Inhibitory effect of recombinant BCG containing the fusion gene BFNA on EB virus-positive tumors.

Abstract

To obtain recombinant Bacillus Calmette-Guérin (rBCG) containing an Epstein-Barr virus (EBV) fusion gene that can inhibit EBV-positive cancer, we obtained BZLF1 and EBNA1 cDNA and overlapped these sequences to assemble the fusion gene BFNA. Then, BFNA was inserted into pMV261, and pMV-BFNA was transfected into BCG-competent cells. Western blotting was performed to detect the target fusion protein, specific antibodies were detected in the serum via enzyme-linked immunosorbent assays (ELISAs), and spleen cell-specific cytokines were detected via enzyme-linked immunospot (ELISPOT) assays. Cytotoxic T lymphocyte (CTL) activity, tumor weight, tumor formation time, and mouse survival were determined in EBV-positive tumor cell (NPRC18) cancer models, and flow cytometry was performed to analyze the quantities of CD8⁺ and CD4⁺ T cells in C57BL/6 J mice. Single-factor analysis of variance was performed with SPSS 19.0 to evaluate rBCG inhibition. The molecular weight of the fusion protein was approximately 55.5 kDa. The titer of the antibody in the rBCG group was highly significant (P ≤ 0.01), which delayed tumorigenesis, and the specific killing ability of rBCG targeting the recombinant target protein was increased. Compared with BCG-EBNA1 and BCG-BZLF1, the rBCG with the BFNA fusion gene presented a greater effect on tumors. Flow cytometric analysis revealed that the numbers of CD4⁺ T and CD8⁺ T cells in the blood of the rBCG group were significantly greater than those in the blood of the control group (P < 0.01). The mice injected with rBCG had greater lymphocyte infiltration in the tumor area. Thus, rBCG exerts a notable immune effect in mice and an inhibitory effect on EBV-positive tumor cell cancer models. KEY POINTS: • rBCG with the BFNA fusion gene induced strong immune responses and delayed EBV tumor growth. • rBCG promoted CD4+ and CD8+ T cell infiltration in EBV-positive tumor models. • The BFNA fusion protein effectively increased CTL activity and prevented tumor progression.