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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

In Vitro Evaluation of Immunogenicity of Recombinant OMP25 Protein Obtained from Endemic Brucella abortus Biovar 3 as Vaccine Candidate Molecule Against Animal Brucellosis

Author(s): Tuğba Atabey, Tayfun Acar, Serap Derman, Emel Ordu, Ayşegül Erdemir, Pakize Neslihan Taşlı, Günseli Kurt Gür, Fikrettin Şahin, Medine Güllüce and Tülin Arasoğlu*

Volume 28, Issue 10, 2021

Published on: 14 June, 2021

Page: [1138 - 1147] Pages: 10

DOI: 10.2174/0929866528666210615104334

Price: $65

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Abstract

Background: Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches.

Objective: The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response.

Methods: The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively.

Results: It was determined that doses of the recombinant Omp25 protein greater than 0.1 μg/mL are toxic to RAW cells. Doses of 1 μg/mL and lower significantly increased inflammation due to Nitric Oxide (NO) levels. ELISA results showed that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 μg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose.

Conclusion: Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.

Keywords: Brucellosis, Brucella abortus, vaccine, OMP25, recombinant protein, fusion protein.

Graphical Abstract
[1]
Bercovich, Z. The use of skin delayed-type hypersensitivity as an adjunct test to diagnose brucellosis in cattle: A review: (Summary of thesis, faculty of veterinary medicine, university of Utrecht, 1999). Vet. Q., 2000, 22(3), 123-130.
[2]
Arasoglu, T.; Gulluce, M.; Ozkan, H.; Adiguzel, A.; Sahin, F. PCR detection of Brucella abortus in cow milk samples collected from Erzurum, Turkey. Turk. J. Med. Sci., 2013, 43(4), 501-508.
[http://dx.doi.org/10.3906/sag-1205-121]
[3]
McDermott, J.J.; Arimi, S.M. Brucellosis in Sub-Saharan Africa: epidemiology, control and impact. Vet. Microbiol., 2002, 90(1-4), 111-134.
[http://dx.doi.org/10.1016/S0378-1135(02)00249-3] [PMID: 12414138]
[4]
Yousefi, S.; Tahmoorespur, M.; Sekhavati, M.H. Cloning, expression and molecular analysis of Iranian Brucella melitensis Omp25 gene for designing a subunit vaccine. Res. Pharm. Sci., 2016, 11(5), 412-418.
[http://dx.doi.org/10.4103/1735-5362.192493] [PMID: 27920824]
[5]
Commander, N.; Spencer, S. Brucellosis DNA vaccine. US Patents US20070224257A1 2014.
[6]
Garrido-Abellan, F; Duran-Ferrer, M; MacMillan, A; Minas, A; Nicoletti, P; Vecchi, G Brucellosis in sheep and goats. Report of the Scientific Committee on Animal Health and Animal Werfare; , 2001, p. 12.
[7]
Cutler, S.J.; Cutler, R.R. Brucellosis, The most common bacterial zoonosis? Biomedical Scientist., 2006, 50(4), 336.
[8]
Buyuktanir, O. Günümüzde biyoteknolojik bakteriyel aşılar. Atatürk Üniv. Vet. Bilim. Derg., 2010, 5(2), 97-105.
[9]
Cassataro, J.; Estein, S.M.; Pasquevich, K.A.; Velikovsky, C.A.; de la Barrera, S.; Bowden, R.; Fossati, C.A.; Giambartolomei, G.H. Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+ T helper 1 response that protects against Brucella melitensis infection. Infect. Immun., 2005, 73(12), 8079-8088.
[http://dx.doi.org/10.1128/IAI.73.12.8079-8088.2005] [PMID: 16299302]
[10]
Siadat, S.D.; Salmani, A.S.; Aghasadeghi, M.R. Brucellosis vaccines: an overview. In: Zoonosis. Salmani, A. Jacob Lorenzo-Morales Eds., Intech Open, 2012, pp. 1908-2445.
[11]
Yang, X.; Skyberg, J.A.; Cao, L.; Clapp, B.; Thornburg, T.; Pascual, D.W. Progress in Brucella vaccine development. Front. Biol., 2013, 8(1), 60-77.
[http://dx.doi.org/10.1007/s11515-012-1196-0] [PMID: 23730309]
[12]
Nascimento, I.P.; Leite, L.C. Recombinant vaccines and the development of new vaccine strategies. Braz. J. Med. Biol. Res., 2012, 45(12), 1102-1111.
[http://dx.doi.org/10.1590/S0100-879X2012007500142] [PMID: 22948379]
[13]
Cardoso, F.C.; Pacífico, R.N.; Mortara, R.A.; Oliveira, S.C. Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomic studies. Clin. Exp. Immunol., 2006, 144(3), 382-391.
[http://dx.doi.org/10.1111/j.1365-2249.2006.03081.x] [PMID: 16734606]
[14]
Sette, A.; Rappuoli, R. Reverse vaccinology: Developing vaccines in the era of genomics. Immunity, 2010, 33(4), 530-541.
[http://dx.doi.org/10.1016/j.immuni.2010.09.017] [PMID: 21029963]
[15]
Plotkin, S.A. Immunologic correlates of protection induced by vaccination. Pediatr. Infect. Dis. J., 2001, 20(1), 63-75.
[http://dx.doi.org/10.1097/00006454-200101000-00013] [PMID: 11176570]
[16]
Montaraz, J.A.; Winter, A.J. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice. Infect. Immun., 1986, 53(2), 245-251.
[http://dx.doi.org/10.1128/IAI.53.2.245-251.1986] [PMID: 3089933]
[17]
Pugh, G.W., Jr; Tabatabai, L.B. Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19. Infect. Immun., 1996, 64(2), 548-556.
[http://dx.doi.org/10.1128/IAI.64.2.548-556.1996] [PMID: 8550206]
[18]
Stevens, M.G.; Tabatabai, L.B.; Olsen, S.C.; Cheville, N.F. Immune responses to superoxide dismutase and synthetic peptides of superoxide dismutase in cattle vaccinated with Brucella abortus strain 19 or RB51. Vet. Microbiol., 1994, 41(4), 383-389.
[http://dx.doi.org/10.1016/0378-1135(94)90034-5] [PMID: 7801538]
[19]
Pasquevich, K.A.; Estein, S.M.; García Samartino, C.; Zwerdling, A.; Coria, L.M.; Barrionuevo, P.; Fossati, C.A.; Giambartolomei, G.H.; Cassataro, J. Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection. Infect. Immun., 2009, 77(1), 436-445.
[http://dx.doi.org/10.1128/IAI.01151-08] [PMID: 18981242]
[20]
Kaushik, P.; Singh, D.K.; Kumar, S.V.; Tiwari, A.K.; Shukla, G.; Dayal, S.; Chaudhuri, P. Protection of mice against Brucella abortus 544 challenge by vaccination with recombinant OMP28 adjuvanted with CpG oligonucleotides. Vet. Res. Commun., 2010, 34(2), 119-132.
[http://dx.doi.org/10.1007/s11259-009-9337-x] [PMID: 20013309]
[21]
Cloeckaert, A.; Baucheron, S.; Vizcaino, N.; Zygmunt, M.S. Use of recombinant BP26 protein in serological diagnosis of Brucella melitensis infection in sheep. Clin. Diagn. Lab. Immunol., 2001, 8(4), 772-775.
[http://dx.doi.org/10.1128/CDLI.8.4.772-775.2001] [PMID: 11427425]
[22]
Gupta, V.; Kumari, R.; Vohra, J.; Singh, S.; Vihan, V. Comparative evaluation of recombinant BP26 protein for serological diagnosis of Brucella melitensis infection in goats. Small Rumin. Res., 2010, 93(2-3), 119-125.
[http://dx.doi.org/10.1016/j.smallrumres.2010.05.009]
[23]
Goel, D.; Bhatnagar, R. Intradermal immunization with outer membrane protein 25 protects Balb/c mice from virulent B. abortus 544. Mol. Immunol., 2012, 51(2), 159-168.
[http://dx.doi.org/10.1016/j.molimm.2012.02.126] [PMID: 22464098]
[24]
Delpino, M.V.; Estein, S.M.; Fossati, C.A.; Baldi, P.C.; Cassataro, J. Vaccination with Brucella recombinant DnaK and SurA proteins induces protection against Brucella abortus infection in BALB/c mice. Vaccine, 2007, 25(37-38), 6721-6729.
[http://dx.doi.org/10.1016/j.vaccine.2007.07.002] [PMID: 17686554]
[25]
Jubier-Maurin, V.; Boigegrain, R-A.; Cloeckaert, A.; Gross, A.; Alvarez-Martinez, M-T.; Terraza, A.; Liautard, J.; Köhler, S.; Rouot, B.; Dornand, J.; Liautard, J.P. Major outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages. Infect. Immun., 2001, 69(8), 4823-4830.
[http://dx.doi.org/10.1128/IAI.69.8.4823-4830.2001] [PMID: 11447156]
[26]
Billard, E.; Dornand, J.; Gross, A. Brucella suis prevents human dendritic cell maturation and antigen presentation through regulation of tumor necrosis factor alpha secretion. Infect. Immun., 2007, 75(10), 4980-4989.
[http://dx.doi.org/10.1128/IAI.00637-07] [PMID: 17635859]
[27]
Commander, N.J.; Spencer, S.A.; Wren, B.W.; MacMillan, A.P. The identification of two protective DNA vaccines from a panel of five plasmid constructs encoding Brucella melitensis 16M genes. Vaccine, 2007, 25(1), 43-54.
[http://dx.doi.org/10.1016/j.vaccine.2006.07.046] [PMID: 17049676]
[28]
Eze, M.O.; Yuan, L.; Crawford, R.M.; Paranavitana, C.M.; Hadfield, T.L.; Bhattacharjee, A.K.; Warren, R.L.; Hoover, D.L. Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro. Infect. Immun., 2000, 68(1), 257-263.
[http://dx.doi.org/10.1128/IAI.68.1.257-263.2000] [PMID: 10603396]
[29]
Zhan, Y.; Cheers, C. Endogenous interleukin-12 is involved in resistance to Brucella abortus infection. Infect. Immun., 1995, 63(4), 1387-1390.
[http://dx.doi.org/10.1128/IAI.63.4.1387-1390.1995] [PMID: 7890399]
[30]
Murphy, E.A.; Parent, M.; Sathiyaseelan, J.; Jiang, X.; Baldwin, C.L. Immune control of Brucella abortus 2308 infections in BALB/c mice. FEMS Immunol. Med. Microbiol., 2001, 32(1), 85-88.
[http://dx.doi.org/10.1111/j.1574-695X.2001.tb00536.x] [PMID: 11750226]
[31]
Ahmed, I.M.; Khairani-Bejo, S.; Hassan, L.; Bahaman, A.R.; Omar, A.R. Serological diagnostic potential of recombinant outer membrane proteins (rOMPs) from Brucella melitensis in mouse model using indirect enzyme-linked immunosorbent assay. BMC Vet. Res., 2015, 11(1), 275.
[http://dx.doi.org/10.1186/s12917-015-0587-2] [PMID: 26530141]
[32]
Bulashev, A.; Jakubowski, T.; Tursunov, K.; Kiyan, V.; Zhumalin, A. Immunogenicity and antigenicity of brucella recombinant outer membrane proteins. Vet. Zootech., 2018, 76(98), 17-24.
[33]
Larkin, MA; Blackshields, G; Brown, NP; Chenna, R; McGettigan, PA; McWilliam, H. Clustal W and Clustal X version 2.0. Bioinformatics, 2007, 23(21), 2947-2948.
[34]
Kumar, S.; Stecher, G.; Tamura, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 2016, 33(7), 1870-1874.
[http://dx.doi.org/10.1093/molbev/msw054] [PMID: 27004904]
[35]
Wiechelman, K.J.; Braun, R.D.; Fitzpatrick, J.D. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal. Biochem., 1988, 175(1), 231-237.
[http://dx.doi.org/10.1016/0003-2697(88)90383-1] [PMID: 3245570]
[36]
Cloeckaert, A.; Vizcaíno, N.; Paquet, J-Y.; Bowden, R.A.; Elzer, P.H. Major outer membrane proteins of Brucella spp.: past, present and future. Vet. Microbiol., 2002, 90(1-4), 229-247.
[http://dx.doi.org/10.1016/S0378-1135(02)00211-0] [PMID: 12414146]
[37]
Sharma, N.; Agrewala, J.N. Potent role of vaccines prepared from macrophages infected with live bacteria in protection against Mycobacterium tuberculosis and Salmonella typhimurium infections. J. Infect. Dis., 2004, 190(1), 107-114.
[http://dx.doi.org/10.1086/421116] [PMID: 15195249]
[38]
Malich, G.; Markovic, B.; Winder, C. The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology, 1997, 124(3), 179-192.
[http://dx.doi.org/10.1016/S0300-483X(97)00151-0] [PMID: 9482120]
[39]
Green, S. Antimicrobial and immunopathologic effects of cytokine-induced nitric oxide synthesis. Curr. Opin. Infect. Dis., 1993, 6, 384-396.
[40]
Gross, A.; Spiesser, S.; Terraza, A.; Rouot, B.; Caron, E.; Dornand, J. Expression and bactericidal activity of nitric oxide synthase in Brucella suis-infected murine macrophages. Infect. Immun., 1998, 66(4), 1309-1316.
[http://dx.doi.org/10.1128/IAI.66.4.1309-1316.1998] [PMID: 9529047]
[41]
Gomez, G.; Pei, J.; Mwangi, W.; Adams, L.G.; Rice-Ficht, A.; Ficht, T.A. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach. PLoS One, 2013, 8(3), e59751.
[http://dx.doi.org/10.1371/journal.pone.0059751] [PMID: 23533646]
[42]
Rock, K.L.; Kono, H. The inflammatory response to cell death. Annu. Rev. Pathol., 2008, 3, 99-126.
[http://dx.doi.org/10.1146/annurev.pathmechdis.3.121806.151456] [PMID: 18039143]
[43]
Brandão, AP; Oliveira, FS; Carvalho, NB; Vieira, LQ; Azevedo, V; Macedo, GC Host susceptibility to Brucella abortus infection is more pronounced in IFN-γ knockout than IL-12/β2-microglobulin double-deficient mice. Clinical and Developmental Immunology, 2011, 2012
[44]
Abamor, E.S.; Allahverdiyev, A.; Tosyali, O.A.; Bagirova, M.; Acar, T.; Mustafaeva, Z. Evaluation of in vitro and in vivo immunostimulatory activities of poly (lactic-co-glycolic acid) nanoparticles loaded with soluble and autoclaved Leishmania infantum antigens: A novel vaccine candidate against visceral leishmaniasis. Asian Pac. J. Trop. Med., 2019, 12(8), 353.
[http://dx.doi.org/10.4103/1995-7645.262564]
[45]
Norman, M.U.; Zbytnuik, L.; Kubes, P. Interferon-γ limits Th1 lymphocyte adhesion to inflamed endothelium: A nitric oxide regulatory feedback mechanism. Eur. J. Immunol., 2008, 38(5), 1368-1380.
[http://dx.doi.org/10.1002/eji.200737847] [PMID: 18412158]
[46]
Wu, C.; Xue, Y.; Wang, P.; Lin, L.; Liu, Q.; Li, N.; Xu, J.; Cao, X. IFN-γ primes macrophage activation by increasing phosphatase and tensin homolog via downregulation of miR-3473b. J. Immunol., 2014, 193(6), 3036-3044.
[http://dx.doi.org/10.4049/jimmunol.1302379] [PMID: 25092892]
[47]
Robinson, C.M.; Nau, G.J. Interleukin-12 and interleukin-27 regulate macrophage control of Mycobacterium tuberculosis. J. Infect. Dis., 2008, 198(3), 359-366.
[http://dx.doi.org/10.1086/589774] [PMID: 18557702]
[48]
Paul, S.; Peddayelachagiri, B.V.; Nagaraj, S.; Kingston, J.J.; Batra, H.V. Recombinant outer membrane protein 25c from Brucella abortus induces Th1 and Th2 mediated protection against Brucella abortus infection in mouse model. Mol. Immunol., 2018, 99, 9-18.
[http://dx.doi.org/10.1016/j.molimm.2018.04.002] [PMID: 29649688]
[49]
Adish, Z; Mukantaev, K; Tursunov, K; Kaukabayeva, G; Kanaev, D; Ramankulov, YM Obtaining and determination of immunogenic properties of TRX-PD-1 recombinant protein. Eurasian J. App. Biotechnol., 2019, 2019(1), 24-32.
[50]
Canali, E.; Bolchi, A.; Spagnoli, G.; Seitz, H.; Rubio, I.; Pertinhez, T.A.; Müller, M.; Ottonello, S. A high-performance thioredoxin-based scaffold for peptide immunogen construction: proof-of-concept testing with a human papillomavirus epitope. Sci. Rep., 2014, 4(1), 4729.
[http://dx.doi.org/10.1038/srep04729] [PMID: 24751665]
[51]
Zhao, J.; Yu, H-Y.; Zhao, Y.; Li, F-H.; Zhou, W.; Xia, B-B.; He, Z.Y.; Chen, J.; Jiang, G.T.; Wang, M.L. Soluble expression, rapid purification, biological identification of chicken interferon-alpha using a thioredoxin fusion system in E. coli and its antiviral effects to H9N2 avian influenza virus. Prep. Biochem. Biotechnol., 2019, 49(2), 192-201.
[http://dx.doi.org/10.1080/10826068.2019.1566150] [PMID: 30734625]
[52]
Nguyen, M.T.; Koo, B-K.; Thi Vu, T.T.; Song, J-A.; Chong, S-H.; Jeong, B.; Ryu, H.B.; Moh, S.H.; Choe, H. Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase. PLoS One, 2014, 9(3), e89038.
[http://dx.doi.org/10.1371/journal.pone.0089038] [PMID: 24614134]
[53]
Mukhija, R.; Rupa, P.; Pillai, D.; Garg, L.C. High-level production and one-step purification of biologically active human growth hormone in Escherichia coli. Gene, 1995, 165(2), 303-306.
[http://dx.doi.org/10.1016/0378-1119(95)00525-B] [PMID: 8522194]

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