Recombinant PBP2a/autolysin conjugate as PLGA-based nanovaccine induced humoral responses with opsonophagocytosis activity, and protection versus methicillin-resistant Staphylococcus aureus infection

Document Type : Original Article

Authors

1 Department of Microbiology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

2 Department of Mycobacteriology & Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran

3 Department of Microbiology, Faculty of Basic Sciences, North Tehran Branch, Islamic Azad University, Tehran, Iran

4 Advanced Therapy Medicinal Product (ATMP) Department, Breast Cancer Research Center, Motamed Cancer Institute, Academic Center for Education, Culture and Research(ACECR), Tehran, Iran

5 Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran

6 Immunotherapy Group, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran

10.22038/ijbms.2022.59992.13303

Abstract

Objective(s): Methicillin-resistant Staphylococcus aureus (MRSA) reasons extreme infections, can resist various conventional antimicrobial agents, and cause morbidity and mortality worldwide. Vaccination seems to help modulate MRSA infections. Nanovaccine is considered a novel strategy in vaccine technology. The primary purpose of the present study was to develop a conjugate vaccine based on recombinant PBP2a and MRSA autolysin formulated in PLGA as a nanoparticle capable of enhancing protective responses against MRSA in the murine model.
Materials and Methods: Recombinant PBP2a and autolysin have been expressed and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column and characterized by SDS-PAGE and western blot. PLGA was bound to recombinant proteins by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and adipic acid dihydrazide (ADH) as a linker and spacer, respectively. Conjugation of recombinant proteins to PLGA was confirmed by the AFM assay, zeta potential, and size distribution, and its efficacy was evaluated in mice. Total IgG, IgG1, IgG2a, IgG2b, and IgM titers were analyzed to assess immune responses. Lastly, the bioactivity of antibodies was tested by using the opsonophagocytosis assay. 
Results: Mice immunized with the r-PBP2a-r-autolysin–PLGA nanovaccine led to increased levels of opsonic antibodies and IgG1, IgG2a, IgG2b, and IgM when compared with other experimental groups. Our results confirmed that vaccination with nanovaccine could reduce the mortality rate against the sub-lethal dose of MRSA challenge. Furthermore, the nanovaccine could eliminate MRSA from the kidney of infected mice. 
Conclusion: This study may provide valuable insights into the protective power of the r-PBP2a-r-autolysin–PLGA conjugate vaccine against MRSA infection.

Keywords


1. Zhao L, Seth A, Wibowo N, Zhao C-X, Mitter N, Yu C, et al. Nanoparticle vaccines. Vaccine 2014; 32:327-337.
2. 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.
3. Sekhon BS, Saluja V. Nanovaccines-an overview. Int J  Pharm Front 2011; 1:101-109.
4. Sridhar R, Ramakrishna S. Electrosprayed nanoparticles for drug delivery and pharmaceutical applications. Biomatter 2013; 3:e24281.
5. Ali A, Shah T, Ullah R, Zhou P, Guo M, Ovais M, et al. Review on recent progress in magnetic nanoparticles: Synthesis, characterization, and diverse applications. Front Chem 2021; 9.
6. Nandedkar T. Nanovaccines: recent developments in vaccination. J Biosci 2009; 34:995-1003.
7. Pati R, Shevtsov M, Sonawane A. Nanoparticle vaccines against infectious diseases. Front Immunol 2018; 9.
8. Alimohammadi YH, Joo SW. PLGA-based nanoparticles as cancer drug delivery systems. Asian Pac J Cancer Prev  2014; 15:517-535.
9. Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 2011; 3:1377-1397.
10. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: An overview of biomedical applications. J Control Release  2012; 161:505-522.
11. Saeidnia S. New approaches to natural anticancer drugs: Springer; 2015.
12. Bharali DJ, Sudha T, Cui H, Mian BM, Mousa SA. Anti-CD24 nano-targeted delivery of docetaxel for the treatment of prostate cancer. Nanomedicine 2017; 13:263-273.
13. Sahay G, Alakhova DY, Kabanov AV. Endocytosis of nanomedicines. J Control Release 2010; 145:182-195.
14. Behzadi S, Serpooshan V, Tao W, Hamaly MA, Alkawareek MY, Dreaden EC, et al. Cellular uptake of nanoparticles: Journey inside the cell. Chem Soc Rev 2017; 46:4218-4244.
15. Mahakalkar A, Hatwar B. Biophysicochemical characteristics & applications of nanoparticles: mini review. Am j drug deliv 2014; 1:035-041.
16. Owens DE, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm  2006; 307:93-102.
17. Salatin S, Maleki Dizaj S, Yari Khosroushahi A. Effect of the surface modification, size, and shape on cellular uptake of nanoparticles. Cell Biol Int  2015; 39:881-890.
18. Tahara K, Sakai T, Yamamoto H, Takeuchi H, Hirashima N, Kawashima Y. Improved cellular uptake of chitosan-modified PLGA nanospheres by A549 cells. Int J Pharm 2009; 382:198-204.
19. Green BN, Johnson CD, Egan JT, Rosenthal M, Griffith EA, Evans MW. Methicillin-resistant Staphylococcus aureus: an overview for manual therapists. J Chiropr Med  2012; 11:64-76.
20. Clegg J, Soldaini E, McLoughlin RM, Rittenhouse S, Bagnoli F, Phogat S. Staphylococcus aureus vaccine research and development: The past, present and future, including novel therapeutic strategies. Front Immunol 2021; 12.
21. Kalali Y, Haghighat S, Mahdavi M. Passive immunotherapy with specific IgG fraction against autolysin: Analogous protectivity in the MRSA infection with antibiotic therapy. Immunol Lett 2019; 212:125-131.
22. Haghighat S, Siadat SD, Sorkhabadi SMR, Sepahi AA, Mahdavi M. Cloning, expression and purification of autolysin from methicillin-resistant Staphylococcus aureus: potency and challenge study in BALB/c mice. Mol Immunol 2017; 82:10-18.
23. Haghighat S, Siadat SD, Sorkhabadi SMR, Sepahi AA, Mahdavi M. Cloning, expression and purification of penicillin binding protein2a (pbp2a) from methicillin resistant Staphylococcus aureus: A study on immunoreactivity in BALB/C mouse. Avicenna J Med Biotechnol 2013; 5:204-211.
24. Lim D, Strynadka NCJ. Structural basis for the [beta] lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus. Nat Struct Mol Biol 2002; 9:870-876.
25. Naghshbandi RZ, Haghighat S, Mahdavi M. Passive immunization against methicillin resistant Staphylococcus aureus recombinant PBP2a in sepsis model of mice: Comparable results with antibiotic therapy. Int Immunopharmacol 2018; 56:186-192.
26. Haghighat S, Siadat SD, Sorkhabadi SMR, Sepahi AA, Mahdavi M. A novel recombinant vaccine candidate comprising PBP2a and autolysin against Methicillin Resistant Staphylococcus aureus confers protection in the experimental mice. Mol Immunol 2017; 91:1-7.
27. Haghighat S, Siadat SD, Rezayat Sorkhabadi SM, Akhavan Sepahi A, Sadat SM, Hossein Yazdi M, et al. Recombinant PBP2a as a vaccine candidate against methicillin-resistant Staphylococcus aureus: Immunogenicity and protectivity. Microb Pathog  2017; 108:32-39.
28. Mortazavi SS, Haghighat S, Mahdavi M. Recombinant PBP2a of methicillin-resistant S. aureus formulation in Alum and Montanide ISA266 adjuvants induced cellular and humoral immune responses with protection in BALB/C mice. Microb Pathog  2020; 140:103945.
29. Varrone JJ, Li D, Daiss JL, Schwarz EM. Anti-glucosaminidase monoclonal antibodies as a passive immunization for methicillin-resistant Staphylococcus aureus (MRSA) orthopaedic infections. Bonekey Osteovision  2011; 8:187-194.
30. Pérez O, Romeu B, Cabrera O, González E, Batista-Duharte A, Labrada A, et al. Adjuvants are Key factors for the development of future vaccines: lessons from the finlay adjuvant platform. Front Immunol  2013; 4:407.
31. Marrack P, McKee AS, Munks M.W. Towards an understanding of the adjuvant action of aluminium. Nat Rev Immunol 2009; 9:287-293.
32. Kashef N, Behzadian-Nejad Q, Najar-Peerayeh S, Mousavi-Hosseini K, Moazzeni M, Djavid GE. Synthesis and characterization of Pseudomonas aeruginosa alginate–tetanus toxoid conjugate. J Med Microbiol  2006; 55:1441-1446.
33. Ames P, DesJardins D, Pier GB. Opsonophagocytic killing activity of rabbit antibody to Pseudomonas aeruginosa mucoid exopolysaccharide. Infect Immun 1985; 49:281-285.
34. Chen L, Li S, Wang Z, Chang R, Su J, Han B. Protective effect of recombinant staphylococcal enterotoxin A entrapped in polylactic-co-glycolic acid microspheres against Staphylococcus aureus infection. Vet Res 2012; 43:20-20.
35. Nascimento IP, Leite LCC. Recombinant vaccines and the development of new vaccine strategies. Braz J Med Biol Res 2012; 45:1102-1111.
36. Andersson C. Production and delivery of recombinant subunit vaccines: Bioteknologi; 2000.
37. Coffman RL, Sher A, Seder RA. Vaccine Adjuvants: Putting Innate Immunity to Work. Immunity 2010; 33:492-503.
38. Mohan T, Verma P, Rao DN. Novel adjuvants & delivery vehicles for vaccines development: A road ahead. Indian J Med Res  2013; 138:779-795.
39. Demento SL, Cui W, Criscione JM, Stern E, Tulipan J, Kaech SM, et al. Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype. Biomaterials 2012; 33:4957-4964.
40. Scully I, Liberator P, Jansen K, Anderson A. Covering all the bases: Preclinical development of an effective Staphylococcus aureus vaccine. Front Immunol 2014; 5.
41. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG. Staphylococcus aureus Infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 2015; 28:603-661.
42. Miller LS, Fowler Jr VG, Shukla SK, Rose WE, Proctor R.A. Development of a vaccine against Staphylococcus aureus invasive infections: Evidence based on human immunity, genetics and bacterial evasion mechanisms. FEMS Microbiol Rev 2020; 44:123-153.
43. Rafiqi SI, Kumar S, Zehra A, Kumar D, Jain S, Sethi K, et al. Nanovaccinology: Dawn of biomimetic vaccine carriers. J Entomol Zool Stud 2017; 5: 795-802. 
44. Silva AL, Soema PC, Slütter B, Ossendorp F, Jiskoot W. PLGA particulate delivery systems for subunit vaccines: Linking particle properties to immunogenicity. Hum Vaccin Immunother 2016; 12:1056-1069.
45. Safari Zanjani L, Shapouri R, Dezfulian M, Mahdavi M, Shafiee Ardestani M. Exotoxin A-PLGA nanoconjugate vaccine against Pseudomonas aeruginosa infection: protectivity in murine model. World J Microbiol Biotechnol 2019; 35:94.
46. Sharp FA, Ruane D, Claass B, Creagh E, Harris J, Malyala P, et al. Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome. Proc Natl Acad Sci 2009; 106:870-875.
47. Genta I, Colonna C, Conti B, Caliceti P, Salmaso S, Speziale P, et al. CNA-loaded PLGA nanoparticles improve humoral response against Staphylococcus aureus-mediated infections in a mouse model: subcutaneous vs. nasal administration strategy. J Microencapsul 2016; 33:750-762.
48. Colonna C, Dorati R, Conti B, Caliceti P, Genta I. Sub-unit vaccine against Staphylococcus aureus-mediated infections: Set-up of nano-sized polymeric adjuvant. Int J Pharm  2013; 452:390-401.
49. Oyewumi MO, Kumar A, Cui Z. Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses. Expert Rev Vaccines 2010; 9:1095-1107.
50. Diwan M, Elamanchili P, Lane H, Gainer A, Samuel J. Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses. J Drug Target 2003; 11:495-507.
51. Thomas C, Gupta V, Ahsan F. Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses. Int J Pharm 2009; 379:41-50.
52. Saini V, Jain V, Sudheesh MS, Jaganathan KS, Murthy PK, Kohli DV. Comparison of humoral and cell-mediated immune responses to cationic PLGA microspheres containing recombinant hepatitis B antigen. Int J Pharm  2011; 408:50-57.
53. Roth DM SJ, Machado DC. Evaluation of the humoral immune response in BALB/c mice immunized with a naked DNA vaccine anti- methicillin-resistant Staphylococcus aureus. Genet Mol Res 2006; 5:503-512.
54. Schaefers MM, Duan B, Mizrahi B, Lu R, Reznor G, Kohane DS, et al. PLGA-encapsulation of the Pseudomonas aeruginosa PopB vaccine antigen improves Th17 responses and confers protection against experimental acute pneumonia. Vaccine 2018; 36:6926-6932.