Vaccination with a Sindbis virus-based DNA vaccine expressing antigen 85B induces protective immunity against Mycobacterium tuberculosis

Abstract

To improve DNA vaccination against Mycobacterium tuberculosis, we evaluated the effectiveness of a Sindbis virus-based DNA construct expressing the tuberculosis antigen 85B (Sin85B). The protective efficacy of Sin85B was initially assessed by aerogenically challenging immunized C57BL/6 mice with virulent Mycobacterium tuberculosis. At 1 and 7 months postinfection, the lung bacterial burdens were considerably reduced and the lung pathology was improved in vaccinated mice compared to naive controls. Furthermore, the mean survival period for Sin85B-immunized mice (305 +/- 9 days) after the tuberculous challenge was extended 102 days relative to the naive mice (203 +/- 13 days) and was essentially equivalent to the survival time of Mycobacterium bovis BCG-vaccinated mice (294 +/- 15 days). The essential role of gamma interferon (IFN-gamma) in Sin85B-mediated protection was established by showing that significantly increased levels of IFN-gamma mRNA were present postinfection in lung cells from vaccinated mice relative to control mice and by demonstrating that IFN-gamma depletion prior to challenge abolished the vaccine-induced protection. The substantial antituberculosis protective responses induced by Sin85B immunization of CD4-/- mice strongly suggested that CD8 cells partially mediate Sin85B-induced protective immunity. Interestingly, Sin85B vaccination did not protect RNase L-/- (a key enzyme in the innate antiviral response) mice while significant protection was detected in RNase L-/- mice immunized with either BCG or a conventional DNA plasmid expressing antigen 85B. These data show that immunization with Sin85B offers protection similar to BCG in a murine model of pulmonary tuberculosis and suggest that Sin85B-induced protection is dependent upon both innate and acquired immune mechanisms.

FIG. 1.

In vitro levels of expression of antigen 85B mRNA from the Sin85B and pVax-85B vectors were similar. RT-PCR analysis of mRNA from RD cells transfected with Sin85B (RD-Sin85B) (lanes 2 and 3, 10 and 5 μl of the PCR product, respectively) or pVax-85B (RD-pVax-85B) (lanes 4 and 5, 5 and 10 μl of the PCR product, respectively) using antigen 85B-specific primers was performed. Using the same mRNA, RT-PCR using GAPDH-specific primers was performed (lane 7, RD-Sin85B mRNA; lane 8, RD-pVax-85B mRNA) as controls. Lanes 1 and 6 contain DNA molecular size markers.

FIG. 2.

Evaluation of antigen 85B antibody titers in mice vaccinated with either the Sin85B or pVax-85B constructs. Recombinant Ag85B was bound to wells of a 96-well plate, and twofold serial dilutions of sera from naive mice or mice vaccinated with either Sin85B or pVax-85B were added to the wells. The secondary antibody was alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G.

FIG. 3.

Histopathological analysis of lungs from vaccinated and naive mice obtained 190 days following an M. tuberculosis aerosol challenge. Lung snips were removed, fixed with formalin, and stained with H&E stain. Representative slides are shown for naive controls and BCG-vaccinated and Sin85B-vaccinated mice. Five mice per group and five sections per each lung were analyzed (magnification, ×172).

FIG. 4.

The survival of mice immunized with BCG or the Sin85B DNA vaccine following an aerogenic challenge with M. tuberculosis Erdman. BCG-vaccinated and naive mice served as controls. Six to eight mice per group were used in this analysis.

FIG. 5.

Vaccine-induced cytokine mRNA responses. Ten days following an aerogenic challenge with M. tuberculosis Erdman, RNA was obtained from lung cells of BCG- and Sin85B-vaccinated mice and used for real-time RT-PCR analysis of IFN-γ, tumor necrosis factor alpha (TNF-alpha), RANTES, IL-2, IL-12, and IL-10 mRNA concentrations relative to mRNA levels of GAPDH. The cytokine levels are expressed as values relative to the mRNA concentrations of the naive controls. *, mRNA levels are significantly elevated compared to the naive controls (P < 0.05).

FIG. 6.

RNase L^−/− mice were immunized with either BCG, pVax-85B, or Sin85B and then challenged aerogenically with virulent M. tuberculosis Erdman. Twenty-eight days postchallenge, the lungs were removed and lung snips were fixed with formalin and stained with Ziehl-Neelsen acid-fast stain. Naive mice served as controls. Magnification, ×186.