Molecular and Cellular Probes 43 (2019) 40–44

Contents lists available at ScienceDirect

Molecular and Cellular Probes

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

Prediction of epitopes of Neisseria Gonorrhoeae against USA human T

leukocyte antigen background: An immunoinformatic approach towards
development of future vaccines for USA population
Malik Siddique Mahmooda, Muhammad Asad-Ullahb, Hina Batoolc, Sana Batoolc,
Naeem Mehmood Ashrafd,∗
a
Institute of Biochemistry and Biotechnology, University of the Punjab, P. O Box No, 54590, Pakistan
b
Department of Biotechnology, Quaid-i-Azam University, P.O Box No, 45320, Islamabad, Pakistan
c
School of Biological Sciences, University of the Punjab, P. O Box No, 54590, Lahore, Pakistan
d
Department of Biochemistry and Biotechnology, University of Gujrat, P. O Box No, 50700, Pakistan

A R T I C LE I N FO A B S T R A C T

Keywords: Gonorrheal infections are the second most prevalent sexually transmitted diseases STDs in the USA populations
Epitope-based vaccines after Chlamydia. These infections have now become an urgent problem to address because Neisseria gonorrhoeae
Neisseria Gonorrheae is capable of developing resistance to multiple antibiotic classes. Infection with these antibiotic-resistant strains
Human leukocyte antigens has become the major public health concern. Although extensive researches are ongoing to control its trans-
USA population
mission and to develop the productive treatments against this pathogen, no effective vaccine could develop till
now. The present study will effectively contribute to the future designing of USA specific epitope-based vaccines.
Through computational approaches, this study has highlighted putative epitopes from Neisseria gonorrhoeae
which restrict maximally to the frequent HLA alleles in the USA populations. Antigenic and non-allergenic
nature of predicted epitopes was verified using vexijen and AllerTOP tools respectively. Total seven epitopes,
four from class-I and three from class-II were antigenic as well as non-allergenic. These epitopes showed USA
population coverage of 99.3% with no allergenic response. Still, additional studies are needed to validate the
immunogenic properties of the predicted epitopes which are likely vaccine target for gonorrhoea in the USA
populations.

1. Introduction
Gonorrhoea, one of the most prevalent sexually transmitted diseases
(STD) globally, is caused by the Neisseria gonorrhoeae [1]. Current sta-
tistics, from the Center for Disease Control and Prevention (CDC), de-
monstrates a progressive increase in the Gonorrheal infections during
the past few years. In the USA, in 2012 total 106.7 cases of gonorrhoea
were reported and this rate increased steadily from 2014 to 2016. In
2016, about 145.8 cases were reported from 100,000 USA populations,
depicting about 18.5% increase from the previous statistics. This per-
sistent increase has ranked gonorrhoea as the second most prevalent
STD in the USA just after Chlamydia. It affects both sexes and results in
severe morbidities if left untreated [2].
Despite active research, effective control options for this disease are
still not available, which is further complicating the situation. One of
the pioneering approaches used for management of the disease was the
use of antimicrobial agents which includes sulfonamides, penicillins,
cephalosporins, tetracyclines, macrolides, and fiuoroquinolone.
However, global emergence and transmission of the resistant strains
have left this treatment strategy inadequate [3]. Another option was the
use of vaccines, and multiple attempts are made, towards the devel-
opment of gonorrhoea's vaccine [4]. Although some vaccines are patent
the problem remains the same [5,6]. Among the many reasons which
result in the lack of potent vaccines against the organism includes the
ability of the pathogen to undergo antigenic variations, unavailability
of proper animal models to try the vaccines, and generation of aller-
genic response against many candidate vaccines [7–9].
Recently epitope-based vaccines are replacing the trivial vaccines.
Instead of focusing on the full antigenic proteins for vaccine develop-
ment, short amino acid sequences from the antigenic protein (epitopes),

∗
Corresponding author.
E-mail addresses: siddique.phd.ibb@pu.edu.pk (M.S. Mahmood), asad_ullah8050@yahoo.com (M. Asad-Ullah), hinabatool198@gmail.com (H. Batool),
sanabatool939@gmail.com (S. Batool), naeem.mahmood@uog.edu.pk (N.M. Ashraf).

https://doi.org/10.1016/j.mcp.2018.11.007
Received 16 September 2018; Received in revised form 9 November 2018; Accepted 22 November 2018
Available online 29 November 2018
0890-8508/ © 2018 Elsevier Ltd. All rights reserved.
M.S. Mahmood et al. Molecular and Cellular Probes 43 (2019) 40–44

likely to induce more directed and potent immune response are under with < 500 nM have intermediate affinity and epitopes
analysis. This strategy of vaccines designing is although safer and target with < 5000 nM low affinity. All predicted T cell epitopes for both
immune response against specific epitopes, but it requires accurate MHC class-I and MHC class-II with IC50 value < 500 nM were con-
knowledge of the amino acid sequence of the immunogenic protein of sidered significant for analysis [16].
interest. An epitope can evoke the significant immune response in the
MHC-restricted individuals, which displays epitope in complex with
2.4. Antigenicity prediction
particular MHC molecule. Human Leukocyte Antigen (HLA) alleles are
remarkably polymorphic and have varying combinations of the alleles
All of the epitopes retrieved in the previous step validated for their
in different populations [10]. Based on this fact, after the identification
antigenic potentials through VaxiJen v.2.0. The server is known for its
of epitopes from the aetiological agent, the binding assessment of the
alignment-free prediction of antigens based solely on their physio-
selected epitopes with population-specific HLA alleles should also be
chemical properties. The threshold value used against bacteria as the
calculated. It would help to design vaccines which show the maximum
targeted organism was 0.5. Epitopes with threshold values of greater
immune response in the targeted populations [11].
than 0.5 considered as antigenic [19].
For the development of epitope-based vaccines against gonorrhoea,
several attempts have made. Most of these identified epitopes are,
however, not suitable vaccine candidates because of their allergenic 2.5. Prediction of non-allergenic nature of antigenic epitopes
effects [11,12]. Recently, non–allergenic and population-specific epi-
topes have reported for various pathogens. A similar work describes To specifically excerpt non-allergenic epitopes from all predicted
non–allergenic HCV epitopes specific for the Pakistani populations epitopes, AllerTOP v.2.0, an allergenicity prediction web-based server
[13]. Such population specific descriptive work is still not available for was used [20]. This server takes advantage of Auto Cross-Covariance
Neisseria gonorrhoeae. (ACC) method and has trained with numerously reported allergens and
The present study is concerned with the investigation of putative non-allergens from different species [21].
epitope candidates from the surface proteins of Neisseria gonorrhoeae
and to examine their interaction with a range of local HLA alleles in the
2.6. Selection of antigenic and non-allergenic epitopes
USA. Furthermore, by considering allergenicity, this study marks epi-
topes capable of eliciting elaborate immune response while lacking the
All predicted epitopes which were antigenic, as well as non-aller-
ability to cause any allergenic or harmful effect in the body. Online
genic, were selected for further analysis. By screening, antigenic and
databases and prediction servers, together with USA HLA backgrounds,
non-allergenic epitopes the possibility of any allergenic responses could
were the primary data resources to carry out this descriptive study.
be reduced.
2. Methodology
2.7. Population coverage
2.1. Selection of antigenic proteins
Non-allergenic epitopes restricted to the broad range of USA HLA
One of the earliest steps of the study was to identify putative anti- alleles would be promising vaccine target. Population coverage of the
genic proteins from Neisseria gonorrhoeae. Formerly published literature epitopes, therefore, was determined using the IEDB tool [22]. All an-
marked some proteins which are well-reported vaccine targets. Amino tigenic and non-allergenic epitopes restricted to the most of the HLA
acid sequences of the selected proteins obtained from the National alleles of the USA populations were selected.
Center of Biotechnology and Information NCBI database [14].
3. Results
2.2. Acquisition of prevalent USA specific HLA alleles
3.1. Selection of antigenic proteins
Most common class-I and class-II HLA alleles in the USA population
retrieved through the online database Allele Frequency Net Database
The extensive literature search resulted in the identification of three
(AFND). In this database allele frequency threshold of more than 3
antigenic proteins from Neisseria gonorrhoeae. All these candidate pro-
provides remarkably recurrent HLA alleles from the USA populations.
teins are considered as antigenic because of the following features.
This threshold value resulted in the retrieval of all HLA alleles sig-
These proteins are present on the surface of the pathogen, and surface
nificantly prevalent in the USA populations [15].
proteins mostly confer virulence to the pathogens. They are involved in
essential metabolic pathways of the pathogen and are non-human
2.3. Epitope scanning from antigenic proteins
homologs. Moreover, these three proteins have sequence and structural
similarity to various known antigens of different organisms [23,24].
Potent immunogenic epitopes from the full-length sequence of an-
Therefore, these three proteins are reported as potential vaccine targets
tigenic proteins were selected. T-cell epitope prediction aims to identify
multiple times and selected for procurement of significant epitopes.
the shortest peptides within an antigenic protein able to stimulate ei-
These proteins include D-alanine—D-alanine ligase (ddl) (Accession No
ther CD4 or CD8 T-cells. Potential epitope regions from the full-length
YP_208579), type IV pilin protein (Accession No YP_207607) and
sequence of antigenic proteins marked via Immune Epitope Data and
competence lipoprotein (Cpl) (Accession No YP_207439).
Analysis Resource (IEDB) server. Moreover, binding affinities of these
highlighted T cell epitopes with common USA HLA alleles accessed by
MHC-I and MHC-II binding prediction tools of the same database [16]. 3.2. Acquisition of prevalent USA specific HLA alleles
MHC-I binding prediction tool used the ANN algorithm. This algorithm
examines all peptides from the input protein and predicts their binding According to AFND analysis, a total of 8 class-I and 11 class-II HLA
affinities with HLA alleles [17]. Binding affinities of epitopes with MHC alleles were found to be widespread in the majority of the USA popu-
class-II molecules predicted by MHC-II binding prediction tool using the lations. Among these eight class-I HLA alleles four belongs to the HLA-
netMHCIIpan algorithm [18]. A, one to the HLA-B and three to HLA-C (Table 1). Likewise, five of the
The prediction results displayed in IC50 nM units. Epitopes having eleven class-II HLA alleles belong to DQB1, four to DRB1, one to the
IC50 values < 50 nM considered to have a high binding affinity, those DPB1 and one to DQA1 (Table 2) (Supplementary file 1).

41
M.S. Mahmood et al. Molecular and Cellular Probes 43 (2019) 40–44

Table 1
Detail of HLA class-I alleles prevalent in USA populations.
Common HLA class-I alleles in USA populations

Class A Frequency Class B Frequency Class C Frequency

HLA-A*01:01 3.19695 HLA-B*35:01 3.105 HLA-C*04:01 5.002

HLA-A*02:01 7.083 HLA-C*07:01 3.39029
HLA-A*11:01 3.736 HLA-C*07:02 4.952
HLA-A*24:02 5.431

3.3. Epitope scanning from antigenic proteins

IEDB marked cogent class-I and class-II epitopes from the full-length
sequences of three antigenic proteins. The binding of these epitopes
with the HLA alleles common in the USA was also confirmed through
IEDB MHC-I and MHC-II binding prediction tools at IC50 < 500 nM.
This prediction ends up with 699 class-I and 685 class-II epitopes from
the three proteins (Fig. 1). Among these D-alanine—D-alanine ligase
(ddl) provided 293 class-I and 288 class-II epitopes. Likewise, type IV
pilin protein furnished, 151 class-I and 152 class-II epitopes and com-
petence lipoprotein (Cpl) contributed 255 class-I and 245 class-II epi-
topes. All these class-I epitopes are nine amino acids long (9 mers),
while class-II epitopes are fifteen amino acids long (15 mers).

3.4. Antigenicity prediction

All predicted class-I and class-II epitopes inquired for their antigenic
behaviour through Vaxijen. From 699 class-I and 685 class-II epitopes,
vexijen marked 407 class I and 398 class II as potent antigenic epitopes
(Fig. 1).

Fig. 1. Epitope statistics: Number of antigenic and non-allergenic epitopes

3.5. Prediction of non-allergenic nature of epitopes obtained from class-I and class-II epitopes after exclusion of allergenic epitopes.

To evacuate probable allergens from the analysis AllerTOP was

used. The results derived from this database displayed 543 class-I and
473 class-II epitopes as likely allergens, thus only 156 class-I and 212
class-II epitopes were significant non-allergenic epitopes. This step ef-
ficiently separated non-allergenic epitopes from the probable allergens.
3.6. Antigenic and non-allergenic epitopes
Highly promising epitopes exhibit significant antigenicity and at the
same time are non-allergenic. By applying these two filters together
total 28 antigenic and non-allergenic class-I epitopes were retrieved.
Out of these 28 epitopes, thirteen were from D-alanine—D-alanine li-
gase, seven from type IV pilin protein and eight from competence li-
poprotein (Supplementary file 2). Similarly, 63 class-II epitopes; 19
from D-alanine—D-alanine ligase, 20 from type IV pilin protein, and 24
from competence lipoprotein were antigenic and non-allergens
(Supplementary file 3).
3.7. Population coverage
The antigenic and non–allergenic epitopes having significant po-
pulation coverage against USA specific HLA alleles are likely vaccine
targets. Population coverage of the antigenic and non-allergenic epi-
topes calculated using the IEDB database. As a result, four class-I and
three class-II epitopes together exhibit 99.2% coverage against the USA
populations (Table 3). These seven epitopes together restrict to most of
the USA HLA alleles a cocktail of these seven antigenic and non-aller-
genic epitopes is, therefore, considered as potential vaccine targets for
these populations (Supplementary file 4).
4. Discussion
Recently, the development of antimicrobial resistant strains of
Neisseria gonorrhoeae, have curtailed the efficacy of this routinely used
treatment option [3]. This catastrophe has transformed the research
trends towards designing of the epitope-based vaccines [25]. Even

Table 2
Detail of HLA class-II alleles prevalent in USA populations.
Common HLA class-II alleles in USA populations

Class DPB1 Frequency Class DQB1 Frequency Class DRB1 Frequency Class DQA1 Frequency

DPB1*04:02 3.0007 DQB1*02:01 3.01 DRB1*03:01 3.1018 DQA1*05:01 7.156

DQB1*03:01 7.156 DRB1*07:01 3.98438
DQB1*03:02 3.49 DRB1*14:02 3.00852
DQB1*05:01 3.372 DRB1*15:01 3.084
DQB1*06:02 3.291

42
M.S. Mahmood et al.
Table 3
Class-I and Class-II antigenic and non-allergenic epitopes having maximum
population coverage to USA specific HLA alleles.
Epitope HLA class Population Population coverage of
coverage all combined epitopes
FSSEREISL Class-I 17.30% 99.22%
LYLLEINTL Class-I 38.73%
WMNSVGDGY Class-I 24.28%
AQITQDWSV Class-I 42.88%
AVTGVGFADLCIEIL Class-II 83.73%
YLLEINTLPGMTGHS Class-II 86.21%
KYAADATARMVKLVD Class-II 86.00%
though typical vaccines are available for Neisseria gonorrhoeae but some
notable restraints like genetic heterogenicity, lack of adequate immune
response and allergenic responses in some individuals, have proved
these vaccines to be less potent [7,26]. Consequently, epitope-based
vaccines are the most recent and reliable choice available these days.
These vaccines can combat with mutating pathogen aptly and therefore
may produce prominent and long-lasting immune response [4,27,28].
Epitope prediction is however quite strenuous and costly as it de-
mands experimental validation of a large number of candidate epitopes.
Fortunately, advances in the bioinformatics have helped researchers to
reduce the burden associated with epitope mapping by subsiding, the
list of potential epitope candidates required for experimental testing.
Moreover, through in-silico analysis, it is possible to identify significant
non-allergenic vaccine targets from the pathogen that restrict to HLA
alleles prevalent in the population of interest. Such in-silico studies thus
help in the production of putative population specific epitope-based
vaccines economically [11,12].
The present study is designed accordingly with the aim to mark
antigenic and non-allergenic class-I and class-II epitopes from the sur-
face proteins of Neisseria gonorrheae, which restrict to the frequent USA
HLA-alleles. The predicted epitopes would, in turn, can be used in fu-
ture designing of gonorrheal vaccines that would be effective for the
USA populations and unable to elicit allergenic responses.
Till date, many vaccine targets have identified in Neisseria gonor-
rhoeae using an in-silico subtractive genomics approach. In this in-silico
based study, three antigenic proteins from the surface of Neisseria go-
norrhoeae selected through an extensive literature review. These pro-
teins are reported to have antigenic abilities. Moreover, these three
antigenic proteins are the non-human homologs involved in pathogen-
specific metabolic pathways and located on the cell surface of the or-
ganism. Therefore these could be considered as potential vaccine can-
didates. Three candidate proteins which were selected include; D-
alanine—D-alanine ligase (ddl), type IV pilin protein and competence
lipoprotein [4,23,24].
After that class-I and class-II, HLA alleles common in the USA po-
pulation retrieved through AFND at allele frequency threshold of
greater than 3 [15]. Eight class-I and eleven class-II HLA alleles were
reported to be widespread, in the majority of the USA populations at
this threshold (Tables 1 and 2). For the identification of putative epi-
topes from a full-length sequence of three antigenic proteins IEDB
server was employed [16]. Total 699 class-I and 685 class-II retrieved
from three selected antigenic proteins. T-cell epitopes presented to-
gether with the MHC-I and MHC-II HLA alleles are known to elicit an
effective immune response [29]. Therefore, epitopes capable of binding
to the common USA HLA alleles selected by MHC-I and MHC-II binding
prediction tools. IEDB results showed that all 699 class-I and 685 class-
II epitopes restricted to the USA specific HLA alleles at IC50 value <
500 nM [16].
Afterwards, antigenicity of the epitopes confirmed through VaxiJen
v.2.0 server [19]. The results at this step marked about 407 class-I and
398 class-II epitopes as antigenic. To omit all allergenic epitopes from
the analysis AllerTOP database was used which helped in the separation
43
Molecular and Cellular Probes 43 (2019) 40–44
of probable allergens from the non-allergens [20]. From 699 class-I
epitopes, 543 were allergenic likewise, from 685 class-II epitopes, 473
were allergenic. After determining antigenicity and allergenicity sepa-
rately, both of these filters applied collectively and 28 class-I and 63
class-II epitopes were retrieved (Fig. 1). These epitopes being antigenic
and non-allergenic are likely to elicit the maximum immune response
without any allergenic side effects. Finally, population coverage of all
antigenic and non-allergenic epitopes calculated through IEDB [22].
Total 7 epitopes from the previous list were showing 99.2% coverage of
the USA populations (Table 3). These epitopes, therefore, could be the
efficient vaccine targets to control gonorrheal infections in these po-
pulations (Supplementary file 4).
5. Conclusion
The epitopes identified through this computational approach are
antigenic, non-allergenic and restrict to the majority of the USA HLA
alleles.
As these epitopes have maximum USA population coverage, there-
fore, they can be used to design an epitope-based vaccine that can scale
down the ongoing prevalence of gonorrheal infections in these popu-
lations. Additionally, by expelling the probable allergens, these vac-
cines can assure non-allergenic immune responses in the infected in-
dividual. The results of the study, therefore, would likely to curtail the
gap between progression and control of infection and help to lower the
excessively high morbidity rate associated with this STD.
Statement of conflict of interest
Authors have declared no confiict of interest.
Acknowledgement
We are thankful for all the online available open access databases
used in this research. The research was self-supported financially by the
authors.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.mcp.2018.11.007.
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