Investigating the Efficiency of Recombinant FliC-Loaded Bacil-lus subtilis Spores in Mice Immunization against Salmonella en-terica Serovar Typhi
,
Abstract
Background: Bacterial spores are among the most efficient vaccine delivery vehicles. Because of their safety and efficacy, Bacillus subtilis spores are increasingly used in this regard. The negatively charged surfaces of the spores allow antigens to be adsorbed onto these structures. In this study, a candidate vaccine against Salmonella enterica serovar Typhi was adsorbed onto B. subtilis spores and the immunogenicity of the formulation was investigated in BALB/c mice.
Methods: This work was performed during 2018-2019 in Islamic Azad University of Lahijan. FliC protein was recombinantly expressed in E. coli BL21 (DE3) cells and purified by affinity chromatography. On the other hand, B. subtilis strain PY79 (ATCC1609) was cultured in DSM medium and after the sporulation, FliC protein was adsorbed onto the spores in three different pH values (4, 7 and 10) and the adsorption was verified using dot-blot assay. FliC-adsorbed spores were then administered to BALB/c mice through the subcutaneous route. Mice immunization was evaluated by serum IgG assessment and challenge study.
Results: FliC protein was successfully expressed and purified. Sporulation was controlled by phase-contrast microscopy. Serum IgG assay showed significant stimulation of the mice's humoral immune system. Immunized mice were able to resist bacterial infection.
Conclusion: The results showed the efficiency of spores as natural adjuvants for the stimulation of mice immune system. The formulation can be exploited for the delivery of recombinant vaccines against bacterial pathogens.
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35. Simon R, Tennant SM, Wang JY, et al (2011). Salmonella enterica serovar Enteritidis core O polysaccharide conjugated to H: g, m flagellin as a candidate vaccine for protection against invasive infection with S. enteritidis. Infect Immun, 79 (10): 4240-4249.
36. Simon R, Wang JY, Boyd MA, et al (2013). Sustained protection in mice immunized with fractional doses of Salmonella Enteritidis core and O polysaccharide-flagellin glycoconjugates. PloS one, 8 (5): e64680.
37. Dai X, Liu M, Pan K, et al (2018). Surface display of OmpC of Salmonella serovar Pullorum on Bacillus subtilis spores. PloS One, 13 (1): e0191627.
38. Yao Y-Y, Chen D-D, Cui Z-W, et al (2019). Oral vaccination of tilapia against Streptococcus agalactiae using Bacillus subtilis spores expressing Sip. Fish Shellfish Immunol, 86: 999-1008.
39. Song M, Hong HA, Huang J-M, et al (2012). Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine. Vaccine, 30 (22): 3266-77.
40. Okamura M, Matsumoto W, Seike F, et al (2012). Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar Enteritidis as a potential vaccine candidate against homologous challenge in chickens. Avian Dis, 56 (2): 354-8.
2. Mohan T, Verma P, Rao DN (2013). Novel adjuvants & delivery vehicles for vaccines development: a road ahead. Indian J Med Res, 138 (5): 779-95.
3. Schijns VE, Lavelle EC (2011). Trends in vaccine adjuvants. Expert Rev vaccines, 10 (4): 539-50.
4. Hong HA, Duc LH, Cutting SM (2005). The use of bacterial spore formers as probiotics. FEMS Microbiol Rev, 29 (4): 813-35.
5. Bader J, Albin A, Stahl U(2012). Spore-forming bacteria and their utilisation as probiotics. Benef Microbes, 3 (1): 67-75.
6. Elshaghabee FM, Rokana N, Gulhane RD, et al (2017). Bacillus as potential probiotics: status, concerns, and future perspectives. Front Microbiol, 8: 1490.
7. Paccez JD, Nguyen HD, Luiz WB, et al (2007). Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles. Vaccine, 25 (24): 4671-80.
8. Paccez JD, Luiz WB, Sbrogio-Almeida ME, et al (2006). Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery. Vaccine, 24 (15): 2935-43.
9. Zhou Z, Xia H, Hu X, et al (2008). Immunogenicity of recombinant Bacillus subtilis spores expressing Clonorchis sinensis tegumental protein. Parasitol Res, 102 (2): 293-7.
10. Huang J-M, Hong HA, Van Tong H, et al (2010). Mucosal delivery of antigens using adsorption to bacterial spores. Vaccine, 28 (4): 1021-30.
11. Mauriello EM, Duc LH, Isticato R, et al (2004). Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine, 22 (9-10): 1177-87.
12. Marks F, von Kalckreuth V, Aaby P, et al (2017). Incidence of invasive salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study. Lancet Glob Health, 5 (3): e310-e323.
13. Yang Y-A, Chong A, Song J (2018). Why is eradicating typhoid fever so challenging: implications for vaccine and therapeutic design. Vaccines (Basel), 6 (3): 45.
14. Ogushi K-i, Wada A, Niidome T, et al (2001). Salmonella enteritidis FliC (flagella filament protein) induces human β-defensin-2 mRNA production by Caco-2 cells. J Biol Chem, 276 (32): 30521-6.
15. Wang L, Rothemund D, Curd H, Reeves PR (2003). Species-wide variation in the Escherichia coli flagellin (H-antigen) gene. J Bacteriol, 185 (9): 2936-43.
16. Monot M, Boursaux-Eude C, Thibonnier M, et al (2011). Reannotation of the genome sequence of Clostridium difficile strain 630. J Med Microbiol, 60 (8): 1193-1199.
17. Chung CT, Miller RH (1993). Preparation and storage of competent Escherichia coli cells. Methods Enzymol, 218: 621-7.
18. Hosseini SA, Nazarian S, Ebrahimi F, et al (2020). Immunogenicity Evaluation of Recombinant Staphylococcus aureus Enterotoxin B (rSEB) and rSEB-loaded Chitosan Nanoparticles Following Nasal Administration. Iran J Allergy Asthma Immunol, 19 (2): 159-171.
19. Hajizade A, Salmanian AH, Amani J, et al (2018). EspA-loaded mesoporous silica nanoparticles can efficiently protect animal model against enterohaemorrhagic E. coli O157: H7. Artif Cells Nanomed Biotechnol, 46 (sup3): S1067-S1075.
20. Sayadmanesh A, Ebrahimi F, Hajizade A, et al (2013). Expression and purification of neurotoxin-associated protein HA-33/A from Clostridium botulinum and evaluation of its antigenicity. Iran Biomed J, 17 (4): 165-170.
21. Hoffmann A, Roeder R (1991). Purification of his-tagged proteins in non-denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Res, 19 (22): 6337-6338.
22. Tavares MB, Souza RD, Luiz WB, et al (2013). Bacillus subtilis endospores at high purity and recovery yields: optimization of growth conditions and purification method. Curr Microbiol,66 (3): 279-85.
23. Frey A, Di Canzio J, Zurakowski D (1998). A statistically defined endpoint titer determination method for immunoassays. J Immunol Methods, 221 (1-2): 35-41.
24. QIAexpressionist A (2002). A handbook for high-level expression and purification of 6xhis-tagged proteins. Valencia, CA: Qiagen, pp.: 1-125.
25. Kruger NJ (2009). The Bradford method for protein quantitation. The protein protocols handbook. 3rd ed. Humana Press, USA, pp.: 17-24.
26. Levine MM, Sztein MB (2004). Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol, 5 (5): 460-4.
27. Abhyankar MM, Orr MT, Lin S, et al (2018). Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica. NPJ vaccines, 3(1):22.
28. Hajizade A, Ebrahimi F, Salmanian A-H, et al (2015). Nanoparticles in vaccine development. J Appl Biotechnol Rep. 1 (4): 125-134.
29. Zhao L, Seth A, Wibowo N, et al (2014). Nanoparticle vaccines. Vaccine, 32 (3): 327-237.
30. Maisonneuve C, Bertholet S, Philpott DJ, et al (2014). Unleashing the potential of NOD-and Toll-like agonists as vaccine adjuvants. Proc Natl Acad Sci U S A, 111 (34): 12294-9.
31. Johnson AG (1994). Molecular adjuvants and immunomodulators: new approaches to immunization. Clin Microbiol Rev, 7 (3): 277-89.
32. Barnes AG, Cerovic V, Hobson PS, et al (2007). Bacillus subtilis spores: a novel microparticle adjuvant which can instruct a balanced Th1 and Th2 immune response to specific antigen. Eur J Immunol, 37 (6): 1538-47.
33. Crump JA, Mintz ED (2010). Global trends in typhoid and paratyphoid fever. Clin Infect Dis, 50 (2): 241-246.
34. Milligan R, Paul M, Richardson M, et al (2018). Vaccines for preventing typhoid fever. Cochrane Database Syst Rev, 5(5):CD001261.
35. Simon R, Tennant SM, Wang JY, et al (2011). Salmonella enterica serovar Enteritidis core O polysaccharide conjugated to H: g, m flagellin as a candidate vaccine for protection against invasive infection with S. enteritidis. Infect Immun, 79 (10): 4240-4249.
36. Simon R, Wang JY, Boyd MA, et al (2013). Sustained protection in mice immunized with fractional doses of Salmonella Enteritidis core and O polysaccharide-flagellin glycoconjugates. PloS one, 8 (5): e64680.
37. Dai X, Liu M, Pan K, et al (2018). Surface display of OmpC of Salmonella serovar Pullorum on Bacillus subtilis spores. PloS One, 13 (1): e0191627.
38. Yao Y-Y, Chen D-D, Cui Z-W, et al (2019). Oral vaccination of tilapia against Streptococcus agalactiae using Bacillus subtilis spores expressing Sip. Fish Shellfish Immunol, 86: 999-1008.
39. Song M, Hong HA, Huang J-M, et al (2012). Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine. Vaccine, 30 (22): 3266-77.
40. Okamura M, Matsumoto W, Seike F, et al (2012). Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar Enteritidis as a potential vaccine candidate against homologous challenge in chickens. Avian Dis, 56 (2): 354-8.
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| Issue | Vol 50 No 7 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijph.v50i7.6638 | |
| Keywords | ||
| Salmonella Typhi Vaccine candidate FliC recombinant protein B. subtilis spores | ||
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How to Cite
1.
Ghorbani N, Assmar M, Amirmozafari N, Issazadeh K. Investigating the Efficiency of Recombinant FliC-Loaded Bacil-lus subtilis Spores in Mice Immunization against Salmonella en-terica Serovar Typhi. Iran J Public Health. 2021;50(7):1474-1482.
