approach. Currently, research is
looking into protease inhibitors, specific for HCV. Although this treatment strategy has had some merit in the
combat against HIV, protease inhibitors against HCV face the same issue as with HIV: the high mutation rate of
these viruses increases the risk of treatment resistance. Other strategies may also be worth looking into,
especially for chronically infected patients: although rare, reversal of T cell exhaustion has been reported in
patients that clear HCV infection spontaneously after years of chronic infection61, making this a viable strategy
to look into. Indeed, in vitro blocking of inhibitory receptors Tim-3 and PD-1, which are markers of T cell
exhaustion reversed proliferation blockades and restored cytokine production of the T cells tested18,19,62. Other
mechanisms will also be worth looking into, such as the immunoregulatory role of NK cells: depleting these
cells in mice lowers viral titers in the blood, and enhances their antiviral T cell responses44. A challenge in this
approach will be to find the level of depletion or NK cell suppresion that is acceptable, as this mechanism also
protects the host from immunopathology, meaning that too much NK cell suppression will lead to excessive
damage in the host.
PREVENTION OF HCV INFECTION
Vaccination is a promising, but very challenging strategy for management of HCV: due to the diversity of the
different HCV genotypes, HCV vaccines will have to induce immunity for all genotypes in order to be effective.
Experiments in monkeys suggest that vaccinations would mainly need to focus on T cell immunity, as antibody
levels start to decrease in the years after successful clearing of HCV, whereas HCV specific memory T cells
remain present for years after infection. Multiple vaccination strategies with different HCV targets have been
11
attempted. These include inactivated viral particles, viral ‘pseudoparticles’ containing HCV proteins E1 and E2
assembled in lipid particles, as well as recombinant vaccines targeting E1 and E2, or HCV core and nonstructural proteins63,64. Most of these vaccines are well tolerated and have effects on viral titers and serum ALT
levels, although they usually do not induce clearance when tested in chronically infected volunteers.63 When
tested in naïve chimpanzees, vaccination is often also not fully protective, as HCV viremia still manifested itself
when the animals were infected after vaccination, and in some cases still led to chronic infection. However, in
animals resolving HCV after vaccination, HCV was cleared earlier without ALT peak and lower viral titers were
seen during the course of infection, similar to rechallenge after a cleared infection65. Although these results
are promising, current vaccination strategies are not effective in preventing infection such as in other vaccines,
but at least speed up recovery and ameliorate HCV symptoms. Future challenges will involve increasing
protection against different HCV genotypes, as well as fully preventing viremia.
In addition to the classic vaccination strategies, DC vaccination has also been suggested as a strategy to
combat HCV infection. By expressing HCV fragments on DCs, these cells are able to stimulate adaptive
immunity, to get a proper immune response against the virus. In this strategy, DCs may be pulsed ex vivo with
HCV pseudoparticles or transduced with viral gene products, which are then processed and presented by the
DCs66. These cells are then transferred back into the host, which hopefully starts an immune response against
the virus. Early results in this strategy seem positive, as mice vaccinated in this fashion against HCV NS3
develop a T cell response against several different epitopes. 67 This strategy may also be promising in fighting
chronic HCV infection. Early results in humans show that this strategy is a safe possibility to treat HCV,
although HCV clearance was not achieved. ‘Vaccinated’ patients developed broad HCV-specific CD8+ T cell
responses after receiving DCs pulsed with lipoprotein particles containing HCV glycoproteins. This
development did not alter viral load or ALT levels, and the T cell response, measured by IFN-γ production,
could not be sustained over time.68 Thus, new targets or adjuvants for DC vaccination against HCV are still
highly sought after. However, despite its potency, DC vaccination is a costly and time-consuming process, as
DCs have to be isolated from the patient’s blood, treated with the vaccine particles, and administered back.
This will limit its use for mass vaccination, as the ideal vaccination should be able to be easily administered in
underdeveloped countries. This does not mean that DC vaccination is a dead end: the strategy may still have
merit in resolving chronic infections. Current treatment strategies based on PEGylated IFN-α and antivirals
require a long and intensive treatment schedule, which is expensive and can have severe side effects. DC
vaccination as a treatment to the disease will be less intensive for the patient and is likely to have far less side
effects, thus being a great step forward compared to current treatment strategies.
DISCUSSION
HCV’s ability to develop viral persistence remains an interesting field of research. Based on current knowledge,
it looks like the development of chronic infection is caused by failure in both the innate and adaptive immune
system. Normally, viral pathogens are cleared within two weeks, in a coordinated process of innate and
adaptive immunity (figure 3). Early viral replication is kept under control by innate immunity, by a combination
of IFN production in the infected tissue, and an influx of NK cells shortly after. Within 4 to 5 days post
infection, virus-specific cytotoxic T lymphocytes become detectable, after which viral titres drop rapidly.
However, HCV shows a completely different course of infection, in which it is known that T cell activity remains
low up to 12 weeks instead of several days69. What happens with other cell types, such as NK cells and DCs, or
the production of interferon is unknown for the course of HCV, due to limited availability of valid animal
models. Based on in vitro data currently available, it seems that T cell deficiencies in HCV infection are the
result of an accumulation of deficiencies early in the immune response, leading to the characteristic
phenotype seen in many patients. Deficiencies in innate immunity, such as attenuation of interferon
production by HCV and disturbed NK cell activity, will lead to failure of innate control over the viral loads, and
provide a ‘wrong start’ situation for the adaptive immune system, where priming activity of DCs is reduced,
and the appropriate cytokine balance is not realized. Combined with immune evasion strategies of HCV, this
leads to the inability of the host to clear HCV, although it remains uncertain how all different deficiencies in
innate immunity form the final complex picture we see after HCV infection in patients.
12
Viral load
IFN-α + IFN-β
NK cells
CTLS
0
1
2
3
4
5
6
7
8
Days post infection
9
10
11
12
13
FIGURE 3 CLASSICAL COURSE OF INFECTION AND IMMUNE RESPONSE. DIRECTLY AFTER VIRAL INFECTION, INTERFERON PRODUCTION
STARTS RISING. COMBINED WITH THE INFLUX OF NK CELLS AT THE SITE OF INFECTION, IFN PRODUCTION SLOWS DOWN VIRAL
REPLICATON LONG ENOUGH TO ALLOW VIRUS-SPECIFIC T CELLS TO DEVELOP, WHICH THEN CLEAR THE INFECTION IN THE FOLLOWING 8-9
DAYS. FIGURE ADAPTED FROM KUBY, IMMUNOLOGY 3RD EDITION, BY T.J. KINDT ET AL.
Research in HCV remains a challenging field, where many in vitro observations have not yet been confirmed as
important during in vivo infection. Since the first stages of infection are mainly asymptomatic, following HCV
infection in its early stages in human patients is a very challenging task, if not an impossible one. Thus, animal
models are required in order to fully understand the viral dynamics, which is problematic due to the fact that
currently, the only suitable animal model requires chimpanzees. New animal models in mice or other small
mammals would be a great step forward in researching HCV infection, although this is hampered by the
species specificity of HCV. Mouse models remain hard to use, since mouse hepatocytes are naturally resistant
to HCV host cell entry and replication, necessitating transgenic mice carrying modified hepatocyte receptors,
or immunodeficient mice carrying human hepatocytes in order to study viral replication. Novel animal models
will be useful for a variety of reasons. First of all, they will help us understand the dynamics of infection,
allowing deeper insight in the replicative mechanism in vivo. They will also allow for better tracking of
infection, perhaps gaining a better understanding on the changes in the state of immunity prior to chronic
infection. Animal models can also be employed to research immunization strategies, although this is mostly
limited to work in chimpanzees due to the same issues plaguing research on HCV’s viral dynamics. Future work
will hopefully arrive at a safe and effective vaccine, as well as treatment that reverse or prevent the issues
seen in the immune system of infected individuals.
13
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