Immunogenicity and protective efficacy of tuberculosis DNA vaccines combining mycolyl-transferase Ag85A and phosphate transport receptor PstS-3

Abstract

DNA vaccines encoding the 32,000 MW mycolyl-transferase Ag85A and the 40,000 MW phosphate-binding protein PstS-3 can elicit protective immune responses against experimental infection with Mycobacterium tuberculosis in C57BL/6 mice. Here we have analysed the vaccine potential of a combination of both antigens using plasmid vectors expressing either a fusion protein of both antigens or the separate proteins driven by two independent promoters (in pBudCE4.1 vector). Comparable levels of Ag85A specific T helper 1 (Th1) type immune responses could be induced by the two combination vaccines and the single vaccine encoding the mycolyl-transferase, whereas induction of PstS-3 specific Th1-mediated responses was impaired in both combination vaccines. In contrast, magnitude of CD8+ mediated responses against the PstS-3 protein was comparable following combination or single DNA vaccination. Antigenic competition was also observed at the antibody level; PstS-3 specific levels being lower in mice vaccinated with the fusion vector and Ag85A specific levels being lower in mice vaccinated with the combination pBudCE4.1 vector (as compared to levels obtained following single plasmid immunization). Protection against M. tuberculosis was only modestly improved in mice vaccinated with the DNA combinations. It is possible that prior activation of Ag85A specific CD4+ T cells directed against this common mycobacterial antigen leads to cross-competition for major histocompatibility complex class II-restricted peptide complexes of the Pst-3 antigen. This may have implications for future combination vaccines using Ag85.

Figure 1

Western blot analysis of equivalent amount of extracts of BHK-21 cells transfected with 2 µg of pV1J.ns-PstS-3-Ag85A (1), pV1J.ns-tPA-PstS-3 (3), pV1J.ns-tPA-Ag85A (4), pBudCE4·1-tPA-Ag85A (5), pBudCE4·1-tPA-PstS-3 (6), pBudCE4·1-tPA-Ag85A-tPA-PstS-3 (7) and pV1J.ns-tPA (8). In lane 2 purified native Ag85A and in lane M protein molecular weight markers were loaded. Primary antibodies used were specific mAbs: anti-Ag85A (a), anti-PstS-3 (b) and a mix of anti-Ag85A and anti-PstS-3 (c).

Figure 2

Antibody and IL-2 responses generated by vaccination of C57BL/6 mice with pV1J.ns-tPA-Ag85A (Ag85DNA), pV1J.ns-tPA-PstS-3 (PstS-3-DNA) or pV1J.ns-PstS-3-Ag85A (fusion-DNA) encoding fusion protein. (a) Ag85A specific total IgG levels in naïve mice, in mice vaccinated with pV1J.ns-tPA-Ag85A or pV1J.ns-PstS-3-Ag85A. (b) PstS-3 specific total IgG levels in naïve mice, mice vaccinated with pV1J.ns-tPA-PstS-3 or pV1J.ns-PstS-3-Ag85A. IgG levels are expressed as mean OD (492 nm) values ± SD of three mice tested individually in each group. (c) IL-2 levels in 24 hr culture supernatant of splenocytes from naïve or DNA vaccinated mice, restimulated with medium (RPMI), native Ag85A (Ag85A-5 µg/ml) and 10 µg/ml of CD4⁺-Ag85A_241–260 or CD4⁺-Ag85A_261–280, recombinant PstS-3 (recPstS-3–5 µg/ml) or 10 µg/ml of CD4⁺-PstS-3_151–170, CD4⁺-PstS-3_191–210 and CD8⁺-PstS-3_281–300. Data are represented as mean c.p.m. ± SD of three animals tested individually in each group. ns: statistically not significant, *P < 0·05, **P < 0·01 and ***P < 0·005 as compared to mice vaccinated with the single plasmid.

Figure 3

IFN-γ and PstS-3-specific CTL responses generated by vaccination of C57BL/6 mice with pV1J.ns-tPA-Ag85A (Ag85A-DNA), pV1J.ns-tPA-PstS-3 (PstS-3-DNA) or pV1J.ns-PstS-3-Ag85A (fusion-DNA) encoding fusion protein. (a) IFN-γ production measured by ELISA, of splenocytes restimulated for 3 days with medium (RPMI), native Ag85A (Ag85A-5 µg/ml) and 10 µg/ml of CD4⁺-Ag85A_241–260 or CD4⁺-Ag85A_261–280, recombinant PstS-3 (recPstS-3–5 µg/ml) or 10 µg/ml of CD4⁺-PstS-3_151–170, CD4⁺-PstS-3_191–210 and CD8⁺-PstS-3_281–300. (b) IFN-γ ELISPOT of splenocytes restimulated for 40 hr with medium (RPMI), recombinant Ag85A (recAg85A-5 µg/ml) and 10 µg/ml of CD4⁺-Ag85A_241–260 or CD4⁺-Ag85A_261–280, CD4⁺-PstS-3_191–210 and CD8⁺-PstS-3_281–300. (c) PstS-3-specific D^b restricted CTL activity induced in mice vaccinated with pV1J.ns-tPA-PstS-3 or pV1J.ns-PstS-3-Ag85A as detected in ⁵¹Cr release assay using unpulsed RMA-S target cells or target RMA-S cells pulsed with PstS-3_285–293 peptide. ns: statistically not significant, *P < 0·05, **P < 0·01 and ***P < 0·005 as compared to mice vaccinated with the single plasmid.

Figure 4

Humoral and cellular immune responses generated by vaccination of C57BL/6 mice with pBud-tPA-Ag85A, pBud-tPA-PstS-3 or pBud-Ag85A-PstS-3. (a) anti-Ag85A total IgG production of naïve mice, mice vaccinated with pBud-Ag85A or pBud-Ag85A-PstS-3. (b) anti-PstS-3 total IgG production of naïve mice, mice vaccinated with pBud-PstS-3 or pBud-Ag85A-PstS-3. (c) IL-2 production of splenocytes restimulated for 1 day with medium (RPMI), recombinant Ag85A (recAg85A-5 µg/ml), and 10 µg/ml of CD4⁺-Ag85A_241–260 or CD4⁺-Ag85A_261–280 or CD4⁺-PstS-3_191–210 or CD8⁺-PstS-3_281–300. (d) IFN-γ ELISPOT of splenocytes incubated for 40 hr with medium, recombinant Ag85A (recAg85A-5 µg/ml) and 10 µg/ml of CD4⁺-Ag85A_241–260, CD4⁺-Ag85A_261–280, CD4⁺-PstS-3_191–210 and CD8⁺-PstS-3-peptide_281–300. Results are represented as net SFC/Mio [(SFC/Mio)_antigen − average(SFC/Mio)_medium]. ns, statistically not significant, *P < 0·05, **P < 0·01 and ***P < 0·005 as compared to mice vaccinated with the single plasmid.

Figure 5

In vivo D^b-PstS-3 specific cytotoxic activity induced in naïve C57BL/6 mice or vaccinated with pV1J.ns-PstS-3-Ag85A or pBud-Ag85A-PstS-3. Results of three individual animals/group are shown. Mean ± SD percentage of specific lysis is given in parentheses.

Figure 6

Replication of luminescent Mtb H37Rv 4 weeks after an intravenous challenge in lungs (a and b) and spleen (c) of C57BL/6 (a) and BALB.B10 (b and c) mice not vaccinated (black bars) or vaccinated with empty vector (white bars, black dots), BCG (black bars, white dots), pV1J.ns-tPA-Ag85A (white bars), pV1J.ns-tPA-PstS-3 (grey bars), pBud-Ag85A-PstS-3 (hatched bars) or pV1J.ns-PstS-3-Ag85A (wavy bars). Data represent the mean log₁₀ RLU ± SD of 3–16 mice per group tested individually. Statistically significant results as compared to the not vaccinated group are represented in the figure by ***(P < 0·005), **(P < 0·01) and *(P < 0·05).