Multi-subunit BCG booster vaccine GamTBvac: Assessment of immunogenicity and protective efficacy in murine and guinea pig TB models

Abstract

New innovative vaccines are highly needed to combat the global threat posed by tuberculosis. Efficient components-antigens and adjuvants-are crucial for development of modern recombinant TB vaccines. This study describes a new vaccine (GamTBvac) consisting of two mycobacterial antigen fusions (Ag85A and ESAT6-CFP10)-with dextran-binding domain immobilized on dextran and mixed with an adjuvant consisting of DEAE-dextran core, and with CpG oligodeoxynucleotides (TLR9 agonists). GamTBvac and its components were assessed for immunogenicity and protective efficacy in GamTBvac-prime/boost and BCG-prime/ GamTBvac-boost in murine and guinea pig TB models. Results show that in both infectious models, GamTBvac has a strong immunogenicity and significant protective effect against Mycobacterium tuberculosis strain H37Rv under aerosol and intravenous challenges. GamTBvac showed a particularly strong protective effect as a BCG booster vaccine.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Expression and purification of GamTBvac components.

Two antigen fusion proteins DBD-ESAT6-CFP10 and DBD-Ag85A (A) were induced by IPTG in E. coli BL21. Coomassie blue staining was used for evaluation of inclusion bodies washing results (B) and column purification process (C); M—molecular weight marker, IB—inclusion bodies, W1-2 –wash of IB with 25 mM Tris pH 7.5 + 50 mM NaCl, W3 –wash of IB with 25 mM Tris pH 7.5 + 50 mM NaCl + 25%EtOH, U– 8M urea wash fractions, T—tail water elution fraction, P—peak water elution fraction, E—elution fractions. Both recombinant fusions were checked by western blotting (D) with rabbit antisera (for rAg85A) or antigen specific monoclonal antibodies (rESAT6 and rCFP10)

Fig 2. Immunogenicity of GamTBvac components in GamTBvac-prime/boost and BCG-prime/GamTBvac-boost regimens.

The popliteal lymph node (A, C) and spleen (B, D) cells (0.5–8×105/well) of the vaccinated mice were stimulated with DBD-Ag85A (10 μg/ml; A, B), DBD-ESAT6-CFP10 (10 μg/ml; C, D) separately in vitro and number of IFNγ secreting cells was assessed by ELISPOT. To estimate the lymphoproliferative potential of vaccine components, the popliteal lymph node cells of the vaccinated mice (2–4×105 cells/well) were stimulated with DBD-Ag85A (10 μg/ml), DBD-ESAT6-CFP10 (10 μg/ml) or PBS separately in vitro (E) five weeks after the final immunization with specific antigen composition. Total IFNγ secretion in splenocytes upon different vaccination regimens was estimated by ELISA (F). To evaluate the antigen-specific antibody titer, sera were collected from each mouse one week after last immunization. Endpoint IgG titers specific to ESAT6, CFP10 and Ag85a were detected by ELISA. Sera from mice treated with PBS or adjuvant (without antigen) were used as a negative control (G). Results were presented as group means ± 95%CIs, n = 4, * P<0.05, ** P<0.01. In graphs F and C asterisk indicate significant differences compare to both PBS and BCG groups.

Fig 3. Protective efficacy of GamTBvac against M. tuberculosis H37Rv infection in murine and guinea pig models.

The mice were immunized subcutaneously with GamTBvac and BCG (5×106 CFU) in different regimens. BCG was administered once at the 1st week and GamTBvac twice (three weeks apart). The control group was injected with PBS. For the acute murine model, the mice were retro-orbitally infected with MTB H37Rv two weeks after the last immunization. The results presented are given as mean Log10 CFU per organ ± 95%CI from groups of seven mice (n = 7), * P<0.05, relative to PBS; ** P<0.05, relative to both PBS and BCG. Thirty days after infection, protective efficacy was measured (n = 6) in lungs (A) and spleens (B). For the aerosol murine model, the mice were infected in aerosol infection chamber to get 103 CFU per animal sixty days after the last immunization. Four weeks later, bacterial load (n = 6) was determined in lungs (C) and spleens (D). Aerosol infection (n = 15) was monitored for more than 400 days until all mice died (E).