Helene Morten Andersen Ahlgreen Maya Gronemann Bonde &

Helene Morten Andersen Ahlgreen Maya Gronemann Bonde &

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 [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].

Andersen
Mads

Simon
Christian

Carlsen
Hjorts

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.

The purpose of this study is to identify possible molecular targets for vaccination in the subtypes 1a and 1b.

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 -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.

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 [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 BLASTed against the
human genome and no significant matches were found [13].
Sequence for the polytope incl. linkers:
mVLVDILAGYrrATDALMTGYhtwrDYPYRLWHYaayctGE
IPFYGKAayyaFRKHPEATYtfyerrSWHINSTALkiMNRLIA
FASRGNHVSrtwSWHINRTALyrwKLGALTGTYqcykILA
GYGAGVakakq
capital letters: epitopes
lower case letters: linkers
italics: epitope that functions as a linker in this polytope

ID

Peptide from protein

HLA supertype

Start position

Sequence

A2-0

NS3

A1

411

ATDALMTGY

A2-1

NS4B

A2

141

ILAGYGAGV

A2-2

NS2

A3, B62

132

KLGALTGTY

A2-3

E2

B27

203

FRKHPEATY

A2-4

NS3

B44

346

GEIPFYGKA

A2-5

NS4B

B62

137

VLVDILAGY

A2-6

E2

A24

227

DYPYRLWHY*

A2-7

E2 1a

A24

36

SWHINSTAL

A2-8

E2 1b

A24

36

SWHINRTAL

NS4B

DR4

208

MHC Class II
A2-9

MNRLIAFASRGNHVS

*) 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.

[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/

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

Recently Viewed Presentations

  • How can urban living be made more sustainable?

    How can urban living be made more sustainable?

    Location of BedZED . Wallington, Greater London, in the SE of England, The UK's largest carbon neutral development. Urban Environments: How can urban living be made more sustainable? CASE STUDY IN A DEVELOPING COUNTRY . Beddington Zero Energy Development (BedZED)...
  • ADvent

    ADvent

    Advent What is Advent? Why do we have Advent? Advent marks the beginning of the Christian year. It is the four weeks before Christmas beginning on November 30 or the nearest Sunday to it.
  • High Quality Wind Retrievals for Hurricanes Isabel and Fabian ...

    High Quality Wind Retrievals for Hurricanes Isabel and Fabian ...

    Rain Rate Product Improved CFRSL rain retrieval algorithm provides earth-gridded 50 km data product Instantaneous integrated rain rates > 2.4 km*mm/hr Resampled to 12.5 km wind vector grid AMSR vs. SRad Path Attenuation, Horizontal Polarization 2003 Hurricane Results QuikSCAT Rev....
  • America Enters World War I

    America Enters World War I

    America Enters World War I Objective By the end of the lesson, SWBAT explain how the sinking of the Lusitania led to the U.S. entering World War I. REVIEW World War I was started because countries had a lot of...
  • The Sphere of the Earth - Administration

    The Sphere of the Earth - Administration

    On back sheet - Make a flip foldable titled Atmospheric Layers. Cut a single sheet of paper into fourths. Stack 3 onto each other leaving about a ½ inch - ¾ inch tab. Fold over - glue together at the...
  • T840 2004K - Open University

    T840 2004K - Open University

    Be careful of plagiarism and collusion - module uses CopyCatch/TurnitIn. Help and support page Referencing properly and getting software to help you manage your references Finding what you need Help and advice Library Help Questions Mark Campbell MBA (Tech Mgmt),...
  • Nonlinear Diffraction and Refraction of Regular and Random ...

    Nonlinear Diffraction and Refraction of Regular and Random ...

    Long-period Harbor Oscillations due to Short Random Waves Meng-Yi Chen & Chiang C. Mei Massachusetts Institute of Technology Typhon Tim 1994: Hualien Harbor,Taiwan Port of Hualien Typhoon Longwang, Oct. 2nd 2005 Past Works Harbor Oscillations - Linear theory Miles &...
  • AND NOW FOR A BRIEF INTRODUCTION TO: THE

    AND NOW FOR A BRIEF INTRODUCTION TO: THE

    DNA is almost completely lacking in catalytic ability ASSEMBLY OF AN INFORMATION BEARING POLYMER: EVOLUTION OF EARLY LIFE FORMS ORIGIN OF EARTH EXTINCT LINEAGES CURRENT TIME ORIGIN OF "LIFE" COMMON ANCESTOR OF ALL LIFE ON EARTH "CENANCESTOR" If the theory...