- Email: [email protected]
Candidate antigenic epitopes for vaccination and diagnosis strategies of Toxoplasma gondii infection: A review
Journal Pre-proof Candidate antigenic epitopes for vaccination and diagnosis strategies of Toxoplasma gondii infection: A review Amirreza Javadi Mamaghani, Anwar Fathollahi, Adel Spotin, Mohammad mehdi Ranjbar, Meisam Barati, Somayeh Aghamolaie, Maryam Karimi, Niloofar Taghipour, Mohammad Ashrafi, Seyyed Javad Seyyed Tabaei PII:
S0882-4010(19)31266-5
DOI:
https://doi.org/10.1016/j.micpath.2019.103788
Reference:
YMPAT 103788
To appear in:
Microbial Pathogenesis
Received Date: 15 July 2019 Revised Date:
5 September 2019
Accepted Date: 8 October 2019
Please cite this article as: Javadi Mamaghani A, Fathollahi A, Spotin A, Ranjbar Mm, Barati M, Aghamolaie S, Karimi M, Taghipour N, Ashrafi M, Tabaei SJS, Candidate antigenic epitopes for vaccination and diagnosis strategies of Toxoplasma gondii infection: A review, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2019.103788. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Candidate antigenic epitopes for vaccination and diagnosis strategies of
2
Toxoplasma gondii infection: A review
3
Amirreza Javadi Mamaghani 1, Anwar Fathollahi2, Adel Spotin3,4, Mohammad mehdi
4
Ranjbar5 , Meisam Barati6, Somayeh Aghamolaie1, Maryam Karimi7, Niloofar Taghipour1,
5
Mohammad Ashrafi8, Seyyed Javad Seyyed Tabaei 1* 1
6
Department of Parasitology and Mycology, School of Medicine, Student Research Committee,
7 8
Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2
Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical,
9 10
Kermanshah, Iran. 3
Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 4
11 5
12
Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension
13 14
Organization (AREEO), Karaj, Iran 6
Department of Cellular and Molecular Nutrition, Shahid Beheshti University of Medical Sciences,
15 16 17
Tehran, Iran. 7
Cellular and Molecular Research Center Kurdistan University of Medical Science, Sanandaj, Iran 8
Faculty of Medicine, Islamic Azad University, Qom, Qom, Iran.
18
Corresponding author:
19
Seyyed Javad Seyyed Tabaei; Department of Parasitology and Mycology, Shahid Beheshti University of
20
Medical Sciences, Tehran, Iran. Fax: +98 21 22439962.; Email: [email protected] ,
21
[email protected]
22 23 24 25 26 27
1
28
Abstract
29
Toxoplasmosis caused by an obligatory intracellular protozoan parasite of Toxoplasma gondii
30
threats a wide spectrum of human and animal hosts. It has been shown that the intensity of
31
the disease in humans depends on the host's immune responses. Immunological investigations
32
on whole protein molecules of T. gondii have shown that these antigens are not fully
33
responsible for the immune response, which leads to a decrease in specificity and affinity of
34
the antigen (epitope)-antibody (paratope) binding. Currently, epitopes have shown promising
35
entities to stimulate B, T, cytotoxic T lymphocyte, and NK cells resulting in enhancement of
36
protective immunity against toxoplasmosis patients. Thus, the accurate designing, prediction,
37
and conducting of antigenic epitopes of T. gondii (with linear and/or spatial structures) can
38
augment our understanding about development of new serological diagnostic kits and
39
vaccines. The current review provides an update on the latest advances of current epitopes
40
described against toxoplasmosis including B cell/T cell epitopes, antigen types, parasite
41
strains, epitope sequences, assay settings (in vitro and/or in vivo), and target strategy. Present
42
results disclosed that the designing of effective multiepitopes of T. gondii by in silico
43
modeling and immunoinformatics tools can strengthen our knowledge about triggering of
44
epitope-based vaccine/diagnosis strategies in future perspectives.
45
Key words: Toxoplasma gondii, Candidate antigenic epitopes, T-cell/B-cell epitope-based
46
vaccine, T-cell/B-cell epitope-based diagnosis strategy.
47 48 49 50 51 52 53 54 55 56 2
57 58
1- Introduction
59
Toxoplasma gondii is an intracellular blood and tissue protozoan parasite that is distributed
60
worldwide and causes toxoplasmosis in humans and other warm-blooded animals [1, 2].
61
Approximately, one-third of the world's populations are infected with Toxoplasma infection
62
[3]. The diagnostic importance of toxoplasmosis is clinically highlighted in pregnant women
63
and immunocompromised patients [4]. The currently available chemical drugs for the
64
treatment of T. gondii are not completely safe and effective [5]. Therefore, by focusing on the
65
high rate of public health concerns and economic impact of toxoplasmosis, it would be
66
essential to develop an effective commercial diagnostic kit and vaccine against toxoplasmosis
67
[6, 7]. Previous studies imply that the design of the toxoplasmosis vaccine and the production
68
of enzyme-linked immunosorbent assay (ELISA) commercial kits should be mainly based on
69
surface antigens of tachyzoites isolated from mice or cell culture [8]. On the other hand, some
70
evidence revealed that each antigen can induce some specific immune cells, which
71
subsequently can result in a specific response [9]. It has been shown that -all of the regions of
72
whole protein molecules of T. gondii are not fully responsible for the immune responses. The
73
presence of these bystander regions leads to a decrease in specificity and affinity of the
74
antigen (epitope)-antibody (paratope) binding. Indeed, an epitope is a region of an antigen
75
that is specifically detected by and stimulate the B cells or T-cells [10].
76
Currently, epitope prediction availability has provided the possibility to electively stimulate
77
B, T, cytotoxic T lymphocyte, and NK cells responses that contribute to providing enhanced
78
protective immunity against toxoplasmosis in patients. Thus, the accurate in silico prediction
79
of antigenic epitopes and design of recombinant antigens of T. gondii (with linear and/or
80
spatial structures) can help to develop more effective serological diagnostic kits and vaccines.
3
81
In addition to epitope mapping methods such as X-ray diffraction, scanning mutagenesis,
82
nuclear
83
immunoinformatics tools are now available to study epitopes. Immunogenic multi-epitope
84
candidates has been developed using bioinformatics online servers to develop the new
85
diagnostic tests and vaccine planning [12]. This comprehensive review represents an updated
86
systematic review on the latest advances of clinical usefulness of epitopes described against
87
toxoplasmosis including B cell/T cell epitopes, antigen types, parasite strains, epitope
88
sequences, assay settings (in vitro and/or in vivo), and target strategy.
89
magnetic
resonance,
overlapping
peptides,
phage
display
[11],
and
2- Database search
90
Four bibliographic databases including Science Direct, PubMed Central (PMC), Scopus, and
91
Google Scholar were searched for articles published up to 2019. The following MeSH
92
(Medical Subject Headings) keywords were considered in the initial search strategy:
93
“Toxoplasmosis”, “Epitope-based vaccines”, “Epitope-based diagnostic”, and “Antigenic
94
multi-epitope of T. gondii (Figure 1).
95
3- Phage display method
96
The method of “phage display” is a tool for the study of antigenic epitopes by using a random
97
peptide library (a source of specific protein binding molecules) [12-14]. Using this method,
98
conformational and linear antigenic epitopes can be obtained at the same time. In this
99
method, a part of the target gene is inserted in the phage coat gene locus; leads to the
100
expression of extrinsic polypeptides that are presented while maintaining specific spatial
101
compatibility are demonstrated on the phage surface. Then, the polypeptides are evaluated for
102
affinity and specificity. This tool has been extensively used in studies of the antigenic
103
epitopes of T. gondii, and various epitopes obtained from MIC, GRA and SAG indicating
4
104
epitope-displaying phage can provide high protective immunity [12, 15, 16] As well, this
105
method can be very helpful in understanding the relationship between host and parasite [17].
106
4- Immunoinformatics tools
107
The use of immunoinformatics tools to predict antigenic epitopes has introduced an
108
interesting method. Epitope prediction by immunoinformatics tools is useful in vaccine
109
development due to eliciting immune responses with enhanced production of specific
110
antibodies, increased specificity and long-lasting memory [18]. Epitopes are classified into
111
two groups of B cell and T cell epitopes based on their cellular immune responses. Designing
112
and predicting epitopes based on physicochemical properties can have high specificity and
113
avidity with the receptors of B and T lymphocytes.
114
4-1- B cell epitope prediction
115
B cells have two types of linear and conformational epitopes, which can be recognized and
116
predicted by using several online bioinformatics servers.
117
4-1-2- Prediction of linear B cell epitopes
118
A linear B-cell epitope is a consecutive sequence of about 10 to 30 amino acids that is
119
detected by B cell receptor (BCR). These epitopes can easily be attached to the floor of the
120
ELISA plate wells and accessible to the paratope regions of the antibodies. Several properties
121
of peptides including antigenicity, flexibility, hydrophilicity, and accessibility have attracted
122
a great attention to select effective B-cell epitopes [19-21]. To predict appropriate epitopes, it
123
is often necessary to analyze the combination of parameters and different algorithms using
124
several methods. Recently, new servers developed to evaluate these parameters including;
125
ABCpred (Artificial neural network based B-cell epitope prediction server), LBtope (Linear
126
B-Cell Epitope Prediction server), IEDB (The Immune Epitope Database), BCpred
127
(Prediction of Continuous B-Cell Epitopes), and SVMTriP (support vector machine to 5
128
integrate tri-peptide). In order to design diagnostic ELISA kits on the basis of SAG, GRA and
129
MIC proteins, the linear B-cell epitopes prediction is more performed by ABCpred server,
130
indicating these epitopes can provide specificity of above 80% [22].
131
4-1-3- Conformational B cell epitopes prediction
132
Unlike linear B cell epitopes, conformational B-cell epitopes are not tandem sequences of
133
amino acids, rather they are comprised of spatial assembly of several short amino acid
134
sequences of a protein that are far away from each other in the initial sequence. This type of
135
epitope comprises 90 percent of B-cell epitopes [23]. In order to detect conformational B-cell
136
epitopes, the three-dimensional antigen structure should be identified. The combination of in-
137
silico analysis and experimental methods has led to improvements in the localization and
138
analysis of conformational epitope. There are several available epitope servers including:
139
CEP [24], CBTope, DiscoTope, and MEPS [25, 26]. Peptide libraries in combination with in
140
silico modeling for conformational epitope prediction is a potential technique for predicting
141
protein conformational epitopes in B cells [27]. For the first time, Scott and Smith used a
142
phage-expressed random peptide library for localization of epitopes in the antigen [12, 28].
143
4-1-3-1 SAG1 antigen
144
At present, many algorithms are available based on prediction models of conformational B
145
cell epitopes. One of the most important antigens is the major surface antigen 1 (SAG1),
146
which consists of 3-5% of the total protein of tachyzoites [29, 30]. SAG1 (P30) is the most
147
immunogenic antigen in the tachyzoite structure in T. gondii. This antigen is the most potent
148
immunologic response in the body against the parasite and induces high titers of IgA, IgM,
149
and IgG [31]. Residues 125–269 include all B cell epitopes predictable on the SAG1 antigen
150
after infection with the T. gondii, and the sequence of residues 125–165 is necessary to
151
complete the structure of these B cell epitopes [32]. 6
152
4-1-3-2- GRA antigens
153
All of the dense granule antigens (GRA) are recognized as excretory/secretory antigen (E/S).
154
GRA1 is a 24-kDa polypeptide called P24. This protein consists of 175 amino acids that the
155
sequence of residues 57–149 is immunologic B cell epitopes [32, 33]. GRA2 antigen reduces
156
the pathogenicity of T. gondii. This protective effect is mediated by CD4+ T cells that
157
respond to this antigen, and provide long-term immunogenicity against these intracellular
158
parasites. The 59 C-terminal region from GRA2 encompasses at least three B cell epitopes.
159
GRA3 is a 30 kDa protein which exists in dense granules. This protein is secreted into the
160
parasitophorous vacuole (PV) and causes the development of the PVcavity to enter the
161
cytoplasm of host cells. Using ELISA method, the reactivity of peptides encoded by
162
fragmented genomic DNA from phage display of T. gondii with a monoclonal antibody was
163
confirmed to be a GRA3 epitope [16]. Amino acids 20 to 30 in the GRA4 antigen structure is
164
known as C protein. The GRA4 protein is involved in nutrition and transferring of proteins
165
between the parasite and the host. The 11 amino acids at the C terminus and the amino acids
166
318–334 from GRA4 proteins contain a major B cell epitope [34-36]. GRA5 is a 21 kDa
167
protein that is secreted during the invasion of parasites into host cells. The gene encoding this
168
protein has 834 bp, which has no intron regions. The GRA5 antigen has five epitopes and the
169
N-terminal region is hydrophobic and contains 25 amino acids. This hydrophobic region of
170
the GRA5 is located inside the membrane of the (PV) and the C-terminal region is inside the
171
PV space [37-39]. GRA6 has been demonstrated to be suitable for designing novel and
172
alternative vaccine candidate for toxoplasmosis and serodiagnostic assays [40]. GRA6
173
localized in the PV narrowly associated with the network. Furthermore, the GRA6 gene has
174
only a single copy in the genome of T. gondii which contains no intron in its sequence. Wang
175
et al. (2016) identified the B cell epitopes of GRA6 protein by bioinformatics prediction
176
techniques[41]. Consequently, they confirmed the prediction through experimental methods 7
177
by using ELISA technique. After the invasion of the parasite into the host cell, GRA7 is
178
secreted into the cytoplasm of bradyzoite-infected cells. The GRA7 coding gene lacks the
179
intron region. Based on previous studies, the specificity and sensitivity of GRA7 antigen to
180
human serum using ELISA method was about 98% and 88%, respectively; in addition,
181
GRA7-ELISA demonstrated the highest positive rate in pregnant women [42, 43].
182
4-1-3-3- ROP1 antigen
183
Rhoptry protein 1 (ROP1) is a soluble protein that is secreted into the PV during entry to the
184
host cell and then rapidly disappears. This protein plays a role during early steps in the
185
process of invasion [44]. The ROP1 protein has been evaluated in designing ELISA method
186
not only for the diagnosis of the toxoplasmosis, but also for differentiation of acute and
187
chronic (IgG, IgG avidity, and IgM ELISA) phases of the disease. Furthermore, ROP1 has
188
been examined as a vaccine candidate against toxoplasmosis in mice and sheep [45].
189
4-1-3-4- P35 antigen
190
The P35 T. gondii antigen is detected by specific IgG at the primary phase of infection. This
191
results in rapid detection and control of acute infection in pregnant women. P35 has been
192
studied to differentiate between chronic and acute toxoplasmosis [46].
193
T. gondii invasion of the host cell is a multi-stage process. One of the first steps is secretion
194
of micronemal (MIC) proteins to attach apical tachyzoite to host cell receptors. MIC1 is a
195
protein with a molecular weight of 60 kDa and a beta-galactoside-binding lectin. In previous
196
studies, it has been shown that MIC1 antigen can be a candidate for the toxoplasmosis
197
diagnosis in early stages and the development of the vaccine [47].
198
4-2- T cell epitope prediction
8
199
T-cell epitopes can be divided into two groups: A) T-helper cells antigenic epitopes including
200
Predicted epitopes that require antigen processing by antigen presenting cells (APCs) and
201
presentation on class II major histocompatibility complex (MHC). B) Cytotoxic T cell
202
antigenic epitopes including predicted epitopes that require antigen processing by nucleated
203
somatic cells and presentation on class I MHC. The basis for predicting T-cell epitopes is that
204
they can be presented on MHC molecules [12, 48].
205
4-3- Prediction of peptide-MHC binding
206
MHC-I and MHC-II molecules have a similar three-dimensional structure. In both of the
207
molecules presenting peptides are bound to the groove region. Groove region is composed of
208
two α-helices overlying a floor of eight antiparallel β-strands. Nevertheless, the binding
209
grooves of MHC I and MHC II have key differences. The peptide bond cleavage in MHC-1
210
molecule is formed only by α chain, while in the MHC-II molecule it is made by a co-
211
assembly of both α and β chains. The MHC-I peptide-binding groove also comprises deep
212
binding bags by physicochemical interactions that are associated to binding properties and
213
help to predictions. Peptides with different amino acid chains are commonly placed in the
214
MHC class I groove and interact with this molecule. The peptide-MHC-I prediction
215
technique requires fixed-length amino acids. It is generally preferable to predict peptides in
216
peptide-MHC-I ligands have amino acid with nine residues. In contrast, the length of peptides
217
that react with class II MHC molecules is more than 30 amino acids [49]. There are five basic
218
prediction approaches for MHC molecular affinity peptides:
219
1; Machine learning technique: The machine learning technique is solving the problem of
220
searching for core binding motifs, and it can complete data from peptide residue interplay to
221
modify the specificity, precision, and applicability of predictions. 2; Binding motif technique:
222
This technique is simple and easy to accomplishment, and it is predominantly appropriate for
223
the prediction of MHC allele-binding peptides without experimental data. 3; Quantitative 9
224
matrix technique: this technique uses linear processing, nevertheless it is problematic to add
225
new experiences data into the prediction model, the difference of predictions using this
226
technique is reduced. 4; sequence similarity prediction: Biochemical experiments that similar
227
protein sequences fold into similar 3D structures provides a foundation for approaches that
228
predict the structural features of a new protein based on the similarity between its sequence
229
and sequences of recognized protein structures. However, this method has relatively low
230
precision and is rarely used. 5; Molecular modeling method: Molecular modeling includes all
231
the computational techniques that simulate the molecular interactions in vitro and therefore
232
predict the structure and behavior of the molecules of the study. Molecular modeling is used
233
in as various as fields of drug design, computational biology, materials science and
234
computational chemistry to study molecular systems of small chemical systems to large
235
biological molecules [12, 50, 51].
236
5- Results
237
5-1- Antigenic multiepitope peptides and chimeric antigens of T. gondii
238
In previous years, several computational methods have been developed which are able to
239
predict the antigenic epitopes. These methods rely on physicochemical properties of amino
240
acids to predict the structural and functional characteristics of peptide chains that
241
consequently determine the arrangement and localization of epitopes. Moreover, numerous
242
experimental methods may be used to recognize epitopes, including epitope mapping and
243
phage display of cDNA libraries.
244
Use of a combination of two, three or more precisely selected epitopes from antigens at the
245
various stages of T. gondii life cycle is the best strategy to overcome the antigen complexity
246
of the parasite. Therefore, the chimeric protein can be more immunogenic antigen than the
247
whole antigen [52]. The chimeric antigens are a new group of recombinant antigens that have
10
248
recently been preferred on native recombinant antigens. To date, many studies have shown
249
that it is beneficial to use these antigens for serological diagnosis and even Toxoplasma
250
vaccine design (Table 1). Indeed, using chimeric antigens containing epitopes from different
251
stages of infection can broaden the diagnostic spectrum of the possible diagnostic methods or
252
provide vaccines with the advantage of response to different phases of the disease.Frickel et
253
al. (2008) recognized two T. gondii–specific H-2Ld–restricted T cell epitopes, one from
254
GRA4 and the other from ROP7 that were involved in the chronic and acute stage of
255
infection with T. gondii (Table 1) (refrence). The progress in bioinformatics,
256
immonoinformatics and synthetic biology provides the possibility to accelerate and improve
257
the design and development of Toxoplasma antigens which consequently contribute to the
258
design of precise vaccine and serodiagnostic tests [53]. Such strategies make possible the
259
design and the further synthesis of recombinant protein with high antigenic features and
260
decreased production costs [54]. Such a perspective, have increased the attention to the
261
research on T. gondii multi-epitope antigens [22, 55]. The best advantage of designing multi-
262
epitope chimeric recombinant antigens is the possibility to select epitopic parts of several
263
highly immunogenic Toxoplasma proteins (Figure 2) based on their location and availability
264
in the protein structure and physico-chemical properties and the complexity of their spatial
265
structure. These epitopes are linked together by linkers that have high solubility and
266
flexibility (Serine has an alcoholic agent that results in high protein solubility and Glycine
267
has a shorter lateral branch in its structure that makes the structure of the protein more
268
flexible). Due to the presence of epitopes which represents several immunogenic proteins of
269
Toxoplasma that are linked by polar and flexible linkers, such a chimeric multi-epitope
270
antigen has high antigenicity, solubility and flexibility levels (Figure 3).
271
6- Discussion
11
272
Many strategies of the vaccine against toxoplasmosis have been tested in animal models;
273
however, these efforts at best only resulted in relative protection against the disease. The use
274
of immunogenic multi-epitope chimeric recombinant antigens in the design of vaccine has
275
many advantages, particularly combination of several immunogenetic epitopes of parasitic
276
antigens to make a highly immunogensic multi epitope recombinant antigen. In addition, it
277
provides the accessibility of peptide regions with high avidity to T-cell and B cell receptors
278
that help to create effective host immune responses with immune memory. Serologic methods
279
play an imperative role in the serodiagnosis of animal and human toxoplasmoses. The most
280
of the current diagnostic ELISA kits use crude tachyzoites antigens. However, the use of the
281
latter antigens in the design of diagnostic tests reduces the specificity and sensitivity of the
282
test. In addition, it is very difficult and costly to standardize and mass produce the potential
283
diagnostic kit. Holec-Ga˛sior et al. (2012) revealed that the sensitivity of the IgG ELISA for
284
the MIC1-MAG1 chimeric antigens was about as much as that for the Toxoplasma lysate
285
antigen (TLA), 90.9% and 91.8%, respectively. This research group also produced the MIC1,
286
MAG1 and SAG1 chimeric antigens containing immunodominant regions from three T.
287
gondii antigens, which obtained better results than the chimeric antigen containing only two
288
segments from the MAG1 and MIC1 antigens [47]. It indicates that, the precise selection of
289
protein segments are of importance in the process of antigen production for the diagnostic
290
kits. Among the various T. gondii proteins, only a specified few numbers of peptide epitopes
291
elicit the dominant CD8 T cell responses that are derived from a fewer number of antigens.
292
This phenomena which are known as immunodominance when be considered during vaccine
293
design can facilitate this process and improve the results. Consistently, Feliu et al. (2013)
294
reported that the CD8 T cell responses induced by immunodominant GRA6 antigen can
295
control the parasite burden, however, the reactions to the epitopes derived from subdominant
296
GRA4 and ROP7 antigens are not protective [56]. The location of the peptide epitope at the
12
297
C-terminus of the GRA6 antigenic precursor is involved in the determining immunodominace
298
of the epitope, however it is not dependent on the peptide affinity for the MHC I molecule. B-
299
cell epitopes can also demonstrate immunodominance effect. Dai et al. (2012) demonstrated
300
that three antigenic recombinant epitopes, which were belonged to antigens SAG1, SAG2,
301
and SAG3, were reactive to human T. gondii-positive serum antibodies with a very high
302
affinity [55]. The results of these studies confirmed the usefulness of ELISA kits developed
303
by the recombinant-multiepitope peptide antigens for the serologic diagnosis of
304
toxoplasmosis in pregnant women [52, 55]. Hajissa et al. (2015) produced a recombinant
305
chimeric protein by using the immunogenic epitopes of SAG1, GRA2, and GRA7 antigens.
306
This recombinant protein was used to develop diagnostic human sera IgG by western blot and
307
ELISA tests with 100% specificity and sensitivity [22]. The increased sensitivity and
308
specificity of the diagnostic test are achieved through the combination of identified parts of
309
antigenic epitopes in several whole antigens of the parasite to produce recombinant multi-
310
epitope antigens. Using this approach, the non-specific regions of the antigen that potentially
311
react with the antibody are removed. Bioinformatics tools are extensively functional for
312
epitope recognition in protein analysis [18, 22, 55]. Frickel et al. (2008) have shown that T
313
cells reactive to GRA4-derived epitopes are predominant two weeks after infection, whereas
314
the ROP7-derived epitope reactive T cells are predominant at 6–8 weeks after infection [57].
315
Feliu et al. (2013) used genetically modified parasites to evaluate the effects of
316
immunodominance against a specified peptide epitope. They found that in spite of epitopes of
317
subdominant GRA4 and ROP7 antigens, specific immunoglobulins and CD8 T cell responses
318
against dominant GRA6 antigen control the burden of the parasite. Interestingly, the location
319
of the epitope which is at the C-terminus of the GRA6 antigenic precursor determines the
320
optimal processing and immunodominance [56]. In a study published in 2013, eleven
321
peptides taken from T. gondii SAG1 were evaluated by pig sera collected at different times
13
322
after infection using ELISA method. Among the 11 peptides tested, four peptides PS11,
323
PS10, PS6, and PS4 were finally introduce for immunodiagnostic purposes.[58]. To
324
determine the relationship between the characteristics toxoplasmosis and infectious strains,
325
Kong et al. (2003) designed an ELISA test for typing strains that uses serum of toxoplasmosis
326
patients against polymorphic peptides from Toxoplasma proteins GRA7, GRA6, GRA3 and
327
SAG2A (Table 1) [59]. Cong et al. (2010) using the synthesis of antigenic epitopes of SAG1,
328
GRA6, GRA7, SAG2C, and SPA proteins (Table 1) enhanced immunity in the body of
329
transgenic mice, which reduced the parasitic burden of toxoplasmosis [60] .
330
7. Conclusions
331
In spite of considerable advances in studying T. gondii antigenic epitopes, many methodical
332
and theoretical obstacles prevent epitope-based vaccines or serodiagnostics from becoming
333
commercial diagnostics or vaccines: (A) Because of the complication of immune reaction
334
mechanisms in the body, the design and construction of epitope-based serodiagnostics or
335
vaccines have been laborious; (B) The usage of epitope-based diagnostics or vaccines is
336
extremely dependent on the precise identification of conformational T-helper epitopes and B
337
cell epitopes. At present, epitope identification remained at the level of simulation and
338
prediction. Indeed, it is not clear how multiple epitopes are organized and compounded to
339
produce optimum performance and there is a lack of theoretical models and empirical
340
evidence about this subject. However, the mentioned problems will be resolved and epitope-
341
based diagnostics or vaccines will be designed and used in the future. In fact, according to the
342
evidence from a large body of studies, it seems that the design of antigenic epitopes of SGA1,
343
SGA2, SGA3, GRA4, GRA6 and GRA7 proteins may be used as a better strategy in the
344
design of T. gondii vaccines and diagnostic kits used in human in the near future.
345
Acknowledgments
14
346
This Review study is equated from the Department of Parasitology and Mycology Shahid
347
Beheshti University of Medical Sciences, Tehran, Iran and the Department of Parasitology,
348
Tabriz University of Medical Sciences, Iran.
349
Conflict of interest
350
None declared
351
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16
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47. 48. 49. 50. 51. 52.
53. 54. 55. 56.
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Cong, H., et al., Comparative efficacy of a multi-epitope DNA vaccine via intranasal, peroral, and intramuscular delivery against lethal Toxoplasma gondii infection in mice. 2014. 7(1): p. 145. Maksimov, P., et al., Peptide-microarray analysis of in-silico predicted epitopes for the serological diagnosis of Toxoplasma gondii in infected humans. 2012: p. CVI. 00119-12. Leyva, R., P. Hérion, and R.J.P.r. Saavedra, Genetic immunization with plasmid DNA coding for the ROP2 protein of Toxoplasma gondii. 2001. 87(1): p. 70-79. Bonhomme, A., et al., Toxoplasma gondii-structure variations of the antigen P30. 1994. 108(3): p. 281-287. Beghetto, E., et al., Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products. 2006. 44(6): p. 2133-2140. Cao, A., et al., Toxoplasma gondii: vaccination with a DNA vaccine encoding T-and B-cell epitopes of SAG1, GRA2, GRA7 and ROP16 elicits protection against acute toxoplasmosis in mice. 2015. 33(48): p. 6757-6762. Cesbron-Delauw, M., et al., Amino acid sequence requirements for the epitope recognized by a monoclonal antibody reacting with the secreted antigen GP28. 5 of Toxoplasma gondii. 1992. 29(11): p. 1375-1382. Duquesne, V., et al., Protection of nude rats against Toxoplasma infection by excretedsecreted antigen-specific helper T cells. 1990. 58(7): p. 2120-2126. Cong, H., et al., Toxoplasma gondii HLA-B* 0702-restricted GRA720-28 peptide with adjuvants and a universal helper T cell epitope elicits CD8+ T cells producing interferon-γ and reduces parasite burden in HLA-B* 0702 mice. 2012. 73(1): p. 1-10. Macêdo, A.G., et al., SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts. 2013. 6(1): p. 163. Lu, G., et al., Epitope analysis, expression and protection of SAG5A vaccine against Toxoplasma gondii. 2015. 146: p. 66-72. Tan, T.G., et al., Identification of T. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans. 2010. 28(23): p. 3977-3989. Wang, Y., et al., Increased survival time in mice vaccinated with a branched lysine multiple antigenic peptide containing B-and T-cell epitopes from T. gondii antigens. 2011. 29(47): p. 8619-8623. Wang, Y., et al., Identification of novel B cell epitopes within Toxoplasma gondii GRA1. 2013. 135(3): p. 606-610. Zhang, T.-E., et al., Protective immunity induced by peptides of AMA1, RON2 and RON4 containing T-and B-cell epitopes via an intranasal route against toxoplasmosis in mice. 2015. 8(1): p. 15.
522 523
Figure legends:
524
Figure 1. Flow chart displaying study collection for the review.
525 526 527
Figure 2: Toxoplasma gondii organelles. The major organelles and hypothetical 3D structure of their antigens designed with by the SWISS-MODEL server of the asexually tachyzoite phase are displayed.
528 529 530
Figure 3: A) Linear epitope prediction of SAG1, 2, 3 antigens using ABCpred server. B) Multi-epitope prediction of linear epitopes predicted SAG1, 2, 3 antigens. C) Hypothetical 3D multi-epitope structure designed using the SWISS-MODEL server.
531
18
532 533 534 535 536 537 538 539 540 541 542
19
Table1: Candidate antigenic epitopes for vaccination and diagnosis strategies of T. gondii.
Antigen type
Antigen form
B cell
T cell
GRA4 ROP7
Secretory proteins
_
+ CD8
SAG1
GPI-linked surface protein
+
SAG2A, GRA3, GRA6, and GRA7
Secretory proteins
SAG1 GRA2 GRA7
Surface protein (SAG1)
SAG1 GRA6 GRA7 SAG2C SPA
Study model (Human/Animal ) BALB/c
Parasite strain
Epitope sequence
T. gondii Pru
GRA4: SPMNGGYYM ROP7: IPAAAGRFF
-
+
[57]
-
Pig sera
Not mentioned
+
-
[30]
+
-
Human
types I (RH), II (Me49 and Prugniaud), and III (VEG and CEP)
PS4-2: TTSSCTSKAVTLSSL PS6-3: DAQSCMVTVTVQARA PS10-3: SPEKHHCTVQLE PS11-2: GTASHVSIFAMVTGLIGSIA GRA6: (6I/III: CLHPERVNVFDY 6II: CLHPGSVNEFDF (d)6II: CLHPGSVNEFD- & (d)6I/III: CLHPERVNVFD-) SAG2A: (2I/III: RNNDG-SSAPC 2II: RNNDGGSSAPC) GRA3: (3I/III: ADQPEAHQNLAEPVC 3II: ADQPGAHQNLAEPVC) GRA7: (7II: CVPESGKDGEDARQ (d)7II: CVPESGKDGEDA-7III: CVPESGEDREDARQ (d)7III: CVPESGEDREDA)
+
-
[59]
+
-
Human
Not mentioned
+
-
[22]
-
+ CD8+
HLAA* 1101 transgenic mice
Type II Prugniaud (Pru) strain20
-
+
[61]
Secretory proteins (GRA2 GRA7)
Surface proteins (SAG1, SAG2C SPA) Secretory (GRA6
SAG1 (KLSAEGPTTMTLVCGK AAVILTPTENHFTLKC TEPPTLAYSPNRQICP) GRA2 (DERQQEPEEPVSQRAS TQAPDSPNGLAETQAP GVVNQGPVDVPFSGKP) GRA7 (AATASDDELMSRIRNS MGLTRTYRHFSPRKNR PELTEEQQRGDEPLTT) SAG1: KSFKDILPK GRA6: AMLTAFFLR GRA7:
Strategy Diagnosis vaccine
Reference
GRA7)
RSFKDLLKK SAG2C: STFWPCLLR SPA: SSAYVFSVK, AVVSLLRLLK
SAG1, GRA1, ROP2, GRA4 SAG2C, SAG2X
Surface protein (SAG1, SAG2C & SAG2X) Secretory (GRA1 GRA4 ROP2)
+
+
BALB/c mice
High virulent RH strain
SAG1-I (TCPDKKSTA) SAG1-II (ILPKLTENPW) GRA1 (DTMKSMQRDED), ROP2 (GDV VIEELFNRIPETS) GRA4 (SGLTGVKDS) SAG 2C (SQFLSLSLL) SAG2X (AAGTTATAV)
-
+
[62]
SAG1 SAG2 SAG3 P35 GRA5 GRA6
Surface protein (SAG1 SAG2 SAG3 P35) Secretory (GRA5 GRA6
+
-
Human
Not mentioned
+
-
[55]
GRA6 GRA1
Secretory
+
-
Human
Not mentioned
SAG1_EP1: QGNASSDKGA SAG1_EP2: GLIGSFAACV SAG2_EP1: SYDGTPEKPQ SAG2_EP2: GRNNDGSSAPTP SAG3_EP1: KDKGDCERNK SAG3_EP2: QPGTEGESQA P35_ EP1: GMPKPENPVR P35_ EP2: QPGTTTTTTS GRA5_ EP1: FVGVAGSTRD GRA5_ EP2: EESKESATAE GRA6_ EP1: GRRSPQEPSG GRA6_ EP2: EGGAEDDRRP Nd
+
-
[39]
SAG1, GRA1, GRA2, GRA4, NTPase1, NTPase2
Surface protein (SAG1)
+
-
Human
Not mentioned
Peptide Microarray Analysis
+
-
[63]
Secretory (GRA1 GRA2 GRA4 NTPas1NTPas2)
21
ROP2
Secretory
-
+
Human
P30
Surface
+
-
-
GRA1
Secretory
+
_
Human
SAG1, GRA2, GRA7 and ROP16
Surface (SAG1) Secretory (GRA2, GRA7 and ROP16)
+
+
GP28.5
Secretory antigen
+
P24
Secretory antigen
-
Wiktor strain
197 to 216 of the ROP2 protein TDPGDVVIEELFNRIPETS
-
+
[64]
256-265, 286-295, 220-229, 58-67, 160169, 244-253 (No amino acids sequence)
Nd
Nd
[65]
RH strain
EEVIDTMKSMQRDED ,DEMKVIDDVQQLEK
+
-
[66]
BALB/c mice
RH strain
-
+
[67]
-
Human
Nd
SAG1 (LGPVKLSAEGPT, VVTCPDKKSTA) GRA2 (TAAKTHTVRGFKV, TQAPDSPNGLAETQAPV) GRA7 (SYFAADRLVP, PELTEEQQRGDEPL, QEVPESGEDGEDARQ) ROP16 ( SPAQERRGSPQRQI, VMMINANGV , TLPENKATVVRRGS, KLNNMMIDV) VPVPDFSQ
+
-
[68]
+
Rat model
RH strain
-
+
[69]
-
+
[62]
-
+
[61]
SAG1 GRA1 ROP2 GRA4
Surface protein (SAG1) Secretory (GRA1 GRA4 ROP2)
+
+
SPF grade BALB/c female mice
RH strain
GRA6 GRA3 SAG2C SAG2D SAG2X
Surface protein (SAG2, SPA) Secretory (GRA6 GRA3 MIC1)
-
+ CD8
Human and HLAA*0201 Kb transgenic mice
type II Prugniaud (Pru) strain
22
(CysLeuSerAlaGlyAlaTyrAlaAlaGluGlyG lyAspAsnGlnSerSerAlaValSerAspArg), (ValGluGluValIleAspThrMetLysSerMet GlnArgAspGluAspllePheLeuArgAlaLeuA snLys) and (GlyGluThrValGluGluAlaIleGluAspValAI aGInAlaGlu) SAG1-I (TCPDKKSTA) SAG1-II (ILPKLTENPWQ) GRA1 (DTMKSMQRDED) ROP2 (PGDVVIEELFNRIPETSV) GRA4 (SGLTGVKDSSS) GRA6 (FMGVLVNSL) GRA3 (FLVPFVVFL) SAG2C (FLSLSLLVI) SAG2D (FMIAFISCFA) SAG2X (FVIFACNFV)
SAG2X SAG3 SPA MIC1
GRA7
Secretory (GRA7)
-
+ CD8
SAG2A
Surface protein (SAG2A)
+
-
Human HLAB*0702 transgenic mice Human
type II T gondii strain, ME49
SAG5A
Surface protein (SAG5A)
+
+
BALB/c mice
T. gondii RH (type I) & ME49(type II) PRU strain
GRA4
Secretory (GRA4)
+
+
C57BL/6 mice
GRA6 GRA 3, GRA6, GRA 7, and Sag 1
Surface protein (SAG1) Secretory (GRA3 GRA6 GRA7)
-
+ CD8
GRA4 GRA1 SAG1
Surface protein (SAG1) Secretory (GRA4 GRA1)
+
GRA1
Secretory
+
SAG2X (FMIVSISLV) SAG3 (FLLGLLVHV) SAG3 (FLTDYIPGA) SPA (ITMGSLFFV) SPA (GLAAAVVAV) MIC1(VLLPVLFGV) GRA7 (LPQFATAAT)
-
+
[70]
Type I/III: RNNDG-SSAPTP Type II: RNNDGGSSAPTP
+
-
[71]
SAG5A (HAPTPSFLGLLAVVF) (Peptide vaccine as booster)
-
+
[72]
Nd
1: (last 11 C-terminal residues of GRA4) 2: (region 318-334 of GRA4)
-
+
[36]
BALB/c mice HLAB07+ transgenic mice and naturally infected HLAB07+ individuals
type II Prugneaud (Pru) strain
HF10 GRA6: (HPGSVNEFDF) in Ld mice HLAA02 GRA6[VVFVVFMGV], GRA6[FMGVLVNSL], GRA3[FLVPFVVFL], HLA-B07 GRA7[LPQFATAAT], GRA3[VPFVVFLVA] HLA-A03 SAG1[KSFKDILPK], GRA7[RSFKDLLKK], GRA6[AMLTAFFLR]
-
+
[73]
+
SPF grade male BALB/c and Kunming mice
GRA4 (STEDSGLTGVKDSSS) GRA1 (DTMKSMQRDED) SAG1 (TCPDKKSTA)
-
+
[74]
-
Pig
highly virulent Gansu Jingtai strain (GJS, type I strain) Gansu Jingtai strain
(YSEVGNVNMEEVIDTMKSMQ), (NKGETVEEAIEDVAQAEGLN)
+
-
[75]
23
(LEKDKQQLKDDIGFLTGERE) GRA4
Secretory
+
-
Pig
Gansu Jingtai strain
AMA1, RON2 and RON4
Secretory
+
+
SPF Female BALB/c mice
T. gondii RH strain
24
(PYADGQQGSPPPQGQL), (EDSGLTVVRDSSSSESTVTP) and (TELDDGYRPPPFNPRPSPYA) AMA1 (CAELCDPSNKPGHLL) RON2 (LTAGGPLPHGSWS WSGTPPEVQTTGG SQIS) RON4 (KEQFFQFLQHLSA DYPKQVQTVYEFL GWVADK)
+
-
[18]
-
+
[76]
25
26
Highlight •
The studies have shown that predicting and constructing antigenic epitopes of surface and secretory Toxoplasma gondii proteins could be suitable for vaccine design and serological tests.
•
The results of other studies have shown that the synthesis of antigenic and immunogenic multi-epitope can enhance the specificity of antigenic and antibody responses.
•
Antigenic multi-epitope are likely to be potential substitutes for the toxic and recombinant Toxoplasma gondii antigens.
S0882-4010(19)31266-5
DOI:
https://doi.org/10.1016/j.micpath.2019.103788
Reference:
YMPAT 103788
To appear in:
Microbial Pathogenesis
Received Date: 15 July 2019 Revised Date:
5 September 2019
Accepted Date: 8 October 2019
Please cite this article as: Javadi Mamaghani A, Fathollahi A, Spotin A, Ranjbar Mm, Barati M, Aghamolaie S, Karimi M, Taghipour N, Ashrafi M, Tabaei SJS, Candidate antigenic epitopes for vaccination and diagnosis strategies of Toxoplasma gondii infection: A review, Microbial Pathogenesis (2019), doi: https://doi.org/10.1016/j.micpath.2019.103788. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Candidate antigenic epitopes for vaccination and diagnosis strategies of
2
Toxoplasma gondii infection: A review
3
Amirreza Javadi Mamaghani 1, Anwar Fathollahi2, Adel Spotin3,4, Mohammad mehdi
4
Ranjbar5 , Meisam Barati6, Somayeh Aghamolaie1, Maryam Karimi7, Niloofar Taghipour1,
5
Mohammad Ashrafi8, Seyyed Javad Seyyed Tabaei 1* 1
6
Department of Parasitology and Mycology, School of Medicine, Student Research Committee,
7 8
Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2
Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical,
9 10
Kermanshah, Iran. 3
Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. 4
11 5
12
Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension
13 14
Organization (AREEO), Karaj, Iran 6
Department of Cellular and Molecular Nutrition, Shahid Beheshti University of Medical Sciences,
15 16 17
Tehran, Iran. 7
Cellular and Molecular Research Center Kurdistan University of Medical Science, Sanandaj, Iran 8
Faculty of Medicine, Islamic Azad University, Qom, Qom, Iran.
18
Corresponding author:
19
Seyyed Javad Seyyed Tabaei; Department of Parasitology and Mycology, Shahid Beheshti University of
20
Medical Sciences, Tehran, Iran. Fax: +98 21 22439962.; Email: [email protected] ,
21
[email protected]
22 23 24 25 26 27
1
28
Abstract
29
Toxoplasmosis caused by an obligatory intracellular protozoan parasite of Toxoplasma gondii
30
threats a wide spectrum of human and animal hosts. It has been shown that the intensity of
31
the disease in humans depends on the host's immune responses. Immunological investigations
32
on whole protein molecules of T. gondii have shown that these antigens are not fully
33
responsible for the immune response, which leads to a decrease in specificity and affinity of
34
the antigen (epitope)-antibody (paratope) binding. Currently, epitopes have shown promising
35
entities to stimulate B, T, cytotoxic T lymphocyte, and NK cells resulting in enhancement of
36
protective immunity against toxoplasmosis patients. Thus, the accurate designing, prediction,
37
and conducting of antigenic epitopes of T. gondii (with linear and/or spatial structures) can
38
augment our understanding about development of new serological diagnostic kits and
39
vaccines. The current review provides an update on the latest advances of current epitopes
40
described against toxoplasmosis including B cell/T cell epitopes, antigen types, parasite
41
strains, epitope sequences, assay settings (in vitro and/or in vivo), and target strategy. Present
42
results disclosed that the designing of effective multiepitopes of T. gondii by in silico
43
modeling and immunoinformatics tools can strengthen our knowledge about triggering of
44
epitope-based vaccine/diagnosis strategies in future perspectives.
45
Key words: Toxoplasma gondii, Candidate antigenic epitopes, T-cell/B-cell epitope-based
46
vaccine, T-cell/B-cell epitope-based diagnosis strategy.
47 48 49 50 51 52 53 54 55 56 2
57 58
1- Introduction
59
Toxoplasma gondii is an intracellular blood and tissue protozoan parasite that is distributed
60
worldwide and causes toxoplasmosis in humans and other warm-blooded animals [1, 2].
61
Approximately, one-third of the world's populations are infected with Toxoplasma infection
62
[3]. The diagnostic importance of toxoplasmosis is clinically highlighted in pregnant women
63
and immunocompromised patients [4]. The currently available chemical drugs for the
64
treatment of T. gondii are not completely safe and effective [5]. Therefore, by focusing on the
65
high rate of public health concerns and economic impact of toxoplasmosis, it would be
66
essential to develop an effective commercial diagnostic kit and vaccine against toxoplasmosis
67
[6, 7]. Previous studies imply that the design of the toxoplasmosis vaccine and the production
68
of enzyme-linked immunosorbent assay (ELISA) commercial kits should be mainly based on
69
surface antigens of tachyzoites isolated from mice or cell culture [8]. On the other hand, some
70
evidence revealed that each antigen can induce some specific immune cells, which
71
subsequently can result in a specific response [9]. It has been shown that -all of the regions of
72
whole protein molecules of T. gondii are not fully responsible for the immune responses. The
73
presence of these bystander regions leads to a decrease in specificity and affinity of the
74
antigen (epitope)-antibody (paratope) binding. Indeed, an epitope is a region of an antigen
75
that is specifically detected by and stimulate the B cells or T-cells [10].
76
Currently, epitope prediction availability has provided the possibility to electively stimulate
77
B, T, cytotoxic T lymphocyte, and NK cells responses that contribute to providing enhanced
78
protective immunity against toxoplasmosis in patients. Thus, the accurate in silico prediction
79
of antigenic epitopes and design of recombinant antigens of T. gondii (with linear and/or
80
spatial structures) can help to develop more effective serological diagnostic kits and vaccines.
3
81
In addition to epitope mapping methods such as X-ray diffraction, scanning mutagenesis,
82
nuclear
83
immunoinformatics tools are now available to study epitopes. Immunogenic multi-epitope
84
candidates has been developed using bioinformatics online servers to develop the new
85
diagnostic tests and vaccine planning [12]. This comprehensive review represents an updated
86
systematic review on the latest advances of clinical usefulness of epitopes described against
87
toxoplasmosis including B cell/T cell epitopes, antigen types, parasite strains, epitope
88
sequences, assay settings (in vitro and/or in vivo), and target strategy.
89
magnetic
resonance,
overlapping
peptides,
phage
display
[11],
and
2- Database search
90
Four bibliographic databases including Science Direct, PubMed Central (PMC), Scopus, and
91
Google Scholar were searched for articles published up to 2019. The following MeSH
92
(Medical Subject Headings) keywords were considered in the initial search strategy:
93
“Toxoplasmosis”, “Epitope-based vaccines”, “Epitope-based diagnostic”, and “Antigenic
94
multi-epitope of T. gondii (Figure 1).
95
3- Phage display method
96
The method of “phage display” is a tool for the study of antigenic epitopes by using a random
97
peptide library (a source of specific protein binding molecules) [12-14]. Using this method,
98
conformational and linear antigenic epitopes can be obtained at the same time. In this
99
method, a part of the target gene is inserted in the phage coat gene locus; leads to the
100
expression of extrinsic polypeptides that are presented while maintaining specific spatial
101
compatibility are demonstrated on the phage surface. Then, the polypeptides are evaluated for
102
affinity and specificity. This tool has been extensively used in studies of the antigenic
103
epitopes of T. gondii, and various epitopes obtained from MIC, GRA and SAG indicating
4
104
epitope-displaying phage can provide high protective immunity [12, 15, 16] As well, this
105
method can be very helpful in understanding the relationship between host and parasite [17].
106
4- Immunoinformatics tools
107
The use of immunoinformatics tools to predict antigenic epitopes has introduced an
108
interesting method. Epitope prediction by immunoinformatics tools is useful in vaccine
109
development due to eliciting immune responses with enhanced production of specific
110
antibodies, increased specificity and long-lasting memory [18]. Epitopes are classified into
111
two groups of B cell and T cell epitopes based on their cellular immune responses. Designing
112
and predicting epitopes based on physicochemical properties can have high specificity and
113
avidity with the receptors of B and T lymphocytes.
114
4-1- B cell epitope prediction
115
B cells have two types of linear and conformational epitopes, which can be recognized and
116
predicted by using several online bioinformatics servers.
117
4-1-2- Prediction of linear B cell epitopes
118
A linear B-cell epitope is a consecutive sequence of about 10 to 30 amino acids that is
119
detected by B cell receptor (BCR). These epitopes can easily be attached to the floor of the
120
ELISA plate wells and accessible to the paratope regions of the antibodies. Several properties
121
of peptides including antigenicity, flexibility, hydrophilicity, and accessibility have attracted
122
a great attention to select effective B-cell epitopes [19-21]. To predict appropriate epitopes, it
123
is often necessary to analyze the combination of parameters and different algorithms using
124
several methods. Recently, new servers developed to evaluate these parameters including;
125
ABCpred (Artificial neural network based B-cell epitope prediction server), LBtope (Linear
126
B-Cell Epitope Prediction server), IEDB (The Immune Epitope Database), BCpred
127
(Prediction of Continuous B-Cell Epitopes), and SVMTriP (support vector machine to 5
128
integrate tri-peptide). In order to design diagnostic ELISA kits on the basis of SAG, GRA and
129
MIC proteins, the linear B-cell epitopes prediction is more performed by ABCpred server,
130
indicating these epitopes can provide specificity of above 80% [22].
131
4-1-3- Conformational B cell epitopes prediction
132
Unlike linear B cell epitopes, conformational B-cell epitopes are not tandem sequences of
133
amino acids, rather they are comprised of spatial assembly of several short amino acid
134
sequences of a protein that are far away from each other in the initial sequence. This type of
135
epitope comprises 90 percent of B-cell epitopes [23]. In order to detect conformational B-cell
136
epitopes, the three-dimensional antigen structure should be identified. The combination of in-
137
silico analysis and experimental methods has led to improvements in the localization and
138
analysis of conformational epitope. There are several available epitope servers including:
139
CEP [24], CBTope, DiscoTope, and MEPS [25, 26]. Peptide libraries in combination with in
140
silico modeling for conformational epitope prediction is a potential technique for predicting
141
protein conformational epitopes in B cells [27]. For the first time, Scott and Smith used a
142
phage-expressed random peptide library for localization of epitopes in the antigen [12, 28].
143
4-1-3-1 SAG1 antigen
144
At present, many algorithms are available based on prediction models of conformational B
145
cell epitopes. One of the most important antigens is the major surface antigen 1 (SAG1),
146
which consists of 3-5% of the total protein of tachyzoites [29, 30]. SAG1 (P30) is the most
147
immunogenic antigen in the tachyzoite structure in T. gondii. This antigen is the most potent
148
immunologic response in the body against the parasite and induces high titers of IgA, IgM,
149
and IgG [31]. Residues 125–269 include all B cell epitopes predictable on the SAG1 antigen
150
after infection with the T. gondii, and the sequence of residues 125–165 is necessary to
151
complete the structure of these B cell epitopes [32]. 6
152
4-1-3-2- GRA antigens
153
All of the dense granule antigens (GRA) are recognized as excretory/secretory antigen (E/S).
154
GRA1 is a 24-kDa polypeptide called P24. This protein consists of 175 amino acids that the
155
sequence of residues 57–149 is immunologic B cell epitopes [32, 33]. GRA2 antigen reduces
156
the pathogenicity of T. gondii. This protective effect is mediated by CD4+ T cells that
157
respond to this antigen, and provide long-term immunogenicity against these intracellular
158
parasites. The 59 C-terminal region from GRA2 encompasses at least three B cell epitopes.
159
GRA3 is a 30 kDa protein which exists in dense granules. This protein is secreted into the
160
parasitophorous vacuole (PV) and causes the development of the PVcavity to enter the
161
cytoplasm of host cells. Using ELISA method, the reactivity of peptides encoded by
162
fragmented genomic DNA from phage display of T. gondii with a monoclonal antibody was
163
confirmed to be a GRA3 epitope [16]. Amino acids 20 to 30 in the GRA4 antigen structure is
164
known as C protein. The GRA4 protein is involved in nutrition and transferring of proteins
165
between the parasite and the host. The 11 amino acids at the C terminus and the amino acids
166
318–334 from GRA4 proteins contain a major B cell epitope [34-36]. GRA5 is a 21 kDa
167
protein that is secreted during the invasion of parasites into host cells. The gene encoding this
168
protein has 834 bp, which has no intron regions. The GRA5 antigen has five epitopes and the
169
N-terminal region is hydrophobic and contains 25 amino acids. This hydrophobic region of
170
the GRA5 is located inside the membrane of the (PV) and the C-terminal region is inside the
171
PV space [37-39]. GRA6 has been demonstrated to be suitable for designing novel and
172
alternative vaccine candidate for toxoplasmosis and serodiagnostic assays [40]. GRA6
173
localized in the PV narrowly associated with the network. Furthermore, the GRA6 gene has
174
only a single copy in the genome of T. gondii which contains no intron in its sequence. Wang
175
et al. (2016) identified the B cell epitopes of GRA6 protein by bioinformatics prediction
176
techniques[41]. Consequently, they confirmed the prediction through experimental methods 7
177
by using ELISA technique. After the invasion of the parasite into the host cell, GRA7 is
178
secreted into the cytoplasm of bradyzoite-infected cells. The GRA7 coding gene lacks the
179
intron region. Based on previous studies, the specificity and sensitivity of GRA7 antigen to
180
human serum using ELISA method was about 98% and 88%, respectively; in addition,
181
GRA7-ELISA demonstrated the highest positive rate in pregnant women [42, 43].
182
4-1-3-3- ROP1 antigen
183
Rhoptry protein 1 (ROP1) is a soluble protein that is secreted into the PV during entry to the
184
host cell and then rapidly disappears. This protein plays a role during early steps in the
185
process of invasion [44]. The ROP1 protein has been evaluated in designing ELISA method
186
not only for the diagnosis of the toxoplasmosis, but also for differentiation of acute and
187
chronic (IgG, IgG avidity, and IgM ELISA) phases of the disease. Furthermore, ROP1 has
188
been examined as a vaccine candidate against toxoplasmosis in mice and sheep [45].
189
4-1-3-4- P35 antigen
190
The P35 T. gondii antigen is detected by specific IgG at the primary phase of infection. This
191
results in rapid detection and control of acute infection in pregnant women. P35 has been
192
studied to differentiate between chronic and acute toxoplasmosis [46].
193
T. gondii invasion of the host cell is a multi-stage process. One of the first steps is secretion
194
of micronemal (MIC) proteins to attach apical tachyzoite to host cell receptors. MIC1 is a
195
protein with a molecular weight of 60 kDa and a beta-galactoside-binding lectin. In previous
196
studies, it has been shown that MIC1 antigen can be a candidate for the toxoplasmosis
197
diagnosis in early stages and the development of the vaccine [47].
198
4-2- T cell epitope prediction
8
199
T-cell epitopes can be divided into two groups: A) T-helper cells antigenic epitopes including
200
Predicted epitopes that require antigen processing by antigen presenting cells (APCs) and
201
presentation on class II major histocompatibility complex (MHC). B) Cytotoxic T cell
202
antigenic epitopes including predicted epitopes that require antigen processing by nucleated
203
somatic cells and presentation on class I MHC. The basis for predicting T-cell epitopes is that
204
they can be presented on MHC molecules [12, 48].
205
4-3- Prediction of peptide-MHC binding
206
MHC-I and MHC-II molecules have a similar three-dimensional structure. In both of the
207
molecules presenting peptides are bound to the groove region. Groove region is composed of
208
two α-helices overlying a floor of eight antiparallel β-strands. Nevertheless, the binding
209
grooves of MHC I and MHC II have key differences. The peptide bond cleavage in MHC-1
210
molecule is formed only by α chain, while in the MHC-II molecule it is made by a co-
211
assembly of both α and β chains. The MHC-I peptide-binding groove also comprises deep
212
binding bags by physicochemical interactions that are associated to binding properties and
213
help to predictions. Peptides with different amino acid chains are commonly placed in the
214
MHC class I groove and interact with this molecule. The peptide-MHC-I prediction
215
technique requires fixed-length amino acids. It is generally preferable to predict peptides in
216
peptide-MHC-I ligands have amino acid with nine residues. In contrast, the length of peptides
217
that react with class II MHC molecules is more than 30 amino acids [49]. There are five basic
218
prediction approaches for MHC molecular affinity peptides:
219
1; Machine learning technique: The machine learning technique is solving the problem of
220
searching for core binding motifs, and it can complete data from peptide residue interplay to
221
modify the specificity, precision, and applicability of predictions. 2; Binding motif technique:
222
This technique is simple and easy to accomplishment, and it is predominantly appropriate for
223
the prediction of MHC allele-binding peptides without experimental data. 3; Quantitative 9
224
matrix technique: this technique uses linear processing, nevertheless it is problematic to add
225
new experiences data into the prediction model, the difference of predictions using this
226
technique is reduced. 4; sequence similarity prediction: Biochemical experiments that similar
227
protein sequences fold into similar 3D structures provides a foundation for approaches that
228
predict the structural features of a new protein based on the similarity between its sequence
229
and sequences of recognized protein structures. However, this method has relatively low
230
precision and is rarely used. 5; Molecular modeling method: Molecular modeling includes all
231
the computational techniques that simulate the molecular interactions in vitro and therefore
232
predict the structure and behavior of the molecules of the study. Molecular modeling is used
233
in as various as fields of drug design, computational biology, materials science and
234
computational chemistry to study molecular systems of small chemical systems to large
235
biological molecules [12, 50, 51].
236
5- Results
237
5-1- Antigenic multiepitope peptides and chimeric antigens of T. gondii
238
In previous years, several computational methods have been developed which are able to
239
predict the antigenic epitopes. These methods rely on physicochemical properties of amino
240
acids to predict the structural and functional characteristics of peptide chains that
241
consequently determine the arrangement and localization of epitopes. Moreover, numerous
242
experimental methods may be used to recognize epitopes, including epitope mapping and
243
phage display of cDNA libraries.
244
Use of a combination of two, three or more precisely selected epitopes from antigens at the
245
various stages of T. gondii life cycle is the best strategy to overcome the antigen complexity
246
of the parasite. Therefore, the chimeric protein can be more immunogenic antigen than the
247
whole antigen [52]. The chimeric antigens are a new group of recombinant antigens that have
10
248
recently been preferred on native recombinant antigens. To date, many studies have shown
249
that it is beneficial to use these antigens for serological diagnosis and even Toxoplasma
250
vaccine design (Table 1). Indeed, using chimeric antigens containing epitopes from different
251
stages of infection can broaden the diagnostic spectrum of the possible diagnostic methods or
252
provide vaccines with the advantage of response to different phases of the disease.Frickel et
253
al. (2008) recognized two T. gondii–specific H-2Ld–restricted T cell epitopes, one from
254
GRA4 and the other from ROP7 that were involved in the chronic and acute stage of
255
infection with T. gondii (Table 1) (refrence). The progress in bioinformatics,
256
immonoinformatics and synthetic biology provides the possibility to accelerate and improve
257
the design and development of Toxoplasma antigens which consequently contribute to the
258
design of precise vaccine and serodiagnostic tests [53]. Such strategies make possible the
259
design and the further synthesis of recombinant protein with high antigenic features and
260
decreased production costs [54]. Such a perspective, have increased the attention to the
261
research on T. gondii multi-epitope antigens [22, 55]. The best advantage of designing multi-
262
epitope chimeric recombinant antigens is the possibility to select epitopic parts of several
263
highly immunogenic Toxoplasma proteins (Figure 2) based on their location and availability
264
in the protein structure and physico-chemical properties and the complexity of their spatial
265
structure. These epitopes are linked together by linkers that have high solubility and
266
flexibility (Serine has an alcoholic agent that results in high protein solubility and Glycine
267
has a shorter lateral branch in its structure that makes the structure of the protein more
268
flexible). Due to the presence of epitopes which represents several immunogenic proteins of
269
Toxoplasma that are linked by polar and flexible linkers, such a chimeric multi-epitope
270
antigen has high antigenicity, solubility and flexibility levels (Figure 3).
271
6- Discussion
11
272
Many strategies of the vaccine against toxoplasmosis have been tested in animal models;
273
however, these efforts at best only resulted in relative protection against the disease. The use
274
of immunogenic multi-epitope chimeric recombinant antigens in the design of vaccine has
275
many advantages, particularly combination of several immunogenetic epitopes of parasitic
276
antigens to make a highly immunogensic multi epitope recombinant antigen. In addition, it
277
provides the accessibility of peptide regions with high avidity to T-cell and B cell receptors
278
that help to create effective host immune responses with immune memory. Serologic methods
279
play an imperative role in the serodiagnosis of animal and human toxoplasmoses. The most
280
of the current diagnostic ELISA kits use crude tachyzoites antigens. However, the use of the
281
latter antigens in the design of diagnostic tests reduces the specificity and sensitivity of the
282
test. In addition, it is very difficult and costly to standardize and mass produce the potential
283
diagnostic kit. Holec-Ga˛sior et al. (2012) revealed that the sensitivity of the IgG ELISA for
284
the MIC1-MAG1 chimeric antigens was about as much as that for the Toxoplasma lysate
285
antigen (TLA), 90.9% and 91.8%, respectively. This research group also produced the MIC1,
286
MAG1 and SAG1 chimeric antigens containing immunodominant regions from three T.
287
gondii antigens, which obtained better results than the chimeric antigen containing only two
288
segments from the MAG1 and MIC1 antigens [47]. It indicates that, the precise selection of
289
protein segments are of importance in the process of antigen production for the diagnostic
290
kits. Among the various T. gondii proteins, only a specified few numbers of peptide epitopes
291
elicit the dominant CD8 T cell responses that are derived from a fewer number of antigens.
292
This phenomena which are known as immunodominance when be considered during vaccine
293
design can facilitate this process and improve the results. Consistently, Feliu et al. (2013)
294
reported that the CD8 T cell responses induced by immunodominant GRA6 antigen can
295
control the parasite burden, however, the reactions to the epitopes derived from subdominant
296
GRA4 and ROP7 antigens are not protective [56]. The location of the peptide epitope at the
12
297
C-terminus of the GRA6 antigenic precursor is involved in the determining immunodominace
298
of the epitope, however it is not dependent on the peptide affinity for the MHC I molecule. B-
299
cell epitopes can also demonstrate immunodominance effect. Dai et al. (2012) demonstrated
300
that three antigenic recombinant epitopes, which were belonged to antigens SAG1, SAG2,
301
and SAG3, were reactive to human T. gondii-positive serum antibodies with a very high
302
affinity [55]. The results of these studies confirmed the usefulness of ELISA kits developed
303
by the recombinant-multiepitope peptide antigens for the serologic diagnosis of
304
toxoplasmosis in pregnant women [52, 55]. Hajissa et al. (2015) produced a recombinant
305
chimeric protein by using the immunogenic epitopes of SAG1, GRA2, and GRA7 antigens.
306
This recombinant protein was used to develop diagnostic human sera IgG by western blot and
307
ELISA tests with 100% specificity and sensitivity [22]. The increased sensitivity and
308
specificity of the diagnostic test are achieved through the combination of identified parts of
309
antigenic epitopes in several whole antigens of the parasite to produce recombinant multi-
310
epitope antigens. Using this approach, the non-specific regions of the antigen that potentially
311
react with the antibody are removed. Bioinformatics tools are extensively functional for
312
epitope recognition in protein analysis [18, 22, 55]. Frickel et al. (2008) have shown that T
313
cells reactive to GRA4-derived epitopes are predominant two weeks after infection, whereas
314
the ROP7-derived epitope reactive T cells are predominant at 6–8 weeks after infection [57].
315
Feliu et al. (2013) used genetically modified parasites to evaluate the effects of
316
immunodominance against a specified peptide epitope. They found that in spite of epitopes of
317
subdominant GRA4 and ROP7 antigens, specific immunoglobulins and CD8 T cell responses
318
against dominant GRA6 antigen control the burden of the parasite. Interestingly, the location
319
of the epitope which is at the C-terminus of the GRA6 antigenic precursor determines the
320
optimal processing and immunodominance [56]. In a study published in 2013, eleven
321
peptides taken from T. gondii SAG1 were evaluated by pig sera collected at different times
13
322
after infection using ELISA method. Among the 11 peptides tested, four peptides PS11,
323
PS10, PS6, and PS4 were finally introduce for immunodiagnostic purposes.[58]. To
324
determine the relationship between the characteristics toxoplasmosis and infectious strains,
325
Kong et al. (2003) designed an ELISA test for typing strains that uses serum of toxoplasmosis
326
patients against polymorphic peptides from Toxoplasma proteins GRA7, GRA6, GRA3 and
327
SAG2A (Table 1) [59]. Cong et al. (2010) using the synthesis of antigenic epitopes of SAG1,
328
GRA6, GRA7, SAG2C, and SPA proteins (Table 1) enhanced immunity in the body of
329
transgenic mice, which reduced the parasitic burden of toxoplasmosis [60] .
330
7. Conclusions
331
In spite of considerable advances in studying T. gondii antigenic epitopes, many methodical
332
and theoretical obstacles prevent epitope-based vaccines or serodiagnostics from becoming
333
commercial diagnostics or vaccines: (A) Because of the complication of immune reaction
334
mechanisms in the body, the design and construction of epitope-based serodiagnostics or
335
vaccines have been laborious; (B) The usage of epitope-based diagnostics or vaccines is
336
extremely dependent on the precise identification of conformational T-helper epitopes and B
337
cell epitopes. At present, epitope identification remained at the level of simulation and
338
prediction. Indeed, it is not clear how multiple epitopes are organized and compounded to
339
produce optimum performance and there is a lack of theoretical models and empirical
340
evidence about this subject. However, the mentioned problems will be resolved and epitope-
341
based diagnostics or vaccines will be designed and used in the future. In fact, according to the
342
evidence from a large body of studies, it seems that the design of antigenic epitopes of SGA1,
343
SGA2, SGA3, GRA4, GRA6 and GRA7 proteins may be used as a better strategy in the
344
design of T. gondii vaccines and diagnostic kits used in human in the near future.
345
Acknowledgments
14
346
This Review study is equated from the Department of Parasitology and Mycology Shahid
347
Beheshti University of Medical Sciences, Tehran, Iran and the Department of Parasitology,
348
Tabriz University of Medical Sciences, Iran.
349
Conflict of interest
350
None declared
351
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522 523
Figure legends:
524
Figure 1. Flow chart displaying study collection for the review.
525 526 527
Figure 2: Toxoplasma gondii organelles. The major organelles and hypothetical 3D structure of their antigens designed with by the SWISS-MODEL server of the asexually tachyzoite phase are displayed.
528 529 530
Figure 3: A) Linear epitope prediction of SAG1, 2, 3 antigens using ABCpred server. B) Multi-epitope prediction of linear epitopes predicted SAG1, 2, 3 antigens. C) Hypothetical 3D multi-epitope structure designed using the SWISS-MODEL server.
531
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532 533 534 535 536 537 538 539 540 541 542
19
Table1: Candidate antigenic epitopes for vaccination and diagnosis strategies of T. gondii.
Antigen type
Antigen form
B cell
T cell
GRA4 ROP7
Secretory proteins
_
+ CD8
SAG1
GPI-linked surface protein
+
SAG2A, GRA3, GRA6, and GRA7
Secretory proteins
SAG1 GRA2 GRA7
Surface protein (SAG1)
SAG1 GRA6 GRA7 SAG2C SPA
Study model (Human/Animal ) BALB/c
Parasite strain
Epitope sequence
T. gondii Pru
GRA4: SPMNGGYYM ROP7: IPAAAGRFF
-
+
[57]
-
Pig sera
Not mentioned
+
-
[30]
+
-
Human
types I (RH), II (Me49 and Prugniaud), and III (VEG and CEP)
PS4-2: TTSSCTSKAVTLSSL PS6-3: DAQSCMVTVTVQARA PS10-3: SPEKHHCTVQLE PS11-2: GTASHVSIFAMVTGLIGSIA GRA6: (6I/III: CLHPERVNVFDY 6II: CLHPGSVNEFDF (d)6II: CLHPGSVNEFD- & (d)6I/III: CLHPERVNVFD-) SAG2A: (2I/III: RNNDG-SSAPC 2II: RNNDGGSSAPC) GRA3: (3I/III: ADQPEAHQNLAEPVC 3II: ADQPGAHQNLAEPVC) GRA7: (7II: CVPESGKDGEDARQ (d)7II: CVPESGKDGEDA-7III: CVPESGEDREDARQ (d)7III: CVPESGEDREDA)
+
-
[59]
+
-
Human
Not mentioned
+
-
[22]
-
+ CD8+
HLAA* 1101 transgenic mice
Type II Prugniaud (Pru) strain20
-
+
[61]
Secretory proteins (GRA2 GRA7)
Surface proteins (SAG1, SAG2C SPA) Secretory (GRA6
SAG1 (KLSAEGPTTMTLVCGK AAVILTPTENHFTLKC TEPPTLAYSPNRQICP) GRA2 (DERQQEPEEPVSQRAS TQAPDSPNGLAETQAP GVVNQGPVDVPFSGKP) GRA7 (AATASDDELMSRIRNS MGLTRTYRHFSPRKNR PELTEEQQRGDEPLTT) SAG1: KSFKDILPK GRA6: AMLTAFFLR GRA7:
Strategy Diagnosis vaccine
Reference
GRA7)
RSFKDLLKK SAG2C: STFWPCLLR SPA: SSAYVFSVK, AVVSLLRLLK
SAG1, GRA1, ROP2, GRA4 SAG2C, SAG2X
Surface protein (SAG1, SAG2C & SAG2X) Secretory (GRA1 GRA4 ROP2)
+
+
BALB/c mice
High virulent RH strain
SAG1-I (TCPDKKSTA) SAG1-II (ILPKLTENPW) GRA1 (DTMKSMQRDED), ROP2 (GDV VIEELFNRIPETS) GRA4 (SGLTGVKDS) SAG 2C (SQFLSLSLL) SAG2X (AAGTTATAV)
-
+
[62]
SAG1 SAG2 SAG3 P35 GRA5 GRA6
Surface protein (SAG1 SAG2 SAG3 P35) Secretory (GRA5 GRA6
+
-
Human
Not mentioned
+
-
[55]
GRA6 GRA1
Secretory
+
-
Human
Not mentioned
SAG1_EP1: QGNASSDKGA SAG1_EP2: GLIGSFAACV SAG2_EP1: SYDGTPEKPQ SAG2_EP2: GRNNDGSSAPTP SAG3_EP1: KDKGDCERNK SAG3_EP2: QPGTEGESQA P35_ EP1: GMPKPENPVR P35_ EP2: QPGTTTTTTS GRA5_ EP1: FVGVAGSTRD GRA5_ EP2: EESKESATAE GRA6_ EP1: GRRSPQEPSG GRA6_ EP2: EGGAEDDRRP Nd
+
-
[39]
SAG1, GRA1, GRA2, GRA4, NTPase1, NTPase2
Surface protein (SAG1)
+
-
Human
Not mentioned
Peptide Microarray Analysis
+
-
[63]
Secretory (GRA1 GRA2 GRA4 NTPas1NTPas2)
21
ROP2
Secretory
-
+
Human
P30
Surface
+
-
-
GRA1
Secretory
+
_
Human
SAG1, GRA2, GRA7 and ROP16
Surface (SAG1) Secretory (GRA2, GRA7 and ROP16)
+
+
GP28.5
Secretory antigen
+
P24
Secretory antigen
-
Wiktor strain
197 to 216 of the ROP2 protein TDPGDVVIEELFNRIPETS
-
+
[64]
256-265, 286-295, 220-229, 58-67, 160169, 244-253 (No amino acids sequence)
Nd
Nd
[65]
RH strain
EEVIDTMKSMQRDED ,DEMKVIDDVQQLEK
+
-
[66]
BALB/c mice
RH strain
-
+
[67]
-
Human
Nd
SAG1 (LGPVKLSAEGPT, VVTCPDKKSTA) GRA2 (TAAKTHTVRGFKV, TQAPDSPNGLAETQAPV) GRA7 (SYFAADRLVP, PELTEEQQRGDEPL, QEVPESGEDGEDARQ) ROP16 ( SPAQERRGSPQRQI, VMMINANGV , TLPENKATVVRRGS, KLNNMMIDV) VPVPDFSQ
+
-
[68]
+
Rat model
RH strain
-
+
[69]
-
+
[62]
-
+
[61]
SAG1 GRA1 ROP2 GRA4
Surface protein (SAG1) Secretory (GRA1 GRA4 ROP2)
+
+
SPF grade BALB/c female mice
RH strain
GRA6 GRA3 SAG2C SAG2D SAG2X
Surface protein (SAG2, SPA) Secretory (GRA6 GRA3 MIC1)
-
+ CD8
Human and HLAA*0201 Kb transgenic mice
type II Prugniaud (Pru) strain
22
(CysLeuSerAlaGlyAlaTyrAlaAlaGluGlyG lyAspAsnGlnSerSerAlaValSerAspArg), (ValGluGluValIleAspThrMetLysSerMet GlnArgAspGluAspllePheLeuArgAlaLeuA snLys) and (GlyGluThrValGluGluAlaIleGluAspValAI aGInAlaGlu) SAG1-I (TCPDKKSTA) SAG1-II (ILPKLTENPWQ) GRA1 (DTMKSMQRDED) ROP2 (PGDVVIEELFNRIPETSV) GRA4 (SGLTGVKDSSS) GRA6 (FMGVLVNSL) GRA3 (FLVPFVVFL) SAG2C (FLSLSLLVI) SAG2D (FMIAFISCFA) SAG2X (FVIFACNFV)
SAG2X SAG3 SPA MIC1
GRA7
Secretory (GRA7)
-
+ CD8
SAG2A
Surface protein (SAG2A)
+
-
Human HLAB*0702 transgenic mice Human
type II T gondii strain, ME49
SAG5A
Surface protein (SAG5A)
+
+
BALB/c mice
T. gondii RH (type I) & ME49(type II) PRU strain
GRA4
Secretory (GRA4)
+
+
C57BL/6 mice
GRA6 GRA 3, GRA6, GRA 7, and Sag 1
Surface protein (SAG1) Secretory (GRA3 GRA6 GRA7)
-
+ CD8
GRA4 GRA1 SAG1
Surface protein (SAG1) Secretory (GRA4 GRA1)
+
GRA1
Secretory
+
SAG2X (FMIVSISLV) SAG3 (FLLGLLVHV) SAG3 (FLTDYIPGA) SPA (ITMGSLFFV) SPA (GLAAAVVAV) MIC1(VLLPVLFGV) GRA7 (LPQFATAAT)
-
+
[70]
Type I/III: RNNDG-SSAPTP Type II: RNNDGGSSAPTP
+
-
[71]
SAG5A (HAPTPSFLGLLAVVF) (Peptide vaccine as booster)
-
+
[72]
Nd
1: (last 11 C-terminal residues of GRA4) 2: (region 318-334 of GRA4)
-
+
[36]
BALB/c mice HLAB07+ transgenic mice and naturally infected HLAB07+ individuals
type II Prugneaud (Pru) strain
HF10 GRA6: (HPGSVNEFDF) in Ld mice HLAA02 GRA6[VVFVVFMGV], GRA6[FMGVLVNSL], GRA3[FLVPFVVFL], HLA-B07 GRA7[LPQFATAAT], GRA3[VPFVVFLVA] HLA-A03 SAG1[KSFKDILPK], GRA7[RSFKDLLKK], GRA6[AMLTAFFLR]
-
+
[73]
+
SPF grade male BALB/c and Kunming mice
GRA4 (STEDSGLTGVKDSSS) GRA1 (DTMKSMQRDED) SAG1 (TCPDKKSTA)
-
+
[74]
-
Pig
highly virulent Gansu Jingtai strain (GJS, type I strain) Gansu Jingtai strain
(YSEVGNVNMEEVIDTMKSMQ), (NKGETVEEAIEDVAQAEGLN)
+
-
[75]
23
(LEKDKQQLKDDIGFLTGERE) GRA4
Secretory
+
-
Pig
Gansu Jingtai strain
AMA1, RON2 and RON4
Secretory
+
+
SPF Female BALB/c mice
T. gondii RH strain
24
(PYADGQQGSPPPQGQL), (EDSGLTVVRDSSSSESTVTP) and (TELDDGYRPPPFNPRPSPYA) AMA1 (CAELCDPSNKPGHLL) RON2 (LTAGGPLPHGSWS WSGTPPEVQTTGG SQIS) RON4 (KEQFFQFLQHLSA DYPKQVQTVYEFL GWVADK)
+
-
[18]
-
+
[76]
25
26
Highlight •
The studies have shown that predicting and constructing antigenic epitopes of surface and secretory Toxoplasma gondii proteins could be suitable for vaccine design and serological tests.
•
The results of other studies have shown that the synthesis of antigenic and immunogenic multi-epitope can enhance the specificity of antigenic and antibody responses.
•
Antigenic multi-epitope are likely to be potential substitutes for the toxic and recombinant Toxoplasma gondii antigens.