The Ag85A signal peptide from M. tuberculosis H37Rv was used to construct a recombinant secretory BCG (Bacillus Chalmette-Guérin) plasmid for the development of safe and effective EBV (Epstein-Barr virus) vaccines. The Ag85A gene was inserted into the pMV261 vector and fused to the EBV LMP2A (latent membrane protein) and hGM-CSF (human granulocyte/macrophage colony-stimulating factor) genes (secretory BCG plasmid). Western blot analysis was used to determine the levels of expression of the hGM-CSF and LMP2A proteins in rBCG (recombinant BCG). A series of experiments were conducted to determine humoral immunity, cellular immunity, and antitumor effects.
After transformation, the recombinant pMVGCA plasmid effectively expressed GCA in BCG, and the rBCG proteins were recognized by antibodies against hGM-CSF and LMP2A. The maximum dose of rBCG resulted in antibody titers of 1:19,800 and 1:21,800 six weeks after immunization. Specific lysis was maximal and approximately two times stronger in mice immunized with the control when the effector:target ratio was 40:1. When compared to the control groups, tumorigenicity was lower in the rBCG treatment group, with a tumor inhibition rate of 0.81 0.09. rBCG expressing a hGM-CSF and LMP2A fusion protein inhibits EB virus-positive tumors.