Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.
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.
Alderson
M R
MR
Department of Immunology, Corixa Corporation, Seattle, Washington 98104, USA. alderson@corixa.com
Bement
T
T
Day
C H
CH
Zhu
L
L
Molesh
D
D
Skeiky
Y A
YA
Coler
R
R
Lewinsohn
D M
DM
Reed
S G
SG
Dillon
D C
DC
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*
D000942:Antigens, Bacterial; D001428:Bacterial Vaccines