PLOS ONE

RESEARCH ARTICLE

Design of a novel multi-epitope vaccine

candidate against hepatitis C virus using
structural and nonstructural proteins: An
immunoinformatics approach
Esmaeil Behmard1, Hussein T. Abdulabbas2, Saade Abdalkareem Jasim3,
Sohrab Najafipour1, Abdolmajid Ghasemian ID4*, Akbar Farjadfar5, Ebrahim Barzegari6,
Amin Kouhpayeh7*, Parviz Abdolmaleki8*

1 School of Advanced Technologies in Medicine, Fasa University of Medical Sciences, Fasa, Iran,
2 Department of Medical Laboratory Techniques, Faculty of Health and Medical Techniques, Imam Ja’afar
a1111111111 Al-Sadiq University, Al Muthanna, Iraq, 3 Medical Laboratory Techniques Department, Al-maarif University
a1111111111 College, Ramadi, Iraq, 4 Noncommunicable Diseases Research Center, Fasa University of Medical
a1111111111 Sciences, Fasa, Iran, 5 Department of Medical Biotechnology, Fasa University of Medical Sciences, Fasa,
Iran, 6 Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical
a1111111111
Sciences, Kermanshah, Iran, 7 Department of Pharmacology, Fasa University of Medical Sciences, Fasa,
a1111111111 Iran, 8 Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

* [email protected] (PA); [email protected] (AK); [email protected] (AG)

OPEN ACCESS
Abstract
Citation: Behmard E, Abdulabbas HT, Abdalkareem
Jasim S, Najafipour S, Ghasemian A, Farjadfar A, et Hepatitis C virus (HCV) infects the liver and causes chronic infection. Several mutations in
al. (2022) Design of a novel multi-epitope vaccine the viral genome have been associated with drug resistance development. Currently, there
candidate against hepatitis C virus using structural
and nonstructural proteins: An immunoinformatics
is no approved vaccine against the HCV. The employment of computational biology is the
approach. PLoS ONE 17(8): e0272582. https://doi. primary and crucial step for vaccine design or antiviral therapy which can substantially
org/10.1371/journal.pone.0272582 reduce the duration and cost of studies. Therefore, in this study, we designed a multi-epi-
Editor: Manas Ranjan Dikhit, Rajendra Memorial tope vaccine using various immunoinformatics tools to elicit the efficient human immune
Research Institute of Medical Sciences, INDIA responses against the HCV. Initially, various potential (antigenic, immunogenic, non-toxic
Received: February 10, 2022 and non-allergenic) epitope segments were extracted from viral structural and non-structural
Accepted: July 21, 2022
protein sequences using multiple screening methods. The selected epitopes were linked to
each other properly. Then, toll-like receptors (TLRs) 3 and 4 agonists (50S ribosomal protein
Published: August 30, 2022
L7/L12 and human β-defensin 2, respectively) were added to the N-terminus of the final vac-
Copyright: © 2022 Behmard et al. This is an open cine sequence to increase its immunogenicity. The 3D structure of the vaccine was mod-
access article distributed under the terms of the
Creative Commons Attribution License, which
eled. Molecular dynamics simulations studies verified the high stability of final free vaccines
permits unrestricted use, distribution, and and in complex with TLR3 and TLR4. These constructs were also antigenic, non-allergenic,
reproduction in any medium, provided the original nontoxic and immunogenic. Although the designed vaccine traits were promising as a
author and source are credited.
potential candidate against the HCV infection, experimental studies and clinical trials are
Data Availability Statement: All relevant data are required to verify the protective traits and safety of the designed vaccine.
within the article and its Supporting Information
files.

Funding: The authors received no specific funding

for this work.

Competing interests: The authors have declared

that no competing interests exist.

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

1. Introduction
Hepatitis C virus (HCV) is one of the most important human infectious diseases threatening
the life of a large part of the international community [1–3]. The World Health Organization
(WHO) has estimated that ~200 million cases are infected with the HCV worldwide, and 3–4
million new cases are diagnosed each year [4–6]. Liver cirrhosis and carcinoma are major con-
sequences of chronic HCV infection. The treatment efficacy is insufficient due to various side
effects of anti-HCV drugs and the development of drug resistance by the virus [7–10]. There-
fore, there is an unmet requirement to develop new strategies such as vaccine design to reduce
the HCV incidence and related mortality.
It is well known that a successful vaccination should be associated with sufficient immunity
(through elicit of immune responses) and protection [11, 12]. Various vaccine candidates have
been developed against the HCV [13–15]. The design of a multi-epitope vaccine derived from
the structural and non-structural protein sequences is promising and preferable than whole
cell or inactivated candidates against viral pathogens [16–20]. These proteins play a major role
in the process of viral penetration and replication within the host cell [9, 10]. Multi-epitope
vaccines advantages over the inactivated or whole protein vaccines include lower costs and
rapidity of production, higher immunity and safety potential, lower non-specific reactions or
cross-reactivity and convenient manipulation [21–23]. Identification of potential epitopes
capable of eliciting immune responses have become a challenge in current safety research [17,
18]. Application of basic structure design techniques and immunoinformatics tools in hybrid
with dynamic mechanistic studies are promising to profoundly understand these mechanisms
[16–18]. Among these methods, computational techniques draw attentions increasingly,
because of their contribution to remarkable reduction of time and costs of vaccine design [14–
20, 24]. With the advent of sequencing technologies, sufficient genomic and proteomic infor-
mation have been provided from various viruses. As a result, peptide-based and epitope-based
vaccines can be designed with the help of various bioinformatics tools [14–20, 24]. The design
of epitope-based vaccines is now a familiar concept. Epitope-based vaccines against Leish-
mania donovani complex [15], Theileria parasites [16], Chikungunya virus [17], Human Her-
pes virus [18], human immunodeficiency virus (HIV) [19], Ebola virus [20], and human
coronavirus [22] have been suggested to date. For successful development of peptide-based
vaccines understanding the molecular details of antigen detection is an important and effective
step. Though the identification of immunogenic epitopes has been facilitated by various algo-
rithms, more computational studies are needed to prove the tendency and detail of interac-
tions between the immune receptor and epitopes. Considering this, docking and molecular
dynamics simulation services are effective sources of molecular-level structural information in
immunology. Various Toll-like receptors (TLRs) such as TLR2, TLR3, TLR4 and TLR6 act as
receptors of the viral proteins [25]. The membrane TLR4 and intracellular TLR3 agonist/adju-
vant has been adopted for ligand and receptor interaction assessment in some studies of multi-
epitope vaccine design [21, 22]. TLR3 plays a crucial role in the antiviral induction of hepatic
macrophages and Kupffer cells via nuclear factor kappa B (NF-KB) pathway and pro-inflam-
matory cytokines CXCL10. TLR4 agonist also provokes substantial immune responses [26].
In this study, a multi-epitope vaccine was designed against the HCV using immunoinfor-
matics and computational methods from sequences of HCV structural and non-structural
proteins.

2. Materials and methods

The basic steps of designing a multi-epitope vaccine are shown in the Fig 1.

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

Fig 1. Systemic flowchart of the multi-epitope subunit vaccines build against HCV.
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2.1. Viral protein sequences

For designing multi-epitope vaccine constructs against HCV, both structural and non-struc-
tural protein sequences were obtained from protein databases. The occurrence of high muta-
tion rate in the HCV genome in infected populations facilitates immune escape and
adaptation of the virus. Therefore, to obtain a vaccine active against wide range of viral strains,
alignment of the viral protein sequences from various strains is needed to gain conserved
sequences in order to select efficient candidate epitopes. To this aim, clustalW2 was used to
perform multiple sequence alignment [27].

2.2. Prediction of MHC-І binding (Cytotoxic T Lymphocyte (CTL))

epitopes
Predicting peptides that are capable of inducing T-cell responses is a crucial step in the design
of epitope-based peptide vaccine. The NetCTL v1.2 server (www.cbs.dtu.dk/services/NetCTL)
was applied to predict the CD8+ T-cell epitopes. In the IEDB MHC class I binding tool (www.
iedb.org), the Artificial Neural Network and human were selected as prediction method and
MHC source species, respectively. The IC50 value was selected 50 nM. In the NetCTL v1.2
server, the threshold value was selected as 0.5.

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

2.3. Prediction of MHC-ІІ binding (Helper T Lymphocyte (HTL)) epitopes

The IEDB tool was used to predict HTL epitopes in structural and non-structural proteins of
the HCV. Then, HTL epitopes with IC50 value of less than 50 nM were selected for further
use.

2.4. Prediction of linear B-cell epitopes

As we know, one of the important factors in the process of immunization of the human body
against pathogens is the secretion of antibodies by B lymphocytes, participating in the humoral
immunity. In this study, BCPred server (http://ailab.ist.psu.edu/bcpred) was utilized to predict
20-mer B-cell epitopes, using a support vector machine (SVM) algorithm and a kernel
technique.

2.5. Estimation of antigenicity, allergenicity, and toxicity of CTL, HTL and

B cells epitopes
The antigenic potential of CTL, HTL and B cells epitopes was predicted using the VaxiJen
v2.0, and epitopes with antigenic values of more than 0.4 were selected for further screening
[28]. The toxicity and allergenicity of these selected epitopes were checked by the ToxinPred
and the AllerTOP servers, respectively [29, 30]. Subsequently, the ability to induce the secre-
tion of various interleukins (interferon-gamma (IFN-γ), interleukin-4 (IL-4) and interleukin-
10 (IL-10) secreted by B and HTL (CD4+) cells epitopes was checked through the IFNepitope,
IL4pred and IL10pred server tools [31–33]. Among the predicted epitopes, those with high
antigenic potential, non-allergenicity and non-toxicity and high solubility at high expression
levels were selected to develop a multi-epitope vaccine.

2.6. Designing of multi-epitope vaccine construct

The multi-epitope vaccine was designed via fusion of CTL, HTL and B cells epitopes with
appropriate linkers (AAY and KK) according to recent studies [15–19]. Two separate adju-
vants were also added to the N termini to increase the immunogenicity of the multi-epitope
vaccines [15–19]. To this aim, a 124- and a 45-amino acid sequences from 50S ribosomal pro-
tein L7/L12 (TLR4 agonist) and human β-defensin 2 (TLR3 agonist) were respectively
obtained and added separately to the N terminus of the vaccine sequence using the appropriate
linker (EAAAK) [15]. The vaccines designed in this route contained 686 and 607 residues,
respectively.

2.7. Estimation of immunogenic, allergenic and physiochemical properties

of multi-epitope vaccines
The VaxiJen v2.0 and SolPro (http://scratch.proteomics.ics.uci.edu/) web tools were used to
predict the antigenic potential and solubility of the multi-epitope vaccines, respectively [28].
The allergenicity of the vaccines was checked using AllerTOP server [29]. In addition, the phy-
siochemical properties of the vaccines, such as amino acid composition, theoretical isoelectric
point (pI), grand average of hydropathicity (GRAVY), molecular weight, aliphatic and instabil-
ity index, and in vitro and in vivo half-life were evaluated using ProtParam server [34].

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

2.8. Modeling the three-dimensional structure of the multi-epitope

vaccines
The secondary structural properties of the multi-epitope vaccine were analyzed using the
SOPMA secondary structure prediction method [35]. Modeling and refinement of the three-
dimensional (3D) structure of the multi-epitope vaccines was performed using the Galaxy
server (http://galaxy.seoklab.org/). The Galaxy server relaxes the model structure using repack-
ing and molecular dynamics simulation. The 3D structure of the multi-epitope vaccines was
then validated using a variety of tools, such as SWISS-MODEL server [36], ProSA-web [37]
and ERRAT (https://servicesn.mbi.ucla.edu/).

2.9. Molecular dynamics simulation of multi-epitope vaccines

High-performance MD simulation is an important method to validate the stability of the mod-
eled structures. The multi-epitope vaccines and vaccine-TLRs complexes were simulated for
100 ns using Gromacs 2020 package [38], implementing similar protocols as in our previous
studies [39]. The simulation trajectories were saved for the multi-epitope vaccines and com-
plexes every 10 ps and root mean square deviations (RMSDs), root mean square fluctuations
(RMSFs) and radius of gyration (Rg) analyses were carried out by Gromacs tools box. The
final structures of multi-epitope vaccines were used as a ligand for docking to TLR3 and TLR4.

2.10. Molecular docking of toll-like receptors and multi-epitope vaccines

The molecular docking of the final vaccines after 100 ns simulations with TLR3 (PDB ID:
2A0Z) and TLR4 (PDB ID: 4G8A, www.rcsb.org) was performed using the ClusPro 2.0 web
server [40] to analyze the binding pattern of vaccines with TLR3 and TLR4. Based on the low-
est global energy, the final vaccine-TLRs complexes models were selected and visualized using
PyMOL package and LIGPLOT software [41, 42].

3. Results
3.1. Sequence alignment
The alignment of various HCV protein sequences was performed using ClustalW2 (https://
embnet.vital-it.ch/software/ClustalW.html). The results of this step has been shown in supple-
mentary data.

3.2. Prediction and evaluation of HTL, CTL and B cell epitopes

The IEDB MHC-II prediction tool, the NetCTL 1.2 and the BCPred servers were used to pre-
dict HTL (15-mer), CTL and B-cell linear epitopes of the HCV proteins, respectively (S1–S9
Tables). The potentially antigenic and non-allergenic epitopes (using VaxiJen v2.0 and Aller-
TOP v.2.0, respectively) were adopted and next, those induced various cytokines such as IFN-
γ, IL-4, and IL-10 (via prediction of B and HTL epitopes) were obtained (Table 1).

3.3. Combining selected epitopes to make the final multi-epitope vaccine

Selected potential epitopes including 18 CTL, 7 HTL and 5 LBL epitopes were fused together
using AAY and KK linkers, respectively (Fig 2A). The 50S ribosomal protein L7/L12 (TLR4
agonist) and human β-defensin 2 (TLR3 agonist) adjuvants were separately added to the N ter-
minal part sequence of the final multi-epitope vaccine using an EAAAK linker to increase
immunogenicity (Fig 2A) giving final multi-epitope vaccines sequences with 686 and 607 resi-
dues (S1 Fig).

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

Table 1. Final selected CTL, HTL and LBL epitopes for multi-epitope vaccines construction.
Protein CD8+-T cell epitope CD4+-T cell epitope B-cell epitope
42 36 32
P7 YAIYGTWPL LVPGMTYAIYGTWPLLLL IKGRLVPGMTYAIYGTWPLL
54
LLALPQRAY
432
NS3 VTQTVDFSLDPTFTI
126
LLSPRPVSY
204
APTGSGKST
362 500 587
GRHLIFCHSKKK AWYELTPAETTVRLR RLKPTLRGPTPLLY
123 386 181
RGSLLSPRPVSYLK INAVAYYRGLDVSVI SPTFTDNSTPPAVP
107 534 86
VTRHADVIPV SVFTGLTHIDAHFLSQT PAPQGTRSLTPCTC
383 554
GLGINAVAY GDNFPYLVAYQATVCARAQAP
262
GAPITYSTY
230
PSVAATLGFGAYMSKAH
454 560 141
NS5B IEPLDLPQI IYHSLSRARPRWFMW KSEVFCVQPEKGGR
489
LRKLGVPPLR
132
TPIDTTIMAKSEVFCVQPEK
560 35
IYHSLSRARPRWFMWCLLLLSVGVGIYL NMVYATTSRSASLRQ
500
WRHRARSVRARLLSQGGRAATCGKYLFNWAVRTKLKLTPI
199
QRVEFLVNAWKSKKSPMGFSYDTRCFDSTVTESDIRV
29
SLLRHHNMVYATTRSASLRQKKVTF
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3.4. Evaluation of antigenicity, allergenicity, physiochemical properties and

secondary structure of the vaccine constructs
The designed vaccines were sufficiently antigenic and non-allergenic. Their molecular weight
included 76.27 kDa and 67.87 kDa, and their theoretical pI included 9.98 and 10.16 (Table 2).
The instability and the aliphatic indices of the final vaccines have been represented in the
Table 2. The final vaccines have a grand average of hydropathicity (GRAVY) values of -0.152
and -0.169 (Table 2 and S1 Fig).

3.5. Modeling, refinement, and validation of vaccine constructs

The three-dimensional structures of the final vaccines were modeled and refined (Fig 2). In
the optimal structural model of construct 1, 91.08% of the residues were in the favored area,
7.17% in the allowed area and 1.75% in the disallowed area of Ramachandran plot (Fig 2B). In
addition, the quality factor obtained from the ERRAT analysis was 76.41% and the Z-score was
−3.28 (Fig 2C). Regarding the construct 2, 91.07% of the residues were in the favored area,
7.28% in the allowed area and 1.65% in the disallowed area of Ramachandran plot (Fig 2D). In
addition, the quality of 72.64% and the Z-score of −3.88 was obtained (Fig 2E). The ERRAT
score included 76.41% and 72.64% for the vaccine candidates (Fig 3F and 3G).

3.6. Prediction of B-cell epitopes in the final vaccines

In general, four discontinuous B-cell epitopes with score of 0.83 to 0.87, and 12 linear B-cell
epitopes with score of 0.83 to 0.93 were selected as the final epitopes in 50S ribosomal protein
L7/L12-multi-epitope vaccine (S2 Fig, Tables 3 and 4). In addition, six discontinuous B-cell
epitopes with score range of 0.82 to 0.88, and 14 linear B-cell epitopes with score range of 0.83
to 0.91 were selected as the final epitopes in human β-defensin 2-multi-epitope vaccine (S2
Fig, Tables 3 and 4). For both multi-epitope vaccines, the PI values of 0.87 and 0.88

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Fig 2. Prediction and assessment of the 3D structure of the multi-epitope vaccines. (A) Schematic image of the final vaccine.
(B) Ramachandran plot analysis of refined construct 1, showing 91.08%, 7.17% and 1.75% residues in favored, allowed and
disallowed region, respectively, (C) ProSA validation of 3D construct 1, showing Z-score -3.28, (D) Ramachandran plot analysis
of refined construct 2, showing 91.07%, 7.28% and 1.65% residues in favored, allowed and disallowed region, respectively, (E)
ProSA validation of 3D construct 2, showing Z-score -3.88, (F and G) 3D structure of vaccines models showing α-helix (red
cartoon), β-strand (yellow cartoon) and loop (green cartoon).
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Table 2. Various features of multi-epitope vaccines.

Features Assessment
Construct 1 Construct 2
Adjuvant 50S ribosomal protein L7/L12 (TLR4) Human β-defensin 2 (TLR3)
Number of amino acids 686 607
Molecular weight 76270.36 Dalton 67872.61 Dalton
Chemical formula C3496H5551N933O938S19 C3104H4887N851O811S24
Theoretical pI 9.98 10.16
Total number of negatively charged residues (Asp + Glu): 43 24
Total number of positively charged residues (Arg + Lys) 107 97
Total number of atoms 10937 9677
Extinction coefficient (at 280 nm in H2O) 126060 M-1cm-1 127550 M-1cm-1
Instability index 35.01 38.13
Aliphatic index 89.55 81.76
Grand average of hydropathicity (GRAVY) -0.152 -0.169
Antigenicity 0.53 (AntigenPro), 0.75 (Vaxijen v.2.0) 0.80 (AntigenPro), 0.78(Vaxijen v.2.0)
Allergenicity Probable non-allergen (AllergenFP v.1.0) Probable non-allergen (AllergenFP v.1.0)
Probable non-allergen (AllerTOP v.2.0) Probable non-allergen (AllerTOP v.2.0)
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demonstrated that 87% and 88% of residues located in the predicted ellipsoid area of the epi-
topes and these epitopes had the highest solvent accessibility.

3.7. Molecular dynamic simulation of multi-epitope vaccines

The RMSD of the constructs Cα atoms raise up over time which highlighted a continues
increasing trend up to 25 ns and next reached a plateau indicating a stable structural confor-
mations, while the RMSD value converged within ~1 nm (Fig 3A) Likewise, the Cα-RMSF
graph of the constructs during 100 ns of MD simulation was identified as stable dynamics
behavior (Fig 3B). However, higher fluctuation rates were observed in the loop regions. The
similar patterns of the RMSD and RMSF and low changes at various time intervals, revealed
good structural stability of the vaccine constructs.

3.8. Molecular docking of the immune receptors (TLRs) with adjuvant and
vaccines
A diverse hydrophobic and hydrophilic interactions participated in the adjuvants-TLRs binding (S3
Fig). Moreover, the molecular docking of the final vaccines after 100 ns simulations with TLR3
(PDB ID: 2A0Z) and TLR4 (PDB ID: 4G8A) was performed (S4 Fig). Next, the complex with best
ClusPro score was selected and further applied for running MD simulation investigations.

3.9. Molecular dynamic simulation of multi-epitope vaccines-TLRs

complexes
The molecular dynamics simulations of the docked complexes were performed for 100 ns.
These simulations studies are capable to address several important queries about the structural
stability of the final complexes. To address these queries, it is warranted to assess several main
statistical factors based on MD simulation outputs (Fig 3C–3G).
The conformational stability of the vaccines and TLRs in the complexes states was investi-
gated based on the calculation of the overall fluctuations of the final vaccines. The RMSD val-
ues were calculated as a time-dependent parameter for decoding the displacement of Cα

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Fig 3. MD simulations outputs of free vaccines and vaccines-TLRs complexes; A and B: The RMSD and RMSF
values of Cα atoms for free vaccines, respectively; C and D: The RMSD values of Cα atoms for vaccines and TLRs in
the complex states, respectively; E and F: The RMSF values of Cα atoms for vaccines and TLRs in the complex
states, respectively; G: The Rg values of vaccines structures in the complex states.
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Table 3. Linear B-cell epitopes of the final vaccines.

Linear B cell epitopes Score
250
Construct 1 AAYGAPITYS259 0.93
306
Construct 1 RAAYTPID313 0.91
62
Construct 1 LKGAGS67 0.91
70
Construct 1 LTVVKRIKDLIGL82 0.90
278
Construct 1 SKAHAAYIEPLDL290 0.90
614
Construct 1 AIYGTWPLLKKRLKPTLRGPT634 0.89
645 651
Construct 1 TDNSTPP 0.88
562
Construct 1 ARAQAPK568 0.88
45
Construct 1 KAEILDKS52 0.87
589
Construct 1 VYATTS594 0.85
356
Construct 1 VGIYLAAYW364 0.85
606
Construct 1 RLVPGM611 0.83
1
Construct 2 GIINTLQKYYCRVRGGRC18 0.91
521
Construct 2 RQKKKGRLVPGMTYAIYGTW540 0.89
199 215
Construct 2 SKAHAAYIEPLDLPQIA 0.88
225
Construct 2 PLRAAYTPI233 0.86
237
Construct 2 IMAKSE242 0.86
94
Construct 2 PVSYAAYAPTGSGKSTA114 0.85
572
Construct 2 PAVPKKPA579 0.85
549
Construct 2 PTLRG553 0.84
339
Construct 2 SKKS342 0.82
511
Construct 2 YATTS515 0.82
66 70
Construct 2 LPQRA 0.81
176
Construct 2 PITYS180 0.81
259
Construct 2 SRARPRW265 0.81
22
Construct 2 SCLPKEEQ29 0.80
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atoms in the final vaccine constructs and TLRs during simulation times (Fig 3C and 3D). Fluc-
tuations in the vaccine’s RMSD graph could be due to the movement of the residues to achieve
a suitable and stable conformation of the vaccines in the complexes states. The RMSD fluctua-
tions of the vaccines structures in the complexes states were lower than free states vaccines,
which indicated the stability of the vaccines structures during complexes simulations (Fig 3).
In addition, the RMSD graph of the TLRs structures in the complexes states were low and
smooth, which indicated their structural stability in the complexes states (Fig 3).
Local structural fluctuations were estimated utilizing the RMSF factor for the vaccines and
TLRs structures during the complexes simulations (Fig 3E and 3F). Loop areas exhibited high-
est fluctuations in the complexes states. The RMSF graph of the TLRs showed low flexibility,
which clarified that TLRs had good structural stability in the complexes states (Fig 3F).
The high value of Rg (Fig 3G) indicates a decrease in protein compactness. Fluctuations in
the Rg diagram indicate the movements of the vaccines domains which induce conformational
changes and thereby select a suitable conformation in the complexes states. Hence, the studied
complexes had a proper structural stability.

3.10. Estimation of binding free energy of immune receptor-vaccine

complex
Both hydrophilic (ΔEpolar) and hydrophobic energy terms (ΔEnonpolar) were participated in the
binding of vaccines to the TLRs (Table 5). However, hydrophilic energy was as main driving

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Table 4. Discontinuous B-cell epitopes of the final vaccines.

Discontinuous B cell epitopes Number of Score
residues
A:A250, A:A251, A:Y252, A:G253, A:A254, A:P255, A:I256, A:T257, A:Y258, A:S259, A:A275, A:S278, A:K279, A:A282, A: 45 0.87 Construct
A283, A:Y284, A:I285, A:E286, A:P287, A:L288, A:D289, A:L290, A:P291, A:Q292, A:R306, A:A307, A:A308, A:Y309, A:T310, 1
A:P311, A:I312, A:D313, A:V356, A:G357, A:I358, A:Y359, A:L360, A:A361, A:A362, A:Y363, A:W364, A:H366, A:R367, A:
A368, A:R369
A:A562, A:R563, A:A564, A:Q565, A:A566, A:P567, A:K568, A:Y571, A:K586, A:P620, A:L621, A:L622, A:K623, A:K624, A: 33 0.87 Construct
R625, A:L626, A:K627, A:P628, A:T629, A:L630, A:R631, A:G632, A:P633, A:T634, A:P635, A:L636, A:T645, A:D646, A:N647, 1
A:S648, A:T649, A:P650, A:P651
A:K45, A:A46, A:E47, A:I48, A:L49, A:D50, A:K51, A:S52, A:D59, A:I61, A:L62, A:K63, A:G64, A:A65, A:G66, A:S67, A:L70, A: 56 0.84 Construct
T71, A:V72, A:V73, A:K74, A:I76, A:K77, A:D78, A:L79, A:I80, A:G81, A:L82, A:G83, A:L84, A:K85, A:E86, A:S87, A:K88, A: 1
D89, A:V91, A:D92, A:K96, A:K100, A:G101, A:L102, A:S103, A:K104, A:E105, A:E106, A:A107, A:E108, A:S109, A:L145, A:
P146, A:Q147, A:R148, A:P177, A:V178, A:S179, A:Y180
A:R580, A:V589, A:Y590, A:A591, A:T592, A:T593, A:S594, A:A597, A:Q601, A:K602, A:A614, A:I615, A:Y616, A:G617, A: 16 0.83 Construct
T618, A:W619 1
A:V510, A:Y511, A:A512, A:T513, A:T514, A:S515, A:A518, A:R521, A:K523, A:K524, A:G526, A:R527, A:L528, A:V529, A: 25 0.88 Construct
P530, A:G531, A:M532, A:T533, A:Y534, A:A535, A:I536, A:G538, A:T539, A:W540, A:R546 2
A:V13, A:G15, A:G16, A:R17, A:C18 5 0.87 Construct
2
A:S180, A:S199, A:K200, A:A203, A:A204, A:Y205, A:I206, A:E207, A:P208, A:L209, A:D210, A:L211, A:P212, A:Q213, A:I214, 35 0.84 Construct
A:A215, A:A216, A:V223, A:P224, A:P225, A:L226, A:A228, A:A229, A:Y230, A:T231, A:P232, A:I233, A:D234, A:T236, A: 2
I237, A:M238, A:A239, A:K240, A:S241, A:E242
A:G1, A:I2, A:I3, A:N4, A:T5, A:L6, A:Q7, A:K8, A:Y10, A:C11, A:R12, A:R14, A:S22, A:C23, A:L24, A:P25, A:K26, A:E27, A: 32 0.84 Construct
E28, A:Q29, A:C41, A:K44, A:K45, A:A65, A:R97, A:P98, A:V99, A:S100, A:Y101, A:A102, A:A103, A:L120 2
A:A485, A:Q486, A:A487, A:P549, A:T550, A:L551, A:R552, A:P572, A:A573, A:V574, A:P575, A:K576, A:K577, A:P578, A: 16 0.83 Construct
A579, A:Q581 2
A:R42, A:Y104, A:A105, A:P106, A:T107, A:G108, A:S109, A:G110, A:K111, A:S112, A:T113, A:A114, A:H124, A:K126, A: 20 0.82 Construct
K128, A:A129, A:L136, A:S137, A:P138, A:R139 2
https://doi.org/10.1371/journal.pone.0272582.t004

force in the interactions between the immune receptors and the vaccines, compared to hydro-
phobic energy. Paramount residues which were participated at the interface regions have been
identified (Fig 4). Accordingly, both polar and non-polar residues played key roles at the bind-
ing process, of the vaccines-TLRs complexes (Fig 4).

Table 5. The contribution of various energy components in the ΔGbind (kJ/mol).

Construct 1-TLR 4 Construct 2-TLR 3
ΔEelea -15786.76 ± 490.57 -11730.95 ± 240.61
ΔEvdWb -460.52 ± 87.34 -247.89 ± 68.97
ΔGPBc 1476.22 ± 249.39 2323.25 ± 429.45
ΔGSAd -64.67 ± 8.02 -45.45 ± 8.03
ΔEnon-polare -525.19 ± 47.68 -9407.7 ± 335.03
ΔEpolarf -14310.54 ± 369.95 -293.34 ± 38.5
ΔGbind -14835.73 ± 208.82 -9701.04 ± 186.76
a
Electrostatic connection
b
van der Waals connection
c
Polar contribution of the solvation effect
d
Non-polar contribution of solvation effect
e
ΔEnon-polar = ΔEvdW + ΔGSA
f
ΔEpolar = ΔEele + ΔGGB

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

Fig 4. The 3D view of the final system conformations. The interface residues between two proteins TLRs (orange cartoon) and vaccines (magenta cartoon)
residues (orange and magenta sticks) are labeled. Hydrogen bonds and hydrophobic contacts are presented as green dashed line and arc with spokes radiating,
respectively. A and B indicate TLR4-construct 1 and TLR3-construct 2 complexes, respectively.
https://doi.org/10.1371/journal.pone.0272582.g004

4. Discussion
Following the HCV protein sequences alignment, we used the IEDB MHC-II prediction tool,
the NetCTL 1.2 and the BCPred servers to predict HTL (15-mer), CTL and B-cell linear epi-
topes, respectively (S1–S9 Tables). Those antigenic and non-allergenic epitopes were adopted
and next those antigenic B and HTL epitopes were used for the construction of our multi-epi-
tope vaccines (Table 1).
Selected epitopes containing 18 CTL, 7 HTL and 5 LBL epitopes were fused together using
AAY and KK linkers, respectively (Fig 2A). Two adjuvants including 50S ribosomal protein
L7/L12 (TLR4 agonist) and human β-defensin 2 (TLR3 agonist) were added separately to the
N terminal domain of the final multi-epitope vaccine using an EAAAK linker to increase
immunogenicity (Fig 2A), synthesizing constructs with 686 and 607 residues (S1 Fig). These
designed vaccines were also potentially antigenic and non-allergenic. Their molecular weight
included 76.27 kDa and 67.87 kDa, and their theoretical pI were 9.98 and 10.16 (Table 2),
which indicates the basic nature of the vaccines. The instability and the aliphatic indices of the
final vaccines have been represented in the Table 2, which indicates their appropriate structure
and high thermo-stability. The final vaccines also deciphered a grand average of hydropathicity
(GRAVY) values of -0.152 and -0.169, confirming their hydrophilic characters (Table 2).

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

Therefore, they can interact with other proteins and are soluble in water. The predicted sec-
ondary structure of the final vaccine has been exhibited in the S1 Fig.
Galaxy server, SWISS-MODEL, ProSA-web and ERRAT servers (Fig 2) demonstrated that
in the best three-dimensional structural model of the construct 1, 91.08% of the residues placed
in the favored area, 7.17% in the allowed area and 1.75% in the disallowed area of Ramachan-
dran plot (Fig 2B). In addition, the quality factor obtained from the ERRAT analysis was
76.41% and its Z-score was −3.28 (Fig 2C). Regarding the construct 2, 91.07% of the residues
were in the favored area, 7.28% in the allowed area and 1.65% in the disallowed area of Rama-
chandran plot (Fig 2D). In addition, the quality factor and Z-score included 72.64% and −3.88,
respectively (Fig 2E). A �90% reliability cut-off is mandatory for residues placing in the
favoured region [43] which was confirmed in our study. The ERRAT score of 76.41% and
72.64% for the vaccine constructs clarified the adequate quality and validity percentage. An
ERRAT score >50 indicates a suitable quality model [44]. All of these indicators confirm the
good quality of the 3D structures of the final vaccines (Fig 3F and 3G).
We observed four discontinuous B-cell epitopes with a score of 0.83 to 0.87, and 12 linear
B-cell epitopes with a score of 0.83 to 0.93, selected as the final epitopes in 50S ribosomal pro-
tein L7/L12-multi-epitope vaccine (S2 Fig, Tables 3 and 4). In addition, six discontinuous B-
cell epitopes with score of 0.82 to 0.88, and 14 linear B-cell epitopes with score of 0.83 to 0.91
were selected as the final epitopes in human β-defensin 2-multi-epitope vaccine (S2 Fig, Tables
3 and 4). For both multi-epitope vaccines, the PI values of 0.87 and 0.88 demonstrated that
87% and 88% of residues located in the predicted ellipsoid area of the epitopes, having the
highest solvent accessibility. Following the MD simulations for 100 ns, in the RMSD of the
constructs, Cα atoms raise up over time which highlighted a continues increasing trend up to
25 ns and then reaching a plateau which indicated a stable structural conformations, while the
RMSD value converged within ~1 nm (Fig 3A). Likewise, the Cα-RMSF graph of the con-
structs deciphered stable dynamics behavior (Fig 3B). However, higher fluctuation rates were
observed in the loop regions. Similar patterns of the RMSD and RMSF and low changes at var-
ious time intervals, revealed good structural stability of the vaccine constructs. These final con-
structs were used as the ligands for docking to the TLRs. The molecular docking demonstrated
the interaction of the TLRs with the adjuvants. Accordingly, a diverse hydrophobic and hydro-
philic interactions participated in the adjuvants-TLRs binding (S3 Fig).
Moreover, the molecular docking of the final vaccines after 100 ns simulations with TLR3
(PDB ID: 2A0Z) and TLR4 (PDB ID: 4G8A) was performed (S4 Fig). The simulations studies
were capable of addressing main queries regarding the structural stability of the final com-
plexes depicted as outputs (Fig 3C–3G).
The conformational stability of the vaccines and TLRs in the complexes states was investi-
gated based on the calculation of the overall fluctuations of the final vaccines. The RMSD val-
ues were calculated as a time-dependent parameter for decoding the displacement of Cα
atoms in the final vaccine constructs and TLRs during simulation times (Fig 3C and 3D). Fluc-
tuations in the vaccine’s RMSD graph could be due to the movement of the residues to form a
suitable and stable conformation of the vaccines in the complexes states, being lower than free
states vaccines, which indicated the stability of the vaccines structures during complexes simu-
lations (Fig 3). In addition, the RMSD graph of the TLRs structures in the complexes states
were low and smooth, which indicated their structural stability in the complexes states (Fig 3).
Local structural fluctuations estimation utilizing the RMSF factor (Fig 3E and 3F) revealed
highest fluctuations of loop areas in the complexes states. These localized fluctuations in vac-
cines residues can facilitate vaccine epitopes’ exposure to TLRs and make sufficient space for
side chains to adopt suitable conformation for binding to TLRs. The RMSF graph of the TLRs

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

showed low flexibility, clarifying their sufficient structural stability in the complexes states (Fig
3F).
The high value of Rg indicates a decrease in protein compactness. Fluctuations in the Rg
diagram indicate the movements of the vaccines domains which induce conformational
changes and thereby select a suitable conformation in the complexes states. According to the
pattern of changes in these factors, our vaccines structures system displayed high stability dur-
ing complexes simulations (Fig 3G). Accordingly, the multi- epitope vaccines-TLRs constructs
achieved a suitable and stable conformational complexes during the simulations times,
highlighting their proper structural stability.
The binding free energy profile of our vaccine candidates (Table 5) [45] revealed that both
hydrophilic (ΔEpolar) and hydrophobic energy terms (ΔEnonpolar) participated in the binding of
vaccines to the TLRs. However, hydrophilic energy was the main driving force in the interac-
tions, compared to hydrophobic energy (Fig 4). Accordingly, both polar and non-polar resi-
dues played key roles in the binding process, of the vaccines-TLRs complexes (Fig 4).
Noticeably, the output of the TLRs-vaccines complexes outlined the high structural stability
of molecular species docked together during the simulation. Hydrophilic residues play a vital
role in the interactions between the vaccines and the TLRs, which in turn determine high sta-
bility of the complex structure. Indeed, these binary interactions cause the efficient adhesion of
molecular species together. Accordingly, the vaccine candidate constructs had the ability to
stimulate the immune system’s responses efficiently though further studies are warranted to
verify these findings in vitro and in vivo. In previous studies multi-epitope vaccine constructs
have been used against Leishmania donovani stimulating CD8+ T cells and INFγ (19 antigenic
proteins, 49 epitopes binding to 40 different MHC class I supertypes) [46], provoking CD8+ T
cells (LeIF and TSA) [47] and induction of CD4+, CD8+, IFN-γ and IL-10 [48]. Major limita-
tions of our survey included lack of experimental assessment of the multi-epitope vaccine in
vitro, in vivo or in clinical trial. A study using multi-epitope DNA- and peptide-based vaccines
provoked CD4+ and CD8+ responses against the HCV and were evaluated in BALB/c mice
model [49].

5. Conclusion
The efficient anti-HCV treatment approaches are limited due to various side effects, recur-
rency and antiviral resistance development. Thereby, it is essential to seek other strategies like
vaccine design. Currently, it is possible to design recombinant and multi-epitope vaccines
more rapid and cost effective using a variety of computational methods. Accordingly, we
attempted to design multi-epitope vaccine candidates based on the conserved areas of the
HCV structural and non-structural protein sequences using the immunoinformatics tools.
According to our findings, the vaccines constructs (including CTL, HTL and B cell epitopes
plus two adjuvants or TLR3 and TLR4 agonists) were efficient in terms of antigenicity, non-
toxicity, solubility, immunogenicity, non-allergenicity and stability. The 3D structures of the
multi-epitope vaccine candidates’ models were highly stable and soluble. Additionally, their
interactions with the TLR3 and TLR4 resulted in efficient vaccine candidates with acceptable
traits in silico. Our computational findings were promising considering the candidate multi-
epitope vaccines potential in controlling the HCV infection.

Supporting information
S1 Table. Linear B cell (LBL) epitopes of the P7 protein.
(DOCX)

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

S2 Table. Linear B cell (LBL) epitopes of the NS3 protein.

(DOCX)
S3 Table. Linear B cell (LBL) epitopes of the NS5B protein.
(DOCX)
S4 Table. Helper T Lymphocyte (HTL) epitopes of the NS5B protein.
(DOCX)
S5 Table. Helper T Lymphocyte (HTL) epitopes of the NS3 protein.
(DOCX)
S6 Table. Helper T Lymphocyte (HTL) epitopes of the P7 protein.
(DOCX)
S7 Table. Cytotoxic T Lymphocyte (CTL) epitopes of the P7protein.
(DOCX)
S8 Table. Cytotoxic T Lymphocyte (CTL) epitopes of the NS3 protein.
(DOCX)
S9 Table. Cytotoxic T Lymphocyte (CTL) epitopes of the NS5B protein.
(DOCX)
S1 Fig. Prediction of the secondary structure of the construct 1 (A) and construct 2 (B) vac-
cines. The predicted results showed that among 686 amino acids in the construct 1, 268
(39.07%), 135 (19.68%), 66 (9.62%) and 217 (31.63%) amino acids are involved in α-helix,
extended strand, beta turn, and random coil, respectively. Our predicted outputs revealed that
among 607 amino acids in the construct 2, 205 (33.77%), 132 (21.75%), 63 (10.38%) and 207
(34.10%) amino acids are involved in α-helix, extended strand, beta turn, and random coil,
respectively.
(DOCX)
S2 Fig. Linear (A and C) and Discontinuous (B and D) B-cell epitopes of the construct 1 (A
and B) and construct 2 (C and D) vaccines (colored spheres).
(DOCX)
S3 Fig. The 3D view of the molecular docking conformations. The interface residues
between two proteins TLRs (orange cartoon) and adjuvants (magenta cartoon) residues
(orange and magenta sticks) are labeled. Hydrogen bonds and hydrophobic contacts are pre-
sented as green dashed line and arc with spokes radiating, respectively. A and B indicate
TLR4-50S ribosomal protein L7/L12 and TLR3-human β-defensin 2 complexes, respectively.
(DOCX)
S4 Fig. A and B indicate TLR4-construct 1 and TLR3-construct 2 complexes, respectively.
TLRs and costructs are depicted as brown and blue cartoons respectively.
(DOCX)
S1 Data. Multiple alignment of the NS3 protein sequences in HCV strains.
(PDF)
S2 Data. Multiple alignment of the P7 protein sequences in HCV strains.
(PDF)
S3 Data. Multiple alignment of the NS5B protein sequences in HCV strains.
(PDF)

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PLOS ONE Multi-epitope vaccine against hepatitis C virus

Acknowledgments
This work was supported by the Iran National Science Foundation.

Author Contributions
Conceptualization: Esmaeil Behmard, Akbar Farjadfar, Ebrahim Barzegari, Amin
Kouhpayeh.
Data curation: Esmaeil Behmard, Sohrab Najafipour, Abdolmajid Ghasemian, Parviz
Abdolmaleki.
Formal analysis: Esmaeil Behmard.
Funding acquisition: Hussein T. Abdulabbas, Saade Abdalkareem Jasim, Ebrahim Barzegari.
Investigation: Esmaeil Behmard, Ebrahim Barzegari.
Methodology: Esmaeil Behmard, Ebrahim Barzegari.
Project administration: Sohrab Najafipour, Ebrahim Barzegari.
Resources: Abdolmajid Ghasemian, Parviz Abdolmaleki.
Software: Abdolmajid Ghasemian.
Supervision: Sohrab Najafipour, Ebrahim Barzegari.
Validation: Abdolmajid Ghasemian, Parviz Abdolmaleki.
Visualization: Abdolmajid Ghasemian.
Writing – original draft: Abdolmajid Ghasemian.
Writing – review & editing: Hussein T. Abdulabbas, Saade Abdalkareem Jasim, Abdolmajid
Ghasemian, Akbar Farjadfar, Amin Kouhpayeh.

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