A novel DNA vaccine for protective immunity against virulent Mycobacterium bovis in mice

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB). The proteins Ag85B, MPB64, and ESAT-6 are the major immunogenic antigens of M. bovis; these proteins play important roles in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed various DNA vaccines with the genes encoding the three antigens mentioned above. This procedure involved the following steps: fusion of two genes (pcDNA-MPB64-Ag85B, pcMA), fusion of three genes (pcDNA-MPB64-Ag85B-ESAT-6, pcMAE), bivalent combinations (pcDNA-Ag85B + pcDNA-MPB64, pcA + M), and trivalent combinations (pcDNA-Ag85B + pcDNA-MPB64 + pcDNA-ESAT-6, pcA + M + E). The immune response to the DNA vaccines was evaluated based on serum antibody titers, lymphocyte proliferation assay, and titers of the cytokines interferon-γ (IFN-γ) and interleukin-2 (IL-2). The protective efficacy following challenge with a virulent M. bovis strain, C68001, was evaluated based on survival rate, bacterial loads in lung tissue, and histopathologic changes. A significant 2-fold increase in serum antibody levels was observed in mice vaccinated with fusion DNA (two or three genes). Furthermore, the lymphocyte proliferation (SI) values and the levels of IFN-γ and IL-2 were higher in mice vaccinated with fusion DNA (two or three genes) than in those immunized with polyvalent combination DNA vaccines (P < 0.05). Additionally, the fusion DNA vaccines provided protection that was superior to that provided by the polyvalent combination DNA vaccines following challenge with M. bovis strain C68001. The protective efficacy of the fusion DNA vaccines in mice immunized three times was equivalent to the protective efficacy in mice immunized once with the Bacillus Calmette–Guerin (BCG) vaccine. This suggests that fusion DNA vaccine represent a promising approach for the prevention of bTB.