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Research Article

volume 52 issue 9 (september 2018) : 1317-1323,   Doi: 10.18805/ijar.B-3428

Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice

L
Losa Rose
B
Bablu kumar
A
Ankita Jain
M
M.K. Singh
A
Abhishek
A
Arockiasamy Arun Prince Milton
1Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, India
Cite article:- Rose Losa, kumar Bablu, Jain Ankita, Singh M.K., Abhishek, Milton Prince Arun Arockiasamy (2018). Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice. Indian Journal of Animal Research. 52(9): 1317-1323. doi: 10.18805/ijar.B-3428.

ABSTRACT

Outer membrane vesicles (OMVs) contain biologically active proteins, lipoolysaccharide (LPS), periplasmic and membrane-bound proteins and are known to perform diverse biological functions. OMVs from Brucella abortus S19 were isolated and characterized by transmission electron microscopy (TEM), SDS-PAGE and immunoreactivity was investigated by western blotting. On TEM, bilayered spherical structures of 50-200 nm were observed. SDS-PAGE of OMVs revealed approximate bands size of 82 kDa, 68 kDa, 38 kDa, 32 kDa, 29 kDa and 18 kDa. Western blot analysis of OMVs revealed a dominant immunoreactive band of 38 kDa that correspond to some major outer membrane proteins. Humoral immune response was measured by indirect ELISA which showed that OMV specific antibodies were detected from 7th day post immunization (DPI) onwards and showed a rising trend up to 35th DPI. Cell mediated immune (CMI) response against OMVs as evidenced by the proliferation of splenocytes have also been observed. Thus OMVs were found to possess immunogenic proteins which had potential to induce both humoral as well as cell mediated immunity. After correlating this immune response with protection it has been concluded that OMV can be used as one of the vaccine candidate against brucellosis.

REFERENCES

  1. Avila-Calder´on, E.D., Lopez-Merino, A., Jain, N., Peralta, H., Lopez-Villegas, E.O., Sriranganathan, N., et al., (2012) Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice. Clin. Dev Immunol ., 352493.
  2. Choi-kim, K., Maheswaran, S.K., Felice, L.J., Molitor, T.W. (1991). Relationship between iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida. Vet. Microbiol., 28:75-92
  3. Colligan, J.E., Kruisbeek, A.H., Margulies, B.H., Shevoch, E.M., Strobee, W. (1994). Current protocols in Immunology Vol. 1 Current protocols., John Wiley and Sons, INC, USA
  4. Collins, B.S. (2011). Gram negative outer membrane vesicles in vaccine development. Discov. Med., 12(62):7-15
  5. Ellis, T.N., Kuehn, M.J. (2010). Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol. Mol Biol R., 74(1): 81–94
  6. Gamazo, C., Moriyon, I. (1987). Release of outer membrane fragments by exponentially growing Brucella melitensis cells. Infec Immun., 55(3) 609–615.
  7. Gamazo, C., Winter, A.J., Moriyon, I., Riezu-Boj, J.I., Blasco, J.M., Diaz, R. (1989). Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains of Brucella ovis and Brucella melitensis. Infec Immun., 57(5):1419–1426.
  8. Jain, L., Rawat, M., Prajapati, A., Tiwari, A.K., Kumar, B., Chaturvedi, V.K., Saxena, H.M., Ramakrishnan, S., Kumar, J., Kerketta, P. (2015). Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544. Biologicals., 3(5): 369-376.
  9. Jain, L., Rawat, M., Ramakrishnan, S., Kumar, B. (2017). Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization. Biologicals., 45:27-32.
  10. Kaparakis-Liaskos, M., Ferrero, R.L. (2015). Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol., 15:375-    387.
  11. Kaur, G., Singh, S., Sunil Kumar, B.V., Mahajan, K., Verma, R. (2016). Characterization and immunogenicity of outer membrane vesicles from Brucella abortus. J. Immunoassay. Immunochem., 37(3):261-72. 
  12. Kimura, A., Toneatto, D., Kleinschmidt, A., Wang, H., Dull, P. (2011). Immunogenicity and safety of a multicomponent meningococcal serogroup B vaccine and a quadrivalent meningococcal CRM197 conjugate vaccine against serogroups A, C, W-135, and Y in adults who are at increased risk for occupational exposure to meningococcal isolates. Clin. Vaccine. Immunol., 18:483-486
  13. Kumar, B., Chaturvedi, V.K., Somrajan, S.R., Kumar, P., Sreedevi, R., Kumar, S., Kaushik, P. (2011). Comparative immune response of purified native OmpH protein derived from Pasteurella multocida P52 and oil adjuvant vaccine against hemorrhagic septicemia in mice. Indian J. Anim. Sci., 81:1193-1196.
  14. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature., 277:680-685.
  15. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol .Chem., 193(1): 265-75.
  16. Mashburn-Warren, L.M., Whiteley, M. (2006). Special delivery: Vesicle trafficking in prokaryotes. Mol.Microbiol., 61(4):839–846.
  17. McBroom, A.J., Kuehn, M.J. (2007). Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol. Microbiol., 63(2):545–558.
  18. Merril, C.R., Dunau, M.L., Goldman, D. (1981). A rapid sensitive silver stain for polypeptides in polyacrylamide gels. Anal Biochem.,110(1):201-7.
  19. Mossman, T. (1983). Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J. Immunol Meth., 65:55-63.
  20. Pasquevich, K.A., Samartino, C.G., Coria, L.M., Estein S.M., Zwerdling, A., Ibanez, A.E., et al. (2010). The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis. J. Immunol. 184(9):5200–5212.
  21. Prados-Rosales, R., Baena, A., Martinez, L.R., Luque-Garcia, J., Kalscheuer, R., Veeraraghavan, U., et al., (2011). Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2 dependent manner in mice. J .Clin. Invest., 121:1471–1483.
  22. Rivera, J., Cordero, R.J.B., Nakouzi, A.S., Frases, S., Nicola, A., Casadevall, A. (2010). Bacillus anthracis produces membrane-    derived vesicles containing biologically active toxins. Proc. Natl. Acad. Sci .USA., 107:19002–19007
  23. Schild, S., Nelson, E.J., Camilli, A. (2008). Immunization with Vibrio cholera outer membrane vesicles induces protective immunity in mice. Infect. Immun.,76:4554-63
  24. Schooling, S.R., Beveridge, T.J. (2006). Membrane vesicles: an overlooked component of the matrices of biofilms. J. Bacteriol., 188:5945–5957
  25. Siadat, S.D., Shirdast, H., Aghasadeghi, M.R., Norouzian, D., Atyabi, S.M., Sadat, S.M., et al., (2010). Determination of cytokine profile following the immunization with Brucella abortus S99 lipopolysaccharide Neisseria meningitidis serogroup B outer membrane vesicle complex in animal model. J. Ardabil. Univ. Med. Sci., 10:223-32 
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Indian Journal of Animal Research
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    Research Article

    volume 52 issue 9 (september 2018) : 1317-1323,   Doi: 10.18805/ijar.B-3428

    Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice

    L
    Losa Rose
    B
    Bablu kumar
    A
    Ankita Jain
    M
    M.K. Singh
    A
    Abhishek
    A
    Arockiasamy Arun Prince Milton
    1Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, Uttar Pradesh, India
    Cite article:- Rose Losa, kumar Bablu, Jain Ankita, Singh M.K., Abhishek, Milton Prince Arun Arockiasamy (2018). Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice. Indian Journal of Animal Research. 52(9): 1317-1323. doi: 10.18805/ijar.B-3428.

    ABSTRACT

    Outer membrane vesicles (OMVs) contain biologically active proteins, lipoolysaccharide (LPS), periplasmic and membrane-bound proteins and are known to perform diverse biological functions. OMVs from Brucella abortus S19 were isolated and characterized by transmission electron microscopy (TEM), SDS-PAGE and immunoreactivity was investigated by western blotting. On TEM, bilayered spherical structures of 50-200 nm were observed. SDS-PAGE of OMVs revealed approximate bands size of 82 kDa, 68 kDa, 38 kDa, 32 kDa, 29 kDa and 18 kDa. Western blot analysis of OMVs revealed a dominant immunoreactive band of 38 kDa that correspond to some major outer membrane proteins. Humoral immune response was measured by indirect ELISA which showed that OMV specific antibodies were detected from 7th day post immunization (DPI) onwards and showed a rising trend up to 35th DPI. Cell mediated immune (CMI) response against OMVs as evidenced by the proliferation of splenocytes have also been observed. Thus OMVs were found to possess immunogenic proteins which had potential to induce both humoral as well as cell mediated immunity. After correlating this immune response with protection it has been concluded that OMV can be used as one of the vaccine candidate against brucellosis.

    REFERENCES

    1. Avila-Calder´on, E.D., Lopez-Merino, A., Jain, N., Peralta, H., Lopez-Villegas, E.O., Sriranganathan, N., et al., (2012) Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice. Clin. Dev Immunol ., 352493.
    2. Choi-kim, K., Maheswaran, S.K., Felice, L.J., Molitor, T.W. (1991). Relationship between iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida. Vet. Microbiol., 28:75-92
    3. Colligan, J.E., Kruisbeek, A.H., Margulies, B.H., Shevoch, E.M., Strobee, W. (1994). Current protocols in Immunology Vol. 1 Current protocols., John Wiley and Sons, INC, USA
    4. Collins, B.S. (2011). Gram negative outer membrane vesicles in vaccine development. Discov. Med., 12(62):7-15
    5. Ellis, T.N., Kuehn, M.J. (2010). Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol. Mol Biol R., 74(1): 81–94
    6. Gamazo, C., Moriyon, I. (1987). Release of outer membrane fragments by exponentially growing Brucella melitensis cells. Infec Immun., 55(3) 609–615.
    7. Gamazo, C., Winter, A.J., Moriyon, I., Riezu-Boj, J.I., Blasco, J.M., Diaz, R. (1989). Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains of Brucella ovis and Brucella melitensis. Infec Immun., 57(5):1419–1426.
    8. Jain, L., Rawat, M., Prajapati, A., Tiwari, A.K., Kumar, B., Chaturvedi, V.K., Saxena, H.M., Ramakrishnan, S., Kumar, J., Kerketta, P. (2015). Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544. Biologicals., 3(5): 369-376.
    9. Jain, L., Rawat, M., Ramakrishnan, S., Kumar, B. (2017). Active immunization with Brucella abortus S19 phage lysate elicits serum IgG that protects guinea pigs against virulent B. abortus and protects mice by passive immunization. Biologicals., 45:27-32.
    10. Kaparakis-Liaskos, M., Ferrero, R.L. (2015). Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol., 15:375-    387.
    11. Kaur, G., Singh, S., Sunil Kumar, B.V., Mahajan, K., Verma, R. (2016). Characterization and immunogenicity of outer membrane vesicles from Brucella abortus. J. Immunoassay. Immunochem., 37(3):261-72. 
    12. Kimura, A., Toneatto, D., Kleinschmidt, A., Wang, H., Dull, P. (2011). Immunogenicity and safety of a multicomponent meningococcal serogroup B vaccine and a quadrivalent meningococcal CRM197 conjugate vaccine against serogroups A, C, W-135, and Y in adults who are at increased risk for occupational exposure to meningococcal isolates. Clin. Vaccine. Immunol., 18:483-486
    13. Kumar, B., Chaturvedi, V.K., Somrajan, S.R., Kumar, P., Sreedevi, R., Kumar, S., Kaushik, P. (2011). Comparative immune response of purified native OmpH protein derived from Pasteurella multocida P52 and oil adjuvant vaccine against hemorrhagic septicemia in mice. Indian J. Anim. Sci., 81:1193-1196.
    14. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature., 277:680-685.
    15. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol .Chem., 193(1): 265-75.
    16. Mashburn-Warren, L.M., Whiteley, M. (2006). Special delivery: Vesicle trafficking in prokaryotes. Mol.Microbiol., 61(4):839–846.
    17. McBroom, A.J., Kuehn, M.J. (2007). Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol. Microbiol., 63(2):545–558.
    18. Merril, C.R., Dunau, M.L., Goldman, D. (1981). A rapid sensitive silver stain for polypeptides in polyacrylamide gels. Anal Biochem.,110(1):201-7.
    19. Mossman, T. (1983). Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J. Immunol Meth., 65:55-63.
    20. Pasquevich, K.A., Samartino, C.G., Coria, L.M., Estein S.M., Zwerdling, A., Ibanez, A.E., et al. (2010). The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis. J. Immunol. 184(9):5200–5212.
    21. Prados-Rosales, R., Baena, A., Martinez, L.R., Luque-Garcia, J., Kalscheuer, R., Veeraraghavan, U., et al., (2011). Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2 dependent manner in mice. J .Clin. Invest., 121:1471–1483.
    22. Rivera, J., Cordero, R.J.B., Nakouzi, A.S., Frases, S., Nicola, A., Casadevall, A. (2010). Bacillus anthracis produces membrane-    derived vesicles containing biologically active toxins. Proc. Natl. Acad. Sci .USA., 107:19002–19007
    23. Schild, S., Nelson, E.J., Camilli, A. (2008). Immunization with Vibrio cholera outer membrane vesicles induces protective immunity in mice. Infect. Immun.,76:4554-63
    24. Schooling, S.R., Beveridge, T.J. (2006). Membrane vesicles: an overlooked component of the matrices of biofilms. J. Bacteriol., 188:5945–5957
    25. Siadat, S.D., Shirdast, H., Aghasadeghi, M.R., Norouzian, D., Atyabi, S.M., Sadat, S.M., et al., (2010). Determination of cytokine profile following the immunization with Brucella abortus S99 lipopolysaccharide Neisseria meningitidis serogroup B outer membrane vesicle complex in animal model. J. Ardabil. Univ. Med. Sci., 10:223-32 
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