[Dimo-thylidioctyl ammonium bromide-BCG polysaccharide nucleic acid adjuvant enhanced the immunogenicity of a Mycobacterium tuberculosis subunit vaccine].

OBJECTIVE: To investigate the activity of a novel adjuvant consisting of dimethyldioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG-PSN). METHODS: BCG-PSN was extracted by hot-phenol method, and combined with DDA and Mycobacterium tuberculosis fusion antigen AMM (Ag85B-MPT64(190-198)-Mtb8.4) to formulate the Mycobacterium tuberculosis subunit vaccine. Mice were immunized subcutaneously with a 2-week interval between the immunizations (0.2 ml/dose), and humoral and cell-mediated immunity were detected by ELISA and ELISPOT respectively. RESULTS: With the stimulation of Ag85B in vitro, the number of antigen specific IFN-gamma producing spleen lymphocytes were 222 +/- 79, 259 +/- 85, 230 +/- 64 per million respectively in the mice immunized with AMM + DDA + BCG-PSN, AMM + DDA, and BCG. Spleen lymphocytes in these 3 groups produced higher levels of IFN-gamma compared to the groups with the adjuvant of IFA or BCG-PSN alone or without adjuvant upon stimulation with Ag85B (t = 2.923-7.118, P < 0.05). Furthermore, the adjuvant consisting of DDA and BCG-PSN increased the ratio of Ig(2a)/IgG1 than DDA alone (0.125 vs. 0.025). Combined with AMM, the adjuvant DDA and the one consisting of DDA and BCG-PSN induced higher level of immunity than incomplete Freund's adjuvant (IFA), NaCl, and BCG-PSN alone. CONCLUSION: Mycobacterium tuberculosis subunit vaccine AMM + DDA + BCG-PSN induced a strong Th1-type immune response, and DDA + BCG-PSN, especially DDA promoted the immune response of the M. tuberculosis subunit vaccine in mice.

Evaluation of a recombinant BCG expressing antigen Ag85B and PPE protein Rv3425 from DNA segment RD11 of Mycobacterium tuberculosis in C57BL/6 mice.

Antigen 85B (Ag85B) is an important immunodominant antigen of Mycobacterium tuberculosis, and is a very promising vaccine candidate molecule. Rv3425 is a member of the subgroup 3 of the PPE family, which does not exist in all BCG strains. In this study we constructed a new rBCG which included this united gene (Ag85B-Rv3425). The level of antigen-stimulated T cells expressing IFN-gamma was significantly higher in the C57BL/6 mice vaccinated with rBCG::Ag85B-Rv3425 than with BCG. In addition, the sera from mice immunized with rBCG::Ag85B-Rv3425 revealed an increase in the specific immunoglobulin G titers than that from mice immunized with BCG. Antigen specific IgG subclass analysis showed that rBCG::Ag85B-Rv3425 tended to facilitate IgG2a production, suggesting enhancement of predominant Th1 response which in turn may facilitate increased production of protective IFN-gamma. These results suggested that this rBCG::Ag85B-Rv3425 could be a strong vaccine candidate for further study.

Chitosan microspheres enhance the immunogenicity of an Ag85B-based fusion protein containing multiple T-cell epitopes of Mycobacterium tuberculosis.

To develop novel delivery system for tuberculosis (TB) subunit vaccine, biodegradable chitosan microspheres were prepared and used to deliver a fusion protein, Ag85B-MPT64(190-198)-Mtb8.4 (AMM for short), made from three Mycobacterium tuberculosis genes. AMM-loaded microspheres were first characterized for their morphology, size, zeta potential, loading efficiency, and in vitro release of AMM. C57BL/6 mice were immunized at weeks 1, 3 and 5 subcutaneously with AMM formulated in chitosan microspheres, in incomplete Freund's adjuvant (IFA), or in phosphate-buffered saline (PBS), respectively. Three weeks after the last immunization, humoral and cell-mediated immune responses were examined. It was shown that the microspheres bound AMM quite efficiently (loading efficiency: >99%). AMM-loaded chitosan microspheres were observed as aggregated shapes with the average particle size of 5.78+/-0.65 microm and zeta potential of 32.77+/-1.51 mV. In vitro release studies revealed that only small amount of antigen was released in 16 days. Following subcutaneous administration, splenocytes immunized with AMM in chitosan microspheres produced higher levels of IFN-gamma compared to administration of AMM in PBS upon stimulation with Ag85B and synthetic peptide MPT64(190-198). The levels of Ag85B-specific IgG (H+L), IgG1 and IgG2a in sera of mice immunized with AMM in chitosan microspheres were also higher than those with AMM in PBS. These results indicate that chitosan microspheres when used as a carrier for fusion protein AMM could elicit strong humoral and cell-mediated immune responses.