advanced search

<< Back To Home

TB VACCINE RESEARCH

Sunday, 31st of July 2011

TB VACCINE RESEARCH

‘The increased research activities into tuberculosis vaccines are the result of increased public and private funding. Within the past decade, funding has at least quadrupled, reaching more than half a billion US dollars for tuberculosis research.31—33 Of this funding, roughly a fifth is devoted to vaccine research and nearly a third to basic research. This effort is highly commendable, but insufficient. Estimates suggest that about four-times this amount of financial support is needed to reach the goal of production of improved vaccines within the next decade.10, 15, 32’

Best viewed at

http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(11)70146-3/fulltext

Fact and fiction in tuberculosis vaccine research: 10 years later

Original Text

Dr Stefan HE Kaufmann PhD a

Tuberculosis is one of the most deadly infectious diseases. The situation is worsening because of co-infection with HIV and increased occurrence of drug resistance. Although the BCG vaccine has been in use for 90 years, protection is insufficient; new vaccine candidates are therefore needed. 12 potential vaccines have gone into clinical trials. Ten are aimed at prevention of tuberculosis and, of these, seven are subunit vaccines either as adjuvanted or viral-vectored antigens. These vaccines would be boosters of BCG-prime vaccination. Three vaccines are recombinant BCG constructs—possible replacements for BCG. Additional vaccine candidates will enter clinical trials in the near future, including postexposure vaccines for individuals with latent infection. In the long term, vaccines that prevent or eradicate infection with Mycobacterium tuberculosis would be the best possible option. Improved knowledge of immunology, molecular microbiology, cell biology, biomics, and biotechnology has paved the way towards an effective and safe vaccine against tuberculosis. The pipeline of new vaccine candidates from preclinical to clinical testing could be accelerated by development of biomarkers that can predict the clinical outcome of tuberculosis.

In the first issue of The Lancet Infectious Diseases, I co-authored a Review called Prospects for better tuberculosis vaccines.1 10 years later, in this follow-up report I discuss the progress. The epidemiological values provided in the 2001 report1 are still valid—every minute of every day, nearly 20 people develop and four people die from tuberculosis globally (figure 1). Morbidity and mortality have decreased; however, this decrease of up to 1% per year is attributable to the fact that the world population is increasing at a faster rate than is incidence of tuberculosis.2 Hence, in absolute numbers, tuberculosis has not decreased in the past decade. Tuberculosis is highly prevalent in most developing countries (eg, 340 cases per 100 000 people in sub-Saharan Africa),3 but industrial nations cannot be complacent. In 1998, the frequency of tuberculosis in London had risen to 32 cases per 100 000 population. Many people thought tuberculosis was under control in developed countries, but a recent report in The Lancet4 suggests the opposite: 45·7 tuberculosis cases per 100 000 population were recorded in London in 2009.5

 

Figure 1 Full-size image (54K) Download to PowerPoint

The life-cycle of Mycobacterium tuberculosis and different vaccination measures

Vaccines in clinical trials are aimed at prevention of active tuberculosis. Future vaccines should aim to prevent or eradicate M tuberculosis infection.

Co-infection with HIV is the driving force of the resurgence of tuberculosis.6 Multidrug-resistant strains continue to emerge, and this problem has been surpassed by extensively drug-resistant tuberculosis, which has been notified in 58 countries.7 Despite the problem of resistance, the same drugs have been used for the past several decades.8 As in 2001, nearly 50% of all tuberculosis cases are diagnosed incorrectly.9 Finally, the only available vaccine against tuberculosis remains BCG.10, 11 BCG protects children against severe forms of the disease but fails to protect against the most prevalent form nowadays, pulmonary tuberculosis in adults.12 10 years ago, researchers predicted that a new vaccine with 50% efficacy could prevent 9 million deaths from tuberculosis by the year 2030.13 This prediction is now invalid since a new vaccine will not become available before 2020. Accordingly, more recent predictions stating that new pre-exposure vaccines could reduce tuberculosis prevalence and mortality by 40—50% are highly speculative.14

In view of these discouraging facts, the target of the StopTB Partnerships to reduce prevalence and mortality of tuberculosis by one-half by 2015, compared with rates in 1990, seems courageous, and to eliminate tuberculosis by 2050—ie, to reduce incidence to less than one new case per million inhabitants globally—seems highly ambitious if not fanciful.15, 16

Despite these disillusions, winds of change are being felt. As of the first quarter of 2001, few potential new vaccines had been developed and tested in preclinical models.1 As of early 2011, 12 vaccine candidates have reached clinical trials at different stages (figure 1 and table 1). All but two of these are canonical vaccines—ie, they are given pre-exposure and aim to prevent active tuberculosis but not Mycobacterium tuberculosis infection. Two (one in a phase 2 assessment and one already passed phase 3) are therapeutic vaccines for immune therapy in adjunct to chemotherapy of individuals who have HIV—M tuberculosis coinfection.29, 30 These two immunotherapeutic vaccines will not be covered further in this Review. Of the ten preventive vaccine candidates, seven are undergoing safety and immunogenicity testing in small groups in phase 1 trials. Three are in phase 2 trials to work out the best dose and route and immune parameters of efficacy. Two vaccines are ready to undergo phase 2b trials for evidence of protective efficacy against natural M tuberculosis infection. Protective vaccine efficacy will only be shown in phase 3 trials.

Table 1Table image  

Most advanced vaccine candidates in clinical trials

The increased research activities into tuberculosis vaccines are the result of increased public and private funding. Within the past decade, funding has at least quadrupled, reaching more than half a billion US dollars for tuberculosis research.31—33 Of this funding, roughly a fifth is devoted to vaccine research and nearly a third to basic research. This effort is highly commendable, but insufficient. Estimates suggest that about four-times this amount of financial support is needed to reach the goal of production of improved vaccines within the next decade.10, 15, 32

A vaccine against tuberculosis, as it passes through the research and development pipeline becomes increasingly expensive, ranging from US$1—2 million for research and discovery to $1—5 million in phase 1, $5—20 million in phase 2, and hundreds of millions of dollars in phase 3 clinical trials.34 Phase 3 trials need many years until completion10, 35 and although they are extraordinarily costly, little planning has gone into these studies thus far. Feedback from phase 1 and 2 trials with new vaccines and previous experience with controlled BCG trials can provide valuable information about important confounding factors, such as coinfection, contact with environmental mycobacteria, and health and nutrition status of trial participants.10,12,36—38 As a further complication, combinations of different vaccines in prime—boost regimens have to be assessed and this process will further raise cost. Increased financial commitment creates the need for data-driven criteria to decide whether a candidate will progress to the next stage of development or not (go or no-go criteria for attrition). In other words, only vaccines most likely to possess better efficacy and safety compared with BCG can be advanced further. Experimental animal models provide helpful guidelines for gating of entry into the clinical trial pipeline. Although they can indicate the protective potential of a vaccine, they cannot predict its protective activity in people. To maximise information from animal experiments, general rules about experimental conditions and animal species of greatest predictive value should be worked out. During clinical trials, biomarkers that could predict clinical outcome of vaccine trials at early stages would be very useful (panel).9, 35, 39 Interferon-γ responses measured in peripheral blood cells are insufficient for prediction of clinical outcomes of vaccine trials.

Panel

Biomarkers for tuberculosis

Markers to monitor trials

Biomarker studies focus on different stages of natural infection with M tuberculosis.39, 40 These studies should be intensified and used for monitoring of early-stage clinical trials to construct a platform of markers that can lead to custom-made signatures, specifically modified for the different types of vaccines.

Immune markers

Test systems for immune markers include whole-blood assays in which unseparated peripheral blood cells are stimulated with defined antigens and cytokines measured in supernatants by ELISA.41 Alternatively, enzyme-linked immunosorbent spot assays are used to identify cytokine-producing cells.42, 43 Finally, flow-cytometric analysis with more advanced equipment, the fluorescence activated cell sorter, has the advantage of combining cytokine staining with surface marker determination—eg, polyfunctional T cells with CD4 versus CD8 phenotype, or effector versus memory phenotype.44

Global biomarkers

Transcriptome analyses have provided proof of concept that latent infection can be discriminated from active tuberculosis disease by a few differentially expressed genes. Different assays and different study populations provided similar results with a remarkable degree of overlap in relevant transcripts.45—48 Next generation analysis will harness deep sequencing technologies.49 Metabolomics and proteomics have been exploited less intensively. In the case of metabolomics, fewer than 20 metabolites with differential serum abundance were sufficient for distinction of latent infection and active tuberculosis (J Weiner, Max Planck Institute for Infection Biology, personal communication). Proteomics revealed a signature composed of four protein markers that allowed distinction between active tuberculosis and latent infection.50

Active tuberculosis is a possible, but not inevitable, result of infection. Latent infection without apparent clinical disease is the far more common outcome—about 90% of people with the bacteria have latent M tuberculosis infection. M tuberculosis is thought to survive in the infected host for long periods of time.51 One survival strategy of M tuberculosis is the downregulation of its metabolic and replicative activity, to a stage termed dormancy.52 Latency is the result of active containment of dormant M tuberculosis by an efficient immune response. Latent tuberculosis can progress to active disease once the immune response becomes weakened or aberrant. Active tuberculosis is preceded by resuscitation of M tuberculosis, which ignites full metabolic and replicative activity as a go signal for unrestricted growth of M tuberculosis. Additionally, albeit less frequently, superinfection with a new strain of M tuberculosis can lead to active tuberculosis in individuals who have a latent infection.53, 54

Progression from latency to active tuberculosis can be caused by endogenous exhaustion or immune suppression, deviation of the immune response (because of infections with helminths or environmental mycobacteria), or immunocompromising exogenous factors such as HIV coinfection (the most common driver of tuberculosis).35, 51

Principally, M tuberculosis are intracellular bacteria that persist within macrophages and perhaps a few other host-cell types,52 which shield it against antibody attack. The role of antibodies in tuberculosis is unclear, although circumstantial evidence suggests a role for antibodies in protection against tuberculosis, which needs further clarification (figure 2).55 T cells are at the centre of protection and pathological changes in tuberculosis, because they detect infected host cells (figure 2).51 The gene products of the major histocompatibility complex class II (MHC II) present antigenic peptides derived from M tuberculosis residing in the phagosome, which leads to the stimulation of CD4 T cells. M tuberculosis possesses many molecular structures that stimulate pattern recognition receptors including Toll-like receptors, nucleotide-binding oligomerisation domain receptors, and others. As a result, CD4 T cells are mainly of T helper 1 (Th1) type (figure 2).51 These Th1 cells produce interferon γ, tumour necrosis factor α (TNFα), and interleukin 2, and are judged the most crucial mediators of protection.10, 35, 51

 

Figure 2 Full-size image (101K) Download to PowerPoint

Targets and effectors of protective immunity in tuberculosis

T cells participate in protection. Cytokines activate macrophages, killer molecules lyse target cells and Mycobacterium tuberculosis. The role of B cells is unknown. Th=T helper, CTL=cytolytic T lymphocytes, TNF=tumour necrosis factor.

Some researchers argue that multifunctional T cells producing these cytokines concomitantly are of particular importance for long-term protection.44, 56 However, debate exists as to whether multifunctional T cells offer better protection than various T cells producing only one or two cytokines, as long as all cytokines are made available.35, 57 In the past decade, a new CD4 T-cell subset with a distinct cytokine profile, namely the Th17 cells, has been described.58 These Th17 cells produce interleukin-17, which mainly stimulates neutrophils. Circumstantial evidence suggests that Th17 cells can contribute to immunity against tuberculosis during early stages of infection (figure 2).59, 60

CD8 T cells are important for protection, first as extra producers of these cytokines and second as cytolytic T lymphocytes. The killer molecule of cytolytic T lymphocytes, granulysin with help of perforin, and granzymes, can directly attack M tuberculosis, even when the pathogen is hidden within cells (figure 2).61 For several decades, the mechanisms by which M tuberculosis antigens could enter the cytosolic MHC class I processing machinery were elusive; however, researchers have generally established that M tuberculosis antigens can be loaded on both MHC II and MHC I molecules leading to stimulation of CD4 and CD8 T cells, respectively.62—64

Although the original understanding was that only CD8 T cells produce killer molecules, evidence suggests that CD4 T cells do so too. Reciprocally, cytokine production is not a unique capacity of CD4 T cells. Thus, CD4 and CD8 T cells share similar biological functions. Are both T-cell types mutually compensatory or do they have unique functions? One obvious difference is the disparate MHC restriction. CD8 T cells recognise antigenic peptides in the context of MHC I with a broad target range of almost all nucleated cells. By contrast, CD4 T cells sense peptide-loaded MHC II that is constitutively expressed only on professional antigen-presenting cells (APCs). However, MHC II can be induced in non-professional APC during inflammation.

γδ T cells and CD1-restricted αβ T cells recognise mycobacterial components and might be participants in protection against tuberculosis, although their precise role is unclear (figure 2).65, 66 Since they express similar functions as MHC-I-restricted and MHC-II-restricted CD8 and CD4 T cells, again, there could be functional overlap and these unconventional T cells might support protection and become particularly important whenever conventional T cells are impaired. Whether immune memory is needed, or whether continuous generation of new effector T cells provide protection during latency, is unknown.67 Because M tuberculosis persists in the host, the role of true memory responses—defined as being present in the absence of the nominal antigen—could be difficult to work out.

Finally, circumstantial evidence suggests that during the long-lasting struggle between the immune response and M tuberculosis to contain the pathogen, exhaustive and suppressive mechanisms might arise that can affect protective immunity and thus facilitate disease reactivation. These inhibitory mechanisms include cytokines, which affect Th1 cells including interleukin-4, interleukin-10, and transforming growth factor-β produced by Th2 cells and regulatory T cells, and co-expression of inhibitory molecules.68, 69 Additionally, coinfections with environmental mycobacteria, HIV, or helminths can affect the M tuberculosis-specific immune response during latent infection.36,70—72 Thus, continuous fine-tuning of the immune response that controls M tuberculosis is needed.73 This notion holds equally true for immunity evoked by vaccination or by natural infection with M tuberculosis, since in either case, reactivation of tuberculosis must be prevented in view of persistent M tuberculosis.

BCG is the only approved vaccine against tuberculosis.74 It has been given 4 billion times with a proven safety and efficacy record and with low cost (a few cents per dose).10, 74 The two major failures of BCG are incomplete protection against pulmonary tuberculosis in adults, the most prevalent form of the disease,12, 37 and safety issues in people who are HIV positive or otherwise immunocompromised.72

Ideally, new vaccines should be better than BCG in terms of both efficacy and safety. That is, they should be safe in individuals who have HIV, and produce reliable protection not only in newborn babies and children but also in adults. However, development of different types of vaccination regimens might be necessary, including vaccines that can be given to newborn babies with efficacy similar to that of BCG but that are safer and hence usable in neonates with HIV, and vaccines that are more efficacious and protect against tuberculosis in adults. Similarly, development of different vaccines used before and after exposure could be needed—ie, for individuals who are uninfected and for those with latent M tuberculosis.75 In animal models, pre-exposure vaccine candidates prevent active tuberculosis, although they do not succeed in eradicating M tuberculosis (table 1). This finding is likely to hold true for people. Thus, risk of tuberculosis reactivation remains a notable result of HIV co-infection.

The first group of vaccine candidates comprises prime vaccines to replace BCG and the second group booster vaccines to be given in addition to BCG. Replacement vaccines in clinical trials are recombinant (r)BCG with the most advanced being rBCGΔureC::hly (VPM 1002) developed in Germany.17 This vaccine showed improved efficacy in preclinical models, most likely by activating a broader immune response, including Th17 and CD8 T cells, in addition to Th1 cells.17, 63, 76 The vaccine has completed a first phase 1 trial in Germany, and a second in South Africa. As a next step this vaccine is being prepared for a phase 2 trial in infants. The underlying mechanism of this vaccine is the membrane-perforating activity of listeriolysin, which results in apoptosis of infected host cells leading to cross-presentation for improved immunity.63 The second vaccine of this group is rBCG30,18 which overexpresses the immunodominant antigen (Ag85B) shared by BCG and M tuberculosis.18 This vaccine has completed a phase 1 trial.77 The most recent addition to this group is Aeras-422, which entered a phase 1 clinical trial at the end of 2010.19 Aeras-422 combines features of both VPM 1002 and rBCG30. This vaccine expresses perfringolysin, which has similar biological activity to listeriolysin, and in addition to Ag85B, expresses a related protein Ag85A, and Rv3407, which is expressed during reactivation.78 Additional rBCG constructs and deletion mutants of M tuberculosis could enter phase 1 trials by the end of 2011 or early 2012 (table 2). Auxotrophic M tuberculosis vaccines are promising because of their good safety profile.84, 85

Table 2Table image  

Next generation vaccines in preclinical testing

One group of booster vaccines are viral-vectored vaccines. The MVA85A/Aeras-485 consists of a modified vaccinia Ankara (MVA) expressing Ag85A, a cognate of Ag85B (table 1 and table 3). The MVA is an attenuated vaccinia virus. The Oxford MVA85A/Aeras-485 is prepared for a phase 2b trial.20, 86 Also, adenoviruses have been used as vaccine vectors. Crucell Ad35/Aeras-402, which is prepared for a phase 2b trial, is based on adenovirus 35 expressing three antigens.21 The AdAg85A, which is undergoing a phase 1 trial, is based on adenovirus 5 expressing only Ag85A.22 Both adenovirus vectors have been rendered replication-deficient.

Table 3Table image  

Antigens of vaccines under investigation

Protein—adjuvant combinations are composed of fusion proteins in appropriate adjuvants (table 1, table 3, and table 4). These include Hybrid 1 in the adjuvant IC-31 or in CAF01,24, 25 M72 in the adjuvant system AS02,26, 27 or in AS01,15 and HyVac4/Aeras-404 in IC-31.23, 28 Hybrid 1 is a fusion of antigens Ag85B and ESAT-6. It has completed phase 1 clinical testing in both adjuvant formulations and is prepared for phase 2 trial.87 In HyVac4, ESAT-6 is replaced by TB10·4 and this vaccine has completed a phase 1 trial. M72, a fusion of Rv1196 and Rv0125, is in phase 2 testing.

Table 4Table image  

Adjuvants of vaccines under investigation

All of these booster vaccines are aimed at improvement of BCG-induced immunity and all are given before exposure to M tuberculosis. Several booster vaccine candidates are being prepared for clinical phase 1 trials in 2011, including a fusion protein consisting of four M tuberculosis antigens, Rv3619, Rv1813, Rv3620, and Rv2608 composed in a synthetic monophoryl lipid A adjuvant, and a vaccine based on heparin-binding hemagglutinin.82, 88 H56, another vaccine candidate soon to enter phase 1 clinical trial (table 2 and table 3), is effective when given before and after exposure75, 83 in a mouse model of tuberculosis. This vaccine is a fusion of antigens of H1 with an additional antigen Rv2660, which is expressed by M tuberculosis during starvation.83

Biomarkers predicting clinical endpoints can reduce the cost of, and accelerate, clinical trials.39 With ten preventive vaccines in clinical trials and more to come, attrition has become a major factor. Few vaccine candidates will be tested in phase 3 clinical trials, which will ultimately define protective efficacy against naturally acquired tuberculosis. Although vaccine trials in phase 2b can provide preliminary efficacy data, these are insufficient. Even for very high incidences of tuberculosis in the order of 0·5% to 1·0%, participant groups in phase 3 trials should be in the order of 20 000 per group to obtain statistically relevant data.

Newborn babies have a higher risk of tuberculosis than do adults, but in neonates, the standard against which a new vaccine will be measured is raised because BCG is partly protective in this population. Hence, biomarkers that can predict progression to active tuberculosis early on in clinical testing would be of utmost value.39, 40 Monitoring of vaccine trials, with appropriate biomarker measurements, could provide information for an educated go or no-go decision. Biomarkers are being sought in different immunological and biomic systems. Immune markers are based on antigen-specific T-cell responses, notably, release of cytokines relevant to protection, such as interferon γ, TNFα, interleukin 2, and killer molecules—ie, perforin, granulysin, and granzyme.41,42,89—91 Antigen-specific interferon γ responses alone are not indicative of protective immunity.57, 92

More recent studies focus on multifunctional T cells, which secrete several cytokines of protective value in tuberculosis.43, 55 However, the relevance of these multifunctional T cells as biomarkers is unclear.57 Global gene expression profiling by use of transcriptomics has led to the definition of biosignatures that distinguish individuals who are latently infected, healthy, or with active tuberculosis.45—48 Less well advanced are biosignatures based on metabolomic and proteomic markers.39, 50 Finally, a longitudinal study has been initiated in which household contacts of patients with newly diagnosed tuberculosis are being followed up for 2 years and blood samples stored for analyses.40 In this study, 3% of household contacts will probably develop active tuberculosis, biomarkers might correlate with progression to active tuberculosis. Results from these studies are expected in the next 2 years.

Whether biomarkers that discriminate latent infection in healthy individuals from active tuberculosis in patients, and even biomarkers that can predict risk of tuberculosis reactivation, can be directly applied to monitoring of vaccine trials is unclear.93 This doubt arises mainly because whether vaccine-induced immunity is based on identical mechanisms as those that contain M tuberculosis in individuals with latent infections is unknown. Some researchers argue that vaccine-induced immunity should be better than immunity against M tuberculosis,93 since the main target population of vaccines are individuals who develop tuberculosis disease and hence fail to maintain latent infection. However a vaccine that stimulates an immune response in susceptible individuals that is similar to the response in people with latent infection could provide a first clue. Subunit vaccines are composed of one or few antigens and markers of their immunogenicity are therefore restricted to these antigens. Despite the difficulties in the development of such markers, their design is almost mandatory, since ultimately different vaccines might need to be combined in heterologous prime—boost regimens to achieve maximum protection.

Would the introduction of a first new vaccine by 2020 bring an end to the search for new vaccines against tuberculosis? The answer is most likely no. Vaccines in the clinical trial pipeline in the best-case scenario will prevent disease but will neither wipe out the pathogen nor prevent superinfection.52, 53 Next generation vaccination strategies should aim to eradicate M tuberculosis. Eradication is needed particularly in view of high incidences of co-infection with HIV. 15 million individuals are already infected with both HIV and M tuberculosis, of whom, each year, a million will develop tuberculosis, and of whom every second person of this million will die.6

HIV disables the immune response and does not distinguish between immunity induced by natural infection or vaccination. Hence, vaccines that sustain latent infection will be rendered ineffective, as will immune deviation caused by helminths, environmental mycobacteria, or endogenous factors.35, 36, 71, 73 In all of these situations, vaccine-induced immunity might no longer be able to contain M tuberculosis and allow for tuberculosis after reactivation or superinfection. Hence, vaccination strategies that succeed in eradicating M tuberculosis would be highly welcome (figure 1). This aim could be achieved by combining the most efficient prime and boost vaccine in a novel regimen. Even more attractive might be a vaccine that prevents infection with M tuberculosis (figure 1). Such a vaccine is hard to envisage on the basis of present immunological knowledge. Immune mechanisms required for prevention of infection, therefore, should be explored in more depth. Antibodies that attack M tuberculosis as it enters the lung by triggering aggressive effector mechanisms while blocking essential functions for the pathogen's survival in the host could be a lead towards such a vaccine.

Although current candidate vaccines are imperfect, clinical trials should be continued. Mathematical modelling has predicted that such vaccines can contribute to tuberculosis control.14 Knowledge from current vaccine trials can provide valuable information for the design of future vaccines. This reverse translation from clinical observation to basic research has not been used up to now. Hence, both clinical testing and basic research of potential vaccines are needed to concomitantly identify new candidates that could follow the first generation of vaccines, because they are qualitatively different and prevent or eliminate M tuberculosis infection. To achieve these ambitious goals, a tighter relation between basic research and clinical studies is called for.

I searched PubMed, restricted to the past 10 years, with the terms “vaccination”, “immune response”, “therapy”, “prevention”, “HIV/AIDS”, “dormancy”, “latency”, “tuberculosis”, “mycobacterium”, “BCG”, “vaccine safety”, “clinical trials”, “subunit vaccines,” and “biomarkers”. Relevant references from articles identified by this search strategy are included, as is information reported by the Treatment Action Group, Stop TB Partnership, WHO, and George Institute for International Health. The date of the last search was May 30, 2011.

Conflicts of Interest

I am co-inventor of, and hold a patent for, the rBCGΔUreC:Hly vaccine and a member of the scientific advisory boards of Intercell and of Vaccine Project Management.

Acknowledgments

I thank ML Grossman for editorial support, D Schad for graphical design, Peter Andersen, Bill Jacobs, Camille Locht, Carlos Martin, Helen McShane, Opokua Ofori-Anyinam, and Jerry Sadoff, for sharing information on their vaccines. Work from my laboratory on tuberculosis vaccine development receives support from NEWTBVAC(FP7 grant number Health-F3-2009-241745), and the Bill & Melinda Gates Foundation Grand Challenges in Global Health GC6-74(number 37772) of which I am Principal Investigator, and GC12-82(number 37885).

1 Collins HL, Kaufmann SHE. Prospects for better tuberculosis vaccines. Lancet Infect Dis 2001; 1: 21-28. Summary | Full Text | PDF(683KB) | CrossRef | PubMed

2 US Census Bureau (International Database). World population growth rates: 1950—2050. http://www.census.gov/population/international/data/idb/worldgrgraph.php. (accessed June 29, 2011).

3 WHO. Global tuberculosis control: epidemiology, strategy, financing. WHO Report 2009. Geneva: World Health Organization, 2009.

4 Zumla A. The white plague returns to London—with a vengeance. Lancet 2011; 377: 10-11. Full Text | PDF(132KB) | CrossRef | PubMed

5 Public Health Action Support Team. London TB service review and health needs assessment: final project report. London: TB Commissioning Board, 2010.

6 Harries AD, Zachariah R, Corbett EL, et al. The HIV-associated tuberculosis epidemic—when will we act?. Lancet 2010; 375: 1906-1919. Summary | Full Text | PDF(226KB) | CrossRef | PubMed

7 Gandhi NR, Nunn P, Dheda K, et al. Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis. Lancet 2010; 375: 1830-1843. Summary | Full Text | PDF(591KB) | CrossRef | PubMed

8 Ma Z, Lienhardt C, McIlleron H, Nunn AJ, Wang X. Global tuberculosis drug development pipeline: the need and the reality. Lancet 2010; 375: 2100-2109. Summary | Full Text | PDF(296KB) | CrossRef | PubMed

9 Wallis RS, Pai M, Menzies D, et al. Biomarkers and diagnostics for tuberculosis: progress, needs, and translation into practice. Lancet 2010; 375: 1920-1937. Summary | Full Text | PDF(394KB) | CrossRef | PubMed

10 Kaufmann SHE, Hussey G, Lambert PH. New vaccines for tuberculosis. Lancet 2010; 375: 2110-2119. Summary | Full Text | PDF(1340KB) | CrossRef | PubMed

11 Calmette A. Sur la vaccination préventive des enfants nouveau-nés contre la tuberculose par le BCG. Annales de l'Institut Pasteur 1927; 41: 201-232. PubMed

12 Colditz GA, Brewer TF, Berkey CS, et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. JAMA 1994; 271: 698-702. PubMed

13 Murray CJ, Salomon JA. Modeling the impact of global tuberculosis control strategies. Proc Natl Acad Sci USA 1998; 95: 13881-13886. PubMed

14 Abu-Raddad LJ, Sabatelli L, Achterberg JT, et al. Epidemiological benefits of more-effective tuberculosis vaccines, drugs, and diagnostics. Proc Natl Acad Sci USA 2009; 106: 13980-13985. PubMed

15 Working group on new TB vaccines. Tuberculosis Vaccine Candidates 2010. StopTB Partnership. http://www.stoptb.org/wg/new_vaccines/assets/documents/TBVaccine Pipeline 10 — 03 21 11.pdf. (accessed June 29, 2011).

16 Dye C, Williams BG. Eliminating human tuberculosis in the twenty-first century. J R Soc Interface 2008; 5: 653-662. CrossRef | PubMed

17 Grode L, Seiler P, Baumann S, et al. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin. J Clin Invest 2005; 115: 2472-2479. CrossRef | PubMed

18 Tullius MV, Harth G, Maslesa-Galic S, Dillon BJ, Horwitz MA. A replication-limited recombinant Mycobacterium bovis BCG vaccine against tuberculosis designed for human immunodeficiency virus-positive persons is safer and more efficacious than BCG. Infect Immun 2008; 76: 5200-5214. CrossRef | PubMed

19 Sun R, Skeiky YA, Izzo A, et al. Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis. Vaccine 2009; 27: 4412-4423. CrossRef | PubMed

20 McShane H, Pathan AA, Sander CR, et al. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat Med 2004; 10: 1240-1244. CrossRef | PubMed

21 Radosevic K, Wieland CW, Rodriguez A, et al. Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon. Infect Immun 2007; 75: 4105-4115. CrossRef | PubMed

22 Santosuosso M, McCormick S, Zhang X, Zganiacz A, Xing Z. Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis. Infect Immun 2006; 74: 4634-4643. CrossRef | PubMed

23 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: 741-746. CrossRef | PubMed

24 Dietrich J, Andersen C, Rappuoli R, et al. Mucosal administration of Ag85B-ESAT-6 protects against infection with MycobacteriuM tuberculosis and boosts prior bacillus Calmette-Guerin immunity. J Immunol 2006; 177: 6353-6360. PubMed

25 Christensen D, Foged C, Rosenkrands I, et al. CAF01 liposomes as a mucosal vaccine adjuvant: in vitro and in vivo investigations. Int J Pharm 2010; 390: 19-24. CrossRef | PubMed

26 Leroux-Roels I, Leroux-Roels G, Ofori-Anyinam O, et al. Evaluation of the safety and immunogenicity of two antigen concentrations of the Mtb72F/AS02(A) candidate tuberculosis vaccine in purified protein derivative-negative adults. Clin Vaccine Immunol 2010; 17: 1763-1771. CrossRef | PubMed

27 Von Eschen K, Morrison R, Braun M, et al. The candidate tuberculosis vaccine Mtb72F/AS02A: tolerability and immunogenicity in humans. Hum Vacc 2009; 5: 475-482. PubMed

28 Dietrich J, Aagaard C, Leah R, et al. Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J Immunol 2005; 174: 6332-6339. PubMed

29 Von Reyn CF, Mtei L, Arbeit R, et al. Prevention of tuberculosis in Bacille Calmette-Guérin-primed, HIV-infected adults boosted with an inactivated whole-cell mycobacterial vaccine. AIDS 2010; 24: 675-685. CrossRef | PubMed

30 Cardona PJ. RUTI: a new chance to shorten the treatment of latent tuberculosis infection. Tuberculosis (Edinb) 2006; 86: 273-289. CrossRef | PubMed

31 Kaufmann SH, Parida SK. Changing funding patterns in tuberculosis. Nat Med 2007; 13: 299-303. CrossRef | PubMed

32 Jimenez-Salazar E. Tuberculosis research and development: 2010 report on tuberculosis research funding trends, 2005—2009, 2nd edn. http://www.stoptb.org/assets/documents/resources/publications/technical/TBR_D. (accessed June 29, 2011).

33 Moran M, Guzman J, Henderson K, et al. G-Finder 2010—neglected disease research and development: is the global financial crisis changing R&D?. London: Policy Cures, 2011.

34 Kaufmann SHE. The new plagues: pandemics and poverty in a globalized world. London: Haus Publishing, 2009.

35 Kaufmann SHE. Future vaccination strategies against tuberculosis: thinking outside the box. Immunity 2010; 33: 567-577. CrossRef | PubMed

36 Hatherill M, Adams V, Hughes J, et al. The potential impact of helminth infection on trials of novel tuberculosis vaccines. Vaccine 2009; 27: 4743-4744. CrossRef | PubMed

37 Fine PE. The BCG story: lessons from the past and implications for the future. Rev Infect Dis 1989; 11 (suppl 2): S353-S359. PubMed

38 Sander C, McShane H. Translational mini-review series on vaccines: Development and evaluation of improved vaccines against tuberculosis. Clin Exp Immunol 2007; 147: 401-411. CrossRef | PubMed

39 Parida SK, Kaufmann SH. The quest for biomarkers in tuberculosis. Drug Discov Today 2010; 15: 148-157. CrossRef | PubMed

40 Kaufmann SH, Parida SK. Tuberculosis in Africa: learning from pathogenesis for biomarker identification. Cell Host Microbe 2008; 4: 219-228. CrossRef | PubMed

41 Black GF, Weir RE, Floyd S, et al. BCG-induced increase in interferon-gamma response to mycobacterial antigens and efficacy of BCG vaccination in Malawi and the UK: two randomised controlled studies. Lancet 2002; 359: 1393-1401. Summary | Full Text | PDF(122KB) | CrossRef | PubMed

42 Lalvani A, Pathan AA, Durkan H, et al. Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Lancet 2001; 357: 2017-2021. Summary | Full Text | PDF(101KB) | CrossRef | PubMed

43 Diel R, Goletti D, Ferrara G, et al. Interferon-gamma release assays for the diagnosis of latent Mycobacterium tuberculosis infection: a systematic review and meta-analysis. Eur Respir J 2011; 37: 88-99. CrossRef | PubMed

44 Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol 2008; 8: 247-258. PubMed

45 Maertzdorf J, Repsilber D, Parida SK, et al. Human gene expression profiles of susceptibility and resistance in tuberculosis. Genes Immun 2011; 12: 15-22. CrossRef | PubMed

46 Jacobsen M, Repsilber D, Gutschmidt A, et al. Candidate biomarkers for discrimination between infection and disease caused by Mycobacterium tuberculosis. J Mol Med 2007; 85: 613-621. CrossRef | PubMed

47 Jacobsen M, Repsilber D, Kleinsteuber K, et al. Suppressor of cytokine signaling (SOCS)-3 is affected in T cells from TB patients. Clin Microbiol Infect 201010.1111/j.1469-0691.2010.03326.x. published online July 29. PubMed

48 Berry MP, Graham CM, McNab FW, et al. An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature 2010; 466: 973-977. CrossRef | PubMed

49 Schadt EE, Turner S, Kasarskis A. A window into third-generation sequencing. Hum Mol Genet 2010; 19: R227-R240. CrossRef | PubMed

50 Agranoff D, Fernandez-Reyes D, Papadopoulos MC, et al. Identification of diagnostic markers for tuberculosis by proteomic fingerprinting of serum. Lancet 2006; 368: 1012-1021. Summary | Full Text | PDF(161KB) | CrossRef | PubMed

51 Cooper AM. Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009; 27: 393-422. CrossRef | PubMed

52 Russell DG. Who puts the tubercle in tuberculosis?. Nat Rev Microbiol 2007; 5: 39-47. CrossRef | PubMed

53 Rustomjee R, Lienhardt C, Kanyok T, et al. A phase II study of the sterilising activities of ofloxacin, gatifloxacin and moxifloxacin in pulmonary tuberculosis. Int J Tuberc Lung Dis 2008; 12: 128-138. PubMed

54 Cohen T, Wilson D, Wallengren K, Samuel EY, Murray M. Mixed-strain Mycobacterium tuberculosis infections among patients dying in a hospital in KwaZulu-Natal, South Africa. J Clin Microbiol 2011; 49: 385-388. CrossRef | PubMed

55 Glatman-Freedman A. The role of antibody-mediated immunity in defense against Mycobacterium tuberculosis: advances toward a novel vaccine strategy. Tuberculosis (Edinb) 2006; 86: 191-197. CrossRef | PubMed

56 Foulds KE, Wu CY, Seder RA. Th1 memory: implications for vaccine development. Immunol Rev 2006; 211: 58-66. CrossRef | PubMed

57 Kagina BM, Abel B, Scriba TJ, et al. Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis after bacillus Calmette-Guérin vaccination of newborns. Am J Respir Crit Care Med 2010; 182: 1073-1079. CrossRef | PubMed

58 Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol 2009; 27: 485-517. CrossRef | PubMed

59 Khader SA, Bell GK, Pearl JE, et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4(+) T cell responses after vaccination and during Mycobacterium tuberculosis challenge. Nat Immunol 2007; 8: 369-377. CrossRef | PubMed

60 Lockhart E, Green AM, Flynn JL. IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J Immunol 2006; 177: 4662-4669. PubMed

61 Stenger S, Hanson DA, Teitelbaum R, et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998; 282: 121-125. CrossRef | PubMed

62 Schaible UE, Winau F, Sieling PA, et al. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat Med 2003; 9: 1039-1046. CrossRef | PubMed

63 Winau F, Weber S, Sad S, et al. Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis. Immunity 2006; 24: 105-117. CrossRef | PubMed

64 van der Wel N, Hava D, Houben D, et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 2007; 129: 1287-1298. CrossRef | PubMed

65 Brigl M, Brenner MB. CD1: antigen presentation and T cell function. Annu Rev Immunol 2004; 22: 817-890. CrossRef | PubMed

66 Scotet E, Nedellec S, Devilder MC, Allain S, Bonneville M. Bridging innate and adaptive immunity through gammadelta T-dendritic cell crosstalk. Front Biosci 2008; 13: 6872-6885. PubMed

67 Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol 2004; 22: 745-763. CrossRef | PubMed

68 Belkaid Y, Tarbell K. Regulatory T cells in the control of host-microorganism interactions (*). Annu Rev Immunol 2009; 27: 551-589. CrossRef | PubMed

69 Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008; 26: 677-704. CrossRef | PubMed

70 Azzopardi P, Bennett CM, Graham SM, Duke T. Bacille Calmette-Guerin vaccine-related disease in HIV-infected children: a systematic review. Int J Tuberc Lung Dis 2009; 13: 1331-1344. PubMed

71 Hesseling AC, Marais BJ, Gie RP, et al. The risk of disseminated Bacille Calmette-Guerin (BCG) disease in HIV-infected children. Vaccine 2007; 25: 14-18. CrossRef | PubMed

72 WHO. Revised BCG vaccination guidelines for infants at risk for HIV infection. Wkly Epidemiol Rec 2007; 82: 193-196. PubMed

73 Dorhoi A, Kaufmann SH. Fine-tuning of T cell responses during infection. Curr Opin Immunol 2009; 21: 367-377. CrossRef | PubMed

74 Zwerling A, Behr MA, Verma A, et al. The BCG World Atlas: A database of global BCG vaccination policies and practises. PLoS Med 2011; 8: e1001012. CrossRef | PubMed

75 Kaufmann SH. Tuberculosis vaccines-a new kid on the block. Nat Med 2011; 17: 159-160. CrossRef | PubMed

76 Desel C, Dorhoi A, Bandermann S, Grode L, Eisele B, Kaufmann SHE. Recombinant BCG induces superior protection over parental BCG by stimulating a balanced combination of type 1 and type 17 cytokine responses. J Infect Dis (in press).

77 Hoft DF, Blazevic A, Abate G, et al. A new recombinant bacille Calmette-Guerin vaccine safely induces significantly enhanced tuberculosis-specific immunity in human volunteers. J Infect Dis 2008; 198: 1491-1501. CrossRef | PubMed

78 Mollenkopf HJ, Grode L, Mattow J, et al. Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis. Infect Immun 2004; 72: 6471-6479. CrossRef | PubMed

79 Sambandamurthy VK, Derrick SC, Hsu T, et al. Mycobacterium tuberculosis DeltaRD1 DeltapanCD: a safe and limited replicating mutant strain that protects immunocompetent and immunocompromised mice against experimental tuberculosis. Vaccine 2006; 24: 6309-6320. CrossRef | PubMed

80 Walker KB, Brennan MJ, Ho MM, et al. The second Geneva consensus: recommendations for novel live TB vaccines. Vaccine 2010; 28: 2259-2270. CrossRef | PubMed

81 Asensio JA, Arbues A, Perez E, Gicquel B, Martin C. Live tuberculosis vaccines based on phoP mutants: a step towards clinical trials. Expert Opin Biol Ther 2008; 8: 201-211. CrossRef | PubMed

82 Locht C, Hougardy JM, Rouanet C, Place S, Mascart F. Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis. Tuberculosis (Edinb) 2006; 86: 303-309. CrossRef | PubMed

83 Aagaard C, Hoang T, Dietrich J, et al. A multistage tuberculosis vaccine that confers efficient protection pre- and post-exposure. Nat Med 2011; 17: 189-194. CrossRef | PubMed

84 Sampson SL, Dascher CC, Sambandamurthy VK, et al. Protection elicited by a double leucine and pantothenate auxotroph of Mycobacterium tuberculosis in guinea pigs. Infect Immun 2004; 72: 3031-3037. CrossRef | PubMed

85 Sampson SL, Mansfield KG, Carville A, et al. Extended safety and efficacy studies of a live attenuated double leucine and pantothenate auxotroph of Mycobacterium tuberculosis as a vaccine candidate. Vaccine 2011; 29: 4839-4847. CrossRef | PubMed

86 Sander CR, Pathan AA, Beveridge NE, et al. Safety and immunogenicity of a new tuberculosis vaccine, MVA85A, in Mycobacterium tuberculosis-infected individuals. Am J Respir Crit Care Med 2009; 179: 724-733. CrossRef | PubMed

87 van Dissel JT, Arend SM, Prins C, et al. Ag85B-ESAT-6 adjuvanted with IC31 promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in naive human volunteers. Vaccine 2010; 28: 3571-3581. CrossRef | PubMed

88 Bertholet S, Ireton GC, Ordway DJ, et al. A defined tuberculosis vaccine candidate boosts BCG and protects against multidrug-resistant Mycobacterium tuberculosis. Sci Transl Med 2010; 2: 53ra74. PubMed

89 Black GF, Thiel BA, Ota MO, et al. Immunogenicity of novel DosR regulon-encoded candidate antigens of Mycobacterium tuberculosis in three high-burden populations in Africa. Clin Vaccine Immunol 2009; 16: 1203-1212. CrossRef | PubMed

90 Harari A, Rozot V, Enders FB, et al. Dominant TNF-alpha(+) Mycobacterium tuberculosis-specific CD4(+) T cell responses discriminate between latent infection and active disease. Nat Med 2011; 17: 372-376. CrossRef | PubMed

91 Semple PL, Watkins M, Davids V, et al. Induction of granulysin and perforin cytolytic mediator expression in 10-week-old infants vaccinated with BCG at birth. Clin Dev Immunol 2011; 2011: 438463. PubMed

92 Mittrucker HW, Steinhoff U, Kohler A, et al. Poor correlation between BCG vaccination-induced T cell responses and protection against tuberculosis. Proc Natl Acad Sci USA 2007; 104: 12434-12439. PubMed

93 Walzl G, Ronacher K, Hanekom W, Scriba TJ, Zumla A. Immunological biomarkers of tuberculosis. Nat Rev Immunol 2011; 11: 343-354. PubMed

a Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany

Correspondence to: Dr Stefan H E Kaufmann, Department of Immunology, Max Planck Institute for Infection Biology, Berlin D-10117, Germany