Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice

( views:92, downloads:0 )
Author:
WANG Li-mei()
SHI Chang-hong()
FAN Xiong-lin()
XUE Ying()
BAI Yin-lai()
XU Zhi-kai()
Journal Title:
CHINESE MEDICAL JOURNAL
Issue:
Volume 120, Issue 14, 2007
DOI:
Key Word:
mycobacterium tuberculosis;Bacillus Calmette-Guérin;ESAT-6;vaccine

Abstract: Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.Methods Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 μl normal saline containing 106 CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses.Results There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P<0.05). The elicited IFN-γ level of rBCG group was (1993±106) pg/ml, which was also significantly higher than that in BCG group ((1463±105) pg/ml, P<0.05). The splenocyte proliferation index of rBCG group reached 4.34±0.31, which was higher than that of BCG group (3.79±0.24, P<0.05).Conclusion rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.

  • [1]Colditz GA,Brewer TF,Berkey CS,Wilson ME,Burdick E,Fineberg HV,et al.Efficacy of BCG vaccine in the prevention of tuberculosis.Meta-analysis of the published literature.JAMA 1994; 271:698-702.
  • [2]Girard MP,Fruth U,Kieny MP.A review of vaccine research and development:tuberculosis.Vaccine 2005; 23:5725-5731.
  • [3]Ohara N,Yamada T.Recombinant BCG vaccines.Vaccine 2001; 19:4089-4098.
  • [4]Hanson MS,Lapcevich CV,Haun SL.Progress on development of the live BCG recombinant vaccine vehicle for combined vaccine delivery.Ann N Y Acad Sci 1995; 754:214-221.
  • [5]Collins DM.New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex.Immunol Cell Biol 2000; 78:342-348.
  • [6]Mahairas GG,Sabo PJ,Hickey MJ,Singh DC,Stover CK.Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M.bovis.J Bacteriol 1996; 178:1274-1282.
  • [7]Behr MA,Wilson MA,Gill WP,Salamon H,Schoolnik GK,Rane S,et al.Comparative genomics of BCG vaccines by whole-genome DNA microarray.Science 1999; 284:1520-1523.
  • [8]Sorensen AL,Nagai S,Houen G,Andersen P,Andersen AB.Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.Infect Immun 1995; 63:1710-1717.
  • [9]Harboe M,Oettinger I,Wiker HG,Rosenkrands I,Andersen P.Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG.Infect Immun 1996; 64:16-22.
  • [10]Andersen P,Andersen AB,Sorensen AL,Nagai S.Recall of long-lived immunity to Mycobacterium tuberculosis infection in mice.J Immunol 1995; 154:3359-3372.
  • [11]Pollock JM,Andersen P.Predominant recognition of the ESAT-6 protein in the first phase of interferon with Mycobacterium bovis in cattle.Infect Immun 1997; 65:2587-2592.
  • [12]Mustafa AS,Amoudy HA,Wiker HG,Abal AT,Ravn P,Offung F,et al.Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis.Scand J Immunol 1998; 48:535-543.
  • [13]Ulrichs T,Munk ME,Mollenkopf H,Behr-Perst S,Colangeli R,Gennaro ML,et al.Differential T cell responses to Mycobacterium tuberculosis ESAT6 in tuberculosis patients patients and healthy donors.Eur J Immunol 1998; 28:3949-3958.
  • [14]Sambrook J,Fritisch EF,Maniatis T.Molecular cloning:a laboratory manual,2nd ed.New York:Cold Spring Harbor Laboratory Press,Cold Spring Harbor; 1989:16-66.
  • [15]Parish T,Stoker NG.Mycobacteria Protocols.New Jersey:Humana Press; 1998:129-145.
  • [16]Shi JH,Zhao YT,Wang JL,Han W,Yan Z,ZhangYQ.Analysis of low molecular peptides by sodium dodecylsulfate-polyacrylamide gel electrophoresis.J Fourth Mil Med Univ (Chin) 2000; 21:761-763.
  • [17]Andersen P,Askgaard D,Ljungqvist L,Bentzon MW,Heron I.T cell proliferative response to antigens secreted by Mycobacterium tuberculosis.Infect Immun 1991; 59:1558-1563.
  • [18]Fine PE.Variation in protection by BCG:implications of and for heterologous immunity.Lancet 1995; 346:1339-1345.
  • [19]Brandt L,Cunha JF,Olsen AW,Chilima B,Hirsch P,Appelberg R,et al.Failure of the Mycobacterium bovis BCG vaccine:some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis.Infect Immun 2002; 70:672-678.
  • [20]Dhar N,Rao V,Tyagi AK.Recombinant BCG approach for development of vaccines:cloning and expression of immunodominant antigens of M.tuberculosis.FEMS Microbiol Lett 2000; 190:309-316.
  • [21]Skjot RLV,Oettinger T,Rosenkrands I,Ravn P,Brock I,Jacobsen S,et al.Comparative evaluation of Low-Molecular-Mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-Cell antigens.Infect Immun 2000; 68:214-220.
  • [22]Brandt L,Oettinger T,Holm A,Andersen AB,Andersen P.Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis.J Immunol 1996; 157:3527-3533.
  • [23]Shi CH,Xu ZK,Zhu DS,Li Y,Bai YL,Xue Y.Screening and construction of recombinant BCG strains expressing the Ag85B-ESAT6 fusion protein.Chin J Tuberc Respir Dis (Chin) 2005; 28:254-257.
  • [24]Bonecini-Almeida MG,Chitale S,Boutsikakis I,Geng JY,Doo H,He SH,et al.Induction of in vitro human macrophage anti-mycobacterium tuberculosis activity:requirement for IFN-γ and primed lymphocytes.J Immunol 1998; 160:4490-4498.
  • [25]Flynn JL,Chan J,Triebold KJ,Dalton DK,Stewart TA,Bloom BR.An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection.J Exp Med 1993; 178:2249-2254.
  • [26]Renshaw PS,Panagiotidou P,Whelan A,Gordon SV,Hewinson RG,Williamson RA,et al.Conclusive evidence that the major T-cell antigens of the mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight,1:1 complex and characterization of the structural properties of ESAT-6,CFP-10,and the ESAT-6 CFP-10 complex.Implications for pathogenesis and virulence.J Biol Chem 2002; 277:21598-21603.
WanfangData CO.,Ltd All Rights Reserved
About WanfangData | Contact US
Healthcare Department, Fuxing Road NO.15, Haidian District Beijing, 100038 P.R.China
Tel:+86-010-58882616 Fax:+86-010-58882615 Email:yiyao@wanfangdata.com.cn