| Tuberculosis is still a serious health problem around the world. Even in some developed countries it is rising with AIDS, immigration, poverty and other social problems. The epidemic and drug resistance situation of tuberculosis in our country is very afraid. The fifth national tuberculosis epidemiological surveillance in 2011 showed that there are about 1.3 million new TB infections in China every year, which accounts for 14.3% of the global incidence, ranking second place in the world. The lack of effective vaccines to prevent the transmission of tuberculosis probably is the main reason of failing to control the epidemics of tuberculosis.BCG, the only available vaccine against tuberculosis, can protect children and infants well. However, its effects on adult are variable. There is an urgent need to develop a new vaccine to improve or replace BCG. Considering the present situation of tuberculosis vaccine, new vaccines should have characteristics that are able to establish long-term immune response in vivo. Because vesicular stomatitis virus(VSV) owns some of unique advantages, such as safety and rare production of anti VSV antibodies in human population, it becomes an ideal vector to deliver antigens for vaccine development.The triple antigen fusion gene p846 and its individual antigen Rv3615 c, Mtb10.4 and Rv2660 c were obtained by PCR and cloned into vector VSV-XN2, respectively. Using VSV reverse genetic system, recombinant VSV virus were generated expressing specific antigens in BHK cells. At the same time, we constructed the prokaryotic expression vectors pGEX-5X-1-Rv3615 c, pGEX-5X-1-Mtb10.4, pGEX-5X-1-Rv2660 c and pet28a-p846, and purified the Rv3615 c, Mtb10.4, Rv2660 c and TFP846 proteins from Escherichia coli. The immunization protocol was shown as following: 6 to 8-week-old female BalB/c mice were randomly divided and administrated with recombinant VSVs. The group of mice receiving BCG was the positive control. The splenic lymphocytes were isolated and stimulated with corresponding antigens in vitro two weeks after the final immunization. The cellular immune response was then detected. The infection course was followed up to 24 weeks and the protective role of VSV-846 to resistant tuberculosis infection was evaluated. Moreover, the potential mechanism that how recombinant VSV perform as delivery vector was also explored.Our results indicated that VSV-846 vaccine could elicit more strong cellular immune response as well as humoral immune responses compared with that of viral vaccines expressing its individual antigen. Importantly, VSV-846 also showed the better protection than BCG 24 weeks post final immunization. Measurements of cell proliferation by Brdu, cytotoxcity by LDH and lung injury by immunohistochemistry, demonstrated that the triple antigen fusion vaccine VSV-846 trigger more strong T cell responses, especially the memory T cell response. Meanwhile, we examined the potential of VSV-846 in priming-boostering strategy. Results showed that the bacteria load in VSV-846 boosting group was significant lower than that of BCG group by reduced CFU 1.10 log10(P<0.01), indicating the feasible of this method. Collectively, the novel recombinant VSV-846 vaccine we generated could induce both strong cellular and humoral immune response. It also could efficiently promote effector memory T cells production, which may benefit the long-term protection of BalB/c mice against BCG challenge. |
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