A Study On The Anti-tuberculosis Of TAT-Ag85B Vaccine Combined With T-bet Adjuvants

ObjectivesTo construct a novel anti-tuberculosis complex vaccine which consists of TAT-Ag85B protein and T-bet adjuvant. Then, a number of mice were immunized with the vaccine to evaluate its immunogenicity and protection. Afterwards, we explore the mechanism of enhanced immune response by the new complex vaccine. Our study also provided a new mentality for the research and development of new TB vaccines and theoretical basis.Methods1. According to the full-length sequence of the target gene Ag85B and TAT-PTD in GenBank, pET-28a-TAT-Ag85B and pET-28a-Ag85B recombinant expression plasmids were constructed. These two recombinant expression plasmids were identified by PCR, and digested by restriction endonucleases and sequencing subsequently.2. Two recombinant expression plasmids pET-28a-Ag85B and pET-28a-TAT-Ag85B were transformed into E.coli BL21(DE3), respectively. These two plasmids express rAg85B and rTAT-Ag85B by the induction of IPTG. The expressed products were analyzed by SDS-PAGE and purified by Ni-IDA His-band resin.3. BALB/c mice were immunized with Ag85B or TAT-Ag85B protein and pcDNA3.1-T-bet. After the last immunization, the anti-Ag85B antibody titers in sera were tested by ELISA. Meanwhile, the mouse spleen lymphocytes were cultured in the context of Ag85B, and then tested the secretion of cytokines in culture fluid by ELISA.4. After the last immunization, mice in all the groups were challenged with MTB H37Rv and the numbers of MTB loads in the spleens and lungs were determined by in vitro colony formation assays. Whereafter, lung tissue pathological were used to evaluate the areas of lung lesion at week8after the infection.5. Abdominal macrophage in mice were stimulated with Ag85B and TAT-Ag85B, respectively. Distribution of Ag85B in macrophage were assayed by immunofluorescence. Next, contents of Ag85B in macrophage were detected by western blot and the expression of CD80/CD86were evaluated by flow cytometry. Results1. PCR, double digestion with restriction endonucleases and sequencing confirmed that the recombinant plasmids pET-28a-TAT-Ag85B and pET-28a-Ag85B were constructed successfully.2. After induction of IPTG, the recombinant protein rAg85B and rTAT-Ag85B were expressed correctly and successfully. Subsequently, two protein were purified by affinity chromatography.3. ELISA results showed that the anti-Ag85B antibody IgG titer in TAT-Ag85B/T-bet were higher than other groups, in comparison with Ag85B group and TAT-Ag85B group, IgG2a titer was significantly increased in TAT-Ag85B/T-bet group, while IgG1level was obviously decreased. As same as antibodies, the levels of IFN-γ/IL-2were significantly higher in TAT-Ag85B/T-bet group when compared with other groups. While in comparison with Ag85B group and TAT-Ag85B group, significantly lower of IL-4and IL-10were found in TAT-Ag85B/T-bet group.4. At the beginning of week1or week2, in both lungs and spleens, the bacterial loads have no significant differences among the mice with five treatments. At week4, mice treated with the combination of TAT-Ag85B and T-bet have lower loads compared with PBS treated mice (p<0.05). At week8, the group of T-bet/TAT-Ag85B treated mice have lower loads compared with PBS treated mice (p<0.01), or compared with T-bet/Ag85B (p<0.05) mice in both lungs and spleens. To estimate the appearance of lung tissue in each group, the lungs of the mice from the TAT-Ag85B/T-bet group appeared almost normal, with few tubercles present in comparison with PBS group. Consistently, mice that received TAT-Ag85B/T-bet vaccinations were characterized by reduced pathology and inflammation. Little Mycobacterium tuberculosis were found in lungs of the mice from the TAT-Ag85B/T-bet group by acid-fast staining.5. In comparison with Ag85B, TAT-Ag85B in large, interspersed distribution. Importantly, TAT-Ag85B stimulated higher expressions of CD80and CD86on macrophage than Ag85B did.ConclusionThe study preliminarily clarified that the combination of TAT-Ag85B proteins and T-bet genetic adjuvant enhances Ag85B-specific antibody responses, which is dominated by Th1immune response. In addition, it appeared an effective protection in the lung and spleen by immunization following M. tuberculosis infection. Furthermore, the increasement of antigen presenting ability induced by TAT-Ag85B protein may be the possible reason for the strong immune response and protection. Therefore, the findings provide a new practical strategy for the improvement of vaccine candidates.