Recombinant bacillus calmette-guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model

Abstract

Tuberculosis (TB) continues to ravage humanity, causing 2 million deaths per year. A vaccine against TB more potent than the current live vaccine, bacillus Calmette-Guérin (BCG), is desperately needed. Using two commercially available strains of BCG as host strains, BCG Connaught and Tice, we have constructed two recombinant BCG vaccines stably expressing and secreting the 30-kDa major secretory protein of Mycobacterium tuberculosis (M. tb.), the primary causative agent of TB. We have tested the efficacy of the two strains in the highly susceptible guinea pig model of pulmonary TB, a model noteworthy for its close resemblance to human TB. Animals immunized with the recombinant BCG vaccines and challenged by aerosol with a highly virulent strain of M. tb. had 0.5 logs fewer M. tb. bacilli in their lungs and 1 log fewer bacilli in their spleens on average than animals immunized with their parental conventional BCG vaccine counterparts. Statistically, these differences were highly significant. Paralleling these results, at necropsy, animals immunized with the recombinant BCG vaccines had fewer and smaller lesions in the lung, spleen, and liver and significantly less lung pathology than animals immunized with the parental BCG vaccines. The recombinant vaccines are the first vaccines against TB more potent than the current commercially available BCG vaccines, which were developed nearly a century ago.

Figure 1

rBCG30 secretes a large amount of r30 even in the absence of selective pressure. Culture filtrate proteins of the wild-type and recombinant BCG Conn strains (Left) and Tice strains (Right) grown for 4 weeks were subjected to SDS/PAGE (lane 1) or immunoblot analysis (lanes 2–4). The rBCG30 strains were subcultured in the presence of hygromycin three times over 12 weeks (lanes 2) or in the absence of hygromycin either three times over 12 weeks (lanes 3) or six times over 24 weeks (lanes 4). By densitometry, the relative amounts of the 30-kDa protein in the four strains were BCG Conn, 1.00; rBCG30 Conn, 2.03; BCG Tice, 1.10; rBCG30 Tice, 5.98.

Figure 2

rBCG30-immunized animals exhibit strong, cutaneous DTH to r30. Guinea pigs were sham-immunized (Sham) or immunized with purified r30, wild-type BCG (Conn or Tice, as indicated), or rBCG30 (Conn or Tice, as indicated) and skin-tested with an intradermal injection of r30. The extent of induration was measured after 24 h. Data are the mean diameter + SE. In experiments 1, 2, and 3, differences between the BCG Conn and rBCG30 Conn were significant at P = 0.03, P = 0.01, and P < 0.0007, respectively, by ANOVA or K-W methods (P < 0.0001 for all three experiments combined). In experiment 3, differences between BCG Tice and rBCG30 Tice were significant at P < 0.0001 and differences between rBCG30 Conn and rBCG30 Tice were significant at P < 0.02 by either statistical method.

Figure 3

Animals immunized with recombinant or wild-type BCG are protected against weight loss after challenge with M. tb. Animals in the immunization groups described in Fig. 2 were challenged with M. tb. by aerosol and weighed weekly for 10 weeks. In experiments 1 and 2, an additional group of control animals was not challenged but weighed weekly (Uninfected controls). Data are the mean net weight gain or loss ± SE for each group of animals compared with their weight immediately before challenge. In experiment 3, daggers (†) indicate deaths of four sham-immunized animals during week 8.

Figure 4

Animals immunized with rBCG30 have significantly fewer M. tb. bacilli in the lung and spleen than animals immunized with BCG. At the end of the 10-week observation period, challenged animals were killed, and CFU of M. tb. in the lung and spleen were assayed. Data are the mean ± SE for all animals in a group (see Table 1). The lower limit of detection was 2.0 logs/organ (1 CFU on a plate seeded with an undiluted 1% sample of an organ, i.e., 100 μl of a total sample volume of 10 ml). In experiment 3, one spleen culture from the rBCG30 Conn and three spleen cultures from the rBCG30 Tice group had 0 CFU on plates seeded with undiluted samples. For statistical purposes, these three organs were scored as 2.0 logs.

Figure 5

Animals immunized with rBCG30 have fewer and smaller lesions in their lung and liver than animals immunized with BCG. These photographs depict a representative, formalin-treated left lung and liver from a sham-, BCG Tice-, and rBCG30 Tice-immunized animal. The organs of the sham-immunized animal are peppered with tubercles (arrows), many of which have coalesced into large lesions (arrowheads). The organs of the BCG and rBCG30 immunized animals have progressively fewer and smaller lesions.