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In silico peptide based vaccine design against non-structural

protein 3 oI hepatitis C
Vikas Kaushik, Joginder Singh*, Ramandeep Kaur and Anupam Kumar
School of Biotechnology and Biosciences, Lovely Professional University, Phagwara, Punjab, India
Designing drug against Hepatitis C Virus (HCV) is not eIIective, as people who are most recently inIected with such inIection
do not show any symptoms and when the condition become Iatal aIter time oI exposure Irom the time oI inIection , the liver
becomes scarred, known as liver Cirrhosis which is incurable. Designing oI peptide based vaccine may overcome this
problem as they are the preIerred candidate Ior designing epitopes because oI their high activity and speciIicity. The
prediction oI epitopes in NS3 protein provides a suitable primary immunodiagnostic antigen Ior the detection oI the HCV. In
our study, one epitope i.e. LLGTIVTSL was selected on the basis oI halI-liIe oI dissociation, isoelectric point and binding
score between predicted epitopes and MHC. The 3D structure oI the epitopes was modeled using homology modeling by
Swiss Model. The predicted epitope LLGTIVTSL was identiIied to be a highly conserved, immunogenic and potential
vaccine candidate, capable Ior generating both CD8 and CD4 responses.
2013 Elsevier Science. All rights reserved.
Keywords: Epitopes, HCJ, NS3, Jaccine
1. Introduction
Hepatitis C virus was identiIied in 1989 through expression cloning oI immunoreactive cDNA derived Irom the inIectious
non-A and non-B Hepatitis agent which had already being recognized as the major cause oI transIusion-acquired hepatitis. It
typically causes persistent hepatotropic inIection, although it is the major challenge to detect viral antigens reliably in inIected
liver tissues. Flavivirus belongs to the Iamily Flaviviridae and genus HCV having positive single stranded RNA as a genomic
material. BeIore the era oI Molecular Biology, members oI the Iamily Flaviviridae had been previously classiIied as
Togaviridae. The HCV genome is an uncapped 9.6-kb RNA containing highly structured 5` and 3` ends. The 5` non coding
region is a well conserved, 341 nucleotide sequence element that Iolds into a complex structure containing Iour major
domains. Flavivirus encodes one large open reading Irame containing a 5` type cap and conserved RNA structures at both the
5` and 3` untranslated regions that are important Ior viral genome translation and replication |1|. The genomic RNA is
translated into a single polyprotein precursor consisting oI three structural Capsid (C), perinuclear membrane (prM), and
Envelop (E) protein and seven non-structural NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 proteins arranged in the order C-
prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5. Only the structural proteins become the part oI the mature, inIectious virion,
whereas the non-structural proteins are involved in the polyprotein processing, viral RNA synthesis and virus morphogenesis.
Non-structural protein 3 is a multi-Iunctional protein with an N-terminal protease domain (NS3pro) that is responsible Ior
proteolytic processing oI the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase
and RNA-stimulated NTPase domain are essential Ior RNA replication. The serine protease domain oI NS3 is a key player in
the replicative cycle oI Flavivirus. The data shows approximately 66 population oI Northern India is Iound to be inIected
with NS3a |2|. In one study, data showed that NS3a also inIects the Hepatitis C in Punjab-Pakistan also to a greater extent |3|.
In the Southern part oI India the data shows that 60 oI population is Iound to be inIected by NS1 and 40 oI population is
Iound to be inIected with NS3 |4|. ThereIore it can be concluded Irom these data that, although NS3 is somewhere aIIecting
with greater extent and somewhere with less extent and it also aIIects the population oI other Asian countries but NS3 is the
most common viral protein which inIects the population, so it is the subject oI choice to study and to design the novel vaccine
Ior human welIare.
In this in silico study, we designed a 9-mer peptide Irom the uncharacterized non-structural 3 protein having subtype 3a
(NS3a) oI the Hepatitis C virus by the CPHmodel-3.2 Server, an online tool Ior homology modeling and the Iinal structure
Proceedings oI International ConIerence on Computing Sciences
ISBN: 98-93-5107-172-3
498 Elsevier Publications, 2013
Corresponding author. Vikas Kaushik
Vikas Kaushik, Joginder Singh, Ramandeep Kaur and Anupam Kumar
aIter its validation using RAMPAGE Ior its secondary structure stability processed Ior docking with both MHC Class-I and
MHC Class-II molecule. The main aim oI this study was to get the epitope that could bind actively and speciIically to both the
MHC molecules and thus by giving raise to memory cells it proves itselI to be a better vaccine. We hope, this vaccine would
get success to clear out all the phases oI clinical trial and it would be eIIective vaccine in the cure oI Hepatitis C.
. Methodology
Uncharacterized protein NS3a sequence:
The sequence oI the uncharacterized NS3 protein, subtype 3a was obtained Irom the UniProt database which was Iurther used
Ior the epitope prediction.
~tr,D6BQ42,D6BQ429HEPC NS3 (Fragment) OSHepatitis C virus subtype 3a GNNS3 PE4 SV1
Epitope prediction:
The epitopes (9-mer peptides) Irom the sequence NS3a protein oI HCV were predicted Ior all the Human Leucocyte Antigen
(HLA) alleles Ior both the MHC Class-I and MHC Class-II molecules. The prediction Ior promiscuous MHC binders was
carried out via ProPred-I |5| and ProPred |10| Ior MHC Class-I and MHC Class-II respectively.
Virtual Screening:
In this step, we screened the promiscuous MHC binders separately Ior both the class oI MHC molecules. From the result table
obtained Irom the epitope prediction, each epitope were counted and arranged in the decreasing order on the basis oI the
number oI predictions obtained Ior multiple alleles as binders. A separate prepared table Ior both MHC Class-I and MHC
Class-II was then used to screen Ior such epitopes which is common in both the MHC molecules that is screened those
epitopes which would bind to both class oI MHC molecule. This will prove to be a better vaccine as epitope binding to only
one oI the MHC molecule will not be so eIIective Ior producing immunity against such viral agent |11|. We obtained six
peptide sequences (epitopes) aIter virtual screening which were the binders to both MHC Class-I and MHC Class-II molecule.
Structure prediction:
The structure prediction oI above six predicted epitopes was carried out by using the CPHmodel-3.2 Server, an online three
dimensional structure prediction sever which is based on automated homology modeling.
Energy simulation:
The predicted structure oI the above peptide sequences were energy optimized using the tool GROMACS (Groningen
Machine Ior Chemical Simulation) |6|, which is itselI a matter oI intense study works well on the LINUX environment. It
uses the Iive dynamic steps that is, pdb2gmx; editconI; genbox; grompp and mdrun. The energy optimization is necessary in
order to decrease the electronic repulsion and steric hindrance and Ior the Iavorable local interactions between the amino acid
side chains so as to get a stable native and Iunctional conIormation.
AIter the energy simulation oI the peptide molecules, they were validated by using the online validation server RAMPAGE
based on the concept oI Ramachandran Plot. The structures were validated to conIirm its stable secondary structure
conIormation. This was validated by analyzing the number oI amino acid residues in Iavored region, allowed region and
disallowed region. It was analyzed that the peptide sequence VLSTATQTF showed the amino acid residues in the Iavored
region with 100 per cent. And at second position, the peptide sequence LLGTIVTSL showed the amino acid residues in the
499 Elsevier Publications, 2013
In silico peptide based vaccine design against non-structural protein 3 of hepatitis C
Iavored region with 83.3 per cent. Other peptide sequences showed either 75 per cent or less than that. This again can be a
step oI screening but instead we used all six epitopes structure Ior docking with MHC Class-I and MHC Class-II molecule.
MHC Class-I & MHC Class-II structure:
Three-dimensional structure oI MHC Class-I and MHC Class-II (PDB ID: 3MGR; PDB ID: 1DLH) respectively oI Human
origin were solved by X-Ray DiIIraction in both the molecules but 3MGR is the crystal structure oI MHC Class-I HLA-A2
molecule complexed with HCV and 1DLH is the crystal structure oI Human MHC Class-II (HLA-DR1, Alpha chain) and
(HLA-DR1, Beta chain) complexed with enterotoxin type B precursor oI InIluenza virus. These complexed ligands were then
removed using three dimensional molecular visualization tools, Pymol to obtain the pure crystal structure oI MHC molecules.
We perIormed the docking oI all the six peptide structures separately each with MHC Class-I (PDB ID: 3MGR) and MHC
Class-II (PDB ID: 1DLH) molecule using an online docking tool named as Patch Dock |7|. For the best result the docking
score should be maximum, with least atomic coordinate energy (ACE) value. In our study, we Iound that the docking oI the
peptide sequence (LLGTIVTSL) with MHC Class-I (PDB ID: 3MGR) showed the top most score oI 8896 with the least ACE
value oI -255.14 KJ/Cal as compared to other peptide sequences. In second case, the docking oI peptide sequence
(LLGTIVTSL) with the MHC Class-II (PDB ID: 1DLH) again showed the highest score oI 9012 with least ACE value oI -
300.73 KJ/Cal, as compared to other peptide sequence.
. Results:
Fig 1- Predicted structures oI all 6 epitopes.
500 Elsevier Publications, 2013
Vikas Kaushik, Joginder Singh, Ramandeep Kaur and Anupam Kumar
Fig 2 3D structure oI the MHC class I and MHC class II molecule.
Figure 3 - The peptide binding to the MHC class I and MHC class II molecule. The peptides (shown in blue color) predicted
to a have very high aIIinity Ior the MHC-I and MHC-II modeled with the help oI Gromacs server. The highest binder peptide
LLGTIVTSL is derived Irom the HCV.
501 Elsevier Publications, 2013
In silico peptide based vaccine design against non-structural protein 3 of hepatitis C
Table-1 Comparison oI HalI liIe oI dissociation, pI value and binding scores between the six epitopes according to Propred soItware
. Discussion
The NS3 is the multiIunctional protein with an N protease domain that is responsible Ior proteolytic processing oI viral
polyprotein and contains RNA helicase, NTPase which are essential Ior RNA replication. ThereIore, NS3 protein is our
imperative choice Ior inhibition to stop the proteolytic processing. With the rapid advancement in computational
methodologies and immune-bioinIormatics, we can design a peptide based vaccine as it is preIerred Ior designing oI epitopes
because oI their high speciIicity and activity. The LLGTIVTSL epitope was selected on the basis oI halI-liIe oI dissociation,
isoelectric point |8| and binding score. The halI-liIe oI dissociation is the predicted halI time oI dissociation to HLA molecule.
More the time oI dissociation, better the candidate epitope. From the table, it can be inIerred that, the average score oI halI-liIe
oI disassociation (t
) Ior LLGTIVTSL is 7.071951, which is the greater than the other epitopes. So on the basis oI (t
) this
one can be taken in to consideration. In case oI the isoelectric point, it accounts Ior the Iact that the MHC molecule constitutes
mostly the amino acids that are basic in nature |9|. So, accordingly the epitope should be more acidic in nature or the pI value
should be less. So, it can be inIerred Irom the table that the same epitope LLGTIVTSL has the average pI value oI 5.52.
Structure based modeling oI epitopes was done with the help oI CPH model and Iurther the energy was optimized with the
Gromacs tool. Then aIter that the binding Score was calculated, which was again best in case oI LLGTIVTSL. However, as
shown in the hypothetical example oI planning a vaccine, the discovery oI suitable antigens could be a small part oI the
problem oI producing an eIIective vaccine, but never the less important. The study shows that the predicted epitope was
highly conserved and potential vaccine candidate.
. Conclusion
Immune-inIormatics is an emergent branch oI inIormatics science that long ago pullulated Irom the tree oI knowledge that
is bioinIormatics. To a great extent, immune-inIormatics is typiIied by epitope prediction methods. The present study was
undertaken with an objective to initiate the use oI Immuno inIormatics. These approaches are currently used Ior prediction oI
antigenic determinants in the protein sequence oI NS3 oI HCV without using their cultures. The prediction oI HCV epitopes
Ior is recognized against MHC molecules. Out oI several epitopes six epitopes were screened out which will bind to both
MHC Class I and MHC Class II with low or higher aIIinity.The predicted epitopes may be served as a useIul diagnostic
reagent Ior evaluating T-cell responses in the context oI natural inIection and also might be helpIul Ior designing a subunit
vaccine against HCV. The eIIective development oI antigen prediction methods would signiIicantly reduce the laboratory
resource required to identiIy pathogenic proteins as candidate subunit vaccines.
502 Elsevier Publications, 2013
. Tables
Vikas Kaushik, Joginder Singh, Ramandeep Kaur and Anupam Kumar
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|3| Abida Shehzadi, Shahid ur Rehman, and Muhammad Idrees, Promiscuous prediction and conservancy analysis oI CTL binding epitopes
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|4| Chandra M. Rekha Thippavuzzula et al, Genotyping oI Hepatitis C virus (HCV) in inIected patients Irom South India, InIection. Genetics
and Evolution, 7:724-730, 2007.
|5| Singh, H. and Raghava, G.P.S. (2001) ProPred: Prediction oI HLA-DR binding sites. BioinIormatics, 17(12), 1236-37.
|6| Christian Blau , Helmut Grubmuller, gcontacts: Fast contact search in bio-molecular ensemble data, Max Planck Institute Ior
Biophysical Chemistry, Am Fassberg 11, 37077 Gttingen, Germany
|7| Schneidman-Duhovny D, Inbar Y, Nussinov R, WolIson HJ. Patch Dock and SymmDock: servers Ior rigid and symmetric docking. Nucl.
Acids. Res. 33: W363-367, 2005.
|8| Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R.D., Bairoch A. ExPASy: the proteomics server Ior in-depth protein knowledge
and analysis Nucleic Acids Res. 31:3784-3788(2003).
|9| Laurie AT, Jackson RM, Q-SiteFinder: an energy-based method Ior the prediction oI protein-ligand binding sites, BioinIormatics. 2005
May 1; 21(9):1908-16. Epub 2005 Feb 8.
|10| Harpreet Singh and G.P.S. Raghava, Prediction oI promiscuous MHC Class-I binding sites. BioinIormatics, 19: 1009-1014, 2003.
|11| R. Parida, M.S. Shaila, S. Mukherjee, N.R. Chandra, R. Nayak, Computational analysis oI proteome oI H5N1 avian inIluenza Virus to
deIine T cell epitopes with vaccine potential Vaccine, 25:7530-7539, 2007.
503 Elsevier Publications, 2013

Dengue virus, 493
methodology, 494
results and discussions, 495496