Comparison of BCG prime-DNA booster and rBCG regimens for protection against tuberculosis

Abstract

Developing an effective adult prophylaxis vaccine is a high priority in the global control of tuberculosis (TB), because TB remains an important public health problem and the current widely used BCG vaccine provides effective protection only for children but variable protection against adult TB. BCG priming-heterologous vaccines booster and recombinant BCG technologies have been thought as two important regimens for inducing effective protection against adult TB. Obviously, defining the protective efficacy of the two regimens would benefit more rational design of the future adult TB vaccines. In this study, a recombinant BCG strain (rBCG::685A) expressing the fusion protein of ESAT-6 and Ag85A (r685A) of Mycobacterium tuberculosis was constructed successfully and the secretion of r685A protein from rBCG strain was confirmed by western blotting with anti-ESAT-6 and anti-Ag85A polyclonal antibodies, respectively. The immune responses and protective effects in rBCG::685A vaccinated C57BL/6 mice were compared with that of our previous reported BCG prime-pcD685A booster regimen. Boosting BCG with pcD685A DNA elicited higher level of r685A protein specific IFN-γ secreted by splenocytes and a more significant increase of both TNF-α and iNOS responses in the lung, thus providing better control of bacterial growth in both lung and spleen of immunized mice challenged with virulent M. tuberculosis, compared with mice vaccinated with rBCG::685A or BCG alone. Our results have implications for development of more effective adult TB vaccines for improved control of TB.

Keywords: DNA vaccine; ESAT-6-Ag85A; prime-boost; recombinant BCG; tuberculosis.

Figure 1. Recombinant Mycobacterium–E.coli shuttle plasmid pM685A and the secretory expression of r685A fusion protein from rBCG::685A. The plasmid pM685A containing the full-fragment of ESAT-6 and Ag85A was engineered to express the secreted r685A protein under the direction of Hsp60 promoter (PHsp60) and α-Ag signal peptide (α-AgSP). The expression of the r685A fusion protein in the culture supernatant from rBCG::685A was confirmed by western blotting with anti-ESAT-6 rabbit antibody or anti-Ag85A chicken polyclonal antibody. The cultural supernatant of BCG was used as negative control.

Figure 2. The level of IFN-γ secreted by splenocytes of different group mice detected by ELISA (n = 5). Nine weeks after immunization, splenocytes were obtained from 5 mice in each group were prepared individually and 10⁶ cells were added into each well of 96-well microtiter plates. PPD (10 μg/ml), r685A protein (5 μg/ml) in complete RPMI-1640 medium were used as the stimulator, respectively. The results were shown as mean ± SD (pg/ml).

Figure 3. mRNA levels of IFN-γ, IL-10, TNF-α, and iNOS in the lung of different vaccinated mice detected by qRT-PCR (n = 5). Tested cDNAs were normalized to the endogenous RNA levels of the internal control GAPDH. The fold increase of gene expression in vaccinated group was calculated using 2^−ΔΔCT method. The results were shown as mean ± SD.

Figure 4. Bacterial load per lung and spleen in C57BL/6 mice (n = 7). Nine weeks later, different vaccinated mice were challenged i.v. with 10⁶ CFU of M. tuberculosis H37Rv. Four weeks after challenge, lungs and spleens were homogenized and cultured for bacterial load. The results were shown as the mean (± SEM) per group.

Figure 5. Representative lung pathology of different vaccinated C57BL/6 mice after infection. Four weeks post-challenge, the right lung from different vaccinated mice was fixed and embedded for HE staining (10 × 4, left row) and acid-fast staining (10 × 40, right row). Arrows indicate acid-fast staining positive bacteria.

Figure 6. Immunization scheme for 2 regimens. For prime-boost regimen, C57BL/6 mice were first immunized s.c. with a dose of 10⁶ CFU of BCG, then 100 μg pcD685A DNA was injected intramuscularly 2 weeks later and repeated twice with 2-week intervals. For recombinant BCG regimen, mice were vaccinated once with 10⁶ CFU of rBCG::685A. PBS was used as negative control. Nine weeks after the immunization, 7 mice were challenged with 10⁶ CFU M. tuberculosis H37Rv and 5 mice were used for the immunological assays. All experiments were repeated twice.