Effects of DNA- and Mycobacterium bovis BCG-based delivery of the Flt3 ligand on protective immunity to Mycobacterium tuberculosis

Abstract

The control of intracellular pathogens such as Mycobacterium tuberculosis is dependent on the activation and maintenance of pathogen-reactive T cells. Dendritic cells (DCs) are the major antigen-presenting cells initiating antimycobacterial T-cell responses in vivo. To investigate if immunization strategies that aim to optimize DC function can improve protective immunity against virulent mycobacterial infection, we exploited the ability of the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) to expand the number of DCs in vivo. A DNA fusion of the genes encoding murine Flt3L and M. tuberculosis antigen 85B stimulated enhanced gamma interferon (IFN-gamma) release by T cells and provided better protection against virulent M. tuberculosis than DNA encoding the single components. Vaccination of mice with a recombinant Mycobacterium bovis BCG strain secreting Flt3L (BCG:Flt3L) led to early expansion of DCs compared to immunization with BCG alone, and this effect was associated with increased stimulation of BCG-reactive IFN-gamma-secreting T cells. BCG and BCG:Flt3L provided similar protective efficacies against low-dose aerosol M. tuberculosis; however, immunization of immunodeficient mice revealed that BCG:Flt3L was markedly less virulent than conventional BCG. These results demonstrate the potential of in vivo targeting of DCs to improve antimycobacterial vaccine efficacy.

FIG. 1.

Flt3L-Ag85B is expressed by DNA vaccines in a functional form. (A) Schematic representation of pCDNA3 vectors expressing Flt3L, M. tuberculosis Ag85B (p85B), and the Flt3L-Ag85B fusion protein (pFlt-85). (B) Secretion of Flt3L by DNA-transfected HEK 293 cells. Flt3L in the culture medium of cells 3 days after transfection was detected by ELISA. (C) Generation of DCs from bone marrow progenitors using culture medium from DNA-transfected HEK 293 cells. The generation of CD11c⁺ MHC-II⁺ cells on day 6 after addition of HEK 293 supernatants to bone marrow cells was determined by flow cytometry. (D) Total number of CD11c⁺ MHC-II⁺ cells in culture.

FIG. 2.

Immunogenicity and protective efficacy of DNA encoding murine Flt3L and M. tuberculosis Ag85B. (A) Splenocytes from immunized mice were cultured with 3 μg/ml Ag85B, and the level of IFN-γ released was determined by ELISA. (B) For determination of protective efficacy, 4 weeks following the final vaccination mice were infected by the aerosol route with 100 CFU of M. tuberculosis H37Rv. Four weeks after challenge, the bacterial load, expressed as the log₁₀ CFU (mean ± standard error of the mean), was analyzed in the lung. The significance of differences between groups was determined by ANOVA. The error bars indicate standard errors of the means, and the data are representative of two separate experiments.

FIG. 3.

Construction and in vivo immunogenicity of BCG:Flt3L. BCG Pasteur was transformed with the control pMV261 plasmid (BCG:Ct) or plasmid pJEX73 (BCG:Flt3L), and the presence of Flt3L in cell lysates was determined by immunoblotting with the anti-c-myc MAb 9E10 (A). The presence of Flt3L in the culture supernatants of rBCG strains was determined by murine Flt3L-specific ELISA (B). To assess the immunogenicity, mice were not vaccinated (striped bar) or were vaccinated with 5 × 10⁵ CFU of BCG:Ct (open bars) or BCG:Flt3L (solid bars), and at the indicated time points splenocytes (C) or DLN cells (D) were stimulated with 1 μg/ml BCG lysate. The number of IFN-γ-secreting cells was determined by an enzyme-linked immunospot assay. The significance of differences between BCG:Flt3L-vaccinated animals and BCG:Ct-vaccinated animals (asterisk, P < 0.05) was determined by ANOVA. The data are representative of two separate experiments.

FIG. 4.

Influence of BCG:Flt3L on DC numbers in vaccinated mice. Mice were vaccinated subcutaneously with 5 × 10⁵ CFU of rBCG, and at the indicated time points the numbers of CD11c⁺ MHC-II⁺ cells in the spleen (A, C, and E) and DLNs (B, D, and F) were determined by flow cytometry. The cells were further defined as cells that were B220⁺ (C and D) or B220⁻ (E and F). Striped bars, unvaccinated mice; open bars, BCG:Ct-vaccinated animals; solid bars, BCG:Flt3L-vaccinated animals. The significance of differences between BCG:Flt3L-vaccinated animals and BCG:Ct-vaccinated animals (asterisk, P < 0.05) was determined by ANOVA. The data are representative of two separate experiments.

FIG. 5.

Protective efficacy of BCG secreting Flt3L. Mice were immunized subcutaneously with 5 × 10⁵ CFU of BCG:Flt3L or BCG:Ct, and 12 weeks after immunization they were challenged by the aerosol route with M. tuberculosis H37Rv. Four weeks postchallenge the bacterial loads, expressed as log₁₀ CFU (means ± standard errors of the means), were analyzed in the (A) lungs and (B) spleens of mice. The statistical significance between naïve and rBCG-vaccinated groups (asterisk, P < 0.01) was analyzed by ANOVA. The data are representative of three individual experiments. Unv, unvaccinated mice. (C and D) For assessment of rBCG growth in vivo, mice were infected i.v. with 1 × 10⁶ CFU of BCG:Flt3L (•) or BCG:Ct (○). At 1, 14, 28, and 56 days postinfection the bacterial loads were assessed in the lungs (C) and spleens (D). The statistical significance between groups (asterisk, P < 0.05) was analyzed by ANOVA.

FIG. 6.

Safety of BCG:Flt3L in immunodeficient mice. (A) RAG-1^−/− mice were infected i.v. with 1 × 10⁶ CFU of BCG:Flt3L (•) or BCG:Ct (○), and survival was monitored over time. The significance of differences in survival was determined by the Mantel-Cox log rank test (asterisk, P < 0.001). (B and C) RAG-1^−/− mice were vaccinated subcutaneously with 5 × 10⁵ CFU of rBCG strains, and the numbers of CD11c⁺ MHC-II⁺ cells in the DLNs (B) and spleens (C) of the vaccinated mice were determined by flow cytometry. Striped bars, unvaccinated mice; open bars, BCG:Ct-vaccinated mice; solid bars, BCG:Flt3L-vaccinated mice. There were no statistically significant differences between groups as determined by ANOVA. The data are representative of one of two individual experiments.