Immunology and Cell Biology (2009) 87, 279286 & 2009 Australasian Society for Immunology Inc.

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REVIEW

TB vaccines: current status and future perspectives

Claus Aagaard, Jes Dietrich, Mark Doherty and Peter Andersen
Vaccines against intracellular pathogens such as Mycobacterium tuberculosis need to induce strong cellular immune responses. Antigen discovery programs have exploited this and used proteome studies and T-cell recognition in PPD-positive individuals to select proteins and after testing for protective efcacy in animals the most promising proteins have been put together in fusion molecules. Three such fusion proteins are currently in clinical trials, the two most advanced have already passed phase I trials and are entering phase II. Immunology and Cell Biology (2009) 87, 279286; doi:10.1038/icb.2009.14; published online 7 April 2009 Keywords: tuberculosis; bacterial; vaccination; BCG; latency

Since the early work on small pox and anthrax vaccines by Edward Jenner and Louis Pasteur, vaccination has been considered the most cost effective control strategy against infectious diseases including tuberculosis (TB). Mycobacterium tuberculosis, the causative agent of TB remains a major worldwide health problem that causes approximately two million deaths annually. The disease can be cured by antibiotic treatment but successful global TB control efforts face many obstacles: the difculty of timely diagnosis, the difculty in reaching the poorest populations where TB is endemic, and the fact that bacterial clearance requires many months of treatment. Vaccination has also been only partially successful, despite the fact that the vaccine against M. tuberculosis, M. bovis Bacillus Calmette-Guerin (BCG), is the most widely used vaccine in the world. In high burden areas, exposure is more or less continuous and so infection in adults cannot be prevented by BCG, which gives protection for only a limited period. Therefore, a new vaccine is urgently needed, that can prevent the estimated 810 million new TB cases per year.1 Further, an estimated 2 billion people are already infected with M. tuberculosis.2 Many of these will develop disease in the future if not treated. This contributes to the complexity of TB control efforts and needs to be considered in TB vaccine design and development programs along with preclinical and clinical considerations. Since 1921, when BCG was introduced, more than 3 billion people have received the vaccine. BCG is cheap and safe and protects children efciently against the early manifestations of TB.36 However, BCG has proven to have limited effect against adult pulmonary TB, particularly in the developing world, apparently because BCG vaccination induces protective memory that lasts for 1020 years.68 The time-frame for the waning of BCG-induced protection through childhood and young adult life coincides with the gradual increase in TB incidence, which in some highly TB-endemic regions, such as sub-Saharan Africa, reaches a peak of 4500 cases per 100 000 individuals in the 25- to 35-year-old age group. As a result, depending on whether they include adults or

not, large, well-controlled vaccine eld trials have given estimates of protection that range from 0 to 80%.911 BCG is a live vaccine and the development of protective immunity after BCG vaccination appears to require vaccine replication in the host, which can be prevented by a pre-existing immune response that can cross-react to BCG.1214 Not surprisingly, therefore, studies have indicated that BCG is ineffective in individuals presensitized to mycobacteria, for example, by exposure to environmental mycobacteria or previous BCG vaccination or M. tuberculosis infection.15 The failure of BCG to replicate in sensitized individuals means that BCG cannot be used as a booster vaccine to counteract the waning effect of the BCG vaccination given after birthso attempts to boost protection by giving multiple doses of BCG have proven ineffective.16 In addition to vaccination, widespread latent TB infection in adults is another signicant barrier to the use of BCG (or BCG-derived) vaccines for boosting immunity. During recent years, the TB situation has even worsened with the advent of M. tuberculosis and human immunodeciency virus (HIV) copandemic and the emergence of multidrug-resistant TB and extremely drug-resistant TB (XDR TB). The threat that drug-resistant strains could become widespread has highlighted the importance of an increased effort against TB. In particular, XDR TB patients are left with treatment options that are much less effective and it is not uncommon that they die even after entering treatment.17 These problems have, therefore, increased efforts to develop novel vaccines that can be used in both sensitized and latently infected individuals, though many daunting technical problems face such attempts. Because of the renewed focus and substantial funding, TB vaccine research has gained momentum in recent years and large antigen discovery programs have provided the basis for several new experimental vaccines that, based on different delivery platforms and prime/ boost strategies, have entered clinical trials.18 In this review we will discuss the complexity of the problems we are facing in our attempts

Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark Correspondence: Dr P Andersen, Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK2300 Copenhagen, Denmark. E-mail: pa@ssi.dk Received 2 February 2009; accepted 15 February 2009; published online 7 April 2009

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to develop a new protein-based subunit vaccine against TB, some of the novel strategies that are presently being pursued, the background for these efforts as well as where we may go from here in terms of new strategies and planned clinical trials. VACCINATION STRATEGIES Pre-exposure vaccination strategies For the development of new protective vaccines against TB it is important to realize that the population can be divided into two main target groups (especially in developing countries) depending on whether they have been infected with M. tuberculosis or not (Figure 1). Both groups could benet from a new protective vaccine against TB but it has been considered unlikely that a single vaccine will be effective in both situations (explained below). As a consequence, there are two vaccination strategies: either a prophylactic vaccine administered before exposure or a postexposure vaccine administered after exposure (Figure 1). One prophylactic vaccine strategy aims at replacing BCG to obtain a longer-lasting and more protective immune response. As it is thought unlikely that a subunit vaccine can replace BCG, this strategy focuses on the development of a novel recombinant BCG or attenuated M. tuberculosis vaccine. The second prophylactic strategy aims at boosting and prolonging BCG-induced immunity in the large group of people that have already been BCG vaccinated. Compared to live mycobacterial vaccines, where it is not clear whether such a vaccine could be virulent enough to overcome the existing immunity due to earlier exposure to environmental mycobacteria or a previous BCG vaccination, subunit vaccine strategies are not complicated by this concern (Figure 2). The obvious choice is, therefore, to use BCG for priming, and a subunit vaccine as a booster, allowing the designers of booster vaccines to take advantage of the widespread use of BCG vaccination and the likelihood that itor a live mycobacterial replacement vaccinewill be in use for many years to come. As a vaccine administered as a booster to children or adolescents may also be given to individuals who did not receive the BCG vaccine, or who received an ineffective BCG vaccination (incorrectly administered, or with vaccine that was

too old or incorrectly stored), a booster vaccine should also be able to prime an effective immune response. As a result, all of the vaccines currently in clinical trials were initially screened in animal models for the ability to prime an efcient protective immune response on their own.19 Several new vaccine strategies involving BCG and subunit vaccines are presently being pursued. Postexposure vaccination strategies Another important target group for a new TB vaccine is the latently infected individuals, who may account for up to a third of the worlds population. This huge number arises because relatively few infected individuals actually develop acute TB after infection. Depending on the hosts immune status, an infection with M. tuberculosis will either develop into clinical disease/active infection or into a latent infection where the mycobacteria are believed to persist in a range of metabolic stages from slowly replicating to practically dormant (Figure 1) and the bias is very heavily tilted towards latent infection. Since the failure of Kochs tuberculin as a therapeutic vaccine against active disease where many of the patients died from treatment with tuberculin,20 vaccination as treatment for active pulmonary TB has been regarded with the greatest caution, and antibiotics are presently the only treatment. However, it is now believed that a vaccination in latently infected individuals with carefully selected antigens will not pose the same safety problems as a therapeutic vaccination against the active form of the disease. Moreover, as non-dividing (or slowly dividing) bacteria have proven difcult to eliminate with antibiotics, vaccination represents a very appealing possibility for the global control of latent TB. However, there are signicant challenges for a postexposure vaccine. As M. tuberculosis adapts to the conditions in the immune

Figure 1 Vaccination strategies. A prophylactic vaccine (currently BCG) is given at birth to prevent infection and clinical disease. Prophylactic vaccination strategies aim at either boosting BCG (BCG booster vaccine) or at replacing BCG (BCG replacement vaccine). A postexposure vaccine is designed to be effective against a latent infection, whereas a therapeutic vaccine targets individuals with active disease. However, presently a therapeutic vaccine given without chemotherapy is not being considered as a realistic option for individuals with active disease. Immunology and Cell Biology

Figure 2 Exposure to mycobacteria sensitizes individuals and generates an immune response that can rapidly eliminate live mycobacterial-based vaccines, such as BCG, before a protective response has been established. As BCG is an attenuated strain, it appears to be less able to resist this response than more aggressively growing pathogenic species.

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host and transforms from a rapidly growing to a state of nonreplicating persistence, these changes in phenotype will be certainly reected in the gene expression prole, and will most likely also lead to a radically different antigenic repertoire. This has been conrmed in human studies showing that the antigens preferentially recognized by the immune system differed in latently infected healthy individuals compared to patients with active disease.21 Proteins that are primarily expressed during the latent stage of infection are obvious candidates for a postexposure vaccine, and screening for recognition of proteins expressed by dormant M. tuberculosis in latently infected individualswhile ongoinghas already identied many potential candidates. The challenge, therefore, is to transform these into a vaccine that effectively protects against reactivation of the disease. Because of the sensibilization due to M. tuberculosis exposure, multiplication of BCG is blocked and BCG cannot effectively protect when used as a postexposure vaccine.14,15,22 For the same reasons it is also unlikely that any new live whole-cell vaccine will be able to establish effective protective immunity. In contrast, subunit vaccines are not restricted by previous immunity to mycobacteria, and their activity in individuals sensitized by previous exposure, such as latently infected individuals, does not appear to be impacted15 (in fact, it may even be enhanced, if it boosts existing immunity) making them highly attractive as postexposure vaccines. In addition, the subunit vaccines under development use either replication-decient viral vectors, or are entirely synthetic, so they pose no safety threat even in HIV-positive individuals and are, therefore, suitable for vaccination programs in TB-endemic regions, where the TB and HIV epidemics are ever more closely intertwined. Immunological challenges for vaccine developers The challenges in the development of a prophylactic TB vaccine are signicant. The detailed mechanisms of the host response to M. tuberculosis, and how M. tuberculosis can counteract the immune system, are still not known. The rst cell to encounter MTB is probably the alveolar macrophage and it is thought that shortly after infection, these antigen-presenting cells migrate to the draining lymph nodes where mycobacterial antigens are presented to T cells, and a T-helper (Th1)-type response is initiated, ultimately resulting in the formation of granulomas in infected organs.2326 The granuloma is a structure of infected macrophages surrounded by primarily CD4 and CD8 T cells, though gd T cells and CD1-restricted ab T cells are also found among the T cells that are involved in the complex immune response generated to contain the infection. Failure to contain the infection may result in caseation and necrosis leading to active clinical disease, release of bacilli and transmission.2730 The T-cell-derived cytokines interferon (IFN)-g and tumour necrosis factor (TNF)-a are central players in the activation of macrophages and containment of M. tuberculosis inside the granuloma.31 The critical role of IFN-g in the control of TB has been clearly demonstrated by the susceptibility to mycobacterial infections of mice with a disrupted IFN-g gene and of humans with defects in IFN-g response or production.3238 As a consequence, the ability to stimulate T-cell release of IFN-g has been used as one of the most important criteria for the initial identication of vaccine antigens in antigen discovery programs. However, it is important to note that a strong vaccine-induced Th1 response (IFN-g release or frequency of IFN-g producing T cells) does not necessarily guarantee a high degree of protection.3943 The ability of a vaccine to induce pluripotent T-cell responses (T cells that produce IFN-g, TNF-a and interleukin-2) has recently been associated with increased protection in a mouse model of Leishmania and has been proposed as

the mechanism for the protective efcacy of M. bovis BCG and the Ag85B-ESAT-6 fusion protein44 (T Lindenstrm, submitted to J Immunol). However, denitive immune correlates of protection in humans are probably only going to emerge in the context of successful vaccine trials, where we can infer the cause and effect. Adding to the task of making a vaccine, several studies indicate that M. tuberculosis actively subverts the hosts immune response at virtually every level and countering the pathogens strategies in the design of novel vaccines is a daunting task. It will require careful design to activate the correct response against important antigenic targets.45 ANTIGEN DISCOVERY Prophylactic vaccines The early attempts to develop a second-generation vaccine against TB used killed bacilli or complex subcellular preparations derived from the bacilli.46,47 However, when tested in animal models these preparations gave rise to strong inammatory responses but very little longterm protection.48 Later, secretory or culture ltrate-based vaccines were found by several groups to impart signicant protection in various animal models.4951 However, the major concern about their use was that these were relatively crude preparations: the antigenic content varied with culture conditions. It could hold both protective as well as numerous inactive or even deleterious molecules, and therefore a more standardized vaccine was needed.52 Consequently, large efforts were focused on antigen discovery programs. As a cellmediated immune response was known to be essential for the control of M. tuberculosis, molecules inducing a strong Th1 cell response during a natural infection were chosen as vaccine targets. Screening for candidates was, therefore, done on the basis of recognition by immune cells in different animal models, or in human subjects during early stages of infection, using biochemical fractionation of M. tuberculosis protein mixtures. The original observations were based on the evaluation of T-cell responses to molecular mass fractions of culture ltrate.53 Most of the dominant T-cell targets from M. tuberculosis that were identied could be grouped into two groups based on size. It was observed in several animal models as well as in TB patients that one immunoreactive fraction contained small proteins with a size below 1012 kDa and the other group contained proteins with a molecular mass of around 3040 kDa.5457 Some of the more prominent antigens found in this fraction included the three members of the Ag85 complex (Rv3804c, Rv1886c, Rv0129c), plus Rv1196/PPE18, Rv0125/PepA, Rv0915c/PPE14 and Rv3616c.58,59 In the lower molecular mass fraction, several proteins belonging to the ESAT-6 family (early secretory antigenic target-6) including ESAT-6/Rv3875, CFP10/ Rv3874, TB9.8/Rv0287 and Mtb9.9A/Rv1793 were isolated.6063 The ESAT-6 family was identied after the completion of the M. tuberculosis genomethis family is not based on homology among the members (which is quite limited) but rather on their organization on the bacterial chromosome where they are found as adjacent pairs close to a PPE or a PE gene.64 The ESAT-6 family holds 23 members, 4 are almost identical paralogues of Rv1793 and 4 other members are paralogues of Rv1792, resulting in a total of 15 different ESAT-6 family proteins encoded from the M. tuberculosis genome. Several of the members identied have been shown to be recognized in TB patients including ESAT-6/Rv3875, CFP10/Rv3874, TB10.4/Rv0288 and Mtb9.9A/Rv1793.63,65,66 A recent study using bioinformatics to select 94 proteins for screening, rst for dominant human T-cell antigens and secondly for protective efcacy in mice, conrmed the strong immunodominance of the 6 ESAT-6 family members, as well as 4 PPE proteins, based on responses in cells from healthy PPD-positive donors.42
Immunology and Cell Biology

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Postexposure vaccines Until recently, little has been known about the conditions that induce dormancy in M. tuberculosis and the bacterial response to those conditions. As mentioned above, it has been known for some time that control of bacterial replication in animal models requires production of IFN-g, TNF-a and nitric oxide.67 Exposure of the bacteria or bacterially infected cells to these agents in vitro leads to a downregulation of genes which are highly recognized by TB patients in the early phase of infectionincluding well-studied immunodominant antigens such as Ag85 and ESAT-6.68 Exposure of the pathogen in vitro to other conditions thought to reect the environment inside the granuloma such as limited access to iron, oxygen or nutrients has a similar effect, Mimicking these conditions and inducing bacterial dormancy in vitro has thus been the subject of intensive research in recent years. O2 depletion has been the most comprehensively studied and it provides a link between the avascular environment of the encapsulated granuloma and the capacity of M. tuberculosis to adapt to hypoxic conditions. Wayne et al. demonstrated in a series of important studies that a gradual depletion of O2 changes bacterial respiration towards nitrate reduction and induces signicant metabolic, chromosomal and structural changes in the bacteria consistent with dormancy.6971 The rst M. tuberculosis gene to be identied as being induced by hypoxia and potentially involved in latency was hspX, (Rv2031) also known as a-crystallin. More recent work using a whole genome microarray has identied more than 100 genes whose expressions are rapidly altered by dened hypoxic conditions and has identied the DosR regulon which consists of 48 genes that are coregulated with hspX.72,73 The DosR regulon is upregulated by bacterial sensing of low, non-toxic concentrations of NO and appears to prepare M. tuberculosis for dormancy.74 Similarly, other conditions thought to reect in vivo infection, such as growth in activated macrophages or within articial granulomas, have been demonstrated to upregulate the DosR genes.75,76 Hypoxia-driven dormancy seems to be reversible, as provision of O2, even after long periods of hypoxiainduced bacteriostasis, results in resuscitation and bacterial replication. Recent data suggest that synchronous resuscitation of surviving dormant bacteria is promoted by pheromone-like substances (the socalled resuscitation promoting factors) secreted from slowly replicating bacteria.77 Some of these substances may also promote bacterial spreading and transmission by dissolving the macrophage cell wall through lysozyme-like activity.78 Nutrient starvation is another factor expected to be encountered by the bacteria in vivo and, therefore, has been used in vitro to induce a state of non-replicating persistence with decreased respiration. Proteome and microarray analysis demonstrated that a large number of transcriptional changes occurred, but interestingly, although some of the DosR genes were also upregulated by starvation; the overall pattern differed signicantly from that induced by hypoxia, which would suggest the involvement of a regulon different from DosR.79 Many of these changes appeared to involve lipid metabolism, consistent with earlier ndings that longterm survival in the murine lung requires that M. tuberculosis express isocitrate lyase, an enzyme essential for the metabolism of fatty acids.80 Importantly this gene was necessary for replication of the bacteria in the late stage of infection in wild-type mice, whereas bacteria with a disruption of the gene still multiplied in IFN-g knockout mice. This suggests that the metabolism of M. tuberculosis in vivo is profoundly inuenced by the host response to infection. It is possible that activated macrophages are more efcient at depriving the bacteria of nutrients by resisting changes to phagosome trafcking81,82 and that the bacteria switch their metabolism to fatty acid degradation in response to this. This hypothesis is supported by the examination of
Immunology and Cell Biology

the transcription prole of M. tuberculosis grown in activated murine macrophages or in the lungs of infected mice which indicates that M. tuberculosis adapts to immune activation by expressing fatty aciddegrading enzymes and secreting siderophores to facilitate the acquisition of iron.76,83 This nding underscores the complexity of the bacterial transcriptional response to the multiple environmental signals encountered during its intracellular lifestyle and studying how to design vaccines that target the bacteria in its latent phase is presently a priority in many laboratories. What is of key importance is obviously the direct demonstration that the genes upregulated in vitro is of similar importance in vivo. Based on the study of transcriptional proles at various stages of M. tuberculosis infection in the mouse lung such a pattern is in fact now emerging and it clearly emphasizes the inuence of the host immune status on this change in pathogen gene expression.84 VACCINES IN DEVELOPMENT Prophylactic adjuvanted subunit vaccines After careful testing of many potential vaccine candidates in animal models for prophylactic vaccination, a total of ve proteins have been selected to be included in three rst-generation adjuvanted subunit vaccines. Based on secreted mycobacterial proteins identied in proteome studies or recognition by human T cells the fusions, Ag85B-ESAT-6, Ag85B-TB10.4, and a fusion of domains from Mtb32 and Mtb39 (Mtb72f) were selected and are presently the most advanced fusion protein prophylactic vaccines. They all protect close to the level obtained with BCG in mice and guinea pigs when formulated in selected Th1 inducing adjuvants,62,85,86 and Ag85BESAT-6 and Mtb72f have in addition been shown to be protective in non-human primates.87,88 Given that subunit vaccines will most likely be administered as booster vaccines to BCG-vaccinated individuals, it is important to test the subunit vaccines after previous BCG vaccination and thus to demonstrate that they can add to the protection induced by BCG. The heterologous prime-boost approach is attractive because it expands pre-existing memory T cells against epitopes shared by both the BCG and the booster vaccines. Mtb72f and the TB10.4-based fusions should all be strong BCG booster vaccines as the antigens present in these are all expressed by BCG. ESAT-6, however, is an important M. tuberculosis-specic antigen that is strongly recognized after M. tuberculosis infection and the Ag85B-ESAT-6 fusion protein, therefore, both boosts pre-existing immunity (due to Ag85B also being expressed by BCG) and induces a response to important new epitopes (ESAT-6 is not expressed by BCG) thereby expanding the recipients immune repertoire. Whether boosting or supplementing BCG is the better strategy is not known and Ag85B-ESAT-6 and Mtb72f have in fact both been shown to improve the protective efcacy of BCG in the mouse TB model.88,89 Another interesting vaccine strategy is to vaccinate with BCG and subunit vaccines, simultaneously. When Mtb72f was coadministrated with BCG, it increased the protective efcacy in guinea pigs compared to BCG alone.90 Similar results have been obtained in mice with the Ag85B-ESAT-6 subunit vaccine, and it was even possible to boost the anti-TB immunity and protective efcacy by subsequent vaccination with Ag85B-ESAT-6 in a Th1 adjuvant.88 Supplementing BCG with subunit vaccines may, therefore, be a strategy to improve the efcacy of BCG in childhood followed by subunit boosting at the age of 1013 years to prevent adult manifestations of pulmonary TB. Importantly for the childhood BCG-positive subunit vaccination strategy, the Ag85B-ESAT-6 fusion protein in the IC31 adjuvant has recently been shown to be able to induce a fully functional antimycobacterial

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T-cell response in neonates91 a major achievement because neonates have been thought to have a limited capacity for inducing Th1 responses and IFN-g expression.92 Prophylactic viral vector vaccines Viral vectors such as adenovirus or vaccinia trigger a Th1-dominated immune response, characterized by elevated induction of IFN-g, and thereby bias the response to the M. tuberculosis antigens they express in the same direction.93,94 Depending on the vector chosen, virally delivered vaccines may also stimulate greater CD8 recognition of the expressed M. tuberculosis antigens. The rst virally vectored TB vaccine to be tested in humans was MVA-85A, a recombinant, replication-decient vaccinia virus expressing Ag85A from M. tuberculosis.95 This vaccine has performed well in animal models and results from initial human trials found that it was also highly immunogenic in humans.96 In BCG-vaccinated individuals, even those who received their BCG vaccination years earlier, the magnitude of the anti-Ag85A response was greater than in naive donors, suggesting that the vaccine was boosting previous immunity.30 Side effects are apparently relatively mild and MVA-85A is now undergoing multiple phase I/II trials in Africa.97 The second virally vectored vaccine is Aeras-402 Ad35, a replication-decient recombinant adenovirus-35 expressing Ag85A, Ag85B and TB10.4.98 All three antigens are present in BCG and all are highly immunogenic in humans. There are as yet, very little data on the efcacy or safety of this vaccine in animal models, but the vaccine has passed initial phase I testing in the United States and is currently in the second stage of phase I testing, in populations from a TB-endemic area; in this case, South Africa. Although these vaccines are not restricted by (and may even benet from) earlier sensitization to mycobacteria, they must still face issues of sensitization, which may impact their use as booster vaccines. For adenovirus-based vaccines, there is evidence that previous humoral responses can reduce vaccine efcacy, and this has dictated the choice of a type 35 adenovirus as the vector: serological responses to type 35 adenovirus have a relatively low frequency (ranging from 3 to 5% in developed countries to 20% in Africa) compared to type 5 adenovirus. The practical effect of this pre-existing immunity remains unknown. In the case of MVA-85A, many adults, especially in TB-endemic areas, will have been vaccinated with the vaccinia vaccine and there are some data to suggest that this can reduce the efcacy of vaccinia-vectored vaccines.99 However, it is not known what effect this will have in a clinical situation where the duration between vaccinations will generally be many years. Postexposure subunit vaccines The potential of postexposure administration of TB vaccines has been the subject of debate for the last 20 years and there has so far been no consistent success in attempts to use prophylactic vaccine candidates as postexposure or therapeutic vaccines.100102 There has, however, been much debate on their potential safety. Robert Koch. more than 100 years ago, reported that injection of tuberculin into animals with active TB to induce protective immunity could trigger acute worsening of existing TB and even induce new immunopathological changes distant from the sites of active disease.20 In some studies, these vaccines even resulted in disease exacerbation.101,103 However, this was in subjects with well-advanced, progressive disease. It is now thought (again, based on animal models) that the low burden of mycobacteria during latent infection and the minimal activation of immunity should prevent the triggering a Koch-like response. As the number of new cases of active TB is by far outnumbered by the more

than 2 billion people estimated to be infected with a latent infection and who are at risk of reactivation of the disease, a postexposure vaccine is of high priority. Although it is possible that postexposure vaccination will be similar to a plain boosting of BCG, data from animal models suggest otherwise. One of the few vaccines with reported protective activity postinfection is the M. leprae Hsp65DNA vaccine. This vaccine was reported to induce dramatic reductions in both pulmonary and splenic bacterial loads in mice with established TB infection.104 However, its therapeutic efciency has been challenged.103 Some recent reports support the therapeutic potential of DNA vaccines,105,106 but Morris and colleagues used a combination of several DNA vaccines (including Ag85A) with demonstrated prophylactic activity107 and saw no effect against reactivation.102 The explanation for these contrasting results is unclear but it is important to keep in mind that in addition to the induction of antigen-specic responses, the therapeutic effects of DNA vaccines can also be due to short-term, non-specic stimulation of the innate immune system via CpG sequences. As described above, the breakthrough in our understanding of the dynamic transition of bacteria from active multiplication, dormancy and resuscitation have recently stimulated attempts to identify components for postexposure vaccines based on antigens characteristic of dormant bacteria such as those expressed by the DosR regulon and the resuscitation promoting factor genes but most of this work is still in the early research stage. CLINICAL STUDIES Assessment of safety and immunogenicity and rst clinical results on subunit vaccines The goal of a vaccine phase I trial is to show that a vaccine is safe to use (with immunogenicity as a frequent secondary consideration). After intradermal BCG vaccination, some individuals will develop an ulcerative lesion at the vaccination site. Despite this local reactogenicity, very severe adverse reactions are rare after BCG vaccination, and even though more than 3 billion doses of BCG have been given to people BCG safety has not been an issue except in severely immunocompromised populations.108 Any new TB vaccine will have to be at least as safe as BCG. In TB vaccine trials, the potential risk for Koch reactionseven if the consensus is that this risk is smallis most likely in individuals already infected with M. tuberculosis before vaccination. The plan being followed for clinical vaccine development by the various groups is, therefore, to complete phase I trials rst in carefully screened individuals without evidence of previous M. tuberculosis infection, before proceeding to phase I trials in healthy PPDpositive individuals. In both steps the optimal dose will be determined to minimize the possibility of triggering a necrotic reaction due to overproduction of proinammatory cytokines (a Koch reaction). Both Ag85B-ESAT-6 and Mtb72f, the two recombinant protein vaccines in clinical trials, have been shown to be well tolerated without any adverse reactions and to be highly immunogenic in humans. Ag85BESAT-6 was given in IC31 adjuvant, a mixture of oligodeoxynucleotides and polycationic amino acids,109 by intramuscular injection in a phase I trial in Holland whereas the Mtb72f was given in AS2, an oilin-water emulsion containing 3-deacylated monophosphoryl lipid A and a puried fraction of Quillaria saponaria, known as Quil A.86 For both fusion proteins, expanded phase I studies testing the vaccines in BCG-vaccinated, latently infected and individuals from TB endemic regions are ongoing. There is also consensus that Th1 cell responses are important for protection32,34,110112 and, therefore, the induction of mycobacteriaspecic T cells capable of producing IFN-g has become an important marker for the new TB vaccines. All of those tested so far induce
Immunology and Cell Biology

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signicant IFN-g responses. However, although protective immunity needs IFN-g production, the correlation between the amount of vaccine-induced IFN-g responses and degree of immune protection is not known.113 In addition, other immune cell subsets may be important for protective TB immunity. Protective efcacy: phases II and III trials Both Ag85B-ESAT-6 and Mtb72f were planned from the start to be tested as BCG booster vaccines, for two reasons. First, in the absence of efcacy data in humans it would be ethically difcult to justify replacing BCG114 because, as discussed above, BCG given at birth has been shown to be effective against disseminated forms of TB.115 Secondly, it is not clear how long immunity will last, so boosting BCG-induced responses in adolescents may offer longer overall protection. Therefore, the vaccines will be tested in adults (for safety and ethical reasons) but age deescalation will be included in phase II trials. Studies involving immunogenicity and protection in animal models can indicate which new TB vaccines and vaccination strategies are the most promising. However, true protective efcacy can only be measured in phase III trials, and without a clear correlate for protective efcacy it will likely be impossible to objectively prioritize among candidates that have passed phases I and II in terms of safety and immunogenicity. As a result of this, despite the cost involved, it is likely that more than one vaccine will progress to phase III testing. Because of the absence of accurate methods to measure infection rates, especially when BCG or related vaccines are given, the long latency of M. tuberculosis infection, and delayed reactivation disease, efcacy trials have used clinical endpoints such as disease to monitor efcacy and, therefore, have needed very large sample sizes with long-term follow-up periods. M. tuberculosis infection, (which is much more frequent than overt disease) could not be used as an endpoint because BCG vaccination induces positive PPD-specic delayed-type hypersensitivity responses, which are the standard responses used to detect asymptomatic latent infection.116 However, new diagnostic tests capable of distinguishing between immunity induced by BCG vaccination and postvaccination asymptomatic M. tuberculosis infection might allow for an infection endpoint to be studied in future vaccine trials.117 PERSPECTIVE AND FUTURE DIRECTIONS Although TB vaccine development has come a long way in the last decade there are still major obstacles ahead of us. For the leading candidates, there is no guarantee that they will progress through phase III clinical trials and registration. Experiences from HIV and malaria vaccine trials have taught us that it is important to continue preclinical research and keep developing new and even better vaccines for the pipeline. Another important point for the development of new vaccines in the future is that the human-adapted members of the M. tuberculosis complex are more genetically diverse than generally recognized and has recently been linked to changes in human demography and to both ancient and recent human migrations.118 Such diversity is most likely to have functional consequences for the mycobacterial strains and could affect the efcacy of new vaccines. Moreover, vaccines may show different protective efcacies against different mycobacterial strains. Consequently the efcacy of new vaccines should ideally be tested against several clinical M. tuberculosis isolates, and preferably against strains from all six major human MTBC lineages. However, although it is clearer than ever that designing a vaccine, which can cope with the many strategies that M. tuberculosis has evolved to escape the hosts immune response will be complex, there remain reasons to be optimistic. The rst new
Immunology and Cell Biology

vaccines against M. tuberculosis in half a century are progressing through clinical trials at a rapid pace. Phase II trials are already underway with two vaccines and at least two more expected to reach that stage over the next year. At the same time, vaccines which have shown detectable activity against the latent form of the disease in animal models are already in late preclinical stages and several new adjuvants, effective at stimulating cell-mediated responses and apparently safe in humans are also in trials. As we dissect the immune response against M. tuberculosis, and the pathogens response, we are becoming capable of designing vaccine strategies, which should let us tip the balance in the hosts favour.

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Maher D, Dye C, Floyd K, Pantoja A, Lonnroth K, Reid A et al. Planning to improve global health: the next decade of tuberculosis control. Bull World Health Organ 2007; 85: 341347. Dolin Pj RM, Kochi A. Global tuberculosis incidence and mortality during 1990-2000. Bull World Health Organ 1994; 72: 213220. al-Kassimi FA, al-Hajjaj MS, al-Orainey IO, Bamgboye EA. Does the protective effect of neonatal BCG correlate with vaccine-induced tuberculin reaction? Am J Respir Crit Care Med 1995; 152: 15751578. Colditz GA, Berkey CS, Mosteller F, Brewer TF, Wilson ME, Burdick E et al. The efcacy of bacillus Calmette-Guerin vaccination of newborns and infants in the prevention of tuberculosis: meta-analyses of the published literature. Pediatrics 1995; 96: 2935. Lanckriet C, Levy-Bruhl D, Bingono E, Siopathis RM, Guerin N. Efcacy of BCG vaccination of the newborn: evaluation by a follow-up study of contacts in Bangui. Int J Epidemiol 1995; 24: 10421049. Sterne JA, Rodrigues LC, Guedes IN. Does the efcacy of BCG decline with time since vaccination? Int J Tuberc Lung Dis 1998; 2: 200207. Comstock GW, Woolpert SF, Livesay VT. Tuberculosis studies in Muscogee County, Gerorgia. Twent-year evaluation of community trial of BCG vaccination. Public Health Rep 1976; 91: 276280. Hart PD, Sutherland I. BCG and vole bacillus vaccines in the prevention of tuberculosis in adolescence and early adult life. Br Med J 1977; 2: 293295. Brewer TF. Preventing tuberculosis with bacillus Calmette-Guerin vaccine: a metaanalysis of the literature. Clin Infect Dis 2000; 31(Suppl 3): S64S67. Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E, Fineberg HV et al. Efcacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. JAMA 1994; 271: 698702. Fine PE, Group KPT. Randomised controlled trial of single BCG, repeated BCG, or combined BCG and killed Mycobacterium leprae vaccine for prevention of leprosy and tuberculosis in Malawi. Karonga Prevention Trial Group. Lancet 1996; 348: 1724. Bjerkedal T. Mycobacterial infections in Norway: a preliminary note on determining their identity and frequency. Am J Epidemiol 1967; 85: 157173. Dickinson JM, Aber VR, Mitchison DA. Studies on the treatment of experimental tuberculosis of the guinea pig with intermittent doses of isoniazid. Tubercle 1973; 54: 211224. Palmer CE, Long MW. Effects of infection with atypical mycobacteria on BCG vaccination and tuberculosis. Am Rev Respir Dis 1966; 94: 553568. Brandt L, Feino Cunha J, Weinreich Olsen A, Chilima B, Hirsch P, Appelberg R et al. Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis. Infect Immun 2002; 70: 672678. Leung CC, Tam CM, Chan SL, Chan-Yeung M, Chan CK, Chang KC. Efcacy of the BCG revaccination programme in a cohort given BCG vaccination at birth in Hong Kong. Int J Tuberc Lung Dis 2001; 5: 717723. Jassal M, Bishai WR. Extensively drug-resistant tuberculosis. Lancet Infect Dis 2008. Doherty TM. Real world TB vaccines: clinical trials in TB-endemic regions. Vaccine 2005; 23: 21092114. Williams A, Hatch GJ, Clark SO, Gooch KE, Hatch KA, Hall GA et al. Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis. Tuberculosis (Edinb) 2005; 85: 2938. Koch R. Fortsetzung der mitteilungen uber ein heilmittel gegen tuberkulose. Dtsch Med Wochenschr 1891; 17: 101102. Demissie A, Leyten EM, Abebe M, Wassie L, Aseffa A, Abate G et al. Recognition of stage-specic mycobacterial antigens differentiates between acute and latent infections with Mycobacterium tuberculosis. Clin Vaccine Immunol 2006; 13: 179186. Buddle BM, Wards BJ, Aldwell FE, Collins DM, de Lisle GW. Inuence of sensitisation to environmental mycobacteria on subsequent vaccination against bovine tuberculosis. Vaccine 2002; 20: 11261133. Muller I, Cobbold SP, Waldmann H, Kaufmann SH. Impaired resistance to Mycobacterium tuberculosis infection after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. Infect Immun 1987; 55: 20372041. Orme IM. The kinetics of emergence and loss of mediator T lymphocytes acquired in response to infection with Mycobacterium tuberculosis. J Immunol 1987; 138: 293298.

TB vaccines in development C Aagaard et al 285

25 Orme IM, Collins FM. Adoptive protection of the Mycobacterium tuberculosis-infected lung. Dissociation between cells that passively transfer protective immunity and those that transfer delayed-type hypersensitivity to tuberculin. Cell Immunol 1984; 84: 113120. 26 Pedrazzini T, Hug K, Louis JA. Importance of L3T4+ and Lyt-2+ cells in the immunologic control of infection with Mycobacterium bovis strain bacillus Calmette-Guerin in mice. Assessment by elimination of T cell subsets in vivo. J Immunol 1987; 139: 20322037. 27 Andersen P. Tuberculosis vaccinesan update. Nat Rev Microbiol 2007; 5: 484487. 28 Reed S, Lobet Y. Tuberculosis vaccine development; from mouse to man. Microbes Infect 2005; 7: 922931. 29 Kaufmann SH. The contribution of immunology to the rational design of novel antibacterial vaccines. Nat Rev Microbiol 2007; 5: 491504. 30 Beveridge NE, Price DA, Casazza JP, Pathan AA, Sander CR, Asher TE et al. Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional Mycobacterium tuberculosis-specic CD4+ memory T lymphocyte populations. Eur J Immunol 2007; 37: 30893100. 31 Kaufmann SH, McMichael AJ. Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis. Nat Med 2005; 11: S33S44. 32 Cooper AM, Dalton DK, Stewart TA, Grifn JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon gamma gene-disrupted mice. J Exp Med 1993; 178: 22432247. 33 Dorman SE, Holland SM. Interferon-gamma and interleukin-12 pathway defects and human disease. Cytokine Growth Factor Rev 2000; 11: 321333. 34 Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993; 178: 22492254. 35 Frucht DM, Holland SM. Defective monocyte costimulation for IFN-gamma production in familial disseminated Mycobacterium avium complex infection: abnormal IL-12 regulation. J Immunol 1996; 157: 411416. 36 Jouanguy E, Lamhamedi-Cherradi S, Lammas D, Dorman SE, Fondaneche MC, Dupuis S et al. A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection. Nat Genet 1999; 21: 370378. 37 Jouanguy E, Altare F, Lamhamedi S, Revy P, Emile JF, Newport M et al. Interferongamma-receptor deciency in an infant with fatal bacille Calmette-Guerin infection. N Engl J Med 1996; 335: 19561961. 38 Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, Williamson R et al. A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 1996; 335: 19411949. 39 Cowley SC, Elkins KL. CD4+ T cells mediate IFN-gamma-independent control of Mycobacterium tuberculosis infection both in vitro and in vivo. J Immunol 2003; 171: 46894699. 40 Elias D, Akuffo H, Britton S. PPD induced in vitro interferon gamma production is not a reliable correlate of protection against Mycobacterium tuberculosis. Trans R Soc Trop Med Hyg 2005; 99: 363368. 41 Majlessi L, Simsova M, Jarvis Z, Brodin P, Rojas MJ, Bauche C et al. An increase in antimycobacterial Th1-cell responses by prime-boost protocols of immunization does not enhance protection against tuberculosis. Infect Immun 2006; 74: 21282137. 42 Bertholet S, Ireton GC, Kahn M, Guderian J, Mohamath R, Stride N et al. Identication of human T cell antigens for the development of vaccines against Mycobacterium tuberculosis. J Immunol 2008; 181: 79487957. 43 Coler RN, Dillon DC, Skeiky YA, Kahn M, Orme IM, Lobet Y et al. Identication of Mycobacterium tuberculosis vaccine candidates using human CD4(+) T-cells expression cloning. Vaccine 2009; 27: 223233. 44 Darrah PA, Patel DT, De Luca PM, Lindsay RW, Davey DF, Flynn BJ et al. Multifunctional TH1 cells dene a correlate of vaccine-mediated protection against Leishmania major. Nat Med 2007; 13: 843850. 45 Stewart GR, Patel J, Robertson BD, Rae A, Young DB. Mycobacterial mutants with defective control of phagosomal acidication. PLoS Pathog 2005; 1: 269278. 46 Weiss DW, Dubos RJ. Antituberculous immunity induced in mice by vaccination with killed tubercle bacilli or with a soluble bacillary extract. J Exp Med 1955; 101: 313330. 47 Barclay WR, Anacker R, Brehmer W, Ribi E. Effects of oil-treated mycobacterial cell walls on the organs of mice. J Bacteriol 1967; 94: 17361745. 48 Orme IM. Induction of nonspecic acquired resistance and delayed-type hypersensitivity, but not specic acquired resistance in mice inoculated with killed mycobacterial vaccines. Infect Immun 1988; 56: 33103312. 49 Pal PG, Horwitz MA. Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect Immun 1992; 60: 47814792. 50 Andersen P. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun 1994; 62: 25362544. 51 Roberts AD, Sonnenberg MG, Ordway DJ, Furney SK, Brennan PJ, Belisle JT et al. Characteristics of protective immunity engendered by vaccination of mice with puried culture ltrate protein antigens of Mycobacterium tuberculosis. Immunology 1995; 85: 502508. 52 Andersen P. TB vaccines: progress and problems. Trends Immunol 2001; 22: 160168. 53 Andersen P, Heron I. Simultaneous electroelution of whole SDS-polyacrylamide gels for the direct cellular analysis of complex protein mixtures. J Immunol Methods 1993; 161: 2939. 54 Andersen P, Heron I. Specicity of a protective memory immune response against Mycobacterium tuberculosis. Infect Immun 1993; 61: 844851. 55 Boesen H, Jensen BN, Wilcke T, Andersen P. Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis. Infect Immun 1995; 63: 14911497. 56 Demissie A, Ravn P, Olobo J, Doherty TM, Eguale T, Geletu M et al. T-cell recognition of Mycobacterium tuberculosis culture ltrate fractions in tuberculosis patients and their household contacts. Infect Immun 1999; 67: 59675971. 57 Pollock JM, Andersen P. Predominant recognition of the ESAT-6 protein in the rst phase of interferon with Mycobacterium bovis in cattle. Infect Immun 1997; 65: 25872592. 58 Borremans M, de Wit L, Volckaert G, Ooms J, de Bruyn J, Huygen K et al. Cloning, sequence determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis. Infect Immun 1989; 57: 31233130. 59 Bassey EO, Life PF, Catty D, Gaston JS, Kumararatne DS. T-cell response to mycobacterial proteins: a comparative study of tuberculous and control immunoblots of Mycobacterium tuberculosis and M. bovis BCG. Tuber Lung Dis 1996; 77: 146153. 60 Brandt L, Elhay M, Rosenkrands I, Lindblad EB, Andersen P. ESAT-6 subunit vaccination against Mycobacterium tuberculosis. Infect Immun 2000; 68: 791795. 61 Brodin P, Majlessi L, Brosch R, Smith D, Bancroft G, Clark S et al. Enhanced protection against tuberculosis by vaccination with recombinant Mycobacterium microti vaccine that induces T cell immunity against region of difference 1 antigens. J Infect Dis 2004; 190: 115122. 62 Dietrich J, Aagaard C, Leah R, Olsen AW, Stryhn A, Doherty TM et al. Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efcient protection and ESAT6-based sensitive monitoring of vaccine efcacy. J Immunol 2005; 174: 63326339. 63 Coler RN, Skeiky YA, Vedvick T, Bement T, Ovendale P, Campos-Neto A et al. Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis. J Immunol 1998; 161: 23562364. 64 Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998; 393: 537544. 65 Skjot RL, Brock I, Arend SM, Munk ME, Theisen M, Ottenhoff TH et al. Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family. Infect Immun 2002; 70: 54465453. 66 Skjot RL, Oettinger T, Rosenkrands I, Ravn P, Brock I, Jacobsen S et al. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun 2000; 68: 214220. 67 Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Immunol 2001; 19: 93129. 68 Rogerson BJ, Jung YJ, LaCourse R, Ryan L, Enright N, North RJ. Expression levels of Mycobacterium tuberculosis antigen-encoding genes versus production levels of antigen-specic T cells during stationary level lung infection in mice. Immunology 2006; 118: 195201. 69 Wayne LG, Hayes LG. An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun 1996; 64: 20622069. 70 Wayne LG, Lin KY. Glyoxylate metabolism and adaption of Mycobacterium tuberculosis to survival under anaerobic conditions. Infect Immun 1982; 37: 10421049. 71 Wayne LG, Sramek HA. Antigenic differences between extracts of actively replicating and synchronized resting cells of Mycobacterium tuberculosis. Infect Immun 1979; 24: 363370. 72 Park HD, Guinn KM, Harrell MI, Liao R, Voskuil MI, Tompa M et al. Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 2003; 48: 833843. 73 Sherman DR, Voskuil M, Schnappinger D, Liao R, Harrell MI, Schoolnik GK. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha-crystallin. Proc Natl Acad Sci USA 2001; 98: 75347539. 74 Voskuil MI, Schnappinger D, Visconti KC, Harrell MI, Dolganov GM, Sherman DR et al. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 2003; 198: 705713. 75 Karakousis PC, Yoshimatsu T, Lamichhane G, Woolwine SC, Nuermberger EL, Grosset J et al. Dormancy phenotype displayed by extracellular Mycobacterium tuberculosis within articial granulomas in mice. J Exp Med 2004; 200: 647657. 76 Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, Monahan IM et al. Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment. J Exp Med 2003; 198: 693704. 77 Downing KJ, Mischenko VV, Shleeva MO, Young DI, Young M, Kaprelyants AS et al. Mutants of Mycobacterium tuberculosis lacking three of the ve rpf-like genes are defective for growth in vivo and for resuscitation in vitro. Infect Immun 2005; 73: 30383043. 78 Cohen-Gonsaud M, Barthe P, Bagneris C, Henderson B, Ward J, Roumestand C et al. The structure of a resuscitation-promoting factor domain from Mycobacterium tuberculosis shows homology to lysozymes. Nat Struct Mol Biol 2005; 12: 270273.

Immunology and Cell Biology

TB vaccines in development C Aagaard et al 286

79 Betts JC, Lukey PT, Robb LC, McAdam RA, Duncan K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression proling. Mol Microbiol 2002; 43: 717731. 80 McKinney JD, Honer zu Bentrup K, Munoz-Elias EJ, Miczak A, Chen B, Chan WT et al. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 2000; 406: 735738. 81 Schaible UE, Sturgill-Koszycki S, Schlesinger PH, Russell DG. Cytokine activation leads to acidication and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. J Immunol 1998; 160: 12901296. 82 Wagner D, Maser J, Lai B, Cai Z, Barry III CE, Honer Zu Bentrup K et al. Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cells endosomal system. J Immunol 2005; 174: 14911500. 83 Timm J, Post FA, Bekker LG, Walther GB, Wainwright HC, Manganelli R et al. Differential expression of iron-, carbon-, and oxygen-responsive mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients. Proc Natl Acad Sci USA 2003; 100: 1432114326. 84 Shi L, Jung YJ, Tyagi S, Gennaro ML, North RJ. Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence. Proc Natl Acad Sci USA 2003; 100: 241246. 85 Olsen AW, van Pinxteren LAH, Okkels LM, Rasmussen PB, Andersen P. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Infect Immun 2001; 69: 27732778. 86 Skeiky YA, Alderson MR, Ovendale PJ, Guderian JA, Brandt L, Dillon DC et al. Differential immune responses and protective efcacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein. J Immunol 2004; 172: 76187628. 87 Langermans JA, Doherty TM, Vervenne RA, van der Laan T, Lyashchenko K, Greenwald R et al. Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Vaccine 2005; 23: 27402750. 88 Dietrich J, Billeskov R, Doherty TM, Andersen P. Synergistic effect of bacillus calmette guerin and a tuberculosis subunit vaccine in cationic liposomes: increased immunogenicity and protection. J Immunol 2007; 178: 37213730. 89 Skeiky YA, Sadoff JC. Advances in tuberculosis vaccine strategies. Nat Rev Microbiol 2006; 4: 469476. 90 Brandt L, Skeiky YA, Alderson MR, Lobet Y, Dalemans W, Turner OC et al. The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs. Infect Immun 2004; 72: 66226632. 91 Kamath AT, Rochat AF, Valenti MP, Agger EM, Lingnau K, Andersen P et al. Adult-like anti-mycobacterial T cell and in vivo dendritic cell responses following neonatal immunization with Ag85B-ESAT-6 in the IC31 adjuvant. PLoS ONE 2008; 3: e3683. 92 Levy O. Innate immunity of the newborn: basic mechanisms and clinical correlates. Nat Rev Immunol 2007; 7: 379390. 93 Drexler I, Staib C, Sutter G. Modied vaccinia virus Ankara as antigen delivery system: how can we best use its potential? Curr Opin Biotechnol 2004; 15: 506512. 94 Wang J, Thorson L, Stokes RW, Santosuosso M, Huygen K, Zganiacz A et al. Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis. J Immunol 2004; 173: 63576365. 95 Goonetilleke NP, McShane H, Hannan CM, Anderson RJ, Brookes RH, Hill AV. Enhanced immunogenicity and protective efcacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vaccine using mucosal administration and boosting with a recombinant modied vaccinia virus Ankara. J Immunol 2003; 171: 16021609. 96 McShane H, Pathan AA, Sander CR, Keating SM, Gilbert SC, Huygen K et al. Recombinant modied vaccinia virus Ankara expressing antigen 85A boosts BCGprimed and naturally acquired antimycobacterial immunity in humans. Nat Med 2004; 10: 12401244. 97 Sander CR, Pathan AA, Beveridge NE, Poulton I, Minassian A, Alder N et al. Safety and immunogenicity of a new TB vaccine, MVA85A, in M. tuberculosis infected individuals. Am J Respir Crit Care Med 2009. 98 Radosevic K, Wieland CW, Rodriguez A, Weverling GJ, Mintardjo R, Gillissen G et al. Protective immune responses to an rAd35 TB vaccine in two mouse strains (H-2d vs H-2b): CD4 and CD8 T-cell epitope mapping and role of IFN{gamma}. Infect Immun 2007; 75: 41054115. 99 Rooney JF, Wohlenberg C, Cremer KJ, Moss B, Notkins AL. Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination. J Virol 1988; 62: 15301534. 100 Dhillon J, Mitchison DA. Effect of vaccines in a murine model of dormant tuberculosis. Tuber Lung Dis 1994; 75: 6164. 101 Turner J, Rhoades ER, Keen M, Belisle JT, Frank AA, Orme IM. Effective preexposure tuberculosis vaccines fail to protect when they are given in an immunotherapeutic mode. Infect Immun 2000; 68: 17061709. 102 Repique CJ, Li A, Collins FM, Morris SL. DNA immunization in a mouse model of latent tuberculosis: effect of DNA vaccination on reactivation of disease and on reinfection with a secondary challenge. Infect Immun 2002; 70: 33183323. 103 Taylor JL, Turner OC, Basaraba RJ, Belisle JT, Huygen K, Orme IM. Pulmonary necrosis resulting from DNA vaccination against tuberculosis. Infect Immun 2003; 71: 21922198. 104 Lowrie DB, Tascon RE, Bonato VL, Lima VM, Faccioli LH, Stavropoulos E et al. Therapy of tuberculosis in mice by DNA vaccination. Nature 1999; 400: 269271. 105 Zhu D, Jiang S, Luo X. Therapeutic effects of Ag85B and MPT64 DNA vaccines in a murine model of Mycobacterium tuberculosis infection. Vaccine 2005; 23: 46194624. 106 Ha SJ, Jeon BY, Youn JI, Kim SC, Cho SN, Sung YC. Protective effect of DNA vaccine during chemotherapy on reactivation and reinfection of Mycobacterium tuberculosis. Gene Therapy 2005; 12: 634638. 107 Delogu G, Li A, Repique C, Collins F, Morris SL. DNA vaccine combinations expressing either tissue plasminogen activator signal sequence fusion proteins or ubiquitinconjugated antigens induce sustained protective immunity in a mouse model of pulmonary tuberculosis. Infect Immun 2002; 70: 292302. 108 Hoft DF, Leonardi C, Milligan T, Nahass GT, Kemp B, Cook S et al. Clinical reactogenicity of intradermal bacille Calmette-Guerin vaccination. Clin Infect Dis 1999; 28: 785790. 109 Lingnau K, Riedl K, von Gabain A. IC31 and IC30, novel types of vaccine adjuvant based on peptide delivery systems. Expert Rev Vaccines 2007; 6: 741746. 110 Bean AG, Roach DR, Briscoe H, France MP, Korner H, Sedgwick JD et al. Structural deciencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin. J Immunol 1999; 162: 35043511. 111 Botha T, Ryffel B. Reactivation of latent tuberculosis infection in TNF-decient mice. J Immunol 2003; 171: 31103118. 112 Denis B, Lefort A, Flipo RM, Tubach F, Lemann M, Ravaud P et al. Long-term followup of patients with tuberculosis as a complication of tumour necrosis factor (TNF)alpha antagonist therapy: safe re-initiation of TNF-alpha blockers after appropriate anti-tuberculous treatment. Clin Microbiol Infect 2008; 14: 183186. 113 Hoft DF, Worku S, Kampmann B, Whalen CC, Ellner JJ, Hirsch CS et al. Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J Infect Dis 2002; 186: 14481457. 114 Snider Jr DE. Ethical issues in tuberculosis vaccine trials. Clin Infect Dis 2000; 30 (Suppl 3): S271S275. 115 Trunz BB, Fine P, Dye C. Effect of BCG vaccination on childhood tuberculous meningitis and miliary tuberculosis worldwide: a meta-analysis and assessment of cost-effectiveness. Lancet 2006; 367: 11731180. 116 Hoft DF, Tennant JM. Persistence and boosting of bacille Calmette-Guerin-induced delayed-type hypersensitivity. Ann Intern Med 1999; 131: 3236. 117 Andersen P, Doherty TM, Pai M, Weldingh K. The prognosis of latent tuberculosis: can disease be predicted? Trends Mol Med 2007; 13: 175182. 118 Hershberg R, Lipatov M, Small PM, Sheffer H, Niemann S, Homolka S et al. High functional diversity in Mycobacterium tuberculosis driven by genetic drift and human demography. PLoS Biol 2008; 6: e311.

Immunology and Cell Biology