Bioinformatic Approach of B and T Cell Epitopes of PLD and CP40 Proteins of Corynebacterium pseudotuberculosis ovis Mexican Isolate 2J-L towards a Peptide-Based Vaccine

Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.


Introduction
Reverse vaccinology is a branch of immunoinformatics, and is also known as the in silico search for vaccine candidates.The computational analysis constitutes an alternative tool, allowing the characterization of potential vaccine candidates, avoiding pathogen microorganism cultures, the use of experimental animals in the early stages of design, and achieving significant savings in research time and costs [1].This technology was first used in the prediction of antigens for the development of a vaccine against Meningococcus serogroup B [2].
Bioinformatics programs allow the prediction based on different antigen characteristics such as structure, adherence, fusion, antigenicity, binding to Major Histocompatibility Complex (MHC) classes I and II, binding sequences to proteasomes, and B and T lymphocyte activators [3].
In silico programs to determine epitopes, specific regions of a protein that can be recognized by antibodies (B cell epitopes), or amino acid sequences that can interact with TCRs in the context of MHC I or MHC II-mediated antigen presentation (T cell epitopes) are considered of great practical interest for vaccine design [4].
Immune Epitope Database and Analysis Resource (IEDB) is a website to allow B and T cell epitope predictions, providing a regularly updated compilation of binding epitopes and their affinities.It constitutes a prediction tool accessible through a single intuitive web interface that incorporates different programs and algorithms for the general characterization of a protein [5].
In addition, B cell epitope mapping is a promising approach to identifying the main antigenic determinants of a protein, linear epitopes for continuous amino acid residues next to each other in the primary structure or discontinuous epitopes, also known as conformational ones, based on residues close to each other in the three-dimensional structure of the protein [6].
In fact, B cell and T cell epitope prediction allows the formulation of vaccines based on peptides to substitute the use of the entire protein with the guarantee of producing specific antibodies or a cellular immune response [3], which represents an alternative to avoid the use of toxigenic nature antigens.
Phospholipase D (PLD) [7] and Endoglycosidase CP40 [8] are virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a gram-positive rod that cause Caseous lymphadenitis (CLA), a source of economic loss to the sheep and goat industries.CLA characteristic lesions are associated with encapsulated cutaneous and visceral abscesses with pyogranulomas formation, a response of the immune system to contain the infections in the host's tissues, but which makes it difficult to eliminate the bacteria [9,10].
Conventional antibiotic treatment and commercial vaccines are not efficient at controlling the disease [11].The fact that C. pseudotuberculosis ovis is an intracellular facultative bacterium makes vaccines' cellular immune response induction an important role for pathogen elimination [12].
Most vaccine formulations from CLA include the PLD toxin [13], because it is considered the principal virulent factor of C. pseudotuberculosis ovis, especially for contributing to bacterial spread in the host and their enzymatic capacity to hydrolyze the sphingomyelin of the cell membrane, causing direct damage to endothelial cells and dermo necrosis [14].
Antibodies against PLD are generated to neutralize the toxin effect, but the intracellular condition of the bacteria makes it necessary to direct the action of the immune system to obtain cell-mediated immunity, which is more difficult using conventional vaccine strategies.Antibodies might help to protect animals against infection, but full protection by any vaccine model for CLA must provide better stimulation of cellular immunity, such as the activation of T CD8+ cells and IFN-γ secretion [12,13].
In that way, virulence factor CP40, a protein with enzymatic activity that can hydrolyze Fc regions of ovine antibodies [8], has been previously identified as a protective antigen against CLA [15,16].Vaccine formulations with CP40 induce a cellular immune response, with the highest survival percentage in animal challenge studies [17].PLD [18,19] and CP40 [17,20] immunogens' potential has been evaluated in some experimental assays, and they are capable of enhancing humoral and cellular immune responses.
Mexico has a sheep and goat industry that is still developing, so it is important to study the disease to improve control measures for future eradication.Previous studies [21,22] have shown the presence of this pathogenic agent in Mexican small ruminant herds, which, added to the fact that no vaccines are commercialized in the country, arouses interest in solving this problem.
Previously, the complete sequence of the pld (GenBank accession number: OL347711) and cp40 (GenBank accession number: OL347712) genes of Corynebacterium pseudotuberculosis ovis isolate 2J-L, obtained from Jalisco State, Mexico, was reported [23].Since those virulence factors have been studied as promising vaccine antigens, this work aimed to in silico identify B and T cell epitopes of PLD 2J-L and CP40 2J-L to determine possible peptides for a vaccine design that combines the specific components that allow effective immune system activation.
A schematic representation of B-cell linear epitopes for PLD 2J-L and CP40 2J-L was performed using the JMol structure visualization program (Figure 2).
For predicting B-cell conformational epitopes, a protein structure PDB format is required.That is why the structure of PLD 2J-L and CP40 2J-L proteins was performed using the Phyre2 web portal, which produces potential 3D models of the proteins, based on the alignment of sequences and crystal structures of known proteins.PLD 2J-L and CP40 2J-L amino acid sequences were used to compare against an updated library of protein sequences, using PSI-BLAST, with the detection of 1000 homologues for each protein and PBD format archives generated and used for structural epitope prediction.
A total of 13 linear epitopes were identified for each protein, and scores above the cut-off value were taken into account for peptide selection.Epitopes were identified with a minimum size of 4 and 5 residues and a maximum size of 33 and 46 for PLD 2J-L and CP40 2J-L, respectively.The number 1 epitope of both proteins presented the highest score value, 0.856 for PLD 2J-L and 0.807 for CP40 2J-L.
A schematic representation of B-cell linear epitopes for PLD 2J-L and CP40 2J-L was performed using the JMol structure visualization program (Figure 2).  2.
For predicting B-cell conformational epitopes, a protein structure PDB format is required.That is why the structure of PLD 2J-L and CP40 2J-L proteins was performed using the Phyre2 web portal, which produces potential 3D models of the proteins, based on the alignment of sequences and crystal structures of known proteins.PLD 2J-L and CP40 2J- B lymphocyte conformational epitopes, composed of non-contiguous amino acid residues in the primary sequence of the protein but joined by a folding structure, were predicted based on the 3D model of the PLD 2J-L and CP40 2J-L proteins.The analysis of the structures allowed for the identification of eight epitopes for PLD 2J-L and three for CP40 2J-L (Table 3).The largest epitopes of the PLD 2J-L protein are 1 and 7, with scores of 0.822 and 0.609, respectively.The CP40 2J-L epitope 2 was estimated with a value of 0.683 spanning 171 residues of the protein, which includes a large part of the molecule as a possible B-cell binding conformational epitope.
In addition, a schematic representation of discontinuous B cell epitope arrangement was performed for both proteins, allowing spatial visualization of antigenic epitopes (Figure 3).Fifty B lymphocyte epitopes for PLD 2J-L and forty-seven for CP40 2J-L (9-12 by BepiPred 2.0, 9-8 by Emini Surface Accessibility Scale, 11-11 by Antigenicity Kolaskar-Tongaonkar scale, and 13-13 linear and 8-3 discontinuous by ElliPro using protein structures in PDB format) were estimated using bioinformatics tools.The resulting data set represents the B cell epitopes for PLD 2J-L and CP40 2J-L estimated by different predictors.Epitope prediction by different programs is not identical, so regions that overlap in each epitope prediction were identified (Table 4).
In this work, the use of various predicted programs allowed the identification of regions in the protein sequence as epitopes, with variation in length and the presence or absence of certain amino acids, but most likely considering an epitope.

T Lymphocyte Epitope Predictions for PLD 2J-L and CP40 2J-L Proteins
The cellular immune response is extremely important, especially the activation of T lymphocytes, for the resolution of intracellular bacterial infections.Since T cell epitopes are bound in a linear form to MHCs, the capacity of PLD 2J-L and CP40 2J-L proteins to activate T cells was evaluated in the context of antigen presentation by MHC classes I (Table 5) and II (Table 6).In addition, the amino acid residues that were identified as part of some B cell epitopes are also shown.The PLD 2J-L protein sequences with the greatest potential to activate T CD8+ cells in the context of different MHC I alleles: position 56 VAIGANAL 63 (alleles H-2-Kb and H-2-Db), 139 LEPAGVRV 146 (alleles H-2-Kd, H-2-KK, and H-2-Qa1) and sequence 202 GYYNI 206 (alleles H-2-Kd, H-2 Kb, and H-2-KK).For the CP40 2J-L protein, the sequences that were recognized by more than one type of allele were sequences 137 RTV-GAQL 143 (alleles H-2-Qa1 and H-2-Kb) and 205 KIMGAFSEL 213 (alleles H-2-Kb and H-2-Qa1).Nevertheless, none of the proteins presented epitopes with the capacity to be presented by MHC class I alleles H-2-Dd, H-2-Ld, or H-2-Qa2.Also, results show sequences that were selected as B cell epitopes (Table 5).
Epitopes predicted for presentation on T CD4+ in the context of MHC II for both proteins were not capable of being recognized by more than one type of allele.Neither of the two proteins present epitopes for T cell activation in the context of the MHC II allele H2-Ied.
However, the analysis made it clear which T epitopes are also considered epitopes of B cells (Table 6).Sequence 24 VHNPASTAN 38 of PLD 2J-L could activate T cells (MHC II-H2-IAb Allele) and B cells as part of epitopes predicted by BepiPred 2.0, Emini Surface scale, Antigenicity scale, and Ellipro programs.Following the same analysis for CP40 2J-L, the sequence 286 YAHPEENDT 300 is considered a T cell epitope (MHC II-H2IAb Allele) and a B cell epitope according to all the programs used.

B Lymphocyte Epitopes Prediction for PLD 2J-L and CP40 2J-L
The results showed that PLD 2J-L and CP40 2J-L are multi-epitopic proteins with the potential as candidates for the development of vaccines and diagnostic tools for Caseous lymphadenitis.Several of the predicted B cell epitopes for PLD 2J-L and CP40 2J-L were classified as exposed epitopes, located on the surface of the respective proteins.These regions can be used as synthetic peptides to stimulate an antibody response, ensuring better targeting of the immune system during a natural infection.Some of the sequences were identified as antigenic and antibody-binding epitopes, all with high conservation and predicted by BepiPred 2.0 and the Antigenicity Kolaskar-Tongaonkar Scale.
Almost the entire sequence of the PLD 2J-L and CP40 2J-L proteins was mapped and included as a B cell epitope, demonstrating their immunogenic potential.Most of the complete set of predicted linear epitopes was included in some conformational epitopes, which can be used as a basis for protein-protein interaction studies using three-dimensional structures [24].
B cell epitope number 2 of PLD 2J-L is formed by the sequence of the catalytic loop, which is a conserved region important for the enzymatic activity of the protein.In addition, we found that the epitopes at position (51-100) of PLD 2J-L, predicted by BepiPred 2.0 and ElliPro, include a highly conserved sequence of the catalytic loop, also forming part of a conformational epitope.Since this region is conserved between PLDs of different strains of C. pseudotuberculosis, the peptides that include it could be a good candidate for vaccine development.In addition, the toxin PLD is the main virulence factor of the bacteria, and usually, vaccine strategies use the complete protein without first considering the inactivation of their biological activity.Toxin inactivation could negatively impact PLD protective potential, a fact that can be exemplified by Hodsong and colleague's experiments, where an attenuated strain called Toxminus was used for vaccine development, and PLD active site modification decreased the protective capacity of the vaccine [7,[25][26][27].More recently, genetic modification for the recombinant PLD active site also did not allow for reaching a survival percentage greater than 57% in vaccinated and challenged animals [28].Other studies have used recombinant PLD as a vaccine antigen evaluated in murine models, presenting different protective percentages of 30% [18], 40% [29], and 42.86% [28] after challenge with virulence strain MIC-6 (10 4 CFU), with the common denominator being the inactivation of the toxin's biological activity with formaldehyde.We could say that peptide 2 of PLD 2J-L, which involves the protein active site, could be a determinant for enhanced protection.PLD anti-exotoxin antibodies protect against tissue damage and bacterial dissemination, which makes it very important to include this antigen in vaccine formulation.
Also, results shown in Table 3 indicate that the region of the PLD protein made up of amino acid residues 50 to 240 has the greatest potential since more epitopes were identified in this region estimated by the different programs.
The immunological potential of CP40 has been previously studied in silico, with the description of six immunodominant epitopes and hydrophobic areas of the protein that could interact with the Toll-like 2 receptor [30].These sequences are conserved in CP40 2J-L, providing evidence for the ability of this protein to bind TLR2.Toll-like receptors (TLR) are involved in the innate and adaptive responses of the immune system [31].
Recently, six peptides from the CP40 protein were evaluated for vaccine formulation.We could identify those peptides as being part of B cell epitopes predicted in our work by the BepiPred 2.0 program.Peptide 1 was included in epitope number 5, peptides 2 and 4 are included in epitope number 8, and peptide 3 is part of epitope number 3. For peptide 5, we could identify the sequence as part of an epitope classified as antigenic and surface-exposed.In addition, peptide 6 was classified as a TLR2 activator.Each peptide was evaluated independently in vaccine formulations and a murine model.Different protective values were obtained; for example, peptides 4 and 6 provided 10% protection, while peptide 5 provided 20%.In addition, peptides 1 and 2 provided 30% and 40%, respectively, the highest protection values.Although all groups were affected during the challenge with a virulence strain, they also showed the production of total IgG antibodies and cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α), indicating a possible activation of the Th1 type response.The authors suggest the use of adjuvants based on PAMPs to improve the immune response offered by these peptides [32].Since peptide sequences are also included on some predicted epitopes in our work, we consider it interesting to evaluate a multi-epitope vaccine with the combination of all peptides in the same formulation to enhance levels of protection.
Previously, no epitope mapping has been performed for these proteins, comparing the results obtained by predictors with different selection criteria such as physicochemical properties, surface epitope exposure, antigenic properties, and three-dimensional structure.In addition, computational prediction tools based on antigen structures constitute an alternative for in silico epitope prediction [33,34].However, structural information is only available for a few antigens, and in the vast majority of cases, one is left with analyzing the primary sequence.Since there is no crystal structure for PLD or CP40, we used the Phyre 2.0 program to predict the PDB format for both proteins.
Our work presents for the first time a summary of the overlapping sequences that were predicted by different criteria and included as B epitopes by more than two predictors.The results in Table 3 provide details of the protein sequences and regions most likely responsible for the humoral response.For peptide selection for vaccine development, we recommended the epitopes that were identified by more than one criterion and have a high score value such as 9 MREKVVLFLSIIMAIMLPVGNAAAAPVVHNPAS 33 of PLD and 9 MHNSPRSVSRLITVGITSALFASTFSAVSASATLSKEPLKASPG 46 of CP40.

T Lymphocyte Epitope Prediction of PLD 2J-L and CP40 2J-L
The intracellular life of C. pseudotuberculosis biovar ovis allows it to survive and multiply within macrophages, so the induction of a cellular immune response is crucial to achieving an effective vaccine that generates complete protection [35].T CD8+ cells are responsible for eliminating intracellular pathogens [36].Therefore, new studies are needed to understand how to modulate this immune response.
MHC class I is expressed in all cells except erythrocytes, neurons, and the fetal trophoblast.Peptides presented by this class of MHC are smaller, between 8 and 10 amino acids.MHC I only presents antigens to the TCR (T cell receptor) of cytotoxic T lymphocytes, cells with CD8+ molecules that specifically interact with MHC I [37,38].
The MCH class II can present peptides between 13 and 17 amino acids.MHC II is only possessed by a very particular group of cells of the immune system, which have been called professional antigen-presenting cells (APCs), and among them are B lymphocytes, monocytes, dendritic cells, epithelial cells of the thymus, Langerhans cells, and cells from Kupffer.These molecules only present antigens to T helper lymphocyte receptors because on their surface there is molecule CD4 that binds to one of the MHC II chains during antigen presentation [38].
MHC genes are highly polymorphic, which means that there are many different alleles in the individuals within a population.Polymorphism is so high that in a mixed population, no two individuals have the same set of genes and MHC molecules.The polymorphic regions in each allele are located in the peptide contact region, which is to be displayed to the lymphocyte.For that reason, the region of contact for each allele of the MHC molecule is highly variable, as polymorphic MHC residues will create specific clefts that only certain types of peptide residues can enter.This imposes a very specific binding between the MHC molecule and the peptide and implies that each MHC variant will be able to specifically bind only those peptides that fit properly into the MHC molecule's cleft, which is variable for each allele [39,40].
Numerous T cell epitope-mapping algorithms have been established and used to rapidly identify putative T cell epitopes [41], mostly directed at the detection of the epitopes for human alleles.We used the IEDB analysis resource database that uses NetMHCpan as a prediction method [42] to generate a high quantitative prediction.Even when this tool constitutes one of the few databases that include a variety of MHC alleles from different organisms, such as humans, chimpanzees, macaques, gorillas, cows, pigs, and mice, there is still limited success for prediction of other species like sheep, caprine, or equine, due to an insufficient training data set.
Since the murine model has been used for experimental CLA vaccine evaluation, constituting a good model for abscess formation and systemic infection [13], we used mouse allele information to search T cell epitopes in the context of presentation by MHC-I and MHC-II.
The selection of regions that share B and T cell activation capacity would be the most opportune for the development of a peptide vaccine, since they would guarantee the stimulation of the humoral and cellular responses.
This study was carried out with MHC alleles as references since the site for prediction does not have any information for ovine or caprine MHC molecules.The genes that code for the MHC in sheep are located on chromosome 20, initially called OLA based on the HLA nomenclature for humans, but later the name Ovar-MHC was unofficially established.In sheep, the class I region is poorly characterized, and different studies have proposed that there are four loci: OLA-A, B, C, and D. Ovine MHC-class I genes are more similar to bovine than to murine class I genes [43]; however, the IEDB database also does not contain information on bovine alleles, so we used the murine allele data.Like MHC class I molecules from other species, those from sheep were found to be distributed in lymphocytes and non-lymphoid tissues [44].The high degree of polymorphism in the genes that encode the MHC impacts the individual immune response during vaccination; for this reason, different alleles were evaluated in the presentation of the T cell epitopes.
The results obtained show that peptides of PLD 2J-L and CP40 2J-L are capable of being presented by MHC I and MHC II alleles in the murine model.The inclusion of these epitopes in a vaccine formulation could produce adequate stimulation of the specific immune response, although it must be taken into consideration that these results were obtained in a murine model.Cytotoxic T lymphocytes (CTLs) recognize antigens presented by the MHC class I receptor found on all nucleated cells.Effector CTL cells have the ability to recognize and kill cells infected with the pathogen, constituting an important component of protective immunity.However, it has been proven that during chronic infections T cells may suffer from functional deterioration, which is one of the main reasons for the inability of the host to eliminate the persistent pathogen.T cells that are generated during the early stages of infection gradually lose function during the course of chronic infection.This phenomenon of exhausted T lymphocyte production has been characterized in mice and humans with established chronic infections by viral agents [45].For these reasons, it is essential to activate memory T cells.Despite some similarities at the molecular, epigenetic, metabolic, and functional levels between effector CD8+ T cells and memory CD8+ T cells, the latter persist for long periods of time and are capable of proliferating after re-encountering the antigen [36].The results achieved in this work correspond to the prediction of epitopes capable of activating the global population of CD8+ T cells; therefore, experimental studies will be required to specifically evaluate the subset of memory CD8+ T cells [36].
There is experimental evidence indicating that formulations with recombinant CP40 protein were more efficient in providing protection associated with a Th1 cell response in the absence of a Th2 profile [20].The identification of a significant increase in the levels of the IgG2 isotype is related to a Th1 cellular response, cells mainly involved in immunity against intracellular pathogens, activation of macrophages and cytotoxic T cells, opsonization production, and complement activation [17].
The role of TLR2 in the homeostasis of memory T CD8+ cells is related to the direct control of the proliferation of these cells and the secretion of IFN- A study demonstrated the participation of TLR2 in the activation of memory T CD8+ cells, during an established chronic viral infection in a murine model, causing an increase in the proliferation and expansion of these cells, induced by IL-7 both in vitro and in vivo [46,47].Another study found that TLR2 increases the activation of T CD8+ cells and allows the generation of functional memory cells in response to low or inefficient TCR signals [48].Taken together, these data support the idea that CP40 2J-L peptides that induce TLR2 signaling could directly contribute to the maintenance of CD8+ T cell memory.
T CD4+ cells activate other cells of the immune system, such as B cells, CD8+ T cells, and macrophages.Under conditions of chronic infection, in which CD8+ T cells take several months or more to clear the infection, CD4+ T cells play an important role in maintaining CD8+ T cell activity [49].Experimental studies have considered the induction of a Th1 response (CD4+ T cells) to be relevant for the activation of CD8+ T cells in the context of MHC I, thus achieving an efficient response against this pathogen [18,19].

PLD 2J-L and CP40 2J-L Proteins Sequences and Structure
The published PLD 2J-L (accession: UYI58181) and CP40 2J-L (accession: UYI58182) protein sequences were retrieved from the National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov/(Accessed on 6 March 2023).Proteins belong to isolate 2J-L of Corynebacterium pseudotuberculosis biovar ovis obtained from a clinical case of Caseous Lymphadenitis in a sheep from the State of Jalisco, Mexico [23].The Phyre2 web portal was used for modeling protein structures and producing structures in PDB format, which is necessary for conformational epitope prediction [50].This web portal includes different programs such as HHpred 1.51 for template detection [51], Psi-pred 2.5 for secondary structure prediction [52], Disopred 2.4 for structure disorder prediction [53], Memsat SVM for transmembrane domain prediction [54], multi-template modeling, Poing 1.0 ab initio for template less structure modeling [55], and the the Jmol viewer for a 3D view of structures [56].

Linear and Conformational B Cell Epitope Prediction Tools
B cell epitope prediction was carried out using available tools in the Immune epitope database and resource website.Linear epitopes were predicted with BepiPred 2.0 (score value: 0.5) [57], the Emini surface scale for surface accessibility prediction (score value: 1.0) [58], and the Kolaskar and Tongaonkar antigenicity scale (score value: 0.5) [59].Ellipro http://tools.iedb.org/ellipro/(Accessed on 5 June 2023) [33] was used for linear and conformational epitopes prediction (score value: 0.5) using 3D protein structures in PDB format.B cell epitopes predicted by Ellipro were visualized in the Jmol program.Manual analyses were done for the identification of sequences predicted by more than one predictor.

Conclusions
The prediction of B and T cell epitopes in an antigen constitutes one of the important steps in the design of multi-epitope-based vaccines.B cell epitopes were predicted independently using selected prediction tools, and the results were compared to obtain conserved sequences by the different programs.T CD8+ and CD4+ cell epitopes and their corresponding MHC restricted alleles were identified by prediction tools provided by the immune epitope database (IEDB).Results confirmed the potential inmunodominant epitopes of PLD and CP40, containing 'promiscuous' or 'universal' epitopes that cover diverse MHC haplotypes.This knowledge can then be utilized to improve peptide-based vaccine design for Caseous Lymphadenitis.

Figure 1 .
Figure 1.B cell epitopes of PLD 2J-L (A) and CP40 2J-L (B) using the IEDB website.Epitopes most likely to interact with antibodies are colored red, surface epitopes are indicated by an underlined sequence, antigenic epitopes are indicated in bold letters, and the sequence that shares the ability to bind antibodies and is further exposed to the surface is shown in blue.

Figure 1 .
Figure 1.B cell epitopes of PLD 2J-L (A) and CP40 2J-L (B) using the IEDB website.Epitopes most likely to interact with antibodies are colored red, surface epitopes are indicated by an underlined sequence, antigenic epitopes are indicated in bold letters, and the sequence that shares the ability to bind antibodies and is further exposed to the surface is shown in blue.

Figure 2 .
Figure 2. A schematic display of B lymphocyte linear epitopes of PLD 2J-L (A) and CP40 2J-L (B) estimated by ElliPro, based on the 3D structure of the proteins.The numbers represent the epitopes of Table2.

Figure 2 .
Figure 2. A schematic display of B lymphocyte linear epitopes of PLD 2J-L (A) and CP40 2J-L (B) estimated by ElliPro, based on the 3D structure of the proteins.The numbers represent the epitopes of Table2.

Figure 3 .
Figure 3.A schematic display of B lymphocyte discontinuous epitopes of PLD 2J-L (A) and CP40 2J-L (B) estimated by ElliPro program, based on the 3D structure of the proteins.Numbers represent each epitope in Table3.Fifty B lymphocyte epitopes for PLD 2J-L and forty-seven for CP40 2J-L (9-12 by Bepi-Pred 2.0, 9-8 by Emini Surface Accessibility Scale, 11-11 by Antigenicity Kolaskar-Tongaonkar scale, and 13-13 linear and 8-3 discontinuous by ElliPro using protein structures in PDB format) were estimated using bioinformatics tools.The resulting data set

Figure 3 .
Figure 3.A schematic display of B lymphocyte discontinuous epitopes of PLD 2J-L (A) and CP40 2J-L (B) estimated by ElliPro program, based on the 3D structure of the proteins.Numbers represent each epitope in Table3.

Table 1 .
B lymphocyte epitopes of PLD 2J-L and CP40 2J-L proteins predicted by the BepiPred 2.0 program.
* Minimum and maximum values within the epitope's entire sequence.

Table 2 .
B lymphocyte linear epitopes of PLD 2J-L and CP40 2J-L proteins determined by the ElliPro program.

Table 2 .
B lymphocyte linear epitopes of PLD 2J-L and CP40 2J-L proteins determined by the ElliPro program.

Table 3 .
B lymphocyte conformational epitopes of PLD 2J-L and CP40 2J-L proteins determined by the ElliPro program.

Table 4 .
Mapping of amino acid residues of the PLD 2J-L and CP40 2J-L proteins predicted as part of B lymphocyte epitopes identified by different prediction programs.

Table 5 .
T lymphocyte epitopes presented in the context of MHC class I, of the PLD 2J-L and CP40 2J-L proteins, and the aa residues that overlap as B cell epitopes.

Table 6 .
T lymphocyte epitopes presented in the context of MHC class II, of the PLD 2J-L and CP40 2J-L proteins and the aa residues that overlap as B cell epitopes.