REVIEWS Drug Discovery Today  Volume 25, Number 6  June 2020

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Epitope-based vaccine design: a

comprehensive overview of
bioinformatics approaches
Sepideh Parvizpour1, Mohammad M. Pourseif1, Jafar Razmara2,
Mohammad A. Rafi3 and Yadollah Omidi1,4
1
Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran
2
Department of Computer Science, Faculty of Mathematical Sciences, University of Tabriz, Tabriz, Iran
3
Department of Neurology, Sidney Kimmel College of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA
4
Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

Computational epitope-based vaccine design is the cornerstone of vaccine development. Owing to the
selection of proper compositions [antigens (Ags), epitopes, peptide linkers, and intramolecular
adjuvants], epitope-based vaccines are considered a cost- and time-effective approach resulting in the
development of vaccines with maximal therapeutic efficacy and minimal adverse reactions. In this
review, we provide insights into in silico epitope-based vaccine design and highlight vaccinology
procedures used for the development of the next-generation vaccines with high effectiveness.

Introduction immune responses by designing specific constructs; (ii) generation

Vaccines are one of the most applicable and cost-effective phar-
maceuticals and have been used for the prevention and/or treat-
ment of diseases for over two centuries. Classically, immunization
against infectious agents was shown to induce humoral and cellu-
lar immunity, resulting in a substantial decrease in mortality and
morbidity rates and improved human life expectancy [1]. Vacci-
nation modalities have been expanded against various diseases
thus far and vaccine development technologies have evolved over
the past few decades. Commonly used traditional methods may
take up to 5–15 years to develop vaccines, with huge associated
costs. A major challenge in the postgenomic era is the determina-
tion of antigenic regions or epitopes for the enhanced stimulation
of the immune response [2]. However, computational in silico
immunoinformatics can aid the vaccine development by rational
design in a time- and cost-effective manner. Despite remarkable
advances in recent decades, there is no generally accepted strategy
for the implementation of approaches for vaccine design. Of the
different methods, epitope-based vaccines have been developed to
deliver both prophylactic and therapeutic effects on pathogen-
specific immunity, with promising outcomes. This strategy offers
several advantages, including (i) elimination of undesirable
Corresponding author: Omidi, Y. (yomidi@tbzmed.ac.ir)
of prolonged immunity with required responses; and (iii) cost- and
time-effectiveness [3]. In 1985, Jacob et al. introduced the first
epitope-based vaccine against infectious diseases caused by Vibrio
cholerae and Escherichia coli [4]. The basic idea behind this strategy
was to recognize the immunodominant epitopes and induce
immune responses. An epitope is an immunogenic region of an
antigen (Ag) that can be recognized by a cognate paratope of an
antibody (Ab), known as B cell epitopes (BCEs), or T cell receptors,
known as T cell epitopes (TCEs), the interaction of which results in
humoral and/or cellular immune responses, respectively. There are
three key steps in rationalized epitope-based vaccine design: (i)
prediction of the immunogenic regions using in silico epitope
mapping; (ii) engineering of the immunogenic construct; and
(iii) evaluation of the vaccine efficiency. Such a step-by-step
approach through computational in silico tools is termed
‘immunoinformatics’.
The low immunogenicity of single-epitope peptides has led to
the idea of designing constructs with multiple epitopes. Vaccine
constructs with the several epitopic regions might improve both
the antigenicity and immunogenicity of the vaccines.
Revolutionized information technology and molecular biolo-
gy, together with growth in genomic data repositories, have
provided the basis for the designing of vaccines using

1359-6446/ã 2020 Elsevier Ltd. All rights reserved.

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Drug Discovery Today  Volume 25, Number 6  June 2020
computational and bioinformatics tools. These tools are used
for in silico mapping of the most appropriate immunogenic
components and for the construction of hypothetical proteins.
The designed vaccine can be then simulated and evaluated
before any experimental validation, enabling the research
and development (R&D) process to be efficiently performed,
and leading to the development of a vaccine with no or few
adverse effects [5]. The impact of these tools on vaccine design
has been highlighted from various viewpoints, including
reverse vaccinology (RV), epitope prediction, and structural
vaccinology. In this review, we provide insights into the pro-
cedures and tools used in epitope-based vaccine design process-
es. We also discuss the step-by-step design and development of
peptide vaccines with desired humoral and cellular immune
responses.
Immunogenicity and immunological responses
Generally, there are two groups of foreign substances that
stimulate immune system responses: (i) exogenous Ags (e.g.,
bacteria and viruses); and (ii) aberrant self-cells presenting
major histocompatibility complex (MHC) proteins, which
originate from either defective Ags or from the virus-infected
and/or tumor cells producing foreign proteins (so-called
‘endogenous Ags’) [6]. The immune system can raise humoral
and/or cellular responses. Humoral immunity triggers Ab-
mediated responses to distinguish Ags or pathogens circulat-
ing in the body. Cellular (or cell-mediated) immune responses
stimulate mainly T cells to respond to the cell presenting
aberrant MHC markers such as cells attacked by pathogens
or tumor cells [7].
(a)
(c)
FIGURE 1
The 3D structure and detail properties of four epitope-based vaccines designed using different vaccine candidate antigens (Ag). (a) Echinococcus granulosus
excretory-secretory Ag enolase. (b) Human melanoma-associated Ag (MAGE). (c) EG95 oncospherical Ag. (d) Secretory Ag Eg14-3-3.
REVIEWS
Figure 1 illustrates the structure of recently designed epitope-
based vaccines against different life-cycle stages of Echinococcus
granulosus and triple-negative breast cancer [8–10].
Reverse vaccinology
RV is a rationalized strategy for the design of vaccines based on the
genomic information and bioinformatics tools that have widely
been used for the identification and examination of different
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immunogenic entities (e.g., bacteria, viruses, cancer cells, para-
sites, or allergens) with the ability to induce immune responses
[11]. So far, research attention has focused on the implementation
of new technologies in vaccine design. In this regard, the integra-
tion of different domains (functional genomics, transcriptomics,
proteomics, vaccinomics, structural biology, immune profiling,
and mathematical simulation and modeling) appears to be vital
for designing effective vaccines [12].
In silico epitope vaccine design
The key notion behind epitope vaccines is to stimulate the im-
mune response through the identification of both BCEs and TCEs.
The efficiency of a subunit vaccine depends on the selection of
appropriate BCEs and TCEs, which are the most significant part of
a vaccine construct, to induce both humoral and cellular immune
responses. The fusion of these epitopes and adjuvants can be
performed using appropriate linkers [13]. Linkers have a critical
role in guaranteeing the functionality of an epitope vaccine.
Furthermore, the proper arrangement of these components also
is important, resulting in the efficient stimulation of the immune
response. Owing to the elimination of various potential adverse
effects (e.g., allergens, toxins, and other undesired domains),
(b)
(d)
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 REVIEWS Drug Discovery Today  Volume 25, Number 6  June 2020

epitope-based vaccines are considered safe. Nevertheless, in de-
signing epitope vaccines, the possible emergence of a cytokine
storm should be taken into account, as reported for other immu-
notherapies, such as monoclonal Abs and T cell therapies. Figure 2
represents rational steps for in silico epitope-based vaccine design.
Antigen(s) selection, discovery, and optimization
The accurate selection of Ags is a key step in designing vaccines,
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the emergence of a cytokine storm or immune function
tolerance.The combination of immunological and genomic in-
formation is often used to identify relevant protein Ags for
diagnostic or vaccine purposes. New Ags can be identified via
epitopes recognized by CD4+ T and/or CD8+ T cells, specially in
the case of emerged, emerging, and re-emerging pathogens
(Figs. 3C and D). In this context, VaxiJen 2.0 was the first RV
server developed for the alignment-independent prediction of

for which the emerging methodology of RV enables the scientifi-
cally rationalized selection of candidate Ags instead of the tradi-
tional process of Ag selection. As shown in Fig. 3, to single out an
Ag of choice, this process can be facilitated through computa-
tional analysis of a potential pathogen genome, high-throughput
screening, and data-profiling techniques [12]. Based on the path-
ogenicity mechanism of each infectious agent, one can target one
or more candidate Ags to trigger effective immune responses
against a designated pathogen [8]. The most challenging issue
is determining which structural properties might render an Ag
with potency to provoke functional immune responses without
protective Ags, and works based on the machine-learning tech-
niques [14]. Whole-protein antigenicity is predicted using the
models derived from bacterial, viral, or tumor protein data-sets,
in which Ag classification is carried out based on the physico-
chemical properties of the proteins. Jenner-Predict is a decision-
tree-based software developed for the prediction of protein
vaccine candidates from the proteomes of bacterial pathogens
[15]. VacSol is a recently developed RV software that works based
on the decision-tree learning algorithm [16], whereas Vacceed,
which is based on RV using machine-learning techniques, is a
high-throughput in silico vaccine candidate discovery pipeline for

(a) Conformational
and linear
BCEs, CD4+,
Epitope CD8+, TCEs Linker
mapping selection/designing

Pathogenicity Agonist
Immunogenicity targeting
Localization throughTLRs,
Allergenicity Computational vaccinology NLRs, RLRs,
Autoimmunity and CLRs
Epitope vaccines

Adjuvant
Antigen(s)
selection/designing
selection

Final vaccine
construct

(b) Formulation (c)

(recombinant, mRNA
Production and DNA vaccines) Production
Animal testing Animal testing
Cloning In vitro and in vivo assessments
Validation
Evaluation Pilot scale production Evaluation
Immunogenecity Clinical trials Immunogenecity
Purification Approval Expression

Marketing
Ag selection Post-marketing evaluation Ag prediction

Microrganism Analysis of genome

cultivation sequences

Traditional vaccinology Reverse vaccinology

Drug Discovery Today

FIGURE 2
Steps for in silico epitope-based vaccine design and validations. The rational design of the vaccine includes the proper construction (or selection) of antigens
(Ags), pattern recognition receptors (PRRs), B cell epitopes (BCEs), T cell epitopes (TCEs), and linkers. Engineered vaccine constructs undergo several validation
processes. (a) In silico epitope vaccine design and development. (b) Traditional approach for vaccine development. (c) Reverse vaccinology approach.
Abbreviations: CLRs, C-type lectin receptors; NLRs, NOD-like receptors; RLRs, ROG-like receptors; TLRs, Toll-like receptors.

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Drug Discovery Today  Volume 25, Number 6  June 2020 REVIEWS

(a)

Cluster
Cluster

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Essential nodes
[Hub proteins]

PPI Network
(b) (c)

Epithope mapping

(d)
MHC allele

T cell epitope
Drug Discovery Today

FIGURE 3
Computational vaccinology has facilitated the process of the identification of antigens (Ags) for vaccine construction. (a) The molecular interaction network (protein–
protein interactions; PPIs), reflecting so-called ‘interactomics’. The PPI network offers crucial holistic results about potential protective Ags of various pathogens. (b)
Analysis of high-throughput (HT) genome/proteome data. HT data not only provide case-specific insights into genome variation but also help to determine aspects of
host–pathogen interactions. (c) The 3D epitope mapping based on artificial intelligence using machine-learning techniques. Such an approach is applied for
determining whether a peptide can be bound to the cleft of major histocompatibility (MHC) molecules. (d) The development of a personalized precision vaccine.

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 REVIEWS
eukaryotic pathogens [17]. AntigenDB is a repository for all
experimentally-determined Ags derived from pathogenic
organisms.
Different aspects of vaccinology should be simulated through
predictive modeling, which is effective for rationalizing the pro-
cedure of vaccine design. By the same token, from systems biology-
based vaccinology emerged the field of vaccinomics, profiling
vaccine immunogenicity in a personalized manner (Fig. 3D). Sys-
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tems vaccinology can pave the way towards the identification of
candidate Ag(s) from numerous pathogenic genomic and/or prote-
omic data [18]. The key limiting step in traditional vaccinology, for
example in the Ag discovery step, is the determination of the
number of Ags to be tested in experimental assays. However, the
application of high-throughput omics data analysis and machine-
learning tools in computational vaccinology enables the scanning
of functional modules in pathogens and critical biological pro-
cesses involved in pathogenesis, resulting in the discovery of
essential protective Ag(s) as well as potential neoantigens (or
neoepitopes) for the development of optimal vaccine candidates
(Fig. 3C).
B and T cell epitope prediction
Traditionally, the epitope identification is carried out through
complex procedures, including Q-TOF and tandem mass spectros-
copy, hydrogen-deuterium exchange, enzyme-linked immunosor-
bent spot (ELISPOT) assay, monoclonal Ab-based identification,
X-ray co-crystallography, and phage peptide libraries. All these
methods are costly, although an alternative way to identify epi-
tope(s) is the use of in silico prediction tools that provide a time-
and cost-effective solution to find epitopes. An important research
field in immunoinformatics is the development of algorithms for
analyzing potential BCEs and TCEs. Additionally, a major goal in
epitope prediction is to analyze the binding affinity of antigenic
peptides to MHC molecules. The most fruitful proposed method
for the prediction of in silico TCEs is based on a data-driven
approach, in which the peptide-binding property of particular
class I or class II MHC alleles is predicted. A variety of tools has
been proposed for the prediction of epitopes, including (i) homol-
ogy modeling; (ii) protein threading; (iii) molecular docking tech-
niques; and (iv) identification of structural binding motifs.
Alternatively, the role of interactions between Ags and Abs in
the human immune system can be studied by analyzing the whole
Ag sequence.
Prediction of linear and conformational B cell epitopes
The activation of memory B cells is based on the recognition of
antigenic determinants (i.e., BCEs) in antigenic proteins. These
antigenic epitopes have a key, yet challenging, role in the devel-
opment of vaccines against extracellular pathogens. The main
challenging topic in this concept refers to the nature and type
of BCEs, which is very important for in silico BCEs mapping. B cell
lymphocytes have high affinity to two types of BCE: continuous
(linear) and discontinuous (conformational) epitopes. Of these,
90% of BCEs are conformational BCEs (CBCEs) but there is no
precise understanding of the length of Ag peptide residues that
structurally contribute to the binding to the paratope site of Abs.
This issue has led to some less reliable (e.g., false negative and false
positive) results in the prediction of CBCEs. X-ray crystallography
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Drug Discovery Today  Volume 25, Number 6  June 2020
and NMR data have shown that Ab–Ag complexes can be formed
with a variable length of residues, in which 15–22 structural
amino acid (aa) residues have a high affinity to 17-aa-long
residues in paratope sites [19]. In this study, it was shown that
short residues (7–12 aa) of BCEs are unable to form an epitope–
paratope complex. In short, to improve the accuracy of in silico
mapping of immunodominant BCEs, several main steps should be
considered, including (i) the elimination of short-length epitopes
[19]; (ii) the analysis of the 3D structure of candidate Ags based on
the structural properties (e.g., hydrophilicity, flexibility, and sur-
face exposure, and solvent accessibility) [20]; (iii) the use of multi-
method BCE prediction procedures based on the results of several
predictors [10]; and (iv) the application of the results of web-based
BCE prediction tools to molecular interaction analysis methods
such as molecular docking and molecular dynamics (MD) simula-
tions. Discontinuous epitope prediction tools have been devel-
oped based on the analysis of the 3D structure of protein Ags.
Among the tools for discontinuous epitope prediction, DiscoTope
2.0 explicitly predicts discontinuous epitopes by combining the
surface accessibility and spatial and aa statistics to differentiate
between epitope and nonepitope sites [21]. PEPOP is another web-
server developed for the prediction of CBCEs, and uses the 3D
coordinates of a protein for clustering the exposed sites of the Ag
that might be related to conformational epitopes. Its improved
version (PEPOP 2.0) is newly developed and showed potential for
designing structural peptides to be detectable via the Abs targeted
for cognate Ags [22]. SEPPA, as a CBCE predictor, looks for the local
spatial context in the protein Ag surface and 3D characteristics of
epitopes using a novel concept of a unit patch of residue triangle
and spatial clustering coefficients [23]. The server develops and
integrates the ratio of glycosylation triangles and related aa index-
es for glycoprotein Ags [24]. Bpredictor is an accurate random
forest-based method that identifies CBCEs based on their 3D
structures [25].
The techniques that predict continuous epitopes use different
physicochemical characteristics of each aa over a small data set.
Although BCEs are mostly discontinuous (90%), the identifica-
tion of continuous epitopes appears to be more challenging than
for other types of epitope. This difficulty might originate from the
variable length of continuous BCEs compared with the almost
fixed-length core of TCEs.
ABCPred, as the first server for the prediction of continuous
BCEs, has two major limitations: the small size of its data set and
the use of random peptides as non-BCEs [26]. BCPRED is another
server that has been developed based on the support vector ma-
chine (SVM) approach [27]. SVMTriP predicts linear antigenic
epitopes based on the SVM technique, combining the tripeptide
similarity and propensity scores [28]. LBtope was the first server to
use experimentally verified BCEs and non-BCEs from the IEDB
database [29], whereas CBtope is an SVM-based predictor using a
combination of traditional features of physicochemical profiles
and sequence-derived inputs, including the composition and
colocation of aas [30].
T cell epitope prediction
The prediction of TCEs has been a challenging research area over
the past three decades. Direct methods have primarily been
designed based on the sequential and structural analysis of TCEs,
Drug Discovery Today  Volume 25, Number 6  June 2020 REVIEWS

whereas indirect methods are mainly based on the prediction of
MHC binders. The high accuracy and specificity of the indirect
methods have resulted in researchers developing and applying
these methods instead of TCE prediction tools. The methods are
generally developed in two groups: MHC class I binders (MHC-I,
so-called ‘CD8+ TCEs’ for endogenous antigen processing), and
MHC class II binders (MHC-II, so-called ‘CD4+ helper TCEs’ for
exogenous Ag processing) [2].
scheme based on the representation of TAP peptide fragments and
composition effects [35].
Several methods and tools have been developed to predict
MHC-I peptide binders. Of these tools, NetMHCcons 1.1 is a server
for the projection of any MHC-I binding peptides [36]. The server
integrates three state-of-the-art predictors (NetMHC, NetMHC-
pan, and PickPocket) to find the most accurate predictions.
NetMHC 4.0 is an allele-specific artificial neural network (ANN)-

Through endogenous Ag processing, Ags are recognized and
cleaved by the proteasome into smaller peptide fragments. These
antigenic peptides are transported to the endoplasmic reticulum
by the transporter associated with antigen-processing (TAP) pro-
tein complex (a heterodimer of TAP1 and 2), and are then pre-
sented by MHC class I molecules to cytotoxic T lymphocytes
(CTLs) [31]. In this regard, PCPS [32] and Netchop 3.1 [33] have
been developed for the prediction of proteasomal cleavage sites.
TAPPred has been established to predict the binding affinity of
peptides towards TAP, the binding prediction of which is impor-
tant for the identification of MHC-I constrained TCEs [34].
TAPhunter predicts TAP-binding peptides using a novel encoding
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based method that has been trained using 94 MHC-I alleles.
NetMHCpan 4.0 is another ANN-based pan-specific method that
is trained utilizing >115 000 quantitative binding data. PickPocket
1.1 is a matrix-based method that relies on the receptor–pocket
similarities among MHC molecules trained on 94 different MHC
alleles. Another tool is NetCTLpan 1.1, which is used to predict
CTL epitopes in protein sequences through the integration of
peptide MHC-I-binding prediction, proteasomal C terminal cleav-
age, and TAP transport efficiency [37]. The method uses a quanti-
tative matrix (QM)-based method on 47 MHC-I alleles with at least
15 binders. The nHLAPred system provides accurate prediction by
applying both the ANN- and QM-based methods.

Exogenous antigen Cd4+ T-cell

Cd8+ T-cell
TCR TCR
CTLPred
Antigen IFNepitope
NetCTLpan
Antigen IL4pred
MHC class I MHC class II

Propred1 Propred
Endosome MHC2pred
NetMHCcons
RANKPEP HLA-DR4Pred
Lysosome MULTIPRED2

Pcleavage Proteasome
NetChop
Antigenic peptides
G
ol

TAPreg MHC class II

gi
ap

TAPPred TAP
pa

TAPhunter
ra
tu
s

MHC class I

Endogenous Exogenous
antigen processing antigen processing

Drug Discovery Today

FIGURE 4
Pathways for the antigen (Ag) processing and presentation by a dendritic cell (DC). Ag processing is carried out in DCs through major histocompatibility (MHC)-I-
binding peptides by proteasomes or MHC-II-binding peptides by the vesicular machinery of endosomes. As a member of the ATP-binding cassette transporter family,
transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum (ER), where they are passed to the
respective MHC-I molecules based on their length and sequence. Once peptides are loaded on the MHC-I molecules, the cascade continues through activation of
trimeric MHC-I–b2-microglobulin–peptide complex and transportation onto the cell surface to be presented to CD8+ T cells through T cell receptors (TCRs).

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MHC-binding peptides recognized by CD8+T cells are presented
to the cell surface. Several direct methods have been developed to
predict the possibility of recognizing MHC-bound peptides by
CD8 + T cells such as CTLpred [38]. As shown in Fig. 4, in exoge-
nous antigen processing, antigenic regions over the MHC-II com-
plex are recognized by CD4+ T cells, known as helper T cells.
The prediction of MHC-II binders is more complicated com-
pared with that of MHC-I binders. This is because of different
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forms of structural grooves in these two classes: MHC-I contains a
closed form of the groove, whereas this is open in MHC-II [39].
Several tools have been developed to predict MHC-II binders, most
of which exploit machine-learning techniques, such as ANNs,
SVMs, and hidden Markov models (HMMs). ProPred predicts
MHC-II-binding regions using a quantitative matrix [40]. The
server simplifies the way of selecting vaccine candidates by locat-
ing promiscuous binding regions. The EpiDOCK tool uses homol-
ogy modeling and molecular docking approaches to predict the
binding affinity of peptides to MHC-II molecules. This webserver
was established using a structure-based algorithm that works by
using docking score-based quantitative matrices (DS-QMs) [41].
NetMHCIIpan 3.1 is an updated version of a quantitative method
[42] that can predict the binding of peptides to MHC-II molecules
in humans and mice. All the aforementioned T-helper epitope-
predicting methods use an indirect scheme to predict MHC-II
binder. Recently, direct methods have also been proposed for
the prediction of T-helper epitopes. Of these methods, IFNepitope
is utilized to predict and design interferon-g (IFN-g)-inducing
peptides, MHC-II binders, or TCEs [43]. Similarly, PREDIVAC
has been established to forecast CD4+TCEs, with substantial
improvements over previous methods [44].
Adjuvant selection
Maximizing the efficiency of a designed vaccine is directly depen-
dent on the discovery, design, and optimization of effectual
adjuvants. Adjuvants are generally defined as molecular com-
plexes that amplify an immune response when managed within
a vaccine construct. Peptide-based vaccines can intrinsically result
in poor immunogenicity even with optimal BCEs and TCEs. The
strength and duration of the immune response to the vaccine are
also vital. Thus, Ags are enhanced by the selection of a suitable
adjuvant, inclusion of which might enhance the immune re-
sponse. Furthermore, the addition of adjuvants into epitope-based
vaccines provides several advantages, including (i) indirectely
extension of the vaccine long-term memory; (ii) expansion of
the Ab repertoire; (iii) refreshing the stunned immune system in
older patients; and (iv) opportunity for dose-sparing [45].
Linker selection
Linkers, or so-called ‘spacers’, are key entities in designing and
engineering protein vaccines. They play an important role regard-
ing the structural stability, interdomain interactions, and func-
tionality of the vaccines [46]. Generally, three major components
of a vaccine construct are linked by spacers: BCEs, TCEs, and
intramolecular adjuvants. The fusion of functional domains with-
out/with an inappropriate linker can yield undesirable effects,
such as a decreased quantity in production, misfolded protein
structures, and/or weakened bioactivity [47]. Various properties of
a linker are considered for their selection, including length, aa
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Drug Discovery Today  Volume 25, Number 6  June 2020
arrangement, hydrophobicity, secondary structure, sensitivity to
proteases, and potential interaction with other components of the
immunogenic construct. SynLinker is the only up-to-date web-
server that facilitates the selection of suitable linker(s) and model-
ing of a fusion protein [48]. The server contains a repository of
2150 naturally occurring peptide linkers plus 110 artificial or
empirical linkers. The server suggests potential linker candidates
based on user-specified criteria, including linker length, aa com-
position, solvent accessibility, and other features that might affect
linker flexibility and function.
Analysis of different aspects of the fusion vaccine
construct
The integration of both BCEs and TCEs can be accomplished
through appropriate aa spacers. The created vaccine sequence is
submitted to a protein structure prediction tool to construct its 3D
structure model. The method searches structural similarity for the
sequence of predicted domains within the Protein Data Bank
(PDB) and produces a list of hits based on four criteria: E-value,
alignment length, identity, and total score. The best hit is selected
as a template structure to build a 3D model using different model-
building tools, including (i) comparative modeling based on the
known homologous proteins with >70% sequence identity; (ii)
threading technique based on construction of a model using a
template from PDB (with sequence identity of 30–70%); and (iii) ab
initio folding based on creation of a novel fold (with <30%
sequence identity). Among a variety of protein structure predic-
tion tools, EasyModeller 4.0 [49], SWISS-MODEL [50], Rosetta [51],
and phyre2 [52] are the most commonly used online servers for the
homology modeling. Furthermore, I-TASSER [53], phyre2, and
RaptorX [54] are used for the threading and/or fold recognition,
whereas I-TASSER and Rosetta are also used for the prediction of ab
initio-based protein structures.
The constructed 3D model is refined by one of two different
strategies: potential energy minimization (PEM) and MD simula-
tions. The PEM strategy is used to tumble down the energy level
towards the closest local minimum that might positively be closer
to the natural form of the molecule. The MD simulation strategy is
computationally more demanding than PEM because it is a robust
method used to study the physical movements of atoms and
molecules and to overcome obstacles in the landscape [55]. Addi-
tionally, structure validation is an important step in homology
modeling, whereas crude models should be evaluated to confirm
the quality of the constructed models. To assess the structural
quality of a constructed vaccine, the predicted 3D structure can be
submitted for assessment by various structure validation tools.
Success paradigms in the development of epitope
vaccines against cancer
Recently, many peptides have been patented and used for the
development of vaccines based on the strategies discussed in this
study. For instance, Itoh and Yamada constructed a vaccine
(US20130164314) comprising 6–13 peptides derived from tumor
Ags, which generated a broad immune response against tumors
[56]. In another patent (US20130122029), a vaccine comprising
two peptides forming a snail protein was developed by Kudo and
Kawami and was shown to provoke remarkable immune responses
against tumors [57]. Tsunoda and Osawa patented a Foxp3
Drug Discovery Today  Volume 25, Number 6  June 2020
peptide-related construct (US8563516), which resulted in the
significant effectiveness of a cancer vaccine for the prevention
and treatment of tumors [58]. In 2015, Nezafat et al. utilized a set of
bioinformatics tools to design an efficient multiepitope peptide
vaccine against lung cancer [46]. They subsequently evaluated the
antitumor efficacy of the vaccine through in vivo preventative and
therapeutic assays. They concluded that their developed epitope-
based vaccine could efficiently result in both preventive and
therapeutic antitumor immunity in TC-1 tumor-bearing mice.
Furthermore, various clinical trials have been reported to validate
the therapeutic profile of the peptide vaccines in patients with
cancer. These efforts have comprehensively been reviewed by Pol
et al. (>60 trials before 2015) and Bezu et al. (>20 trials during
2015–2018) [59,60]. These trials were mostly phase I and II to test
their safety and immunogenicity, which revealed that the peptide-
based vaccines were mainly well tolerated without any severe
adverse effects.
Concluding remarks
Much attention has recently been paid to computational methods
used for the epitope mapping and rational design of immunogenic
and safe vaccines. Now, a variety of databases, web-servers, and
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2 Dhanda, S.K. et al. (2017) Novel in silico tools for designing peptide-based subunit
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standalone softwares are available for the computation and auto-
mation of different stages of vaccine design and validation. Accord-
ing to several reports, the epitope-based vaccine design using in silico
computational methods is an effective strategy that can result in the
development of vaccines with the ability to induce the required
immunogenicity without the emergence of a cytokine storm or
immune tolerance. Based on several in vitro and in vivo studies, if
designed scientifically and critically, the epitope-based vaccines
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offer several advantages over other types of vaccines, including fast
and accurate design, time-/cost-effective formulations, and desired
immunogenicity with minimized adverse effects.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgments
The authors like to acknowledge the Research Center for
Pharmaceutical Nanotechnology (RCPN), Tabriz University of
Medical Sciences (TUOMS) for supporting this study and Prof.
Jaleh Barar for critical reading.
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