RECOMBINANT MYCOBACTERIUM-BOVIS BCG SECRETING FUNCTIONAL INTERLEUKIN-2 ENHANCES GAMMA-INTERFERON PRODUCTION BY SPLENOCYTES

Mycobacterium bovis BCG was genetically engineered to express and secr ete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the contro l of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) wi th the mycobacterial alpha-antigen secretion signal sequence at the am ino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequenc e and the epitope tag. When expressed with the alpha-antigen signal se quence, biologically active IL-2 was secreted into the extracellular m edium. Western blot (immunoblot) analysis of the intracellular IL-2 an d extracellular IL-2 revealed that the secretion signal was appropriat ely cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a s plenocyte population, mouse splenocytes were cultured together with wi ld-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated su bstantial increases in gamma interferon production, which could be rep roduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-1 0, tumor necrosis factor alpha, and granulocyte-macrophage colony-stim ulating factor were not significantly changed, while IL-4 and IL-5 rem ained undetectable (less than 50 pg/ml). The enhanced production of ga mma interferon in response to IL-2-secreting BCG was strain independen t. Recombinant BCG expressing mammalian cytokines provides a novel mea ns to deliver cytokines and may augment the immunostimulatory properti es of BCG in immunization and cancer therapy.