The results of this study show that mice vaccinated with BCG and then challenged with M. tuberculosis
H37Rv are significantly protected, as anticipated, and that this protection is sustained. In contrast, however, while mice infected with the W-Beijing strains HN878 and SA161 were equally well protected on day 30 post-aerosol infection, by day 60, the numbers of CFU in the lungs of control and vaccinated mice were not significantly different. Examination of the lungs of these animals showed an initial reduction in inflammation and lung consolidation and evidence of a better lymphocyte influx, as is usually seen when a vaccine is protective, but these elements were gradually lost as the disease process continued. Thus, while BCG vaccination clearly had an initially positive protective effect against the two W-Beijing strains tested here, this effect was transient and resulted only in a degree of prolonged survival rather than long-lived protection. Many of the W-Beijing and other families of strains we have now tested in our laboratory are of equal or even far higher virulence than the HN878 and SA161 strains used here, and this raises the possibility that BCG-based vaccines may be ineffective in areas of the world where W-Beijing and potentially other families of high-virulence isolates are of increasing prevalence. Our serious concerns on this matter are discussed in more detail elsewhere (24
Analysis of effector T cell responses in animals with prior BCG vaccination and infected with HN878 and SA161 showed evidence of a considerable expansion of CD4+
effector T cells, but this decayed significantly after 20 to 30 days. Given our earlier observation that mice infected with HN878 potently induce CD4+
regulatory T cells (22
), we further investigated this possibility and found evidence of a progressive increase in this cell population during the course of both W-Beijing infections, concomitant with worsening of the disease process. In mice infected with HN878 and SA161 that were first vaccinated with BCG, the expansion of CD4+
cells was minimal on day 30, but thereafter began increasing and peaked on day 60. In SA161-infected mice, robust increases were observed for all the organs in control and BCG-vaccinated animals, with levels of such cells being only somewhat lower in the vaccinated mice on day 60. This may suggest that regulatory T cells are induced by the highly virulent, highly inflammatory SA161 infection very early on in the disease process, and indeed the capacity of the animal to rapidly generate such cells has recently been observed by others (30
). Overall, these observations are thus consistent with the hypothesis that while BCG vaccination might reduce or delay the emergence or influx of CD4+
regulatory T cells in response to these W-Beijing infections, it cannot prevent this from eventually happening.
As for TH17 cells, regarded by some studies as a counterbalance, results were more ambivalent. In HN878-infected mice, both control and vaccinated mice exhibited a steady influx of CD4+
cells, whereas this rate of cellular influx was initially much higher in the lungs of BCG-vaccinated mice infected with SA161. Studies by others (15
) have suggested that vaccination induces a population of TH17 cells that facilitate the influx of memory immune effector T cells in tuberculosis by accelerating the needed chemokine response. If so, then this does not explain the low survival rates seen for BCG-vaccinated mice exposed to SA161 compared to those of animals infected with HN878, in which the TH17 response in the lungs was not potentiated by prior vaccination. Since both groups of animals were vaccinated with the same inoculum of BCG, then this result implies not only that individual isolates may differ in their capacity to trigger the lung TH17 response but also that a strong TH17 response, as seen to SA161, might induce increased CD4+
expansion to counterbalance the proinflammatory effects of IL-17 cells and thus somewhat decrease the subsequent survival seen for these animals.
At this time, we can only conclude that the expansion or influx of CD4+
regulatory T cells seems to parallel the decline of the more rapidly emerging effector T cell response to the two W-Beijing strains. Further studies involving regulatory T cell depletion are under way in our laboratory and will help further address this possibility. Our current working hypothesis is that the emergence of the CD4+
population may be directly in response to lung inflammation and damage mediated by these two clearly highly virulent clinical strains, with suppression or at least interference with the existing protective effector T cell response as an unfortunate side effect. Given our observation (27
) that many of the newly emerging strains are capable of inducing severe lung damage, this is of great concern, especially if prior BCG vaccination as seen here can only slow this process down but not eventually prevent it.
Our observations here regarding the effects of BCG on the growth of clinically relevant isolates in the mouse lung are highly consistent with observations by others. Jeon et al. (14
) tested a panel of clinical isolates in the mouse model and found that BCG was protective in mice infected with HN878 at 4 weeks, but this protection was then rapidly reduced. This study also examined the protective activity of BCG vaccination against several other clinical strains. In three examples, BCG protection remained relatively sustained after infection with the clinical strains, but protection declined substantially when other strains were used. Similarly, Grode et al. (11
) demonstrated that BCG was only transiently protective against a modestly virulent W-Beijing strain and, interestingly, a recombinant BCG vaccine was more potent.
Our results are completely inconsistent, however, with the results of studies published by Sun et al. (34
). In an earlier study (22
), we clearly demonstrated that mice infected with HN878 by low-dose aerosol survived on average about 80 days, dying from the substantial lung pathology similar to that shown above. In the current study, we further demonstrated that BCG was able to slow the growth and subsequent lung pathology caused by the two W-Beijing strains, resulting in prolonged survival of these animals but no evidence of sustained protection. In contrast, Sun et al. (34
) reported the mean survival of control mice infected with HN878 as being close to 300 days, an observation inconsistent with those in other reports demonstrating the high virulence (“hypervirulence”) of HN878 (20
). In the Sun et al. study, BCG extended survival to ~375 days, and this was improved further to ~410 days in mice vaccinated with a lead recombinant BCG vaccine candidate.
The Sun et al. study and the induction of (negative regulator) CD8αα T cells in vaccinated macaques (18
) support the usage of recombinant BCG vaccines, and these vaccines are the current focus of vaccine development today (2
). As we have discussed elsewhere (25
), one can make a series of arguments on why this may be a serious mistake. In addition to our data here that indicate that BCG-based vaccines will not establish immunity capable of overcoming the regulatory T cell response many of the virulent clinical strains seem to potently generate, we have also recently suggested other caveats. The first is that BCG seems to very poor at generating central memory (12
); the consequence of this is that while the effector memory response is faster than primary immunity, it still takes a finite time to be expressed (7
) and this probably still allows virulent strains enough time to grow to the point that the regulatory T cell response can then get triggered. Thus, a vaccine candidate designed to induce central memory might be more effective, and one such example is a recombinant M. smegmatis
(“Δike-plus”), which in a protective study (35
) and in preliminary studies in our hands seems capable of inducing CD44hi
central memory T cells that expand and respond in as little as 7 days after M. tuberculosis
challenge (I. M. Orme and W. R. Jacobs, unpublished observations); this potentially could be fast enough to contain virulent infections before CD4+
cells become expanded.
A further issue regards the demonstration by Comas et al. that multiple families of M. tuberculosis
have selectively conserved genes that encode T cell epitopes, including those expressed by immunodominant antigens (6
). This is very concerning, because one explanation for this is that it ensures a potent T cell immune response, thus driving the granulomatous process but then also the caseous necrosis which eventually has the potential to lead to cavitation and hence transmission. If so, then overexpressing these antigens in a new recombinant BCG vaccine could potentially cause worsening clinical disease and increased transmission. Moreover, as we recently discussed (39
), such recombinant vaccines would actually look superior in short-term animal model challenge experiments, especially if tested against the less-virulent laboratory strains. To avoid such problems, it might be wiser to focus on vaccines that express other protective antigens but not the primary dominant antigens, for example, ID93 (3