Increased B and T Cell Responses in M. bovis Bacille Calmette-Guérin Vaccinated Pigs Co-Immunized with Plasmid DNA Encoding a Prototype Tuberculosis Antigen

Abstract

The only tuberculosis vaccine currently available, bacille Calmette-Guérin (BCG) is a poor inducer of CD8(+) T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation. As the induction of strong CD8(+) T cell responses is a hallmark of DNA vaccines, a combination of BCG with plasmid DNA encoding a prototype TB antigen (Ag85A) was tested. As an alternative animal model, pigs were primed with BCG mixed with empty vector or codon-optimized pAg85A by the intradermal route and boosted with plasmid delivered by intramuscular electroporation. Control pigs received unformulated BCG. The BCG-pAg85A combination stimulated robust and sustained Ag85A specific antibody, lymphoproliferative, IL-6, IL-10 and IFN-γ responses. IgG1/IgG2 antibody isotype ratio reflected the Th1 helper type biased response. T lymphocyte responses against purified protein derivative of tuberculin (PPD) were induced in all (BCG) vaccinated animals, but responses were much stronger in BCG-pAg85A vaccinated pigs. Finally, Ag85A-specific IFN-γ producing CD8(+) T cells were detected by intracellular cytokine staining and a synthetic peptide, spanning Ag85A131-150 and encompassing two regions with strong predicted SLA-1*0401/SLA-1*0801 binding affinity, was promiscuously recognized by 6/6 animals vaccinated with the BCG-pAg85A combination. Our study provides a proof of concept in a large mammalian species, for a new Th1 and CD8(+) targeting tuberculosis vaccine, based on BCG-plasmid DNA co-administration.

Fig 1. BCG-pAg85A combination induces strong mycobacteria-specific proliferative and IFN-γ responses.

Evolution of Ag85A (Fig 1A) and PPD (Fig 1B) specific proliferation throughout the vaccination experiment in group 1 animals vaccinated with only BCG (black symbols), group 2 animals vaccinated with BCG-control vector (blue symbols) and group 3 animals vaccinated with BCG-pAg85A (green symbols). Each point represents mean cpm values of quintuplicate cultures of individual animals. Cells were stimulated in vitro with bovine PPD (M. bovis strain AN5, ex-Pasteur Institute of Brussels, 5 μg/mL final) or recombinant E. coli derived Ag85A (5 μg/mL final). Evolution of Ag85A (Fig 1C) and PPD (Fig 1D) specific IFN-γ production throughout the vaccination experiment. Bars represent mean IFN- γ levels ± SEM of six animals (pg/mL).

Fig 2. BCG-pAg85A combination induces Ag85A-specific IgG antibodies.

A. Ag85A-specific antibodies as detected at day -1 before BCG priming and at days 20, 41, 63 and 118 of the vaccination protocol. Mean O.D. values (490/620nm) of individual sera diluted 1:200 are shown for group 1 (black symbols), group 2 (blue symbols) and group 3 (green symbols). B. IgG1:IgG2 ratio of Ag85A specific antibodies measured at day 63 of vaccination on sera diluted 1:50.

Fig 3. BCG-pAg85A combination induces an IFN-γ producing CD8⁺ T cell compartment.

A: Intracellular IFN-γ staining of Ficoll purified PBMC cells following 3 day culture of purified PBMC from group 1 (black symbols), group 2 (blue symbols) and group 3 (green symbols) animals, cultured in the presence of rAg85A. B: Percentage of total CD8⁺ and C: IFN-γ producing CD8⁺ T cells as detected by intracellular cytokine staining following 3 days of culture in presence of rAg85A.

Fig 4. Ag85A T cell epitope mapping in group 3 pigs vaccinated with BCG-pAg85A combination.

Ag85A T cell epitope mapping in group 3 pigs vaccinated with BCG-pAg85A combination, as tested on day 84, six weeks after the second pAg85A boost. Each point represents mean cpm values ± SEM of quintuplicate cultures stimulated with the overlapping synthetic 20-mer peptides spanning the mature 294 aa Ag85A protein [34] (Innogenetics, Ghent, Belgium 10μg/mL final), bovine PPD (5μg/mL) or recombinant Mtb Ag85A protein (5μg/mL). Different symbols were used to identify the 6 animals. For technical reasons, peptide 10 spanning aa 91–110 of Ag85A was replaced by the corresponding peptide of Ag85B spanning aa* 91–108. This sequence is identical to the Ag85A sequence except for a Gly107Gln shift.

Fig 5. Ag85A T cell epitope mapping in vaccinated pigs.

Lymphoproliferative responses at day 118 in animals vaccinated with BCG (left Fig), with BCG combined with empty vector (middle Fig) or BCG combined with pAg85A (right Fig) of diluted whole blood cultures stimulated for 7 days with medium (T), bovine PPD, recombinant Ag85A of M.tuberculosis, recombinant Ag85A of M. avium subsp. paratuberculosis (Map), 85A _111–130, 85A _131–150 or 85A _231–250. Each point represents mean cpm values of quintuplicate cultures of individual animals.