The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette-Guèrin) in protecting against systemic Mycobacterim avium infection

Abstract

Sub-unit vaccines utilizing purified mycobacterial proteins or DNA vaccines induce partial protection against mycobacterial infections. For example, immunization with DNA vaccines expressing the gene for the immunodominant 35000 MW protein, common to Mycobacterium avium and Mycobacterium leprae but absent from the Mycobacterium tuberculosis complex, conferred significant protection against infection with either virulent M. avium or M. leprae in mice. However, the level of protection was equivalent to that obtained with the viable, attenuated vaccine, Mycobacterium bovis, bacille Calmette-Guèrin (BCG). The cytokine, interleukin (IL)-12, is essential for priming naïve CD4+ T lymphocytes to differentiate into interferon-gamma (IFN-gamma)-secreting T cells. We have used a novel self-splicing vector expressing both chains of murine IL-12 to determine if plasmid IL-12 would increase the efficacy of a vaccine expressing the M. avium 35000 MW protein (DNA-Av35). Co-immunization with p2AIL-12 and DNA-Av35 led to a significant increase in the number of antigen-specific IFN-gamma secreting cells and total amount of IFN-gamma released, but a concomitant fall in the antibody response to the 35000 MW protein. This pattern of response was associated with enhanced clearance of M. avium from the liver and spleen of coimmunized mice, and was significantly more effective than BCG or DNA-Av35. alone. Following M. avium challenge there was significant increase in the expansion of the 35000 MW antigen-reactive T cells in the coimmunized mice. Therefore, plasmid-delivered IL-12 acts as an effective adjuvant to increase the protective efficacy of a single DNA vaccine against M. avium infection above that achieved by BCG, and this strategy may improve the efficacy of subunit vaccines against M. leprae and M. tuberculosis.

Figure 1

Co-immunization with plasmid IL-12 increases the antigen-specific IFN-γ T-cell responses following DNA immunization. Two weeks following the third immunization with DNA-Av35 or the empty vector, DNA-Neg, with and without p2AIL12, splenocytes were stimulated overnight with M. avium 35 000 MW protein and the frequency of IFN-γ secreting cells (a) or amount of IFN-γ secreted (b) analysed. The data represent the means (± SEM) for five mice and are representative of three separate experiments. The significance of the differences between DNA-Av35 alone or with p2AIL12 were ***, P = 0·001 (a) and **, P =0·005 (b).

Figure 2

Co-immunization with plasmid IL-12 reduces antigen-specific antibody following DNA immunization. Mice (n = 5) were immunized with DNA-Av35 or the empty vector, DNA-Neg, with and without p2AIL12. Two weeks following the third immunization the serum geometric mean titre (± SEM) of IgG anti-35 000 MW antibodies was determined by ELISA. The significance of the difference between immunization with DNA-Av35 alone and with pIL-12 was *, P = 0·05. The results are representative of three separate experiments.

Figure 3

Plasmid IL-12 enhances the protection against systemic M. avium infection in the spleen and liver following DNA immunization. Mice (n = 5) were immunized i.m. with DNA-Av35 or the empty vector, DNA-Neg, with and without p2AIL12, or by a single s.c. injection with BCG, with or without pIL-12 i.m. Six weeks following the last DNA immunization and 12 weeks after the BCG immunization, mice were challenged with 1 × 10⁶ M. avium by the intravenous route. Four weeks later, the bacterial loads (c.f.u. ± SEM) were determined in the spleen (a) and liver (b). The significance of the differences between individual groups were determined by anova and were ***, P < 0·001; **, P < 0·01, and ns, not significant. The results are representative of three separate experiments.

Figure 4

Co-administration of plasmid IL-12 with the DNA-Av35 vaccine enhances the 35 000 MW protein-specific IFN-γ T-cell response following M. avium challenge. Mice (n = 5) were immunized as described in Fig. 3. Following M. avium challenge the number of splenocytes secreting IFN-γ (mean ± SEM) (a) and the amount of IFN-γ secreted (mean ± SEM) (b) in response to M. avium 35 000 MW antigen were determined. The significance of differences between groups immunized with DNA-35 alone or with p2AIL12 were ***, P < 0. 0·001; **, P < 0·01; and *, P < 0·05. The results are representative of three separate experiments.