Oral delivery of Mycobacterium bovis BCG in a lipid formulation induces resistance to pulmonary tuberculosis in mice

Abstract

A lipid-based formulation has been developed for oral delivery of Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine. The formulated M. bovis BCG was fed to BALB/c mice to test for immune responses and protection against M. bovis infection. The immune responses included antigen-specific cytokine responses, spleen cell proliferation, and lymphocyte-mediated macrophage inhibition of M. bovis. Oral delivery of formulated M. bovis BCG to mice induced strong splenic gamma interferon levels and macrophage inhibition of virulent M. bovis compared with results with nonformulated M. bovis BCG. Formulated oral M. bovis BCG significantly reduced the bacterial burden in the spleen and lungs of mice following aerosol challenge with virulent M. bovis. Our data suggest that oral delivery of formulated M. bovis BCG is an effective means of inducing protective immune responses against tuberculosis. Lipid-based, orally delivered mycobacterial vaccines may be a safe and practical method of controlling tuberculosis in humans and animals.

FIG. 1.

Influence of temperature on viability of lipid-formulated M. bovis BCG following storage. The number of CFU of M. bovis BCG was determined by plating serial dilutions of formulated M. bovis BCG which had been stored at 4°C (diamonds) or at room temperature (10 to 25°C) (squares) The data shown are the means of M. bovis BCG CFU ± standard errors. The experiment was repeated three times with similar results.

FIG. 2.

Bovine PPD induced IFN-γ responses following oral vaccination with various doses of formulated M. bovis BCG. Mice were sacrificed at 8 weeks after oral immunization with formulated M. bovis BCG (circles), nonformulated M. bovis BCG (triangles), or formulation material only (diamonds). Splenocytes were incubated with bovine PPD for 72 h. Supernatants were then collected and analyzed using a sandwich ELISA. Each treatment group contained six mice. Spleens were individually processed. Results are expressed in picograms/milliliter and are presented as means of triplicate determinations. Bars indicate standard errors.

FIG. 3.

Antigen-induced splenic IFN-γ responses to M. bovis BCG vaccination in BALB/c mice. Mice were euthanatized at 2, 4, 6, and 8 weeks after vaccination with 10⁶ CFU of subcutaneous M. bovis BCG (squares), oral delivery of 10⁷ CFU of formulated M. bovis BCG (circles), nonformulated M. bovis BCG (triangles), or formulation material only (diamonds). Splenocytes were incubated with bovine PPD for 72 h. Supernatants were then collected and analyzed using a sandwich ELISA. Each treatment group contained five to six mice. Spleens were individually processed. Results are expressed in picograms/milliliter and are presented as means of triplicate determinations. Results at 8 weeks are from two separate experiments. P value was <0.05 (Student t test). Bars indicate standard errors.

FIG. 4.

Growth inhibition of M. bovis by macrophages cocultured with NPEC. Macrophages were infected with M. bovis at a multiplicity of infection of two bacilli per macrophage. Nonadherent autologous NPEC were added at a ratio of 10 NPEC per macrophage. Incorporation of [³H]uracil was then assessed at 72 h postinfection. The mean [³H]uracil uptake by cell cultures which did not contain M. bovis was 460 cpm. Growth of intracellular bacilli from cocultured macrophages and NPEC was expressed as means of triplicates. The results are representative of two experiments. The asterisk represents a mean which is significantly different from the mean of the formulation-only control group; bars indicate standard errors.