Enhancing the protective efficacy of Mycobacterium bovis BCG vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein

Abstract

Tuberculosis continues to ravage humanity, killing 2 million people yearly. Most cases occur in areas of the world to which the disease is endemic, where almost everyone is vaccinated early in life with Mycobacterium bovis BCG, the currently available vaccine against tuberculosis. Thus, while more-potent vaccines are needed to replace BCG, new vaccines are also needed to boost the immune protection of the 4 billion people already vaccinated with BCG. Until now, no booster vaccine has been shown capable of significantly enhancing the level of protective immunity induced by BCG in the stringent guinea pig model of pulmonary tuberculosis, the "gold standard" for testing tuberculosis vaccines. In this paper, we describe a booster vaccine for BCG comprising the purified recombinant Mycobacterium tuberculosis 30-kDa protein, the major secreted protein of this pathogen. In the guinea pig model of pulmonary tuberculosis, boosting BCG-immunized animals once with the 30-kDa protein greatly increased cell-mediated and humoral immune responses to the protein in three consecutive experiments. Most importantly, boosting BCG-immunized animals once with the 30-kDa protein significantly enhanced protective immunity against aerosol challenge with highly virulent M. tuberculosis, as evidenced by a significantly reduced lung and spleen burden of M. tuberculosis compared with those for nonboosted BCG-immunized animals (mean additional reduction in CFU of 0.4 +/- 0.1 log in the lung [P = 0.03] and 0.6 +/- 0.1 log in the spleen [P = 0.002]). This study suggests that administering BCG-immunized people a booster vaccine comprising the 30-kDa protein may enhance their level of immunoprotection against tuberculosis.

FIG. 1.

Cutaneous DTH after immunization. Guinea pigs in groups of six were sham-immunized (Sham), immunized with only BCG or rBCG30, or immunized first with BCG or rBCG30 and then boosted with r30. Ten weeks later, the animals were skin tested with an intradermal injection of r30, and the extent of induration was measured after 24 h. One method was used to measure induration in experiment 1, and a different method was used in experiments 2 and 3, as described in the text. Data are the mean diameter ± SE. In experiment 3, BCG- or rBCG30-immunized animals were boosted with r30 in the presence or absence of adjuvant (no Adj), as indicated.

FIG. 2.

Antibody titer after immunization. Immediately after the skin test was read in the animals described in Fig. 1, three to five of the animals in each group were euthanized and their serum assayed for titer of antibody to r30 by ELISA. Data are the reciprocal antibody titer for each individual animal (closed circles) and the geometric mean titer (bar) for each group. For statistical purposes, titers of ≤125 are scored as 125.

FIG. 3.

Weight loss after M. tuberculosis challenge. Guinea pigs were sham immunized (Sham), immunized with only BCG or rBCG30, or immunized first with BCG or rBCG30 and then boosted with r30. Ten weeks later, the animals were challenged by aerosol with highly virulent M. tuberculosis and weighed weekly for 10 weeks. An additional group of control animals was not challenged but weighed weekly (Uninfected). Data are the mean net weight gain or loss ± SE for each group of animals compared with their weight immediately before challenge.

FIG. 4.

Organ burden of bacteria after M. tuberculosis challenge. At the end of the 10-week observation period, the challenged animals analyzed in Fig. 3 were euthanized, and numbers of CFU of M. tuberculosis in the lung and spleen were determined. Data are the mean ± SE for all animals in a group. The lower limit of detection was 1.0 log/organ in experiments 1 and 2 and 2.0 logs/organ in experiment 3.