Protection of BALB/c mice against pathogenic Brucella abortus and Brucella melitensis by vaccination with recombinant Omp16

Document Type: Original Article

Authors

1 Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran

2 Department of Microbiology, Zanjan Branch, Islamic Azad University, Zanjan, Iran

3 Lister Laboratory of Microbiology, Tehran, Iran

4 Department of Microbiology and Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

5 Applied Virology ResearchCenter, Baqiyatallah University of Medical Sciences, Tehran, Iran

Abstract

Objective(s): Prevention of the globally spread zoonotic infection, brucellosis which affects an extensive range of hosts is still challenging researchers. There are no approved vaccines for the prevention of human disease and those used for animal brucellosis have adverse properties, which limit their application. We investigated the immunological and protective effects of recombinant 16 kDa outer membrane protein of Brucella abortus (Omp16) which introduced a new candidate for brucellosis subunit vaccine.
Materials and Methods: Brucella Omp16 gene was cloned in pET-23a and expressed in Escherichia coli BL21 (DE3). Recombinant Omp16 (rOmp16) was purified using nickel resin and confirmed by Western blot analysis. BALB/c mice were immunized with rOmp16, afterward, specific serum antibodies and cytokine responses were evaluated. Protection of immunized mice against pathogenic B. abortus 544 and B. melitensis 16M was evaluated by the intraperitoneal bacterial challenge.
Results: Sequencing results of the recombinant plasmid vector along with Western blotting confirmed the cloning procedure. Recognition of rOmp16 by specific IgG from serum samples of infected cases suggests the stimulation of immune response to this protein. Significant total serum IgG along with remarkable IgG1 and IgG2a response to the protein was recorded. A significant increase in IFN-γ, and IL-4 levels were observed from splenocyte cultures of immunized mice which were stimulated with rOmp16 suggesting the development of T-lymphocyte mediated immunity against the recombinant antigen.
Conclusion: The intraperitoneal challenge with B. abortus 544 and B. melitensis 16M confirmed that rOmp16 is able to elicit efficient protective immune responses in the animal host.

Keywords

Main Subjects


1. Hasanjani-Roushan MR, Ebrahimpour S. Human brucellosis: an overview. Caspian J Intern Med 2015; 6:46-47.

2. Lynch JS. Brucellosis: A zoonotic infection and worldwide public health concern. JAAPA 2014; 27:1-5.

3. Smits HL. Brucellosis in pastoral and confined livestock: prevention and vaccination. Rev Sci Tech 2013; 32:219-228.

4. Poester FP, Samartino LE, Santos RL. Pathogenesis and pathobiology of brucellosis in livestock. Rev Sci Tech 2013; 32:105-115.

5. Singh BB, Dhand NK, Gill JP. Economic losses occurring due to brucellosis in Indian livestock populations. Prev Vet Med 2015, 119:211-215.

6. Doganay M. Human Brucellosis: Importance of Brucellosis. Recent patents on anti-infective drug discovery 2013, 8:2-3.

7. Ramin B, MacPherson P. Human brucellosis: Brucellose beim Menschen. PARAXIS 2011; 100:305-307.

8. Taleski V. An overview of introducing various laboratory tests for diagnosis of human brucellosis in the republic of Macedonia. MJMS 2010; 3:239-245.

9. Olsen SC. Recent developments in livestock and wildlife brucellosis vaccination. Rev Sci Tech 2013; 32:207-217.

10. Blasco JM, Diaz R. Brucella melitensis Rev-1 vaccine as a cause of human brucellosis. Lancet 1993; 342:805.

11. Squarcione S, Maggi P, Lo Caputo S, De Gennaro M, Carbonara S. A case of human brucellosis caused by accidental injection of animal vaccine. G Ital Med Lav 1990; 12:25-26.

12. Nielsen K, Yu WL. Serological diagnosis of brucellosis. Prilozi 2010; 31:65-89.

13. Schurig GG, Sriranganathan N, Corbel MJ. Brucellosis vaccines: past, present and future. Vet Microbiol 2002; 90:479-496.

14. Shumilov KV, Sklyarov O, Klimanov A. Designing vaccines against cattle brucellosis. Vaccine 2010; 28: 31-34.

15. Ficht TA, Kahl-McDonagh MM, Arenas-Gamboa AM, Rice-Ficht AC. Brucellosis: the case for live, attenuated vaccines. Vaccine 2009; 27:40-43.

16. Abadi AH, Mahdavi M, Khaledi A, Esmaeili A, Esmaeili D, Sahebkar A. Study of serum bactericidal and splenic activity of Total-OMP-CagA combination from Brucella abortus and Helicobacter pylori in BALB/c mouse model. Microb Pathog 2018; 121: 100-105.

17. Zheng WY, Wang Y, Zhang ZC, Yan F. Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. GMR 2015; 14:11965-11974.

18. Clausse M, Diaz AG, Ibanez AE, Cassataro J, Giambartolomei GH, Estein SM. Evaluation of the efficacy of outer membrane protein 31 vaccine formulations for protection against Brucella canis in BALB/c mice. Clin Vaccine Immunol 2014; 21:1689-1694.

19. Sung KY, Jung M, Shin MK, Park HE, Lee JJ, Kim S, Yoo HS. Induction of immune responses by two recombinant proteins of Brucella abortus, outer membrane proteins 2b porin and Cu/Zn superoxide dismutase, in mouse model. J Microbiol Biotechnol 2014; 24:854-861.

20. Ibanez AE, Smaldini P, Coria LM, Delpino MV, Pacifico LG, Oliveira SC, et al. Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow's Milk Proteins. PLoS One 2013; 8:1-13.

21. Gomez G, Pei J, Mwangi W, Adams LG, Rice-Ficht A, Ficht TA. Immunogenic and invasive properties of Brucella melitensis 16M outer membrane protein vaccine candidates identified via a reverse vaccinology approach. PLoS One 2013; 8:59751-59759.

22. Goel D, Bhatnagar R. Intradermal immunization with outer membrane protein 25 protects Balb/c mice from virulent B. abortus 544. Mol Immunol 2012; 51:159-168.

23. Lim JJ, Kim DH, Lee JJ, Kim DG, Min W, Lee HJ, et al. Evaluation of recombinant 28 kDa outer membrane protein of Brucella abortus for the clinical diagnosis of bovine brucellosis in Korea. J Vet Med Sci 2012; 74:687-691.

24. Mythili T, Rajendra L, Thiagarajan D, Srinivasan VA. Immunogenecity of a Brucella abortus S19 glyco-conjugate vaccine consisting of lipo-polysaccharide and outer membrane protein in cattle calves. Curr. Trends Biotechnol. Pharm. 2010; 4:510-518.

25. Pasquevich KA, Estein SM, Garcia Samartino C, Zwerdling A, Coria LM, Barrionuevo P, Fossati CA, Giambartolomei GH, 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; 77:436-445.

26. Paquet JY, Vinals C, Wouters J, Letesson JJ, Depiereux E. Topology prediction of Brucella abortus Omp16 and Omp16 porins after critical assessment of transmembrane beta strands prediction by several secondary structure prediction methods. J Biomol Struct Dyn 2000; 17:747-757.

27. Mobasheri H, Ficht TA, Marquis H, Lea EJ, Lakey JH. Brucella Omp16 and Omp16 porins: single channel measurements and topology prediction. FEMS Microbiol Lett 1997; 155:23-30.

28. Cloeckaert A, Verger JM, Grayon M, Vizcaino N. Molecular and immunological characterization of the major outer membrane proteins of Brucella. FEMS Microbiol Lett 1996; 145:1-8.

29. Aghababa H, Mohabati Mobarez A, Khoramabadi N, Behmanesh M, Mahdavi M, Tebianian M, et al. A comparative approach to strategies for cloning, expression, and purification of Mycobacterium tuberculosis mycolyl transferase 85B and evaluation of immune responses in BALB/c mice. Mol. Biotechnol. 2014; 56:487-497.

30. Guide for the Care and Use of Laboratory Animals. Washington DC, 8 edn. National Academies Press 2011.

31. Leylabadlo HE, Bialvaei AZ, Kafil HS. Brucellosis in Iran: Why Not Eradicated? Clin Infect Dis 2015; 61:1129-1167.

32. Mangalgi SS, Sajjan AG, Mohite ST, Kakade SV. Serological, Clinical, and Epidemiological Profile of Human Brucellosis in Rural India. Indian J Community Med 2015; 40:163-167.

33. Musallam, II, Abo-Shehada MN, Hegazy YM, Holt HR, Guitian FJ. Systematic review of brucellosis in the Middle East: disease frequency in ruminants and humans and risk factors for human infection. Epidemiol Infect 2016; 144:671-685.

34. Ducrotoy M, Bertu WJ, Matope G, Cadmus S, Conde-Alvarez R, Gusi AM, et al. Brucellosis in Sub-Saharan Africa: Current challenges for management, diagnosis and control. Acta Trop 2017; 165: 179-193.

35. Hotez PJ, Savioli L, Fenwick A. Neglected tropical diseases of the Middle East and North Africa: review of their prevalence, distribution, and opportunities for control. PLoS Negl Trop Dis 2012; 6:1-8.

36. Shalmali, Panda AK, Chahota R. Sero-prevalence of brucellosis in occupationally exposed human beings of Himachal Pradesh (India). J Commun Dis 2012; 44:91-95.

37. Haque N, Bari MS, Hossain MA, Muhammad N, Ahmed S, Rahman A, et al. An overview of Brucellosis. Mymensingh Med J 2011; 20:742-747.

38. Nicoletti P. Brucellosis: past, present and future. Prilozi 2010; 31:21-32.

39. Dean AS, Crump L, Greter H, Schelling E, Zinsstag J. Global burden of human brucellosis: a systematic review of disease frequency. PLoS Negl Trop Dis 2012; 6:1-13.

40. Blasco JM, Molina-Flores B. Control and eradication of Brucella melitensis infection in sheep and goats. Vet Clin North Am Food Anim Pract 2011; 27:95-104.

41. Olsen SC, Stoffregen WS. Essential role of vaccines in brucellosis control and eradication programs for livestock. Expert Rev Vaccines 2005; 4:915-928.

42. Ko KY, Kim JW, Her M, Kang SI, Jung SC, Cho DH, et al. Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis. Vet Microbiol 2012; 156:374-380.

43. Siadat SD, Aghasadeghi MR, Karami S, Sadat SM, Moshiri A. Biological and immunological characteristics of Brucella abortus S99 major outer membrane proteins. Jundishapur J.Microbiol 2011; 4:29-36.

44. Connolly JP, Comerci D, Alefantis TG, Walz A, Quan M, Chafin R, et al. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development. Proteomics 2006; 6:3767-3780.

45. Cloeckaert A, Kerkhofs P, Limet JN. Antibody response to Brucella outer membrane proteins in bovine brucellosis: immunoblot analysis and competitive enzyme-linked immunosorbent assay using monoclonal antibodies. J Clin Microbiol 1992; 30:3168-3174.

46. Gonzalez D, Grillo MJ, De Miguel MJ, Ali T, Arce-Gorvel V, Delrue RM, et al. Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export. PLoS One 2008; 3:1-11.

47. Jain S, Kumar S, Dohre S, Afley P, Sengupta N, Alam SI. Identification of a protective protein from stationary-phase exoproteome of Brucella abortus. PATHOG DIS 2014; 70:75-83.

48. Tibor A, Wansard V, Bielartz V, Delrue RM, Danese I, Michel P, et al. Effect of omp10 or omp19 deletion on Brucella abortus outer membrane properties and virulence in mice. Infect Immun 2002; 70:5540-5546.

49. Cloeckaert A, Vizcaino N, Paquet JY, Bowden RA, Elzer PH. Major outer membrane proteins of Brucella spp.: past, present and future. Vet Microbiol 2002; 90:229-247.

50. Tibor A, Decelle B, Letesson JJ. Outer membrane proteins Omp10, Omp16, and Omp19 of Brucella spp. are lipoproteins. Infect Immun 1999; 67:4960-4962.