Revolutionizing leishmaniasis control: leveraging immunoinformatics for precision-driven in-silico vaccine design
Main Article Content
Keywords
immunoinformatics, leishmaniasis, multi-epitope vaccine pteridine reductase
Abstract
Leishmaniasis is an endemic disease in many countries that affects vulnerable populations of humans, dogs, and cats. This study employs immunoinformatics to design a multi-epitope vaccine construct for leishmaniasis by identifying vaccine targets on the protein Pteridine Reductase. T-cell and B-cell epitopes were screened for antigenicity, toxicity, and allergenicity. Shortlisted T-cell targets were confirmed to have appropriate IC values (≤ 50 nM and ≤ 500 nM for MHC I and MHC II, respectively). One B-cell epitope, six MHC class I epitopes, and 25 MHC class II epitopes were selected for inclusion in the vaccine construct, which was linked with EAAAK, CPGPG, and AAY linkers, along with a CPG Oligodeoxynucleotide adjuvant. The vaccine construct had a Ramachandran score of 89.9% and an Errat score of 98.4615. HLA alleles were predicted to produce an immune response in 81.8% of the global population, indicating encouraging potential for broad immunogenicity. Successful binding of the vaccine construct with TLR-9 was confirmed through molecular docking, and the docked complex exhibited a low eigenvalue of 2.06e-0.6 and a ΔG of -11.8 kcal mol-1, indicating stable binding and a high level of flexibility. Codon optimization was carried out, followed by in-silico cloning of the vaccine in Escherichia coli K12 strain using the vector pET-21a (+). These results suggest that the vaccine is stable and capable of eliciting a promising immune response against Leishmania, making it a favorable candidate for experimental trials.
References
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6 Pradhan S, Schwartz RA, Patil A, Grabbe S, Goldust M. Treatment options for leishmaniasis. Clin Exp Dermatol. 2022;47(3):516-521. 10.1111/ced.14919
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9 Abdellahi L, Iraji F, Mahmoudabadi A, Hejazi SH. Vaccination in Leishmaniasis: A Review Article. Iran Biomed J. 2022;26(1):1-35.
10 Qureshi H, Basheer A, Faheem M, Arshad MW, Rai SK, Jamal SB. Designing a multi-epitope vaccine against Shigella dysenteriae using immuno-informatics approach. Front Genet. 2024;15:1361610. 10.3389/fgene.2024.1361610
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14 López-Blanco JR, Aliaga JI, Quintana-Ortí ES, Chacón P. iMODS: Internal coordinates normal mode analysis server. Nucleic Acids Res. 2014;42(W1):W271-W276. 10.1093/nar/gku339
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21 Bhattacharya P, Gannavaram S, Ismail N, Saxena A, Dagur PK, Akue A, et al. Toll-like Receptor-9 (TLR-9) Signaling Is Crucial for Inducing Protective Immunity following Immunization with Genetically Modified Live Attenuated Leishmania Parasites. Pathogens. 2023;12(4):534. 10.3390/pathogens12040534
22 Melisi D, Frizziero M, Tamburrino A, Zanotto M, Carbone C, Piro G, et al. Toll-Like Receptor 9 Agonists for Cancer Therapy. Biomedicines. 2014;2(3):211-228. 10.3390/biomedicines2030211
23 Bauer JA, Pavlović J, Bauerová-Hlinková V. Normal Mode Analysis as a Routine Part of a Structural Investigation. Molecules. 2019;24(18):3293. 10.3390/molecules24183293
24 Adhikari UK, Tayebi M, Rahman MM. Immunoinformatics Approach for Epitope-Based Peptide Vaccine Design and Active Site Prediction against Polyprotein of Emerging Oropouche Virus. J Immunol Res. 2018;2018:6718083. 10.1155/2018/6718083
25 Naz A, Awan FM, Obaid S, Rasool N, Parveen Z, Ahmed A, et al. Identification of putative vaccine candidates against Helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach. Infect Genet Evol. 2015;32:280-291. 10.1016/j.meegid.2015.03.027
26 Basheer A, Jamal SB, Alzahrani B, Faheem M. Development of a tetravalent subunit vaccine against dengue virus through a vaccinomics approach. Front Immunol. 2023;14:1273838. 10.3389/fimmu.2023.1273838
27 Pappalardo F, Flower D, Russo G, Pennisi M, Motta S. Computational modelling approaches to vaccinology. Pharmacol Res. 2015;92:40-45. 10.1016/j.phrs.2014.08.006
28 Brown F, Hoey EM, Martin SJ, Rima BK, Trudgett A. Vaccine Design. Trends Biotechnol. 1994;12(6):251.
29 Kanwal M, Basheer A, Bilal M, Faheem M, Aziz T, Alamri AS, et al. In silico vaccine design for Yersinia enterocolitica: a comprehensive approach to enhanced immunogenicity, efficacy and protection. Int Immunopharmacol. 2024;143:113241. 10.1016/j.intimp.2024.113241
30 Bello AR, Nare B, Freedman D, Hardy L, Beverley SM. PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc Natl Acad Sci U S A. 1994;91(24):11442–11446. 10.1073/pnas.91.24.11442
31 Boakye A, Gasu EN, Mensah JO, Borquaye LS. Computational studies on potential small molecule inhibitors of Leishmania pteridine reductase. J Biomol Struct Dyn. 2023;41(21):12128-12141. 10.1080/07391102.2023.2166119
32 Faria MS, Reis FCG, Lima APC. Toll-Like Receptors in Leishmania Infections: Guardians or Promoters? J Parasitol Res. 2012;2012:930257. 10.1155/2012/930257
33 Fu H, Liang Y, Zhong X, Pan Z, Huang L, Zhang H, et al. Codon optimization with deep learning to enhance protein expression. Sci Rep. 2020;10(1):17617. 10.1038/s41598-020-74091-z
34 Jalal K, Abu-Izneid T, Khan K, Abbas M, Hayat A, Bawazeer S, et al. Identification of vaccine and drug targets in Shigella dysenteriae sd197 using reverse vaccinology approach. Sci Rep. 2022;12:251. 10.1038/s41598-021-03988-0
35 Sacramento L, Trevelin SC, Silva Júnior DS, Costa DL, Almeida RP, Pereira C, et al. Toll-Like Receptor 9 signaling in dendritic cells regulates neutrophil recruitment to inflammatory foci following Leishmania infantum infection. Infect Immun. 2015;83(12):4604-4616. 10.1128/IAI.00975-15
36 Sasidharan S, Saudagar P. Leishmaniasis: Where Are We and Where Are We Heading? Parasitology Res. 2021;120(5):1541–1554. 10.1007/s00436-021-07139-2
37 Stacey KJ, Blackwell JM. Immunostimulatory DNA as an adjuvant in vaccination against Leishmania major. Infect Immun. 1999;67(8):3719-3726. 10.1128/IAI.67.8.3719-3726.1999
38 Tarrahimofrad H, Rahimnahal S, Zamani J, Jahangirian E, Aminzadeh S. Designing a multi-epitope vaccine to provoke the robust immune response against influenza A H7N9. Sci Rep. 2021;11(1):24485. 10.1038/s41598-021-03932-2
39 Yang Z, Bogdan P, Nazarian S. An in silico deep learning approach to multi-epitope vaccine design: a SARS-CoV-2 case study. Sci Rep. 2021;11(1):3238. 10.1038/s41598-021-81749-9
40 Fakruddin M, Mazumdar RM, Mannan KS, Chowdhury A, Hossain MN. Critical factors affecting the success of cloning, expression, and mass production of enzymes by recombinant E. coli. ISRN Biotechnol. 2013;2013:1–7. 10.5402/2013/590587
41 Liese J, Schleicher U, Bogdan C. TLR9 signaling is essential for the innate NK cell response in murine cutaneous leishmaniasis. Eur J Immunol. 2007 Dec;37(12):3424-3434. 10.1002/eji.200737182
42 Kaye PM, Matlashewski G, Mohan S, Le Rutte E, Mondal D, Khamesipour A, et al. Vaccine value profile for leishmaniasis. Vaccine. 2023;41(S2):S153-S175. 10.1016/j.vaccine.2023.01.057
43 Shilling PJ, Mirzadeh K, Cumming AJ, Widesheim M, Köck Z, Daley DO. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol. 2020;3:214. 10.1038/s42003-020-0939-8
44 Park SW, Lee BH, Song SH, Kim MK. Revisiting the Ramachandran plot based on statistical analysis of static and dynamic characteristics of protein structures. J Struct Biol. 2023 Mar;215(1):107939. 10.1016/j.jsb.2023.107939
45 Kumar P, Kumar P, Shrivastava A, Dar MA, Lokhande KB, Singh N, et al. Immunoinformatics-based multi-epitope containing fused polypeptide vaccine design against visceral leishmaniasis with high immunogenicity and TLR binding. Int J Biol Macromol. 2023 Dec;253(Pt 8):127567. 10.1016/j.ijbiomac.2023.127567
46 Naz S, Aroosh A, Caner Ş, Atalay EŞ, Toz S, Ozbel Y, et al. Immunoinformatics approach to design a multi-epitope vaccine against cutaneous leishmaniasis. Vaccines. 2023;11(2):304. 10.3390/vaccines11020304
47 Begum M, Khurshid R, Saleem A, Nagra SA. MUC1 based breast cancer vaccines: role of post translational modifications. J Ayub Med Coll Abbottabad. 2008 Oct-Dec;20(4):130-133
48 eBioMedicine, “Leishmania: An Urgent Need for New Treatments,” eBioMedicine, 2023.
49 Ramprasadh SV, Rajakumar S, Srinivasan S, Susha D, Sharma S, Chourasiya R. Computer-Aided Multi-Epitope Based Vaccine Design Against Monkeypox Virus Surface Protein A30L: An Immunoinformatics Approach. Protein J. 2023 Dec;42(6):645-663. 10.1007/s10930-023-10150-4
50 Srivastava S, Saudagar P, Bhowmick S, Khadem S, Jha BA, Saha B, et al. Leishmania expressed lipophosphoglycan interacts with Toll-like receptor (TLR)-2 to decrease TLR-9 expression and reduce anti-leishmanial responses. Clin Exp Immunol. 2013;172(3):403-409. 10.1111/cei.12084
51 Dhanushkumar T, Santhosh ME, Prasanna K Selvam, Rambabu M, Dasegowda KR, Vasudevan K, et al. Advancements and hurdles in the development of a vaccine for triple-negative breast cancer: a comprehensive review of multi-omics and immunomics strategies. Life Sci. 2024 Jan 15;337:122360. 10.1016/j.lfs.2023.122360
52 Mandal S, Khushi, Waribam PC, Srivastava R, Patgiri SJ, Natarajaseenivasan K. Development of a multi-epitope vaccine candidate targeting blood-stage of malaria through immunoinformatics approach. Hum Immunol. 2025;86(4):111346. 10.1016/j.humimm.2025.111346
2 Baneth G, Solano-Gallego L. Leishmaniasis. Vet Clin North Am Small Anim Pract. 2022 Nov; 52(6):1359-1375. 10.1016/j.cvsm.2022.06.012
3 de Vries HJC, Schallig HD. Cutaneous Leishmaniasis: A 2022 Updated Narrative Review into Diagnosis and Management Developments. Am J Clin Dermatol. 2022;23(6):823–840. 10.1007/s40257-022-00726-8
4 Mann S, Frasca K, Scherrer S, Henao-Martínez AF, Newman S, Ramanan P, et al. A Review of Leishmaniasis: Current Knowledge and Future Directions. Curr Trop Med Rep. 2021;8(2):121–132. 10.1007/s40475-021-00232-7
5 Montaner-Angoiti E, Llobat L. Is Leishmaniasis the New Emerging Zoonosis in the World? Vet Res Commun. 2023;47:1777-1799. 10.1007/s11259-023-10171-5
6 Pradhan S, Schwartz RA, Patil A, Grabbe S, Goldust M. Treatment options for leishmaniasis. Clin Exp Dermatol. 2022;47(3):516-521. 10.1111/ced.14919
7 Chen X, Shi Y, Zhou S, Geng M, Tu H, Song J, et al. Risk factors of visceral leishmaniasis in the world: a review. Chinese J Schistosomiasis Control. 2024;36(4):412-421. 10.16250/j.32.1374.2024079
8 Husein-ElAhmed H, Gieler U, Steinhoff M. Evidence supporting the enhanced efficacy of pentavalent antimonials with adjuvant therapy for cutaneous leishmaniasis: a systematic review and meta-analysis. J Eur Acad Dermatol Venereol. 2020;34(10):2216-2228. 10.1111/jdv.16333
9 Abdellahi L, Iraji F, Mahmoudabadi A, Hejazi SH. Vaccination in Leishmaniasis: A Review Article. Iran Biomed J. 2022;26(1):1-35.
10 Qureshi H, Basheer A, Faheem M, Arshad MW, Rai SK, Jamal SB. Designing a multi-epitope vaccine against Shigella dysenteriae using immuno-informatics approach. Front Genet. 2024;15:1361610. 10.3389/fgene.2024.1361610
11 Kayraklioglu N, Horuluoglu B, Klinman DM. CpG Oligonucleotides as Vaccine Adjuvants. Methods Mol Biol. 2021;2197:51-85. 10.1007/978-1-0716-0872-2_4
12 Molteni M, Gemma S, Rossetti C. The Role of Toll-Like Receptor 4 in Infectious and Noninfectious Inflammation. Mediators Inflamm. 2016;2016:6978936. 10.1155/2016/6978936
13 Kazemi B, Tohidi F, Bandehpour M, Yarian F. Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania. Iran Biomed J. 2010 Jul;14(3):97-102. PMid: 21079660 (PMCID: PMC3904060)
14 López-Blanco JR, Aliaga JI, Quintana-Ortí ES, Chacón P. iMODS: Internal coordinates normal mode analysis server. Nucleic Acids Res. 2014;42(W1):W271-W276. 10.1093/nar/gku339
15 Biogene C. pET-21a-d(+). [Internet]. Available from: https://www.creative-biogene.com/pet-21a-item-vet-1482-441351.html
16 Akhoundi M, Downing T, Votýpka J, Kuhls K, Lukeš J, Schoenian G, et al. Leishmania infections: Molecular targets and diagnosis. Mol Aspects Med. 2017;57:1-29. 10.1016/j.mam.2016.11.012
17 Shah JA, Darrah PA, Ambrozak DR, Turon TN, Mendez S, Kirman J, et al. Dendritic cells are responsible for the capacity of CpG oligodeoxynucleotides to act as an adjuvant for protective vaccine immunity against Leishmania major in mice. J Exp Med. 2003 Jul 21;198(2):281-291. 10.1084/jem.20030645
18 das Neves GM, Kagami LP, Gonçalves IL, Eifler-Lima VL. Targeting pteridine reductase 1 and dihydrofolate reductase: the old is a new trend for leishmaniasis drug discovery. Future Med Chem. 2019;11(16):2107-2130. 10.4155/fmc-2018-0512
19 Dashti F, Raisi A, Pourali G, Razavi ZS, Ravaei F, Sadri Nahand J, et al. A computational approach to design a multiepitope vaccine against H5N1 virus. Virol J. 2024;21(1):67. 10.1186/s12985-024-02337-7
20 Wang H, Wang S, Fang R, Li X, Xing J, Li Z, et al. Enhancing TB vaccine efficacy: current progress on vaccines, adjuvants and immunization strategies. Vaccines. 2023;12(1):38. 10.3390/vaccines12010038
21 Bhattacharya P, Gannavaram S, Ismail N, Saxena A, Dagur PK, Akue A, et al. Toll-like Receptor-9 (TLR-9) Signaling Is Crucial for Inducing Protective Immunity following Immunization with Genetically Modified Live Attenuated Leishmania Parasites. Pathogens. 2023;12(4):534. 10.3390/pathogens12040534
22 Melisi D, Frizziero M, Tamburrino A, Zanotto M, Carbone C, Piro G, et al. Toll-Like Receptor 9 Agonists for Cancer Therapy. Biomedicines. 2014;2(3):211-228. 10.3390/biomedicines2030211
23 Bauer JA, Pavlović J, Bauerová-Hlinková V. Normal Mode Analysis as a Routine Part of a Structural Investigation. Molecules. 2019;24(18):3293. 10.3390/molecules24183293
24 Adhikari UK, Tayebi M, Rahman MM. Immunoinformatics Approach for Epitope-Based Peptide Vaccine Design and Active Site Prediction against Polyprotein of Emerging Oropouche Virus. J Immunol Res. 2018;2018:6718083. 10.1155/2018/6718083
25 Naz A, Awan FM, Obaid S, Rasool N, Parveen Z, Ahmed A, et al. Identification of putative vaccine candidates against Helicobacter pylori exploiting exoproteome and secretome: a reverse vaccinology based approach. Infect Genet Evol. 2015;32:280-291. 10.1016/j.meegid.2015.03.027
26 Basheer A, Jamal SB, Alzahrani B, Faheem M. Development of a tetravalent subunit vaccine against dengue virus through a vaccinomics approach. Front Immunol. 2023;14:1273838. 10.3389/fimmu.2023.1273838
27 Pappalardo F, Flower D, Russo G, Pennisi M, Motta S. Computational modelling approaches to vaccinology. Pharmacol Res. 2015;92:40-45. 10.1016/j.phrs.2014.08.006
28 Brown F, Hoey EM, Martin SJ, Rima BK, Trudgett A. Vaccine Design. Trends Biotechnol. 1994;12(6):251.
29 Kanwal M, Basheer A, Bilal M, Faheem M, Aziz T, Alamri AS, et al. In silico vaccine design for Yersinia enterocolitica: a comprehensive approach to enhanced immunogenicity, efficacy and protection. Int Immunopharmacol. 2024;143:113241. 10.1016/j.intimp.2024.113241
30 Bello AR, Nare B, Freedman D, Hardy L, Beverley SM. PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major. Proc Natl Acad Sci U S A. 1994;91(24):11442–11446. 10.1073/pnas.91.24.11442
31 Boakye A, Gasu EN, Mensah JO, Borquaye LS. Computational studies on potential small molecule inhibitors of Leishmania pteridine reductase. J Biomol Struct Dyn. 2023;41(21):12128-12141. 10.1080/07391102.2023.2166119
32 Faria MS, Reis FCG, Lima APC. Toll-Like Receptors in Leishmania Infections: Guardians or Promoters? J Parasitol Res. 2012;2012:930257. 10.1155/2012/930257
33 Fu H, Liang Y, Zhong X, Pan Z, Huang L, Zhang H, et al. Codon optimization with deep learning to enhance protein expression. Sci Rep. 2020;10(1):17617. 10.1038/s41598-020-74091-z
34 Jalal K, Abu-Izneid T, Khan K, Abbas M, Hayat A, Bawazeer S, et al. Identification of vaccine and drug targets in Shigella dysenteriae sd197 using reverse vaccinology approach. Sci Rep. 2022;12:251. 10.1038/s41598-021-03988-0
35 Sacramento L, Trevelin SC, Silva Júnior DS, Costa DL, Almeida RP, Pereira C, et al. Toll-Like Receptor 9 signaling in dendritic cells regulates neutrophil recruitment to inflammatory foci following Leishmania infantum infection. Infect Immun. 2015;83(12):4604-4616. 10.1128/IAI.00975-15
36 Sasidharan S, Saudagar P. Leishmaniasis: Where Are We and Where Are We Heading? Parasitology Res. 2021;120(5):1541–1554. 10.1007/s00436-021-07139-2
37 Stacey KJ, Blackwell JM. Immunostimulatory DNA as an adjuvant in vaccination against Leishmania major. Infect Immun. 1999;67(8):3719-3726. 10.1128/IAI.67.8.3719-3726.1999
38 Tarrahimofrad H, Rahimnahal S, Zamani J, Jahangirian E, Aminzadeh S. Designing a multi-epitope vaccine to provoke the robust immune response against influenza A H7N9. Sci Rep. 2021;11(1):24485. 10.1038/s41598-021-03932-2
39 Yang Z, Bogdan P, Nazarian S. An in silico deep learning approach to multi-epitope vaccine design: a SARS-CoV-2 case study. Sci Rep. 2021;11(1):3238. 10.1038/s41598-021-81749-9
40 Fakruddin M, Mazumdar RM, Mannan KS, Chowdhury A, Hossain MN. Critical factors affecting the success of cloning, expression, and mass production of enzymes by recombinant E. coli. ISRN Biotechnol. 2013;2013:1–7. 10.5402/2013/590587
41 Liese J, Schleicher U, Bogdan C. TLR9 signaling is essential for the innate NK cell response in murine cutaneous leishmaniasis. Eur J Immunol. 2007 Dec;37(12):3424-3434. 10.1002/eji.200737182
42 Kaye PM, Matlashewski G, Mohan S, Le Rutte E, Mondal D, Khamesipour A, et al. Vaccine value profile for leishmaniasis. Vaccine. 2023;41(S2):S153-S175. 10.1016/j.vaccine.2023.01.057
43 Shilling PJ, Mirzadeh K, Cumming AJ, Widesheim M, Köck Z, Daley DO. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol. 2020;3:214. 10.1038/s42003-020-0939-8
44 Park SW, Lee BH, Song SH, Kim MK. Revisiting the Ramachandran plot based on statistical analysis of static and dynamic characteristics of protein structures. J Struct Biol. 2023 Mar;215(1):107939. 10.1016/j.jsb.2023.107939
45 Kumar P, Kumar P, Shrivastava A, Dar MA, Lokhande KB, Singh N, et al. Immunoinformatics-based multi-epitope containing fused polypeptide vaccine design against visceral leishmaniasis with high immunogenicity and TLR binding. Int J Biol Macromol. 2023 Dec;253(Pt 8):127567. 10.1016/j.ijbiomac.2023.127567
46 Naz S, Aroosh A, Caner Ş, Atalay EŞ, Toz S, Ozbel Y, et al. Immunoinformatics approach to design a multi-epitope vaccine against cutaneous leishmaniasis. Vaccines. 2023;11(2):304. 10.3390/vaccines11020304
47 Begum M, Khurshid R, Saleem A, Nagra SA. MUC1 based breast cancer vaccines: role of post translational modifications. J Ayub Med Coll Abbottabad. 2008 Oct-Dec;20(4):130-133
48 eBioMedicine, “Leishmania: An Urgent Need for New Treatments,” eBioMedicine, 2023.
49 Ramprasadh SV, Rajakumar S, Srinivasan S, Susha D, Sharma S, Chourasiya R. Computer-Aided Multi-Epitope Based Vaccine Design Against Monkeypox Virus Surface Protein A30L: An Immunoinformatics Approach. Protein J. 2023 Dec;42(6):645-663. 10.1007/s10930-023-10150-4
50 Srivastava S, Saudagar P, Bhowmick S, Khadem S, Jha BA, Saha B, et al. Leishmania expressed lipophosphoglycan interacts with Toll-like receptor (TLR)-2 to decrease TLR-9 expression and reduce anti-leishmanial responses. Clin Exp Immunol. 2013;172(3):403-409. 10.1111/cei.12084
51 Dhanushkumar T, Santhosh ME, Prasanna K Selvam, Rambabu M, Dasegowda KR, Vasudevan K, et al. Advancements and hurdles in the development of a vaccine for triple-negative breast cancer: a comprehensive review of multi-omics and immunomics strategies. Life Sci. 2024 Jan 15;337:122360. 10.1016/j.lfs.2023.122360
52 Mandal S, Khushi, Waribam PC, Srivastava R, Patgiri SJ, Natarajaseenivasan K. Development of a multi-epitope vaccine candidate targeting blood-stage of malaria through immunoinformatics approach. Hum Immunol. 2025;86(4):111346. 10.1016/j.humimm.2025.111346