Proceedings oI International ConIerence on Computing Sciences

WILKES100 ICCS 2013

ISBN: 978-93-5107-172-3
Development oI peptide-based vaccine Ior dengue virus
variants using in-silico method
Krishna Kumar and Vikas Kaushik
*

1
Department of Electronics and Communication Engineering, Lovely Professional University, PB, India
Abstract
Dengue virus is one oI the inIectious agents oI Flaviviridae Iamily that aIIects the human beings around the world at a great
extent. It causes various types oI diseases by inIecting human body, one oI which is dengue Iever. This research is Iocused on
In- silico designing oI dengue virus vaccines, by considering all the 7 types oI non-structural(NS1, NS2A, NS2B, NS3, NS4A,
NS4B and NS5) proteins Iound in that Iour dengue virus serotypes (DENV 1-4) as they play major roles in diIIerent stages oI
dengue virus liIe cycle during viral inIection. The sequences oI all the Dengue viral Non-Structural proteins were retrieved
Irom SwissProt database and T-cell epitopes were predicted by using ProPred and ProPred 1 server, which predicted some
highest binding aIIinity epitopes against MHC molecules. The hydrophobicity and molecular weight oI the highly conserved
epitopes were predicted by 'Compute PI/MW tool and the epitope having lowest PI value was selected as the binding pocket
oI MHC molecule is rich in basic residues that will interact with acidic residues oI ligand molecule. The selected epitope was
docked with MHC molecules by using 'AutoDock 4.2. The docking results indicated that they were stable as the selected
epitope was eIIiciently bound to the MHC molecule. As a whole, Non-Structural protein epitopes studied in this research
could be used as suitable vaccine candidate against Dengue virus inIections.
2013 Elsevier Science. All rights reserved.
Keywords: Non-structural proteins, T-cell epitopes, MHC molecules, Dengue vaccine
1. Introduction
Dengue virus is one oI the inIectious agents that belong to Flaviviridae Iamily. Dengue virus has Iour types oI
strains named as DENV-1, DENV-2, DENV-3 and DENV-4; all oI them are capable to cause disease by inIecting
the human body |10|. These viruses are transmitted to humans when an inIective mosquito known as Aedes
aegypti bite humans. The incubation period Ior dengue virus is 4-7 days.
The genome oI Dengue virus contains a positive-sense RNA consisting oI an open reading Irame that code Ior
polyprotein. The polypeptide is processed co-translationally by dengue viral and various other cellular proteases
to generate only 3 types oI structural proteins, capsid, that binds to positive-sense RNA oI viral genome
producing a nucleocapsid, and two lipid containing membrane proteins: prM having molecular mass, 7-8 kDa and
an envelope protein E oI molecular mass, 55-60 kDa. The Iunction oI envelop protein is to control the receptor
binding and Iusion with the host cell |5|.
Dengue viral polyprotein also Iorm 7 types oI Non-structural proteins, named as NS1, NS2A, NS2B, NS3,
NS4A, NS4B and NS5 |2|.The most important non-structural proteins are NS1 having molecular mass, 44-49
kDa; NS3 oI 67-76 kDa molecular mass and NS5 (molecular mass, 91-98 kDa) and these proteins are expressed
in the viral inIected cells |8|. Among these, the most immunogenic is NS1. NS1 is involved in virus replication
and regulation oI the innate immune responses. The soluble and membrane associated NS1 can activate human
complement and induce host vascular leakage |1|. NS3 is a type oI serine protease. It displays three enzymatic
activities: serine protease, RNA helicase and NTPase. The NS3 serine protease, in association with NS2B, is
responsible Ior its auto cleavage. It cleaves the polyprotein at dibasic sites in the cytoplasm, i.e.; C-prM, NS2A-
NS2B, NS2B-NS3, NS3-NS4A, NS4A-2K and NS4B-NS5 |9|. NS5 is the most conserved oI all the non-
structural proteins in dengue virus serotypes. It Iunctions as an RNA-dependent RNA polymerase. NS5 is
responsible Ior the replication oI the viral positive and negative genome, and perIorms the capping oI genome in
493 Elsevier Publications, 2013
*
Corresponding author. Krishan kumar
Krishna Kumar and Vikas Kaushik
the cytoplasm. Besides its role in genome replication, it also prevents the development oI cellular antiviral state
by blocking the interIeron- alpha/beta (IFN-u/) signaling pathway. It also inhibits phosphorylation oI TYK2 and
STAT2 in host that prevents the activation oI JAK-STAT signaling pathway |4, 6|.
The use oI immunoinIormatics methods in development oI a saIe subunit vaccine against dengue virus reduces
the time and the eIIorts |3|. T cell mediated immunity can be one oI the options in neutralizing the antigenic
inIection. In T cell mediated immunity, MHC molecules play an important role by recognizing the Ioreign
antigenic peptides and its expression to the cell surIace, where these peptides are processed and degraded by the
recruitment oI HTLs and CTLs |7|. In this research, we have considered the T cell mediated processing and
degradation oI antigenic peptide in the host and developed a new conserved epitope in dengue virus Non-
Structural proteins as these proteins play an important role in diIIerent stages oI viral liIe cycle in host. This new
epitope can be suitable and used as a saIe subunit vaccine against dengue inIections.
2. Methodology
2.1. Retrieving all nonstructural proteins sequences of Dengue virus serotypes:
All non-structural proteins sequences in Iour dengue virus serotypes (DENV 1-4) were retrieved by accessing
UniProtKB/SwissProt |16| protein sequence database. The sequences were in FASTA Iormat. These sequences
were used as query Ior carrying out Iurther analysis.
2.2. Prediction of T-cell epitopes for all nonstructural proteins in DENV (1-4) serotypes
The prediction oI T cell epitopes was done with ProPred |11| and ProPred I |12| servers. ProPred predicts
MHC class II binding peptides Ior 51 alleles and ProPred I predicts MHC class I binding peptides (CTL epitopes)
Ior 47 alleles.
2.3. Screening of Predicted T cell Epitopes for nonstructural protein in DENV (1-4) serotypes:
The predicted T cell epitopes were screened on the basis oI number oI alleles on which they bound and their
average binding score. Top Iive conserved epitopes (Ior both MHC-II and MHC-I) were considered and selected
as an eIIicient MHC class II and MHC class I binding peptides.
2.4. Retrieval of 3D structure of MHC molecule:
The 3 Dimensional structure Iiles oI MHC class-I (PDB ID: 1HSA) and MHC class- II (PDB ID: 1DLH) were
retrieved Irom PDB (Protein Data Bank) |17|.
2.5. Tertiary Structure prediction of epitopes:
The 3D structure prediction oI various epitopes oI dengue virus serotype was perIormed with PEP-FOLD
server |13|.
2.6 Calculating isoelectric point (PI value) and Molecular weight of selected epitopes:
The Isoelectric values (PI) and Molecular weight oI each selected epitopes were calculated using 'Compute
PI/Mw tool at ExPASy web server. The epitope having lowest PI value was selected.
2.7 Docking epitopes with MHC molecules:
The screened epitope was docked with MHC II (HLA DR1) and MHC I (HLA B2705) molecules by using
AutoDock 4.2 |14| tool to see the possible interactions and best conIormation was selected on the basis oI lowest
energy.
494 Elsevier Publications, 2013
Development of peptide-based vaccine for dengue virus variants using in-silico method
\

3. Results and Discussions
The protein sequences oI all the 7 types oI Non-structural proteins Iound in Dengue virus serotype (DENV 1-
4) were retrieved Irom SwissProt Protein sequence database. The T cell epitopes were predicted by using
ProPred and ProPred1 server that predicted some highest binding aIIinity epitopes against MHC II and MHC I
molecules respectively. The peptides (T cell epitopes) having highest score were screened out as it is well thought
out that higher the score oI any peptide; greater is the probability oI its binding to the MHC molecules. Then, the
conserved peptides among all the serotypes oI dengue virus were searched out, because the conserved peptides
are considered to be used as a better subunit vaccines candidate. Then top Iive peptides were screened against
MHC II and MHC I molecules each on the basis oI number oI alleles on which they bound and their average
binding scores. The 3D structure prediction oI these epitopes was perIormed with PEP-FOLD server. The PEP-
FOLD server had generated 5 best models Ior each epitope sequence on the basis oI sOPEP (Optimized Potential
Ior Ecient structure Prediction) Ior diIIerent clusters. The PEP-FOLD cluster Iile also calculates Apollo TM-
Score Ior diIIerent clusters. By considering these scores (sOPEP & TM-Score), two best structure models, Iirst on
the basis oI sOPEP and second having largest TM-Score, were selected out oI 5 models that PEP-FOLD had
generated. These two screened models were docked with MHC molecule using PatchDock |15| soItware to
determine which one is binding eIIiciently to the MHC molecule. On the basis oI their binding score, the best
model was selected. Isoelectric Point (PI values) and Molecular wt. oI these selected epitopes, on the basis oI
Patch Dock result, were calculated by using 'Compute PI/MW soItware at ExPASy web server. The peptide
having highest score in Patch Dock result and showing less PI value was selected to dock with MHC molecules
with Autodock 4.2. The less PI valued peptide was selected because the binding pocket oI the MHC molecules
contains positively charged exposed side groups that would interact with ligand having negative charge
Fig 1: Tertiary structure oI MHC binding peptides (Epitopes).
495 Elsevier Publications, 2013
Krishna Kumar and Vikas Kaushik
Table 1: MHC Binding Peptides Showing Binding Score, PI value, and Mol. Wt.:
Sequence Binding Score
(Patch Dock)
PI Value Mol.Wt.
LILAPTRVV 8880 9.75 981.25
LMCHATFTM 9362 6.73 1054
LRGLPIRYQ 9480 10.84 1115.34
MRLLSPVRV 8636 12 1070.36
YNMMGKREK 9084 9.7 1156.38
DARTYSDPL 8174 4.21 1037.09
FEPEREKSA 7976 4.09 1092.17
FQADSPKRL 8118 8.75 1061.21
RRCLKPVIL 8356 10.86 1097.43
RRGDLPVWL 7578 9.6 1111.31
From above table, LMCHATFTM was selected as an eIIicient T cell epitope as it exhibited lesser PI value as
well as a high binding aIIinity to MHC II molecule. This epitope also showed better aIIinity, when it was docked
with MHC I (Binding score 8600) molecule using Patch Dock. Finally, the selected epitope (LMCHATFTM)
was docked with both MHC class II and MHC class I molecules by using AutoDock 4.2 and the results were
analysed to see the interactions.
Fig 2: AutoDock result oI Dengue virus peptide (Selected epitope) docked with (A) MHC- II (HLA DR1) and (B) MHC- I (HLA B2705).
AutoDock 4.2 result showed that the MHC-II-peptide complex was stabilized by three hydrogen bonds Iormed
between MHC-II and selected epitope (MHC binding peptide). The hydrogen bonds were Iormed between
THR90 oI MHC II and LEU1 oI ligand (MHC binding peptide). Other hydrogen bonds were Iormed between
ASP171 oI receptor and THR6 oI ligand at two diIIerent atoms. The selected epitope was also docked to the
(A) (B)
496 Elsevier Publications, 2013
Development of peptide-based vaccine for dengue virus variants using in-silico method

MHC class I molecule to analyze the binding aIIinity oI that epitope against MHC class I molecule. The
AutoDock soItware generated a binding conIormation in which 3 hydrogen bonds were Iormed between the
ligand and the receptor molecules. One hydrogen bond was Iormed between LEU1 oI ligand and GLU 212
residues oI MHC I molecule. Other two hydrogen bonds were between LYS 58 oI MHC I and MET 2 oI ligand &
between HIS 4 oI ligand molecule and LEU 230 residues oI MHC I. The docking results oI dengue virus peptide
with MHC molecules showed that the dengue virus peptide easily bound to the active site oI MHC molecules
with a high binding energy.
4. Conlcusion
In this study, we have Iound that LMCHATFTM peptide could be the better vaccine candidate against
inIection oI dengue virus as it showed the best binding aIIinity against both the MHC class I and MHC class II
molecules. We have predicted the highest binding aIIinity epitopes against MHC II and MHC I by considering
the Non-Structural proteins oI Dengue virus serotypes. Selection oI conserved epitopes among all the Iour
serotypes oI Dengue virus (DENV1, DENV2, DENV3 and DENV4) has played a signiIicant role to select a
better vaccine candidate against dengue virus. Docking was useIul to explore the binding mechanism and studies
on the novel ligand against the NS3 protein showed that the Iree binding energy Ior the inhibitor was less,
signiIying that the ligand binds Iavorably to the active site. As a result, the ligand thus developed is likely to
inhibit viral inIections, and it may be concluded that this research work can be useIul Ior vaccine designing
against dengue virus serotype.
References
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|13| Maupetit J, Derreumaux P, TuIIery P. PEP-FOLD: an online resource Ior de novo peptide structure prediction. Nucleic Acid Res., 2009.
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|16| www.uniprot.org.
|17| www.RCSB.org.
497 Elsevier Publications, 2013
Index

C
Cournot's model
dynamic perspective, 487488
equilibrium stage, 489

S
Stackelberg's equilibrium, 489490