title: Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.
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.
authors.affiliation: Department of Immunology, Corixa Corporation, Seattle, Washington 98104, USA. alderson@corixa.com
authors.forename: M R
authors.initials: MR
authors.lastname: Alderson
authors.forename: T
authors.initials: T
authors.lastname: Bement
authors.forename: C H
authors.initials: CH
authors.lastname: Day
authors.forename: L
authors.initials: L
authors.lastname: Zhu
authors.forename: D
authors.initials: D
authors.lastname: Molesh
authors.forename: Y A
authors.initials: YA
authors.lastname: Skeiky
authors.forename: R
authors.initials: R
authors.lastname: Coler
authors.forename: D M
authors.initials: DM
authors.lastname: Lewinsohn
authors.forename: S G
authors.initials: SG
authors.lastname: Reed
authors.forename: D C
authors.initials: DC
authors.lastname: Dillon
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001428:Bacterial Vaccines; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D001428:Bacterial Vaccines