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FIG. 1. Induction of anti-Ag85 antibodies by vaccination with Ag85A DNA. BALB/c mice were injected with 5 ^g or 100 ^g Ag85A DNA and serum samples were collected at eight (hatched bars) and 21 weeks (solid bars). Anti-Ag85 antibodies were measured by ELISA and data shown represent geometric mean ELISA titre + S.E.M. for groups of 10 mice.

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FIG. 1. Induction of anti-Ag85 antibodies by vaccination with Ag85A DNA. BALB/c mice were injected with 5 ^g or 100 ^g Ag85A DNA and serum samples were collected at eight (hatched bars) and 21 weeks (solid bars). Anti-Ag85 antibodies were measured by ELISA and data shown represent geometric mean ELISA titre + S.E.M. for groups of 10 mice.

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FIG. 2. Induction of lymphoproliferative responses by vaccination with Ag85A DNA. BALB/c mice were injected with 5 ^g or 100 ^g Ag85A DNA and spleen cells were prepared for antigen restimulation at eight (hatched bars) and 21 weeks (solid bars). Lymphocyte proliferation is shown as stimulation index for pools of three spleens per group.

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FIG. 2. Induction of lymphoproliferative responses by vaccination with Ag85A DNA. BALB/c mice were injected with 5 ^g or 100 ^g Ag85A DNA and spleen cells were prepared for antigen restimulation at eight (hatched bars) and 21 weeks (solid bars). Lymphocyte proliferation is shown as stimulation index for pools of three spleens per group.

potent anti-mycobacterial properties. IFN-y levels in the culture supernatants approached 6 ng/ml, similar to those levels achieved by non-specific activation by the mitogen concanavalin A (not shown). Other cytokines secreted included IL-2, granulocyte macrophage colony-stimulating factor (GM-CSF) and tumour necrosis-a (TNF-a), but little or no IL-4, IL-6 and IL-10 (Huygen et al 1996). Therefore, as suggested by the IgG subtype profile of anti-Ag85 antibodies, Ag85A DNA induced a strong Th1-type response.

Potential induction of CTL responses by Ag85A DNA was assessed in two ways. First, spleen cells were restimulated in vitro with complete Ag85 protein then tested for lytic activity against stably transfected target cells expressing Ag85 (Huygen et al 1996). Second, a specific H-2d-restricted CTL epitope was identified in Ag85A contained within the 20-mer peptide corresponding to amino acids 61—80 (Denis et al 1998). Peptide restimulation of spleen cells (from mice injected with 100 p.g Ag85A DNA one year previously) activated Ag85A-specific effector cells capable of lysing target cells expressing Ag85A protein or pulsed with specific peptide, but not untreated cells or cells pulsed with a different Ag85A peptide (Fig. 3).

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