A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis

Abstract

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine, Mycobacterium bovis BCG, is urgently needed. We describe a recombinant BCG vaccine (rBCG30) expressing and secreting the 30-kDa major secretory protein of Mycobacterium tuberculosis, the primary causative agent of TB, that affords greater survival after challenge than parental BCG in the highly demanding guinea pig model of pulmonary TB. Animals immunized with rBCG30 and then challenged by aerosol with a highly virulent strain of M. tuberculosis survived significantly longer than animals immunized with conventional BCG. The parental and recombinant vaccine strains are comparably avirulent in guinea pigs, as they display a similar pattern of growth and clearance in the lung, spleen, and regional lymph nodes. The pMTB30 plasmid encoding the 30-kDa protein is neither self-transmissible nor mobilizable to other bacteria, including mycobacteria. The pMTB30 plasmid can be stably maintained in Escherichia coli but is expressed only in mycobacteria. The recombinant and parental strains are sensitive to the same antimycobacterial antibiotics. rBCG30, the first vaccine against TB more potent than nearly century-old BCG, is being readied for human clinical trials.

FIG. 1.

Plasmid pMTB30. Derived from the mycobacterium-E. coli shuttle vector pSMT3 (13), the plasmid contains a full-length copy of the M. tuberculosis 30-kDa major secretory protein gene and flanking regions, including the 5′ promoter region. The insert is placed into the HindIII and BamHI restriction enzyme sites in the pSMT3 vector in an orientation opposite that of the vector-encoded promoter of heat shock protein 60 (15).

FIG. 2.

rBCG30-immunized animals exhibit strong cutaneous DTH to r30. Guinea pigs were sham immunized (Sham) or immunized with parental BCG 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 diameters (in millimeters) + standard errors (SE). The differences between BCG Conn and rBCG30 Conn were significant at a P value of 0.000007, differences between BCG Tice and rBCG30 Tice were significant at a P value of 0.00001, and differences between rBCG30 Conn and rBCG30 Tice were significant at a P value of 0.0001 by the Kruskal-Wallis nonparametric method. (By analysis of variance, P is <0.0001 for all three comparisons.)

FIG. 3.

rBCG30-immunized animals survive longer than BCG-immunized animals. Animals immunized with parental or recombinant BCG Conn (a) or Tice (b) were challenged with virulent M. tuberculosis and monitored for survival (n = 20 or 21/group). Sham-immunized animals and uninfected animals served as additional controls (n = 12/group). Differences in survival between sham-immunized animals and animals immunized with either one of the parental BCG strains were statistically significant (P = 0.0005 for BCG Conn and P = 0.0009 for BCG Tice, by the log rank test). Differences between animals immunized with BCG Conn and rBCG30 Conn were not statistically significant. However, differences between animals immunized with BCG Tice and rBCG30 Tice were statistically significant (P = 0.0003), as were differences between animals immunized with rBCG30 Tice and rBCG30 Conn (P = 0.007). When all rBCG30-immunized animals (Conn and Tice) were compared with all BCG-immunized animals, differences in survival were also statistically significant (P = 0.0004). Within groups, there was no difference in survival between animals that were skin tested before challenge and those that were not.

FIG. 4.

Parental and recombinant BCG Tice strains are comparably avirulent in guinea pigs. (a) Weight gain after immunization. Guinea pigs were immunized intradermally with 10³ CFU of BCG or rBCG30 Tice and weighed weekly. Both groups (n = 12/group) gained weight normally after immunization. (b) Clearance of vaccine strains. Guinea pigs were immunized intradermally as described for panel a and then euthanized 2 to 26 weeks later (n = 3/group/timepoint), as indicated, and CFUs per organ were assayed in the right lung, spleen, and inguinal lymph nodes. The insets show total CFU per organ at 10 to 26 weeks after immunization plotted against a vertical scale appropriate for low bacterial counts. SE, standard error.