Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6

Abstract

In this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dose-dependent immune response was induced to both single components as well as to the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level of Mycobacterium bovis BCG-induced protection over a broad dose range. The vaccine induced efficient immunological memory, which remained stable 30 weeks postvaccination.

FIG. 1

SDS-PAGE analysis of purified recombinant M. tuberculosis antigens. One microgram of protein was loaded in each lane. Lane 1, molecular weight standard; lane 2, recombinant ESAT-6; lane 3, recombinant Ag85B; lane 4, Ag85B-ESAT-6 fusion protein; lane 5, ESAT-6-Ag85B fusion protein. Protein bands were visualized by Coomassie blue staining.

FIG. 2

Antigen-specific responses by PBMC 1 week after the last immunization with the fusion proteins between Ag85B and ESAT-6. C57BL/6J mice were immunized three times with either Ag85B-ESAT-6 (A) or ESAT-6-Ag85B (B) emulsified in MPL-DDA. As a negative control, a group of mice received the adjuvant combination alone (C). The IFN-γ responses were measured in cell cultures pooled from eight animals in each group. Each point represents the mean of triplicate values ± standard error of the mean. The experiment was performed twice with similar results.

FIG. 3

Efficacy of different doses of a subunit vaccine based on Ag85B-ESAT-6. C57BL/6J mice were immunized s.c. three times with different doses of Ag85B-ESAT-6 emulsified in MPL-DDA. Mice immunized with the adjuvant alone were included. Ten weeks after the first vaccination, the mice received an aerosol challenge with M. tuberculosis Erdman, and the numbers of bacteria (CFU) were quantified in the lungs and spleens 6 weeks later. The values are shown as log₁₀ CFU in the lung and spleen. All data represent the mean of 5 to 20 individual mice ± standard error of the mean. Numbers in parentheses indicate the number of animals in each group. CFU counts in naive mice were 5.74 ± 0.04 and 4.65 ± 0.22 (n = 10) in the lung and spleen, respectively. ∗∗, P < 0.01; ∗∗∗, P < 0.001 compared to naive mice.