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 Journal of Biomedical Informatics 93 (2019) 103160

Contents lists available at ScienceDirect

Journal of Biomedical Informatics

journal homepage: www.elsevier.com/locate/yjbin

Towards the first multi-epitope recombinant vaccine against Crimean-Congo T

hemorrhagic fever virus: A computer-aided vaccine design approach
Mokhtar Nosrati, Mandana Behbahani, Hassan Mohabatkar
⁎

Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran

ARTICLE INFO ABSTRACT

Keywords: Crimean-Congo hemorrhagic fever (CCHF) is considered one of the major public health concerns with case
Crimean-Congo hemorrhagic fever fatality rates of up to 80%. Currently, there is no effective approved vaccine for CCHF. In this study, we used a
Computer-aided vaccine design computer-aided vaccine design approach to develop the first multi-epitope recombinant vaccine for CCHF. For
Multi-epitope vaccine this purpose, linear B-cell and T-cell binding epitopes from two structural glycoproteins of CCHF virus including
Gc and Gn were predicted. The epitopes were further studied regarding their antigenicity, allergenicity, hy-
drophobicity, stability, toxicity and population coverage. A total number of seven epitopes including five T-cell
and two B-cell epitopes were screened for the final vaccine construct. Final vaccine construct composed of 382
amino acid residues which were organized in four domains including linear B-cell, T-cell epitopes and cholera
toxin B-subunit (CTxB) along with heat labile enterotoxin IIc B subunit (LT-IIc) as adjuvants. All the segments
were joined using appropriate linkers. The physicochemical properties as well as the presence of IFN-γ inducing
epitopes in the proposed vaccine, was also checked to determining the vaccine stability, solubility and its ability
to induce cell-mediated immune responses. The 3D structure of proposed vaccine was subjected to the prediction
of computational B-cell epitopes and molecular docking studies with MHC-I and II molecules. Furthermore,
molecular dynamics stimulations were performed to study the vaccine-MHCs complexes stability during sti-
mulation time. The results suggest that our proposed vaccine was stable, well soluble in water and potentially
antigenic. Results also demonstrated that the vaccine can induce both humoral and cell-mediated immune re-
sponses and could serve as a promising anti-CCHF vaccine candidate.

1. Introduction Therefore, development of an effective vaccine against CCHFV is ne-

cessary. Generally, vaccine development is a complex, lengthy and
Crimean-Congo hemorrhagic fever (CCHF) is a wide spread zoonotic accurate process that can be accelerated with detailed understanding of
viral disease with case fatality rates of up to 80% caused by CCHF virus the molecular mechanism of diseases [7].
(CCHFV) [1]. Recently, there is a drastic increase of CCHFV infection Designing an effective vaccine requires an adequate understanding
around the world, especially in Eastern Mediterranean region [2]. of the structure and function of the immune system as well as the target
CCHFV is an enveloped single-stranded, ambisense sense RNA virus pathogen. Generally, to initiate a specific immune response to an in-
with a diameter of 90–100 nm belonging to the Nairovirus genus of vasive pathogen, the immune system must be able to recognize in-
the Bunyaviridae family, which includes five genera, and over 350 virus fectious agent. After recognition, inducing adaptive immune responses
species. The virus genome organized in three segments including small needs antigen processing and presentation through Antigen Presenting
(S), a medium (M), and a large (L) segment which encoding the nu- Cells (APCs). Based on the origin of antigen there are two different
cleoprotein (N), glycoproteins (Gn and Gc), and the RNA-dependent pathways for antigen processing including cytosolic (for tumor and
RNA polymerase (RdRp) (L), respectively [3–5]. viral antigens) and endocytic (for exogenous antigens). Finally, the
Unfortunately, until now there is no licensed vaccine or approved fragmented antigens expressed on the surface of APCs through specific
targeted therapies for prevention or treatment of CCHF, although sup- glycoproteins named MHCs molecules. There are two different types of
portive care and Ribavirin can help a lot to treat the disease. Despite MHC molecules including MHC-I (MHC-Class I) and MHC-II (MHC-Class
high fatality rate, widespread distribution and lack of treatment and II). Simultaneous interactions between the MHC, processed antigen and
vaccine for CCHF, but there are limit studies about the disease [1,6]. T-Cell Receptor (TCR) trigger the cellular and humoral responses.

⁎
Corresponding author.
E-mail address: h.mohabatkar@ast.ui.ac.ir (H. Mohabatkar).

https://doi.org/10.1016/j.jbi.2019.103160
Received 23 July 2018; Received in revised form 17 March 2019; Accepted 27 March 2019
Available online 27 March 2019
1532-0464/ © 2019 Elsevier Inc. All rights reserved.
M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Understanding of these processes can be very helpful for designing an computational methods have highly considered as alternative or com-
effective vaccine [8,9]. Nowadays, due to the rapid development of plementary approaches for epitopes determination and vaccine design.
molecular immunology and determination of the molecular mechan- Computer-aided vaccine design, which also called computational im-
isms of pathogenesis, much research is aimed on vaccine design against munology, can provide an integrated pipeline for mapping potential B-
different lethal diseases. and T-cell epitopes, allergenic sites in studied antigens and prediction
Recently, some efforts have been made to introduce a novel vaccine of HLA-epitope affinity. Thus, the approach could reduce the time and
against the disease. In this regard, Garrison et al developed a DNA cost required for vaccine design [27–29]. In recent decade, computa-
vaccine expressing the M-segment glycoprotein precursor gene of tional design of epitope-based vaccine have been used for vaccine de-
CCHFV with the ability to provoke strong humoral immune responses velopment against many infectious disease and even cancer. For this
and prevention against CCHF and lethal in the studied animal model purpose, recently many bioinformatics tools have been developed that
[10]. In another report, Dowall et al developed a recombinant candi- facilitates the development of epitope-based vaccines.
date vaccine expressing the CCHFV nucleoprotein which was im- Bioinformatics and computational tools can provide converting of
munogenic but fails to create protection against the disease [11]. Kor- large-scale immunological data such as antigen presentation and pro-
tekaas and colleague introduced a subunit vaccine with the ability to cessing, antigen-antibody interactions and antigen determination to
inducing high levels of humoral responses, but no protection was ob- obtain expressive interpretations. Therefore, currently vaccine design
served in mice after CCHFV challenge infection [12]. process especially epitope based vaccine development is facilitated
Generally, vaccines can be considered as effective immunity inducer along with introducing applied bioinformatics tools such as epitope
against pathogens as well as an active way to control infectious dis- mapping (software/web service), protein modeling software and pro-
eases. Due to the wide variety and extensive genetic variation among tein-protein/ligand interaction analysis software [30].
different CCHFV serotypes, conventional vaccine platforms such as live- Although not much time has passed since the introduction of the
attenuated and inactivated vaccine cannot protect persons against dif- first report about computational vaccine design, in the recent years,
ferent CCHFV strains. Furthermore, there are some drawbacks asso- a lot of progress has been made in this regard. Subsequently, numerous
ciated with conventional vaccines including weak immunity, multi-dose vaccines were developed based on computational approaches that in-
administration, allergenic potential and low safety. Consequently cur- clude efficient vaccine against Toxoplasma gondii [31], Rickettsia pro-
rently, new vaccine platforms such as Subunit Vaccines, DNA vaccine, wazekii [32], Streptococcus pneumoniae [33], Leishmania infantum [34],
and multi-epitope vaccine (MEV) are highly regarded [13]. Among Chlamydia pneumoniae [35], Brucella abortus [36], Staphylococcus aureus
them, due to high specificity, safety and low-cost production of MEVs [15], Escherichia coli [37], Vibrio cholera [38], Human immunodeficiency
these vaccines have attracted attention. Commonly, MEVs are en- virus-1 [39], Hepatitis C virus [40] and many others. In many empirical
gineered based on dominant and conserved B- and T-cell epitopes from studies, the efficacy of computationally designed vaccines is approved
desired antigens without potential IgE-epitopes. Accordingly, an effec- [34,39,41,42].
tive MEV can provide an effective immunization against different ser- There is a common procedure to in silico vaccine design, which is
otypes of a pathogen. Despite mentioned advantages of MEVs, poor used in most of the previous studies. The procedure, includes antigen
immunogenicity is considered as a major drawback to development of selection, epitope prediction, vaccine engineering and vaccine evalua-
MEVs [14–16]. To overcome the mentioned blind spot use of some tion. The results from the common protocol may be limited by some
natural protein-based adjuvants such as diphtheria toxin (DT), cholera drawbacks such as inappropriate physicochemical properties of pre-
toxin B-subunit (CTxB), heat-labile enterotoxin B subunit (LTB) and dicted epitopes or final construct, toxic epitopes, instability of final
toll-like receptors (TLRs) ligands in final construct of proposed vaccines vaccine and unable to effective expression of designed vaccine in a
is suggested [17–19]. In the study, CTxB and LT-IIc were selected as desired host [43,44]. Therefore, in the present study, we used a special
protein adjuvants. CTxB is a 124 amino acids, nontoxic, homo- multi-steps procedure to decrease mentioned obstacles. In our protocol
pantameric, commercially available and membrane-binding subunit of linear B cell epitopes were selected based on their antigenicity, aller-
cholera toxin that could increase homoral and mocusal immunity re- genicity, toxicity, and water solubility as decisive parameters. Simi-
sponse. Furthermore, CTxB receptor is widely expressed in the plasma larly, antigenicity, hydrophobicity, population coverage and allergeni-
membrane of immune cells; therefor the protein is highly regarded as city were considered as determining parameters to T-cell epitope
suitable natural adjuvant [20]. LT-IIc is a member of the type II sub- prediction. The screened epitopes were then merged to each other as
family of LTBs that composed by 121 amino acid residues. Moreover, well as to two natural adjuvants using appropriate linkers for organi-
LT-IIc is one of the best-studied type II LTB which has been shown to zation of final vaccine construct. Furthermore, the efficacy and stability
enhance antigen-specific CD8+ T cell immune responses when co-ad- of the vaccine were evaluated by a set of bioinformatics approaches
ministered with a model antigen [21]. including molecular docking, molecular dynamics and in silico cloning.
The protein adjuvants can increase the level of mean antibody titers,
the generation of immune memory (especially T cell memory) and 2. Material and methods
seroconversion rates in populations. The adjuvants also can reduce
amounts of antigen in vaccine formulation as well as permit im- The procedures used for the multi-epitope vaccine development are
munization with fewer doses of vaccine [22,23]. These biological ac- depicted in Fig. 1.
tivities can be achieved through induce of multiple chemokines and
cytokines in bone marrow-derived dendritic cells (BMDCs) including 2.1. Primary data collection
keratinocyte-derived chemokine (KC), eotaxin, IL-27, IFN-γ, Th2-asso-
ciated cytokine IL-5 and Th1-type cytokines IL-12. Furthermore, acti- As the first stage of our study, the reference amino acid sequences of
vating Toll-like receptors (TLRs) signaling pathway, inducing pro-in- CCHFV proteins including nucleoprotein, glycoprotein precursor, and
flammation cytokines (such as IL-6, IL-10, and IL-12), promotion of the RNA-dependent RNA polymerase were retrieved from NCBI (https://
infiltration of antigen-presenting cells (APC) and induction of antigen- www.ncbi.nlm.nih.gov) in FASTA format with accession numbers of
specific CD8+ T cells are other mechanisms of action of the protein NP_950237.1, NP_950235.1 and ACM78472.1 respectively. In addition,
adjuvants associated biological activities [24–26]. Furthermore, pre- the primary amino acid sequence of CTxB and LT-IIc were obtained
sence of linear and conformational B- and T-cell epitopes in protein from Uniprot (http://www.uniprot.org) with accession entry of
adjuvant can improve immune responses against main antigens. Q57193 and H6W8F2 respectively. Also, the three-dimension structure
Experimental determination of dominant epitopes and evaluation of of MHC-I and II were retrieved from Protein Data Bank (https://www.
potential vaccines efficacy, are tedious and costly. Therefore, recently rcsb.org/) in PDB format with PDB entry of 1E27 and 1AQD

2
M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Fig. 1. Schematic presentation of the procedures used for multi-epitope vaccine development for CCHFV.

respectively. based on antigenicity, Hydrophobicity, allergenicity and population

coverage using a set of online tools including VaxiJen, peptide2
2.2. Multiple sequence alignment and antigen selection (https://www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.
php), AllerTOP (http://www.pharmfac.net/allertop/) and IEDB ana-
To determine conservancy level of the virus proteins between dif- lysis resource (http://tools.iedb.org/population/) respectively. Finally,
ferent CCHFV serotypes protein BLAST was performed (https://blast. the epitope(s) with appropriate properties were selected for the vaccine
ncbi.nlm.nih.gov/Blast.cgi). Furthermore, for determining the con- construct.
served region(s) in the protein sequences multiple sequence alignment
was performed by Clustal Omega server (https://www.ebi.ac.uk/Tools/ 2.5. Linear B-cell epitope prediction and screening
msa/clustalo). Additionally, antigenicity of the CCHFV proteins was
evaluated using VaxiJen 2.0 server (http://www.ddg-pharmfac.net/ The linear B-cell epitope prediction was carried out using BCPREDS
vaxijen/VaxiJen/VaxiJen.html) and Predicting Antigenic Peptides server at (http://ailab.ist.psu.edu/bcpred/predict.html) with fixed
(http://imed.med.ucm.es/Tools/antigenic.pl). Finally, most conserved length epitope (20 amino acids) prediction method and specificity of
and antigenic protein was selected for further analysis. 80%. The server predicts linear B-cell epitopes by using SVM combined
with subsequence kernel (SSK) attitude with an accuracy of 74.57%.
2.3. Antigen analysis and input preparation Furthermore, for cross-validation of the predicted epitopes, the pre-
dicted epitopes were checked through BepiPred-2.0 at (http://www.
The selected antigen was further analyzed to determine antigenic cbs.dtu.dk/services/BepiPred/), SVMTrip at (http://sysbio.unl.edu/
regions, conserved domain(s), sequence features and physicochemical SVMTriP/prediction.php) and ABCpred at (http://crdd.osdd.net/
properties. For this purposes, a set of online tools including Predicting raghava/abcpred/ABC_submission.html). Later, high-ranked and
Antigenic Peptides (http://imed.med.ucm.es/Tools/antigenic.pl), shared B-cell epitopes were selected for further study. Furthermore, the
NCBI's conserved domain database (https://www.ncbi.nlm.nih.gov/ B-cell epitopes were further evaluated in term of antigenicity, aller-
Structure/cdd/wrpsb.cgi), Uniprot (http://www.uniprot.org) and genicity, toxicity, and solubility using VaxiJen, AlgPred (http://crdd.
ProtParam (https://web.expasy.org/protparam/) were used. osdd.net/raghava/algpred/submission.html), Toxin pred (http://crdd.
osdd.net/raghava/toxinpred/) and PepCalc (https://pepcalc.com/)
2.4. T-cell epitope prediction and selection online servers respectively. Finally, the predicted linear B-cell epitopes
with appropriate properties were selected for locating in the vaccine
The MHC-I restricted epitopes were predicted using ProPred-1 structure.
server (http://crdd.osdd.net/raghava/propred1/index.html) with de-
fault parameters. The server uses special matrices for 47 MHC-I alleles. 2.6. Vaccine engineering and physicochemical properties
Similarly, the MHC-II restricted epitopes were predicted through
ProPred server (http://crdd.osdd.net/raghava/propred/) with a The determined appropriate B- and T-cell epitopes were organized
threshold of 5%. The server uses special matrices for 51 HLA-DR alleles in the final vaccine construct. For this purpose, the screened B- and T-
that cover more than 90% of MHC-II molecules expressed on antigen cell epitopes were merged using GPGPGPG amino acid linker in a
presenting cells. Furthermore, the predicted epitopes were screened random pattern. The joined epitopes were considered as a multi-epitope

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

segment or core of the proposed vaccine. The final construct of the 2.11. Molecular dynamics (MD) simulation of ligand-receptor complex
proposed vaccine was prepared after merging the multi-epitope part
with two natural adjuvants including CTxB and LT-IIc at N and C Molecular dynamic is a computational approach, which is used to
terminal using EAAAK linker respectively. Furthermore, amino acid describe properties of the molecules' behavior, ligand-receptor inter-
composition and some physicochemical properties of the proposed actions, solvation, stability and fluctuations as well as conformational
vaccine including molecular weight, iso-electric point, net charge at pH changes of molecules. In the present study, MD simulation was per-
7, estimated solubility in water, estimated half-life in the mammalian formed on the vaccine-MHC model complexes using NAMD graphical
reticulocytes and instability index were determined using Protparam interface module incorporated visual molecular dynamics (VMD)
(http://web.expasy.org/cgi-bin/protparam/protparam), Pepcalc during 15 ns stimulation. The protein structure file (PSF) of the com-
(http://pepcalc.com/), plexes was built using automatic PSF generator module in VMD. The
MD simulations were done under NPT equivalent conditions at 1 bar
and 300 K and using accessing PSF and PDB files. Afterward, DCD
2.7. Secondary and tertiary structure prediction
trajectory files were generated by NAMD. Finally, the MD simulations
results were analyzed based on root mean square deviation (RMSD) and
The secondary structure of the proposed vaccine was predicted
Radius of gyration (Rg).
using Prabi server at (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.
pl?page=/NPSA/npsa_gor4.html) through GOR4 secondary structure
prediction method. The method uses all possible pair frequencies within 2.12. Revers translation, codon optimization and in silico cloning
a window of 17 amino acid residues with a mean accuracy of 64.4%. In
addition, the three-dimension structure of the vaccine was generated To clone and express the vaccine in a suitable expression vector, the
using I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I- amino acid sequence of the vaccine model was back translated into
TASSER/). I-TASSER is a ranked approach to protein structure and nucleotide sequence using Gene infinity server (http://www.
function prediction that relies on the level of similarity between the geneinfinity.org/sms/sms_backtranslation.html) based on codon usage
input and available template structures in PDB. table of Escherichia coli K12. The optimized DNA was further evaluated
in term of Codon Adaptation Index (CAI), GC content and Codon
Frequency Distribution (CFD) using GenScript Rare Codon Analysis
2.8. Model refinement and quality assessment Tool at (https://www.genscript.com/tools/rare-codon-analysis). These
parameters have key roles in optimized protein expression in the host
To reduce possible structural mistakes in the predicted tertiary expression system. In latest step, restriction sites in the DNA were
structure of the vaccine, refinement of the predicted model was done mapped using NEBcutter V2.0 at (http://nc2.neb.com/NEBcutter2/)
using 3Drefine server (http://sysbio.rnet.missouri.edu/3Drefine/). then appropriate restriction sites were added to 5′ and 3′-OH of the
Additionally, the geometry quality of the vaccine was validated based optimized DNA.
on Ramachandran plot using RAPAGE server (http://mordred.bioc.
cam.ac.uk/~rapper/rampage.php) and Z-score of ProSA server at
3. Results
(https://prosa.services.came.sbg.ac.at/prosa.php) also ERRAT quality
factor (http://services.mbi.ucla.edu/ERRAT/).
3.1. Multiple sequence alignment and antigen selection

2.9. Conformational B-cell and IFN-γ inducing epitopes prediction To designing an effective multi-epitope vaccine for cross protection
against CCHFV, firstly all protein encoded by the virus were studied for
The conformational B-cell epitopes in the vaccine model were pre- determining more conserved protein between different serotypes of the
dicted using ElliPro server at (http://tools.iedb.org/ellipro/) with fol- virus. For this end, protein BLAST was done. The results of protein
lowing epitope prediction parameters: minimum score 0.5 and max- BLAST for CCHFV proteins are summarized in Table 1. The results
imum distance seven (angstrom). Furthermore, IFN-γ inducing epitopes showed that RNA polymerase has most conservancy level between
in the amino acid sequence of the vaccine were predicted using different CCHFV serotypes. Furthermore, average antigenicity and
IFNepitope server (http://crdd.osdd.net/raghava/ifnepitope/scan.php) VaxiJen score of the proteins were determined (Table1). The results
with motif and SVM hybrid prediction approach. demonstrated that Glycoprotein precursor has the highest antigenicity
followed by RNA polymerase and Nucleoprotein respectively. Due to
2.10. Molecular docking study high antigenicity, good conservancy and higher exposure probability to
immune system, Glycoprotein precursor was selected for further ana-
To validate the binding affinity of the vaccine to MHC-I and MHC-II lysis and the vaccine design.
molecules, molecular docking was performed between the proposed
candidate vaccine and HLA-A0201 with PDB entry of 4UQ3, and HLA- 3.2. Antigen analysis and input preparation
DRB1_01:01 with PDB entry of 1AQD. Molecular docking studies were
carried out using Patchdock server (https://bioinfo3d.cs.tau.ac.il/ The potential antigenic regions in the glycoprotein precursor sequence
PatchDock/) with default complex type and clustering RMSD of 4 Å. are illustrated in Fig. 2. The results showed that there are 72 antigenic
The server uses object recognition and image segmentation techniques determinants in the glycoproteins. Results also confirmed that six regions
in its algorithm. in the glycoprotein sequence including 414–469, 654–720, 798–841,

Table 1
Results of CCHFV proteins BLAST and antigenicity prediction. Glycoprotein precursor had highest average antigenicity (1.3096) and VaxiJen score (5207), while
sequence identity of the glycoprotein between different serotypes of CCHFV was least value in compare to other studied antigens.
Protein Average antigenicity VaxiJen score Minimum identity (%) Maximum identity (%)

Nucleoprotein 1.0138 0.3330 92 99

Glycoprotein precursor 1.3096 0.5207 84 99
RNA polymerase 1.0324 0.4348 96 99

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Fig. 2. Antigenicity plot of glycoprotein precursor from CCHFV. There are 72 antigenic determinants in the glycoproteins. Six regions including 414–469, 654–720,
798–841, 1012–1043, 1290–1325 and 1441–1470 were found to be high antigenic potential with average antigenic propensity more than 1.15.

Table 2 form both Gc and Gn were predicted using BCPREDS. A total number of
Molecular features of glycoprotein precursor from CCHFV. After processing of 11 and six epitopes were predicted from Gc and Gn respectively.
the precursor six structural and non-structural elements and a signal peptide are Furthermore, to determine share epitopes and increasing the con-
originated. fidence, BCPREDS outputs were cross-checked using three different
Feature key Position(s) Description Sequence length servers including ABCpred, SVMTrip and, BepiPred-2.0 (Table 5). Since
in the mentioned servers linear B-cell epitope prediction is performed
Short sequence 1–18 Signal peptide 18 through different algorithms, sequence similarity and score of predicted
Chain 19–1684 Envelopment polyprotein 1666
Chain 19–247 Mucin-like variable region 229
epitopes were considered as decisive factors for screening share epi-
Chain 248–519 GP38 272 topes. Subsequently, the epitopes with a high score and sequence si-
Chain 520–842 Glycoprotein N 323 milarity in the used server were selected for final screening. Con-
Chain 843–1040 Non-Structural protein M 198 sidering the mentioned conclusive factors, five epitopes were selected
Chain 1041–1684 Glycoprotein C 644
from the antigens. The screened epitopes were further evaluated re-
garding antigenicity, allergenicity, toxicity, solubility and hydro-
philicity for defining final B-cell epitopes. The results showed that one
1012–1043, 1290–1325 and 1441–1470 have high antigenic potential
epitope from each studied antigens (Table 6) has appropriate proper-
with average antigenic propensity more than 1.15. Results also showed
ties, so considered as final B-cell epitopes for the vaccine construct.
that there are three conserved domains in the Glycoprotein including
Nairovirus M polyprotein-like, Herpes virus major outer envelope glyco-
protein (BLLF1) and Hantav rus glycoprotein G2 which were located in 3.5. Vaccine engineering and physicochemical properties
241–884, 32–234 and 1177–1250 regions in the glycoprotein sequence
respectively. Furthermore, the results demonstrated that after processing Based on the immuno-informatics analysis finally, one epitope from
of the precursor six structural and non-structural features including the each Gc and Gn were considered as linear B-cell epitopes. Furthermore,
signal peptide, Mucin-like variable region, non-structural glycopro- five epitopes from mentioned antigens were selected as T-cell epitopes.
tein GP38, glycoprotein N, Non-Structural protein M and glycoprotein C The final vaccine composed of 382 amino acids, which organized in
originated (Table 2). Therefore, glycoprotein N and glycoprotein C were four domains: CTxB and LT-IIc as adjuvants, Linear B-cell epitopes and
selected to further analysis due to their high antigenicity along with good T-cell epitopes, which were joined using appropriate linkers. A graphic
conservancy level and more probabilities to expose the immune system. diagram of the vaccine domain structures with linker's sites designed is
Finally, amino acid sequences of the glycoproteins were considered as depicted in Fig. 3. Furthermore, physicochemical properties and amino
inputs for more analysis and final vaccine construct. acid composition of the final construct were predicted using Protparam
and Pepcalc. The results revealed that the vaccine protein was a
3.3. T-cell epitope prediction and selection 40.7 kDa stable soluble protein with pI 8.74, a net charge of 5.4 and
estimated half-life was found to be 30 h. The Protein half-life is defined
In order to prediction of T-cell epitopes, we used a two-step special as the time it takes for half of the amount of protein in a cell to dis-
screening. Briefly, MHC-I and II restricted epitopes were predicted and appear after its synthesis in the cell. Therefore, more half-life can
ranked according to their score using ProPred1 and ProPred respec- provide more time for our proposed chimeric protein to exposure to the
tively. A total number of 44 and 47 MHC-I restricted epitopes were immune system [45]. Final amino acid sequence and amino acid
predicted for Gc and Gn respectively. The predicted MHC-I restricted composition of the vaccine is depicted in Fig. 4.
epitopes were compared to MHC-II restricted epitopes to determine
shared epitopes. In the next step, 12 and 13 share epitopes from Gc and 3.6. Secondary and tertiary structure prediction
Gn were selected for further screening respectively (Table 3). Finally,
the epitopes with the ability to binds most MHC-I and II alleles along The secondary structure of the final vaccine construct was predicted
with high antigenicity, hydrophobicity, population coverage and non- using GOR IV method on the basis the amino acid sequence of the
allergenicity properties were considered as the final T-cell epitopes. protein. The results showed that 29.06, 22.25 and 48.69% of total 382
After final screening, a total number of three and two T-cell epitopes amino acids were organized in alpha helix, extended strand and random
were determined from Gc and Gn respectively (Table 4). coil respectively (Fig. 5). Furthermore, the primary 3D model of the
proposed vaccine was predicted by I-TASSER online server. The ten best
3.4. Linear B-cell epitope prediction and screening threading templates were selected (PDB entry: 3jc8Q, 5gaoE, 4l6t,
5jcsS, 1ltrF, 4btgA, 1ltrF, 5voxO, 1ltrA, and 5yfpH) through Local Meta-
Prediction of linear B-cell epitopes from the antigens was performed Threading-Server program to model our proposed vaccine. Finally, the
through a cross-checking method. For this, firstly linear B-cell epitope top five models for the protein vaccine were suggested with C-score of

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Table 3 and GDT-HA), division score and physical realism score respectively.
Results of T-cell epitope prediction. The labled sequences with (*) are shared T- Consequently, the refined model with appropriate properties was se-
cell epitopes between MHC-I and II risterected epitopes. The share epitopes lected for further evaluations concerning mentioned factors (Table 7).
with high affinity to more MHC-I and MHC-II alleles were screened for further Furthermore, the geometric quality of primary and refined models was
evaluation. A total number of 44 and 47 MHC-I restricted epitopes were pre-
evaluated by applying Ramachandran plot, ERRAT quality factor, and
dicted for Gc and Gn respectively which among them 12 and 13 shared epitopes
ProSA z-score. The results showed that in the raw model 68.7%, 22.1%
were selected for further evaluation.
and 9.2% of residues were located in favored, allowed, and outlier re-
MHC-I restricted MHC-I restricted Number of MHC-I Number of MHC-II gions, respectively (Fig. 6-a) while, in the refined model, 73.4%, 18.9%,
epitopes in Gc epitopes in Gn bonded alleles for bonded alleles for
and 7.6% of residues were positioned in favored, allowed, and outlier
predicted epitopes predicted epitopes
regions, respectively (Fig. 6-b). Moreover, the quality factor and z-score
Gc Gn Gc Gn of primary model were 71.5517 and −1.74 while after refinement
mentioned parameters were determined 75.4098 and −2.02 that in-
LSEPRNIQQ TAEIHGDNY 1 1 0 0
dicates improving in geometric quality of the primary model after re-
FLFLAPFIL* ILCKAIFYL* 5 4 46 22
FILLILFFM* LNLERIPWV* 2 2 46 13
finement. The refinement and primary 3D model of proposed vaccine
KLPPEIITL YLLIIVGTL 2 7 0 41 are illustrated in Fig. 5.
KVNGHLIHK YVITCILCK 2 3 0 50
MYSPVFEYL* NYGGPGDKI 4 2 2 0 3.8. Conformational B-cell and IFN-γ inducing epitopes prediction
GLFKYRHLK FLFWFSFGY* 2 6 0 38
ILFFMFGWR* RLTSDGLAR 1 1 5 0
ESIMKLEER ESTGVALKR 1 2 0 0 Due to the key role of conformational B-cell epitopes as well as IFN-
CVELTSQER YVITCILCK* 2 3 0 50 γ inducing epitopes in adaptive and innate immune responses, the
VKWKVEYIK* GKMAIYICR 1 1 3 0 protein vaccine was subjected to prediction of the mentioned epitopes.
FLAPFILLI* LLRTETAEI* 2 1 25 48
IFN-γ is the signature cytokine of both the innate and adaptive immune
ERLADRRIA KRLKQYREL 1 6 0 0
RRTRGLFKY CRQGYCLRI 3 2 0 0
systems with ability to provok antiviral immune responses and pro-
GRSESIMKL ARHVIQCPK 4 1 0 0 tection against reinfection. The release of IFN-γ is the important step of
EDASESKLL IPKGTGDIL 1 6 0 0 the Th1 response that induce the production of protective virus-neu-
IHVDEPDEL* CDTSCEIMI 2 2 11 0 tralizing IgG and enhance the magnitude of antiviral cytotoxic T lym-
NHASFVNLL NHPKTTMAF 3 1 0 0
phocytes (CTLs) responses [46]. To prediction of conformational B-cell
LQVYHIGNL* QHFLKDNLI 1 2 26 7
KEWPHSRNW QQHFLKDNL 1 3 0 0 epitopes, the 3D structure of the proposed vaccine was used as an input
LESVKSFFY* QEGRGHVKL 1 2 13 0 for prediction of probable conformational B-cell epitopes via ElliPro. A
EPDELTVHV CEIMIPKGT 2 2 0 0 total number of seven conformational B-cell epitopes were predicted
SGISCKVRI IPLLGKMAI* 1 7 0 40
from the vaccine, which included 207 residues out of 382 residues.
GAGEITVLV IPWVVRKLL 1 8 0 0
QQKLPPEII SATGKNCEI 1 3 0 0
Results also showed that most of the residues, which were located in the
WPSCTYTGV FGYVITCIL* 4 6 0 36 multi-epitope region in our proposed vaccine, were included in the
FGWRILFCF* FWFSFGYVI* 3 5 19 9 predicted conformational B-cell epitopes (Table 8). The predicted
TEAIVCVEL YLLIIVGTL* 2 7 0 37 conformational B-cell epitopes are depicted in Fig. 7. Furthermore, the
GEITVLVEV IPWVVRKLL 1 8 0 0
amino acid sequence of the final proposed vaccine was applied for
ILLILFFMF* KTTMAFLFW 1 1 23 0
EPRNIQQKL QEGRGHVKL 4 2 0 0 prediction of 15-mer IFN-γ inducing epitopes using MERCI method in
HPRIEEGFF CETTPVNAI 3 2 0 0 IFNepitope server. The results showed that there were 40 positive IFN-γ
EPRNIQQKL RLGSELGCY 4 1 0 0 inducing epitopes with a score of greater than or equal to one (Table 9).
VFMGIFLFL* ASRLTSDGL 3 1 13 0
Results also revealed that the predicted epitopes were located in three
GYRRIIEKL HPKTTMAFL 4 6 0 0
NHASFVNLL INRVRSFKL* 3 1 0 19
regions including 20–62, 125–149 and 283–356, which were related to
MPKTSLCFY KRLKQYREL 4 6 0 0 CTxB, multi-epitope region and LT-IIc respectively.
KNLLNSTSL HPKTTMAFL 2 6 0 0
ASFVNLLNI YLLIIVGTL* 2 7 0 37 3.9. Molecular docking study
RGLFKYRHL VPVKCRQGY* 3 4 0 10
– ITICNGSTI* – 2 – 9
– LGCPKIPLL – 2 – 0 The binding affinity and interaction between the proposed vaccine
– EETELYLNL – 4 – 0 and MHC-I and II molecules were evaluated using molecular docking
– NYGGPGDKI – 2 – 0 via Patchdock server. PatchDock docking output is a list of candidate
– SEEPSDDCI – 3 – 0
complexes including specified receptor and ligand molecule that ranked
– IPWVVRKLL – 8 – 0
based on Geometric shape complementarity score, approximate inter-
face area of the complex and Atomic contact energy (ACE). The results
−1.66, −0.56, −3.34, −3.62 and −2.42 respectively. The C-score is of molecular docking studies between our proposed vaccine and MHC
defined in the range of −5 to 2 and usually higher score indicate a molecules revealed that the proposed vaccine has high affinity to both
model with higher confidence. Accordingly, the model with C-score of MHC-I and II. The best-docked model for vaccine-MHC-I and vaccine-
−0.56 was selected for further analysis. MHC-II complexes showed a docking score of 17,810 and 18,498 re-
spectively. Furthermore, approximate interface areas of the mentioned
complexes were found to be 3338.70 and 3749.50 Å correspondingly.
3.7. Model refinement and quality assessment Also, ACE of the selected top rank complexes were calculated as −266
and −419.48. The high ranked complexes between both MHC mole-
The selected model of the vaccine was then subjected to refinement cules and the vaccine were selected for further analysis through mole-
processes by 3Drefine server. For this purpose, the whole protein cular dynamics.
structure refinement including secondary structure elements, loop re-
gions and protein side-chains were refined. Selection of refined model 3.10. MD simulation results
in 3Drefine server is based on five factors including 3Drefine score,
GDT-TS, GDT-HA, RMSD, RWplus and MolProbity which indicated To validate the stability of the vaccine-MHC complexes molecular
potential energy (3Drefine score and RWplus), similarity score (GDT-TS dynamics was performed. For this purpose, the high ranked complexes

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Table 4
The results of final T-cell epitope screening. A total number of three and two T-cell epitopes (labeled with *) were screened from Gc and Gn respectively based on
their antigenicity, hydrophobicity, allergenicity and population coverage. Generally, predicted epitopes from Gc have better properties than Gn derived ones.
Protein T-cell epitopes Antigenicity Hydrophobicity (%) Allergenicity Population coverage (%) Final decision

Gc FLFLAPFIL 0.2586 100 No 29.10 –

FILLILFFM 0.2343 100 No 10.06 –
MYSPVFEYL 0.0270 55.56 No 11.21 –
ILFFMFGWR 1.6738 77.78 Yes 71.04 –
VKWKVEYIK −0.2568 44.44 Yes 24.32 –
FLAPFILLI 0.6746 100 No 76.69 *
IHVDEPDEL −0.0627 44.44 Yes 32.05 –
LQVYHIGNL 1.0338 44.44 No 59.74 *
LESVKSFFY −0.1855 44.44 No 23.12 –
FGWRILFCF 0.6046 66.67 No 89.12 *
ILLILFFMF 0.3806 100 No 24.76 –
VFMGIFLFL −0.0320 88.89 No 13.12 –

Gn ILCKAIFYL −0.1887 66.67 No 62.54 –

LNLERIPWV 1.3691 66.67 Yes 88.54 –
FLFWFSFGY 1.3227 66.67 Yes 33.32 –
YVITCILCK −0.1778 44.44 No 19.54 –
LLRTETAEI 0.0964 44.44 No 79.54 *
IPLLGKMAI 0.3897 77.78 No 43.22 –
FGYVITCIL −0.0073 55.56 Yes 27.12 –
FWFSFGYVI 1.5147 66.67 Yes 41.05 –
YLLIIVGTL 0.6623 66.67 No 45.32 *
INRVRSFKL −0.3028 44.44 Yes 18.03 –
YLLIIVGTL −0.5074 66.67 No 36.12 –
VPVKCRQGY 1.2904 33.33 No 35.32 –
ITICNGSTI −0.2864 33.33 No 10.04 –

formed by PatchDock were subjected to MD simulation. As the first Table 6

step, energy values, pressure, temperature, volume and density of the The final screening of selected linear B-cell epitopes from Gc and Gn. One linear
system were checked at the equilibration steps and the end of the si- B-cell epitope from each antigen was selected for final vaccine construct. The
mulation. The results revealed that MD simulation steps were per- selected epitopes were found to be good water soluble, high antigenic (anti-
formed appropriately. The backbone RMSD of the vaccine-MHC com- genicity score more than 0.8) and without allergenicity and toxicity potential.
plexes are depicted in Fig. 8. The results demonstrated that in both Epitope Antigenicity Allergenicity Toxicity Solubility
studied complexes after a gradual increase in backbone RMSD until the
DELTVHVKSDDPDVVAASSS* 0.8272 No No Good
6th ns the systems almost reached steady state. In fact, the protein
KLQSCTHGVPGDLQVYHIGN 0.2226 Yes No Poor
vaccine and MHC molecules try to get the best conformation relative to LIHKIEPHFNTSWMSWDGCD 0.2253 No No Poor
each other during the MD simulation. Radius of gyration is an im- GLARHVIQCPKRKEKVEETE 0.5288 Yes No Good
portant indicator of protein compactness and stability. Therefore, the VDQRLGSELGCYTINRVRSF* 0.8924 No No Good
Rg values of the vaccine-MHC alleles were monitored during MD sti-
mulations (Fig. 9). The results showed that the Rg values of both the
vaccine-MHC-I and MHC-II complexes decreased significantly after more compactness and stability during MD simulations. In other words,
three and four ns respectively, which indicated the complexes gained this trend revealed suitable interactions between our proposed vaccine
and MHC alleles.

Table 5
Results of linear B-cell epitope prediction using three different servers. The score and sequence similarity were considered as determining factors for comparison of
the server outputs and epitope selection. A total number of three and two epitopes from Gc and Gn were screened for final evaluation respectively. Selected epitopes
are labeled with (*).
Protein Epitope Score Sequence similarity (%)

BCPREDS ABCpred SVMTrip BepiPred-2.0 ABCpred SVMTrip BepiPred-2.0

Gc DELTVHVKSDDPDVVAASSS* 0.998 0.85 0.516 High 85 60 60

GDRQVGEWPKATCTGDCPER 0.996 0.69 – High 70 – 90
EETGYRRIIEKLNNKKGKNK 0.994 0.74 0.386 Low 90 25 45
IEHKGTIIGKQNSTCTAKAS 0.978 0.79 0.238 High 95 20 50
KLQSCTHGVPGDLQVYHIGN* 0.957 0.69 0.528 High 100 85 55
NMGDWPSCTYTGVTQHNHAS 0.935 0.74 – Low 60 – 50
RNWRCNPTWCWGVGTGCTCC 0.915 0.76 – Low 70 – 25
LIHKIEPHFNTSWMSWDGCD* 0.915 0.68 0.295 High 90 20 100
APWGAINVQSTYKPTVSTAN 0.901 0.87 0.238 Intermediate 90 20 50
SCSEEDTKKCVNTKLEQPQS 0.849 0.70 – High 65 – 100
QKLPPEIITLHPRIEEGFFD 0.835 0.87 – High 90 – 95

Gn IHGDNYGGPGDKITICNGST 0.999 0.90 – Intermediate 75 – 55

GLARHVIQCPKRKEKVEETE* 0.995 0.86 0.603 High 100 60 100
RLKQYRELKPQTCTICETTP 0.993 0.60 – Intermediate 65 – 45
PKGTGDILVDCSGGQQHFLK 0.959 0.74 – Intermediate 80 – 55
IDLGCPKIPLLGKMAIYICR 0.908 0.85 – Low 85 – 30
VDQRLGSELGCYTINRVRSF* 0.841 0.65 0.533 High 95 50 85

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

a highly conserved epitope in RNA-dependent RNA polymerase of

CCHFV and introduced it as a potential epitope-based vaccine for
CCHFV [48]. In Oany et al study degree of conservancy was considered
as determining factor while along with conservancy some other factors
such as immunogenicity level, allergenicity, population coverage and
physicochemical properties of an epitope must be considered for epi-
Fig. 3. Graphical presentation of the purposed vaccine for CCHF. The vaccine is tope-based vaccine design. The mentioned parameters were considered
organized in four different domains including CTxB and LT-IIc as adjuvants, in the present study. Furthermore, in the mentioned study envelope
Linear B-cell epitopes (blue regions) and T-cell epitopes (pink regions). The glycoprotein of CCHFV was also evaluated for determination of con-
used linker for merging mentioned domains were GPGPGPG (red regions) and served epitopes. The results were contrary to our study, because high
EAAAK (yellow regions). (For interpretation of the references to colour in this
variation in the envelope glycoprotein was observed, while this varia-
figure legend, the reader is referred to the web version of this article.)
tion is focused in the N terminal mucin-like domain, which is not part of
mature glycoprotein [49].
3.11. Revers translation, codon optimization and in silico cloning To develop an efficient vaccine for CCHF we used both im-
munoinformatics and structural vaccinology strategies. In recent years,
To evaluate the cloning and expression of the vaccine within the the strategies are used to design many vaccines. In this study, we made
expression vector in silico cloning was performed through Gene infinity some modification in the common method. In conventional method,
server. For this, the amino acid sequence of the vaccine was reverse after selecting the desired antigen, an epitope mapping is performed
translated according to the codon usage of E. coli expression system. and some evaluation such as affinity to MHCs, physicochemical prop-
Furthermore, the designed gene was evaluated in term of GC-content, erties and mRNA prediction [27,30]. However, in our study along with
CAI and CFD to determine codon usage frequency and codon usage the mentioned steps multi-step epitope screenings besides more in silico
distribution using GenScript rare codon analysis tool. The results validations are performed.
showed that GC-content, CAI and CFD of the improved sequence were As illustrated in Fig. 2 our proposed multi-epitope vaccine is com-
found to be 57.46%, 1 and 100% that were satisfactory. Furthermore, posed of two natural adjuvants (CTxB and LT-IIc), two B-cell epitopes
after determination of restriction sites within the optimized DNA, XhoI and five T-cell epitopes, which were merged to each other by appro-
and NdeI restriction sites were created in 5′ and 3′-OH of the DNA re- priate linkers. In our vaccine construct, after analysis of all CCHFV
spectively, followed by adding six histidine residues on both ends. virulence factors, Gc and Gn glycoproteins were selected as main an-
tigens and the sources of final B- and T-cell epitopes due to its high level
of conservancy (up to 84%) and antigenicity.
4. Discussion Generally, viral glycoproteins play a vital role in the catalysis of
virus fusion to host cells as well as assist in the invasion [50,51].
Due to error-prone nature of CCHFV polymerase, high infectious Consequently, recently, they are considered as crucial components for
rate and lack of animal model, the development of a vaccine against vaccine development against viral infections such as HIV-1 [52], HCV
CCHFV is very challenging. However, recently, with the development of [53], Ebola virus [51], human coronavirus [54], Dengue virus [55] and
bioinformatics and computational methods, these limitations are di- many others.
minished. In this regard, using computational method, epitope map- Determination of immunogenic B- and T-cell epitopes is a crucial
ping, design of recombinant proteins and the evaluation of physico- step in epitope-based vaccine design. T-cells have the key role in anti-
chemical properties as well as candidate vaccines efficacy can be body production through polarization to Th2 and antigen presenting
available [40,47]. Therefore, this study was planned to design an effi- during viral infections [56,57]. Accordingly, dominant T-cell epitopes
cient multi-epitope recombinant vaccine against CCHFV using a special of both Gc and Gn were predicted with the help of a multi-step
multi-step bioinformatics approach. screening procedure. To screening T-cell epitopes, firstly MHC-I re-
Based on our best knowledge there is only one study about com- stricted T-cell epitopes were predicted. In the next step, the predicted
putational design epitope-based vaccine for CCHF. Oany et al identified

Fig. 4. Amino acid sequence and composition of final vaccine construct. The Amino acid sequence and composition can affect the protein 2D and 3D structures as
well as its function. The results reflected that the protein is composed by 382 amino acids. Glycine and Tryptophan are the most and the least residues in the final
vaccine construct.

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Fig. 5. Secondary structure of the final vaccine construct predicted by GOR4 method. Most amino acid residues of the vaccine were organized in random coil
(48.69%) followed by alpha helix (29.06) and extended strand (22.25%) respectively.

Table 7
Results of the model refinement. The low 3Drefine, MolProbity and RWplus scores indicate better quality model. On the contrary, higher GDT-TS, RMSD and GDT-HA
scores indicate conservative refinement and higher quality. Based on the mentioned parameters, model No.1 was selected as the best-refined model.
Model 3Drefine score GDT-TS GDT-HA RMSD RWplus MolProbity

1 29,866 0.9993 0.9666 0.376 −53,727.135 3.461

2 30,223.2 1 0.9758 0.351 −53,610.179 3.413
3 30,879 1 0.9863 0.318 −53,450.421 3.390
4 31,941 1 0.9935 0.275 −53,327.261 3.381
5 35,185 1 0.9993 0.203 −53,166.064 3.339

Fig. 6. 3D structure of primary (a) and refined model (b) of the final vaccine construct. Ramachandran plots showed that in the raw model 68.7%, 22.1% and 9.2% of
residues were located in favored, allowed, and outlier regions, respectively (Fig. 5-a) while, in the refined model, 73.4%, 18.9%, and 7.6% of residues were
positioned in favored, allowed, and outlier regions, respectively.

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Table 8
Conformational B-cell epitopes from 3D model of proposed vaccine. A total number of seven epitopes were predicted. Epitopes No. 3 and 4 were considered as the
broadest and smallest conformational B- cell epitopes with 78 and 5 amino acid residues.
Number Residues Number of residues score

1 I2, L4, F6, G7, V8, F9, F10, T11, V12, L13, L14, S15, S16, A17, Y18, A19, H20, G21, T22, P23, Q24, N25, I26, T27, D28, L29, C30, A31, 31 0.787
E32, H34, N35
2 V308, N309, I310, S311, S312, D313, V314, N315, K316, D317, S318, K319, G320, I321, Y322, I323, S324, S325, S326, A327, G328, 25 0.746
K329, T330, F332, I333
3 D80, Q82, K83, K84, A85, I86, E87, R88, M89, K90, D91, T92, L93, R94, I95, A96, Y97, L98, T99, E100, A101, K102, V103, E104, K105, 78 0.714
L106, C107, V108, W109, N110, N111, K112, T113, P114, H115, A116, A117, A118, A119, I120, S121, M122, A123, N124, E125, A126,
A127, A128, K129, D130, E131, L132, K137, S138, D139, D140, P141, D142, V143, V144, A145, A146, G181, P182, G183, F184, L185,
A186, P187, F188, I189, L190, L191, I192, G193, P194, G195, P196
4 Y339, P340, D341, N342, Y343 5 0.690
5 T282, Y283, A284, G285, V286, S287, K288, T289, F290, K291, D292, K293, C294, A295 14 0.688
6 L334, S345, N346, E347, M348, R349, K350, I351, A352, M353, A354, A355, V356, L357, S358, N359, V360, R361, V362, N363, L364, 39 0.686
C365, A366, S367, E368, A369, Y370, T371, P372, N373, H374, V375, W376, A377, I378, E379, L380, A381, P382
7 I240, G241, P242, G243, P244, G245, P246, G247, Y248, L249, L250, I251, I252, V253, G254 15 0.592

epitopes was checked for their ability to binding to MHC-II alleles. After weaknesses such as low immunogenicity and instability have faced the
that, as primary screening, the T-cell epitopes with ability to bind to vaccine platform to serious challenge. Incorporating natural adjuvants
both MHC-I and II were selected for further analysis. Finally, five T-cell in epitope-based vaccine construct can notably diminish these obstacles
epitopes were screened based on antigenicity, population coverage, [19]. Hence, we merged the immunodominant epitopes with two nat-
hydrophobicity and allergenicity. Chowell et al demonstrated that hy- ural adjuvants including CTxB and LT-IIc at N and C terminal using
drophobicity is a hallmark of immunogenic T-cell epitopes and marks a EAAAK linker. The EAAAK can increase stability as well as decrease the
step toward removing the requirement for experimental epitope testing interaction between the vaccine domains and cause more effective se-
for vaccine development [58]. Therefore, in the study, for first time we paration [61,62].
considered hydrophobicity of the predicted T-cell epitopes as a de- After the organization of different domains of the vaccine, physi-
termining factor in epitope screening process. cochemical, immunological and structural properties of our proposed
B-cell epitopes have a pivotal role in boosting neutralizing-antibody vaccine were evaluated using different bioinformatics tools. The results
responses in different infections. Consequently, currently identification revealed that the vaccine is stable, water-soluble, non-allergenic and
of B-cell epitopes is highly considered for epitope-driven vaccine de- highly antigenic.
velopment and diagnostic reagents design [59,60]. With the aim of From an empirical attitude, to achieve a high-level protein expres-
achieving a potent protective immunity against CCHF two top-ranked sion in E. coli, some key parameters such as CAI, GC, and CFD content
linear B-cell epitopes were selected based on their antigenicity, aller- of the gene should be optimized. Generally, a gene with CAI of > 0.8 is
genicity, toxicity and hydrophilicity. The screened B- and T-cell epi- rated as good for expression in the desired host. The GC content of 30%
topes were combined into our vaccine construct as the final im- to 70% is considered as ideal percentage range. The CFD value of 100
munodominant epitopes using GPGPGPG linker to retain both supports maximum protein expression in the desired host [19]. All the
structural features and conformational dependent immunogenicity of mentioned parameters of the optimized gene showed that our proposed
the epitope vaccine. vaccine could be expressed efficiently in E. coli host.
Besides the various advantages of epitope-based vaccines, some After organization of the vaccine elements, the 3D structure of it

Fig. 7. Predicted conformational B-cell epitopes of the final vaccine construct. A total number of seven conformational B-cell epitopes were predicted from the
vaccine, which included 207 out of 382 residues.

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Table 9 was predicted using I-TASSER webserver. The primary 3D structure was
The predicted IFN-γ inducing epitopes from the proposed vaccine. A total subjected to the refining process to achieve a high-quality 3D structure.
number of 40 positive IFN-γ inducing epitopes with a score of greater tha- Quality of the refined model was assigned by Ramachandran plot,
n or equal to one were predicted from the vaccine. The amino acid residues of ERRAT quality factor, and ProSA z-score, and the results demonstrated
the vaccine, which are located in 126–141 region showed highest score (1.21).
that the quality of the refined model was significantly improved.
Number Sequence Start-End score Since discontinuous B-cell epitopes as well as IFN-γ inducing epitopes
have a crucial role in humoral and cell-mediated adaptive im-
1 GTPQNITDLCAEYHN 20–35 1
mune responses by producing antibodies and stimulating macrophages
2 TPQNITDLCAEYHNT 21–36 1
3 PQNITDLCAEYHNTQ 22–37 1 and natural killer cells, prediction of the epitopes currently are highly
4 QNITDLCAEYHNTQI 23–38 1 considered for vaccine design [13]. Subsequently, prediction of dis-
5 NITDLCAEYHNTQIH 24–39 1 continuous B-cell epitopes and IFN-γ inducing epitopes were performed.
6 ITDLCAEYHNTQIHT 25–40 1.02
For this, the refined 3D model was subjected to prediction as input, and
7 TDLCAEYHNTQIHTL 26–41 1.09
8 NDKIFSYTESLAGKR 41–56 1
results showed that 207 residues out of 382 residues of the vaccine were
9 DKIFSYTESLAGKRE 42–57 1 defined as a conformational B-cell epitope. Furthermore, the results in-
10 KIFSYTESLAGKREM 43–58 1 dicated that there were 40 positive IFN-γ inducing epitopes in our vaccine,
11 IFSYTESLAGKREMA 44–59 1 which shows the vaccine can provoke a strong antibody secretion, cell-
12 FSYTESLAGKREMAI 45–60 1
mediated responses and long-running protection against CCHFV.
13 SYTESLAGKREMAII 46–61 1
14 YTESLAGKREMAIIT 47–62 1 Antigen presentation is a required step in the recognition of anti-
15 AAAKDELTVHVKSDD 125–140 1 gens by immune system. APCs presenting the antigen using MHC I and
16 AAKDELTVHVKSDDP 126–141 1.21 II molecules [63]. Accordingly, the high affinity of a multi-epitope
17 AKDELTVHVKSDDPD 127–142 1 vaccine to MHC molecules can be considered as a determining factor.
18 KDELTVHVKSDDPDV 128–143 1
19 LTVHVKSDDPDVVAA 131–146 1
Therefore, we evaluated the binding affinity of the vaccine to MHC-I
20 TVHVKSDDPDVVAAS 132–147 1 and II using molecular docking study. The results confirmed that our
21 VHVKSDDPDVVAASS 133–148 1 vaccine has high affinity to both MHC molecules. To gain more insight
22 HVKSDDPDVVAASSS 134–149 1 into the vaccine-MHCs interaction and evaluate their stability, top-
23 AGVSKTFKDKCASTT 283–298 1
ranked complexes of the vaccine-MHC-I as well as vaccine-MHC-II were
24 SVQLVNISSDVNKDS 303–318 1
25 VQLVNISSDVNKDSK 304–319 1 subjected to MD stimulations. The results confirmed the suitable sta-
26 QLVNISSDVNKDSKG 305–320 1 bility of the studied complexes demonstrating persistent interactions
27 LVNISSDVNKDSKGI 306–321 1 between MHC molecules and our proposed vaccine.
28 VNISSDVNKDSKGIY 307–322 1 The results of our study showed that the proposed vaccine could be
29 NISSDVNKDSKGIYI 308–323 1
30 IPGGQYYPDNYLSNE 332–347 1
considered as a good candidate for more in vitro and in vivo evalua-
31 PGGQYYPDNYLSNEM 333–348 1 tions. In general, after proposing an in silico designed vaccine candidate
32 GGQYYPDNYLSNEMR 334–349 1 some in vitro and in vivo validation such as potential toxicity, probable
33 GQYYPDNYLSNEMRK 335–350 1 allergenicity, cross-reactivity, stability, determination of effective dose,
34 QYYPDNYLSNEMRK 336–351 1
total antibody titers and proliferation of lymphocytes, challenge in
35 YYPDNYLSNEMRKIA 337–352 1.05
36 YPDNYLSNEMRKIAM 338–353 1 animal model must be done to clarify the vaccine efficacy[64]. Vaccine
37 PDNYLSNEMRKIAMA 339–354 1 development is a time-consuming, complex and costly process that can
38 DNYLSNEMRKIAMAA 340–355 1 be simplified in to two main stages including pre-clinical and clinical
39 NYLSNEMRKIAMAAV 341–356 1 developments. In two mentioned stages selection of the right antigen(s),
40 FTVLLSSAYAHGTPQ 9–24 1
vaccine efficacy in test tubes and animals, formulation, safety for
human use, good manufacturing practice standards, immunity against

Fig. 8. RMSD plots of docked vaccine-MHCs complexes. The results indicated the stable microscopic interaction between the vaccine and MHCs molecules.

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M. Nosrati, et al. Journal of Biomedical Informatics 93 (2019) 103160

Fig. 9. Rg plots of docked the vaccine-MHCs complexes. The results reflected that the complex were compact and stable during stimulations.

artificial infection and clinical disease, vaccine efficacy under natural Acknowledgment
disease conditions and the long-term efficacy are assessed. However,
the vaccine development time, may be affected by some other para- The authors would like to acknowledge the University of Isfahan for
meters such as nature of target pathogen, equipment and facilities the financial support of the study.
available, technical and manufacturing hurdles [65,66]. Therefore,
determining the exact time to introduce a vaccine is very difficult; but References
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