Immunogenicity and protective efficacy against murine tuberculosis of a prime-boost regimen with BCG and a DNA vaccine expressing ESAT-6 and Ag85A fusion protein

Abstract

Heterologous prime-boost regimens utilizing BCG as a prime vaccine probably represent the best hope for the development of novel tuberculosis (TB) vaccines. In this study, we examined the immunogenicity and protective efficacy of DNA vaccine (pcD685A) expressing the fusion protein of Ag85A and ESAT-6 (r685A) and its booster effects in BCG-immunized mice. The recombinant r685A fusion protein stimulated higher level of antigen-specific IFN-γ release in tuberculin skin test- (TST-) positive healthy household contacts of active pulmonary TB patients than that in TST-negative population. Vaccination of C57BL/6 mice with pcD685A resulted in significant protection against challenge with virulent Mycobacterium tuberculosis H37Rv when compared with the control group. Most importantly, pcD685A could act as a BCG booster and amplify Th1-type cell-mediated immunity in the lung of BCG-vaccinated mice as shown the increased expression of IFN-γ. The most significant reduction in bacterial load of both spleen and lung was obtained in mice vaccinated with BCG prime and pcD685A DNA booster when compared with BCG or pcD685A alone. Thus, our study indicates that pcD685A may be an efficient booster vaccine against TB with a strong ability to enhance prior BCG immunity.

Figure 1

Construction of recombinant plasmids. Genes encoding ESAT-6, Ag85A, the fusion protein of ESAT-6, and Ag85A with a 45 bp linker, were cloned from M. tuberculosis H37Rv genomic DNA, respectively (A). The PCR products of the fusion genes were inserted into the BamHI and EcoRI sites of pProExHTb or pcDNA3.1, resulting in the recombinant plasmids pPro685A and pcD685A, respectively. The recombinant plasmids were identified by enzyme digestion (B). Lane M: DNA molecular marker; lane A1: PCR product of esat-6; lane A2: PCR product of fbpA; lane A3: PCR product of esat-6-fbpA; lane B1: products of pPro685A digested with enzymes; lane B2: products of pcD685A digested with enzymes.

Figure 2

Expression, purification, and identification of recombinant fusion protein. E. coli BL21 (DE3) harboring pPro685A was cultured with IPTG. The expression (a) and the purification procession of r685A fusion protein (b) were confirmed by SDS-PAGE and Western blotting (c). Lane M: protein molecular size marker (kDa); lane a1: E. coli strain without IPTG; lane a2: E. coli strain 2 h after IPTG induction; lane a3: E. coli strain 4 h after IPTG induction; lane a4: E. coli strain 6 h after IPTG induction; lane b1: cell lysis; lane b2: fraction from Ni-NTA column after wash with denaturing binding buffer; lanes b3 and b4: fraction after wash with wash buffer; lane b5: r685A protein eluted with native elution buffer; lane c1: anti-ESAT-6 antibody; lane c2: anti-Ag85A antibody.

Figure 3

Western blot analysis of cell lysates from Hela cells transfected with the pcD685A construct (lane 2) or empty vector (lane 1). The primary antibody used was mouse antisera after pcD685A DNA vaccination.

Figure 4

Immunogenicity of r685A fusion protein in healthy population. A total of 17 household contacts with active TB patients were tested by TST and r685A-WBIA, respectively. Each square represents the IFN-γ concentration in a sample, and median values for TST− (n  =  7) and TST+ (n  =  10) groups are indicated by horizontal lines.

Figure 5

r685A-specific IgG antibody-induced in immunized mice. Mice were immunized and bled at day 14 following the last immunization; serum antibody levels were assessed by ELISA. Sera from five animals in each group were evaluated individually at the dilutions indicated. Results shown are the mean and standard error.

Figure 6

Differential expression of IFN-γ and IL-10 in the lungs of vaccinated mice (n  =  3). Two weeks after DNA vaccination, RNA extracted from lung of each mouse in different groups was used for the detection of cytokines mRNA concentrations by qRT-PCR analysis. The levels of the cytokine mRNAs for each group were normalized based on the levels of GAPDH. Cytokine mRNA levels were expressed as values relative to the control group.

Figure 7

Bacterial load per lung and spleen in C57BL/6 mice at 4 weeks after challenge (n  =  7). Vaccinated C57BL/6 mice (n  =  7) were challenged i.v. with 10⁶ CFU virulent M. tuberculosis H37Rv strain. Four weeks after challenge, lungs and spleens were harvested aseptically and numbers of bacterial CFU per organ were enumerated. Results are shown as the mean (±SEM) log₁₀ CFU/organ. *P < .05 versus control; **P < .05 versus BCG. This experiment was repeated twice with similar results.

Figure 8

Representative lung pathology of C57BL/6 mice after challenge. Vaccinated C57BL/6 mice were challenged i.v with 10⁶ CFU virulent Mycobacterium tuberculosis H37Rv strain. Four weeks after infection, lung tissue sections from different vaccine groups were prepared for HE staining (amplification 10  ×  4), (a) control; (b) vector control; (c) pcD685A; (d) BCG; (e) BCG plus vector; (f) BCG plus pcD685A.