Helene Morten Andersen Ahlgreen Maya Gronemann Bonde &
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 .
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 .
Figure 1, Polyprotein of HCV : The HCV polyprotein consists of 10 proteins. The core protein, E1, and E2 are structural proteins and
the seven other proteins are non-structural.
The purpose of this study is to identify possible molecular targets for vaccination in the subtypes 1a and 1b.
The HCV Sequence Database  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  and NetCTL  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 
predicted several epitopes in both proteins, but CPHmodels  was not able to identify a total structure of
the protein. The only motif identified was an E1 -helix predicted by TMHMM  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 : 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
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 .
Figure 3, Global distribution of different HCV genotypes : 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.
MHC Class I
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 .
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  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 BLASTed against the
human genome and no significant matches were found .
Sequence for the polytope incl. linkers:
capital letters: epitopes
lower case letters: linkers
italics: epitope that functions as a linker in this polytope
Peptide from protein
MHC Class II
*) functions as a linker in this case
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.
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
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.
RNA viruses have relative high mutation rates and thus constructing the ideal vaccine which covers the entire worlds 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.
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