Figure (6): Hepatitis C virus (HCV) infection and immune response (Asselah et al., 2009). HCV can induce several signalling pathways. (A) Toll-like receptor (TLR) signaling, (B) Retinoic acid-inducible gene-I (RIG-I)-like RNA helicase signaling and (C) IFN signaling pathway. Toll-like receptor (TLR) signaling is launched when TLR3 is activated by foreign viral RNA, subsequently it will recruits IkB kinase (IKK)-related kinases, IKKi (also known as IKKe) and TANK-binding kinase 1 (TBK1, also known as T2K and NAK). These kinases, collectively with adaptors TANK and NAP1, catalyse the phosphorylation of interferon (IFN) regulatory factor-3 (IRF-3). TLR3 attachment will start the activation of transcription factors AP-1 and nuclear factor-kB (NF-kB). Phosphorylated IRF-3 forms a dimer, translocates into the nuclei, binds to DNA and regulates the expression of IFN β in collaboration with AP-1 and NF-kB. The HCV NS3-4A serine protease may block the phosphorylation and effector action of IRF-3. After recognition of viral RNA, (RIG-I) and Mda5 (not shown) recruit IFN β promoter stimulator-1 (IPS-1, also known as MAVS, Cardif and VISA) via CARD–CARD (caspase recruitment domain) interaction. IPS-1 is localised to mitochondria and acts as an adaptor that plays a critical role in the activation of IRF-3 and IRF-7 in a TBK1- and IKKi-dependent manner. IPS-1 also interacts with the Fas-associated death domain protein (FADD), which is required for the activation of IRF-3 and NF-kB. IRF-7 forms a dimer, translocates into the nucleus to induce IFN α/β; homodimers of IRF-3 collaborate with NF-κ-B to induce IFN β. IPS-1 is targeted and inactivated by NS3-4A, a serine protease from HCV known to block IFN production (Asselah et al., 2009).
It is worth to note that exogenous recombinant IFN α supplement stimulate the same cellular receptors, creating the same cascades that take place with endogenous production. Perhaps the exogenous IFN can fulfill maximal effectiveness with larger concentrations can be applied (Santantonio, 2005; Soza et al., 2005).
2.4.2 Adaptive Immune Responses.
In contradiction to the innate immune responses that are triggered within hours to days after infection, More or less 6–8 weeks were needed before adaptive immune responses become detectable (Thimme et al., 2002, Logvinoff et al., 2004). Many constituents of the adaptive immune system are implicated in viral clearance, including T cell responses and humoral antibody responses (produced by B cells).
In fact, most acutely HCV-infected individuals produce antibodies against epitopes within the structural as well as non-structural proteins. Only a small fraction of antibodies can inhibit virus binding or entry which called ‘neutralizing antibodies’ while the majority have no relevant antiviral activity (Su et al., 2002; Logvinoff et al., 2004). The successful adaptive immune response against HCV includes all components of the adaptive immune system, specifically antibodies, CD4+ T cells, and CD8+ T cells. All three components have been shown to be associated with viral clearance (Chang et al., 1997; Thimme et al., 2002).
2.4.2.1 Neutralizing Antibodies
Linear and conformational discontinuous epitopes is the principal target of neutralizing antibodies. This epitopes is located within the envelope glycoproteins E1 and E2. A hotspot of neutralizing epitopes is standed beside the hypervariable region 1 (HVR1) of E2, and the high viral quasispecies variability in this region, which is a consequence of replication by the error-prone RNA-dependent RNA polymerase (Sabo et al., 2011), has been attributed to viral evasion from the neutralizing antibody response. The viral regions embattled by neutralizing epitopes have imperative functions in virus binding and entry, like binding to the host cell receptors CD81 and SRB1, however it may also be implicated in critical post-attachment steps (Haefelin and Thimme, 2013).
The ultimate protective role of neutralizing antibodies could not be validated in HCV-infected patients with acute-resolving infection for a long time, since neutralizing antibodies were detectable in patients after persistent infection had been launched (Bartosch et al. 2003; Haefelin and Thimme, 2013). It is important to note that viral clearance can also occur in the absence of neutralizing antibodies, and even in agammaglobulinaemic patients demonstrating that neutralizing antibodies do not certainly have a crucial role in HCV clearance (Adams et al., 1997).
2.4.2.2 CD4+ T Cells:
HCV-specific CD4+ T cells that apprehend viral antigens in the context of HLA class II molecules perform a central role in viral control. This is confirmed by diverse observations such as: CD4+ depletion studies in the chimpanzee model have revealed strong evidence for the important role of HCV-specific CD4+ T cells in viral control. Indeed, after the antibody-mediated depletion of CD4+ T cells, chimpanzees temporarily controlled viremia, indicating that this initial control was mediated by CD8+T cells. However, viral titers increased again and this was associated with the evolution of viral escape mutations in CD8+ T cell epitopes (Gruener et al., 2000; Grakoui et al., 2003). These data confirm the conception that HCV-specific CD8+ T cells are the fundamental antiviral effector cells, whereas HCV-specific CD4+ T cells perform key helper functions for preventing viral rid from the CD8+ T cell response (Haefelin and Thimme, 2013).
2.4.2.3 CD8+ T Cells:
Virus-specific CD8+ T cells are key adaptive effector cells involved in HCV clearance. In fact, in patients with acute HCV infection, viral load remains at high titers during the first weeks of infection. After 6–8 weeks, virus specific CD8+ T cells imitated in the peripheral blood and this is transitionally related to clinical symptoms, the rise of liver enzymes, and a sharp drop in viral load (Adams et al., 1997; Gruener et al., 2000).
HCV-specific CD8+ T cells stimulate their antiviral effects through two diverse effector mechanisms. First, they can act cytotoxically, e.g., they destroy infected target cells (mostly hepatocytes) that carry viral antigens by HLA class I molecules on their cell surface. Indeed, cytotoxicity can be shown either by cell-bound receptors like FAS and its ligand FAS-L or by paracrine secretory factors like perforin (Jo et al., 2009). Second, HCV-specific CD8+ T cells mediate to viral control by non-cytolytic mechanisms, e.g., excretion of antiviral cytokines like Tumor Necrosis Factor-alpha (TNF-α) and Interferon-gamma (IFN-γ). In fact, experiments carried out in a HLA-A2 expressing HCV replicon cell model illustrated that HCV-specific CD8+ T cells execute their antiviral effects mainly through non cytolytic mechanisms, particularly IFN-γ secretion, while cytolytic effector functions were only observed at high effector-to target ratios. Also, HCV replicon cells are sensitive to cytolysis by perforin; nevertheless, the majority of HCV-infected patients present virus-specific CD8+T cells with shortage in perforin secretion, which emphasize that cytotoxic pathway may play a slight role in natural HCV infection (Jo et al., 2009; Haefelin and Thimme, 2013).
2.5 What are the mechanisms of HCV Evasion from Immune Response?
It is well recognized that nearly all viral infections stimulate immune responses and do not persist. HCV has emerged mechanisms to escape immune eradication, thus allowing it to persist in the infected liver.
2.5.1 Variability of HCV Sequences:
Wide variability of HCV, which mutates not only between patients, but also at any instant within the same patient, is an utmost challenge. HCV subsists in each infected patient as a swarm of genetically related variants called quasispecies. Proficient mathematical models of viral kinetics show that up to 1012 virions are assembled every day in single chronically HCV infected human (Neumann et al., 1998). By the high level of virion turnover, in conjunction with the lack of proofreading by the HCV RNA polymerase huge number of viral mutations will be generated permitting virus evasion from immune response (Burke and Cox, 2010).
2.5.2 Mutations allow T cell evasion:
Virus escape is also represented by decline in T cell responses to HCV infection. In contrast to immune responses which require several weeks to develop, the pathogen needs only few hours or days to replicate; it is well-recognized that immune escape mutations may blunt the role of the immune response successfully (Weiner et al., 1995). Mutation of class I or II major histocompatibility complex (MHC) restricted T cell epitopes reduce probability of infected hepatocytes clearance (Eckels et al., 2000). Against strong multispecific cytotoxic lymphocyte (CTL) response, simultaneously mutation of some epitopes would be indispensable for survival of the virus. Few cases of amino acid substitution in CTL epitopes were elucidated by longitudinal analysis through chronic infection suggested that CTL escape that occurs may be restricted only to early infection (Burke and Cox, 2010).
2.5.3 T Cell Inhibitory Receptors:
Up on T cells activation, family of inhibitory receptors is over expressed such as; programmed death-1 (PD-1) that is an ITIM-containing inhibitory receptor. Surface PD-1 levels have recently showed higher expression on HCV specific T cells in individuals who fail to beat infection than T cells from those who successfully eradicate it in both early and later infection (Burke and Cox, 2010).
2.6 Current treatment approaches and limitations:
Pegylated interferon (PegIFN)-α together with ribavirin for 24 or 48 weeks was the approved combined treatment for chronic hepatitis C till 2011. With this regimen, HCV infected patients with genotype 1 had sustained virological response (SVR) rates of about 50% in North America and Western Europe. Patients infected with HCV genotypes 2, 3, 5 and 6 were attained higher SVR rates (up to about 80%), while intermediate SVR rates were achieved in patients with HCV genotype 4 (Antaki et al., 2010; EASL, 2011). Three novel HCV direct-acting antivirals (DAAs) have been recently authorized. Sofosbuvir, was the first pangenotypic nucleotide analogue inhibitor of HCV RNA-dependent RNA polymerase. Secondly, Simeprevir was the first-generation NS3-4A protease inhibitor mainly active against genotypes 1 and 4. Daclatasvir, is the third pangenotypic NS5A inhibitor (EASL, 2015).
Each one of these three licensed DAAs can be used as a constituent of a triple combination regimen with PegIFN-α and ribavirin. SVR rates were achieved ranged from 60–100% according to the DAA utilized, the occurrence of detectable preexisting amino acid substitutions providing resistance to the DAA taken, the HCV genotype, and liver disease severity (recommended in advanced fibrosis scores). However these combinations are the best nowadays, the study of their side effects still challenging due to PegIFN-and of ribavirin action (Meissner et al., 2014).
2.6.1 Predictors of Progression of HCV Infection:
Hundreds of attempts and experimental drugs have been directed against liver fibrosis. Only very few drugs have attained phase III (clinical trials in patients) or become available in market (Polestra, 2016). Assessment of fibrosis not only contributes to stage the severity of disease, it permits serial evaluation of disease progression. Moreover not only viral and patient characteristics are the sole determinants to the outcome of infection and treatment, the genetic diversity of the host has a big importance too.
Gene expression profiling approach provides a way to analyze detailed pictures of molecular events occurring in blood samples (Honda et al., 2001). This allowed us to correlate the dynamics of gene expression changes with clinical findings .High efficient techniques now achieve rapid and accurate attribution and evaluation of gene expression in blood and in tissues (Shackel et al., 2002).
2.6.1.1 Liver Biopsy
Percutaneous liver biopsy is the gold standard test has been known to measure fibrosis. Although there is great experience with this test, its invasive procedure is time consuming, painful, has wide sampling error range and makes both patients and expert physicians solicitous. Recent studies showed a difference of at least one histologic stage between the right and left hepatic lobes (Rockey and Friedman, 2006). Moreover, validated grading systems, such as METAVIR, Scheuer, Knodell or Ishak, are rarely been concordant (Regev et al., 2002).
2.6.1.2 Overview of Non-Invasive Markers of Fibrosis:
Wide categories of blood, plasma or serum routine tests ‘markers’ for fibrosis have been proved. For example, some detect abnormalities in serum chemistries, Including aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), bilirubin, platelet count, albumin and α2-macroglobulin, among others. The AST/ALT ratio has been applied for several years as a noninvasive technique for assessing the severity of liver disease. Recent study has showed the low diagnostic precision of AST/ALT percentage in detecting fibrosis as compared to other models, as APRI (AST and platelet count) and FIB-4 (Age, AST, ALT and platelet count)( Amorim et al., 2012). However, non invasive tests permit frequent reevaluation as well as being accepted by patients it allows poorly discrimination between intermediate stages of fibrosis (Schiavon et al., 2015).