Vaccine Reports 6 (2016) 1322

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Vaccine Reports
journal homepage: www.elsevier.com/locate/vacrep

Review Article

T-cell epitope mapping for the design of powerful vaccines

Tarek A. Ahmad a,b,, Amrou E. Eweida c, Laila H. El-Sayed b,d
a
Special Projects Department, Bibliotheca Alexandrina, Alexandria, Egypt
b
SeptivaK Research Group, Immunology and Allergy Department, Medical Research Institute, Alexandria University, Alexandria, Egypt
c
Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt
d
Immunology and Allergy Department, Medical Research Institute, Alexandria University, Alexandria, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Epitope mapping has emerged as a powerful tool to design vaccines. It proved itself a number of times to
Received 5 April 2016 reveal the building blocks of potent immuno-prophylactics. T-cell epitope mapping aims to identify the
Revised 7 July 2016 shortest amino acid sequence of an epitope of a specific antigen that is recognized by CD4+ and/or CD8+
Accepted 13 July 2016
T-cells. T-cell epitopes have the potential to stimulate both long lasting and exclusive cytotoxic immune
response. Therefore, they are crucial for vaccine development against emerging intracellular pathogens
such as Plasmodium malaria, Mycobacterium spp., genital Chlamydia, HIV, HCV as well as cancer cells.
Keywords:
This review documents and discusses the different methods used in T-cell epitope mapping, and the role
Epitope mapping
Vaccines
of each method to identify the potent epitopes in viruses, bacteria, parasites, as well as human diseases.
Cellular response The comment of the article guides the researchers to identify the most suitable mapping techniques for
Molecular immunology their antigens.
T-cells  2016 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2. Methods of T-cell epitope mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.1. MHC Multimers (MM). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2. Solid phase MHC-peptide complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3. Lymphoproliferation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.4. Intracytoplasmic cytokine staining (ICS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.5. Cell ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.6. ELISPOT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.7. Activation markers induced on specific T-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.8. Cell free system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.9. High throughput T-epitope mapping (HTP). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.10. Pepscan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3. Comment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1. Introduction usually initiate two major immune responses. The B-cells lympho-
cytes engulf the foreign body and differentiated into plasma cells,
When a foreign immunogen is exposed to the immune system, that secrete immunoglobulin antibodies. For this reason this acti-
it is first encountered by the antigen presenting cells (APCs) that vation is referred as B-cell humoral response. Antibodies recognize
foreign molecules called antigens or immunogens and bind to a
Corresponding author at: 34 Memphis St., 21525 Alexandria, Egypt. specific site on the antigen surface called the epitope or the
E-mail addresses: Tarekadnan@yahoo.com, tarek.adnan.ahmad@gmail.com antigenic determinant [1,2]. Unlike B-cells, T-cells do not directly
(T.A. Ahmad). recognize free floating antigens. However, antigens should be first

http://dx.doi.org/10.1016/j.vacrep.2016.07.002
2405-7843/ 2016 Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
14 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322
up-taken by an APC such as B-cells or dendritic cells or macro-
phages, and presented to the T-cells by the major histocompatibil-
ity complex (MHC). Then the T-cells receptor (TCR) recognizes the
fragments exposed by the APCs and initiates the T-cell response
[3,4].
The T-cell-mediated immune response depends on four differ-
ent types of T-cells lymphocytes. The cytotoxic T- lymphocytes
(CTL), or killer T-cells play a unique role to destroy cells displaying
foreign motifs on their surfaces. Whereas helper T-cells (HTC), con-
tribute to both the humoral and the cellular immune responses by
stimulating the proliferation and differentiation of B-cells and
killer T-cells. In addition, regulatory T-cells function in maintaining
tolerance toward self-antigens, playing an important role in
autoimmune diseases. While, memory T-cells are responsible for
maintaining a relatively long lasting immunological memory
towards the same antigen [1,2,5].
The CTL expresses a cluster of differentiation (CD) receptor gly-
coprotein termed CD8+ on its surface, which recognizes the MHC
class I-peptide complex displayed by the APCs. Association of
MHC class I-peptide complex with the cytotoxic TCR, and other
accessory proteins, aids in activating a series of reactions that
result in apoptosis of the infected cell by forcing the cell to suicide
[4,6,7]. On the other hand, the HTC expresses a specific
glycoprotein on its surface termed CD4+ that has the ability to
recognize class II MHC-peptide complex. Association of class II
MHC-peptide complex with the helper TCR gives signals that a
pathogen is invading the cell. The initiation, differentiation and
regulation of those responses trigger the secretion of small cell-
signal molecules referred to as cytokines [3,8]. Cytokines (CK) are
functionally classified into a T-cell helper 1 (Th1) group that
enhances the cellular immune response such as interferon-c
(IFN-c) and interleukins (IL-12 and IL-18), and a Th2 group that
enhances the humoral immune response such IL-2, IL-4 and IL-10
[9]. Those can be measured to monitor the cellular immune
response [10].
The design of vaccines and diagnostics is a recent art that
attracted the attention of scientists worldwide [11]. Epitope
mapping is a new branch of immunology which focuses on the
selection of the most potent epitopes that could serve as potential
targets for the production of epitope-based immune-preparations.
Several occasions highlighted the advantages of following the
route to design vaccines and diagnostics [1215]. T-cell epitope
mapping is a term that refers to the process of identifying
the shortest amino acid sequence of the epitope of a specific anti-
gen that is recognized by CD4+ and/or CD8+ T-cell receptors, and at
the same time has the potential to stimulate a long lasting and a
cytotoxic immune response [16,17]. Therefore, T-cell epitope
mapping is critical for constructing epitope-based vaccines as well
as potent therapeutics for intracellular pathogens and cancer cells
[9,1719]. Unlike the majority of B-cell epitopes, T-cell epitopes
are linear protein molecules that consist of 12 to 20 amino acids.
This fact facilitates their cloning and their synthesis as peptide
vaccines [16]. Some methods may be used for both T- and B-cell
epitope mapping such as pepscan, in silico, ELISA and ELISPOT that
corresponds to the microarray in B-cell epitoping [14]. Simultane-
ously, new techniques such as mRNA [20] and random phage
display library [21] are under investigation.
The selection of a target epitope that has the ability to induce a
protective B-cell dependent immune response is crucial for vaccine
development [5,7,18], however this will not always lead to the
establishment of long lasting protective memory inside the host
[22]. Moreover, in the case of intracellular pathogens and tumors
the cellular arm of the immune response managed by T-cell
activity, is highly involved in the process of fighting the foreign
bodies as well as inducing a cytotoxic activity [4,23]. The genera-
tion of memory T-cells enables the rapid recovery of the host
and the prevention of re-infection by the same pathogen [23]. In
addition, it was shown that the integration of the action of cell-
mediated immunity effectors such as IFN-c, Natural Killer (NK)
cells, as well as Th1 helper T-cells are required in the control of
the pathogenesis of intracellular microbes such as Rickettsia [24],
Salmonella [25], Listeria [26], Mycobacteria [27], and Chlamydia
[28,29], as well as allergens [16]. On the other hand, CD8+ T-cells
are highly involved in providing immune protection against
respiratory viruses [30], Vaccinia virus [31], Plasmodium spp.
[3235], Toxoplasma gondii [36], and intracellular bacteria such as
Mycobacterium tuberculosis and Listeria monocytogenes [7,37,38].
Moreover it was noted that, B-cell-deficient mice were able to
sustain complete recovery from genital tract infection with
Chlamydia trachomatis [39], which ensures the essential role of
T-cells in the recovery from some diseases.
Although the development of a T-cell protection is the corner-
stone for constructing potent vaccines [5,10,18], more recent
studies showed that cellular response is not sufficient for the
complete irradiation of intracellular pathogens such as Chlamydia
and M. tuberculosis. A comprehensive immunological study showed
that long lasting persistence and increased susceptibility of
Chlamydia and Mycobacteria infections appeared in the B-cell
deficient models [38,39]. Therefore, it was found that antibodies,
as well, play an indirect role in T-cell activation, thus resulting in
early and effective clearance of the pathogen in the process of
secondary infection. In addition in small pox and Ebola virus [40]
it was found that although the elucidation of potent cellular
immune response is required for the rapid recovery of infected
individuals, the activation of the humoral response is essential
for protection [41,42]. Therefore, the novel approach in the design
of vaccines is to implement strategies capable of activating both
arms of the immune system, the cellular as well as the humoral
immune responses [13]. The different techniques followed to
map the T-cell epitope are described hereafter, in order to integrate
them to the mapping techniques of the B-cells [14].
2. Methods of T-cell epitope mapping
Several techniques depending on in silico and binding methods
developed to point the epitopes that activate the cellular response.
The following paragraphs discuss those methods and document
the majority of the practical trials that involved their use. The
T-cell epitope mapping techniques were the corner-stone to reveal
the potent antigenic determinants of many intracellular bacteria,
viruses, cancer, autoimmune, allergen and parasites antigens.
2.1. MHC Multimers (MM)
This method depends on synthetic MHC molecules that bind to
the tested peptide and propose it to the TCRs. Hence it isolates and
identifies the bound T-lymphocytes, in case the tested peptide is an
appropriate epitope [43]. The most widely spread multimers, are
MHC tetramers composed of four MHC molecules covalently
attached to biotin [44]. The first approach that used this technique
allowed the peptide antigen to attach to the tetramer-complex.
Further coupling was detected by a suitable fluorescent tag that
has a high affinity to biotin such as streptavidin, then the emitted
fluorescence was measured [45]. Although this method is simple
and recoverable as the cells can be reused for flow cytometry stud-
ies, it has some drawbacks. Since, the antigens had to be known in
advance using some other methods of epitope mapping, and the
prior knowledge of MHC-peptide interactions is needed in order
to design suitable tetramer components [46]. This method devel-
oped to apply a reversible binding between the peptide and the
MM. In this later approach, a peptide that has a UV cleavage site
 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322
is left to bind to the MM. Once exposed to the UV light, it dissoci-
ates from the MM allowing other peptides with the same binding
capacity, and with different binding specificity to bind the MM.
This allows the construction of many MM for T-cell epitope map-
ping [47]. It was first thought that this technique is only limited
to few MHC class I alleles due to the limited number of MHC class
II molecules created so far. Therefore, it was used for CTL epitopes
detection and not the HTCs [46]. However, more recent studies
addressed the use of this approach for MHC class II tetramers as
an important method of T-cell epitope mapping [45].
MM proved several times to be a successful tool for epitope
mapping when combined with other mapping techniques. The
approach was combined with pepscan and the lymphoproliferation
assay to map the epitopes of M. tuberculosis [48], or with a in silico-
based method and flow cytometry to identify the potent epitopes
of Chlamydia trachomatis [49]. It was simultaneously combined
with pepscan, the flow cytometry, and cell ELISA to highlight the
epitopes of Mycobacterium leprae [50] or with pepscan and an
algorithm-based method to screen the epitopes of HIV-1 [51]. It
was applied with pepscan and flow cytometry to highlight the
epitopes of Herpes Simplex Virus (HSV-2) [52], or with ELISPOT
to identify the epitopes of small pox virus [53]. Moreover, it was
used with pepscan and IFN-c ELISPOT to investigate the epitopes
of hexon, an adenoviral capsid protein antigen [54], or with the
lymphoproliferation approach and the flow cytometry to investi-
gate the ability of carcino-embryonic antigen (CEA) to elucidate
potent T-cell response in cancer patients [55].
2.2. Solid phase MHC-peptide complexes
The solid phase MHC-peptides complexes can be created by
spotting the MHC class I molecules on a suitable microarray slide,
then allowing it to bind to the peptides that can be recognized by
the T-lymphocytes [56]. However, a novel approach developed by
applying an anti-cytokine antibody to the microarray so that the
cytokine secreted by the target T-cell can be captured as it appears
as a spot and this later can be identified using immune-fluorescent
techniques [57]. The technique allows the screening of large num-
ber of T-cell epitopes. Although the technique was used to reveal
the activation of human cluster of differentiation types CD8+ and
CD4+ T-cells corresponding to Vaccinia, Epstein-Barr viruses
(EBV), Influenza, and HIV-1 [57], it is not widely practiced due to
the complexity of the preparations and the instrumentation [16].
2.3. Lymphoproliferation assay
Lymphocyte-proliferation assay or the so-called 3H-thymidine
uptake assay is used to determine the lymphocytes activation,
and hence gives an overall idea on the cell-mediated immune
response. In this approach, the proliferation of the T-lymphocytes
can be determined by the fact that cells undergoing activation
increase their rate of DNA and protein synthesis [16]. The specimen
of activated purified peripheral blood mononuclear cells (PBMCs)
or T-cells is mixed with the antigen gradient. The 3H-thymidine
is added after 72 h to 120 h and DNA synthesis is quantified using
a gamma counter to calculate the amount of radio-labeled thymi-
dine integrated into the DNA [16,58]. Although the technique was
widely practiced due to its sensitivity, it has some drawbacks such
as the limited number of well plates (only 96), the use of a radioac-
tive label, the lack of data on the frequency and secreted cytokines
obtained, and long culture days (35 days). In addition, unlike the
MM technique, cells cannot be reused for future work. However,
new procedures appeared to reduce the time of the experiment
and to depend on safer cheap colorimetric detections [59].
Some mapping protocols followed the lymphoproliferation
assays to map T-cell epitopes. The approach proved to be successful
15
in identifying the epitopes of the flagella filament protein FliC of
Salmonella typhimurium [60], and the Smith antigen commonly
detected in Systemic Lupus Erythematosus (SLE) patients [61]
when used alone. It proved to be successful as well when combined
with pepscan and in silico methods in mapping the epitopes of
Babesia bovis parasite [62], and the epitopes of CEA in mice [63].
Together with pepscan it highlighted several epitopes of
M. tuberculosis [64,65], two MHC class II epitopes of prostate cancer
[66], several epitopes of Coxsackie virus B4 structural proteins [67],
several CD4+ T-cell epitopes of ORF2 and ORF3 proteins of human
Hepatitis E virus [68], and to investigate the potency of a peptide
cancer vaccine [69]. Moreover, with IFN-c ELISPOT assay it was
able to identify a novel epitope of the flagella filament protein FliC
of S. typhimurium [70] or with flow cytometry to highlight a
potential cancer antigen [71]. In combination with cell-ELISA it
identified a promising renal cell carcinoma-associated antigen
[72], while with pepscan, flow cytometry, and TEPITOPE software
it highlighted several CD4+ T-cell epitopes of MAGE-3, a potential
tumor antigen [73]. The lymphoproliferation assay data were
consolidated with pepscan and flow cytometry to identify eleven
potent T-cell epitopes of HIV-1 [74] or with in silico approach to
identify novel epitopes of peanut allergy [75]. Moreover, as
discussed before the lymphoproliferation assay could be combined
with the MM approach [48], or the MM approach and the flow
cytometry method [55].
2.4. Intracytoplasmic cytokine staining (ICS)
In the late 1990, ICS or the so-called flow cytometry technique
developed [76] on the principle that stimulated T-lymphocytes
could be re-activated in the presence of a suitable antigen that is
bound to MHC molecule in the presence of large number of APCs.
Once T-lymphocytes are re-activated, specific cytokines should
be released. A specific protein inhibitor is added to allow retaining
of cytokines within their releasing T-lymphocytes. Later on cells
are permeabilized using a suitable detergent, stained with fluores-
cent antibodies, and the emitted fluorescence is detected [58,77].
The technique has the ability to identify and to quantify different
cytokines using monoclonal antibodies labeled with different col-
ors in a single cell, and the ability to test different phenotypic
markers on a specific T-cell. However, it has some drawbacks such
as the professional expertise and high cost to use the device and its
reagents [18,58].
The ICS proved to be successful in the mapping of the epitopes
of HER-2/neu, a potential tumor antigen [78] when used
exclusively. However, the technique was implemented in many
occasions in combination with other mapping methods. When
combined with pepscan, the approach managed to identify the
epitopes of human cytomegalovirus (HCMV) [79], Nef protein of
HIV [80], human herpes virus 8 that cause Kaposis sarcoma [81],
P53 cancer antigen [82], parathyroid hormone-related protein
(PTH-rP), a potent cancer antigen [83], and M. tuberculosis
[8486]. Furthermore, the ICS was implemented with the IFN-c
ELISPOT to identify the epitopes of Nef protein of HIV [87], the
matrix protein of respiratory syncytial virus (RSV) [88], Salmonella
enterica serovars [89], and dengue virus-2 E domain III [90]. Also
the technique was combined with cell-ELISA to detect the epitopes
of Influenza A/California/7/2009 (pH1N1) [91], and Ebola viruses
(EBOVs) glycoproteins [40].
It was combined with pepscan and the IFN-c ELISPOT assay to
highlight the epitopes of P53 [92], or with IFN-c ELISPOT assay
and in silico-based method to investigate the epitopes of mice hep-
aranase, a universal tumor associated antigen [93] and of HIV-1
[94], or with ELISA and in silico-based method to screen the
epitopes of the avian Influenza virus [95]. In addition to that, the
approach was implemented with pepscan, and in silico-based
16 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322
approach to identify the epitopes of prostate specific antigen (PSA)
[96], and M. tuberculosis [97,98]. Additionally, it was combined
with pepscan, the IFN-c ELISPOT and in silico-based approach to
identify the epitopes of the HIV Nef protein [99], the Human
Papilloma virus type 16 (HPV-16) E5 protein [100], and the cancer
testis antigen HOM-TES-14/SCP1 [101].
As mentioned before, the flow cytometry approach was com-
bined with the MM technique [52], the MM approach and a
bioinformatics-based method [49], the MM approach and cell
ELISA [50], the lymphoproliferation assay [71,73,74]. The flow
cytometry was integrated with binding methods of B-cell epitope
mapping to map EBV [102].
2.5. Cell ELISA
In ELISA the 96 or 384 or 1536 [58,103,104] wells are coated
with the first primary anti-cytokine antibody (captured on the
solid wells), and then the tested antigens and the cell medium
are introduced into the wells, followed by the addition of the
stimulated T-lymphocytes. After an incubation period that ranges
between 18 h and 36 h, specific T-cells recognizing a certain anti-
gen will release a specific cytokine that could be captured by the
solid phase antibody [58]. The technique could be used for quanti-
tative as well as qualitative detection of cytokines. It has the
advantage of using frozen PBMC without losing effectiveness
[18,58]. A major limitation is the inability to define the phenotype
of the T-cells that secrete the cytokine without testing purified CD4
+ and CD8+ cells [105]. Therefore, the assay cannot estimate the
frequency of an antigen recognized by a specific T-cell [106].
The ELISA method was practiced to quantify the cellular
response of several pathogens such as HIV [107], Filaria [108],
and M. avium [109]. It was combined with pepscan to highlight
the epitopes of the Lck protein kinase, a potential tumor antigen
[110], the glycoprotein B homolog of human herpes virus 8
[111], and the kallikrein 4, a prostate specific antigen [112]. As
discussed before, the ELISA technique could be combined with
other mapping protocols, such as the flow cytometry, MM, and
lymphoproliferation.
2.6. ELISPOT
T-cells secrete different cytokines, especially IFN-c, which can
be detected as a result of antigenic recognition on antigen present-
ing cells (APCs). The technique is very similar to ELISA, but with
slight variations. A ninety-six well plates with polyvinylidene
fluoride or nitrocellulose membrane lined bottom portion is coated
with a primary anti-cytokine antibody [58,77]. The plate is
incubated as in ELISA and the anti-cytokine antibody captures
the target T-cell cytokine. The primary antibody is conjugated with
a secondary biotinylated antibody that is linked to streptavidin-
enzyme conjugate and the suitable substrate [113]. Therefore,
antigen specific T-lymphocytes can be seen as a spot that can be
counted using a low magnification instrument. The technique is
widely practiced since it requires simple devices and it allows
the use of delicate scanners to detect and count spots [58,114].
Moreover, it enables the detection of two cytokine that are
secreted by the same cell simultaneously using two different
fluorochrome-labeled reagents [114]. However, the assay is not
able to determine the T-cell phenotype and cells cannot be reused
neither studied using flow cytometry [115]. In addition, the spot
may contain many different cells if more than one type of T-cells
binds to the same APC [16].
The ELISPOT technique was recognized in several occasions
such as the mapping of the epitopes of HPV16 [116], H5N1
[53,117], Influenza A [118,119], HIV-1 [120,121], tumor hep-
aranase [122], humans tuberculosis [123], house dust mite
allergen [124], and a human inhibitor of apoptosis [125]. However,
the technique is usually combined with pepscan to highlight the
epitopes HIV-1 [126129], the IE1 protein of the HCMV [130],
H5N1 vaccination [131], the pneumonia virus of mice glycoprotein
G [132], the outer surface protein A of Borrelia garinii [133],
DNA-binding protein 1 (MDP1) of M. tuberculosis [134], bladder
cancer [135], prostate stem cell antigen [136], over-expressed
tumor antigen calcium-activated chloride channel 2 [137], The
NY-ESO-1 tumor antigen [138], and Plasmodium falciparum
circum-sporozoite protein [139]. Moreover, it was used with in
silico-based method to identify the potent epitopes of Ebola virus
[140], Influenza A virus [141], Hepatitis C virus (HCV) [142],
Vaccinia virus [17], M. tuberculosis [143,144], mammaglobin-A, a
highly expressed breast cancer tumor antigen [145], cervical
cancer [146], and human heparanase that is normally over-
expressed in the majority of tumors [147]. As highlighted before,
the ELISPOT approach could be combined with the MM approach
[53,54], the lymphoproliferation assay [70], the flow cytometry
approach, and the flow cytometry and in silico-based method.
Simultaneously, ELISPOT was used in combination with B-cell
epitope mapping to reveal the potent antigenic determinants of
ovarian cancer [148], bovine milk allergen [149], and tau protein
of the Alzheimers disease [150].
2.7. Activation markers induced on specific T-cells
In the previously demonstrated methods, the T-helper (Th)-cell
response was assessed by measuring cytokines or MHC-II
multimers. Although these techniques provide quantitative and
phenotypic information, they do not give complete results for
non-Th1 CD4+ T-cell recall antigens such as tetanus, Mycobacteria,
CMV, HSV-1, Influenza, Candida, and HIV-1. Since tetanus for
example, yields a vigorous proliferation response that does not
correspond to the cytokines assay measurement [151,152].
Therefore, the activation markers induced-method was applied to
allow the identification, counting, and selection of specific CD4+
and CD8+ for T-lymphocytes recall antigens. The technique is
based on culturing the whole blood cells stimulated with an
antigen or mitogen for 4048 h only. Thereafter, the desired CD
was measured by surface staining with specific antibodies [151].
The cells retained their viability since the methodology is not
dependent on cells permeabilization, and they frequently undergo
phenotypic and functional characterization by flow cytometry
[151,153]. It was noticed that the detected CD expression was
sometimes associated with specific cytokines measurement [153].
The activation marker technique is simple and time saving
[151,152]. This technique may be also applied for quantitative
and qualitative detection of specific CDs [152]. Although, some
findings claimed that some CDs are only transiently expressed
during the course of Th-lymphocytes activation, intracellular
stabilizers for CD were applied to overcome this defect [152].
Several markers were proposed for use by this technique such as
CD25 (IL-2Ra)/CD134 (OX40) assay [151] and CD154 assay [152]
for the detection of CD4+ cells, while CD137 (TNF4-1BB) [153]
was applied for the detection of both CD8+ and CD4+ cells. To
the best of the authors knowledge no epitope was practically
described by this method.
2.8. Cell free system
Hartman et al. used the cell free system (CFS) approach to iden-
tify Influenza epitopes. This approach could be briefly summarized
in three main steps: (1) incubating the whole antigens with HLA to
allow binding, (2) application of proteases to the antigens/HLA
mixture, (3) then using mass spectrometry to sequence HLA bound
peptides [154]. Although this methods did not find its way for
 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322
frequent application since it only detects antigens of known
structure and it is expensive [155], few researchers used it to
map the epitope of HIV/HSV as being previously well studied [156].
2.9. High throughput T-epitope mapping (HTP)
These are a group of in silico prediction based on algorithmic
immuno-informatics approaches. They offer the preliminary step
aiming to screen and reduce the number of antigenic peptides
for further mapping by any of the binding methods previously
described [157]. Moreover, in silico approaches overcome the
shortage in the number of functional T-lymphocytes available for
laboratory work [19], saves time and effort by focusing only on
targeted antigenic peptides which in turn will reduce the cost of
the overall process of epitope mapping [158], and increases the
specificity by only screening short panels of antigenic peptides as
well as using small number of cells [106,159]. The in silico methods
offer accurate results since the majority of T-cell epitopes are linear
[160]. Several software showed to perform in silico T-cell epitope
prediction [161] such as iVax [162], Tepitope [163], EpiMatrix,
[164167], SYPPEITHI [168], EpiMer [144], Conservatrix [167],
Rankpep [169], Epi-Assembler and Vaccine-CAD [170], iTopia assay
platform [171], EpiMatrixs extension JanusMatrix [172], DNAStar
[173], Proped [174], and PEPFR [175]. However, T-cell epitopes
predicted by HTP needs further confirmation by binding ones
[158].
The approach demonstrated success especially when combined
with other binding methods. The success was very obvious in the
identification of several potent HIV epitopes as being an obvious
challenge for vaccinologists due to HLA polymorphism and high
mutation rate [160]. In 1997, De Groot et al. [164] identified 134
T-cell epitopes of HIV using EpiMatrix/HIV algorithm, then
discovered several other epitopes of HIV-1 using the ELISPOT
approach and the EpiMatrix algorithm [144], then revealed 31
novel epitopes by the same method [165], thereafter identified 5
epitopes that were selected to serve as building blocks for
epitope-driven cross-clade HIV-1 vaccine (the GAIA vaccine)
against HIV [166]. In 2005, the results were enriched by combining
the EpiMatrix with the Epi-Assembler and Vaccine-CAD methods
[170]. Furthermore in 2006, Koita et al. [176] evaluated 21
immunogenic consensus sequence. In 2012, Levitz et al. [177] used
the same technique to identify 11 HLA-A2 epitopes of HIV,
followed by De Groot et al. [178] that marked 27 HLA-A3 epitopes
of HIV, while Mori et al. [179] used a novel in silico approach to
identify HLA-restricted epitopes of HIV.
Simultaneously, the technique was used alone in mapping the
epitopes of HCV and Influenza A virus [180], swine-origin Influenza
A (H3N2) variant virus [181,182], 2009-H1N1 and 2004-H5N1
Influenza virus [183], Leptospira borgpetersenii [184] and Eimeria
[173]. As discussed before, the EpiMatrix approach was combined
with the MM technique [51]. In addition, bioinformatics-based
approaches were combined with the lymphoproliferation assay
[62,63,75,185], the flow cytometry approach [9698], the ELISPOT
technique, the MM approach and the flow cytometry analysis
[49], the flow cytometry and the ELISPOT approaches
[90,93,94,100,101], the cell-ELISA assay and the flow cytometry
approach [95], as previously mentioned. New approaches based
on the detection of both B-cell binding techniques, and T-cell
epitope by in silico methods appeared for Eimeria [173,186], human
[187,188] and avian [174] tuberculosis, H1N1[189], and foot-
mouth disease virus [190].
2.10. Pepscan
In 1984 Mario Geysen, introduced pepscan as a new method for
epitope mapping, where the target antibody is examined against a
17
library of peptides from the protein of interest (antigen) to test
their ability to bind to the desired antibody [191]. Pepscan
technique was able to identify B-cell epitopes [9,14], as well as
T-cell epitopes [16].
The pepscan technique proved its success to screen the epitopes
of the hemagglutinin molecule of Influenza A virus [192], JC
polyoma virus [193], and the tumor-associated antigen, a potent
antigen that is highly expressed in breast cancer patients [194].
The technique was usually used as a preliminary step for T-cell
epitope mapping as mentioned before.
3. Comment
Epitope mapping has emerged as a powerful tool for the design
and production of vaccines. Although the selection of target B-cell
epitopes is crucial for vaccine development, it does not necessarily
lead to the design of potent long lasting or intracellular vaccines.
T-cell response demonstrated its involvement in eliminating intra-
cellular pathogens, destroying abnormal cells, and inducing long
term protection. Although T-cell epitope mapping proved its
importance for the development of vaccines, its integration with
B-cell mapping demonstrated its necessity to control several
pathogens. Unlike B-cell epitopes, the T-cells are linear. This
offered the privilege to the design and production of T-cell epitopes
based vaccines, especially the new trend DNA and recombinant
preparations. A special concern in research was dedicated to
emerging influenza viruses, HIV, HCV, M. tuberculosis, M. avium,
malarias parasite and cancers.
Although the majority of T-cell epitope mapping are based on
binding methods, some are bioinformatics based or integrated
with physical techniques. Pepscan was the first method to be intro-
duced for epitope mapping since the mid 1980s, and found several
applications in B-cell epitoping. It was simultaneously used in the
1990s as a preliminary step to reveal the T-cell target epitopes in
combination with other binding and bioinformatics methods. In
the mid-1990s T-cell epitoping flourished by using the MHC mul-
timers. This technique was applied to reveal the T-cell epitopes for
Mycobacteria, Chlamydia, HIV, HSV, small pox and cancer.
Although it is simple and recoverable, the prior knowledge of the
antigenic structure and its first exclusive use for MHC class I
epitopes limited its application as a sole ideal method for T-cell
epitope mapping. The limited number of tests allowed by MM,
introduced the solid phase MHC-complexes method that depends
on microarray. However, the limitation of the method to MHC class
I epitopes, and expensive instrumentation restricted its use as well.
Later on the lympho-proliferation assay appeared. It was simple,
cheap, and sensitive. However, it was time consuming, lacked the
ability to measure CK types and quantity, as well as using risky
radio-labeling for sometimes and cells were not recoverable.
However, it developed to safer, cheaper, but less sensitive
colorimetric-based methods. Despite those primary limitations it
was widely practiced to unravel the epitopes of intracellular bacte-
ria, viruses, tumor markers, autoimmune disease, and the peanut
allergy epitopes. In the late 1990s the flow cytometry method
appeared. It overcame the defects of time consumption, CK quali-
tative and quantitative measurement, number of tested samples,
but lacked the advantage of being cost effective. It had unique
advantages of testing different CKs in one sample, and measuring
different phenotypic markers. The ICS advantages allowed the
method to map several new viruses like Ebola, avian flu, HPV and
CMV. Moreover it was applied to map the epitopes of other
intracellular bacteria, viruses, and tumor markers.
In the beginnings of the new millennium, cell-ELISA adaptation
in T-cell epitope mapping evolved. It was simple, relatively cheap,
tested large number of samples, saved times, and had the
advantage of measuring CK qualitatively and quantitatively.
18 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322

Table 1
Comparison between the different methods of T-cell epitope mapping.

Pepscan MM Solid Lympho- ICS (Flow Cell-ELISA ELISPOT Activation CFS In silico (HTP)
phase proliferation cytometry) markers
MHC
Method Idea Screen Signal for Signal for Detect Signal for Signal for a Signal for Marker Identify Algorithms
against a coupling of CK on a PMNCs specific CK specific CK specific detects bound based
protein MHC with microarray activation CKs specific CD epitopes
library TCR by mass-
spec.
Role/Use Preliminary General General General Screen Quantify a CK Screen up Detects Rough Preliminary
several CKs to 2-CKs Th-1 or -2
activity
Markers No No No No Yes No No Yes No No
phenotype
Identify CK No No No No Yes Yes Yes No No No
Quantify CK No No No No Yes Yes Semi No No No
Antigen Structure Known Known No No No No No No No Known
Genetically Yes No No No No No No No No Yes
mapped
Application Number of Large Limited Large Limited Large Large Large Limited Limited Large
samples
Recoverable Yes No No No No No Yes Yes
cells
Combined EL; ICS; EL; EP; EL; EP; ICS; EL; EP; LY; EP; ICS; LY; ICS; LY; ICS EL; EP; ICS;
with other LY; MM. ICS; LY; MM; PC; PP. MM, PC; PP. MM, PC; PP. MM, PC; LY; MM.
T-methods PC; PP. PP.
Previous Bt; Cs; Pa; Bt; Cs; vs. vs. Al; Bt; Cs; Bt; Cs; vs. Bt; Cs; Pa; vs. Al; Bt; Cs; vs. Al; Bt; Cs;
uses vs. Ds; Pa; vs. Pa; vs. Pa; vs.
Combined vs. Al; Cs; Ds Bt; Pa; vs.
with B-
methods
Pros./Cons. Detect B Easy, Complex & Easy & very Fast, test Simple Simple, Simple, Costs Save time
and T- simple, but costs cheap several CK at device, cheap, cheap & commercial and efforts,
epitopes each once, need save time & can kit that but
peptide expertise &is can use measure 2 costs confirmatory
needs a expensive. freezed cells CKs at test needed
MHC once

Key: Al = Allergens; Bt = Bacteria; CD = Cluster of differentiation; CK = Cytokine; Cs = Cancer; Ds = Disease; EL = Cell ELISA; EP = ELISPOT; PC = in silico; LP = Lymphoproliferation;
MHC = Major Histocompatibility Complex; Pa = Parasite; PMNCs = Poly-morpho-nuclear cells; PP = Pepscan; Vs = Virus.

However, it lacked the ability to measure different CKs at the same
time. ELISPOT appeared to ameliorate the defects of ELISA and the
expensive ICS. ELISPOT technique has lower price, and was able to
measure different CK types in one sample, without losing the
major privileges already owned by ELISA. In 2005, a commercial
method called activation markers was introduced to measure
the CK quantitatively. Although the idea of the method shows to
be very promising, until now it has no frequent applications.
Simultaneously the CFS method did not show any advantages
when compared to other methods such as the immuno-
bioinformatics ones.
Despite the fact that the majority of T-cell epitope mapping
techniques are binding methods, recent advances in bioinformatics
and reverse vaccinology strategies allowed scientists to implement
in silico-based approaches for the large scale investigations of new
target antigens. Especially, that T-cell epitope are linear, and hence
accurate results are obtained from HTP methods. These methods
focus only on promising target epitopes and it is now known that
they speed the discovery process by about 1020-fold. Therefore,
they were usually introduced as primary step to unravel the
potency of novel unknown epitopes, especially those causing out-
breaks such as HIV, HCV, H1N1, H5N1, H3N2, Influenza A, Ebola,
and Listeria. Pepscan was used as well to narrow the number of
tests.
(Table 1) displays the major criteria that can be used to com-
pare the appropriateness of a method to be used for T-cell epitope
mapping. Preliminary screening of antigens of knows structures
were performed by in silico and pepscan methods. The general
detection of T-cell response against a specific epitope was per-
formed by the low cost lymphoproliferation assay, while the
screening of cytokine responses was fulfilled by ICS or ELISPOT,
depending on the available facilities and expertise. The cell-ELISA
was usually applied when an accurate evaluation of a specific cyto-
kine response was necessary to identify, especially when ICS was
not an introduced method for cost reasons. Since nowadays the
majority of antigens have known structures pepscan and HTP
methods are frequently applied to screen the full genomes for T-
cell epitopes in order to save time, control cost and reduce efforts.
As long as the accurate quantification of cytokines is not a neces-
sary target in low resource research projects, the urge to use cell-
ELISA and ICS shifted towards the semi-quantitative ELISPOT. This
confirms that the decision to select the best method to be used to
map an antigen is a case matter. It depends on the previous knowl-
edge of the antigens structure, the availability of sophisticated
equipment and experienced professionals, the available fund, the
number of samples, the urgency to get results, and the type of cel-
lular responses desired from the designed vaccine. All the mapping
methods integrated to offer sufficient information to design a
potent vaccine.
Acknowledgments
The authors would like to thank Mr. Ahmed Issa and Mr. Derek
Gulbranson for their efforts to edit this review.
 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322 19

References [33] N.S. Butler, N.W. Schmidt, J.T. Harty, Differential effector pathways regulate
memory CD8 T cell immunity against Plasmodium berghei versus P. yoelii
sporozoites, J. Immunol. 184 (2010) 25282538.
[1] P. Cresswell, M.J. Androlewicz, B. Ortmann, Assembly and transport of class I
[34] N.W. Schmidt, N.S. Butler, V.P. Badovinac, J.T. Harty, Extreme CD8 T cell
MHC-peptide complexes, Ciba Found. Symp. 187 (1994) 150162.
requirements for anti-malarial liver-stage immunity following immunization
[2] P. Cresswell, Assembly, transport, and function of MHC class II molecules,
with radiation attenuated sporozoites, PLoS Pathog. 6 (2010) e1000998.
Annu. Rev. Immunol. 12 (1994) 259293.
[35] A. Trimnell, A. Takagi, M. Gupta, T.L. Richie, S.H. Kappe, R. Wang, Genetically
[3] A.M. Lennon-Dumnil, A.H. Bakker, P. Wolf-Bryant, H.L. Ploegh, C.
attenuated parasite vaccines induce contact-dependent CD8+ T cell killing of
Lagaudrire-Gesbert, A closer look at proteolysis and MHC-class-II-
Plasmodium yoelii liver stage-infected hepatocytes, J. Immunol. 183 (2009)
restricted antigen presentation, Curr. Opin. Immunol. 14 (2002) 1521.
58705878.
[4] N. Ismail, J.P. Olano, H.M. Feng, D.H. Walker, Current status of immune
[36] I.A. Khan, K.H. Ely, L.H. Kasper, A purified parasite antigen (p30) mediates CD8
mechanisms of killing of intracellular microorganisms, FEMS Microbiol. Lett.
+ T cell immunity against fatal Toxoplasma gondii infection in mice, J.
207 (2002) 111120.
Immunol. 147 (1991) 35013506.
[5] K. Kedzierska, S.A. Valkenburg, P.C. Doherty, M.P. Davenport, V. Venturi, Use it
[37] J.S. Woodworth, S.M. Behar, Mycobacterium tuberculosis-specific CD8+ T cells
or lose it: establishment and persistence of T cell memory, Front. Immunol. 3
and their role in immunity, Crit. Rev. Immunol. 26 (2006) 317352.
(2012) 111.
[38] J.S. Woodworth, S.M. Fortune, S.M. Behar, Bacterial protein secretion is
[6] H.G. Rammensee, K. Falk, O. Rtzschke, Peptides naturally presented by MHC
required for priming of CD8+ T cells specific for the Mycobacterium
class I molecules, Annu. Rev. Immunol. 11 (1993) 213244.
tuberculosis antigen CFP10, Infect. Immun. 76 (2008) 41994205.
[7] T. Nagata, Y. Koide, Induction of specific CD8 T cells against intracellular
[39] M. Johansson, M. Ward, N. Lycke, B-cell-deficient mice develop complete
bacteria by CD8 T-cell-oriented immunization approaches, J. Biomed.
immune protection against genital tract infection with Chlamydia
Biotechnol. 2010 (2010) 764542.
trachomatis, Immunology 92 (1997) 422428.
[8] R.F. Wang, The role of MHC class II-restricted tumor antigens and CD4+ T cells
[40] S. Wu, T. Yu, X. Song, S. Yi, L. Hou, W. Chen, Prediction and identification of
in antitumor immunity, Trends Immunol. 22 (2001) 269276.
mouse cytotoxic T lymphocyte epitopes in Ebola virus glycoproteins, Virol. J.
[9] P.A. Castric, F.J. Cassels, Peptide epitope mapping in vaccine development:
9 (2012).
introduction, J. Ind. Microbiol. Biotechnol. 19 (1997) 5657.
[41] F. Fenner, D.A. Henderson, I. Arita, Z. Jezek, I.D. Ladnyi, Smallpox and its
[10] S.C. De Rosa, F.X. Lu, J. Yu, S.P. Perfetto, J. Falloon, S. Moser, et al., Vaccination
Eradication, The World Health Organization, Geneva, Switzerland, 1988.
in humans generates broad T cell cytokine responses, J. Immunol. 173 (2004)
[42] D.M. Knipe, P.M. Howley, in: J.J. Esposito (Ed.), Fields Virology, Lippincott
53725380.
Williams & Wilkins, Philadelphia, 2001.
[11] J.M. Gershoni, A. Roitburd-Berman, D.D. Siman-Tov, N.T. Freund, Y. Weiss,
[43] S.R. Hadrup, A.H. Bakker, C.J. Shu, R.S. Andersen, J. van Veluw, P. Hombrink,
Epitope mapping: the first step in developing epitope-based vaccines,
et al., Parallel detection of antigen-specific T-cell responses by
Biodrugs 21 (2007) 145156.
multidimensional encoding of MHC multimers, Nat. Methods 6 (2009) 520
[12] L.M. Childs, E.B. Baskerville, S. Cobey, Trade-offs in antibody repertoires to
526.
complex antigens, Philos. Trans. R. Soc. Lond. B Biol. Sci. 370 (2015).
[44] A.H. Bakker, T.N. Schumacher, MHC multimer technology: current status and
[13] T.A. Ahmad, The development of immunization trials, Edorium J. Biotechnol.
future prospects, Curr. Opin. Immunol. 17 (2005) 428433.
1 (2015) 16.
[45] G.T. Nepom, MHC class II tetramers, J. Immunol. 188 (2012) 24772482.
[14] T.A. Ahmad, A.E. Eweida, S.A. Sheweita, B-cell epitope mapping for the design
[46] V. Cecconi, M. Moro, S. Del Mare, P. Dellabona, G. Casorati, Use of MHC class II
of vaccine and effective diagnostics, Trials Vaccinol. 5 (2016) 7183.
tetramers to investigate CD4+ T cell responses: problems and solutions,
[15] A.S. De Groot, Immunomics: discovering new targets for vaccines and
Cytometry A 73A (2008) 10101018.
therapeutics, Drug Discov. Today 11 (2006) 203209.
[47] W.W. Kwok, J.A. Gebe, A. Liu, S. Agar, N. Ptacek, J. Hammer, et al., Rapid
[16] L. Malherbe, T-cell epitope mapping, Ann. Allergy Asthma Immunol. 103
epitope identification from complex class-II-restricted T-cell antigens, Trends
(2009) 7679.
Immunol. 22 (2001) 583588.
[17] P. Gilchuk, C.T. Spencer, S.B. Conant, T. Hill, J.J. Gray, X. Niu, et al., Discovering
[48] A. Geluk, K.E. van Meijgaarden, K.L. Franken, J.W. Drijfhout, S. DSouza, A.
naturally processed antigenic determinants that confer protective T cell
Necker, et al., Identification of major epitopes of Mycobacterium tuberculosis
immunity, J. Clin. Invest. 123 (2013) 19761987.
AG85B that are recognized by HLA-A0201-restricted CD8+ T cells in HLA-
[18] R.A. Seder, P.A. Darrah, M. Roederer, T-cell quality in memory and protection:
transgenic mice and humans, J. Immunol. 165 (2000) 64636471.
implications for vaccine design, Nat. Rev. Immunol. 8 (2008) 247258.
[49] W. Kuon, H.G. Holzhtter, H. Appel, M. Grolms, S. Kollnberger, A. Traeder,
[19] F. Kern, G. LiPira, J.W. Gratama, F. Manca, M. Roederer, Measuring Ag-specific
et al., Identification of HLA-B27-restricted peptides from the Chlamydia
immune responses: understanding immunopathogenesis and improving
trachomatis proteome with possible relevance to HLA-B27-associated
diagnostics in infectious disease, autoimmunity and cancer, Trends
diseases, J. Immunol. 167 (2001) 47384746.
Immunol. 26 (2005) 477484.
[50] A. Geluk, S.J. van den Eeden, K. Dijkman, L. Wilson, H.J. Kim, K.L. Franken,
[20] J.D. Doucet, D. Gauchat, R. Lapointe, MRNA PCR-based epitope chase method,
et al., ML1419c peptide immunization induces Mycobacterium leprae-specific
Methods Mol. Biol. 969 (2013) 305320.
HLA-A0201-restricted CTL in vivo with potential to kill live Mycobacteria, J.
[21] T. Midoro-Horiuti, R.M. Goldblum, Epitope mapping with random phage
Immunol. 187 (2011) 13931402.
display library, Methods Mol. Biol. 1131 (2014) 477484.
[51] K.B. Bond, B. Sriwanthana, T.W. Hodge, A.S. De Groot, T.D. Mastro, N.L. Young,
[22] P.H. Lambert, M. Liu, C.A. Siegrist, Can successful vaccines teach us
et al., An HLA-directed molecular and bioinformatics approach identifies new
how to induce efficient protective immune responses?, Nat Med. 11 (2005)
HLA-A11 HIV-1 subtype E cytotoxic T lymphocyte epitopes in HIV-1-infected
S54S62.
Thais, AIDS Res. Hum. Retroviruses 17 (2001) 703717.
[23] J.U. Igietseme, F.O. Eko, Q. He, C.M. Black, Antibody regulation of T-cell
[52] E.J. Novak, A.W. Liu, J.A. Gebe, B.A. Falk, G.T. Nepom, D.M. Koelle, et al.,
immunity: implications for vaccine strategies against intracellular pathogens,
Tetramer-guided epitope mapping: rapid identification and characterization
Expert Rev. Vaccines 3 (2004) 2334.
of immunodominant CD4+ T cell epitopes from complex antigens, J. Immunol.
[24] D.H. Walker, J.P. Olano, H.M. Feng, Critical role of cytotoxic T lymphocytes in
166 (2001) 66656670.
immune clearance of rickettsial infection, Infect. Immun. 69 (2001) 1841
[53] M. Terajima, L. Orphin, A.M. Leporati, P. Pazoles, J. Cruz, A.L. Rothman, et al.,
1846.
Vaccinia virus-specific CD8(+) T-cell responses target a group of epitopes
[25] J. Hess, C. Ladel, D. Miko, S.H. Kaufmann, Salmonella typhimurium
without a strong immunodominance hierarchy in humans, Hum. Immunol.
aroA-infection in gene-targeted immunodeficient mice: major role of
69 (2008).
CD4+ TCR-alpha beta cells and IFN-gamma in bacterial clearance
[54] A.M. Leen, A. Christin, M. Khalil, H. Weiss, A.P. Gee, M.K. Brenner, et al.,
independent of intracellular location, J. Immunol. 156 (1996) 33213326.
Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and
[26] E.R. Unanue, Inter-relationship among macrophages, natural killer cells and
immunotherapy, J. Virol. 82 (2008) 546s554s.
neutrophils in early stages of Listeria resistance, Curr. Opin. Immunol. 9
[55] L. Fong, Y. Hou, A. Rivas, C. Benike, A. Yuen, G.A. Fisher, et al., Altered peptide
(1997) 3543.
ligand vaccination with Flt3 ligand expanded dendritic cells for tumor
[27] J.L. Casanova, L. Abel, Genetic dissection of immunity to mycobacteria: the
immunotherapy, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 88098814.
human model, Annu. Rev. Immunol. 20 (2002) 581620.
[56] M. Schena, R.A. Heller, T.P. Theriault, K. Konrad, E. Lachenmeier, R.W. Davis,
[28] R.P. Morrison, C. HD, Immunity to murine chlamydial genital infection, Infect.
Microarrays: biotechnologys discovery platform for functional genomics,
Immun. 70 (2002) 27412751.
Trends Biotechnol. 16 (1998) 301316.
[29] J.U. Igietseme, F.O. Ekp, B. CM, Contemporary approaches to designing and
[57] J.D. Stone, W.E. Demkowicz Jr., S. LJ, HLA-restricted epitope identification and
evaluating vaccines against Chlamydia, Expert Rev. Vaccines 2 (2003) 129
detection of functional T cell responses by using MHC-peptide and
146.
costimulatory microarrays, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 3744
[30] J.E. Kohlmeier, D.L. Woodland, Immunity to respiratory viruses, Annu. Rev.
3749.
Immunol. 27 (2009) 6182.
[58] T.M. Clay, A.C. Hobeika, P.J. Mosca, H.K. Lyerly, M.A. Morse, Assays for
[31] J. Goulding, R. Bogue, V. Tahiliani, M. Croft, S. Salek-Ardakani, CD8 T cells are
monitoring cellular immune responses to active immunotherapy of cancer,
essential for recovery from a respiratory vaccinia virus infection, J. Immunol.
Clin. Cancer Res. 7 (2001) 11271135.
189 (2012) 24322440.
[59] G. Repetto, A. del Peso, J.L. Zurita, Neutral red uptake assay for the estimation
[32] J.E. Epstein, K. Tewari, K.E. Lyke, B.K. Sim, P.F. Billingsley, M.B. Laurens, et al.,
of cell viability/cytotoxicity, Nat. Protoc. 3 (2008) 11251131.
Live attenuated malaria vaccine designed to protect through hepatic CD8+ T
[60] M.A. Bergman, L.A. Cummings, R.C. Alaniz, L. Mayeda, I. Fellnerova, B.T.
cell immunity, Science 334 (2011) 475480.
Cookson, CD4+-T-cell responses generated during murine Salmonella enterica
20 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322
serovar Typhimurium infection are directed towards multiple epitopes within
the natural antigen FliC, Infect. Immun. 73 (2005) 72267235.
[61] B.L. Talken, K.R. Schfermeyer, C.W. Bailey, D.R. Lee, R.W. Hoffman, T cell
epitope mapping of the Smith antigen reveals that highly conserved Smith
antigen motifs are the dominant target of T cell immunity in systemic lupus
erythematosus, J. Immunol. 167 (2001) 562580.
[62] R.A. Court, K. Sitte, J.P. Opdebeeck, I.J. East, Mapping the T cell epitopes of the
Babesia bovis antigen 12D3: implications for vaccine design, Parasite
Immunol. 20 (1998) 18.
[63] H. Kobayashi, R. Omiya, M. Ruiz, E. Huarte, P. Sarobe, J.J. Lasarte, et al.,
Identification of an antigenic epitope for helper T lymphocytes from
carcinoembryonic antigen, Clin. Cancer Res. 8 (2002) 32193225.
[64] B.Y. Lee, M.A. Horwitz, T-cell epitope mapping of the three most abundant
extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs,
Infect. Immun. 67 (1999) 26652670.
[65] P. Launois, R. DeLeys, M.N. Niang, A. Drowart, M. Andrien, P. Dierckx, et al.,
T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in
tuberculosis and leprosy, Infect. Immun. 62 (1994) 36793687.
[66] D.G. McNeel, L.D. Nguyen, M.L. Disis, Identification of T helper epitopes from
prostatic acid phosphatase, Cancer Res. 61 (2001) 51615167.
[67] J. Marttila, H. Hyty, P. Vilja, T. Hrknen, A. Alho, M. Roivainen, et al., T cell
epitopes in coxsackievirus B4 structural proteins concentrate in regions
conserved between enteroviruses, Virology 293 (2002) 217224.
[68] R. Aggarwal, R. Shukla, S. Jameel, S. Agrawal, P. Puri, V.K. Gupta, et al., T-cell
epitope mapping of ORF2 and ORF3 proteins of human hepatitis E virus, J.
Viral Hepat. 14 (2007) 283292.
[69] K.L. Knutson, K. Schiffman, M.L. Disis, Immunization with a HER-2/neu helper
peptide vaccine generates HER-2/neu CD8 T-cell immunity in cancer patients,
J. Clin. Invest. 107 (2001) 477484.
[70] S.J. McSorley, B.T. Cookson, M.K. Jenkins, Characterization of CD4+ T cell
responses during natural infection with Salmonella typhimurium, J. Immunol.
164 (2000) 986993.
[71] J. Schmitz, E. Reali, J.W. Hodge, A. Patel, G. Davis, J. Schlom, et al.,
Identification of an interferon-gamma-inducible carcinoembryonic antigen
(CEA) CD8(+) T-cell epitope, which mediates tumor killing in CEA transgenic
mice, Cancer Res. 62 (2002) 50585064.
[72] J.L. Vissers, I.J. De Vries, L.P. Engelen, N.M. Scharenborg, J. Molkenboer, C.G.
Figdor, et al., Renal cell carcinoma-associated antigen G250 encodes a
naturally processed epitope presented by human leukocyte antigen-DR
molecules to CD4(+) T lymphocytes, Int. J. Cancer 100 (2002) 441444.
[73] G. Consogno, S. Manici, V. Facchinetti, A. Bachi, J. Hammer, B.M. Conti-Fine,
et al., Identification of immunodominant regions among promiscuous HLA-
DR-restricted CD4+ T-cell epitopes on the tumor antigen MAGE-3, Blood 101
(2003) 10381044.
[74] C.C. Wilson, B. Palmer, S. Southwood, J. Sidney, Y. Higashimoto, E. Appella,
et al., Identification and antigenicity of broadly cross-reactive and conserved
human immunodeficiency virus type 1-derived helper T-lymphocyte
epitopes, J. Virol. 75 (2001) 41954207.
[75] M. Pascal, G.N. Konstantinou, M. Masilamani, J. Lieberman, H.A. Sampson, In
silico prediction of Ara h 2 T cell epitopes in peanut-allergic children, Clin.
Exp. Allergy 43 (2013) 116127.
[76] S.L. Waldrop, C.J. Pitcher, D.M. Peterson, V.C. Maino, L.J. Picker, Determination
of antigen-specific memory/effector CD4+ T cell frequencies by flow
cytometry: evidence for a novel, antigen-specific homeostatic mechanism
in HIV-associated immunodeficiency, J. Clin. Invest. 99 (1997) 17391750.
[77] A. Letsch, C. Scheibenbogen, Quantification and characterization of specific
T-cells by antigen-specific cytokine production using ELISPOT assay or
intracellular cytokine staining, Methods 31 (2003) 143149.
[78] A. Scardino, P. Alves, D.A. Gross, S. Tourdot, S. Graff-Dubois, E. Angevin, et al.,
Identification of HER-2/neu immunogenic epitopes presented by renal cell
carcinoma and other human epithelial tumors, Eur. J. Immunol. 31 (2001)
32613270.
[79] F. Kern, I.P. Surel, N. Faulhaber, C. Frmmel, J. Schneider-Mergener, C.
Schnemann, et al., Target structures of the CD8(+)-T-cell response to human
cytomegalovirus: the 72-kilodalton major immediate-early protein revisited,
J. Virol. 73 (1999) 81798184.
[80] V. Pancr, B. Georges, G. Angyalosi, F. Castelli, A. Delanoye, M. Delacre, et al.,
Novel promiscuous HLA-DQ HIV Nef peptide that induces IFN-gamma-
producing memory CD4+ T cells, Clin. Exp. Immunol. 129 (2002) 429437.
[81] F. Micheletti, P. Monini, C. Fortini, P. Rimessi, M. Bazzaro, M. Andreoni, et al.,
Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8,
Immunology 106 (2002) 395403.
[82] Y. Umano, T. Tsunoda, H. Tanaka, K. Matsuda, H. Yamaue, H. Tanimura,
Generation of cytotoxic T cell responses to an HLA-A24 restricted epitope
peptide derived from wild-type p53, Br. J. Cancer 84 (2001) 10521057.
[83] G. Francini, A. Scardino, K. Kosmatopoulos, F.A. Lemonnier, G. Campoccia, M.
Sabatino, et al., High-affinity HLA-A()02.01 peptides from parathyroid
hormone-related protein generate in vitro and in vivo antitumor CTL
response without autoimmune side effects, J. Immunol. 169 (2002) 4840
4849.
[84] N. Caccamo, S. Milano, C. Di Sano, D. Cigna, J. Ivanyi, A.M. Krensky, et al.,
Identification of epitopes of Mycobacterium tuberculosis 16-kDa protein
recognized by human leukocyte antigen-A0201 CD8(+) T lymphocytes, J.
Infect. Dis. 186 (2002) 991998.
[85] A. Rafiei, Y. Kiutade, Identification of Mycobacterium tuberculosis CTL epitopes
restricted by HLA-A0201 in HHD mice, Iran. Biomed. J. 11 (2007) 2331.
[86] M.G. Chaitra, M.S. Shaila, N.R. Chandra, R. Nayak, HLA-A0201-restricted
cytotoxic T-cell epitopes in three PE/PPE family proteins of Mycobacterium
tuberculosis, Scand. J. Immunol. 67 (2008) 411417.
[87] R. Draenert, M. Altfeld, C. Brander, N. Basgoz, C. Corcoran, A.G. Wurcel, et al.,
Comparison of overlapping peptide sets for detection of antiviral CD8 and
CD4 T cell responses, J. Immunol. Methods 275 (2003) 1929.
[88] J.A. Rutigliano, M.T. Rock, A.K. Johnson, J.E. Crowe Jr., B.S. Graham,
Identification of an H-2D(b)-restricted CD8+ cytotoxic T lymphocyte
epitope in the matrix protein of respiratory syncytial virus, Virology 337
(2005) 335343.
[89] M. Maybeno, A. Redeker, S.P. Welten, B. Peters, S.M. Loughhead, S.P.
Schoenberger, et al., Polyfunctional CD4+ T cell responses to immuno-
dominant epitopes correlate with disease activity of virulent Salmonella,
PLoS One 7 (2012) e43481.
[90] S. Li, L. Peng, W. Zhao, H. Zhong, F. Zhang, Z. Yan, et al., Synthetic peptides
containing B- and T-cell epitope of dengue virus-2 E domain III provoked
B- and T-cell responses, Vaccine 29 (2011) 36953702.
[91] L.E. Wagar, L. Rosella, N. Crowcroft, B. Lowcock, P.C. Drohomyrecky, J. Foisy,
et al., Humoral and cell-mediated immunity to pandemic H1N1 influenza in a
Canadian cohort one year post-pandemic: implications for vaccination, PLoS
One 6 (2011) e28063.
[92] T.K. Hoffmann, D.J. Loftus, K. Nakano, M.J. Maeurer, K. Chikamatsu, E. Appella,
et al., The ability of variant peptides to reverse the nonresponsiveness of T
lymphocytes to the wild-type sequence p53(264272) epitope, J. Immunol.
168 (2002) 13381347.
[93] X.D. Tang, Y. Wan, L. Chen, T. Chen, S.T. Yu, Z. Xiong, et al., H-2Kb-restricted
CTL epitopes from mouse heparanase elicit an antitumor immune response
in vivo, Cancer Res. 68 (2008) 15291537.
[94] M.A. Altfeld, B. Livingston, N. Reshamwala, P.T. Nguyen, M.M. Addo, A. Shea,
et al., Identification of novel HLA-A2-restricted human immunodeficiency
virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-
A2 supertype peptide-binding motif, J. Virol. 75 (2001) 13011311.
[95] Y. Hou, Y. Guo, C. Wu, N. Shen, Y. Jiang, J. Wang, Prediction and identification
of T Cell epitopes in the H5N1 influenza virus nucleoprotein in chicken, PLoS
One 7 (2012) e39344.
[96] J. Lu, E. Celis, Recognition of prostate tumor cells by cytotoxic T lymphocytes
specific for prostate-specific membrane antigen, Cancer Res. 62 (2002) 5807
5812.
[97] L.X. Wang, T. Nagata, K. Tsujimura, M. Uchijima, S. Seto, Y. Koide,
Identification of HLA-DR4-restricted T-cell epitope on MPT51 protein, a
major secreted protein derived from Mycobacterium tuberculosis using MPT51
overlapping peptides screening and DNA vaccination, Vaccine 28 (2010)
20262031.
[98] T. Aoshi, T. Nagata, M. Suzuki, M. Uchijima, D. Hashimoto, A. Rafiei, et al.,
Identification of an HLA-A0201-restricted T-cell epitope on the MPT51
protein, a major secreted protein derived from Mycobacterium tuberculosis,
by MPT51 overlapping peptide screening, Infect. Immun. 76 (2008) 1565
1571.
[99] H. Gahery, S. Figueiredo, C. Texier, S. Pouvelle-Moratille, L. Ourth, C. Igea,
et al., HLA-DR-restricted peptides identified in the Nef protein can induce HIV
type 1-specific IL-2/IFN-gamma-secreting CD4+ and CD4+/CD8+ T cells in
humans after lipopeptide vaccination, AIDS Res. Hum. Retroviruses 23 (2007)
427437.
[100] D.W. Liu, Y.C. Yang, H.F. Lin, M.F. Lin, Y.W. Cheng, C.C. Chu, et al., Cytotoxic
T-lymphocyte responses to human papillomavirus type 16 E5 and E7
proteins and HLA-A0201-restricted T-cell peptides in cervical cancer
patients, J. Virol. 81 (2007) 28692879.
[101] F. Neumann, C. Wagner, K. Preuss, B. Kubuscho, C. Schormann, S. Stevanovic,
et al., Identification of an epitope derived from the cancer testis antigen
HOM-TES-14/SCP1 and presented by dendritic cells to circulating CD4 T cells,
Blood 106 (2006) 31053113.
[102] D. Cossu, G. Mameli, G. Galleri, E. Cocco, S. Masala, J. Frau, et al., Human
interferon regulatory factor 5 homologous epitopes of Epstein-Barr virus and
Mycobacterium avium subsp. paratuberculosis induce a specific humoral and
cellular immune response in multiple sclerosis patients, Mult. Scler. 21
(2015) 984995.
[103] G. Li Pira, F. Ivaldi, C. Dentone, E. Righi, V. Del Bono, C. Viscoli, et al.,
Evaluation of antigen-specific T-cell responses with a miniaturized and
automated method, Clin. Vaccine Immunol. 15 (2008) 18111818.
[104] D.M. McKinney, R. Skvoretz, M. Qin, G. Ishioka, A. Sette, Characterization of an
in situ IFN-gamma ELISA assay which is able to detect specific peptide
responses from freshly isolated splenocytes induced by DNA minigene
immunization, J. Immunol. Methods 237 (2000) 105117.
[105] G. Li Pira, F. Ivaldi, L. Bottone, F. Manca, High throughput functional
microdissection of pathogen-specific T-cell immunity using antigen and
lymphocyte arrays, J. Immunol. Methods 326 (2007) 2232.
[106] G. Li Pira, F. Ivaldi, P. Moretti, F. Manca, High throughput T epitope mapping
and vaccine development, J. Biomed. Biotechnol. 2010 (2010) 325720.
[107] N. Gohain, W.D. Tolbert, P. Acharya, L. Yu, T. Liu, P. Zhao, et al., Cocrystal
structures of antibody N60i3 and antibody JR4 in complex with gp120
define more cluster A epitopes involved in effective antibody-dependent
effector function against HIV-1, J. Virol. 89 (2015) 88408854.
[108] A. Ramanathan, C. Immanuel, D.N. Rao, P. Kaliraj, Dissecting the immune
response elicited by WbALT-2, ALT MAP in clinical populations and mouse
model: a prophylactic measure against lymphatic filariasis, Lymphat. Res.
Biol. 13 (2015) 120125.
 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322
[109] D. Cossu, S. Masala, J. Frau, G. Mameli, M.G. Marrosu, E. Cocco, et al., Antigenic
epitopes of MAP2694 homologous to T-cell receptor gamma-chain are highly
recognized in multiple sclerosis Sardinian patients, Mol. Immunol. 57 (2014)
138140.
[110] N. Harashima, K. Tanaka, T. Sasatomi, K. Shimizu, Y. Miyagi, A. Yamada, et al.,
Recognition of the Lck tyrosine kinase as a tumor antigen by cytotoxic T
lymphocytes of cancer patients with distant metastases, Eur. J. Immunol. 31
(2001) 323332.
[111] Q.J. Wang, X.L. Huang, G. Rappocciolo, F.J. Jenkins, W.H. Hildebrand, Z. Fan,
et al., Identification of an HLA A0201-restricted CD8(+) T-cell epitope for the
glycoprotein B homolog of human herpesvirus 8, Blood 99 (2002) 3360
3366.
[112] J.A. Hural, R.S. Friedman, A. McNabb, S.S. Steen, R.A. Henderson, M. Kalos,
Identification of naturally processed CD4 T cell epitopes from the prostate-
specific antigen kallikrein 4 using peptide-based in vitro stimulation, J.
Immunol. 169 (2002) 557565.
[113] C. Czerkinsky, G. Andersson, H.P. Ekre, L.A. Nilsson, L. Klareskog, O.
Ouchterlony, Reverse ELISPOT assay for clonal analysis of cytokine
production. I. Enumeration of gamma-interferon-secreting cells, J.
Immunol. Methods 110 (1988) 2936.
[114] A. Gazagne, E. Claret, J. Wijdenes, H. Yssel, F. Bousquet, E. Levy, et al., A
Fluorospot assay to detect single T lymphocytes simultaneously producing
multiple cytokines, J. Immunol. Methods 283 (2003) 9198.
[115] J.D. Altman, P.A. Moss, P.J. Goulder, D.H. Barouch, M.G. McHeyzer-Williams, J.
I. Bell, et al., Phenotypic analysis of antigen-specific T lymphocytes, Science
274 (1996) 9496.
[116] S.H. van der Burg, M.E. Ressing, K.M. Kwappenberg, A. de Jong, K. Straathof, J.
de Jong, et al., Natural T-helper immunity against human papillomavirus
type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-
positive cervical lesions: identification of 3 human leukocyte antigen
class II-restricted epitopes, Int. J. Cancer 91 (2001) 612618.
[117] M. Terajima, J. Cruz, G. Raines, E.D. Kilpatrick, J.S. Kennedy, A.L. Rothman,
et al., Quantitation of CD8+ T cell responses to newly identified HLA-A0201-
restricted T cell epitopes conserved among vaccinia and variola (smallpox)
viruses, J. Exp. Med. 197 (2003) 927932.
[118] P.T. Tan, A.T. Heiny, O. Miotto, J. Salmon, E.T. Marques, F. Lemonnier, et al.,
Conservation and diversity of influenza A H1N1 HLA-restricted T cell epitope
candidates for epitope-based vaccines, PLoS One 5 (2010) e8754. 8710.1371/
journal.pone.0008754.
[119] P.A. Gillis, N. Hernandez-Alvarado, J.S. Gnanandarajah, F. Wussow, D.J.
Diamond, M.R. Schleiss, Development of a novel, guinea pig-specific IFN-
gamma ELISPOT assay and characterization of guinea pig cytomegalovirus
GP83-specific cellular immune responses following immunization with a
modified vaccinia virus Ankara (MVA)-vectored GP83 vaccine, Vaccine 32
(2014) 39633970.
[120] C.A. Semeniuk, R.E. Capina, M.G. Mendoza, J. Kimani, T.B. Ball, M. Luo, et al.,
Identification and characterization of HLA-A0301 epitopes in HIV-1 gag
proteins using a novel approach, J. Immunol. Methods 352 (2010) 118125.
[121] B. Ondondo, S. Abdul-Jawad, A. Bridgeman, T. Hanke, Characterization of
T-cell responses to conserved regions of the HIV-1 proteome in BALB/c mice,
Clin. Vaccine Immunol. 21 (2014) 15651572.
[122] J. Zhang, J. Yang, D. Fan, H. Tao, H. Wang, T. Yu, Peptide FLNPDVLDI of
heparanase is a novel HLA-A2-restricted CTL epitope and elicits potent
immunological antitumor effects in vitro with an 8-branched design, Oncol.
Rep. 29 (2013) 19551961.
[123] K. Horvati, S. Bosze, H.P. Gideon, B. Bacsa, T.G. Szabo, R. Goliath, et al.,
Population tailored modification of tuberculosis specific interferon-gamma
release assay, J. Infect. 72 (2016) 179188.
[124] D. Hinz, C. Oseroff, J. Pham, J. Sidney, B. Peters, A. Sette, Definition of a pool of
epitopes that recapitulates the T cell reactivity against major house dust mite
allergens, Clin. Exp. Allergy 45 (2015) 16011612.
[125] M.H. Andersen, L.O. Pedersen, B. Capeller, E.B. Brcker, J.C. Becker, Thor
Straten P, Spontaneous cytotoxic T-cell responses against survivin-derived
MHC class I-restricted T-cell epitopes in situ as well as ex vivo in cancer
patients, Cancer Res. 61 (2001) 59645968.
[126] V. Novitsky, N. Rybak, M.F. McLane, P. Gilbert, P. Chigwedere, I. Klein, et al.,
Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-,
Rev-, and Nef-specific elispot-based cytotoxic T-lymphocyte responses for
AIDS vaccine design, J. Virol. 75 (2001) 92109228.
[127] J.R. Currier, M. deSouza, P. Chanbancherd, W. Bernstein, D.L. Birx, J.H. Cox,
Comprehensive screening for human immunodeficiency virus type 1
subtype-specific CD8 cytotoxic T lymphocytes and definition of degenerate
epitopes restricted by HLA-A0207 and -C(W)0304 alleles, J. Virol. 76 (2002)
49714986.
[128] N. Frahm, B.T. Korber, C.M. Adams, J.J. Szinger, R. Draenert, M.M. Addo, et al.,
Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in
human immunodeficiency virus across multiple ethnicities, J. Virol. 78 (2004)
21002187.
[129] J.R. Currier, U. Visawapoka, S. Tovanabutra, C.J. Mason, D.L. Birx, F.E.
McCutchan, et al., CTL epitope distribution patterns in the Gag and Nef
proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple
peptide sets increases the detectable breadth of the CTL response, BMC
Immunol. 7 (2006) 8.
[130] N. Frankenberg, S. Pepperl-Klindworth, R.G. Meyer, B. Plachter, Identification
of a conserved HLA-A2-restricted decapeptide from the IE1 protein (pUL123)
of human cytomegalovirus, Virology 295 (2002) 202216.
21
[131] J.W. Zinckgraf, M. Sposato, V. Zielinski, D. Powell, J.J. Treanor, E. von Hofe,
Identification of HLA class II H5N1 hemagglutinin epitopes following
subvirion influenza A (H5N1) vaccination, Vaccine 27 (2009) 53935401.
[132] E.A. Claassen, G.M. van Bleek, Z.S. Rychnavska, R.J. de Groot, E.J. Hensen, E.J.
Tijhaar, et al., Identification of a CD4 T cell epitope in the pneumonia virus of
mice glycoprotein and characterization of its role in protective immunity,
Virology 368 (2007) 1725.
[133] M. Widhe, C. Ekerfelt, S. Jarefors, B.H. Skogman, E.M. Peterson, S. Bergstrm,
et al., T-cell epitope mapping of the Borrelia garinii outer surface protein A in
lyme neuroborreliosis, Scand. J. Immunol. 70 (2009) 141148.
[134] D. Suzuki, T. Nagata, G. Eweda, S. Matsumoto, M. Matsumoto, K. Tsujimura,
et al., Characterization of murine T-cell epitopes on mycobacterial
DNA-binding protein 1 (MDP1) using DNA vaccination, Vaccine 28 (2010)
20202025.
[135] E. Ferris, F. Connan, F. Pags, J. Gaston, A.M. Hagnr, A. Vieillefond, et al.,
Identification of p53 peptides recognized by CD8(+) T lymphocytes from
patients with bladder cancer, Hum. Immunol. 62 (2001) 791798.
[136] A. Kiessling, M. Schmitz, S. Stevanovic, B. Weigle, K. Hlig, M. Fssel, et al.,
Prostate stem cell antigen: identification of immunogenic peptides and
assessment of reactive CD8+ T cells in prostate cancer patients, Int. J. Cancer
102 (2002) 390397.
[137] R. Konopitzky, U. Knig, R.G. Meyer, W. Sommergruber, T. Wlfel, T.
Schweighoffer, Identification of HLA-A0201-restricted T cell epitopes
derived from the novel overexpressed tumor antigen calcium-activated
chloride channel 2, J. Immunol. 169 (2002) 540547.
[138] H.M. Zarour, B. Maillere, V. Brusic, K. Coval, E. Williams, S. Pouvelle-Moratille,
et al., NY-ESO-1 119143 is a promiscuous major histocompatibility complex
class II T-helper epitope recognized by Th1- and Th2-type tumor-reactive
CD4+ T cells, Cancer Res. 62 (2002) 213218.
[139] M. Sedegah, Y. Kim, H. Ganeshan, J. Huang, M. Belmonte, E. Abot, et al.,
Identification of minimal human MHC-restricted CD8+ T-cell epitopes within
the Plasmodium falciparum circumsporozoite protein (CSP), Malar. J. 185
(2013), http://dx.doi.org/10.1186/1475-2875-1112-1185.
[140] K. Sundar, A. Boesen, R. Coico, Computational prediction and identification of
HLA-A2.1-specific Ebola virus CTL epitopes, Virology 360 (2007) 257263.
[141] M. Wang, M.V. Larsen, M. Nielsen, M. Harndahl, S. Justesen, M.H. Dziegiel,
et al., HLA class I binding 9mer peptides from influenza A virus induce CD4 T
cell responses, PLoS One 5 (2010) e10533.
[142] Z. Guo, H. Zhang, H. Rao, D. Jiang, X. Cong, B. Feng, et al., DCs pulsed with
novel HLA-A2-restricted CTL epitopes against hepatitis C virus induced a
broadly reactive anti-HCV-specific T lymphocyte response, PLoS One 7 (2012)
e38390.
[143] M. Wang, S.T. Tang, A. Stryhn, S. Justesen, M.V. Larsen, M.H. Dziegiel, et al.,
Identification of MHC class II restricted T-cell-mediated reactivity against
MHC class I binding Mycobacterium tuberculosis peptides, Immunology 132
(2011) 482491.
[144] A.S. De Groot, A. Bosma, N. Chinai, J. Frost, B.M. Jesdale, M.A. Gonzalez, et al.,
From genome to vaccine: in silico predictions, ex vivo verification, Vaccine 19
(2001) 43854395.
[145] A. Jaramillo, K. Majumder, P.P. Manna, T.P. Fleming, G. Doherty, J.F. Dipersio,
et al., Identification of HLA-A3-restricted CD8+ T cell epitopes derived from
mammaglobin-A, a tumor-associated antigen of human breast cancer, Int. J.
Cancer 102 (2002) 499506.
[146] A. Kather, A. Ferrara, M. Nonn, M. Schinz, J. Nieland, A. Schneider, et al.,
Identification of a naturally processed HLA-A0201 HPV18 E7 T cell epitope
by tumor cell mediated in vitro vaccination, Int. J. Cancer 104 (2003) 345353.
[147] T. Chen, X.D. Tang, Y. Wan, L. Chen, S.T. Yu, Z. Xiong, et al., HLA-A2-restricted
cytotoxic T lymphocyte epitopes from human heparanase as novel targets for
broad-spectrum tumor immunotherapy, Neoplasia 10 (2008) 977986.
[148] S.D. Xiang, Q. Gao, K.L. Wilson, A. Heyerick, M. Plebanski, Mapping T and B
cell epitopes in sperm protein 17 to support the development of an ovarian
cancer vaccine, Vaccine 33 (2015) 59505959.
[149] X. Li, S. Yuan, S. He, J. Gao, H. Chen, Identification and characterization of the
antigenic site (epitope) on bovine beta-lactoglobulin: common residues in
linear and conformational epitopes, J. Sci. Food Agric. 95 (2015) 29162923.
[150] M.L. Selenica, H. Davtyan, S.B. Housley, L.J. Blair, A. Gillies, B.A. Nordhues,
et al., Epitope analysis following active immunization with tau proteins
reveals immunogens implicated in tau pathogenesis, J. Neuroinflammation
11 (2014) 152.
[151] J.J. Zaunders, M.L. Munier, N. Seddiki, S. Pett, S. Ip, M. Bailey, et al., High levels
of human antigen-specific CD4+ T cells in peripheral blood revealed by
stimulated coexpression of CD25 and CD134 (OX40), J. Immunol. 183 (2009)
28272836.
[152] M. Frentsch, O. Arbach, D. Kirchhoff, B. Moewes, M. Worm, M. Rothe, et al.,
Direct access to CD4+ T cells specific for defined antigens according to CD154
expression, Nat. Med. 11 (2005) 11181124.
[153] N.H.R. Litjens, E.A. de Wit, C.C. Baan, M.G.H. Betjes, Activation-induced CD137
is a fast assay for the identification and multi-parameter flow cytometric
analysis of alloreactive t cells, Clin. Exp. Immunol. 174 (2013) 179191.
[154] I.Z. Hartman, A. Kim, R.J. Cotter, K. Walter, S.K. Dalai, T. Boronina, et al., A
reductionist cell-free major histocompatibility complex class II antigen
processing system identifies immunodominant epitopes, Nat. Med. 16
(2010) 13331340.
[155] T.J. Messitt, F. Terry, L. Moise, W. Martin, A.S. De Groot, A comparison of two
methods for T cell epitope mapping: cell free in vitro versus
immunoinformatics, Immunome Res. 7 (2011) e6.
22 T.A. Ahmad et al. / Vaccine Reports 6 (2016) 1322
[156] M. Marcilla, I. Alvarez, A. Ramos-Fernandez, M. Lombardia, A. Paradela, J.P.
Albar, Comparative analysis of the endogenous peptidomes displayed by
HLA-B27 and mamu-B08: Two MHC class I alleles associated with elite
control of HIV/SIV infection, J. Proteome Res. 15 (2016) 10591069.
[157] M.N. Davies, D.R. Flower, Harnessing bioinformatics to discover new
vaccines, Drug Discov. Today 12 (2007) 389395.
[158] A. Patronov, I. Doytchinova, T-cell epitope vaccine design by
immunoinformatics, Open Biol. 3 (2013) 120139.
[159] A.S. De Groot, V. Nene, N.R. Hegde, S. Srikumaran, J. Rayner, W. Martin, T cell
epitope identification for bovine vaccines: an epitope mapping method for
BoLA A-11, Int. J. Parasitol. 33 (2003) 641653.
[160] W. Martin, H. Sbai, A.S. De Groot, Bioinformatics tools for identifying
class I-restricted epitopes, Methods 29 (2003) 289298.
[161] R. Vita, J.A. Overton, J.A. Greenbaum, J. Ponomarenko, J.D. Clark, J.R. Cantrell,
et al., The immune epitope database (IEDB) 3.0, Nucleic Acids Res. 43 (2015)
D405D412.
[162] L. Moise, A. Gutierrez, F. Kibria, R. Martin, R. Tassone, R. Liu, et al., IVAX: An
integrated toolkit for the selection and optimization of antigens and the
design of epitope-driven vaccines, Hum. Vaccin. Immunother. 11 (2015)
23122321.
[163] T. Sturniolo, E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, et al.,
Generation of tissue-specific and promiscuous HLA ligand databases using
DNA microarrays and virtual HLA class II matrices, Nat. Biotechnol. 17 (1999)
555561.
[164] A.S. De Groot, B.M. Jesdale, E. Szu, J.R. Schafer, R.M. Chicz, G. Deocampo, An
interactive Web site providing major histocompatibility ligand predictions:
application to HIV research, AIDS Res. Hum. Retroviruses 13 (1997) 529531.
[165] A.S. De Groot, B. Jesdale, W. Martin, C. Saint Aubin, H. Sbai, A. Bosma, et al.,
Mapping cross-clade HIV-1 vaccine epitopes using a bioinformatics
approach, Vaccine 21 (2003) 44864504.
[166] A.S. De Groot, E.A. Bishop, B. Khan, M. Lally, L. Marcon, J. Franco, et al.,
Engineering immunogenic consensus T helper epitopes for a cross-clade HIV
vaccine, Methods 34 (2004) 476487.
[167] A.S. De Groot, W. Martin, From immunome to vaccine: epitope mapping and
vaccine design tools, Novartis Found. Symp. 254 (2003) 5772.
[168] H. Rammensee, J. Bachmann, N.P. Emmerich, O.A. Bachor, S. Stevanovic,
SYFPEITHI: database for MHC ligands and peptide motifs, Immunogenetics 50
(1999) 213219.
[169] P.A. Reche, J.P. Glutting, E.L. Reinherz, Prediction of MHC class I binding
peptides using profile motifs, Hum. Immunol. 63 (2002) 701709.
[170] A.S. De Groot, L. Marcon, E.A. Bishop, D. Rivera, M. Kutzler, D.B. Weiner, et al.,
HIV vaccine development by computer assisted design: the GAIA vaccine,
Vaccine 23 (2005) 21362148.
[171] A. Fridman, A.C. Finnefrock, D. Peruzzi, I. Pak, N. La Monica, A. Bagchi, et al.,
An efficient T-cell epitope discovery strategy using in silico prediction and
the iTopia assay platform, Oncoimmunology 1 (2012) 12581270.
[172] L. Moise, A.H. Gutierrez, C. Bailey-Kellogg, F. Terry, Q. Leng, K.M. Abdel Hady,
et al., The two-faced T cell epitope: examining the host-microbe interface
with JanusMatrix, Hum. Vaccin. Immunother. 9 (2013) [Epub ahead of print].
[173] X. Song, L. Xu, R. Yan, X. Huang, X. Li, Construction of Eimeria tenella multi-
epitope DNA vaccines and their protective efficacies against experimental
infection, Vet. Immunol. Immunopathol. 166 (2015) 7987.
[174] P. Carlos, V. Roupie, S. Holbert, F. Ascencio, K. Huygen, G. Gomez-Anduro,
et al., In silico epitope analysis of unique and membrane associated proteins
from Mycobacterium avium subsp. paratuberculosis for immunogenicity and
vaccine evaluation, J. Theor. Biol. 384 (2015) 19.
[175] R.S. Salvat, A.S. Parker, Y. Choi, C. Bailey-Kellogg, K.E. Griswold, Mapping the
Pareto optimal design space for a functionally deimmunized biotherapeutic
candidate, PLoS Comput. Biol. 11 (2015) e1003988.
[176] O.A. Koita, D. Dabitao, I. Mahamadou, M. Tall, S. Dao, A. Tounkara, et al.,
Confirmation of immunogenic consensus sequence HIV-1 T-cell epitopes in
Bamako, Mali and Providence, Rhode Island, Hum. Vaccin. 2 (2006).
View publication stats
[177] L. Levitz, O.A. Koita, K. Sangare, M.T. Ardito, C.M. Boyle, J. Rozehnal, et al.,
Conservation of HIV-1 T cell epitopes across time and clades: validation of
immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine, Vaccine 30
(2012) 75477560.
[178] A.S. De Groot, L. Levitz, M.T. Ardito, G. Skowron, K.H. Mayer, S. Buus, et al.,
Further progress on defining highly conserved immunogenic epitopes for a
global HIV vaccine: HLA-A3-restricted GAIA vaccine epitopes, Hum. Vaccin.
Immunother. 8 (2012) 9871000.
[179] M. Mori, K. Matsuki, T. Maekawa, M. Tanaka, B. Sriwanthana, M. Yokoyama,
et al., Development of a novel in silico docking simulation model for the fine
HIV-1 cytotoxic T lymphocyte epitope mapping, PLoS One 7 (2012) e41703.
[180] C.M. Diez-Rivero, P.A. Reche, CD8 T cell epitope distribution in viruses reveals
patterns of protein biosynthesis, PLoS One 7 (2012) e43674. 43610.41371/
journal.pone.0043674.
[181] V.R. Duvvuri, A. Marchand-Austin, A. Eshaghi, S.N. Patel, D.E. Low, J.B.
Gubbay, T. Potential, Cell epitopes within swine-origin triple reassortant
influenza A (H3N2) variant virus which emerged in, an immunoinformatics
study, Vaccine 30 (2012) (2011) 60546063.
[182] F. Wen, J.H. Ma, H. Yu, F.R. Yang, M. Huang, Y.J. Zhou, et al., A novel M2e
multiple antigenic peptide providing heterologous protection on mice, J. Vet.
Sci. (2015).
[183] C.T. Su, C. Schnbach, C.K. Kwoh, Molecular docking analysis of 2009-H1N1
and 2004-H5N1 influenza virus HLA-B4405-restricted HA epitope
candidates: implications for TCR cross-recognition and vaccine
development, BMC Bioinformatics 14 (Suppl. 2:S21) (2013), http://dx.doi.
org/10.1186/1471-2105-1114-S1182-S1121. Epub 2013 Jan 1121.
[184] S. Nitipan, T. Sritrakul, A. Kunjantarachot, S. Prapong, Identification of
epitopes in Leptospira borgpetersenii leucine-rich repeat proteins, Infect.
Genet. Evol. 14 (2013) 4657.
[185] M. Panigada, T. Sturniolo, G. Besozzi, M.G. Boccieri, F. Sinigaglia, G.G. Grassi,
et al., Identification of a promiscuous T-cell epitope in Mycobacterium
tuberculosis Mce proteins, Infect. Immun. 70 (2002) 7985.
[186] T.A. Ahmad, B.A. El-Sayed, L.H. El-Sayed, Development of immunization trials
against Eimeria spp, Trials Vaccinol. 5 (2016) 3847.
[187] A. Rana, A. Rub, Y. Akhter, Proteome-wide B and T cell epitope repertoires in
outer membrane proteins of Mycobacterium avium subsp. paratuberculosis
have vaccine and diagnostic relevance: a holistic approach, J. Mol. Recognit.
28 (2015) 506520.
[188] A. Rana, Y. Akhter, A multi-subunit based, thermodynamically stable model
vaccine using combined immunoinformatics and protein structure based
approach, Immunobiology 221 (2016) 544557.
[189] M. Bello, R. Campos-Rodriguez, S. Rojas-Hernandez, A. Contis-Montes de Oca,
J. Correa-Basurto, Predicting peptide vaccine candidates against H1N1
influenza virus through theoretical approaches, Immunol. Res. 62 (2015)
315.
[190] S. Momtaz, A. Rahman, M. Sultana, M.A. Hossain, Evolutionary analysis and
prediction of peptide vaccine candidates for Foot-and-Mouth-Disease Virus
types A and O in Bangladesh, Evol. Bioinform. Online 10 (2014) 187196.
[191] H.M. Geysen, R.H. Meloen, S.J. Barteling, Use of peptide synthesis to probe
viral antigens for epitopes to a resolution of a single amino acid, Proc. Natl.
Acad. Sci. 81 (1984) 39984002.
[192] B.C. Barnett, D.S. Burt, C.M. Graham, A.P. Warren, J.J. Skehel, D.B. Thomas, I-Ad
restricted T cell recognition of influenza hemagglutinin. Synthetic peptides
identify multiple epitopes corresponding to antibody-binding regions of the
HA1 subunit, J. Immunol. 143 (1989) 26632669.
[193] I. Jelcic, L. Aly, T.M. Binder, I. Jelcic, S. Bofill-Mas, R. Planas, et al., T cell epitope
mapping of JC polyoma virus-encoded proteome reveals reduced T cell
responses in HLA-DRB104:01+ donors, J. Virol. 87 (2013) 33933408.
[194] S. Tu, H. Huang, S.I. Lin, H.Y. Liu, Y. Sher, S. Chiang, et al., A novel
HLA-A2 -restricted CTL epitope of tumor-associated antigen L6 can inhibit
tumor growth In Vivo, J. Immunother. 35 (2012) 235244.