HCV VACCINE

1. Helene Andersen Maya Bonde Andersen Simon Carlsen Morten Ahlgreen Gronemann & Mads Christian Hjortsø HCV VACCINE INTRODUCTION Hepatitis C Virus (HCV) is a blood-borne virus and a major cause of chronic liver inflammation, liver cirrhosis and hepatocellular carcinoma throughout the world. WHO estimates 180 million infected individuals worldwide and about 4 million newly infected each year [1]. The HCV is a RNA virus with a single stranded 9kb genome encoding a polyprotein of about 3000 amino acids cleaved to 10 functional polypeptides including three structural proteins. The core protein capsules the RNA genome, and envelope glycoproteins E1 and E2 are responsible for entry into host cell. Six HCV genotypes are found, but genotype 1 and its subtypes 1a and 1b are responsible for more than 70 % of all cases of HCV and are the major subtypes observed in the western world [2]. The purpose of this study is to identify possible molecular targets for vaccination in the subtypes 1a and 1b. Figure 1, Polyprotein of HCV [3]: The HCV polyprotein consists of 10 proteins. The core protein, E1, and E2 are structural proteins and the seven other proteins are non-structural. HCV PROTEINS AND EPITOPES The HCV Sequence Database [5] contains sequences for all HCV genotypes. Based on five consensus sequences aligned in ClustalX (figure 4), we found the non-structural protein NS3 to be the most conserved. NS3 exhibits three enzymatic activities: the N-terminal of NS3 predominantly functions as a serine protease and the C-terminal comprises the NTPase and RNA helicase functions. Of course, this protein is not possible to target with antibodies. This protein seemed the first choice for search of epitope candidates for MHC Class I presentation. However, SYFPEITHI [6] and NetCTL [7] analysis showed that to target a large amount of HLA supertypes, epitopes from this protein alone would be insufficient. In the analysis high affinity (>0.5) and C-terminal cleavage (>0.8) and a SYFPEITHI score >19 was considered.Therefore, conserved regions from other HCV proteins were submitted to the same searches in SYFPEITHI and NetCTL. The obvious choice for B-cell recognition would be the viral surface proteins E1 and E2. BepiPred [9] predicted several epitopes in both proteins, but CPHmodels [10] was not able to identify a total structure of the protein. The only motif identified was an E1 α-helix predicted by TMHMM [11] to be in a transmembrane (TM) region. Although a BepiPred predicted epitope was located outside of the TM region, the data supports no conclusions at present time. Figure2, Model structure of HCV [3]: The HCV is a ssRNA virus. Its genome is protected by the Core protein and on the surface of the virus two envelope proteins E1 and E2 are found. These envelope proteins form a heterodimer (no structure available), which has shown to be necessary for viral entry into the host cell and hence an obvious target for antibody treatment. Figure 4, Alignment of the NS3 protein: Alignment of NS3 performed using ClustalX. Five consensus sequences for HCV subtypes 1a, 1b, 2a, 2b and 3a are used [5]. Figure 3, Global distribution of different HCV genotypes [4]: The HCV subtypes 1a and 1b are the most commonly found HCV type, infecting 73,2 % of the total number of infected individuals. In Europe and North America these subtypes are the cause of about 80% of all HCV cases. POLYTOPE A polytope was constructed consisting of nine peptides from four different proteins, as epitopes from NS3 alone was inadequate. The HLA range covered the supertypes A1, A2, A3, A24, B27, B44 and B62, which are represented by either one or two alleles in about 90 % of Caucasian population [8]. No suitable epitope could be chosen for B7 and B8. Besides, some of the less commonly occurring supertypes were not investigated, e.g. B58. Optimizing the polytope the program polytope_cont3 [12] was used. Table 1 shows from which protein the different epitopes in the polytope are derived. The polytope includes peptides from four of the ten HCV proteins, covering seven different MHC Class I supertypes and one MHC Class II supertype. All individual epitopes were BLAST’ed against the human genome and no significant matches were found [13]. Sequence for the polytope incl. linkers: mVLVDILAGYrrATDALMTGYhtwrDYPYRLWHYaayctGEIPFYGKAayyaFRKHPEATYtfyerrSWHINSTALkiMNRLIAFASRGNHVSrtwSWHINRTALyrwKLGALTGTYqcykILAGYGAGVakakq capital letters: epitopes lower case letters: linkers italics: epitope that functions as a linker in this polytope *) functions as a linker in this case Figure5, Epitope atlas of the polytope: Atlas of the polytope showing MHC binding affinity, proteasomal cleavage and position of the different epitopes (see table 1). The affinity bar only show the affinity of the epitopes binding to the HLA supertype A2 Table 1: Schematic view of the origin of epitopes in the polytope. For each sequence in the polytope is given the protein from where it was selected, its start position and the HLA supertypes it binds to. ‘ID’ refers to the position in the polytope showed in figure 5; the epitope atlas. NetCTL also identified A2-0, A2-1 and A2-3 as possible epitopes for HLA supertype B62, A2-0 and A2-5 as possible epitope for HLA supertype A3 and also A2-5 as a possible epitope for HLA supertype A2. However these epitopes was not further examined. DISCUSSION RNA viruses have relative high mutation rates and thus constructing the ideal vaccine which covers the entire world’s population a broad spectred polytope would be preferred. For simplicity reasons we aimed at the widely distributed HCV genotypes 1a and 1b and the HLA supertypes A2, A3 and B7 covering most of the people in the world. Unfortunately, no suitable epitope was found to cover the B7 and B8 supertypes. A polytope comprising more rare supertypes was constructed capable to cover roughly 70 % of Caucasian people. The polytope had to include epitopes from several different HCV proteins, since a spectrum broad enough to target all HLA supertypes was impossible to achieve from a single protein. The polytope included one MHC Class II directed epitope that might work in case the DNA fragment is not cleaved intracellular but by normal phagocytosis. The next step in HCV vaccine research would be epitope binding affinity measurements and then – in a few years time – clinical trials might be possible. We consider this an initial step in the direction of achieving a functional vaccine against HCV. Similar methods may be applied to other genotypes and subtypes. REFERENCES ACKNOWLEDGMENTS The poster is produced as part of the course 27685 Immunological Bioinformatics at CBS, DTU. A great thanks to our teachers: Ole Lund, Morten Nielsen and Claus Lundegaard [1] www.who.int (June 21st 2006), [2] Virology. 2006 Apr 25;348(1):1-12. Epub 2006 Feb 7,[3] www-ermm.cbcu.cam.ac.uk (June 21st 2006), [4] http://hcv.lanl.gov, [5] http://hcv.lanl.gov, [6] www.syfpeithi.de, [7] http://www.cbs.dtu.dk/services/NetCTL, [8]March et al: The HLA Facts book, [9] http://www.cbs.dtu.dk/services/BepiPred/, [10] http://www.cbs.dtu.dk/services/CPHmodels/, [11] http://www.cbs.dtu.dk/services/TMHMM/, [12] Polytope_cont3 (Morten Nielsen), [13] http://www.ncbi.nih.gov/blast/