Enhancing immunogenicity of novel multistage subunit vaccine of Mycobacterium tuberculosis using PLGA:DDA hybrid nanoparticles and MPLA : subcutaneous administration

Document Type : Original Article


1 Department of Microbiology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran

2 Department of Medical Bacteriology and Virology, Qaem University Hospital, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

3 Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran

4 Department of Pharmaceutics, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): A new strategy in recent studies is using effective tuberculosis (TB) subunit vaccines combined with appropriate carriers and adjuvants which have shown promising results in preclinical and clinical studies. The aim of the present study was to evaluate the PLGA:DDA hybrid nanoparticles (NPs) for subcutaneous delivery of a novel multistage subunit vaccine along with MPLA adjuvant against Mycobacterium tuberculosis (M. tuberculosis).
Materials and Methods: PLGA and PLGA:DDA NPs containing HspX/EsxS fusion protein and MPLA were prepared by double emulsion method (w/o/w). After characterization, these NPs were subcutaneously administered to BALB/c mice aged 6-8 weeks old. Immunogenicity of formulations were assessed by measuring the level of IFN-γ, IL-4, IL-17 and TGF-β cytokines as well as IgG1, IgG2a and IgA antibodies using ELISA.
Results: Both particles had spherical shape and smooth surface with 316.7 ± 12.7 nm in size, surface charge of -33 ± 1.7 mV, and encapsulation efficiency of 92.2 ± 2% for PLGA NPs and 249.7 ± 16.7 nm in size, surface charge of 39 ± 1.8 mV, and encapsulation efficiency of 35.7 ± 1.4% for PLGA:DDA NPs. The highest IFN-γ response and also IgG2a and IgG1 antibodies titers were observed in groups immunized with PLGA:DDA/HspX/EsxS/MPLA and PLGA:DDA/HspX/EsxS/MPLA as booster as well as PLGA:DDA/HspX/EsxS and PLGA:DDA/HspX/EsxS as booster.
Conclusion: With regard to effective induction of IFN-γ and IgG2a immune responses, PLGA:DDA hybrid NP along with MPLA adjuvant have good potentials for improving the immunogenicity of HspX/EsxS multistage subunit vaccine as well as promoting BCG efficacy as a BCG prime-boost.


Main Subjects

1. Bivas-Benita M, Lin MY, Bal SM, van Meijgaarden KE, Franken KL, Friggen AH, et al. Pulmonary delivery of DNA encoding Mycobacterium tuberculosis latency antigen Rv1733c associated to PLGA–PEI nanoparticles enhances T cell responses in a DNA prime/protein boost vaccination regimen in mice. Vaccine 2009; 27: 4010-4017.
2. WHO. Tuberculosis. Global Tuberculosis Report 2016. http://www.who.int/tb/publications/factsheets/en/.
3. Karimi SM, Sankian M, Khademi F, Tafaghodi M. Chitosan (CHT) and trimethylchitosan (TMC) nanoparticles as adjuvant/delivery system for parenteral and nasal immunization against Mycobacterium tuberculosis (MTb) ESAT-6 antigen. Nanomed J 2016; 3: 223-229.
4. Khademi F, Derakhshan M, Sadeghi R. The role of toll-like receptor gene polymorphisms in tuberculosis susceptibility: a systematic review and meta-analysis. Rev Clin Med 2016; 3: 133-140.
5. Khademi F, Yousefi-Avarvand A, Derakhshan M, Vaez H, Sadeghi R. Middle East Mycobacterium tuberculosis Antibiotic Resistance: A Systematic Review and Meta-Analysis. Infect Epidemiol Microbiol 2017; 3: 25-35.
6. Niu H, Hu L, Li Q, Da Z, Wang B, Tang K, et al. Construction and evaluation of a multistage Mycobacterium tuberculosis subunit vaccine candidate Mtb10. 4–HspX. Vaccine 2011; 29: 9451-948.
7. Ziv E, Daley CL, Blower S. Potential public health impact of new tuberculosis vaccines. Emerg Infect Dis 2004; 10: 1529-1535.
8. Dey B, Jain R, Gupta UD, Katoch V, Ramanathan V, Tyagi AK. A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis. PloS one 2011; 6: e23360
9. Brennan MJ, Clagett B, Fitzgerald H, Chen V, Williams A, Izzo AA, et al. Preclinical evidence for implementing a prime-boost vaccine strategy for tuberculosis. Vaccine 2012; 30: 2811-2823.
10. Xin Q, Niu H, Li Z, Zhang G, Hu L, Wang B, et al. Subunit vaccine consisting of multi-stage antigens has high protective efficacy against Mycobacterium tuberculosis infection in mice. PloS one 2013; 8: e72745.
11. Khademi F, Yousefi-Avarvand A, Derakhshan M, Meshkat Z, Tafaghodi M, Ghazvini K, et al. Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli. Rep Biochem Mol Biol 2017; 6: 15-21.
12. Yousefi-Avarvand A, Tafaghodi M, Soleimanpour S, Khademi F. HspX protein as a candidate vaccine against Mycobacterium tuberculosis: an overview. ‎Front Biol 2018; 13: 293-296.
13. Marongiu L, Donini M, Toffali L, Zenaro E, Dusi S. ESAT-6 and HspX improve the effectiveness of BCG to induce human dendritic cells-dependent Th1 and NK cells activation. PloS one 2013; 8: e75684.
14. Ottenhoff TH, Kaufmann SH. Vaccines against tuberculosis: where are we and where do we need to go? PLoS Pathog 2012; 8: e1002607.
15. Junqueira-Kipnis AP, Neto LMM, Kipnis A. Role of fused Mycobacterium tuberculosis immunogens and adjuvants in modern tuberculosis vaccines. Front Immunol 2014; 5: 1-9.
16. Peek LJ, Middaugh CR, Berkland C. Nanotechnology in vaccine delivery. Adv Drug Deliv Rev 2008; 60: 915-928.
17. Ilyinskii PO, Roy CJ, O’Neil CP, Browning EA, Pittet LA, Altreuter DH, et al. Adjuvant-carrying synthetic vaccine particles augment the immune response to encapsulated antigen and exhibit strong local immune activation without inducing systemic cytokine release. Vaccine 2014; 32: 2882-2895.
18. Kim M-G, Park JY, Shon Y, Kim G, Shim G, Oh Y-K. Nanotechnology and vaccine development. Asian J Pharm Sci 2014; 9: 227-235.
19. Rose F, Wern JE, Ingvarsson PT, van de Weert M, Andersen P, Follmann F, et al. Engineering of a novel adjuvant based on lipid-polymer hybrid nanoparticles: A quality-by-design approach. J Control Release 2015; 210: 48-57.
20. Jensen DK, Jensen LB, Koocheki S, Bengtson L, Cun D, Nielsen HM, et al. Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA. J Control Release 2012; 157: 141-148.
21. Tafaghodi M, Jaafari MR, Eskandari M, Khamesipour A. Immunization against leishmaniasis by PLGA nanospheres loaded with an experimental autoclaved Leishmania major (ALM) and Quillaja saponins. Trop Biomed 2010; 27: 639-650.
22. Mohajer M, Khameneh B, Tafaghodi M. Preparation and characterization of PLGA nanospheres loaded with inactivated influenza virus, CpG-ODN and Quillaja saponin. Iran J Basic Med Sci 2014; 17: 722-726.
23. Mohaghegh M, Tafaghodi, M. Dextran microspheres could enhance immune responses against PLGA nanospheres encapsulated with tetanus toxoid and Quillaja saponins after nasal immunization in rabbit. ‎Pharm Dev Technol 2011; 16: 36-43.
24. Khademi F, Derakhshan M, Yousefi-Avarvand A, Tafaghodi M. Potential of polymeric particles as future vaccine delivery systems/adjuvants for parenteral and non-parenteral immunization against tuberculosis: A systematic review. Iran J Basic Med Sci 2018; 21: 116-123.
25. Hu Y, Ehrich M, Fuhrman K, Zhang C. In vitro performance of lipid-PLGA hybrid nanoparticles as an antigen delivery system: lipid composition matters. Nanoscale Res Lett 2014; 9: 1-10.
26. Sayın B, Somavarapu S, Li XW, Sesardic D, Şenel S, Alpar OH. TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines. Eur J Pharm Sci 2009; 38: 362-369.
27. Vyas SP, Quraishi S, Gupta S, Jaganathan K. Aerosolized liposome-based delivery of amphotericin B to alveolar macrophages. Int J Pharm 2005; 296: 12-25.
28. Khademi F, Taheri RA, Momtazi-Borojeni AA, Farnoosh G, Johnston TP, Sahebkar A. Potential of Cationic Liposomes as Adjuvants/Delivery Systems for Tuberculosis Subunit Vaccines. Rev Physiol Biochem Pharmacol 2018; 175: 47-69.
29. Kaufmann SH. Tuberculosis vaccines: time to think about the next generation. Semin Immunol 2013; 25: 172-181.
30. Khademi F, Derakhshan M, Yousefi-Avarvand A, Tafaghodi M, Soleimanpour M. Multi-stage subunit vaccines against Mycobacterium tuberculosis: An alternative to the BCG vaccine or a BCG-prime boost?. Expert Rev Vaccines 2018; 17: 31-44.
31. Villarreal DO, Walters J, Laddy DJ, Yan J, Weiner DB. Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG. Hum Vaccin Immunother 2014; 10: 2188-2198.
32. Yuan W, Dong N, Zhang L, Liu J, Lin S, Xiang Z, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine expressing a fusion protein of Ag85B-Esat6-HspX in mice. Vaccine 2012; 30: 2490-2497.
33. Shi C, Chen L, Chen Z, Zhang Y, Zhou Z, Lu J, et al. Enhanced protection against tuberculosis by vaccination with recombinant BCG overexpressing HspX protein. Vaccine 2010; 28: 5237-5244.
34. Jeon B-Y, Kim S-C, Eum S-Y, Cho S-N. The immunity and protective effects of antigen 85A and heat-shock protein X against progressive tuberculosis. Microb Infec 2011; 13: 284-290.
35. Spratt JM, Britton WJ, Triccas JA. In vivo persistence and protective efficacy of the Bacille Calmette Guerin vaccine overexpressing the HspX latency antigen. Bioeng bugs 2010; 1: 61-65.
36. Khademi F, Sahebkar A, Fasihi‐Ramandi M, Taheri RA. Induction of strong immune response against a multicomponent antigen of Mycobacterium tuberculosis in BALB/c mice using PLGA and DOTAP adjuvant. Apmis 2018; 126: 509-514.
37. Richter WF, Jacobsen B. Subcutaneous absorption of biotherapeutics: knowns and unknowns. ‎Drug Metab Dispos 2014; 42:1881-1889.
38. Kirby DJ, Rosenkrands I, Agger EM, Andersen P, Coombes AG, Perrie Y. PLGA microspheres for the delivery of a novel subunit TB vaccine. J Drug Target 2008; 16: 282-293.
39. Khademi F, Taheri RA, Avarvand AY, Vaez H, Momtazi-Borojeni AA, Soleimanpour S. Are chitosan natural polymers suitable as adjuvant/delivery system for anti-tuberculosis vaccines?. Microb Pathog 2018; 121: 218-223.
40. Kim I-S, Lee S-K, Park Y-M, Lee Y-B, Shin S-C, Lee KC, et al. Physicochemical characterization of poly (L-lactic acid) and poly (D, L-lactide-co-glycolide) nanoparticles with polyethylenimine as gene delivery carrier. Int J Pharm 2005; 298: 255-262.
41. He C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010; 31: 3657-3666.
42. Verwaerde C, Debrie A-S, Dombu C, Legrand D, Raze D, Lecher S, et al. HBHA vaccination may require both Th1 and Th17 immune responses to protect mice against tuberculosis. Vaccine 2014; 32: 6240-6250.
43. Hoft DF, Worku S, Kampmann B, Whalen CC, Ellner JJ, Hirsch CS, et al. Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J Infect Dis 2002; 186: 1448-1457.