The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents.
Allergy and Immunology ; Antibodies ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Epitopes, B-Lymphocyte* ; Indicators and Reagents ; Peptides ; Toxoplasma* ; Vaccines

HLA-A * 0201, HLA-A * 1101, and HLA-A * 2401 CTL restricted epitopes of platelet membrane glycoprotein II b/III a antibody of human and mice were predicted by use of SYFPEITHI, RANKPEP, BIMAS, SVMHC, PREDEP, MHCPRED, and PROPRED predictive programs. In the results, the peptides (found in HLAPRED) that can lead to autoimmune disease and have been published were removed; and the epitopes of HLA-A * 0201 must cover the epitopes of HLA-A * 1101 and HLA-A * 2401 being combined to predTAP and TAPPred for predicting the binding affinity of peptides toward the TAP transporter and NetChop, MAPPP, PAProc for predicting cleavages; HLA-DR Th restricted epitopes of GPII b/III a antibody were predicted by SYFPEITHI, RANKPEP, MHCPRED, and HLAPRED, after removal of the peptides (found in HLAPRED) that can lead to autoimmune disease and have been published, the Th epitopes must cover the CTL mixed epitopes as being stated above. The secondary structure, hydrophobic regions, flexibility, surface probability and the B cell epitope were predicted by using various methods. Ten mixed peptides of T cell epitopes were selected from more than 1 740 peptides. They were located at the aa9-115, aa24-38, aa50-64, aa65-81, aa109-121 of anti-GP II b/III a-Human and the aal-15, aa26-40, aa46-60, aa68-82, aa93-107 of anti-GP II b/III a-Mice. B cell epitopes of anti-GP II b/III a-Human might locate at aa5-9, aa22-30, aa40-46, aa55-71, aa80-90, aa100-105, aa110-115; and the epitopes of anti-GP II b/III a-Human might locate at aa5-10, aa38-43, aa58-70, aa77-84, and aa99-105.
Animals ; Antibodies ; immunology ; Epitopes, B-Lymphocyte ; immunology ; Epitopes, T-Lymphocyte ; immunology ; Humans ; Mice ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; Vaccines ; immunology

Objectives To develop a multi-stage and multi-epitope vaccine, which consists of epitopes from the early secretory and latency-associated antigens of Mycobacterium tuberculosis (MTB). Methods The B-cell, cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes of 12 proteins were predicted using an immunoinformatics. The epitopes with antigenicity, without cytotoxicity and sensitization, were further screened to construct the multi-epitope vaccine. Furthermore, the proposed vaccine underwent physicochemical properties analysis and secondary structure prediction as well as 3D structure modeling, refinement and validation. Then the refined model was docked with TLR4. Finally, an immune simulation of the vaccine was carried out. Results The proposed vaccine, which consists of 12 B-cell, 11 CTL and 12 HTL epitopes, had a flexible and stable globular conformation as well as a thermostable and hydrophilic structure. A stable interaction of the vaccine with TLR4 was confirmed by molecular docking. The efficiency of the candidate vaccine to trigger effective cellular and humoral immune responses was assessed by immune simulation. Conclusion A multi-stage multi-epitope MTB vaccine construction strategy based on immunoinformatics is proposed, which is expected to prevent both active and latent MTB infection.
Mycobacterium tuberculosis/metabolism* ; Molecular Docking Simulation ; Toll-Like Receptor 4 ; Epitopes, T-Lymphocyte/chemistry* ; Epitopes, B-Lymphocyte/chemistry* ; Vaccines, Subunit/chemistry* ; Computational Biology/methods*

Epitopes, T-Lymphocyte ; immunology ; HLA Antigens ; immunology ; Hepatitis B virus ; immunology ; Humans ; T-Lymphocytes, Cytotoxic ; immunology

To establish a relation between an protein amino acid sequence and its tendencies to generate antibody response, and to investigate an improved in silico method for linear B-cell epitope (LBE) prediction. We present a sequence-based LBE predictor developed using deep maxout network (DMN) with dropout training techniques. A graphics processing unit (GPU) was used to reduce the training time of the model. A 10-fold cross-validation test on a large, non-redundant and experimentally verified dataset (Lbtope_Fixed_ non_redundant) was performed to evaluate the performance. DMN-LBE achieved an accuracy of 68.33% and an area under the receiver operating characteristic curve (AUC) of 0.743, outperforming other prediction methods in the field. A web server, DMN-LBE, of the improved prediction model has been provided for public free use. We anticipate that DMN-LBE will be beneficial to vaccine development, antibody production, disease diagnosis, and therapy.
Amino Acid Sequence ; Computational Biology ; methods ; Epitopes, B-Lymphocyte ; chemistry ; immunology ; ROC Curve

<b>OBJECTIVEb>To predict and identify B-cell linear epitopes of hepatitis B e antigen (HBeAg).

<b>METHODSb>The B-cell linear epitopes of HBeAg were predicted using the software provided by NCBI Database and Immune Epitope Database (IEDB) and synthesized by a solid-phase method followed by conjugation with keyhole limpet hemocyanin (KLH). The KLH conjugates were used for immunization of New Zealand white rabbits, and the immune response of the rabbits was monitored by direct ELISA using a bovine serum albumin conjugate of the predicted epitopes. RESULTS Four new B-cell linear epitopes of HBeAg were identified, namely (1)MDIDPYKEFG(10), (37)LYREALESPEHCSP(50), (74)SNLEDPAS(81) and (127)RTPPAYRPPNAPIL(140). The rabbits immunized with the KLH conjugate showed an antibody titer over 1:512 000. The antisera of B-cell linear epitopes collected could specifically react with HBeAg as shown by ELISA.

<b>CONCLUSIONb>Four B-cell linear epitopes of HBeAg have been confirmed using bioinformatics methods, which provides new evidence for further functional studies of HBeAg in hepatitis B.

<b>OBJECTIVEb>To identify HLA-A0201 restricted cytotoxic T lymphocyte (CTL) epitopes derived from the hepatitis B virus e (HBe) antigen, for future use in a specific immunotherapy based on the identified epitope(s).

<b>METHODSb>HBe gene sequences from the hepatitis B virus serotypes with the highest frequencies in China were analyzed by bioinformatic web-based interfaces for quantitative motif prediction, extended motif prediction, and peptide super-motif prediction. Four candidate peptides were identified: HBe1, HBe2, HBe3, and HBe4. The affinities of each were tested in vitro with T2 cells, which lack the transporter-associated with antigen transport (TAP) protein but express low levels of the MHC class I surface molecule, and measured by the T2 binding assay and DC50 assay. Flow cytometry was used to detect the fluorescence index of control and experimental groups.

<b>RESULTSb>The peptides HBe1 (LLWFHISCL), HBe2 (YLVSFGVWI), HBe3 (CLTFGRETV), and HBe4 (DLLDTASAL) were identified and tested as candidate targets. HBe2 and HBe3 showed higher HLA-A0201 affinity. HBe1, HBe2, and HBe3 showed better binding stability.

<b>CONCLUSIONb>Two peptides based on HBe antigen, YLVSFGVWI and CLTFGRETV, possess both sufficient binding affinity and stability and may represent useful HLA-A0201-restricted CTL epitopes. Further study is needed to determine the immunogenic properties of these two peptides in vivo.

Epitopes, T-Lymphocyte ; HLA-A2 Antigen ; immunology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; Humans ; Mutation

Due to considerably high degree of sequence homology between bacterial and human heat shock proteins (hsp), it has been widely thought that this protein might be involved in autoimmune disease mechanisms in humans.To elucidate how stress proteins contribute in the immunopathogenesis of periodontitis, the present study was performed to evaluate the T cell immune responses specific to Porphyromonas gingivalis (P. gingivalis) heat shock protein (hsp)60 and T-cell epitope specificities for P. gingivalis hsp60 in periodontitis. Anti-P. gingivalis IgG antibody titers were elevated in all patients. We could establish P. gingivalis hsp-specific T cell lines from the peripheral blood of periodontitis, a mixture of CD4+ and CD8+ cells. Of 108 overlapping synthetic peptides spanning whole P. gingivalis hsp60 molecule, ten peptides with epitopes specifities for T-cell were showed. Interestingly, ten epitopes were also identified as T-cell epitopes in the present study as well as B-cell epitopes in periodontitis. Therefore, all the ten representative epitopes were designated as common T-and Bcell epitopes for periodontitis. It is critical in developing a peptide vaccine strategy for potential prevention of periodontitis. It was concluded that P. gingivalis hsp60 might be involved in the immunoregulatory process of periodontitis with heat shock protein specificities.
Autoimmune Diseases ; Cell Line ; Epitopes ; Epitopes, B-Lymphocyte ; Epitopes, T-Lymphocyte* ; Heat-Shock Proteins* ; Hot Temperature* ; Humans ; Immunoglobulin G ; Peptides ; Periodontitis* ; Porphyromonas gingivalis* ; Porphyromonas* ; Sensitivity and Specificity* ; Sequence Homology ; T-Lymphocytes*

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.
Animals ; Cloning, Molecular ; Computational Biology ; Computer Simulation* ; Epitopes ; Epitopes, B-Lymphocyte ; Fusobacterium nucleatum* ; Fusobacterium* ; Hydrophobic and Hydrophilic Interactions ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Membrane Proteins ; Mice ; Periodontitis ; Solubility ; Vaccination