Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes

Abstract

Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2. Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter. To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag. When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium. Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion. To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG. IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG. Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml). The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent. Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.

Mycobacterium bovis bacillus Calmette–Guérin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo

“…MV261 BCG (BCG), a Pasteur strain transfected previously with the kanamycin resistance plasmid pMV261 [27], was used in the in vitro experiments. This strain has demonstrated the similar immunostimulatory property to that of commercial lyophilized BCG preparations.…”

RecombinantMycobacterium bovisbacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity

“…Individual colonies containing pmIL-18 verified by PCR were picked and grown in Middlebrook 7H9 medium as described in Reagents . Construction of BCG Pasteur containing control vector (MV261 BCG) was described previously [39].…”

Immunological Properties of Recombinant <italic>Mycobacterium bovis</italic> Bacillus Calmette-Guérin Strain Expressing Fusion Protein IL-2-ESAT-6

“…Making the BCG strain expressing the immunodominant or specific antigens, such as Ag85B [2], Ag85 complex [3], 38 kDa [4], 19 kDa [5], RD1 (region of difference 1) [6], early secreted antigenic target-6 kDa (ESAT-6) [7], and Ag85B-ESAT-6 [8], is a widely used approach. Expressing the Th1-type cytokines, namely interleukin (IL)-2 [9], IL-12 [10], granulocyte macrophage colony stimulating factors (GM-CSFs) [10], IL-18 [11], and IFN-γ [12] is the other modality. It was reported that two recombinant BCG strains expressing antigen Ag85B or RD1 resulted in enhanced protective efficacy.…”