Construction of A Synthetic Gene Encoding the Multi-Epitope of Toxoplasma gondii and Demonstration of the Relevant Recombinant Protein Production: A Vaccine Candidate
DOI:
https://doi.org/10.31661/gmj.v9i.1708Keywords:
Toxoplasma gondii; DNA Vaccine; Multi-Epitope; BioinformaticsAbstract
Background: Toxoplasma gondii is a widely-distributed parasite all over the world whose attributed severe afflicting complications in human necessitate the development of serodiagnostic tests and vaccines for it. Immunological responses to monovalent vaccines and the application of diagnostic reagents including single antigens are not optimally effective. Bioinformatics approaches were used to introduce these epitopes, predict their immunogenicity and preliminarily evaluate their potential as an effective DNA vaccine and for serodiagnostic goals. Materials and Methods: A 3D structure of proteins was predicted by I-TASSER server, and linear and conformational B cell and T cell epitopes were predicted using the online servers. Then, the predicted epitopes were constructed and called Toxoeb, and their expression in the prokaryotic and eukaryotic cells was demonstrated using SDS-PAGE. In the next step, Western blotting with pooled sera of mice infected with T. gondii was done. Results: The current in silico analysis revealed that the B cell epitopes with high immunogenicity for GRA4 protein were located in the residues 34-71, and 230-266, for GRA14 in 308-387, for SAG1 in 182-195, 261-278, and for GRA7 in residues 101-120, 160-176. The T cell epitopes were selected in overlapping regions with the B cell epitopes. The immunogenic region for GRA4 are in the residues 245-253, 50-58, and 40-54, for GRA14 in 307-315, 351-359, and 308-322, for SAG1 261-269, and 259-267, and for GRA7 in the residues 103-112, and 167-175. The results of the western blotting showed that the expressed protein had immunogenicity. Conclusion: Our constructed multi-epitope of T. gondii could be considered as a candidate for diagnostic and vaccination purposes. [GMJ.2020;9:e1708]
References
Raizman RE, Neva FA. Detection of circulating antigen in acute experimental infections with Toxoplasma gondii. J Infect Dis. 1975;132(1):44-48. https://doi.org/10.1093/infdis/132.1.44PMid:808579 Dubey JP. The history of Toxoplasma gondii-the first 100 years. J Eukaryot Microbiol. 2008;55(6):467-475. https://doi.org/10.1111/j.1550-7408.2008.00345.xPMid:19120791 Dubey JP. Toxoplasmosis of animals and humans. CRC press; 2016. https://doi.org/10.1201/9781420092370 Lindsay D, Dubey J. Toxoplasma gondii: the changing paradigm of congenital toxoplasmosis. Parasitology. 2011;138(14):1829-1831. https://doi.org/10.1017/S0031182011001478PMid:21902872 Sensini A. Toxoplasma gondii infection in pregnancy: opportunities and pitfalls of serological diagnosis. Clin Microbiol and Infect. 2006;12(6):504-512. https://doi.org/10.1111/j.1469-0691.2006.01444.xPMid:16700697 Alibakhshi A, Bandehpour M, Nafarieh T, et al. In silico Analysis of Immunologic Regions of Surface Antigens (Sags) of Toxoplasma gondii. NBM. 2017;5(3):109-118. Hiszczyńska-Sawicka E, Olędzka G, Holec-Gąsior L, et al. Evaluation of immune responses in sheep induced by DNA immunization with genes encoding GRA1, GRA4, GRA6 and GRA7 antigens of Toxoplasma gondii. Vet Parasitol. 2011;177(3-4):281-289. https://doi.org/10.1016/j.vetpar.2010.11.047PMid:21251760 Foroutan M, Ghaffarifar F, Sharifi Z, et al. Bioinformatics analysis of ROP8 protein to improve vaccine design against Toxoplasma gondii. Infect Genet Evol. 2018;62:1-204-93. https://doi.org/10.1016/j.meegid.2018.04.033PMid:29705360 Kulkarni R, Sapkal G, Mahishi L, et al. Design and characterization of polytope construct with multiple B and TH epitopes of Japanese encephalitis virus. Virus res. 2012;166(1-2):77-86. https://doi.org/10.1016/j.virusres.2012.03.006PMid:22445688 Karpenko LI, Bazhan SI, Antonets DV, et al. Novel approaches in polyepitope T-cell vaccine development against HIV-1. Expert rev vaccines. 2014;13(1):155-173. https://doi.org/10.1586/14760584.2014.861748PMid:24308576 Gershoni JM, Roitburd-Berman A, Siman-Tov DD, et al. Epitope mapping. BioDrugs. 2007;21(3):145-156. https://doi.org/10.2165/00063030-200721030-00002PMid:17516710 PMCid:PMC7100438 Pomés A. Relevant B cell epitopes in allergic disease. In arch of allergy immunol. 2010;152(1):1-11. https://doi.org/10.1159/000260078PMid:19940500 PMCid:PMC2956005 Gazzinelli RT, Hakim F, Hieny S, et al. Synergistic role of CD4+ and CD8+ T lymphocytes in IFN-gamma production and protective immunity induced by an attenuated Toxoplasma gondii vaccine. J Immunol. 1991;146(1):286-292. Van MR. Structural and functional approaches to the study of protein antigenicity. Immunol Today. 1989;10(8):266-272. https://doi.org/10.1016/0167-5699(89)90140-0 Wang Y, Wang M, Wang G, 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. Vaccine. 2011;29(47):8619-8623. https://doi.org/10.1016/j.vaccine.2011.09.016PMid:21939715 Dziadek B, Brzostek A. Recombinant ROP2, ROP4, GRA4 and SAG1 antigen-cocktails as possible tools for immunoprophylaxis of toxoplasmosis: what's next?. Bioengineered. 2012;3(6):358-364. https://doi.org/10.4161/bioe.21541PMid:22892593 PMCid:PMC3489714 Garcia JL, Innes EA, Katzer F. Current progress toward vaccines against Toxoplasma gondii. Vaccine (Auckl). 2014;4(1). https://doi.org/10.2147/VDT.S57474 Quan J-H, Chu J-Q, Ismail HAHA, et al. Induction of protective immune responses by a multiantigenic DNA vaccine encoding GRA7 and ROP1 of Toxoplasma gondii. Clin Vaccine Immunol. 2012:CVI. 05385-11. https://doi.org/10.1128/CVI.05385-11PMid:22419676 PMCid:PMC3346315 Yang C-S, Yuk J-M, Lee Y-H, et al. Toxoplasma gondii GRA7-induced TRAF6 activation contributes to host protective immunity. Infect Immun. 2016;84(1):339-350. https://doi.org/10.1128/IAI.00734-15PMid:26553469 PMCid:PMC4693986 Rome ME, Beck JR, Turetzky JM, et al. Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii. Infect Immun. 2008;76(11):4865-4875. https://doi.org/10.1128/IAI.00782-08PMid:18765740 PMCid:PMC2573327 Gasteiger E, Hoogland C, Gattiker A, et al. Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook: Springer; 2005. p. 571-607. https://doi.org/10.1385/1-59259-890-0:571 Garnier J, Gibrat J-F, Robson B. GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol. Vol. 266: Elsevier; 1996. p. 540-553. https://doi.org/10.1016/S0076-6879(96)66034-0 Krogh A, Larsson B, Von Heijne G, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001;305(3):567-580. https://doi.org/10.1006/jmbi.2000.4315PMid:11152613 Armenteros JJA, Tsirigos KD, Sønderby CK, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol. 2019: 37(4):420-423. https://doi.org/10.1038/s41587-019-0036-zPMid:30778233 Yang J, Zhang Y. I-TASSER server: new development for protein structure and function predictions. Nucleic acids research. 2015;43(W1):W174-W181. https://doi.org/10.1093/nar/gkv342PMid:25883148 PMCid:PMC4489253 Zhang C, Freddolino PL, Zhang Y. COFACTOR: improved protein function prediction by combining structure, sequence and protein-protein interaction information. Nucleic Acids res. 2017;45(W1):W291-W299. https://doi.org/10.1093/nar/gkx366PMid:28472402 PMCid:PMC5793808 Wu S, Skolnick J, Zhang Y. Ab initio modeling of small proteins by iterative TASSER simulations. BMC Biol. 2007;5(1):17. https://doi.org/10.1186/1741-7007-5-17PMid:17488521 PMCid:PMC1878469 Laskowski RA, MacArthur MW, Moss DS, et al. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst. 1993;26(2):283-291. https://doi.org/10.1107/S0021889892009944 Saha S, Raghava G, editors. BcePred: prediction of continuous B-cell epitopes in antigenic sequences using physico-chemical properties. International Conference on Artificial Immune Systems; 2004: Springer. https://doi.org/10.1007/978-3-540-30220-9_16 Singh H, Ansari HR, Raghava GP. Improved method for linear B-cell epitope prediction using antigen's primary sequence. PloS one. 2013;8(5):e62216. https://doi.org/10.1371/journal.pone.0062216PMid:23667458 PMCid:PMC3646881 Yao B, Zhang L, Liang S, et al. SVMTriP: a method to predict antigenic epitopes using support vector machine to integrate tri-peptide similarity and propensity. PloS one. 2012;7(9):e45152. https://doi.org/10.1371/journal.pone.0045152PMid:22984622 PMCid:PMC3440317 Chou P, Fasman G. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol 1978: 47: 45-148.. https://doi.org/10.1002/9780470122921.ch2 Karplus P, Schulz G. Prediction of chain flexibility in proteins. Naturwissenschaften. 1985;72(4):212-213. https://doi.org/10.1007/BF01195768 Emini EA, Hughes JV, Perlow D, et al. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol. 1985;55(3):836-839. https://doi.org/10.1128/JVI.55.3.836-839.1985PMid:2991600 PMCid:PMC255070 Parker J, Guo D, Hodges R. New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites. Biochemistry. 1986;25(19):5425-5432. https://doi.org/10.1021/bi00367a013PMid:2430611 Kolaskar A, Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS letters. 1990;276(1-2):172-174. https://doi.org/10.1016/0014-5793(90)80535-Q Kringelum JV, Lundegaard C, Lund O, et al. Reliable B cell epitope predictions: impacts of method development and improved benchmarking. PLoS Comput Biol. 2012;8(12):e1002829. https://doi.org/10.1371/journal.pcbi.1002829PMid:23300419 PMCid:PMC3531324 Ansari HR, Raghava GP. Identification of conformational B-cell Epitopes in an antigen from its primary sequence. Immunome Res. 2010;6(1):6. https://doi.org/10.1186/1745-7580-6-6PMid:20961417 PMCid:PMC2974664 Zhao L, Zhang M, Cong H. Advances in the study of HLA-restricted epitope vaccines. Human vaccines & immunotherapeutics. 2013;9(12):2577-66. https://doi.org/10.4161/hv.26088PMid:23955319 PMCid:PMC4162067 Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990;211(4):943-958. https://doi.org/10.1016/0022-2836(90)90085-Z Grote A, Hiller K, Scheer M, et al. JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 2005;33(suppl_2):W526-W531. https://doi.org/10.1093/nar/gki376PMid:15980527 PMCid:PMC1160137 Kingston RE, Chen CA, Rose JK. Calcium phosphate transfection. Current protocols in molecular biology. 2003;63(1):9.1.1- 9.1. 11 https://doi.org/10.1002/0471142727.mb0901s63PMid:18265332 Wang Y, Wang G, Cai J, et al. Review on the identification and role of Toxoplasma gondii antigenic epitopes. Parasitol Res. 2016;115(2):459-468. https://doi.org/10.1007/s00436-015-4824-1PMid:26581372 Lu G, Wang L, Zhou A, et al. Epitope analysis, expression and protection of SAG5A vaccine against Toxoplasma gondii. Acta Trop. 2015;146:66-72. https://doi.org/10.1016/j.actatropica.2015.03.013PMid:25792417 Wang Y, Wang G, Zhang D, et al. Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1. Parasit Vectors. 2013;6(1):125. https://doi.org/10.1186/1756-3305-6-125PMid:23631709 PMCid:PMC3655890 Romano P, Giugno R, Pulvirenti A. Tools and collaborative environments for bioinformatics research. Brief Bioinform. 2011;12(6):549-561. https://doi.org/10.1093/bib/bbr055PMid:21984743 PMCid:PMC3220874 Wang Y, Zhang D, Yin H, et al. Advances in predicting methods of antigen epitopes. Chinese Veterinary Science/Zhongguo Shouyi Kexue. 2009: 39(10):938-940 Garnier J, Robson B. The GOR method for predicting secondary structures in proteins. Prediction of protein structure and the principles of protein conformation: Springer; 1989. p. 417-465. https://doi.org/10.1007/978-1-4613-1571-1_10 Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. 1981;78(6):3824-3828. https://doi.org/10.1073/pnas.78.6.3824PMid:6167991 PMCid:PMC319665 Saha S, Raghava G. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins : Structure, Function, and Bioinformatics. 2006;65(1):40-48. https://doi.org/10.1002/prot.21078PMid:16894596 Liu S, Shi L, Cheng Y-b, Fan G-x, Ren H-x, Yuan Y-k. Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice. Parasitol.Res 2009;105(1):267. https://doi.org/10.1007/s00436-009-1393-1PMid:19288132 Hajissa K, Zakaria R, Suppian R, Mohamed Z. Immunogenicity of Multiepitope Vaccine Candidate against Toxoplasma gondii Infection in BALB/c Mice. Iran J PARASITOL. 2018;13(2):215.
