Mycobacterial Lipid Components Enhanced The Protective Efficacy Of Tuberculosis Subunit Vaccine

Objective:Mycobacterial cell wall is rich in lipid components, and some of them have antigenicity or play an important role in the process of immune regulation. This experiment aimed at screening effective lipids against TB and investigating the application of lipids from mycobacterial cell wall in tuberculosis subunit vaccine.Methods:We extracted total lipids from Bacille Calmette-Guerin (BCG) cell wall and separated them with silica column into three groups:the nonpolar lipids, the intermediate polarity lipids and the polar lipids. Enzyme linked immunosorbent assay (ELISA) analyzed the immunogenicity of three groups of lipid in the population. Enzyme-linked immunosorbent spot (ELISPOT) assayed the immune reactivity of T cells in the population to lipid components. Furthermore, total lipids and the three groups of lipid were mixed with fusion protein LT70 (ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c) in adjuvant of N, N’-dimethyl-N,N’-dioctadecylammonium bromide (DDA) and Polyinosinic-polycytidylic acid (Poly I:C), respectively. The protective efficacy, including in the enhanced efficacy after BCG prime, of the subunit vaccines containing lipid were evaluated in mouse model and rabbit skin model. For vaccine containing lipid prime strategy, mice were immunized three times with subunit vaccine containing different groups of lipid at Ost,3rd, and 6th week. For BCG prime-vaccine containing lipid boost strategy, mice were primed by BCG at the Ost week and boosted twice with vaccine containing different groups of lipid at the 9st and the 12th week. Mice vaccinated PBS or BCG as the control groups. After the last injected weeks 5, all mice were challenged nasally with 2.5×106 CFU of BCG After the infected weeks 3, the CFU in mice lung were measured. In rabbit skin liquefaction model, the rabbits skin were immunized three times with subunit vaccine containing different groups of lipid at the Ost,3rd, and 6th week, respectively. Rabbits vaccinated with PBS or BCG as the control groups. After the last injected weeks 6, all rabbits were injected intradermally at two sites on each flank with 5×106 CFU of BCG From then on, the lesions were observed every day on their sizes and the liquefaction and healing process. We recorded the time when the lesions formed granulomas, time of ulceration, time of liquefaction peak, onset of healing and time of the lesions healed. When the lesions at the stage of liquefaction peak, liquefied caseum were collected and the number of tubercle bacilli within the liquefied caseum was determined.Results:The result showed that the level of lipid-specific IgG in TB patients were significantly higher than that of healthy people (P<0.05). For the protective efficacy, the number of bacteria harbored in mice immunized with LT70-total lipids apparently declined contrast to BCG vaccination (P<0.05). In BCG prime-subunit vaccine boost strategy, LT70-total lipids and LT70-intermediate polarity lipids vaccine resulted in significantly low bacterial number in them than in BCG group (P<0.01) and LT70 group (P<0.05). For the protective efficacy in rabbit skin model, the number of bacteria at peak of liquefaction in LT70-total lipids group and LT70-intermediate polarity lipids group declined mildly contrast to LT70 group (P>0.05). Results of the observation of lesions showed that lipids could accelerate the liquefaction and healing process of tubercle following BCG challenge. BCG and LT70-total lipids induced skin lesions came to the stages of ulceration and the liquefaction peak earlier than other groups. In BCG group, the day of lesion begining liquefaction was the 3rd day after BCG challenge and the day lesion reached their liquefaction peak was from the 6th to 8th day after BCG challenge. We found that the maximum size of the lesion was at the time of liquefaction peak. The lesion size at the peak of liquefaction in BCG group was 368.19±78.31 mm3, the largest in all groups. The liquefaction and healing process of tubercle in LT70-total lipids group follow on BCG group, the day of lesion begining liquefaction was the 4th day after BCG challenge and the day lesion reached their liquefaction peak was from the 5th to 8th day after BCG challenge, the lesion size at the peak of liquefaction was 332.77±61.19 mm3. The progress of tubercle in single total lipids adjuvant group and LT70-intermediate polarity lipids group were mediate, the day lesion reached their liquefaction peak was from the 7th to 10th and the 10th to 17th day respectively after BCG challenge. The day LT70 group and PBS group reached their stage of liquefaction peak were from the 11 th to 15th day and the 13th to 17th day respectively after BCG challenge.Conclusion:In conclusion, lipids from mycobacterial cell wall have high immunogenicity and accelerate the liquefaction and healing process of tubercle following BCG challenge; LT70 combining with, especially, the total lipids or intermediate polarity lipids can enhance the protective efficacy of subunit vaccine.