The ability of M. tuberculosis
to manipulate the host's immune response is not, of course exclusively one-sided and the host can respond effectively, as proved by the fact that most individuals infected with the bacteria do not succumb to the disease. More encouragingly, increasing the host's immune response by vaccination is also possible, as shown by the partial success of BCG vaccination. The use of the word “partial” to describe BCG's effect is based on its very variable efficacy in different trials (46
). In the developed world, BCG has generally shown very high and consistent efficacy, and it is often credited with helping end the TB epidemics in Europe and Japan (60
). In contrast, its effect in developing countries has been far less consistent (in some studies, the vaccine had no measurable effect on TB incidence at all). The reasons for this may have more to do with vaccine trial design and the local environment than the vaccine itself (47
The implications of this for vaccine design are clear: if BCG vaccination at birth can give only short-lived immunity, the options are either to replace BCG with a vaccine that gives a longer duration of protection or to design a vaccine that can be given at a later time point to boost existing immunity and provide protection in adults. Both of these approaches have advantages and disadvantages, and the vaccines now entering clinical trials include proponents of both approaches. However, one important caveat of administering the vaccine at late time points (referred to as the late booster strategy) is the fact that in highly TB-endemic regions such a vaccine will in many cases be given to already latently infected individuals. It is therefore necessary not only to develop such a vaccine as a BCG booster vaccine but also as a postexposure vaccine -an approach that brings its own set of problems and challenges. Eventually it is possible that the only practical outcome might be a hybrid approach, what can be termed a multiphase vaccine that can be administered regardless of the infectious status of the individual and with activity both in naïve and already-infected individuals (Fig. ).
Replacing BCG (Priming Vaccines)
Replacing BCG will not be an easy task; it is perhaps the most used vaccine in the world, and despite its limitations, is cheap, safe, and well established. As mentioned above, neonatal vaccination with BCG seems to consistently provide significant protection against the most severe childhood manifestations of the disease, such as TB meningitis. However, most studies have reported that the efficacy of BCG lasts for no more than 10 to 20 years (28
). In Europe, where BCG vaccination was combined with campaigns to identify and isolate adults with TB in sanatoria (or once chemotherapy was developed, to treat them), this appears to have been enough to cause a rapid decrease in incidence. However, in much of the world where TB remains endemic, the resources to track and isolate or treat new cases are lacking and BCG vaccination alone has proven unable to decrease the number of cases in adults. Thus, in hyperendemic regions such as crowded shantytowns and urban slums, the incidence is so high (in the worst cases, reaching 1,000 per 100,000) that most adults are continually exposed to infection. In such an environment, any individual without solid immunity would soon be infected. To prevent this with vaccination alone requires the stimulation of an immune response that persists for decades.
It remains unclear if this is a realistic goal. Animal models (and current research funding paradigms) are ill suited to measure vaccine efficacy over years or even decades. That means that what is most often attempted prior to human trials is to exceed the efficacy of BCG in short-term experiments, in the hope that a stronger primary immune response will also lead in turn to a longer-lasting memory response. In contrast to its record in human trials, BCG has generally provided high levels of protection in animal models (in particular in the guinea pig model) of TB (152
) and although considerable progress has been made in the development of efficient subunit vaccines, there is still no reported evidence for efficacies exceeding that of BCG in naïve individuals. Therefore, by this measure, the vaccines that have showed most promise so far as a replacement for BCG are to be found among the live mycobacterial vaccines.
The first of these to enter the clinic, the rBCG30 vaccine, has recently passed phase I clinical trials (67
). This vaccine was derived from a vaccine strain of BCG, which has been genetically modified to overexpress the immunodominant antigen Ag85B (67
). The idea behind this vaccine was to improve BCG by expressing higher levels of an antigen that had already been shown to be protective. Furthermore, based on the observation that BCG appeared to perform well in the earliest recorded trials but poorly in later trials (25
), it was suggested that “modern” strains of BCG were overattenuated and had reduced immunogenicity due to a continued loss of genes from the vaccine stock (10
). This may help explain the long period of protection afforded by BCG found on reanalysis of early vaccination campaigns (6
While it could be argued that variations in efficacy could be explained by the fact that later trials were done under different circumstances and in different populations from the earlier trials, two lines of research support the “attenuation” hypothesis. The first is the rBCG30 vaccine itself: it was reported as having a clearly improved efficacy compared to the parental vaccine strain (67
). The improved efficacy of rBCG30 came as something of a surprise, since BCG possesses a functional gene for Ag85B. However, recent work has shown that overexpression of genes can alter the immune response to the antigens they encode (127
), reminding us that the mere presence of a gene tells us little about its expression in vivo and that a gene's effects may be modulated by the presence or absence of other genes.
The second piece of supporting evidence is the observation that new vaccines based on attenuation of Mycobacterium bovis
(the parental strain of BCG) are both more virulent and give greater protective efficacy than BCG (34
). In addition, highly attenuated mutants of M. tuberculosis
, though still protective when used as vaccines, have proven less virulent—and less protective—than BCG (137
). These results are compatible with the contention that BCG has become “too attenuated.” Building on the same concept, BCG into which the RD1 locus was reintroduced (allowing it to secrete the ESAT-6-CFP10 complex) was reported to be both more virulent but also more protective than the parental BCG strain (124
). Interestingly, this vaccine strain (designated BCG::RD1) has recently been shown to trigger an immune response that is qualitatively more similar to that of M. tuberculosis
than BCG. Specifically, vaccination with BCG::RD1 induced the recruitment of activated/effector T cells and dendritic cells to the lungs of subsequently infected mice far more efficiently than the parental BCG strain (99
). This suggests that antigens encoded by RD1 (and lost during the attenuation of BCG) can significantly modify the type of immune response generated by a mycobacterial vaccine or that a certain level of virulence (perhaps more than that of current vaccine strains) is necessary for the optimal induction of strong immune responses by live vaccines. Efficiency aside, it is questionable if such hypervirulent vaccine strains would have the safety profile necessary for mass vaccination, especially in areas of high human immunodeficiency virus incidence (77
The last of the BCG-based vaccines which is near clinical testing takes a different and more technically sophisticated approach. The rBCG::ΔureC-llo+
vaccine is a urease-deficient BCG mutant which expresses the listeriolysin O gene from Listeria monocytogenes
. These bacteria are unable to arrest phagosome maturation, due to the urease deficiency, and are less virulent than wild-type BCG in immunodeficient scid
mice. This may make it safer in populations where human immunodeficiency virus infection is widespread (which, of course, also tend to be the areas where TB incidence is highest), and where the BCG vaccine can cause occasional cases of disseminated BCGosis (21
). In addition, the decreased pH in the maturing phagosome provides optimal conditions for the listeriolysin, which is thought to damage the phagosome membrane, allowing leakage into the cytosol and potentially increasing the amount of bacterially derived antigen available for presentation to CD8 T cells via the cytosolic scavenger pathways as previously described (29
Other groups have attempted to make new vaccines by attenuating M. tuberculosis
instead of augmenting gene expression in BCG, reasoning that this would give the closest simulation of natural immunity occurring after M. tuberculosis
infection. However, as noted above, M. tuberculosis
is remarkably adept at escaping eradication by the immune response; even a cured natural M. tuberculosis
infection does not necessarily confer life-long protection (32
). It should also be noted that (in animal models) the protection induced by a cured M. tuberculosis
infection is not always superior to that from BCG vaccination of a naïve recipient (108
). Nonetheless, at least two TB vaccines based on attenuated M. tuberculosis
are under development. One of the most important issues such a strategy needs to address is the risk that a vaccine strain based on M. tuberculosis
could revert to a virulent form, and obviously the question of how much attenuation is sufficient (18
The preferred approach has been to make mutations in two crucial genes to reduce the potential for reversion to a virulent form. One such vaccine, developed at the Albert Einstein Hospital in New York, a PanD−
auxotroph of M. tuberculosis
), has demonstrated both safety in immunodeficient scid
mice and protective efficacy in the highly susceptible guinea pig infection model. It is hoped that this vaccine will enter clinical trials within the next 12 months. The ΔphoP
/R mutant, in which the phoP
virulence factor is inactivated by the insertion of an antibiotic gene, is another vaccine that has showed significant promise in animal models (128a
). The hope is that these vaccines will generate better, perhaps longer-lasting immune responses than BCG, but with the possibility of boosting that immunity at a later stage, if necessary. However, if boosting is necessary, these vaccines are unlikely to be successful in that role.
Like BCG, all of these vaccines are intended to be given to immunologically naïve recipients (essentially, that restricts their use to neonates in developing country settings). Boosting BCG-generated immunity by a second dose of BCG given at a later time point has repeatedly failed to show any beneficial effect (89
). This failure is consistent with results from animal studies, which have shown that BCG (and therefore presumably any mycobacterial live vaccine) requires a period of multiplication and dissemination in the host to stimulate a protective immune response. If this is blocked by chemotherapy shortly after vaccination (51
), then the protective effect of vaccination is abrogated. An existing immune response (whether due to prior BCG vaccination or cross-reactive immune responses arising from exposure to other mycobacteria in the environment) appears to have the same effect, leading to the rapid clearance of BCG (11
). This may explain both the demonstrated effect of BCG vaccination in neonates and in adult populations where skin-test-positive individuals have been excluded (25
) and its failure in trials where adult (mycobacterially sensitized) individuals were included. If, as anticipated, new mycobacterial vaccines are unable to stimulate life-long immunity, neonatal vaccination may need to be augmented by nonmycobacterial vaccines, so-called booster vaccines.
Augmenting BCG (Late Booster Vaccines or Postexposure Vaccines)
In contrast to vaccines designed to replace BCG, late booster vaccines aim to take advantage of the widespread use of BCG by boosting immunity in young adults already primed by earlier vaccination in childhood. Approximately 3 billion people, half the world's population, have received BCG (153
), and most of these live in areas where TB is endemic. Even if the protective effect of the new vaccines lasts no longer than that induced by BCG, it would still greatly reduce the number of new TB cases by reducing incidence over the peak age for TB (25 to 35 years). The “priming/boosting” dichotomy is slightly misleading. Even in areas where BCG vaccination is routine, not every person will be BCG vaccinated and not every vaccinee will have effective immunological memory. So booster vaccines will also need to be able to stimulate effective primary responses as well. However, the requirements are slightly different from those for vaccines intended to replace or compete with BCG for neonatal vaccination.
Whereas a vaccine intended to replace BCG needs to demonstrate superior efficacy to BCG to be seriously considered, booster vaccines are often no more effective than BCG at generating primary immune responses (42
). However, they have the additional requirement that they need to be effective in sensitized as well as in naïve recipients, a test which BCG signally fails (11
). Furthermore, with the high prevalence of latent TB in TB-endemic areas, the vaccines need to be designed with their potential activity in already infected individuals in mind (postexposure vaccines). This may demand the inclusion of a completely different set of gene products to target the genes that may be upregulated by the mycobacteria in response to long-term exposure to the hostile environment of the activated macrophage. This will be dealt with later in this review.
A variety of live vaccines have been developed as booster vaccines, including a recombinant adenovirus (179
) and a recombinant strain of Shigella
designed to act as a delivery system for a DNA vaccine (50
). These vaccines are still in the developmental stage and it is not clear when they will be ready for clinical trials. However, one live booster vaccine is now in clinical trials (104
). This is MVA-85A, a recombinant, replication-deficient vaccinia virus expressing the strongly immunogenic antigen 85A from M. tuberculosis
). The vaccine has performed well in animal models (56
) and now data from the first clinical trials show that it is immunogenic and apparently safe in humans (104
Also among the first wave of new TB vaccines entering clinical trials are products based on recombinant proteins. In the past, recombinant protein vaccines have not been terribly successful at stimulating strong Th1 responses, due to the lack of adjuvants suitable for generating strong cell-mediated immunity responses in humans without generating unacceptable side effects (58
). For many years, the only adjuvant approved for human use was alum, which was only effective for vaccines that required a humoral response (e.g., diphtheria, tetanus, and hepatitis B vaccines). Indeed, since this adjuvant biases the immune response towards the Th2 pole (14
) it has actually been shown to decrease the protection generated by vaccination against M. tuberculosis
). More recently, MF59 was approved for human use, but this adjuvant has so far also been restricted to vaccines where generating humoral responses is key, such as influenza (123
However, new adjuvants are now entering the clinic (Table ) and several of these generate strong Th1 responses, making them good candidates for vaccines against M. tuberculosis
. These new vaccines owe their success in animal models to an improved understanding of the activation of the immune system in response to conserved molecules on pathogens (41
) being built around bacterially derived lipids, bacterial toxins, or analogues of these molecules. In some cases (monophosphoryl lipid A, for example) the receptors are known and are part of the Toll-like receptor family. In other words, we are starting to use some of the same techniques to manipulate the immune responses that M. tuberculosis
uses which were described above. We can expect to see more vaccines using this approach in the future, since both recombinant antigens and the adjuvants can easily be synthesized on a large scale, lending themselves to vaccine production.
Adjuvants for use with human subunit vaccinesa
The first of these subunit TB vaccines to enter clinical trials is the 72f vaccine (150
), jointly developed by Corixa and GSK. This vaccine is a fusion molecule comprised of two proteins, with the PPE family member Rv1196 inserted into the middle of the putative serine protease Rv0125, which is thus present as two fragments. The adjuvant used contains the saponin derivative QS21 mixed with the TLR4 ligand monophosphoryl lipid A (150
). In addition to its activity for priming, this vaccine has also been demonstrated to have a BCG booster effect (12
). The second recombinant protein, although developed independently by the Statens Serum Institute, is very similar in its design philosophy. It is a fusion molecule comprised of two immunodominant, secreted proteins from M. tuberculosis
(ESAT-6 and Ag85B) and has proven highly efficacious in animal models ranging from mice to primates, more effective in fact, than the single antigens (42
). The vaccine has also proven effective as a booster for BCG and augments its efficacy even though the ESAT-6 component of the vaccine is not present in BCG (authors' unpublished data).
The Ag85B-ESAT-6 fusion protein is slated for two clinical trials in 2005-2006. The first of these will test the vaccine in a conventional parenteral vaccination strategy, using a mixture of oligodeoxynucleotides and polycationic amino acids as the adjuvant (93
). The second trial, running in parallel, will test the same antigen by the nasal route, using LTK63, a modified, heat-labile enterotoxin from Escherichia coli
as an adjuvant (121
). ESAT-6 is currently also being applied as a diagnostic tool to detect cell-mediated immunity responses that specifically signal the presence of ongoing infection (40
). New fusion molecules that do not contain ESAT-6 are therefore the subject of ongoing research and interestingly, the only antigen so far found which may be able to replace ESAT-6 is also a member of the same small gene family but is, in contrast to ESAT-6, present in BCG (39
In some ways these two recombinant vaccines can be viewed as M. tuberculosis in miniature—they contain two immunodominant antigens and use as adjuvants modified molecules derived from human pathogens that stimulate Th1 responses. This minimalist approach offers certain advantages. By choosing a limited number of carefully tested immunodominant antigens, we should generate a strong protective response, while avoiding the possibility of inducing unwanted modulation of the response or of antigen presentation. By using adjuvant molecules (slightly modified to decrease their toxicity) derived from human pathogens, we should generate exactly the type of inflammatory response the body normally invokes to deal with a bacterial infection.
All of the vaccines described above are prophylactic vaccines, intended to be given to individuals prior to M. tuberculosis
infection, and hopefully prior to exposure to the disease. It is not clear whether they will be efficacious or even safe if given to individuals already infected with M. tuberculosis
. For booster vaccines this is a serious issue, since in TB-endemic areas, the target population can be expected to include many latently infected subjects. What few data are available from animal studies do not suggest that the leading vaccine candidates induce a Koch phenomenon-like effect in infected recipients, but neither do they suggest these vaccines are effective against the latent phase of the disease (167
; authors' unpublished work). It is likely that handling the billions who are potentially latently infected with M. tuberculosis
will require a combined strategy, using a multiphase vaccine effective against both acute and latent infection (Fig. ).
The acute phase of M. tuberculosis
infection is characterized by rapid bacterial growth and the development of an immune response targeted towards bacterial antigens actively secreted in the first growth phase, such as ESAT-6 (154
). The vaccines so far developed against these acute-phase antigens can reduce the severity of the initial bacteremia and disease but they do not prevent the establishment of infection (42
). However, as M. tuberculosis
adapts to the hypoxic and hostile environment of host macrophages, it has been shown to undergo a dramatic change in gene transcription thought to be characteristic of latency (65
) and this change in gene expression may enable the pathogen to persist in the face of strong memory immune responses. This latent stage of infection is normally associated with a few bacteria surviving in a so-called “dormant state” with low or altered metabolic activity.
When grown in vitro under conditions mimicking those thought to exist in vivo, M. tuberculosis
up-regulates overlapping, characteristic sets of genes (145
). Increased expression of genes such as HspX (also called α-crystallin, Rv2031c, etc.) during the stationary growth phase (192
) appears to be crucial for the survival of the organism (193
). In agreement with these in vitro observations it has been demonstrated that the transcription of a number of genes is down-regulated while others are strongly up-regulated after the initiation of a strong immune response in infected animals (146
). Together with the abundance of regulatory proteins in the M. tuberculosis
), this indicates the importance of the pathogen's being able to adapt to different environments during infection. This regulatory flexibility may underlie its ability to shift between acute progressive disease and long-lived latent infection.
In addition to the experimental evidence described above, studies in humans are beginning to offer evidence to support the hypothesis that immune responses in healthy but latently infected individuals are targeted to different antigens than the response to an acute infection (Demissie et al., submitted; Klein et al., submitted). It is hoped that vaccinating with other antigens, such as HspX, identified by the in vitro methods described above may promote an immune response that will help prevent reactivation of disease. There is some evidence that such an approach may indeed work. Lowrie and colleagues successfully employed an HSP65-based DNA vaccine in latently infected mice (95
), but this finding has been somewhat controversial (168
) and emphasizes the need for more research in this field, in particular, the need for rigorously validated animal models.
One area attracting such interest is the “resuscitation-promoting factor” or rpf
genes. Micrococcus luteus
expresses an rpf
gene whose product is required to resuscitate the growth of dormant cultures of Micrococcus luteus
and which is essential for the growth of this organism. It has recently been demonstrated that M. tuberculosis
has at least five rpf
), again suggestive of the importance of latency and resuscitation to the survival of M. tuberculosis
. Given that they appear to be essential for recovery from the dormant state and are immunogenic, secreted antigens, they make tempting targets for vaccination (190
The postexposure vaccination area is now being intensively pursued, and although a vaccine is still in the future, it offers the possibility of a multiphase vaccine that induces responses both against acute phase antigens, thus providing protection if the recipient is subsequently infected, and against latency-associated antigens, so that disease is held in remission even if the vaccine recipient is already infected.