Bioinformatics Analysis to Designing a Multi-epitope-based Peptide Vaccine Combat Leishmania major

Karimi Rouzbahani, Arian; Kheirandish, Farnaz; Hashemzadeh, Pejman

doi:10.30699/ijmm.16.5.430

year 16, Issue 5 (September - October 2022) Iran J Med Microbiol 2022, 16(5): 430-446 | Back to browse issues page

‎ 10.30699/ijmm.16.5.430

Mendeley

Zotero

RefWorks

Karimi Rouzbahani A, Kheirandish F, Hashemzadeh P. Bioinformatics Analysis to Designing a Multi-epitope-based Peptide Vaccine Combat Leishmania major. Iran J Med Microbiol 2022; 16 (5) :430-446
URL: http://ijmm.ir/article-1-1596-en.html

Bioinformatics Analysis to Designing a Multi-epitope-based Peptide Vaccine Combat Leishmania major

Arian Karimi Rouzbahani¹

, Farnaz Kheirandish²

, Pejman Hashemzadeh

1- Student Research Committee, Lorestan University of Medical Sciences, Khorramabad, Iran
2- Department of Medical Biotechnology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran
3- Department of Medical Biotechnology, School of Medicine, Lorestan University of Medical Sciences, Khorramabad, Iran , pejman7genetian@gmail.com

Abstract: (1693 Views)

Background and Aim: Cutaneous leishmaniasis is a significant public health issue worldwide. Cutaneous leishmaniasis is the most prevalent in the world among the different types of leishmaniasis. Currently, available medications have had no discernible influence on the disease's progression. Up to now, there has been no approved cutaneous leishmaniasis vaccine. New developments in vaccination might be a potential way to come up with a vaccination that is successful for the treatment of cutaneous leishmaniasis.
Materials and Methods: This research was conducted to learn more about an effective vaccine for Leishmania major, the ailment's primary cause of CL, which was designed using computational methods. Thus, a multiepitope protein was designed by utilizing potential immune system epitopes, including predicted MHC class I, MHC class II, Cytotoxic T lymphocytes, B-cell, and Interferon-gamma epitopes of Cysteine protease b (CPB), Leishmania homologue of activated C kinase (LACK), and Kinetoplastid membrane protein-11 (KMP-11) antigenic proteins. In order to enhance vaccine immunogenicity, two resuscitation-promoting factors of Mycobacterium tuberculosis were used as adjuvants. Final epitopes were matched with suitable linkers to construct the recombinant structure. The physicochemical and immune-based characteristics of the designed vaccine have been forecasted by using different tools. Moreover, homogeneity modeling was performed to obtain a high-quality 3D structure, followed by refinement and validation. Finally, the codon optimization based on E. coli resulted in a higher CAI value and optimal GC content, followed by combining it in the pET-14b cloning vector.
Results: Evaluation of the various characteristics of the designed vaccine showed that it is an immunogenic and non-allergenic antigen that can induce immune responses against Leishmania major infection, which could be promising for cutaneous leishmaniasis.
Conclusion: Research shows that a recombinant vaccine can be an effective candidate against cutaneous leishmaniasis.

Keywords: Bioinformatics, Cutaneous leishmaniasis, Immunoinformatics, Leishmania major, Multi-epitope vaccine

Full-Text [PDF 1713 kb] (696 Downloads) | | Full-Text (HTML) (1156 Views)

Type of Study: Original Research Article | Subject: Microbial Bioinformatics
Received: 2021/12/25 | Accepted: 2022/06/4 | ePublished: 2022/08/8

References

1. Kevric I, Cappel MA, Keeling JH. New world and old world Leishmania infections: a practical review. Dermatol Clin. 2015;33(3):579-93. [DOI:10.1016/j.det.2015.03.018] [PMID]

2. Kayani B, Sadiq S, Rashid HB, Ahmed N, Mahmood A, Khaliq MS, et al. Cutaneous Leishmaniasis in Pakistan: a neglected disease needing one health strategy. BMC Infect Dis. 2021;21(1):1-10. [DOI:10.1186/s12879-021-06327-w] [PMID] [PMCID]

3. Seyed N, Taheri T, Vauchy C, Dosset M, Godet Y, Eslamifar A, et al. Immunogenicity evaluation of a rationally designed polytope construct encoding HLA-A* 0201 restricted epitopes derived from Leishmania major related proteins in HLA-A2/DR1 transgenic mice: steps toward polytope vaccine. PLoS One. 2014;9(10): e108848. [DOI:10.1371/journal.pone.0108848] [PMID] [PMCID]

4. Miramin-Mohammadi A, Javadi A, Eskandari SE, Mortazavi H, Rostami MN, Khamesipour A. Immune response in cutaneous leishmaniasis patients with healing vs. non-healing lesions. Iran J Microbiol. 2020;12(3):249-55. [DOI:10.18502/ijm.v12i3.3243] [PMID] [PMCID]

5. Mousavi P, Rahimi Esboei B, Pourhajibagher M, Fakhar M, Shahmoradi Z, Hejazi SH, et al. Anti-leishmanial effects of resveratrol and resveratrol nanoemulsion on Leishmania major. BMC Microbiol. 2022;22(1):1-14. [DOI:10.1186/s12866-022-02455-8] [PMID] [PMCID]

6. Ezra N, Ochoa MT, Craft N. Human immunodeficiency virus and leishmaniasis. J Glob Infect Dis. 2010;2(3):248. [DOI:10.4103/0974-777X.68528] [PMID] [PMCID]

7. Lockard RD, Wilson ME, Rodríguez NE. Sex-Related Differences in Immune Response and Symptomatic Manifestations to Infection with Leishmania Species. J Immunol Res. 2019; 2019:4103819. [DOI:10.1155/2019/4103819] [PMID] [PMCID]

8. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PloS one. 2012; 7(5):e35671. [DOI:10.1371/journal.pone.0035671] [PMID] [PMCID]

9. Global Burden of Disease Study C. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386(9995):743-800. [DOI:10.1016/S0140-6736(15)60692-4]

10. Madusanka RK, Silva H, Karunaweera ND. Treatment of Cutaneous Leishmaniasis and Insights into Species-Specific Responses: A Narrative Review. Infect Dis Ther. 2022:1-17. [DOI:10.1007/s40121-022-00602-2] [PMID] [PMCID]

11. Aoun K, Kalboussi Y, Sghaier IB, Souissi O, Hammami H, Bellali H, et al. Assessment of Incubation Period of Cutaneous Leishmaniasis due to Leishmania major in Tunisia. Am J Trop Med Hyg. 2020;103(5):1934-7. [DOI:10.4269/ajtmh.20-0439] [PMID] [PMCID]

12. Alghounaim M, Chivinski J, Barkati S. Cutaneous leishmaniasis in a 12-year-old Syrian immigrant. CMAJ. 2022;194(3):E93-E4. [DOI:10.1503/cmaj.210847] [PMID] [PMCID]

13. Rabienia M, Roudbari Z, Ghanbariasad A, Abdollahi A, Mohammadi E, Mortazavidehkordi N, et al. Exploring membrane proteins of Leishmania major to design a new multiepitope vaccine using immunoinformatics approach. Eur J Pharm Sci. 2020;152:105423. [DOI:10.1016/j.ejps.2020.105423] [PMID]

14. Erber AC, Sandler PJ, de Avelar DM, Swoboda I, Cota G, Walochnik J. Diagnosis of visceral and cutaneous leishmaniasis using loop-mediated isothermal amplification (LAMP) protocols: a systematic review and meta-analysis. Parasites Vectors. 2022;15(1):1-16. [DOI:10.1186/s13071-021-05133-2] [PMID] [PMCID]

15. Shams M, Nourmohammadi H, Basati G, Adhami G, Majidiani H, Azizi E. Leishmanolysin gp63: Bioinformatics evidences of immunogenic epitopes in Leishmania major for enhanced vaccine design against zoonotic cutaneous leishmaniasis. Inform Med Unlocked. 2021;24: 100626. [DOI:10.1016/j.imu.2021.100626]

16. Kedzierski L. Leishmaniasis vaccine: where are we today? J Glob Infect Dis. 2010;2(2):177. [DOI:10.4103/0974-777X.62881] [PMID] [PMCID]

17. Vélez ID, Gilchrist K, Martínez S, Ramírez-Pineda JR, Ashman JA, Alves FP, et al. Safety and immunogenicity of a defined vaccine for the prevention of cutaneous leishmaniasis. Vaccine. 2009;28(2):329-37. [DOI:10.1016/j.vaccine.2009.10.045] [PMID]

18. Okwor I, Mou Z, Dong L, UZONNA JE. Protective immunity and vaccination against cutaneous leishmaniasis. Front Immunol. 2012;3:128. [DOI:10.3389/fimmu.2012.00128] [PMID] [PMCID]

19. Adu-Bobie J, Capecchi B, Serruto D, Rappuoli R, Pizza M. Two years into reverse vaccinology. Vaccine. 2003;21(7-8):605-10. [DOI:10.1016/S0264-410X(02)00566-2]

20. Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2002;2(11):845-58. [DOI:10.1038/nri933] [PMID]

21. Lakhal-Naouar I, Koles N, Rao M, Morrison EB, Childs JM, Alving CR, et al. Transcutaneous immunization using SLA or rLACK skews the immune response towards a Th1 profile but fails to protect BALB/c mice against a Leishmania major challenge. Vaccine. 2019;37(3):516-23. [DOI:10.1016/j.vaccine.2018.11.052] [PMID]

22. Dariushnejad H, Ghorbanzadeh V, Akbari S, Hashemzadeh P. Design of a Novel Recombinant Multi-Epitope Vaccine against Triple-Negative Breast Cancer. Iran Biomed J. 2022;26(2):160-74.

23. Dariushnejad H, Ghorbanzadeh V, Hashemzadeh P. Prediction of B-and T-cell epitopes using in-silico approaches: a solution to the development of recombinant vaccines against covid-19. Minerva Biotechnol Biomol Res. 2021;33(1):36-42. [DOI:10.23736/S2724-542X.20.02652-X]

24. Chen H-Z, Tang L-L, Yu X-L, Zhou J, Chang Y-F, Wu X. Bioinformatics analysis of epitope-based vaccine design against the novel SARS-CoV-2. Infect Dis Poverty. 2020;9(1):1-10. [DOI:10.1186/s40249-020-00713-3] [PMID] [PMCID]

25. Foroutan M, Ghaffarifar F, Sharifi Z, Dalimi A, Pirestani M. Bioinformatics analysis of ROP8 protein to improve vaccine design against Toxoplasma gondii. Infect Genet Evol. 2018;62: 193-204. [DOI:10.1016/j.meegid.2018.04.033] [PMID]

26. Hashemzadeh P, Rouzbahani Ak, Bandehpour M, Kheirandish F, Dariushnejad H, Mohamadi M. Designing a recombinant multiepitope vaccine against Leishmania donovani based immuno-informatics approaches. Minerva Biotecnol. 2020;32(2):52-7. [DOI:10.23736/S1120-4826.20.02610-5]

27. Marciani DJ. Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discov Today. 2003;8(20): 934-43. [DOI:10.1016/S1359-6446(03)02864-2]

28. Liljeroos L, Malito E, Ferlenghi I, Bottomley MJ. Structural and computational biology in the design of immunogenic vaccine antigens. J Immunol Res. 2015;2015. [DOI:10.1155/2015/156241] [PMID] [PMCID]

29. Todolí F, Solano-Gallego L, De Juan R, Morell P, del Carmen Núñez M, Lasa R, et al. Humoral and in vivo cellular immunity against the raw insect-derived recombinant Leishmania infantum antigens KMPII, TRYP, LACK, and papLe22 in dogs from an endemic area. Am J Trop Med Hyg. 2010; 83(6):1287-94. [DOI:10.4269/ajtmh.2010.09-0784] [PMID] [PMCID]

30. Russo D, Turco S, Burns J, Reed S. Stimulation of human T lymphocytes by Leishmania lipopho-sphoglycan-associated proteins. J Immunol. 1992;148(1):202-7.

31. Mortazavidehkordi N, Fallah A, Abdollahi A, Kia V, Khanahmad H, Najafabadi ZG, et al. A lentiviral vaccine expressing KMP11-HASPB fusion protein increases immune response to Leishmania major in BALB/C. Parasitol Res. 2018;117(7):2265-73. [DOI:10.1007/s00436-018-5915-6] [PMID]

32. Rodríguez-Cortés A, Ojeda A, López-Fuertes L, Timón M, Altet L, Solano-Gallego L, et al. Vaccination with plasmid DNA encoding KMPII, TRYP, LACK and GP63 does not protect dogs against Leishmania infantum experimental challenge. Vaccine. 2007;25(46):7962-71. [DOI:10.1016/j.vaccine.2007.08.023] [PMID]

33. Banuls A-L, Hide M, Prugnolle F. Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans. Adv Parasitol. 2007;64: 1-458. [DOI:10.1016/S0065-308X(06)64001-3]

34. Mottram JC, Coombs GH, Alexander J. Cysteine peptidases as virulence factors of Leishmania. Curr Opin Microbiol. 2004;7(4):375-81. [DOI:10.1016/j.mib.2004.06.010] [PMID]

35. He J, Huang F, Li J, Chen Q, Chen D, Chen J. Bioinformatics analysis of four proteins of Leishmania donovani to guide epitopes vaccine design and drug targets selection. Acta tropica. 2019;191:50-9. [DOI:10.1016/j.actatropica.2018.12.035] [PMID]

36. Joshi S, Rawat K, Yadav NK, Kumar V, Siddiqi MI, Dube A. Visceral leishmaniasis: advancements in vaccine development via classical and molecular approaches. Front Immunol. 2014;5:380. [DOI:10.3389/fimmu.2014.00380] [PMID] [PMCID]

37. Lee S, Nguyen MT. Recent advances of vaccine adjuvants for infectious diseases. Immune Netw. 2015;15(2):51-7. [DOI:10.4110/in.2015.15.2.51] [PMID] [PMCID]

38. Reed SG, Hsu F-C, Carter D, Orr MT. The science of vaccine adjuvants: advances in TLR4 ligand adjuvants. Curr Opin Immunol. 2016;41:85-90. [DOI:10.1016/j.coi.2016.06.007] [PMID]

39. Black M, Trent A, Tirrell M, Olive C. Advances in the design and delivery of peptide subunit vaccines with a focus on toll-like receptor agonists. Expert rev vaccines. 2010;9(2):157-73. [DOI:10.1586/erv.09.160] [PMID] [PMCID]

40. Hashemzadeh P, Ghorbanzadeh V, Valizadeh Otaghsara SM, Dariushnejad H. Novel predicted B-cell epitopes of PSMA for development of prostate cancer vaccine. Int J Pept Res Ther. 2020;26(3):1523-5. [DOI:10.1007/s10989-019-09954-9]

41. Hashemzadeh P, Ghorbanzadeh V, Lashgarian HE, Kheirandish F, Dariushnejad H. Harnessing Bioinformatic Approaches to Design Novel Multi-epitope Subunit Vaccine Against Leishmania infantum. Int J Pept Res Ther. 2019:1-12. [DOI:10.1007/s10989-019-09949-6]

42. Dariushnejad H, Ghorbanzadeh V, Akbari S, Hashemzadeh P. Designing a Multiepitope Peptide Vaccine against COVID-19 Variants Utilizing In-silico Tools. Iran J Med Microbiol. 2021;15(5):7. [DOI:10.30699/ijmm.15.5.592]

43. Pruitt KD, Tatusova T, Maglott DR. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2007; 35(suppl_1):D61-D5. [DOI:10.1093/nar/gkl842] [PMID] [PMCID]

44. Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook: Springer; 2005. p. 571-607. [DOI:10.1385/1-59259-890-0:571]

45. Magnan CN, Zeller M, Kayala MA, Vigil A, Randall A, Felgner PL, et al. High-throughput prediction of protein antigenicity using protein microarray data. Bioinformatics. 2010;26(23):2936-43. [DOI:10.1093/bioinformatics/btq551] [PMID] [PMCID]

46. Geourjon C, Deleage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Bioinformatics. 1995;11(6):681-4. [DOI:10.1093/bioinformatics/11.6.681] [PMID]

47. Reche PA, Glutting J-P, Zhang H, Reinherz EL. Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles. Immunogenetics. 2004;56(6):405-19. [DOI:10.1007/s00251-004-0709-7] [PMID]

48. Andreatta M, Nielsen M. Gapped sequence alignment using artificial neural networks: application to the MHC class I system. Bioinformatics. 2016;32(4):511-7. [DOI:10.1093/bioinformatics/btv639] [PMID] [PMCID]

49. Dhanda SK, Mahajan S, Paul S, Yan Z, Kim H, Jespersen MC, et al. IEDB-AR: immune epitope database-analysis resource in 2019. Nucleic Acids Res. 2019;47(W1):W502-W6. [DOI:10.1093/nar/gkz452] [PMID] [PMCID]

50. Chen J, Liu H, Yang J, Chou K-C. Prediction of linear B-cell epitopes using amino acid pair antigenicity scale. Amino acids. 2007;33(3):423-8. [DOI:10.1007/s00726-006-0485-9] [PMID]

51. Bhasin M, Raghava GP. Prediction of CTL epitopes using QM, SVM and ANN techniques. Vaccine. 2004;22(23-24):3195-204. [DOI:10.1016/j.vaccine.2004.02.005] [PMID]

52. Dhanda SK, Vir P, Raghava GP. Designing of interferon-gamma inducing MHC class-II binders. Biology direct. 2013;8(1):30. [DOI:10.1186/1745-6150-8-30] [PMID] [PMCID]

53. Xia F, Dou Y, Lei G, Tan Y. FPGA accelerator for protein secondary structure prediction based on the GOR algorithm. BMC Bioinform. 2011;12(1): 1-9. [DOI:10.1186/1471-2105-12-S1-S5] [PMID] [PMCID]

54. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER Suite: protein structure and function prediction. Nat Methods. 2015;12(1):7-8. [DOI:10.1038/nmeth.3213] [PMID] [PMCID]

55. Heo L, Park H, Seok C. GalaxyRefine: protein structure refinement driven by side-chain repacking. Nucleic Acids Res. 2013;41(W1): W384-W8. [DOI:10.1093/nar/gkt458] [PMID] [PMCID]

56. Lovell SC, Davis IW, Arendall III WB, De Bakker PI, Word JM, Prisant MG, et al. Structure validation by Cα geometry: ϕ, ψ and Cβ deviation. Proteins: Struct Funct Genet. 2003;50(3):437-50. [DOI:10.1002/prot.10286] [PMID]

57. Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 2007;35(suppl_2):W407-W10. [DOI:10.1093/nar/gkm290] [PMID] [PMCID]

58. Colovos C, Yeates TO. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci. 1993;2(9):1511-9. [DOI:10.1002/pro.5560020916] [PMID] [PMCID]

59. Eisenberg D, Lüthy R, Bowie JU. [20] VERIFY3D: assessment of protein models with three-dimensional profiles. Meth Enzymol. 277: Elsevier; 1997. p. 396-404. [DOI:10.1016/S0076-6879(97)77022-8]

60. Kim JS, Kim WS, Choi HG, Jang B, Lee K, Park JH, et al. Mycobacterium tuberculosis RpfB drives Th1‐type T cell immunity via a TLR4‐dependent activation of dendritic cells. J Leukoc Biol. 2013;94(4):733-49. [DOI:10.1189/jlb.0912435] [PMID]

61. Depledge DP, MacLean LM, Hodgkinson MR, Smith BA, Jackson AP, Ma S, et al. Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition. PLoS Negl Trop Dis. 2010;4(9):e829. [DOI:10.1371/journal.pntd.0000829] [PMID] [PMCID]

62. Reithinger R, Dujardin J-C. Molecular diagnosis of leishmaniasis: current status and future applications. J Clin Microbiol. 2007;45(1):21-5. [DOI:10.1128/JCM.02029-06] [PMID] [PMCID]

63. Zahedifard F, Lee H, No JH, Salimi M, Seyed N, Asoodeh A, et al. Anti-leishmanial activity of Brevinin 2R and its Lauric acid conjugate type against L. major: In vitro mechanism of actions and in vivo treatment potentials. PLoS Negl Trop Dis. 2019;13(2):e0007217. [DOI:10.1371/journal.pntd.0007217] [PMID] [PMCID]

64. Khatoon N, Pandey RK, Prajapati VK. Exploring Leishmania secretory proteins to design B and T cell multiepitope subunit vaccine using immunoinformatics approach. Sci Rep. 2017; 7(1):1-12. [DOI:10.1038/s41598-017-08842-w] [PMID] [PMCID]

65. Moafi M, Rezvan H, Sherkat R, Taleban R. Leishmania vaccines entered in clinical trials: A review of literature. Int J Prev Med. 2019;10. [DOI:10.4103/ijpvm.IJPVM_116_18] [PMID] [PMCID]

66. Das A, Ali N. Combining cationic liposomal delivery with MPL-TDM for cysteine protease cocktail vaccination against Leishmania donovani: evidence for antigen synergy and protection. PLoS Negl Trop Dis. 2014;8(8):e3091. [DOI:10.1371/journal.pntd.0003091] [PMID] [PMCID]

67. Basu R, Bhaumik S, Basu JM, Naskar K, De T, Roy S. Kinetoplastid membrane protein-11 DNA vaccination induces complete protection against both pentavalent antimonial-sensitive and-resistant strains of Leishmania donovani that correlates with inducible nitric oxide synthase activity and IL-4 generation: evidence for mixed Th1-and Th2-like responses in visceral leishmaniasis. J Immunol. 2005;174(11):7160-71. [DOI:10.4049/jimmunol.174.11.7160] [PMID]

68. Jain K, Jain N. Vaccines for visceral leishmaniasis: A review. J Immunol Methods. 2015;422:1-12. [DOI:10.1016/j.jim.2015.03.017] [PMID]

69. Lari A, Lari N, Biabangard A. Immunoinformatics Approach to Design a Novel Subunit Vaccine Against Visceral Leishmaniasis. Int J Pept Res Ther. 2022;28(1):1-14. [DOI:10.1007/s10989-021-10344-3] [PMID] [PMCID]

70. Khatoon N, Ojha R, Mishra A, Prajapati VK. Examination of antigenic proteins of Trypanosoma cruzi to fabricate an epitope-based subunit vaccine by exploiting epitope mapping mechanism. Vaccine. 2018;36(42):6290-300. [DOI:10.1016/j.vaccine.2018.09.004] [PMID]

71. Kalita P, Padhi A, Zhang KY, Tripathi T. Design of a peptide-based subunit vaccine against novel coronavirus SARS-CoV-2. Microb Pathog. 2020:104236. [DOI:10.1016/j.micpath.2020.104236] [PMID] [PMCID]

72. Zheng J, Lin X, Wang X, Zheng L, Lan S, Jin S, et al. In silico analysis of epitope-based vaccine candidates against hepatitis B virus polymerase protein. Viruses. 2017;9(5):112. [DOI:10.3390/v9050112] [PMID] [PMCID]

73. Delany I, Rappuoli R, Seib KL. Vaccines, reverse vaccinology, and bacterial pathogenesis. Cold Spring Harb perspect med. 2013;3(5):a012476. [DOI:10.1101/cshperspect.a012476] [PMID] [PMCID]

74. Shahbazi M, Haghkhah M, Rahbar MR, Nezafat N, Ghasemi Y. In silico sub-unit hexavalent peptide vaccine against an Staphylococcus aureus biofilm-related infection. Int J Pept Res Ther. 2016;22(1):101-17. [DOI:10.1007/s10989-015-9489-1]

75. Scott P, Novais FO. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. Nat Rev Immunol. 2016;16(9):581-92. [DOI:10.1038/nri.2016.72] [PMID]

76. Kemp K. Cytokine-producing T cell subsets in human leishmaniasis. Arch Immunol Ther Exp. 2000;48(3):173-6.

77. Modolell M, Choi B-S, Ryan RO, Hancock M, Titus RG, Abebe T, et al. Local suppression of T cell responses by arginase-induced L-arginine depletion in nonhealing leishmaniasis. PLoS Negl Trop Dis. 2009;3(7):e480. [DOI:10.1371/journal.pntd.0000480] [PMID] [PMCID]

78. Dubie T, Mohammed Y. Review on the Role of Host Immune Response in Protection and Immunopathogenesis during Cutaneous Leishmaniasis Infection. J Immunol Res. 2020;2020:2496713. [DOI:10.1155/2020/2496713] [PMID] [PMCID]

79. Aurora R, Creamer TP, Srinivasan R, Rose GD. Local interactions in protein folding: lessons from the α-helix. J Biol Chem. 1997;272(3):1413-6. [DOI:10.1074/jbc.272.3.1413] [PMID]

80. 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):943-58. [DOI:10.1016/0022-2836(90)90085-Z]

81. Huang Z, Zhang C, Xing X-H. Design and construction of chimeric linker library with controllable flexibilities for precision protein engineering. Meth Enzymol. 647: Elsevier; 2021. p. 23-49. [DOI:10.1016/bs.mie.2020.12.004] [PMID]

82. Rodrigues V, Cordeiro-da-Silva A, Laforge M, Silvestre R, Estaquier J. Regulation of immunity during visceral Leishmania infection. Parasites Vectors. 2016;9(1):118. [DOI:10.1186/s13071-016-1412-x] [PMID] [PMCID]

83. Baghbeheshti S, Hadadian S, Eidi A, Pishkar L, Rahimi H. Effect of flexible and rigid linkers on biological activity of recombinant tetramer variants of S3 antimicrobial peptide. Int J Pept Res Ther. 2021;27(1):457-62. [DOI:10.1007/s10989-020-10095-7]

84. Bai Y, Ann DK, Shen W-C. Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent. Proc Natl Acad Sci U S A. 2005;102(20): 7292-6. [DOI:10.1073/pnas.0500062102] [PMID] [PMCID]

85. Takamatsu N, Watanabe Y, Yanagi H, Meshi T, Shiba T, Okada Y. Production of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA vector. FEBS letters. 1990;269(1):73-6. [DOI:10.1016/0014-5793(90)81121-4]