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-gamma (IFN-gamma) 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-gamma 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.

A novel fusion protein-based indirect enzyme-linked immunosorbent assay for the detection of bovine tuberculosis.

Enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine tuberculosis has been widely explored over the years. Three Mycobacterium bovis-specific antigen genes, namely, mpb70, mpb83, and esat-6 were recombined in tandem by spliced overlap extension technology and expressed in Escherichia coli to obtain the fusion protein (rM70-83-E6). Western blot analysis showed that rM70-83-E6 can specifically react with bovine tuberculosis-positive sera but not those from cattle infected with other bovine diseases such as bovine paratuberculosis. An indirect ELISA (iELISA) method was established with rM70-83-E6 as the diagnostic antigen. The diagnostic criteria were determined using 150 serum samples from healthy cattle. Analyses of 85 serum samples from cattle with bovine tuberculosis and 100 serum samples from healthy cattle demonstrated that the sensitivity of the iELISA was 69.4% (59/85) and the specificity was 96.0% (96/100). Moreover, 46 out of 67 purified protein derivative (PPD) skin test-positive samples were also positive by iELISA, giving a positive coincidence of 68.7%, while all 50 PPD skin test-negative samples were negative by iELISA, giving a negative coincidence of 100%. The total coincidence between iELISA and the PPD skin test was 82.1%. This study demonstrated that iELISA using rM70-83-E6 antigen is simple, sensitive and easy to perform and can be used to analysis of a large number of samples for serodiagnosis of bovine tubercuiosis.

The protective immune response induced by B cell epitope of classical swine fever virus glycoprotein E2.

Classical swine fever virus (CSFV) envelope glycoprotein E2 is a major protective immunogen responsible for eliciting neutralizing antibodies and conferring protective immunity against the virus. Based on the core sequence (TAVSPTTLR, 829-837 aa) of the B cell linear epitope of the CSFV E2 protein identified by Lin et al., two oligonucleotides MF and MR were synthesized and used to construct by PCR a gene cassette encoding a 15 amino acid polypeptide M (CTAVSPTTLRTEVVK), which spans 828-842 amino acids of E2. The gene cassette was fused in-frame to 3' terminal of glutathione S transferase gene (GST) of the prokaryotic expression vector pGEX-6p-1, resulting in the recombinant plasmid pGEX-M. After transformation into Escherichia coli BL21 a soluble fusion protein GST-M with expected size of 28 kDa was expressed after inducing with isopropyl-beta-d-thiogalactoside (IPTG). Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the purified GST-M had good reactivity with swine anti-CSFV serum and rabbit anti-CSFV E2 serum. Further vaccination trials showed that the fusion protein GST-M could elicit effectively immune response protecting rabbits and pigs from virulent challenge. This study showed a possibility for developing epitope-based vaccines against CSFV.