The Journal of Experimental Medicine
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© The Rockefeller University Press, 0022-1007/2000/2/551/ $5.00
The Journal of Experimental Medicine, Volume 191, Number 3, February 7, 2000 551-560

Original Article

Expression Cloning of an Immunodominant Family of Mycobacterium tuberculosis Antigens Using Human CD4+ T Cells

Mark R. Aldersona, Teresa Bementa, Craig H. Dayb, Liqing Zhua, David Moleshb, Yasir A. W. Skeikyb, Rhea Colerc, David M. Lewinsohnc,d, Steven G. Reedb,c, and Davin C. Dillonb
a Department of Immunology, Corixa Corporation, Seattle, Washington 98104
b Department of Antigen Discovery, Corixa Corporation, Seattle, Washington 98104
c Infectious Disease Research Institute, Seattle, Washington 98104
d Division of Pulmonary and Critical Care Medicine, University of Washington, Seattle, Washington 98104

Correspondence to: Mark R. Alderson, Department of Immunology, Corixa Corporation, 1124 Columbia St., Suite 200, Seattle, WA 98104. Tel:206-754-5744 Fax:206-754-5715 E-mail:alderson{at}corixa.com.

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.

Key Words: Mycobacterium tuberculosis, intracellular pathogens, antigen presentation, interferon {gamma}, expression cloning


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