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Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.

Alderson MR, Bement T, Day CH, Zhu L, Molesh D, Skeiky YA, Coler R, Lewinsohn DM, Reed SG, Dillon DC - J. Exp. Med. (2000)

Bottom Line: A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C.Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A.The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Corixa Corporation, Seattle, Washington 98104, USA. alderson@corixa.com

ABSTRACT
Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

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Southern blot analysis of Mtb9.9 genes. Genomic DNA from various mycobacterial strains was digested with PstI, separated by agarose gel electrophoresis, and blotted on Nytran®. The mtb9.9a gene was labeled with 32P and used as a probe. Size markers (M) are in kb.
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Figure 5: Southern blot analysis of Mtb9.9 genes. Genomic DNA from various mycobacterial strains was digested with PstI, separated by agarose gel electrophoresis, and blotted on Nytran®. The mtb9.9a gene was labeled with 32P and used as a probe. Size markers (M) are in kb.

Mentions: To further characterize the mtb9.9 gene family, Southern blot analysis was performed on a variety of mycobacterial strains using the mtb9.9a gene as a probe (Fig. 5). The data demonstrated the presence of a highly related gene family containing between three and five members within different isolates of Mtb, including two from clinical sources. The data also indicate that these genes are well conserved in M. bovis BCG, but significantly less conserved or absent in all other mycobacterial species tested.


Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.

Alderson MR, Bement T, Day CH, Zhu L, Molesh D, Skeiky YA, Coler R, Lewinsohn DM, Reed SG, Dillon DC - J. Exp. Med. (2000)

Southern blot analysis of Mtb9.9 genes. Genomic DNA from various mycobacterial strains was digested with PstI, separated by agarose gel electrophoresis, and blotted on Nytran®. The mtb9.9a gene was labeled with 32P and used as a probe. Size markers (M) are in kb.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195809&req=5

Figure 5: Southern blot analysis of Mtb9.9 genes. Genomic DNA from various mycobacterial strains was digested with PstI, separated by agarose gel electrophoresis, and blotted on Nytran®. The mtb9.9a gene was labeled with 32P and used as a probe. Size markers (M) are in kb.
Mentions: To further characterize the mtb9.9 gene family, Southern blot analysis was performed on a variety of mycobacterial strains using the mtb9.9a gene as a probe (Fig. 5). The data demonstrated the presence of a highly related gene family containing between three and five members within different isolates of Mtb, including two from clinical sources. The data also indicate that these genes are well conserved in M. bovis BCG, but significantly less conserved or absent in all other mycobacterial species tested.

Bottom Line: A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C.Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A.The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Corixa Corporation, Seattle, Washington 98104, USA. alderson@corixa.com

ABSTRACT
Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

Show MeSH
Related in: MedlinePlus