Optimizing a Subunit Vaccine of Mycobacterium tuberculosis Using In-Silico and In-Vitro Approaches
Abstract
Background: The present study addresses the development of a novel subunit vaccine (SV) to combat tuberculosis (TB).
Methods: The research used immunoinformatics to develop a subunit vaccine with 7 MHC-I, 3 MHC-II, and 7 B-cell epitopes joined by AAV, GPGPG, and KK linkers. It involved Mtb protein Rv0577 and PADRE sequence as an adjuvant. TLR2 binding affinity (Kd, nM) was determined through PRODIGY. In-silico evaluations determined allergenicity, antigenicity, and physicochemical properties. The vaccine was presented in an AAVDj/8 system, intracellular expression was verified, and the copy number was identified using qPCR and qRT-PCR.
Results: The web tools confirmed the stability, non-allergenicity, and high immunogenicity of the vaccine (0.5673 < 0.4). PRODIGY tool depicted good SV-TLR2 binding (ΔG = -8.8 kcal/mol, Kd = 330 nM) with 59 intermolecular contacts, indicating possible TLR2 activation. Indirect immunofluorescence showed the expression of intracellular proteins. Viral titers, determined by 10-fold serial dilution up to 10³, showed a detectable titer, and copy numbers (10⁹/mL–10¹¹/mL) proved productive viral replication and significant vaccine effectiveness.
Conclusion: This comprehensive methodology, from epitope selection to in-vitro testing, establishes a robust foundation for further exploring and advancing this SV.
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3. Stephanie F, Saragih M, Tambunan USF (2021). Recent progress and challenges for drug-resistant tuberculosis treat-ment. Pharmaceutics, 13(5): 592.
4. Sethi G, Varghese RP, Lakra AK, et al (2024). Immunoinformatics and struc-tural aided approach to develop multi-epitope based subunit vaccine against Mycobacterium tuberculosis. Sci Rep, 14(1): 15923.
5. Awasthi A, Sharma G, Agrawal P (2022). Computational approaches for vaccine design-ing. In: Bioinformatics. Academic Press, pp. 317-335.
6. Crampin AC, Glynn JR, Fine PE (2009). What has Karonga taught us? Tuberculo-sis studied over three decades. Int J Tu-berc Lung Dis, 13(2):153–64.
7. Glick BR, Delovitch TL, Patten CL (2014). Medical Biotechnology. Washington DC: ASM Press, pp. 632-663.
8. Naeem MA, Adeel MM, Kanwal A, et al (2021) Reconnoitering Mycobacterium tuberculosis lipoproteins to design sub-unit vaccine by immunoinformatics ap-proach. Adv Life Sci, 8(3): 300-306.
9. Pal R, Bisht MK, Mukhopadhya S (2022). Secretory proteins of Myco-bacterium tuberculosis and their roles in modulation of host immune re-sponses: focus on therapeutic targets. FEBS J, 289(14): 4146-4171.
10. da Costa C, Benn CS, Nyirenda T, et al (2024). Perspectives on development and advancement of new tuberculosis vaccines. Int J Infect Dis, 141S: 106987.
11. Braunstein M, Bensing BA, Sullam PM (2019). The two distinct types of Se-cA2-dependent export systems. Micro-biology Spectrum, 7(3): 10-1128.
12. Renshaw PS, Lightbody KL, Veverka V, et al (2005). Structure and function of the complex formed by the tuberculo-sis virulence factors CFP-10 and ESAT-6. EMBO J, 24(14): 2491-8.
13. Jackson M, Stevens CM, Zhang L, et al (2020). Transporters involved in the bio-genesis and functionalization of the mycobacterial cell envelope. Chem Rev, 121(9): 5124-5157.
14. Zhu B, Dockrell HM, Ottenhoff THM, et al (2018). Tuberculosis vaccines: Opportu-nities and challenges. Respirology, 23(4): 359-368.
15. Lenz LL, Mohammadi S, Geissler A, et al (2003). SecA2-dependent secretion of au-tolytic enzymes promotes Listeria mon-ocytogenes pathogenesis. Proc Natl Acad Sci USA, 100(21): 12432-7.
16. Saint-Joanis B, Demangel C, Jackson M, et al (2006). Inactivation of Rv2525c in-creases β-lactam susceptibility and viru-lence in Mycobacterium tuberculosis via the twin arginine translocation (Tat) sys-tem substrate pathway. J Bacteriol, 188(18): 6669-6679.
17. DiGiuseppe Champion PA, Cox JS (2007). Protein secretion systems in Mycobacte-ria. Cell Microbiol, 9(6): 1376-84.
18. Singh H, Ansari HR, Raghava GP (2013). Improved method for linear B-cell epitope prediction using antigen’s pri-mary sequence. PLoS One, 8(5): e62216.
19. Rahlwes KC, Dias BRS, Campos PC, et al (2023). Pathogenicity and virulence of Mycobacterium tuberculosis. Virulence, 14(1): 2150449.
20. Khalid K, Hussain T, Jamil Z, et al (2022). Vaccinomics-Aided Development of a Next-Generation Chimeric Vaccine against an Emerging Threat: Mycoplasma genitalium. Vaccines, 10(10): 1720.
21. Fadilah F, Paramita RI, Erlina L, et al (2022). Linker optimization in breast cancer multiepitope peptide vaccine design based on molecular study. In: 4th International Con-ference on Life Sciences and Biotech-nology (ICOLIB 2021), Atlantis Press, pp. 528-538.
22. Arai R, Ueda H, Kitayama A, et al (2001). Design of the linkers which effectively separate domains of a bifunctional fu-sion protein. Protein Eng, 14(8): 529-32.
23. Livingston B, Crimi C, Newman M, et al (2002). A Rational Strategy to Design Multiepitope Immunogens Based on Multiple Th Lymphocyte Epitopes. J Immunol, 168(11): 5499-506.
24. Lennon-Duménil A-M, Bakker AH, Wolf-Bryant P, et al (2002). A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Curr Opin Immunol, 14(1): 15-21.
25. Obaidullah AJ, Alanazi MM, Alsaif NA, et al (2021). Immunoinformatics-guided design of a multi-epitope vaccine based on the structural proteins of severe acute respiratory syndrome coronavirus 2. RSC Adv, 11(29): 18103-18121.
26. Byun EH, Kim WS, Kim JS, et al (2012). Mycobacterium tuberculosis Rv0577, a novel TLR2 agonist induces maturation of dendritic cells and drives Th1 im-mune response. FASEB J, 26(6): 2695-2711.
27. Hasan M, Azim KF, Begum A, et al (2019). Vaccinomics strategy for developing a unique multi-epitope monovalent vac-cine against Marburg marburgvirus. Infect Genet Evol, 70: 140-157.
28. Wu CY, Monie A, Pang X, et al (2010). Im-proving therapeutic HPV peptide-based vaccine potency by enhancing CD4+T help and dendritic cell activation. J Bio-med Sci, 17(1): 88.
29. Gasteiger E, Hoogland C, Gattiker A, et al (2005). Protein identification and analysis tools on the ExPASy server. In: The proteomics protocols handbook. Humana Press, pp. 571-607.
30. Saha S, Raghava GPS (2006). AlgPred: pre-diction of allergenic proteins and map-ping of IgE epitopes. Nucleic Acids Res, 34 (suppl_2): W202-9.
31. Drage MG, Pecora ND, Hise AG, et al (2009). TLR2 and its co-receptors deter-mine responses of macrophages and dendritic cells to lipoproteins of Myco-bacterium tuberculosis. Cell Immunol, 258(1): 29-37.
32. Schneidman-Duhovny D, Inbar Y, Nussi-nov R, et al (2005). PatchDock and SymmDock: servers for rigid and sym-metric docking. Nucleic Acids Res, 33 (suppl_2): W363-7.
33. Grote A, Hiller K, Scheer M, et al (2005). JCat: a novel tool to adapt codon usage of a target gene to its potential expres-sion host. Nucleic Acids Res, 33 (suppl_2):W 526-531.
34. Faust SM, Bell P, Cutler BJ, et al (2013). CpG-depleted adeno-associated virus vectors evade immune detection. J Clin Invest, 123(7): 2994-3001.
35. Rabinowitz J, Chan YK, Samulski RJ (2019). Adeno-associated virus (AAV) versus immune response. Viruses, 11(2): 102.
36. Zhu J, Huang X, Yang Y (2009). The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice. J Clin Invest, 119(8): 2388-98.
37. Wiederstein M, Sippl MJ (2007). ProSA-web: interactive web service for the recogni-tion of errors in three-dimensional structures of proteins. Nucleic Acids Res, 35 (suppl_2): W407-10.
38. Khatoon N, Pandey RK, Prajapati VK (2017). Exploring Leishmania secretory proteins to design B and T cell multi-epitope subunit vaccine using im-munoinformatics approach. Sci Rep, 7(1): 8285.
39. Majlessi L, Prados-Rosales R, Casadevall A, et al (2015). Release of mycobacterial antigens. Immunol Rev, 264(1): 25-45.
40. Rothbard JB, Taylor WR (1988). A sequence pattern common to T cell epitopes. EMBO J, 7(1): 93-100.
41. Hewitt EW (2003). The MHC class I antigen presentation pathway: strategies for viral immune evasion. Immunology, 110(2): 163-9.
42. Makrides SC (1996). Strategies for achieving high-level expression of genes in Esche-richia coli. Microbiol Mol Biol Rev, 60(3): 512-38.
43. Brument N, Morenweiser R, Blouin V, et al (2002). A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and -5. Mol Ther, 6(5): 678-86.
44. Grimm D, Lee JS, Wang L, et al (2008). In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated vi-ruses. J Virol, 82(12): 5887-911.
45. World Health Organization (2022). WHO Global Task Force on TB Impact Meas-urement: report of a subgroup meeting on methods used by WHO to estimate TB disease burden, 11-12 May 2022, Ge-neva, Switzerland. World Health Organ-ization.
46. Fallahi MJ, Nazemi M, Zeighami A, et al (2024). Changes in incidence and clini-cal features of tuberculosis with regard to the COVID-19 outbreak in Southern Iran. BMC Infect Dis, 24(1): 1043.
47. Sharafi M, TalebiMoghaddam M, Narouee S, et al (2024). Estimating the impact of the first 2 years of the COVID-19 pan-demic on tuberculosis diagnosis and treatment outcomes in Southeastern City in Iran: an interrupted time series analy-sis of the preceding 10 years of ecologi-cal data. BMC Health Services Research, 24(1): 1489.
48. Sharifi Y, Ebrahimpur M, Payab M, et al (2022). The Syndemic Theory, the COVID-19 Pandemic, and The Epidem-ics of Non-Communicable Diseases (NCDs). Medical Journal of the Islamic Re-public of Iran, 36:177.
49. Shahnavazi M, Rigi F, Heydarikhayat N (2022). Treatment Adherence and Influ-encing Factors in Patients with Tubercu-losis during the COVID-19 Pandemic: A Mixed Method Study. Health Education and Health Promotion, 10(4): 633-642.
50. Aghajani J, Farnia P, Farnia P, et al (2022). Effect of COVID-19 pandem-ic on incidence of mycobacterial dis-eases among suspected tuberculosis pulmonary patients in Tehran, Iran. Int J Mycobacteriol, 11(4): 415-422.
2. Behera D (2010). Textbook of pulmonary medi-cine. 2nd ed. New Delhi: Jaypee Brothers Medical Publishers.
3. Stephanie F, Saragih M, Tambunan USF (2021). Recent progress and challenges for drug-resistant tuberculosis treat-ment. Pharmaceutics, 13(5): 592.
4. Sethi G, Varghese RP, Lakra AK, et al (2024). Immunoinformatics and struc-tural aided approach to develop multi-epitope based subunit vaccine against Mycobacterium tuberculosis. Sci Rep, 14(1): 15923.
5. Awasthi A, Sharma G, Agrawal P (2022). Computational approaches for vaccine design-ing. In: Bioinformatics. Academic Press, pp. 317-335.
6. Crampin AC, Glynn JR, Fine PE (2009). What has Karonga taught us? Tuberculo-sis studied over three decades. Int J Tu-berc Lung Dis, 13(2):153–64.
7. Glick BR, Delovitch TL, Patten CL (2014). Medical Biotechnology. Washington DC: ASM Press, pp. 632-663.
8. Naeem MA, Adeel MM, Kanwal A, et al (2021) Reconnoitering Mycobacterium tuberculosis lipoproteins to design sub-unit vaccine by immunoinformatics ap-proach. Adv Life Sci, 8(3): 300-306.
9. Pal R, Bisht MK, Mukhopadhya S (2022). Secretory proteins of Myco-bacterium tuberculosis and their roles in modulation of host immune re-sponses: focus on therapeutic targets. FEBS J, 289(14): 4146-4171.
10. da Costa C, Benn CS, Nyirenda T, et al (2024). Perspectives on development and advancement of new tuberculosis vaccines. Int J Infect Dis, 141S: 106987.
11. Braunstein M, Bensing BA, Sullam PM (2019). The two distinct types of Se-cA2-dependent export systems. Micro-biology Spectrum, 7(3): 10-1128.
12. Renshaw PS, Lightbody KL, Veverka V, et al (2005). Structure and function of the complex formed by the tuberculo-sis virulence factors CFP-10 and ESAT-6. EMBO J, 24(14): 2491-8.
13. Jackson M, Stevens CM, Zhang L, et al (2020). Transporters involved in the bio-genesis and functionalization of the mycobacterial cell envelope. Chem Rev, 121(9): 5124-5157.
14. Zhu B, Dockrell HM, Ottenhoff THM, et al (2018). Tuberculosis vaccines: Opportu-nities and challenges. Respirology, 23(4): 359-368.
15. Lenz LL, Mohammadi S, Geissler A, et al (2003). SecA2-dependent secretion of au-tolytic enzymes promotes Listeria mon-ocytogenes pathogenesis. Proc Natl Acad Sci USA, 100(21): 12432-7.
16. Saint-Joanis B, Demangel C, Jackson M, et al (2006). Inactivation of Rv2525c in-creases β-lactam susceptibility and viru-lence in Mycobacterium tuberculosis via the twin arginine translocation (Tat) sys-tem substrate pathway. J Bacteriol, 188(18): 6669-6679.
17. DiGiuseppe Champion PA, Cox JS (2007). Protein secretion systems in Mycobacte-ria. Cell Microbiol, 9(6): 1376-84.
18. Singh H, Ansari HR, Raghava GP (2013). Improved method for linear B-cell epitope prediction using antigen’s pri-mary sequence. PLoS One, 8(5): e62216.
19. Rahlwes KC, Dias BRS, Campos PC, et al (2023). Pathogenicity and virulence of Mycobacterium tuberculosis. Virulence, 14(1): 2150449.
20. Khalid K, Hussain T, Jamil Z, et al (2022). Vaccinomics-Aided Development of a Next-Generation Chimeric Vaccine against an Emerging Threat: Mycoplasma genitalium. Vaccines, 10(10): 1720.
21. Fadilah F, Paramita RI, Erlina L, et al (2022). Linker optimization in breast cancer multiepitope peptide vaccine design based on molecular study. In: 4th International Con-ference on Life Sciences and Biotech-nology (ICOLIB 2021), Atlantis Press, pp. 528-538.
22. Arai R, Ueda H, Kitayama A, et al (2001). Design of the linkers which effectively separate domains of a bifunctional fu-sion protein. Protein Eng, 14(8): 529-32.
23. Livingston B, Crimi C, Newman M, et al (2002). A Rational Strategy to Design Multiepitope Immunogens Based on Multiple Th Lymphocyte Epitopes. J Immunol, 168(11): 5499-506.
24. Lennon-Duménil A-M, Bakker AH, Wolf-Bryant P, et al (2002). A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Curr Opin Immunol, 14(1): 15-21.
25. Obaidullah AJ, Alanazi MM, Alsaif NA, et al (2021). Immunoinformatics-guided design of a multi-epitope vaccine based on the structural proteins of severe acute respiratory syndrome coronavirus 2. RSC Adv, 11(29): 18103-18121.
26. Byun EH, Kim WS, Kim JS, et al (2012). Mycobacterium tuberculosis Rv0577, a novel TLR2 agonist induces maturation of dendritic cells and drives Th1 im-mune response. FASEB J, 26(6): 2695-2711.
27. Hasan M, Azim KF, Begum A, et al (2019). Vaccinomics strategy for developing a unique multi-epitope monovalent vac-cine against Marburg marburgvirus. Infect Genet Evol, 70: 140-157.
28. Wu CY, Monie A, Pang X, et al (2010). Im-proving therapeutic HPV peptide-based vaccine potency by enhancing CD4+T help and dendritic cell activation. J Bio-med Sci, 17(1): 88.
29. Gasteiger E, Hoogland C, Gattiker A, et al (2005). Protein identification and analysis tools on the ExPASy server. In: The proteomics protocols handbook. Humana Press, pp. 571-607.
30. Saha S, Raghava GPS (2006). AlgPred: pre-diction of allergenic proteins and map-ping of IgE epitopes. Nucleic Acids Res, 34 (suppl_2): W202-9.
31. Drage MG, Pecora ND, Hise AG, et al (2009). TLR2 and its co-receptors deter-mine responses of macrophages and dendritic cells to lipoproteins of Myco-bacterium tuberculosis. Cell Immunol, 258(1): 29-37.
32. Schneidman-Duhovny D, Inbar Y, Nussi-nov R, et al (2005). PatchDock and SymmDock: servers for rigid and sym-metric docking. Nucleic Acids Res, 33 (suppl_2): W363-7.
33. Grote A, Hiller K, Scheer M, et al (2005). JCat: a novel tool to adapt codon usage of a target gene to its potential expres-sion host. Nucleic Acids Res, 33 (suppl_2):W 526-531.
34. Faust SM, Bell P, Cutler BJ, et al (2013). CpG-depleted adeno-associated virus vectors evade immune detection. J Clin Invest, 123(7): 2994-3001.
35. Rabinowitz J, Chan YK, Samulski RJ (2019). Adeno-associated virus (AAV) versus immune response. Viruses, 11(2): 102.
36. Zhu J, Huang X, Yang Y (2009). The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice. J Clin Invest, 119(8): 2388-98.
37. Wiederstein M, Sippl MJ (2007). ProSA-web: interactive web service for the recogni-tion of errors in three-dimensional structures of proteins. Nucleic Acids Res, 35 (suppl_2): W407-10.
38. Khatoon N, Pandey RK, Prajapati VK (2017). Exploring Leishmania secretory proteins to design B and T cell multi-epitope subunit vaccine using im-munoinformatics approach. Sci Rep, 7(1): 8285.
39. Majlessi L, Prados-Rosales R, Casadevall A, et al (2015). Release of mycobacterial antigens. Immunol Rev, 264(1): 25-45.
40. Rothbard JB, Taylor WR (1988). A sequence pattern common to T cell epitopes. EMBO J, 7(1): 93-100.
41. Hewitt EW (2003). The MHC class I antigen presentation pathway: strategies for viral immune evasion. Immunology, 110(2): 163-9.
42. Makrides SC (1996). Strategies for achieving high-level expression of genes in Esche-richia coli. Microbiol Mol Biol Rev, 60(3): 512-38.
43. Brument N, Morenweiser R, Blouin V, et al (2002). A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and -5. Mol Ther, 6(5): 678-86.
44. Grimm D, Lee JS, Wang L, et al (2008). In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated vi-ruses. J Virol, 82(12): 5887-911.
45. World Health Organization (2022). WHO Global Task Force on TB Impact Meas-urement: report of a subgroup meeting on methods used by WHO to estimate TB disease burden, 11-12 May 2022, Ge-neva, Switzerland. World Health Organ-ization.
46. Fallahi MJ, Nazemi M, Zeighami A, et al (2024). Changes in incidence and clini-cal features of tuberculosis with regard to the COVID-19 outbreak in Southern Iran. BMC Infect Dis, 24(1): 1043.
47. Sharafi M, TalebiMoghaddam M, Narouee S, et al (2024). Estimating the impact of the first 2 years of the COVID-19 pan-demic on tuberculosis diagnosis and treatment outcomes in Southeastern City in Iran: an interrupted time series analy-sis of the preceding 10 years of ecologi-cal data. BMC Health Services Research, 24(1): 1489.
48. Sharifi Y, Ebrahimpur M, Payab M, et al (2022). The Syndemic Theory, the COVID-19 Pandemic, and The Epidem-ics of Non-Communicable Diseases (NCDs). Medical Journal of the Islamic Re-public of Iran, 36:177.
49. Shahnavazi M, Rigi F, Heydarikhayat N (2022). Treatment Adherence and Influ-encing Factors in Patients with Tubercu-losis during the COVID-19 Pandemic: A Mixed Method Study. Health Education and Health Promotion, 10(4): 633-642.
50. Aghajani J, Farnia P, Farnia P, et al (2022). Effect of COVID-19 pandem-ic on incidence of mycobacterial dis-eases among suspected tuberculosis pulmonary patients in Tehran, Iran. Int J Mycobacteriol, 11(4): 415-422.
| Files | ||
| Issue | Vol 54 No 9 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijph.v54i9.19861 | |
| Keywords | ||
| Tuberculosis Bacillus Calmette-Guérin (BCG) vaccine Immunoinformatics Subunit vaccine Adeno-associated virus (AAV) vector | ||
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How to Cite
1.
Ling Z, Naeem M. Optimizing a Subunit Vaccine of Mycobacterium tuberculosis Using In-Silico and In-Vitro Approaches. Iran J Public Health. 2025;54(9):1938-1953.
