The Influence Of DDA-based Different Adjuvant On The Protective Effect Of A Tuberculosis Subunit Vaccine AMM

Tuberculosis is one of the leading causes of mortality from an infectious disease worldwide. Mycobacterium bovis Bacilli Calmette-Guerin (BCG) is the only vaccine currently available against tuberculosis(TB). BCG is an attenuated, strain of M. bovis that was derived from a virulent strain at the start of the last century, after more than 13 years of continuous in vitro passage. Over the past years, BCG vaccine has maintained its position as the world's most widely used vaccine, despite showing highly variable efficacy in different places. The efficacy of BCG in adults is particularly poor. Accordingly, a new safe and effective vaccine to prevent tuberculosis is necessary to combat this disease. The subunit protein vaccine of these new development vaccines is one of the most promising new vaccines, because subunit vaccines have the advantage of defined composition and easy reception.The current vaccine strategy included priming with BCG or BCG replacement vaccine and boosting with subunit vaccine. In addition, subunit vaccine can enhance the protective efficiency of BCG.Our previous study showed that a fusion protein Ag85B-MPT64190-198-Mtb8.4 (AMM) elicited strong humoral and cell-mediated immune responses in mice in combination with dimethyl dioctadecyl ammonium bromide (DDA) plus BCG polysaccharide nucleic acid (BCG PSN). To compare the effect of differet adjuvants in immunity and protection, we investigated the mix of DDA and one or two kind of immunostimulation components including BCG polysaccharide nucleic acid (BCG PSN), trehalose 6,6-dimycolate (TDM), and polyriboinosinic:polyribocytidylic acid (Poly I:C), which were combined with a Mycobacterium tuberculosis fusion antigen AMM to boost BCG primed immunity.1. DDA and BCG polysaccharide nucleic acid improved the immunogenicity and protective efficacy of tuberculosis subunit vaccine AMM against Mycobacterium tuberculosis infection in mice Objective:To investigate the adjuvant character of DDA or DDA-BCG polysaccharide nucleic acid (BCG PSN), which was combined with a Mycobacterium tuberculosis fusion antigen AMM (Ag85B-MPT64190-198-Mtb8.4) to boost BCG primed immunity.Methods:DDA with or without BCG PSN was mixed with the M. tuberculosis fusion protein AMM to construct subunit vaccine. Mice were immunized with BCG and then boosted twice with AMM formulated with the adjuvant DDA with or without BCG PSN. The mice were vaccinated respectively with phosphate buffered saline (PBS) or BCG without boosting as control. The humoral and cell-mediated immune responses were analyzed at the 4 weeks after the last immunization by ELISA and ELISPOT. In addition, the immunized mice were challenged twelve weeks after the last immunization by i.v. injection of H37Rv strain. Six weeks after the infection, the load of M. tuberculosis in mice and the pathological sections were detected, then the protective efficacy of BCG priming and the AMM subunit vaccine in different adjuvants boosting against Mycobacterium tuberculosis infection was analyzed.Results:With the stimulation of Ag85B and PPD, the number of IFN-γsecreting cells from the mice boosted twice by AMM/DDA/BCG PSN and AMM/DDA were higher than BCG and PBS group (p<0.05). The colony-forming units(CFU) in lungs of mice boosted with AMM/DDA was less than BCG and PBS group (p<0.05), while the CFU in spleens of mice boosted with AMM/DDA/BCG PSN was less than BCG and PBS group (p<0.05). In addition, the mice immunized by BCG and boosted with AMM/DDA/BCG PSN presented fewer lesions in lungs than PBS control, and there is great individual variation in lesions of mice boosted with AMM/DDA.Conclusion:DDA is an idea adjuvant for tuberculosis subunit vaccine; BCG PSN might play a role to alleviate the pathology induced by stronger immunity.2. The comparative study of the adjuvant efficacy of DDA-based different adjuvants in tuberculosis subunit vaccine at the BCG-prime and subunit vaccine-boost strategy.Objective:To investigate the effect of adjuvant containing TDM, PolyI:C, BCG PSN, and cationic liposomes(DDA), which was combined with a Mycobacterium tuberculosis fusion antigen AMM(Ag85B-MPT64190-198-Mtb8.4) to boost BCG-primed immunity.Methods:The mice were grouped based on the fusion antigen AMM in DDA mixed with different adjuvants, including AMM in DDA alone (A/D), AMM in DDA plus TDM(A/D/T), AMM in DDA plus TDM and Poly I:C(A/D/T/P), AMM in DDA plus PolyI:C(A/D/P), AMM in DDA plus BCG PSN(A/D/B), and BCG and PBS vaccine as control. The experimental groups were boosted with the AMM subunit vaccines in different adjuvants in 12th week and 14th week respectively after primed with BCG. The humoral and cell-mediated immune responses were analyzed at the four weeks after boosting by ELISA and ELISPOT. Then, H37Rv strain challenge was performed ten weeks after the boost immunizations. The body weight (mean) of each group(10 mice) were recorded weekly after the challenge. Six weeks after the infection, the load of M. tuberculosis in mice and the pathological sections were detected. The immunology and protective efficacy of different adjuvants in tuberculosis subunit vaccine were analyzed. Results:Mice primed with BCG vaccination followed by boosting with A/D/T and A/D/T/P produced stronger antigen-specific humoral and gamma interferon immune response compared with BCG alone. The CFU in lungs of mice boosted with fusion protein AMM in different adjuvant were less than PBS controls, while the mice primed with BCG and boosted with the subunit vaccine comprised with BCG PSN or PolyI:C in DDA presented fewer lesions in lungs than BCG and BCG prime-AMM in DDA alone boost group (P<0.05); The average weight of mice vaccined with different vaccine were monitored before and after challenging with M. tuberculosis H37Rv, and the weight of mice immunited with PBS decreased obviously, while other groups gained weight during the infection period, the weight of mice in the groups boosting with A/D/T/P and A/D/B increased significantly.Conclusion:The adjuvants Poly I:C and BCG PSN plus DDA combined with AMM to boost BCG primed immunity might alleviate the pathology induced by M. tuberculosis infection in mice, which might cause by some unknown immunologic mechanism.