A novel DNA vaccine containing multiple TB-specific epitopes casted in a natural structure (ECANS) confers protective immunity against pulmonary mycobacterial challenge

Abstract

Epitope-based DNA vaccines designed to induce T cell responses specific for Mycobacterium tuberculosis (M. tb) are being developed as a means of addressing vaccine potency. In this study, we predicted 4 T cell epitopes from ESAT-6, Ag85A/B and CFP-10 antigens and constructed an ECANS (epitopes casted in a natural structure) DNA vaccine by inserting the epitope DNA segments separately into the gene backbone of M. tb-derived HSP65 (heat shock protein 65) carrier. The immunogenicity and protective efficacy of pECANS DNA vaccine were assessed in BALB/c mice after intramuscular immunization with 4 doses of 50 microg ECANS DNA and followed by mycobaterial challenge 4 weeks after the last immunization. Compared to plasmid encoding HSP65, pECANS DNA immunization elicited remarkably higher levels of IFN-gamma production by both CD4(+) and CD8(+) T cells, which were coupled with higher frequencies of antigen-specific T cells and higher CTL activity. Significantly enhanced levels of Th1 cytokines (IFN-gamma and IL-12) and increased serum IgG2a/IgG1 ratio were also noted, indicating a predominant Th1 immune response achieved by pECANS DNA immunization. In the consequence, a better protection against Mycobacterium bovis BCG challenge was achieved which was evidenced by reduced bacterial loads in lungs and spleens and profound attenuation of lung inflammation and injury. Our results suggested that multi-T cell-epitope based ECANS gene vaccine induced T cell response to multiple T cell epitopes and led to enhanced protection against mycobacterial challenge. This strategy might be a useful platform to design multi-T cell epitope-based vaccine against M. tb infection.

Fig. 1

Construction and expression of pHSP65 and pECANS. (A) Schematic representation of pHSP65 and pECANS. (B) Western Blot analysis to characterize the expressions of the plasmids. 293T cells were transfected with the indicated plasmids and the cell lysis was subjected to Western Blot with anti-HSP65 or anti-GAPDH antibody. Individual experiments were conducted three times, with one representative shown for each group.

Fig. 2

TB-specific antibody responses raised by pECANS intramuscular immunization. Mice were immunized with 4 doses of 50 μg pECANS, pHSP65, or mock vaccine at 2 weeks intervals. (A) Two weeks after the final immunization, TB-specific serum IgG responses were analyzed by ELISA assays. (B) Subclasses of serum IgG were detected at the same time. Each bar represents average values of 6 mice, measured induplicate. *P < 0.05; ND, not detected.

Fig. 3

Specific IFN-γ ELISPOT responses elicited by pECANS immunization. Two weeks after the final immunization, splenocytes were collected and stimulated with various TB antigens for 48 h, and then IFN-γ-secreting lymphocytes were quantified by ELISPOT assays. (A) Representative images of splenic ELISPOT responses in the immunized mice. (B) The frequency of IFN-γ-secreting cells in spleen following TB antigen-specific stimulation in vitro. Data are from one representative experiment of three performed and presented as the mean value ±SD (n = 6). *P < 0.05.

Fig. 4

Increased frequencies of IFN-γ-secreting CD4⁺ and CD8⁺ T cell by intramuscular immunization with pECANS. Two weeks following the final immunization, splenocytes were collected and stimulated with the pool of 4 epitopes (A) or inactivated H37Rv (B) for 48 h, then the percentages of CD4⁺IFN-γ⁺ and CD8⁺IFN-γ⁺ T cells were analyzed by flow cytometry. Individual experiments were conducted three times, with one representative shown for each group.

Fig. 5

TB-specific CTL activity elicited by pECANS. Unpulsed splenocytes (CFSE^low) and peptides-pulsed splenocytes (CFSE^high) from naive mice were transferred to the immunized mice. The percentages (A) and representative histograms (B) of splenic TB-specific lysis in the immunized mice were compared. Data are from one representative experiment of three performed and presented as the mean ± SD (n = 6). *P < 0.05.

Fig. 6

Promoted Th1 immune responses by pECANS immunization. The concentrations of Th1/Th2 cytokines in the cell culture supernatant were determined by ELISA after incubation for 48 h with the pool of 4 mixed T cell epitopes (A) or with the inactivated H37Rv bacteria (B). Data are from one representative experiment of three performed and presented as the mean ± SD (n = 6). *P < 0.05.

Fig. 7

Protection against mycobacterial infection by pECANS immunization. Four weeks following the last immunization, mice received an intranasal 1 × 10⁷ CFU of BCG. Six weeks post-challenge, the bacterial loads in the lungs (A) and spleens (B) were measured. The data are presented as the mean ± SD (n = 6) and are one representative of three separate experiments. *P < 0.05. Paraffin sections from lung tissues were stained with HE, and evaluated for the level of lung inflammation (C). Representative histology sections depicted the lung tissue of the immunized mice, using lung section of normal mice as a negative control (D). Magnification: 100×. Individual experiments were conducted three times, with one representative shown.