Hepatitis C Virus

                                                                                                                      Hepatitis C Virus Revision of  T Cell & Memory Cell Knowledge T Lymphocytes bring about the cellular arm of the adaptive immune response and mature in the thymus B Lymphocytes bring about the humoral arm of the adaptive immune response and mature in the bone marrow  T Cells have receptors which specifically bind cognate antigens. These receptors are only able to interact with epitopes attached to a MHC protein.  CD4+ Helper T Cells bind to MHC-II proteins (only exist on antigen presenting cells e.g. dendritic cells) CD8+ Cytotoxic T Cells bind to MHC-I proteins (exist on all cells; if a cell doesn't express MHC-I proteins then an NK cell is instructed to kill it) MHC-II Process: Foreign substance engulfed by antigen presenting cell (e.g. dendritic cell), storing them in a vesicle for breakdown via lysosymes, before being presented to a vesicle containing MHC-II proteins. The potein-epitope complexes are then transferred onto the surface of cytoplasmic membranes for inspection by CD4+ T Cells. MHC-I Process: MHC-I proteins capture epitopes for presentation and store them in a vesicle, before then being transferred onto the surface of cytoplasmic membrane for inspection by CD8+ T Cells. If CD4+ cells recognise a foreign body, they differentiate into either T Helper I or T Helper II cells.  T Helper I Cells generate a response against intracellular foreign bodies such as bacteria and viruses by producing IL-2, which then activates CD8+ to destroy those foreign bodies. Th-I cells also activate macrophages and B cells (the latter in lower amounts tha Th-2 cells) T Helper II Cells generate a response against helminths and other extracellular foreign structures by producing IL-4, IL-5, IL-6 and IL-10, which mainly activates B Cells.  CD8+ T cells bring about cell death by first releasing perforins to tunnel into the cell, before releasing granzymes to enter the cell via the perforins, in turn activating apoptotic enzymes. The T cell also binds to CD95 (AKA 'Fas') death receptor on the cell which triggers apoptosis.  Memory T Cells are T cells which have been activated and encountered their respective foreign body, destroying it. It has been proven that these cells are better equipped for fighting their respective pathogens and can do with a reduce lag-time. Memory B Cells exist as a sub-type of cells which are generated in the germinal centres after a primary infection. These B cells differentiate into memory cells instead of the plasma B cells which produce antibodies to fight the active infection. The exact mechanism by which the differentiation into B memory cells occurs is currently unknown, but it is hypothesised that NF-kB and IL-24 may play key roles.   General Viral hepatitis is one of the major causes of deaths worldwide Hepacivirus C is a virus within the Flaviviridae family, existing as an enveloped, positive sense single stranded RNA virus.  It is the cause of Hepatitis C and a number of carcinomas and lymphomas associated with the human liver.  It is estimated that around 170 million people are infected with HCV worldwide, with 80 million chronically affected. In those chronically infected, around 10^12 new virions are produced each day Blood-borne virus which is primarily transmitted via IVDUs and needle-stick injuries (also occasionally blood transfusion and haemodialysis patients) HCV is also commonly transmitted nosocomially when sanitation protocols are not followed.  In those infected, 20% of people are able to clear the acute infection, whereas 80% of people develop chronic infection. More than 90% of deaths of those with Hep C and Hep B are other complications which occur as a result of the Hepatitis (e.g. infections)  Chimpanzees are the only known animal model of Hepatitis C   Structure Hep C virion is approximately 60nm in diameter It has two envelope glycoproteins (E1 and E2) which are situated on the outer surface of the virion.  There are also three hypervariable regions on the HCV virion (HVR1, HVR2 and IgVR) Hypervariable region 1 (HVR1) exists on the E2 glycoprotein, aiding the virus evade the immune system. Hypervariable regions 2 (HVR2) and 3 (IgVR) are been proven to be essential for the structural integrity of the HCV virion, whereas the deficiency of HVR1 simply makes the virus more prone to virus neutralising antibodies The genome of HCV exists as a single open reading frame of ~9.6kbps This single ORF is translated into one long protein, which is then cleaved into smaller functional proteins  The structural proteins of the virus are Core Protein, E1 and E2 The non-structural proteins are NS1, NS2, NS3, NS4a, NS4b, NS5a and NS5b 10 proteins in total from the original pre-protein NS2 protein has protease activity NS3 protein has, at one end, protease activity specific to serine and at the other end NTPase and Helicase activity. Also prevents immune response by inhibiting interferon production. NS4a protein binds to NS3 as a cofactor to facilitate the serine protease function NS4b protein drives the formation of the 'membranous web', which comprises host cell membranes which have been rearranged following viral protein generation. The membranous web is responsible for “sponge-like inclusions” found in the hepatocytes of those infected with HCV.  NS5a is vital in the replication and assembly of HCV. It is also able to manipulate the interferon response of the host cell, inhibiting its anti-viral activity. NS5b is the RNA Polymerase that replicates the HCV RNA to be packaged into new HCV virions. This Polymerase makes manymistakes when replicating the RNA, thus facilitating HCV as a quasi-species.    Replication Replication of HCV occurs mainly in human hepatocytes, but has also been known to replicate in the mononuclear blood cells. This latter infection is thought to be the cause for many of the immunological conditions known to affected those chronically afflicted by HCV.  The HCV virions are able to access hepatocytes via the sinusoids (blood supply to the liver). Entry into host cells is via a multitude of receptors: CD81, LDL receptor, SR-BI, among others. CD81 is the most important for virion entry and is the most widely targeted by bNabs for this very reason. Once inside the early endosome of the host cell (as it is being endocytosed into the cells), the viral envelope fuses with it and releases its RNA contents.  The virus conducts its replicative cycle on the membranes of intracellular lipids within the host cell. HCV utilises host machinery to replicate, first utilising the translation machinery to produce the large HCV pre-protein which is then cleaved into 3 structural and 7 non structural proteins (as above).   Genotypes HCV is split into 8 separate genotypes, despite being a quasi-species. Subtypes 1a and 1b are found all over the globe and are known to cause approximately 60% of all hepatitis C cases.  Genotype 5 is most common in Southern Africa Genotype 2 is most common in West Africa Genotype 1 is most common in the UK and US Clinical significance of genotype: Genotypes 1 and 4 are not as responsive to interferon-based treatments as the other genotypes are   T Cell Response To HCV Infection HLA-B27, HLA-B57 and HLA-A3 are all associated with spontaneous clearance of HCV In terms of HLA-B27, there is an immunodominant epitope liable to T cell targeting which is situated on NS5b (the polymerase). This epitope is present in nearly all Genotype I HCV infections and is thus a promising target for potential treatment, given that Genotype is the most common genotype in the developed world (UK and US) A 2009 study found that the main mechanism of T cell killing of virally infected cells is not via cytotoxicity, but via the production of cytokines IFN gamma which inactivate viral replication The CD8+ T cells of chronically infected individuals had a reduced ability to produce interferon gamme, therefore raising the question whether an inability to produce this cytokine has a direct influence on whether an individuals infection is cleared or becomes persistent.   Evasion of the Immune System HCV is adept when it comes to evading the immune system: First of all, both glycoproteins (E1 and E2) are very widely glycoslyated to reduce the efficacy of virus neutralising antibodies binding to the virion surface.  HCV viruses are also very structurally similar to VLDL and LDL and are thus able to surround themselves with apolipoproteins, thus shielding their E1 and E2 glycoproteins from bNabs. Also, given the variation in HCV populations allowing it to be classified as a quasi-species, the virus is able to evade the immune response by selecting for mutations within the quasi-species population which facilitate this HVR1 directly shields epitopes that are vulnerable to neutralising antibodies, thus reducing the likelihood of the virus being neutralised The NS3/NS4a protease cleaves MAVS and TRIF host cell proteins which, in turn, inhibits the host cell ability to produce interferons. The NS5a non-structural protein within HCV modulates the host cell's natural interferon response to infection, thus inhibiting cell signalling pathways which lead to the activation of the immune system.  It has been hypothesized that the core protein of the HCV proteome may act as a decoy target for T cells, distracting them from the other proteins driving replication and viral dispersion.  T Cell Exhaustion due to persistent stimulation by antigens is also a key mediator of HCV persistence in the body. This can, however be prevented, by regulating pathways such as PD1 and CTLA4 which are become overexpressed thus causing exhaustion, thus allowing the T cells to effectively continue to perform their function.   Global Elimination Initiative The aim is to rid the world of Hepatitis C Virus by the year 2030  The goal is 80% incidence reduction and 65% mortality reduction Countries such as France, Spain and the UK are currently on track to meet this goal, although the majority of countries are not.  This relies on four main pillars, which all countries much stringently strive to achieve: The implementation of new and improved infection control measure when it comes to blood safety protocol The expansion of services providing clean needles and opioid treatments to those using intravenously administered drugs.  The expansion of treatment services for those already infected with Hepatitis C irrespective of the stage of their disease Widespread increase in HCV testing to meet the goal of 90% of people infected with HCV being diagnosed and provided treatment. Studies Mathematical study in 2019 suggested that the goal was achievable, though it would take until 2032 at the earliest if all the aforementioned interventions are successfully implemented in each country. If achieved, this would prevent 15.1 million new infections and 1.5 million deaths attributed to HCV.  Unfortunately, countries are not meeting their goals and thus the 2030 goal is not likely to be met. It has been suggested that the goal may be pushed back to a more achievable goal such as 2050 (as suggested by this paper).

Hepatitis C Vaccination At the current moment, there are three strong candidates for a HCV vaccine: One uses adenovirus vectors which encode NS3 and NS5B. Another uses adenovirus vectors in combination with MVA vectors to encode the same non structural proteins. Therefore, the body is able to produce antibodies against the NS3 protein, which directly interferes with Interferon production and is responsible for many of the protease actions required for HCV assembly and replication, and the NS5b protein, which is the polymerase responsible for replicating the RNA of the HCV virus to be then packaged into new virions.  Both of the above are currently in phase II clinical trials There is another type of vaccine currently in phase I trials, the HCV E1E2-MF59  vaccine, which exposes the individual to the glycoproteins expressed on the surface of the molecule. It is thought that if an individual can express antibodies against these proteins, they could be destroyed if a HCV infection were to occur, thus meaning the viruses would not be able to enter hepatocytes and would simply be destroyed in the blood.    Treatment of HCV For treating mild chronic hepatitis C peginterferon alfa-2a or 2b and ribavirin. This is also the treatment of choice for children with chronic infection. For the treatment of chronic hepatitis C of genotype 1 or 4, 12 weeks dual therapy using Elbasvir–grazoprevir is recommended by NICE.   Is A Screening Programme Preferable to a Vaccine? France screens pregnant women, high risk groups (such as PWIDs) and those with abnormal LFTs and as a result have subsequently identified approximately 75% of carriers of HCV.  This would be a positive step for the UK to take towards the 2030 goal. Whilst there is no screening programme, in 2017 the UK Government released a report calling for those at increased risk to attend GPs and GUM clinics for free testing. Such high risk groups included those that had received a transfusion before September 1991, those who had had sex with someone with HCV and those who had shared needles with another person.    Argument for a Vaccine: It is estimated that even a vaccine of 50% effectiveness would increase the number of countries on track to reach the 2030 target from 7 to 87 (out of 167 countries studied).   Difficulties in Producing Vaccine: Live attenuated vaccines traditionally made by passing virus through multiple cell lines or deleting virulence factors No virulence factors have been identified HCV only grows in a few human cell lines  Due to high mutation rate it would be far too risky to introduce a live virus into a human host due to risk of resistance development Culturing HCV in vitro is a newly developed process which yields very low titres insufficient for producing a whole inactivated virus. Also, in most countries HCV is a containment level 3 pathogen and therefore cannot be utilised in many research environments.  Chimpanzee model no longer acceptable for use. Certain mouse models exists, e.g. human liver xenograft but the mice have to be immunocompromised to prevent organ rejection immune responses so the immune response to a potential vaccine can't actually be studied. Other mouse models expressing HCV receptors are far too expensive and thus rarely used. The most useful cohorts for clinical trial are PWIDs who have been monitored before they were infected with HCV. Obviously this is fairly niche and thus few cohorts of this type are in existence. A way around this might be to test developed vaccines in those at a lower risk in order to try and understand what a protective immune response to the vaccine entails.  Complete structures of the E1 and E2 glycoproteins have not yet been reported and thus it is difficult to know whether specific targeting is possible, or whether there are better and more conserved targets yet to be found.  The lack of standardised assays across labs working i different countries leads to some potential vaccine candidates being missed due to the lack of available comparison.  The newly developed HCVpp system is a step in the right direction towards rectifying this problem   B Cell Vaccine (bNabs) This would have to involve the discovery of viral regions which are highly conserved among the quasi-species before learning how to effectively target them. Candidates for this include the E1 and E2 glycoproteins which facilitate entry of the HIV virion into the hepatocyte. E2 is the main target of neutralising antibodies, but it is important to note that both of these proteins are very widely glycoslyated to reduce the binding efficacy of virus neutralising antibodies. Most bNabs target E2-CD81 binding, as this binding is absolutely essential for entry into the hepatocyte. The binding residues for E2 on CD81 are W420 in the N-terminal as an alpha helix and bi-lobed binding loop Highly conserved amino acids within the HCV structure are 437W, 533D and N540. It is therefore possible that a vaccine using bNabs which target these amino acids could be developed and would theoretically be possible. This seems like one of the best options currently.