ISOLATION OF RECOMBINANT PHAGE CLONES EXPRESSING MYCOBACTERIAL T-CELLANTIGENS BY SCREENING A RECOMBINANT-DNA LIBRARY WITH HUMAN CD4(+) TH1CLONES

A lambda g11 recombinant DNA library of Mycobacterium leprae ac was sc reened to isolate recombinant phage clones expressing mycobacterial an tigens important for T cell reactivity. The library was plated on a la wn of Escherichia coli Y1090 and recombinant antigens were expressed f rom isolated phage clones in 96-well plates. Pools of recombinant anti gens from 12 wells were tested in T cell proliferation assays with MHC class II restricted human CD4(+) Th1 clones secreting interferon-gamm a and cytotoxic for antigen pulsed antigen presenting cells. By screen ing 1750 pools of recombinant antigens with a mixture of eight Th1 clo nes, we identified two recombinant phage clones that expressed recombi nant mycobacterial antigens stimulatory for T cells. MHC restriction a nalysis and reactivity to a battery of mycobacterial antigens suggeste d that the two responding Th1 clones recognized mycobacterial antigens /epitopes with different MHC class II(HLA-DR) restriction requirements . Our results suggest that the methodology described in this paper is suited to isolate recombinant phage clones expressing mycobacterial re combinant antigens stimulatory for T cells of protective phenotype, Su ch antigens may be useful in designing new vaccines and diagnostic rea gents against mycobacterial diseases. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All righ ts reserved.