Isolation of recombinant phage clones expressing mycobacterial T cell antigens by screening a recombinant DNA library with human CD4+ Th1 clones.
A lambda gt11 recombinant DNA library of Mycobacterium leprae was screened to isolate recombinant phage clones expressing mycobacterial antigens important for T cell reactivity. The library was plated on a lawn of Escherichia coli Y1090 and recombinant antigens were expressed from isolated phage clones in 96-well plates. Pools of recombinant antigens from 12 wells were tested in T cell proliferation assays with MHC class II restricted human CD4+ Th1 clones secreting interferon-gamma and cytotoxic for antigen pulsed antigen presenting cells. By screening 1750 pools of recombinant antigens with a mixture of eight Th1 clones, we identified two recombinant phage clones that expressed recombinant mycobacterial antigens stimulatory for T cells. MHC restriction analysis and reactivity to a battery of mycobacterial antigens suggested 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 recombinant antigens stimulatory for T cells of protective phenotype. Such antigens may be useful in designing new vaccines and diagnostic reagents against mycobacterial diseases.
Mustafa
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Department of Microbiology, Faculty of Medicine, Kuwait University, Safat. abusalim@hsc.kuniv.edu.kw
Lundin
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Oftung
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D000938:Antigen-Presenting Cells / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002999:Clone Cells; D015723:Gene Library; D006801:Humans; D008213:Lymphocyte Activation; D008242:Lysogeny; D009160:Mycobacteriophages / Q000235:genetics* / Q000378:metabolism; D009161:Mycobacterium / Q000276:immunology*; D009166:Mycobacterium leprae / Q000276:immunology / Q000821:virology; D011994:Recombinant Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D018417:Th1 Cells / Q000276:immunology*
D000942:Antigens, Bacterial; D011994:Recombinant Proteins