Construction of Salmonella Pullorum ghost by co-expression of lysis gene E and the antimicrobial peptide SMAP29 and evaluation of its immune efficacy in specific-pathogen-free chicks
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摘要
In this study,a safety enhanced Salmonella Pullorum(S.Pullorum)ghost was constructed using an antimicrobial peptide gene,and evaluated for its potential as a Pullorum disease(PD)vaccine candidate.The antimicrobial peptide SMAP29 was co-expressed with lysis gene E to generate S.Pullorum ghosts.No viable bacteria were detectable either in the fermentation culture after induction of gene E-and SMAP29-mediated lysis for 24 h or in the lyophilized ghost products.Specific-pathogenfree(SPF)chicks were intraperitoneally immunized with ghosts at day 7 of age and no mortality,clinical symptoms or signs of PD such as anorexia,depression and diarrhea were observed.On challenge with a virulent S.Pullorum strain at 4 wk post-immunization,a comparatively higher level of protection was observed in the S.Pullorum ghost immunized chickens with a minimum of pathological lesions and bacterial loads compared to the birds in inactivated vaccine groups.In addition,immunization with the S.Pullorum ghosts induced a potent systemic Ig G response and was associated with significantly increased levels of cytokine IFN-γand IL-4 and relative percentages of CD4~+ and CD8~+ T lymphocytes.Our results indicate that SMAP29 can be employed as a new secondary lethal protein to enhance the safety of bacterial ghosts,and to prepare a non-living bacterial vaccine candidate that can prevent PD in chickens.
In this study,a safety enhanced Salmonella Pullorum(S.Pullorum)ghost was constructed using an antimicrobial peptide gene,and evaluated for its potential as a Pullorum disease(PD)vaccine candidate.The antimicrobial peptide SMAP29 was co-expressed with lysis gene E to generate S.Pullorum ghosts.No viable bacteria were detectable either in the fermentation culture after induction of gene E-and SMAP29-mediated lysis for 24 h or in the lyophilized ghost products.Specific-pathogenfree(SPF)chicks were intraperitoneally immunized with ghosts at day 7 of age and no mortality,clinical symptoms or signs of PD such as anorexia,depression and diarrhea were observed.On challenge with a virulent S.Pullorum strain at 4 wk post-immunization,a comparatively higher level of protection was observed in the S.Pullorum ghost immunized chickens with a minimum of pathological lesions and bacterial loads compared to the birds in inactivated vaccine groups.In addition,immunization with the S.Pullorum ghosts induced a potent systemic Ig G response and was associated with significantly increased levels of cytokine IFN-γand IL-4 and relative percentages of CD4~+ and CD8~+ T lymphocytes.Our results indicate that SMAP29 can be employed as a new secondary lethal protein to enhance the safety of bacterial ghosts,and to prepare a non-living bacterial vaccine candidate that can prevent PD in chickens.
In this study,a safety enhanced Salmonella Pullorum(S.Pullorum)ghost was constructed using an antimicrobial peptide gene,and evaluated for its potential as a Pullorum disease(PD)vaccine candidate.The antimicrobial peptide SMAP29 was co-expressed with lysis gene E to generate S.Pullorum ghosts.No viable bacteria were detectable either in the fermentation culture after induction of gene E-and SMAP29-mediated lysis for 24 h or in the lyophilized ghost products.Specific-pathogenfree(SPF)chicks were intraperitoneally immunized with ghosts at day 7 of age and no mortality,clinical symptoms or signs of PD such as anorexia,depression and diarrhea were observed.On challenge with a virulent S.Pullorum strain at 4 wk post-immunization,a comparatively higher level of protection was observed in the S.Pullorum ghost immunized chickens with a minimum of pathological lesions and bacterial loads compared to the birds in inactivated vaccine groups.In addition,immunization with the S.Pullorum ghosts induced a potent systemic Ig G response and was associated with significantly increased levels of cytokine IFN-γand IL-4 and relative percentages of CD4~+ and CD8~+ T lymphocytes.Our results indicate that SMAP29 can be employed as a new secondary lethal protein to enhance the safety of bacterial ghosts,and to prepare a non-living bacterial vaccine candidate that can prevent PD in chickens.
引文
Barrow P A,Freitas Neto O C.2011.Pullorum disease and fowl typhoid-new thoughts on old diseases:A review.Avian Pathology,40,1-13.
Brogden K A.2005.Antimicrobial peptides:Pore formers or metabolic inhibitors in bacteria?Nature Reviews Microbiology,3,238-250.
Chappell L,Kaiser P,Barrow P,Jones M A,Johnston C,Wigley P.2009.The immunobiology of avian systemic salmonellosis.Veterinary Immunology and Immunopathology,128,53-59.
Chaudhari A A,Jawale C V,Kim S W,Lee J H.2012.Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens.Veterinary Research,43,44.
Chong C,Bost K L,Clements J D.1996.Differential production of interleukin-12 m RNA by murine macrophages in response to viable or killed Salmonella spp.Infection and Immunity,64,1154-1160.
Flynn J L,Goldstein M M,Triebold K J,Koller B,Bloom B R.1992.Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection.Proceedings of the National Academy of Sciences of the United States of America,89,12013-12017.
Gómez-Verduzco G,Tellez G,Quintana A L,Isibasi A,OrtizNavarrete V.2010.Humoral immune response in breeding hens and protective immunity provided by administration of purified Salmonella Gallinarum porins.Poultry Science,89,495-500.
Haidinger W,Mayr U B,Szostak M P,Resch S,Lubitz W.2003.Escherichia coli ghost production by expression of lysis gene E and staphylococcal nuclease.Applied and Environmental Microbiology,69,6106-6113.
Harty J T,Bevan M J.1992.CD8+T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo.Journal of Experimental Medicine,175,1531-1538.
Hensel A,Huter V,Katinger A,Raza P,Strnistschie C,Roesler U,Brand E,Lubitz W.2000.Intramuscular immunization with genetically inactivated(ghosts)Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state.Vaccine,18,2945-2955.
Jalava K,Hensel A,Szostak M P,Resch S,Lubitz W.2002.Bacterial ghosts as vaccine candidates for veterinary applications.Journal of Controlled Release,85,17-25.
Jawale C V,Chaudhari A A,Lee J H.2014.Generation of a safety enhanced Salmonella Gallinarum ghost using antibiotic resistance free plasmid and its potential as an effective inactivated vaccine candidate against fowl typhoid.Vaccine,32,1093-1099.
Jechlinger W,Glocker J,Haidinger W,Matis A,Szostak M P,Lubitz W.2005.Modulation of gene expression by promoter mutants of the lambda c I857/p RM/p R system.Journal of Biotechnology,116,11-20.
Kwon S R,Kang Y J,Lee D J,Lee E H,Nam Y K,Kim S K,Kim K H.2009.Generation of Vibrio anguillarum ghost by coexpression of Phi X 174 lysis E gene and staphylococcal nuclease A gene.Molecular Biotechnology,42,154-159.
Li Q,Hu Y,Chen J,Liu Z,Han J,Sun L,Jiao X.2013.Identification of Salmonella enterica serovar Pullorum antigenic determinants expressed in vivo.Infection and Immunity,81,3119-3127.
Lo W F,Ong H,Metcalf E S,Soloski M J.1999.T cell responses to Gram-negative intracellular bacterial pathogens:A role for CD8+T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules.Journal of Immunology,162,5398-5406.
Lu S,Manges A R,Xu Y,Fang F C,Riley L W.1999.Analysis of virulence of clinical isolates of Salmonella enteritidis in vivo and in vitro.Infection and Immunity,67,5651-5657.
Lundin B S,Johansson C,Svennerholm A M.2002.Oral immunization with a Salmonella enterica serovar typhi vaccine induces specific circulating mucosa-homing CD4+and CD8+T cells in humans.Infection and Immunity,70,5622-5627.
Mader H J,Szostak M P,Hensel A,Lubitz W,Haslberger A G.1997.Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines.Vaccine,15,195-202.
Mayr U B,Haller C,Haidinger W,Atrasheuskaya A,Bukin E,Lubitz W,Ignatyev G.2005.Bacterial ghosts as an oral vaccine:A single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge.Infection and Immunity,73,4810-4817.
Mittrucker H W,Kaufmann S H.2000.Immune response to infection with Salmonella typhimurium in mice.Journal of Leukocyte Biology,67,457-463.
Nguyen L T,Haney E F,Vogel H J.2011.The expanding scope of antimicrobial peptide structures and their modes of action.Trends in Biotechnology,29,464-472.
Panthel K,Jechlinger W,Matis A,Rohde M,Szostak M P,Lubitz W,Haas R.2003.Generation of Helicobacter pylori ghosts by Phi X protein E-mediated inactivation and their evaluation as vaccine candidates.Infection and Immunity,71,109-116.
Peng W,Si W,Yin L,Liu H,Yu S,Liu S,Wang C,Chang Y,Zhang Z,Hu S,Du Y.2011.Salmonella enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks.Immunobiology,216,558-565.
Salerno-Goncalves R,Pasetti M F,Sztein M B.2002.Characterization of CD8+effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine.Journal of Immunology,169,2196-2203.
Sancho P,Tejedor C,Sidhu-Munoz R S,Fernandez-Lago L,Vizcaino N.2014.Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B.ovis infection.Veterinary Research,45,61.
Shivaprasad H L.2000.Fowl typhoid and pullorum disease.Revue Scientifique et Technique-Office International des Epizooties,19,405-424.
Si W,Yu S,Chen L,Wang X,Zhang W,Liu S,Li G.2014.Passive protection against Salmonella enterica serovar Enteritidis infection from maternally derived antibodies of hens vaccinated with a ghost vaccine.Research in Veterinary Science,97,191-193.
Sood S,Rishi P,Dhawan V,Sharma S,Ganguly N K.2005.Protection mediated by antibodies to iron-regulated outermembrane proteins of S.typhi in a mouse peritonitis model.Molecular and Cellular Biochemistry,273,69-78.
Szostak M P,Hensel A,Eko F O,Klein R,Auer T,Mader H,Haslberger A,Bunka S,Wanner G,Lubitz W.1996.Bacterial ghosts:Non-living candidate vaccines.Journal of Biotechnology,44,161-170.
Sztein M B,Tanner M K,Polotsky Y,Orenstein J M,Levine M M.1995.Cytotoxic T lymphocytes after oral immunization with attenuated vaccine strains of Salmonella typhi in humans.Journal of Immunology,155,3987-3993.
Tabrizi C A,Walcher P,Mayr U B,Stiedl T,Binder M,Mc Grath J,Lubitz W.2004.Bacterial ghosts--biological particles as delivery systems for antigens,nucleic acids and drugs.Current Opinion in Biotechnology,15,530-537.
White D W,Harty J T.1998.Perforin-deficient CD8+Tcells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-γbut requires TNF-α.Journal of Immunology,160,898-905.
Wigley P,Berchieri A,Page K L,Smith A L,Barrow P A.2001.Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent,disease-free carriage in chickens.Infection and Immunity,69,7873-7879.
Won G,Chaudhari A A,Lee J H.2016.Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated Salmonella Gallinarum vaccine candidate.Clinical and Experimental Vaccine Research,5,148-158.
Yu S,Zhao H,Wang H,Wang X,Shao G,Xu L,Si W,Chen L,Zhang W,Liu S.2013.Production and characterization of mouse monoclonal antibodies against lysis protein E of phi X174.Journal of Virological Methods,189,355-361.
Yu S Y,Peng W,Si W,Yin L,Liu S G,Liu H F,Zhao H L,Wang CL,Chang Y H,Lin Y Z.2011.Enhancement of bacteriolysis of shuffled phage Phi X174 gene E.Virology Journal,8,doi:10.1186/1743-422X-8-206
Zhu J,Yamane H,Paul W E.2010.Differentiation of effector CD4 T cell populations.Annual Review of Immunology,28,445-489.
Brogden K A.2005.Antimicrobial peptides:Pore formers or metabolic inhibitors in bacteria?Nature Reviews Microbiology,3,238-250.
Chappell L,Kaiser P,Barrow P,Jones M A,Johnston C,Wigley P.2009.The immunobiology of avian systemic salmonellosis.Veterinary Immunology and Immunopathology,128,53-59.
Chaudhari A A,Jawale C V,Kim S W,Lee J H.2012.Construction of a Salmonella Gallinarum ghost as a novel inactivated vaccine candidate and its protective efficacy against fowl typhoid in chickens.Veterinary Research,43,44.
Chong C,Bost K L,Clements J D.1996.Differential production of interleukin-12 m RNA by murine macrophages in response to viable or killed Salmonella spp.Infection and Immunity,64,1154-1160.
Flynn J L,Goldstein M M,Triebold K J,Koller B,Bloom B R.1992.Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection.Proceedings of the National Academy of Sciences of the United States of America,89,12013-12017.
Gómez-Verduzco G,Tellez G,Quintana A L,Isibasi A,OrtizNavarrete V.2010.Humoral immune response in breeding hens and protective immunity provided by administration of purified Salmonella Gallinarum porins.Poultry Science,89,495-500.
Haidinger W,Mayr U B,Szostak M P,Resch S,Lubitz W.2003.Escherichia coli ghost production by expression of lysis gene E and staphylococcal nuclease.Applied and Environmental Microbiology,69,6106-6113.
Harty J T,Bevan M J.1992.CD8+T cells specific for a single nonamer epitope of Listeria monocytogenes are protective in vivo.Journal of Experimental Medicine,175,1531-1538.
Hensel A,Huter V,Katinger A,Raza P,Strnistschie C,Roesler U,Brand E,Lubitz W.2000.Intramuscular immunization with genetically inactivated(ghosts)Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state.Vaccine,18,2945-2955.
Jalava K,Hensel A,Szostak M P,Resch S,Lubitz W.2002.Bacterial ghosts as vaccine candidates for veterinary applications.Journal of Controlled Release,85,17-25.
Jawale C V,Chaudhari A A,Lee J H.2014.Generation of a safety enhanced Salmonella Gallinarum ghost using antibiotic resistance free plasmid and its potential as an effective inactivated vaccine candidate against fowl typhoid.Vaccine,32,1093-1099.
Jechlinger W,Glocker J,Haidinger W,Matis A,Szostak M P,Lubitz W.2005.Modulation of gene expression by promoter mutants of the lambda c I857/p RM/p R system.Journal of Biotechnology,116,11-20.
Kwon S R,Kang Y J,Lee D J,Lee E H,Nam Y K,Kim S K,Kim K H.2009.Generation of Vibrio anguillarum ghost by coexpression of Phi X 174 lysis E gene and staphylococcal nuclease A gene.Molecular Biotechnology,42,154-159.
Li Q,Hu Y,Chen J,Liu Z,Han J,Sun L,Jiao X.2013.Identification of Salmonella enterica serovar Pullorum antigenic determinants expressed in vivo.Infection and Immunity,81,3119-3127.
Lo W F,Ong H,Metcalf E S,Soloski M J.1999.T cell responses to Gram-negative intracellular bacterial pathogens:A role for CD8+T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules.Journal of Immunology,162,5398-5406.
Lu S,Manges A R,Xu Y,Fang F C,Riley L W.1999.Analysis of virulence of clinical isolates of Salmonella enteritidis in vivo and in vitro.Infection and Immunity,67,5651-5657.
Lundin B S,Johansson C,Svennerholm A M.2002.Oral immunization with a Salmonella enterica serovar typhi vaccine induces specific circulating mucosa-homing CD4+and CD8+T cells in humans.Infection and Immunity,70,5622-5627.
Mader H J,Szostak M P,Hensel A,Lubitz W,Haslberger A G.1997.Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines.Vaccine,15,195-202.
Mayr U B,Haller C,Haidinger W,Atrasheuskaya A,Bukin E,Lubitz W,Ignatyev G.2005.Bacterial ghosts as an oral vaccine:A single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge.Infection and Immunity,73,4810-4817.
Mittrucker H W,Kaufmann S H.2000.Immune response to infection with Salmonella typhimurium in mice.Journal of Leukocyte Biology,67,457-463.
Nguyen L T,Haney E F,Vogel H J.2011.The expanding scope of antimicrobial peptide structures and their modes of action.Trends in Biotechnology,29,464-472.
Panthel K,Jechlinger W,Matis A,Rohde M,Szostak M P,Lubitz W,Haas R.2003.Generation of Helicobacter pylori ghosts by Phi X protein E-mediated inactivation and their evaluation as vaccine candidates.Infection and Immunity,71,109-116.
Peng W,Si W,Yin L,Liu H,Yu S,Liu S,Wang C,Chang Y,Zhang Z,Hu S,Du Y.2011.Salmonella enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks.Immunobiology,216,558-565.
Salerno-Goncalves R,Pasetti M F,Sztein M B.2002.Characterization of CD8+effector T cell responses in volunteers immunized with Salmonella enterica serovar Typhi strain Ty21a typhoid vaccine.Journal of Immunology,169,2196-2203.
Sancho P,Tejedor C,Sidhu-Munoz R S,Fernandez-Lago L,Vizcaino N.2014.Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B.ovis infection.Veterinary Research,45,61.
Shivaprasad H L.2000.Fowl typhoid and pullorum disease.Revue Scientifique et Technique-Office International des Epizooties,19,405-424.
Si W,Yu S,Chen L,Wang X,Zhang W,Liu S,Li G.2014.Passive protection against Salmonella enterica serovar Enteritidis infection from maternally derived antibodies of hens vaccinated with a ghost vaccine.Research in Veterinary Science,97,191-193.
Sood S,Rishi P,Dhawan V,Sharma S,Ganguly N K.2005.Protection mediated by antibodies to iron-regulated outermembrane proteins of S.typhi in a mouse peritonitis model.Molecular and Cellular Biochemistry,273,69-78.
Szostak M P,Hensel A,Eko F O,Klein R,Auer T,Mader H,Haslberger A,Bunka S,Wanner G,Lubitz W.1996.Bacterial ghosts:Non-living candidate vaccines.Journal of Biotechnology,44,161-170.
Sztein M B,Tanner M K,Polotsky Y,Orenstein J M,Levine M M.1995.Cytotoxic T lymphocytes after oral immunization with attenuated vaccine strains of Salmonella typhi in humans.Journal of Immunology,155,3987-3993.
Tabrizi C A,Walcher P,Mayr U B,Stiedl T,Binder M,Mc Grath J,Lubitz W.2004.Bacterial ghosts--biological particles as delivery systems for antigens,nucleic acids and drugs.Current Opinion in Biotechnology,15,530-537.
White D W,Harty J T.1998.Perforin-deficient CD8+Tcells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-γbut requires TNF-α.Journal of Immunology,160,898-905.
Wigley P,Berchieri A,Page K L,Smith A L,Barrow P A.2001.Salmonella enterica serovar Pullorum persists in splenic macrophages and in the reproductive tract during persistent,disease-free carriage in chickens.Infection and Immunity,69,7873-7879.
Won G,Chaudhari A A,Lee J H.2016.Protective efficacy and immune responses by homologous prime-booster immunizations of a novel inactivated Salmonella Gallinarum vaccine candidate.Clinical and Experimental Vaccine Research,5,148-158.
Yu S,Zhao H,Wang H,Wang X,Shao G,Xu L,Si W,Chen L,Zhang W,Liu S.2013.Production and characterization of mouse monoclonal antibodies against lysis protein E of phi X174.Journal of Virological Methods,189,355-361.
Yu S Y,Peng W,Si W,Yin L,Liu S G,Liu H F,Zhao H L,Wang CL,Chang Y H,Lin Y Z.2011.Enhancement of bacteriolysis of shuffled phage Phi X174 gene E.Virology Journal,8,doi:10.1186/1743-422X-8-206
Zhu J,Yamane H,Paul W E.2010.Differentiation of effector CD4 T cell populations.Annual Review of Immunology,28,445-489.