The genetic and phenotypic differences that have been identified among M. tuberculosis
isolates during the past decade have raised concerns that the protective responses elicited by new TB vaccines may be strain specific and, therefore, that these novel TB vaccine preparations may not protect against all M. tuberculosis
strains. In fact, it has been speculated that the geographic variability in the efficacy of BCG vaccination may be due to BCG's inability to protect against the various types of M. tuberculosis
strains that are endemic in specific regions of the world (1
). Consistent with this hypothesis, intriguing studies in mice and rabbits have suggested that BCG vaccination is not effective at controlling infections by M. tuberculosis
W-Beijing strains (19
). Selected epidemiologic data have also predicted that the W-Beijing strains may be resistant to BCG-induced protective immunity (1
). However, in contrast to these findings, the results of our experiments did not support the hypothesis that specific M. tuberculosis
strains are resistant to the anti-TB immunity evoked by BCG. In our studies, mice immunized with BCG were protected following aerosol infections with a classic laboratory strain, four Beijing clinical isolates, and four non-Beijing strains. After aerosol infections with all nine of these strains, statistical differences in the lung bacillary burdens and the lung inflammation values were detected between vaccinated and naïve mice at the 4-week time point. Relative growth of the infecting organisms was also statistically lower in BCG-vaccinated animals, relative to naïve mice, for six of the strains (including the Erdman strain and three of the W-Beijing isolates) at 12 weeks postchallenge. Interestingly, BCG immunization induced better protective responses against two of the Beijing lineage strains than against the standard Erdman strain at the 12-week time point. Although pulmonary bacterial burdens were reduced in vaccinated mice only when they were challenged with three of these strains at 20 weeks postchallenge, significant differences in the lung inflammation values and lung pathology between vaccinated and naïve animals were detected at the end of the study for all of the test strains. Importantly, the BCG-induced protection against two of these strains, as measured by decreases in pulmonary mycobacterial growth and reductions in lung pathology, correlated with extended survival periods for the vaccinated animals reported in earlier studies. Mice immunized with BCG and then aerogenically challenged with either the virulent M. tuberculosis
HN878 or Erdman strains survived significantly longer than naïve controls infected with the same virulent strains (6
). Additionally, in this study, while all naïve mice infected with the NY669 strain died by 20 weeks postchallenge, all of their BCG-immunized counterparts survived until the 20-week time point.
The factors that have contributed to the different BCG immunization protection results seen in our study compared to earlier published reports remain uncertain but may include differences in animal models, M. tuberculosis
challenge methods, lung pathology analyses, and strains used for infection and vaccination. Regarding the strains, various production methods can yield mycobacterial strain preparations with contrasting immunogenic activities because of different ratios of live and dead organisms, different bacterial concentrations, and altered surface compositions. For example, the failure to standardize production protocols can result in M. tuberculosis
challenge strains with inconsistent levels of virulence. In a previous comparative study, the use of a subpotent M. tuberculosis
Erdman preparation for murine infections led to improper initial assessments of the virulence of the CDC1551 strain (18
). By contrast, the impact of immunizing with various live attenuated vaccine strains (including different BCG preparations) is unclear. Although different BCG strains have been shown to induce unique immune responses in animal models and humans, both preclinical study results and data from clinical trials have strongly suggested that different BCG preparations usually yield similar levels of protection when given at equivalent doses by the same route of administration (4
). In fact, we have shown that the BCG Pasteur and SSI BCG strains induce statistically indistinguishable protective responses against the M. tuberculosis
HN878 and M. tuberculosis
Erdman strains (S. Derrick, unpublished data). To reduce this strain heterogeneity with the aim of improving the TB vaccine testing process, our laboratory has collaborated with the World Health Organization to provide standard BCG vaccine preparations and M. tuberculosis
challenge strains to researchers throughout the world. Overall, the availability of these reference strains has facilitated and improved the comparative evaluation of new TB vaccines.
In recent reports, phenotypically different M. tuberculosis
isolates have been shown to have various levels of virulence in animal models (9
). In our experiments, the growth profiles for eight of nine M. tuberculosis
strains were similar, and the pulmonary CFU values for most of these isolates at 20 weeks postchallenge were nearly equivalent. However, the importance of the number of culturable M. tuberculosis
bacilli in the lungs at several months postinfection is unclear, and whether the lung CFU values correlate with virulence (as measured in survival studies) is uncertain. In an earlier report, North et al. concluded that the growth rate of mycobacteria in mice is not a reliable indicator of mycobacterial virulence (27
). More recently, Palanisamy et al. showed that organ pathology is a better correlate of virulence than the number of viable organisms in animal tissues (30
). At 16 or 20 weeks postinfection in our study, five of the test strains had elevated lung inflammation values including three strains—HN878, NY669, and Erdman strains—that have been shown to be virulent in mice in this and earlier studies (6
). Moreover, the modestly virulent CDC1551 strain was among the isolates with low inflammation values at the later time points (18
). Therefore, our data suggest that virulence may correlate with the extent of postinfection lung pathology but is not necessarily directly related to pulmonary CFU levels. Obviously, survival studies involving all of the strains in this study are needed to further support the association between virulence and lung inflammation.
In sum, we have evaluated nine different M. tuberculosis strains in a mouse model of pulmonary TB and have shown that while organ mycobacterial growth profiles were generally similar for eight of nine strains, various lung pathological responses were induced after the infection. Most importantly, we showed that BCG vaccination induced significant protective immune responses against all of these strains including four W-Beijing strains. The levels of BCG-induced protective immunity against the eight clinical isolates and the standard Erdman strain were generally similar, especially at the 4-week time point. Overall, we could not demonstrate the strain-specific resistance to BCG-induced protective immunity in our mouse model that has been suggested by other studies. It should be noted, however, that the specific protective immune responses induced by live attenuated vaccines such as BCG vaccine may differ from the protective immunity induced by protein-based, viral vectored, or DNA vaccines against TB. Further studies are needed to assess whether the anti-TB immune responses induced by nonliving TB vaccines also protect against various M. tuberculosis phenotypes.