Heat Shock Protein Gp96 As A New Adjuvant Enhances T Cell Responses Of BCG Vaccine

Tuberculosis (TB) is second only to AIDS as the greatest killer worldwide due to a single infectious agent. Bacillus Calmette-Guerin (BCG) is a vaccineagainst tuberculosis. Although BCG vaccine is also used in many areas, its efficiency continually declines. Its protective effect appears to vary in different area or at different age. About 0 to 80% of immunized individuals could be protected from tuberculosis. So its immunization efficiency needs to be improved.Our previous study showed that heat shock protein gp96 can active T cells by presenting antigens. It also has natural adjuvant function. By insulting many studies, we found some studies that lactoferrin as an adjuvant effects protective efficiency of BCG vaccine. Lactoferrin has a low expression and difficult of separation, so there are not mass produced. Inspired by these studies, we investigated the effect of gp96 on specific T cell response of BCG by immunizing C57BL/6 mice with gp96 adjuvant and BCG. Our results provide new insights into improving efficiency of BCG immunity. In this study, we firstly used gp96 pFastBacTM1 vector we do have to express gp96 protein. Then we purified gp96and identified it by SDS-PAGE and Western Blot. The result suggested that the purity of gp96 is about 95%.By ELISPOT (enzyme linked immunospot assay), IFN-y intracellular staining, we showed that immunization with gp96 induced significant increase about 50% of BCG specific cell immunity. Then according to ELISA (enzyme linked immunosorbent assay), analyzing inflammatory cytokines IFN-y, IL-2, TNF-a, IL12, IL-10, we concluded that gp96 immunization promotes Thl type cytokines secretion and suppresses suppressive cytokine production.Further, we explored the role of gp96 in immune protection against BCG infection. Three groups of mice were immunized with PBS (negative control), BCG alone or BCG plus gp96 for three times at weeks 1,2 and 4, respectively. The mice from each group were challenged with BCG by intrapulmonary dose at week 5 and then were sacrificed after 48 hours. Lung tissues were collected, ground and coated onto plates. The result showed that the live tuberculosis number in the lung of the group vaccinated with BCG and gp96 decreased about 36% than that vaccinated with BCG alone. H&E staining assay showed that mice immunized with BCG and gp96 exhibited a decrease in lung injury. These results above suggested that heat shock protein gp96 immunization efficiently suppresses lung impairment from tuberculosis. At last, three groups of mice were immunized for three times at weeks 1,2 and 4, respectively and were challenged with BCG on the day 7 after immunization. During the next 18 days, the mice vaccinated with BCG demonstrated significantly decreased weight than those vaccinated with BCG and gp96.In conclusion, heat shock protein gp96 as a new adjuvant enhances T cell responses of BCG vaccine. Our results therefore provide new insights into improving efficiency of BCG vaccine immunity, and a novel strategy for designing new generation of gp96 adjuvant vaccine on clinic.