IMMUNE EVASION STRATEGIES OF HEPATITIS C VIRUS

Transcription

1 IMMUNE EVASION STRATEGIES OF HEPATITIS C VIRUS Master Thesis Masters Program Infection & Immunity Kevin Budding, October 2010 Supervisor: Dr. Debbie van Baarle

2 About the front cover: The picture is a fluorescence microscope image of a GFP (Green fluorescent protein) labeled HCV protein, which associates to lipid droplets (red). The lipid mechanism is important for HCV and the association gives an indication of that importance. Picture is courtesy of Mr. Torsten Schaller, Immunology & Molecular Pathology department, University College London, 15 th April 2010 Reference: 1

3 OUTLINE 1. Introduction 3 2. Hepatitis C virus Epidemiology Hepatitis C pathogenesis Genomic organization and protein function Viral life cycle 9 3. The innate immune response against HCV and viral evasion Pattern recognition and NS3/4A Interferon- β signaling and viral evasion Interferon stimulation genes and viral evasion Natural killer cells and HCV The adaptive immune response against HCV and viral evasion The humoral immune response and viral evasion The cellular immune response and dendritic cell dysfunction T cell failure Regulator T cells T cell homing capacity Immune evasion through viral mutations Therapies and vaccination Current and future therapies Vaccine development References 28 2

4 1. INTRODUCTION In this thesis I will discuss the tug of war between hepatitis C virus (HCV) and our innate and adaptive immune system. A chronic HCV infection is the leading causative agent for liver cirrhosis, and currently over 200 million people are infected worldwide. According to epidemiological studies, HCV infects around three till four million people per year. I will start with elaborating on the epidemiology and pathogenesis of the virus, followed by the viral characteristics of HCV, taxonomy, replication cycle, structure, and genome. The focus of this paper will be on the aspect of viral persistence. In order to understand the immune evasive strategies of HCV I have divided the immunological response in two different components, the innate and adaptive component. I have tried to integrate the type of immune response with the immune evasion strategies of HCV. In the therapy section of the thesis I will elaborate on the currently used treatments, their mechanisms of action, the possible potential for vaccine development, and the reaction of the virus on the various therapeutic compounds. With this thesis I have tried to give a coherent, comprehensive insight into the clinical relevance of an HCV infection, the viral characteristics, the immune responses against HCV and HCVs strategies to evade these, and initiate a persistent infection. Kevin Budding, October

5 2. HEPATITIS C VIRUS 2.1 EPIDEMIOLOGY The hepatitis C virus (HCV) was discovered in 1989 via molecular cloning instead of virus purification 1. The first signs of a different form of hepatitis (known as non- A, non- B hepatitis) were already present around the 1970s 2. Data up to 2005 indicate that the seroprevalence (number of persons who test positive for a specific disease based on blood serum specimens) of HCV is 2%, accounting for 123 million people in the world, making it the major causative agent of chronic liver diseases worldwide 3. Although the prevalence of HCV infection is rather high worldwide, there is geographical spreading Figure 1. Worldwide prevalence of HCV 3. Seen in the seroprevalence of the virus (see figure 1). Although the figure depicted above shows quite a clear figure of HCV seroprevalence throughout the world, with the virus mostly present in both Africa and Asia and less in the developed world. It is very difficult to obtain the data and to interpret this. This is due to the fact that most studies are conducted within non- representative populations, such as blood donors or patients who already suffer from chronic liver disease. However, mathematical models have been used in developed countries to measure the seroprevalence of HCV. Via these models it is shown that in the USA there is an increasing 4

6 seroprevalence of HCV over the past decades, 0-44 per before 1965, increasing to around the 1980s 4. An Australian model based upon new HCV infections also shows a steady increase in new HCV cases over the past decades 5. Although there seems to be a decline in newly acquired HCV infection in the 1990s, al mathematical models used for the prediction of HCV seroprevalence support the idea that this seroprevalence will rise in the coming decades. Transmission of HCV occurs via blood- to- blood contact. Important factors that contribute to this transmission are blood transfusions from unscreened donors, drug injections, and non- sterile therapeutical procedures. The most important factor contributing to HCV transmission in developed countries is the usage of injection drugs. However, in developing countries non- sterile usage of syringes in a therapeutic setting is the primary source of HCV transmission 6. According to studies conducted in the USA the majority of HCV injections, 68%, is caused by injection drug usage 7. An important difference between the transmission of HCV and other viruses spread via blood- to- blood contact is that fewer sharing partners are necessary for a successful transmission of the virus. Also, indirect drug sharing, usage of the same cooker and rinse water, can contribute to HCV transmission 8. Non- sterile injections are the primary source of HCV transmission in the developing world. Often sterile syringes are not available, or the injections are supplied by medical staff that lacks the necessary training and/or education. Another factor contributing to the higher transmission rate in a therapeutic setting is that often medication is injected into the patient, whereas in the western world it would be supplied orally 9. A terrible example of HCV transmission via therapeutic injections is the kala- azar (Leishmaniasis, a parasitic disease) treatment program in India. The HCV infection rate among multiple injected patients was up to 31.1% 10. Another example is the parenteral therapy for schistosomiasis in Egypt. It is believed that the high seroprevalence in Egypt is due to contamination during this therapy program 10. In order to tackle this contribution to HCV transmission in 3 rd world countries the World Health Organization (WHO) has set up the SIGN program (Safe Injection Global Network). Within this network, governments, international health agencies, corporations, and individuals collaborate for safer therapeutic injections 3. The last important factor contributing to HCV transmission is blood transfusion. In the western world the contribution of this factor can almost be neglected due to strict rules and regulations in the all- volunteer donor system. Blood of the donors is tested for multiple blood- borne viruses, including HCV. Figures in the USA show a drop of HCV infections acquired after blood transfusion from 91% to 4% 11. However, in most of the countries in the developing world there are no centralized rules and regulations, and no nation- wide blood- testing program is available. According to figures from the WHO, 43% of the donated blood in third world countries is not screened for HCV infection 3. It has always been believed that In contrast to other blood- born viruses sexual transmission of HCV hardly occurs 12. A 5

7 recent study by van Laar et al. that focused on acute HCV in HIV- infected men shows that that there is a substantial increase in acute HCV infection in men who have sex with men (MSM) who denied injecting drug use. These findings give new insights into the discussion whether or not sexual contact plays a significant role on HCV transmission HEPATITIS C PATHOGENESIS A persistent HCV infection can lead to serious clinical symptoms. When faced with a HCV infection, the majority of the patients will develop chronic liver diseases (50% - 80) 14. Steatosis, increased fat content of the liver, is the first clinical symptom during a HCV infection. When developing steatosis the patient will be able to react less effectively to antiviral therapy, and is prone to develop liver fibrosis 15. Both liver fibrosis and cirrhosis are common liver disorders following HCV infection. Different risk factors influence the development of liver fibrosis, which can eventually lead to liver cirrhosis. These risk factors are age, sex, alcohol intake, diabetes, and co- infection with other viruses. The morbidity and mortality of chronic HCV infection is related to progressive fibrosis and the development of cirrhosis 16. Patients who suffer from chronic HCV infection are prone to develop different types of liver cancers such as hepatocellular carcinoma (HCC). HCC is the fifth most common cancer in the world 17, and chronic infection with HCV accounts for 20% of all HCCs worldwide 18 Also, chronic HCV infection seems to be a risk factor for the development of cholangiocarcinomas, carcinomas that originate from the extra- hepatic biliary tree and the intrahepatic bile ducts GENOMIC ORGANIZATION AND PROTEIN FUNCTION The hepatitis C virus is an enveloped single stranded positive RNA (+ ssrna) virus and it belongs to the family of Flaviviridae. The genome length is 9.7 kb 20. There are six different genotypes. Each genotype is further divided into different subtypes (a, b, c, etc.). The different genotypes are differentiated according to worldwide distribution and sequence variation 21. The genome consists of a single open reading frame (ORF) with 2 untranslated regions (UTR) on both sides. These regions are important for viral protein and RNA synthesis and the coordination of these two processes 22. Since the genome does not have a 5 cap the internal ribosomal entry site (IRES) in the 5 UTR is essential for translation. The IRES of HCV binds to the 40s ribosomal subunit and induces a conformational change within the subunit making it mrna bound like. Due to this conformational change eukaryotic initiation factor (eif) 3 and other essential factors for translation are recruited and the complete complex can than be coupled to the active 80s ribosomal subunit, initiating translation of the ORF 23 (see figure 2). 6

8 The ORF codes for a single polyprotein of 3,000 amino acids. The polyprotein is both co- and post- translationally processed by cellular and virally encoded proteases leading to the formation of both nonstructural and structural proteins, seven and three respectively, ten in total 24, 25. The structural proteins are the conserved core protein and the glycosylated E1 and E2 envelope proteins. The proteins are cleaved via a cellular signal peptidase. The mature core protein is essential in viral nucleocapsid formation and it can be separated in two domains, the N- Terminal D1 and the C- Terminal D2 26. The D1 domain consists of a high amount of positively charged amino acids, and has homology to capsid proteins of other Flaviviridae. Domain D2 is essential for the folding of D1, necessary for membrane characteristics of the core 27. Both E1 and E2 glycoproteins are type- 1 transmembrane proteins with an N- Terminal ectodomain and a C- Terminal transmembrane domain and they form heterodimers. The proteins are of high importance in various steps of the viral replication cycle (detailed explanation in section 2.3), since these proteins are on the surface of the viral particle they will be in first contact with the potential target for infection. In between the loci of structural and non- structural proteins lies the small p7 protein. This is a small integral membrane protein and it seems to function as an ion channel 28. The polyprotein is further cleaved into the following nonstructural (NS) proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Cleavage between p7 and NS2 is also conducted by the cellular signal peptidase. Cleavage of the other NS proteins is carried out by two viral enzymes, the NS2 autoprotease, which cleaves NS2 from NS3, and a NS3-4A serine protease, which cleaves the remaining NS proteins within the ORF 24 (see figure 2). All nonstructural proteins are necessary for the intracellular processes of the viral lifecycle. NS2 is a cysteine protease involved in the cleaving of the NS2 NS3 junction. Besides that it acts as a protease, there is also evidence that it is an essential assembly cofactor during viral particle formation 29. The NS3 protein has a broad variety of functions. It has two active domains, an N- Terminal serine protease domain and a C- Terminal RNA helicase/ntpase domain. The serine protease domain requires the NS4A protein to anchor onto the cellular membrane of the host 30. Other factors are also necessary for proper folding and activation of the serine protease domain. The functional NS3 protein is required to cleave the other nonstructural proteins in the ORF. The function of the C- Terminal RNA helicase is not exactly known. The general thought is that it is involved in the initiation of RNA synthesis on the genomic RNA of HCV. Not much research has been conducted into the function of NS4B, however some researchers proposed that it is a component of a membrane associated cytoplasmic HCV replication complex 31. The specific function of NS5A remains rather obscure since many different host proteins interact 7

9 Figure 2. Schematic overview of the 3,000 amino acid polyprotein of HCV. On top the ORF and both the 5 and 3 UTR are visible. After translation the polyprotein of HCV is generated (middle). The cleavage sites as well as the different structural and nonstructural proteins are also depicted in the middle. The bottom illustration shows the function of the various proteins 22. with this viral protein 32, 33. Within the host cell, NS5A is phosphorylated on multiple serine residues. Among the different cellular kinases responsible for hyperphosphorylation of NS5A are AKT and camp- dependent protein kinase A- α 34. The functional and structural characteristics of the, up to now discovered, three functional domains of NS5A remain unclear, although they might influence the efficiency of RNA replication via NS5B 35. NS5B is the most important gene involved in the viral replication cycle. It encodes the RNA- dependent RNA polymerase (RdRP). The protein synthesizes RNA from an RNA template. Experimental evidence indicates that this synthesis of RNA can occur de novo. This has been shown in vitro, and it is postulated that this also occurs in vitro 36. Since NS5B is a polymerase it shows a high structural homology to other polymerases. It consists of a palm- like structure, where the palm contains the active site and the thumb and finger structure of the enzyme provide stabilization and modulation of contact with the RNA template 37. The total enzyme contains a tunnel- like structure, due to interaction of the finger and thumb domain, where through the single stranded RNA template is brought to the active site on the palm structure of the enzyme. NTPs, building blocks necessary for transcription are brought to the active site of the enzyme via another tunnel- like structure. Final fine- tuning of the transcription process is regulated by a beta- hairpin loop that is present in the thumb structure of the enzyme 38, 38. 8

10 2.4 VIRAL LIFE CYCLE The viral life cycle of HCV consists of five major steps; viral entry, uncoating of the viral genome, translation of the viral proteins, genomic replication, and the assembly and release of the newly synthesized virions. In order for the virus to infect a host- cell it needs to attach itself to this cell. For the attachment at least two components are necessary and extracellular host- cell component and a receptor on the virus itself. For HCV various factors on the host cell seem to attribute to a successful viral entry. The first factor identified was CD81. The peculiar thing about CD81 is that it is present on a broad variety of host- cell types. It is not solely present on hepatic cell types. However, experiments have shown that HCV viral entry is reduced on liver cells that lack CD81, or on liver carcinoma cells with downregulated expression of CD81. The viral glycoprotein E2 is able to bind to CD Before the binding of E2 to CD81 various attachment molecules on the cell surface such as glycosamineglycans (heparine sulphate) 40, and LDLR (low density lipoprotein receptor) 41 mediate this initial interaction. A recent study in showed that CD81 independent viral entry is also possible in vitro 42. Besides CD81 other interactions are necessary for successful viral entry. Research has shown that the human scavenger receptor class B type I (SR- BI) might mediate the HCV entry process 43. After the interactions of E2 with LDLR, SR- BI, and CD81, further steps need to be taken by the viral particle. Interaction between CD81 and tight junction protein claudin- 1 (CLDN- 1) is essential for viral entry 44. Interactions between CD81 and CLDN- 1 have also been observed, which suggests the formation of a co- receptor complex between CD81 and CLDN A second tight junction protein has also been identified as a key player in HCV entry in vitro, Occludin (OCLN). OCLN can interact with E2, and suppression of OCLN with sirna inhibits HCV entry in vitro 46. Binding to the tight junction proteins induces receptor- mediated endocytosis. When the virus particle is inside the endosome it becomes acidified due to the influx of H +. This results in the fusion of the viral glycoproteins and the release of the nucelocapsid. The genomic RNA is then released and translation can begin 47. It is possible that new membrane proteins will be identified essential in viral entry, but up till now most of these studies are conducted in vitro. The great challenge will be to proof the entry model in an in vivo experimental setting. As mentioned in paragraph 2.3 the genomic RNA of HCV does not contain a 5-7meG- cap, which is necessary for eukaryotic translation. This 5 - cap is necessary because the cap recruits eif4f (eukaryotic initiation factor 4F) to the mrna, which results in the binding of the 40S ribosomal subunit to the 5 - end of the mrna and translation can commence. HCV uses the IRES domain in the 5 NTR to recruit the 40S ribosomal subunit. The IRES domain consists of several subunits that are each necessary for proper initiation of the translation process. These subunits are SLII, SLIII, and SLIV and the first codons of the ORF 48 (see figure 3). Research has shown that the IRES bound to the 40S 9

11 Figure 3. A) Functional domains within the IRES structure in the 5 UTR of the HCV genome 47 B) Schematic representation of the replication complex of HCV. After the recruitment of the various proteins on the membrane of the ER invagination occurs resulting in a protected environment for the RC 47. ribosomal subunit and eif3 induces conformational changes in 40S resulting in the proper recruitment of 80S and the initiation of translation without the presence of a 5 - cap 49. After the translation of the 10 viral proteins from the ORF, viral RNA replication can commence. Since HCV is a positive strand RNA virus, replication occurs on intracellular membranes (as is the case for all +RNA viruses) 50. The exact replication complex (RC) is not known but the overall thought is that it includes the viral genome, the non- structural proteins, and some cellular cofactors. The model that is proposed nowadays shows invagination of the RC on the endoplasmatic reticulum (ER). Nucleotides necessary for RNA replication can enter the RC, whereas proteases or nucleases cannot. This will protect the RC from degradation, which would occur if the RC was presented to the cytosol 51. It seems likely that the positive RNA genome of the virus serves as the template to synthesize negative strand RNA. The two RNA strains remain paired with each other followed by copying of this complex. This results in the formation of multiple viral (+)RNA genomes 52. As mentioned in paragraph 2.3 the workhorse of the HCV proteins is NS5B, the catalytic subunit of the RC with RNA dependent RNA polymerase activity. This enzyme lacks proof reading function, which leads to the formation of genomic copies with a high genetic variance 53. According to recent studies many cellular cofactors regulate HCV RNA replication in vitro. Host proteins that interact with the non- structural proteins of HCV, but also components of the RNAi pathway and mirnas seem to play a role in the efficiency of RNA replication 54. Other factors that influence RNA replication efficiency in a negative way are nucleotide starvation, temperature shifts, and reactive oxidative species

12 For the release of viral particles, an important role is served for lipoproteins. Within released virus particles the human Apos (apolipoprotein) is found, and experiments with ApoE inhibition have showed a reduction in released HCV particles. Also, HCV assembly seems to occur on lipid droplets and lipids also seem to influence viral infectivity. According to recent studies, viral particles that are associated with cholesterol play a major role in viral infectivity. Research showed that viral particles enriched with cholesterol had a higher infectivity rate compared to the control viruses 56. The blockade of fatty acid synthase (essential in the process of fatty acid metabolism) down regulates the expression of CLDN- 1, mentioned above as crucial for viral entry, and thus reduces viral entry, and tempers the viral infection rate 57. So far, there is no over- all model created for viral assembly and release. Also, the specific roles for the non- structural proteins are not completely understood. It has been shown that mutations in NS5A, NS2, and NS3 do not affect the RNA replication machinery, but do have a significant effect the production of new viruses. It seems logical to assume that there must be a specific role in the assembly and release process for these non- structural proteins. Their exact roles, however, are under heavy debate. More research needs to be conducted to unravel the specific functions of the proteins in the viral life cycle. The most up- to- date proposed model is shown in figure Figure 4. Model for HCV viral assembly and release as proposed by Shimotohno; core protein and non- structural protein complex (NLC) interact on lipid droplet (LD). Association with various lipoproteins determines the density of the virions, low density correlates with higher infectivity. Microsomal triacylglycerol transfer protein (MTP) and triglyceride are important in ApoB/ApoE formation

13 3. THE INNATE IMMUNE RESPONSE AGAINST HCV AND VIRAL IMMUNE EVASION The immune response in humans against HCV consists of two parts; the non- specific, innate, and the specific, adaptive, component. Different parts of both components of the immune response will be discussed in more detail in the following paragraphs. Several components of the non- specific immune system are thought to play an essential role in the clearance of HCV. Since this system is named non- specific, the following host defence mechanisms can be used against a broad spectrum of pathogens. A very important component is the interferon system. Already for a long time it is known that mice with target deletions in their type I interferon receptor are not able to fight viral infections, although their adaptive immune system is in perfect condition 58. The type 1 interferon system makes use of two kinds of interferon, IFN- α and IFN- β. Production of IFN in a cell leads to a stimulus for neighbouring cells to produce antiviral proteins. This will eventually lead to the slow- down of viral production and thus infection, buying the adaptive immune system time to develop a specific immune response 59. The production of IFN- β needs activation by viral proteins in the cell. Several sentinel molecules are active, and will be elaborated on in more detail in the following paragraphs. The viral evasion strategies will be discussed together with the different levels of the immune response in order to give a comprehensive overview of both the immune response and the viral immune evasion. 3.1 PATTERN RECOGNITON AND NS3/4A One of the most important aspects of the non- specific immune system is that it makes use of pattern recognition. Several pathogen- associated molecular patterns (PAMPs) are recognized by specific pattern recognition receptors (PRR). Examples of HCV PAMPs are the single stranded RNA, and double stranded RNA. The PRRs involved in HCV recognition are Toll- like receptors, NOD- like receptors and RIG- like helicases 60. The TLRs are a family of 11 receptors in human. The major part of these receptors is present at the plasma membrane of the cell, to detect the extracellular PAMPs. The TLR signalling cascade involves many adapter proteins, Myd88, TRIF, TRAM, and TIRAP, eventually leading to the activation of transcription factors and the production of IFN- β. In humans, TLR3, TLR7, and TLR9 are present intracellulary, for example on endosomes, to scan for PAMPs. dsrna is recognized by TLR3, ssrna by TLR7, and the non- methylated CpG motifs by TLR9 61. Recently, a TLR3 independent dsrna recognition system has been postulated. The two helicases, retinoic acid inducible gene- I (RIG- 1) and melanoma differentiation associated gene 5 (MDA5), have two CARD signalling domains that move into there activated state upon the binding of dsrna to the helicase domain 62. Both TLR3 and RIG1 are thought to be most important in the recognition of 12

14 intracellular HCV. The signalling cascade followed by endosomal dsrna recognition by TLR3 starts with the recruitment of TRIF. Autophosphorylation takes place on both TRIF proteins, leading to the recruitment of TBK- 1 and IKKε, both kinases that are thought to be essential in the phosphorylation, and thus activation of interferon regulatory factor 3 (IRF- 3), which translocates to the nucleus upon phosphorylation and acts as a transcription factor for IFN- β 63. Another transcription enhancer that becomes activated via TLR3 is NFκB. Activation of NFκB follows after the recruitment of TRAF6 (tumor necrosis factor receptor (TNFR) associated factor 6) 64. Also, an important role is played by receptor interacting factor RIP1, which in its turn, is modulated in activity by RIP3 65. RIG1, as mentioned above, acts completely separate from TLR3 on the same PAMP, dsrna, however, RIG1 recognizes cytoplasmic dsrna. When the helicase domain of RIG- I/MDA- 5 binds dsrna, the CARD domains can freely interact with the CARD domain adapter protein CARDIF. CARDIF needs to be attached to the outer membrane of the mitochondrion; otherwise it cannot recruit the proteins necessary for the downstream signalling cascade 66. The exact signalling cascade and the proteins involved remain unknown. Both TRAF3/6 seem to play a role to activate IRF- 3 and NFκB respectively. The last RIG- 1/MDA5 activated pathway results in the activation and translocation of the AP1 complex, which consists of C- jun and ATF2. Together with NFκB this is also called the enhanceosome. This enhanceosome facilitates chromatin remodelling by HATs, leading to the uncovering of the promoter, and the production of IFN- β. The non- structural protein NS3/4A of HCV is able to cleave CARDIF from the mitochondrial membrane; thereby inhibiting RIG1 mediated IFN- β synthesis. Secondly, the protein is also capable to cleave the TRIF adapter protein. Due to this cleavage TRIF is unable to recruit TBK- 1, which in turn cannot Figure 5. INF- β production is mediated via two independent pathways: TLR3 mediated (depicted on the left side), and RIG- 1 mediated, depicted on the right side. Translocation of transcription factors IRF- 3, ATF2/c- JUN leads to initiation of INF- β transcription. NS3/4A is capable to cleave TRIF, in the TLR3 mediated pathway, and CARDIF, in the RIG- 1 mediated pathway, thereby inhibiting IFN- β synthesis

15 phosphorylate IRF- 3, which as a consequence cannot migrate through the nuclear membrane to act as a transcription factor. Concluding, this protein is capable to shut down IFN- β synthesis, both RIG- 1 and TLR3 mediated (see figure 5). 3.2 INTERFERON- Β SIGNALLING AND VIRAL EVASION The newly synthesized IFN- β can act in a paracrine or autocrine manner. In both cases, IFN- β binds to a transmembrane type I interferon- receptor. Upon binding, a Jak- STAT pathway becomes activated. The two intracellular parts of the interferon- receptor are Janus kinases and phosphorylate each other. Due to this phosphorylation step STAT2 is recruited and associates with IRF- 9. STAT1 is also recruited and due to the phosphorylation of both STAT1 and STAT2 the two proteins heterodimerize via SH2 binding domains, resulting in the formation of the interferon stimulated gene factor (ISGF)3 61. A negative regulator of this process is SOCS3 (suppressor of cytokine signalling 3) 67. When ISGF- 3 translocates to the nucleus it can bind to the IFN- stimulated response element (ISRE), initiating the transcription of interferon stimulated genes (ISGs). One of these genes codes for the transcription factor IRF- 7, which acts as a positive feedback system, due to the enhancing, together with IRF- 3 (mentioned in the previous paragraph), of IFN- α transcription 68. Besides interfering with the signalling cascades that result in IFN- β production, HCV is also capable to interfere with the IFN- β signalling, upon IFN- β receptor stimulation. The first anti- IFN signalling action is undertaken by the core protein of HCV. This protein is thought to interfere directly at the point of Jak- STAT pathway. Whether it does so via direct degradation of STAT1, up- regulation of PIAS (protein inhibitor of activated STAT), or up- regulation of SOCS3 (suppressor of cytokine signalling), remains unknown. Experimental evidence has been gathered in favour of the various mechanism described above 61. Recently, also the mechanism of epigenetic silencing by HCV, discovered with the usage of (partially) IFN- resistant infected cells, has been proposed as a tool used by HCV to become IFN- signalling resistant INTERFERON STIMULATION GENES AND VIRAL EVASION Other ISGs are ADAR (RNA- specific adenosine deaminase) 1, protein kinase R (PKR), 2-5 oligoadenylate synthetases (2-5 OAS), and P Three of these proteins, PKR, ADAR 1, and OAS, are always expressed at a certain base level in every cell. Interferon signalling enhances the production and activity of these proteins. PKR phosphorylates the alpha subunit of eif2. Since this factor is essential to initiate translation, this will result in the blockade of both viral and cellular translation 71. ADAR 1 is an enzyme that interferes with the synthesis of the building blocks of RNA. It deaminates adenosine on target dsrnas resulting in less stable inosine uracil base pairs compared 14

16 to adenosine uracil base pairs. Due to this, mutations accumulate in the viral genome leading to the production of non- functional viral particles OAS activates RNaseL via the formation of short oligoadenylates. This RNaseL degrades viral, but also cellular, RNA. This inhibits translation of the viral proteins 72. The last antiviral protein is P56. This protein also interferes with the translation initiation complex by binding to eif3. As mentioned in chapter 2, this is an essential protein to initiate translation. This process will thus be blocked resulting in the inhibition of viral RNA translation. As explained above, the interferon- stimulated genes (ISG) are important anti- viral defence mechanisms. Several proteins of HCV are, besides being essential for replication and viral assembly, capable to interfere with these protein functions. The first example is the ISG PKR. Both the structural protein E2 as well as the non- structural protein NS5A can inhibit the function of PKR. The structural protein E2 exists in the cytoplasm of the host cell as unglycosylated. This form of E2 shows a high homology with the PKR- eif2α binding domain. This domain is the site where PKR binds and phosphorylates eif2, resulting in a shutdown of the transcription machinery. Due to the competition of unglycosylated E2 for this binding domain with eif2, less eif2 becomes phosphorylated, and transcription can still take place. The exact binding sequence differs between the various genotypes of HCV, making E2 able to block a broad variety PKR- eif2α binding sites 73. NS5A is believed to bind with its IFN sensitivity- determining region (ISDR) to the central part of PKR, thereby inactivating the functional protein. However, the findings on these interactions remain highly speculative since different models in different studies generate different experimental results 61. NS5A also interferes with another ISG, namely the 2-5 OAS. It can bind to 2-5 OAS, with 2 different amino acid sequences on OAS involved. This binding is, however, ISDR independent, giving the virus another option to interfere with IFN- stimulated antiviral proteins 74. Besides interfering directly with the protein, it is shown that more virulent genomic strains (HCV genotype 1 vs 2 or 3) have less RNase L cleavage sites, making them less susceptible for RNAase L / 2-5 OAS activity. The protein P56 also seems to be affected to a certain degree by HCV. NS3/4A, and also other (non) structural proteins, interfere with the signalling from IFN to the P56 promoter. The different IFN- signalling pathways and the immune evasion proteins of HCV are illustrated in figure 6 (page 16). 3.4 NATURAL KILLER CELLS AND HCV Another component of the innate immune system is the natural killer cell (NK cell). NK cells are very important in the primary immune response against viral infections. Cells that do not express MHC class I on their cell surface are recognized by NK cells, and immediate cell lysis induced by the NK cell will follow. Besides MHC class I, many other inhibiting and activating receptors that mediate the binding between NK cells and MHC class I have been discovered; making this system more 15

17 sophisticated than it was thought to be. In HCV infection NK cells are thought to be necessary for optimal priming and cytolytic function of virus specific T cells due to their production of IFN- γ. NK cells can also induce apoptosis of infected hepatocytes 46. Since NK cells are thought to play an essential role in the primary anti- viral response, it seems logical to conclude that HCV must possess characteristics to evade the function of NK cells 75. The binding of viral E2 to CD81 can inhibit the NK cell activity. This interaction inhibits NK cell activation but, in time, will activate B and T cells. This could be regarded as a strategy to establish a high viral load and viral turnover before the specific immune response will be initiated 76. NK cells also have an immune- regulatory role, via different cytokines, and interfering with NK cells could lead to a delayed specific immune response. NK cells are primary activated when MHC class I presentation is abundant. The core protein of HCV can upregulate MHC I expression in hepatocytes, which results in inactive NK cells. These immune evasion strategies aimed specifically on NK cells have only recently been discovered and need more extensive research in order to be understood completely 77. Figure 6. IFN- β binds to the interferon receptor and signaling via Jak- STAT commences. The downstream signaling cascades are shown, eventually leading to the production of ISGs. The different proteins of HCV that interfere with these pathways at multiple levels, and the proteins that directly affect the function of the ISGs are also shown

18 4. THE ADAPTIVE IMMUNE RESPONSE AGAINST HCV AND VIRAL EVASION When the innate immune response against HCV is unable to completely clear the virus, via the measurements described above, the slower but more specific adaptive immune response starts with the generation of a specific immune response against HCV. In the following paragraphs both the cellular mechanisms involved in this specific immune response, as well as the counter measurements taken by the virus to evade this specific immune response, will be described in more detail. 4.1 THE HUMORAL IMMUNE RESPONSE AND VIRAL EVASION The specific immune response against HCV consists of both a humoral component and a cellular component. The humoral component of the specific immune response makes use of viral specific antibodies produced by B- cells. These antibodies bind to the antigens upon the virus, which marks them for destruction by, for example, macrophages. During acute HCV infection and chronic HCV infection neutralizing antibody levels can be detected. These antibodies often attack the envelope glycoproteins E1 and E2, and there is one region that seems especially important, the hyper- variable region 1 (HVR- 1). The epitopes around and in this region have a very important function in various steps of the viral entry process, such as the binding of the virus to CD81 and membrane fusion. Besides the high mutation rate of the virus, which I will discuss in more detail, other factors negatively influence the effect of neutralizing antibodies 70. The glycoproteins of HCV interact with host high- density lipoproteins and the HCV receptor scavenger receptor B1. These interactions can lead to a conformational change, making it impossible for the antibody to bind the specific epitope. Also steric hindrance could negatively influence the binding capacity for the antibody on the epitope 78. There are also specific glycans present on the envelope protein E2 that, beside their function in cell entry, also function as protection for the virus against antibodies. 4.2 THE CELLULAR IMMUNE RESPONSE AND DENDRITIC CELL DYSFUNCTION The cellular immune response is activated mainly by Type 1 helper cells (Th1). They play a crucial role in starting various cellular immune responses, such as cytotoxic T lymphocytes (CTL), and macrophages. Upon cellular uptake of HCV, antigens of HCV are presented in the liver. These antigens are taken up myeloid dendritic cells (DCs). After the migration of the DCs to a draining lymph node, they mature, and express HLA and various co- stimulatory molecules, such as CD80 and CD86. Via these stimulating molecules, naïve helper T cells become activated (binding of CD28 on Th1 cells to CD80 and CD86). Upon activation Th cells express the ligand CD40. The mature DC binds this ligand via the CD40 receptor that is present on the surface of the mature DC. Together with the expression of CD40 ligand various cytokines, such as tumour necrosis factor α (TNF- α). Sequentially, 17

19 DCs secrete IL- 12, a cytokine that promotes the maturation of Th1 cells from the naïve Th cells. When Th1 cells are fully maturated they express IL- 2 and IFN- γ. These cytokines induce the activation and the proliferation of other cells, such as CTLs. The naïve CTL expresses CD28 on its surface and via the CD28 - CD80/CD86 interaction and MHC class I on the DC the viral specific antigen is presented. The HCV specific CTL migrates to the liver recognizes the presented antigens via MHC class 1 on infected hepatocytes, and mediates specific killing of these cells. Besides the activation of CTLs via dendritic cells and Th cells, CTLs can also become activated in a direct manner. In the process of direct activation of CTLs, cross presentation plays an essential role. Dendritic cells can take up proteins via endocytic mechanisms. Two different mechanisms lead to presentation of the (partial) protein on MHC class I. Either via the transfer of the protein from the phagosome to the cytosol, followed by hydrolization, transport to the associated MHC 1 molecule in the endoplasmatic reticulum or phagosome, and extracellular presentation to patrolling CTLS, or via cleavage of the protein by endosomal proteases, followed by the binding to MHC 1 within the endosome, and extracellular presentation 79. Up to now, twenty different CTL epitopes on the HCV polyprotein have been identified 80. The virus- specific CTL mediated killing is thought to play an essential role in viral eradication; this is due to the fact that CTLs inhibit viral replication 81. As stated above, CTL mediated killing involves the recognition of peptides (8 11 amino acids in length) presented by MHC class 1 molecules. Upon recognition of the specific peptide, the CTL releases perforin. Perforin is capable to form a pore in the cellular membrane of the infected cell. Through this pore, granzyme can migrate into the cytoplasm, and induce apoptosis. Besides the induction of apoptosis via a granzyme mediated pathway, CTL can also directly induce apoptosis via the expression of Fas- ligand (Fas- L) or TNF- α. Studies have shown that the expression of Fas is upregulated in HCV infected hepatocytes. Besides the upregulation of Fas, also CTLs that have infiltrated the liver express FasL. The presence of both Fas and FasL in the liver has resulted in the conclusion that this system is important to induce apoptosis in HCV infected liver cells. The binding of Fas to FasL results in the formation of the death- inducing signalling complex. This complex activates caspase- 8. Active caspase- 8 can start 2 independent signalling pathways. In the first pathway the molecule Bid is cleaved, and cytochrome C is released from the mitochondria into the cytoplasm. The presence of cytochrome C results in the cleavage of various caspase molecules, the final one being caspase- 7, resulting in cell death. The second pathway is the presence of active caspase- 8 itself in the cytoplasm, resulting in the cleavage of the previous mentioned caspase molecules and the induction of apoptosis 82. The most important aspect of the apoptosis induction via Fas- L or TNF- α is that it happens without cell- to- cell contact 81. An overview of the different adaptive immune responses is shown in figure 7. 18

20 Figure 7. All components involved in the adaptive immune response are shown in this picture. Neutralizing antibodies can attack extracellular viral particles. They are also capable to block viral entry. Th1 helper cells (CD4 + ) activate cytotoxic T lymphocytes (CD8 + ) via DCs and antigen presentation. CTLs use Perforin, Fas/FasL, or TNF- α to induce apoptosis. The secretion of IFN- γ results in non- cytotoxic clearance of the virus 86. The exact mechanisms underlying the adaptive immune evasion capacities of HCV are poorly understood. This is due to the fact that the chronic HCV infection characteristics differ from one patient to another. If we look chronological to the events that lead to a specific immune response it al starts with the antigen- presenting DCs. Impairments in the interactions between the DCs and T cells can lead to a non- functional selective immune response. Many in vitro studies have been conducted, but gave conflicting results 52. One of the studies states that HCV alters dendritic cell function. DCs that were cultured in presence of HCV peptides were less potent in stimulating an antigen specific immune response. This results in less functional Th1 cells and CTLs. A possible mechanism underlying this phenomenon could be the alteration of the cytokine secretion profile 83. The alteration in cytokine secretion can have massive effects on the immune system, since cytokines are the primary signalling molecules involved in maturation and proliferation of all the cells of the immune system. The proteins of HCV also seem to play a crucial role in interfering with DC function. In a study by Krishnadas et al. researchers have shown that the core protein, NS3, NS4, NS5, and the fused polyprotein that consists of core, NS3, and NS4, can impair DC function on multiple levels. The proteins seem capable to regulate the expression of co- stimulatory and antigen presentation molecules, reduce IL- 12 secretion, induce the expression of FasL to mediate DC apoptosis, interfere with allo- stimulatory capacity, inhibit toll- like receptor signaling and inhibit nuclear translocation of NFkappaB. Both the HCV core protein and E2 can inhibit DC maturation 84. The recognition of the core proteins and NS3 seems to involve TLR2. Research has shown that DCs that are activated by ligands of TLRs derived from viruses have reduced cross- presentation ability, which plays an important role in the initiation of an immune response. This ability to evade DC signaling is seen in other viruses and 19

21 bacteria. Evidence that supports this hypothesis is found recently. From studies it was concluded that TLR2 expression on DCs is reduced in HCV patients and that TLR2 stimulated DCs are less potent in stimulating T cells to proliferate 85. The activation of NK cells by DCs involves both IFN- α signaling and the expression of major histocompatibility complex class- I related Chain A and B (MICA/B). Both signals seem to be reduced in patients suffering from chronic HCV, leading to the activation of less NK cells. The structural protein E2 shows high affinity to bind two DC specific receptors, DC- SIGN and L- SIGN. The binding of E2 to these receptors could interfere with intracellular pathways in DCs 81. It is still under heavy debate whether the findings described above are essential for HCV persistence or that they are consequences of an already persisted HCV infection. Another discrepancy lies in the fact that the above findings are not present in every patient. For example, it has been reported that DCs that do express the core protein and NS3 showed normal cytokine production and that they were perfectly capable to stimulate T cells. Further research needs to be conducted to clarify the exact mechanisms underlying disrupted DC functioning and impaired T cell or NK cell activation by DCs T- CELL FAILURE As is the case with defective antigen presentation, or DC signaling, the mechanisms involved in T- cell failure are poorly understood. One of the factors that could lead to impaired T cell function is the chronic antigenic stimulation. This phenomenon can be observed in other chronic viral infections. The constant presentation of antigen results in chronic T cell activation, but also loss of function. This often starts with less IL- 2 production, followed by the decline of cytotoxicity, TNF- α, and IFN- γ production respectively. In HCV specific CD4 + and CD8 + T cells T cell failure is expressed by less IFN- γ production. It is also observed that less IL- 2 activated T cell mediated killing precedes the development of chronic hepatitis C 77. When a T cell becomes dysfunctional it starts to express the protein- programmed death 1 (PD- 1). This protein needs its ligand PD- L1, and upon binding, this protein will activate an intracellular pathway that will result in T cell apoptosis 22. Due to the fact that these processes affect the CD4+ T helper cells, they will also have an effect on the proliferation and maturation of cytotoxic CD8 + T cells. Recently a human cytokine has also been found as a mediator of HCV- specific T cell failure. Studies have shown that IL- 10 levels are often elevated in chronic HCV infection. IL- 10 has multiple effects on the immune response. It inhibits IFN- α production, and it can downregulate the effector T cell responses

22 4.4 REGULATOR T CELLS Regulator T cells (Tregs) are a subpopulation of T cells and they are often elevated during chronic HCV infection. They are identified by the constant expression of factor forkhead box P3 (FoxP3), CD25 + and CD24 +, and glucocorticoid- induced TNFR- α family- related protein (GITR). These cells can inhibit both proliferation and/or cytokine production of responding effector T cells (Teffs). Via inhibiting signals, Tregs are thought to play an important role in balancing the immune response to infection, and act as an extra mechanism to evoke auto immunity and an excessive immune response, which could lead to extensive tissue damage 87. The exact mechanism of Teffs regulation is not known, but it probably involves, cytokine signaling via IL- 10 or TGF- β, or by acting as an IL- 2 sink, depleting IL- 2 for Teffs 88. The inhibitory signals can be delivered to the target cell via direct cell- to- cell contact. This pathway involves the cellular proteins CTLA- 4, GITR, and PD- 1. Signaling over distance can occur via TGF- β or IL- 10. Tregs express the T cell receptor complex, as all other T cells, and Tregs are thought to be antigen specific during HCV infection. Research into protein derived peptides from HCV and the antigen specificity of Tregs showed that only a few peptides, with an overlapping region, were able to stimulate Tregs. This indicates that there could be a dominant region on the core protein of HCV that is capable to activate Tregs, and thus suppress the immune response 89. Not much research has been conducted into the co- stimulatory signaling molecules but the PD- 1 system may be for a part responsible for the inhibitory function of Tregs during chronic HCV infection 88, 90. The mechanism behind the generation of Tregs is currently thought to be primarily a DC- dependent mechanism. It is known for a long time that DCs are capable to trigger the proliferation of Tregs. Different factors influence the proliferation of Tregs, IL- 10 production, an immature DC phenotype and less expression of co- stimulatory DC molecules (such as CD80), to name a few. This information in combination with the fact that HCV interferes with the functionality of DCs on multiple levels, can lead to the conclusion that HCV suppresses the CD4 + and CD8 + mediated immune response via a DC dependent upregulation of Tregs. Whether there is a DC independent pathway to upregulate Tregs remains to be discovered. 4.5 T CELL HOMING CAPACTITY An experiment with HCV infected chimpanzees conducted by Thimme et al. showed that chimps that progressed to the state of viral persistence, without any temporary viral control, did not have any virus specific CD8 + T cell response. The researchers hypothesized that this could be attributed to the fact that the T cells lacked homing capacity to the site of infection, in this case, the liver 91. Again, the results in studies that compared the presence of virus specific CD8 + T cells in the liver and the blood were conflicting, studies in patients showed that sometimes the CD8 + T cell levels in the liver where 21