The role of oxidative stress during inflammatory processes

Biol. Chem., Vol. 389, pp. 497–511, May 2008 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.057

2008/315

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Review

Not to wake a sleeping giant: new insights into host-pathogen interactions identify new targets for vaccinationagainst latent Mycobacterium tuberculosis infection

May Young Lin1,2 and Tom H.M. Ottenhoff1,2,*1 Department of Immunohaematology and BloodTransfusion, Leiden University Medical Centre,NL-2300 RC Leiden, The Netherlands2 Department of Infectious Diseases, Leiden UniversityMedical Centre, NL-2300 RC Leiden, The Netherlands

* Corresponding authore-mail: [email protected]

Abstract

Mycobacterium tuberculosis is one of the worlds’ mostsuccessful and sophisticated pathogens. It is estimatedthat over 2 billion people today harbour latent M. tuber-culosis infection without any clinical symptoms. As mostnew cases of active tuberculosis (TB) arise from this(growing) number of latently infected individuals, urgentmeasures to control TB reactivation are required, includ-ing post-exposure/therapeutic vaccines. The currentbacille Calmette-Guerin (BCG) vaccine and all new gen-eration TB vaccines being developed and tested areessentially designed as prophylactic vaccines. Unfortu-nately, these vaccines are unlikely to be effective in indi-viduals already latently infected with M. tuberculosis.Here, we argue that detailed analysis of M. tuberculosisgenes that are switched on predominantly during latentstage infection may lead to the identification of new anti-genic targets for anti-TB strategies. We will describeessential host-pathogen interactions in TB with particularemphasis on TB latency and persistent infection. Sub-sequently, we will focus on novel groups of late-stagespecific genes, encoded amongst others by the M. tuber-culosis dormancy (dosR) regulon, and summarise recentstudies describing human T-cell recognition of these dor-mancy antigens in relation to (latent) M. tuberculosisinfection. We will discuss the possible relevance of thesenew classes of antigens for vaccine development againstTB.

Keywords: bacille Calmette-Guerin (BCG); dormancy;dosR regulon; M. bovis persistence; post-exposurevaccines; T-cells.

Introduction

Mycobacterium tuberculosis, the causative agent oftuberculosis (TB, also known as the ‘white plague’), wasidentified by Robert Koch in 1882. The highly complex

interactions of M. tuberculosis with the human host havebeen studied intensely ever since, but despite theseefforts many critical gaps in our knowledge remain, pre-cluding successful control of the TB pandemic. M. tuber-culosis is one of the world’s most successful andsophisticated pathogens, as it causes persistent infec-tion in what is estimated to be over 2 billion people, yetlargely without causing clinical symptoms (asymptomaticor ‘latent infection’) (Comstock et al., 1974; Wayne andSohaskey, 2001; Lillebaek et al., 2002).

Human beings represent the only known natural res-ervoir of the bacillus. Following primary infection, only5–10% of those infected will ever develop active TB dis-ease, mostly within 2 years and commonly presenting aspulmonary TB in the adult. Approximately 8–10 millionindividuals newly develop active TB each year, and 2–3million die from the disease. The remaining 90–95% ofinfected cases develop latent M. tuberculosis infection,which can be maintained for the lifetime of the personunless the immunological balance between pathogenand host is perturbed; this can trigger reactivation ofM. tuberculosis and result in active TB. The enormousreservoir of latently infected individuals represents themain source of new TB cases (Lillebaek et al., 2002),although it has become clear that new TB cases can alsoarise due to exogenous re-infection with M. tuberculosisin areas with high TB endemicity (Fine and Small, 1999;van Rie et al., 1999). The best-known factor driving pro-gression of latent towards active infection is humanimmunodeficiency virus (HIV) co-infection: this increasesthe proportion of TB disease reactivation from 5–10% ina lifetime to 5–10% per life-year (Corbett et al., 2003).Due to the expanding HIV/AIDS pandemic, the numberof TB reactivation related casualties is growing. Otherhost and environmental factors involved in compromisingthe host’s ability to control M. tuberculosis infectioninclude malnutrition, ageing, stress (Tufariello et al.,2003), type 2 diabetes (Alisjahbana et al., 2007), the useof immunosuppressive agents and likely also geneticallycontrolled host factors. On the pathogen’s side, the bac-terial factors essential to the waking up this sleeping‘giant’ and their precise interplay with the host remain tobe identified. It is likely that new molecular geneticexpression technologies will help uncover these in thenearby future.

Anti-tuberculous chemotherapy has been highly effec-tive in treating TB cases and in interrupting transmissionof infection. An operational problem is that effective anti-TB chemotherapy requires long-term treatment with acombination of at least three drugs. Despite the enor-

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498 M.Y. Lin and T.H.M. Ottenhoff


mous impact of TB chemotherapy, recently there hasbeen an alarming increase of multi-drug resistant andeven extensively drug resistant M. tuberculosis strains.This is largely due to lack of compliance to the lengthytreatment schedules and the use of suboptimal drugcombinations, thus facilitating the selection of drug-resistant strains. If these escape variants will turn out tobe infection- and transmission-competent, which is sup-ported by current data, this will pose serious challengesto the control of TB in the future.

New anti-tuberculosis strategies

The most efficient and cost-effective intervention strate-gy against any infectious disease is vaccination. Sincethe 1920s, M. bovis bacille Calmette-Guerin (BCG)strains have been widely used as prophylactic TB vac-cines. BCG vaccines are live attenuated bacterial strainsthat share a high degree of genetic and genomic homol-ogy ()95%) with M. tuberculosis (Behr et al., 1999).Despite its wide use in massive vaccination programmes,the benefits of the BCG vaccine and efficacy remain top-ics of debate. BCG has proven to be highly efficaciousagainst severe TB in children, including TB meningitisand miliary TB. In addition, BCG protects adults againstleprosy, which is caused by the related species Myco-bacterium leprae (Ponnighaus et al., 1992; Fine, 1995).Unfortunately, this does not translate to sufficient protec-tion of adults against the main and contagious form ofTB, which is pulmonary TB in adults. Several randomisedcontrolled trials and observational studies in adults haverevealed rather discrepant results, with protective effi-cacies ranging from 0 to 80% (Fine, 1995). Many expla-nations have been put forward to account for thisconundrum, including differences in trial methodologies,host population genetics, regional differences inM. tuberculosis strains and differences in the BCG vac-cine strains used. It is currently believed that heterolo-gous immunity induced by environmental mycobacteriaplays a significant role in blocking or masking the pro-tective efficacy of the BCG vaccine (Palmer and Long,1966; Andersen and Doherty, 2005). BCG also clearlyfails to prevent TB reactivation from latent infection.Moreover, BCG revaccination offers no additive protec-tion against TB (Rodrigues et al., 2005). Thus, BCG hashad little impact on the global prevalence and epidemi-ology of TB.

Developing better TB vaccines that can complementor replace BCG constitute a major global research area.Basically, there are three main strategies:

1) TB subunit vaccines, based upon immunodominantantigens delivered in selected platforms, includingnon-replicating viruses (MVA, adenoviruses) andadjuvants;

2) live mycobacterial vaccines based upon geneticallyimproved BCG; and

3) highly attenuated live M. tuberculosis.

The subunit based approaches aim to induce high lev-els of cell mediated immunity and memory to a single orrestricted number of antigens, preferably those that areimmunodominant and essential for mycobacterial viru-

lence. The concept behind the use of live mycobacterialvaccines is the induction of a broader cellular immuneresponse against a plethora of antigens, which may helpachieving optimal protective immunity. These differentapproaches have been recently discussed in detail (Skei-ky and Sadoff, 2006; Andersen, 2007a; Kaufmann, 2007).

Almost all new generation TB vaccines that are cur-rently in clinical development have been designed as pre-exposure vaccines. Prophylactic subunit vaccines aregenerally considered to be particularly effective in boost-ing immunity induced by prior BCG vaccination, whereaslive vaccines aim to replace BCG by more efficient vac-cine strains. Whilst these vaccines aim to increase hostresistance prior to infection, they are unlikely to be effec-tive as post-exposure or therapeutic vaccines in latentlyinfected individuals (Turner et al., 2000; Fine, 2001). Thisis underlined by the above-mentioned inefficacy of BCGrevaccination in affording added protection compared tosingle BCG administration. It is clear that post-exposureor ‘therapeutic’ vaccines that control, or even better,eradicate dormant persistent bacteria will protect againstTB reactivation and contribute to TB control.

Phases and faces of M. tuberculosis

TB vaccine discovery approaches rest on the assumptionthat the vaccine antigens administered are alsoexpressed by infected host cells, where they are recog-nised by T-cells that execute the desired effectorresponse, either assisting phagocytes in controlling oreliminating live bacteria, or – alternatively – by cytolysisof infected cells. Despite significant advances, relativelylittle is known about the M. tuberculosis antigen reper-toire that is truly expressed on the surface of infectedcells, and which is considered relevant to human T-cells.This is particularly evident for molecules encoded byrecently discovered ‘groups’ of M. tuberculosis genesexpressed during specific phases – or stages – ofM. tuberculosis infection. Proteins that are stronglyexpressed during the early phase of infection can behighly efficacious vaccine targets in models of acuteinfection. Traditionally, TB vaccine discovery has focusedmostly on such antigens, several of which have shownstrong protective efficacy in animal models, including thewell known early stage antigens we.g., early secreted anti-genic target-6 (ESAT-6), TB10.4, Ag85A and Ag85Bx.

However, one caveat of this approach is that antigensthat are highly expressed during early infection – or underlaboratory conditions of log phase growth – may not nec-essarily induce optimal immunity and concomitant pro-tection during the later stages of infection, because theymight not be expressed optimally during later stages ofM. tuberculosis infection. Adequate control of late stageinfection may require different T-cell subsets, includingT-cells specific for antigens expressed during late stageTB infection. Several factors may contribute to the lackof ‘early’ antigens in inducing protection against latestage TB infection, including exhaustion of relevant T-cellmemory populations, insufficient expression of early anti-gens on infected cells and immune regulatory effectsaffecting the T-cells specific for early immunodominantantigens.



Immunity to Mycobacterium tuberculosis dosR genes 499


Another important issue that we will address is thatneither natural infection nor BCG vaccination may beable to achieve optimal activation of T-cells specific forlate stage antigens. Detailed analysis of the M. tubercu-losis genes that are switched on selectively (or at leastpredominantly) during late stage infection may help toidentify novel antigens to activate T-cells with the poten-tial to control such stages of infection. Targeting and re-directing the human immune response to these antigensmay thus help preventing TB reactivation.

One of the best established and controlled laboratorymodels to study adaptive responses of M. tuberculosisduring non-proliferating conditions is the ‘Wayne’ model(Wayne and Hayes, 1996). This model is based on thegradual depletion of oxygen from M. tuberculosis cul-tures, which triggers M. tuberculosis to enter a state ofnon-replicating persistence (NRP) (Wayne and Sohaskey,2001). Two different NRP stages can be discriminated:NRP1 and NRP2. NRP1 is characterised by slow rate ofturbidity without corresponding increase in CFU or syn-thesis of DNA. NPR2 is an anaerobic stage in which nofurther increase in turbidity is observed. The use of thisand other models combined with genome wide trans-criptome profiling has led to the identification of M. tuber-culosis genes that are particularly expressed duringconditions thought to replicate bacterial dormancy. Vos-kuil et al. (2003) initially studied M. tuberculosis during invitro conditions of nitric oxide (NO) and hypoxia, andidentified a regulon called the dosR or devR (dormancy)regulon, which consists of 48 genes which are co-ordi-nately up-regulated in M. tuberculosis under these con-ditions. Soon afterwards, several studies showedexpression and up-regulation of many of the dosR/devRregulon genes by M. tuberculosis in the Wayne model(Muttucumaru et al., 2004; Voskuil et al., 2004). DuringNRP1, M. tuberculosis shows reduced general mRNAsynthesis (Wayne and Hayes, 1996). Sherman et al.(2001) showed that the response of M. tuberculosis tohypoxia was characterised by significant alterations inapproximately 100 genes. Over 40 genes, mainly includ-ing members of the dosR regulon, were induced, where-as expression of most other genes was significantlyrepressed. It is known that expression of early stagegenes, such as ESAT-6 and Ag85B is repressed duringstationary phase (Rogerson et al., 2006).

These studies above indicate that M. tuberculosisshows phase specific gene expression. This may be rel-evant to TB vaccinology, as it provides new directions forantigen discovery and strategies for TB vaccine design.In this review we will first focus on essential host-path-ogen interactions in TB. Thereafter, we will discussM. tuberculosis phase specific gene expression and theirpossible relevance for TB vaccination. We will reviewrecent work describing characteristics of the M. tuber-culosis dosR regulon, as well as evidence documentinghuman T-cell recognition of new late stage dosR/devRregulon encoded antigens in relation to (latent) M. tuber-culosis infection.

Infection and disease

Tuberculosis infection is acquired through inhalation ofaerosolised infectious particles containing M. tuberculo-

sis, which can reach the alveoli in the distal airways (Frie-den et al., 2003). The bacteria are generally taken up byalveolar macrophages where they persist and replicateslowly. Interestingly, it has been reported that M. tuber-culosis is also capable of invading and replicating in typeII alveolar epithelial cells (Bermudez and Goodman,1996). Moreover, such type II pneumocytes are capableof producing proinflammatory cytokines that can influ-ence macrophage host resistance to mycobacteria (Satoet al., 2002).

M. tuberculosis infection will activate both the innate(see below) and the adaptive immune response, whichincludes helper (CD4q) T-cells, cytotoxic (CD8q) T-cells,gd T-cells and the production of interferon g (IFNg) andtumour necrosis factor a (TNFa), both key cytokines inimmunity to TB. As a result, the infected individual willtypically develop a positive tuberculin skin test (TST),based on the acquisition of delayed-type hypersensitivityto M. tuberculosis. Detection of latent TB has relied onthis principle for almost a century now. More recently, thelimited specificity of the TST has urged the developmentof new, IFNg release assays with higher specificity for thedetection of latent TB (Pai et al., 2007), even though it isnot clear whether the sensitivities of the tests are highenough to replace the TST.

In addition, current evidence is accumulating thatIFNg, even though essential in host resistance ofM. tuberculosis, is not an ideal correlate – or biomarker– of protective immunity to M. tuberculosis. This is likelyexplained in part by the fact that many other componentsof the immune system contribute to immunity toM. tuberculosis. Synergistic interactions between differ-ent pathways, such as evident for IFNg and TNFa, maymask the essential role of single individual componentsand illustrate the necessity of determining more complex,multi-factorial biomarker profiles in protection of TB.Thus, future tests will need to measure more complexpatterns of host immunity to capture the full breadth ofimmune responses to infection and vaccination to estab-lish better signatures of protection.

More complex profiling approaches are also needed toidentify the host genetic factors controlling host resis-tance vs. susceptibility to TB. Numerous host genes arelikely involved, but their identification has been difficult,and large scale studies with sufficient power are probablyneeded to identify the significant but smaller effects ofsingle genetic polymorphisms, in analogy to successfulstudies in, e.g., Crohn’s disease. Moreover, it is likely thatsynergistic interactions between different genes anddownstream pathways exist, which may mask the con-tribution of single individual components. Despite theselimitations, consistent associations have been reportedfor several genes: natural resistance associated macro-phage protein-1 or NRAMP1 (Bellamy et al., 1998; Gal-lant et al., 2007; Schurr, 2007), the vitamin D receptor(VDR) gene and MHC genes, particularly HLADRB1 (Bel-lamy, 2003) and SP110 (Tosh et al., 2006). Deficienciesin signalling pathways of adaptive and innate immunityalso impact on TB susceptibility, as will be discussed inthe following sections. Of importance here, most geneticfactors identified to date have significant roles in con-trolling innate and adaptive components of the cell medi-ated immune response in general, and to intracellular





pathogens in particular (reviewed by Ottenhoff et al.,2002; van de Vosse et al., 2004). Thus, genetic factorsthat control the balance of the cell mediated immuneresponse are likely to impact on TB resistance andsusceptibility.

Host response to M. tuberculosis

Innate immunity to M. tuberculosis

Macrophages and dendritic cells (DCs) are the first hostcells targeted by invading M. tuberculosis bacteria. Theyare the key mediators of innate immunity to M. tuber-culosis and recognise the pathogen through conservedfamilies of pattern recognition receptors (PRRs), includ-ing members of the Toll-like receptor family (TLR). TLRactivation induces 1) release of cytokines wsuch as inter-leukin 12 (IL-12) and chemokinesx; 2) differentiation ofDCs; 3) regulation of phagocytosis; and 4) triggering ofanti-microbial activities (Koul et al., 2004; Krutzik et al.,2005). Other relevant PRRs include C-type lectins, suchas DC-specific ICAM grabbing non-integrin (DC-SIGN),mannose receptors (MR) and intracellular NOD/NLRreceptors (Ferwerda et al., 2005). Furthermore, macro-phages express Fcg receptors and complement recep-tors, which promote recognition of opsonised bacteria(Ernst, 1998; Gordon, 2002).

In the course of infection, additional macrophages andresident DCs are recruited to the site of infection. MatureDCs re-locate to the lymph nodes where they produceinflammatory cytokines and prime CD4q and CD8q T-cells against mycobacteria (Tufariello et al., 2003). It hasbeen reported that M. tuberculosis is able to prevent DCmaturation by the binding of mannosylated LAM (Man-LAM) (as well as other components) to DC-SIGN, therebyinducing the production of IL-10 (Geijtenbeek et al.,2003), which suppresses immunity and T-cell activation.

The T-cell response to M. tuberculosis

Ample evidence points to the indispensable role of CD4q

lymphocytes in protective immunity to M. tuberculosis. Inhumans, loss of CD4q T-cells due to progressing HIV dis-ease greatly increases the chance of TB reactivation andTB re-infection (Corbett et al., 2003). Mice deficient inCD4qT-cells have impaired ability to control infection andeventually die from TB (Caruso et al., 1999). Adoptivetransfer of CD4q T-cells taken at the height of the primaryimmune response to M. tuberculosis conferred protectionagainst M. tuberculosis in T-cell deficient mice (Orme andCollins, 1983).

CD4q T-cells comprise several subclasses, amongstwhich T helper 1 (Th1) cells are the most abundant andprominent. Th1 cells are characterised by the productionof IFNg and TNFa (Ottenhoff et al., 1986, 1991; Haanenet al., 1991); other CD4q T-cell subsets encompass Th2cells, T-regulatory cells (Tregs) and Th17 cells. Th2 cellsproduce IL-4, IL-10 and TGFb and influence immunity toM. tuberculosis possibly by antagonising Th1 cellsthrough IL-4 (Rook, 2007). Tregs in M. tuberculosis infec-tion were shown to be associated with active TB disease(Hanekom, 2005), but are also induced efficiently by BCG

(Joosten et al., 2007) and M. leprae (Ottenhoff and deVries, 1987). More recently, Th17 cells (characterised bythe production of the pro-inflammatory cytokine IL-17)(Acosta-Rodriguez et al., 2007) were described. As yet,the exact roles and modes of action of these latter threepopulations in human TB remain to be established.

The involvement of CD8q T-cells in the containment ofM. tuberculosis is suggested by the rapid migration ofsuch cells to the sites of infection (Serbina and Flynn,1999) and their presence in granulomas (Gonzalez-Juarrero et al., 2001). Mycobacteria specific CD8qT-cellscan produce IFNg, but are mostly recognised for theircytotoxic function (Geluk et al., 2000). It has been sug-gested that CD8q T-cells control later stage M. tubercu-losis infection, whereas CD4qT-cells are important duringearly stages (van Pinxteren et al., 2000).

TCRabq CD8q T-cells and CD4qT-cells recognise anti-gens in the context of MHC class I and II, respectively,yet bacterial glycolipid antigens are typically presentedto T-cells by CD1 molecules that are abundantlyexpressed on DCs (Kaufmann, 2001). gdq T-cells recog-nise phosphate-group containing non-protein antigens inthe absence of an antigen presenting molecule (Kauf-mann, 2007). These latter two populations are commonlyreferred to as unconventional T-cells and make up asmaller fraction of circulating T-cells. UnconventionalT-cells, however, may contribute to antibacterial immu-nity, particularly in the early phase of infection.

Upon activation, both CD4q and CD8q T-cells produceseveral effector molecules. As mentioned, IFNg syner-gises with TNFa in activating M. tuberculosis infectedmacrophages and subsequent killing of M. tuberculosis(Boom et al., 2003). The vital role of TNFa in the controlof (latent) TB infection was recently underlined again bythe use of TNFa blockers in chronic inflammatory dis-eases, such as rheumatoid arthritis and Crohn’s disease:blocking TNFa, however, led to disproportional reacti-vation of latent M. tuberculosis infection and disseminat-ing TB in affected patients (Ehlers, 2003; Jacobs et al.,2007). M. tuberculosis infected mice with neutralisedTNFa or deficient in TNF receptor signalling also rapidlydied with markedly higher bacterial burden (Flynn et al.,1995), similar to humans.

Activated T-cells also produce granzymes, Fas-L(CD95L), granulysins and perforins which assist in killingM. tuberculosis infected macrophages (Kaleab et al.,1990a,b; Canaday et al., 2001).

The granuloma: M. tuberculosis’ hideout

In most cases, the cell mediated response is able tocontain M. tuberculosis in well-organised granulomas, inwhich it can persist for decades without causing anysymptoms. It is possible that in these lesions truly dor-mant bacilli are present that persist in a metabolicallyreduced or inactive state (Kaufmann, 2001). Alternatively,(a subpopulation of) persisting bacteria might continue toreplicate slowly. Regardless, in both cases latent infec-tion arises, representing a balance between host defenceand bacterial dormancy/slow replicating persistence,which can be maintained for many decades. Granulomaformation is a hallmark of latent M. tuberculosis infectionand provides a microenvironment in which interactions





between T-cells, macrophages and cytokines are facili-tated and bacteria are sequestered from spreading fur-ther into the host. The granuloma is a highly dynamicstructure in which CD4q and CD8q T-cells occupy dis-crete and different sites that are most likely associatedwith their functions in different stages of infection anddisease (Tsai et al., 2006; Ulrichs and Kaufmann, 2006).Disruption of these organised structures is a typical fea-ture of TB reactivation.

Granulomas can be formed anywhere in the lung, yet90% of the post-primary TB cases occur in the upperlobes (Balasubramanian et al., 1994). Previously, it wasreported that in fewer than 10% of the tuberculouslesions viable bacteria could be detected, whereas in50% of normal lung tissue these bacteria could be recov-ered (Opie and Aronson, 1927). In concordance, M.tuberculosis DNA was detected in the upper lobes of nor-mal lung tissue during latency (Hernandez-Pando et al.,2000). Together, these studies suggest that M. tubercu-losis may also persist in human lung tissue outside theclassic tuberculous granulomas, but this important issueneeds substantial further investigation.

Besides proper TNFa signalling, also adequate IL-12/IL-23/IFNg signalling is required for both the control ofM. tuberculosis infections and for development of maturegranulomas, as subjects genetically deficient in this axisoften fail to develop mature granulomas (Casanova andAbel, 2002; Ottenhoff et al., 2002). Likewise, mice defi-cient in IFNg signalling fail to form granulomas followingM. tuberculosis infection (Pearl et al., 2001).

The containment of the bacteria and the local intra-granuloma interactions between the host and M. tuber-culosis eventually determines the outcome of disease.

Response of M. tuberculosis to the host:evasive manoeuvres from immune killinginside the hostile human macrophage

The alveolar macrophage’s phagosome is the favouredmilieu for replication and persistence of M. tuberculosis,and perhaps also for the survival of dormant TB bacilli.Intracellular bacilli are usually degraded in highly bacte-ricidal acidic phagolysosomes. However, M. tuberculosishas adopted powerful strategies to resist, or escapefrom, these otherwise microbicidal pathways.

Escape from reactive nitrogen intermediates

Activation of macrophages by IFNg and TNFa inducesthe activity of NO synthase 2 (NOS2). NOS2 catalysesthe conversion of L-arginine into NO and related nitrogenintermediates (RNI) (MacMicking et al., 1997), which canact as potent cytotoxic agents. In vitro studies with mac-rophages have shown that the L-arginine-dependent pro-duction of RNI is the principal effector mechanism inactivated murine macrophages, responsible for inhibitingvirulent M. tuberculosis (Chan et al., 1992). Not surpris-ingly, therefore, M. tuberculosis appears to have armeditself against such oxidative and nitrosative hostresponses through the presence of noxR1 (Ehrt et al.,1997), noxR3 (Ruan et al., 1999) and ahpC (Chen et al.,

1998) genes, which all have been shown to aid in resis-tance to RNIs.

Escape of M. tuberculosis from phagolysosomalfusion

After uptake of intracellular bacteria by professionalphagocytes into classical phagosomes, the latter typi-cally fuse with lysosomes to form phagolysosomes.These compartments are highly acidic, deprived of nutri-ents (‘starvation’ stress), hypoxic and rich in hydrolyticenzymes that degrade and kill bacteria (Vieira et al.,2002). Already in the early 1970s it was recognised thatin order to evade killing, M. tuberculosis arrested phago-some maturation by preventing fusion with lysosomes(Armstrong and Hart, 1975). Proteins and molecularmechanisms associated with this phagosomal matura-tion block include LAMP1, GTPases of the Rab familyand the calcium binding protein calmodulin, coronin-1 (orTACO) (Russell, 2001; Schuller et al., 2001). More recent-ly, using chemical genetics, a network of human kinaseswas demonstrated to be essential in phagosome matu-ration. Functional disruption of kinases in the Akt1 net-work by RNAi or chemical inhibitors led to enhancedkilling of intracellular bacteria, including M. tuberculosis(Kuijl et al., 2007). Additional pathways have recentlybeen reviewed in detail (Russell, 2001; Hestvik et al.,2005; Deretic et al., 2006).

A recent provocative study has suggested thatM. tuberculosis and M. leprae – but not BCG – canescape from the phagosome, at least upon longer-term culture in human cells: M. tuberculosis containingphagosomes fused with lysosomes, but subsequentlyM. tuberculosis translocated from the phagolysosomesinto the cytosol. This process was dependent on a bacil-lary secretion system involving CFP-10 (van der Wel etal., 2007). The subject of possible translocation of M.tuberculosis remains a matter of debate, and furtherstudies are needed to dissect this process further.

M. tuberculosis genome unveiled: noveltargets for anti-tuberculous drugs?

The availability of several complete M. tuberculosisgenome sequences (Cole et al., 1998) (www.TBDB.org)combined with the possibility to study genome-wideexpression profiles under controlled conditions that arethought to be representative of distinct phases ofM. tuberculosis infection may pave the way towards dis-secting how M. tuberculosis is able to persist in humansand help identifying novel targets for intervention in(latent) TB. Using post-genomic approaches, several newcandidate genes have been identified, including two largemultigene families encoding so-called PE or PPE glycine-rich proteins. These large gene families account forapproximately 10% of the entire coding capacity of M.tuberculosis genome (Cole et al., 1998). Not much isknown yet about the function of these proteins, whichhave virtually no equivalent in any other species. Recentstudies have demonstrated their immunogenic and vac-cine potential (Chaitra et al., 2005, 2007). In addition,genes expressed by M. tuberculosis during oxygen dep-





rivation have been identified, as will be discussed in moredetail in the following section. Besides oxygen starvation,intracellular mycobacteria are also deprived from nutri-ents, such as iron and carbon, and thus exposed to nutri-ent starvation. Interestingly, M. tuberculosis adapts toboth of these specific limitations by uniquely altering itsgene expression patterns (Timm et al., 2003).

Each of these gene families offers interesting new anti-biotic targets. Obviously, molecules involved in M. tuber-culosis’ remarkable and abundant lipid metabolism arealso highly attractive targets. These include methyltrans-ferases, cyclopropane synthases (involved in the synthe-sis of mycobacterium specific mycolic acids) (Yuan andBarry, 1996; Huang et al., 2002), as well as lipolytic mol-ecules (degradation of lipids) (Cotes et al., 2007). One ofthe most effective anti-TB drugs, isoniazid, indeed tar-gets cell wall biosynthesis.

As mentioned earlier, the bacterial factors that alter thebalance from NRP to reactivation and proliferation remainunknown. Recently, a family of M. tuberculosis growthfactors (resuscitation promotion factors, Rpfs) was iden-tified, which appeared to be required for the resuscitationof dormant bacteria (Mukamolova et al., 2002). Deletionsof rpf genes resulted in defective growth of the bacteriain vivo, defective resuscitation in vitro (Downing et al.,2005), and even in delayed reactivation from chronic TBin vivo (Tufariello et al., 2006). The role of Rpfs and themechanisms controlling their expression in TB are stillunclear, but are most likely to be of relevance to bacterialpersistency and reactivation. Moreover, identification ofthe M. tuberculosis genes that permit its characteristicpersistence in humans will be essential to developingnovel anti-TB strategies directed against M. tuberculosis’persistence (Murphy and Brown, 2007). This is all themore a challenging task, as there are currently no animalmodels that replicate the full characteristics of latentM. tuberculosis infection, with the possible exception ofcertain non-human primates (Flynn and Chan, 2001).

Immunity to DosR regulon-encoded genes

The M. tuberculosis dosR regulon: stage orphase-specific gene expression

TB disease control is impaired by a fundamental lack ofunderstanding of M. tuberculosis’ unusual ability to sur-vive in the human host for many decades. Tuberculouslesions are essentially avascular and deprived of oxygenand nutrients (Wayne and Sohaskey, 2001). Given thefact that M. tuberculosis needs oxygen and is able topersist despite these low oxygen levels in lesions,M. tuberculosis must be able to adapt to gradual oxygendepletion. Studies have demonstrated that M. tubercu-losis’ adaptation to hypoxic shift down is characterisedby nitrate reduction, altered metabolism and chromoso-mal and structural changes of the non-replicating bac-teria (Wayne and Sramek, 1979; Wayne and Lin, 1982;Wayne and Hayes, 1998). The dosR regulon was foundto play a critical role in preparing M. tuberculosis for thismetabolic shift-down, as an essential step towards dor-mancy (Schnappinger et al., 2003; Voskuil et al., 2003).

Hypoxia directly affects the expression of M. tuber-culosis genes, resulting in altered metabolism which likelycontributes to dormancy. During hypoxia, M. tuberculosiswas first found to massively up-regulate expression ofthe 16 kDa wa-crystallin (acr), Rv2031c, HspXx protein(Yuan et al., 1996). These initial observations led to thesearch for similarly up-regulated genes using wholegenome microarray approaches. Voskuil et al. (2003)showed that low concentrations of NO induced expres-sion of a 48-gene (dormancy or latency) regulon, includ-ing HspX, in M. tuberculosis. The induction of all 48 dosRgenes occurred swiftly, as after only 5 min of NO expo-sure mRNA of all genes was detectable. Hypoxia similarlyinduced expression of this regulon. The complete48-gene regulon appeared to be under the control of theRv3133c (dosR/devR) gene. The importance of the dosRregulon was further underscored by altered M. tubercu-losis survival rates in in vitro hypoxia models, as survivalof wild-type M. tuberculosis was superior to that ofM. tuberculosis dosR mutants. NO was found to revers-ibly inhibit aerobic respiration and bacterial replication(Voskuil et al., 2003).

In vivo regulation of the dosR regulon was studied bytranscriptional profiling of M. tuberculosis in infectedmurine lung tissue. Five dormancy genes were selectedfor closer study, based on their strong expression in IFNg

activated macrophages. All five genes were highlyexpressed in mouse lungs 21 days post-infection andremained abundantly expressed there after (Schnappin-ger et al., 2003). The same study showed that most dor-mancy regulon genes were also strongly induced byM. tuberculosis in IFNg activated wild-type macrophagesbut not in IFNg activated macrophages from NOS2 defi-cient mice. In line with this, expression of three M. tuber-culosis dormancy genes (HspX, Rv2623 and Rv2626c)was delayed in the lungs of IFNg KO mice compared towild-type mice. Together, these studies show that host(type-1) immunity is essential in inducing the expressionof bacterial transcription patterns characteristic of NRP(Shi et al., 2003). Indeed, immune-compromised humansand animals succumb rapidly from TB infection, probablywithout a phase of latency, suggesting that also thehuman immune response is involved in triggering TBlatency. Without immune pressure, e.g., hypoxia or NO,M. tuberculosis may not be activated to up-regulate itsdosR/devR regulon, and thus fails to transit from repli-cating to non-replicating persistence.

Many of the dosR genes are conserved hypotheticalproteins (CHP) or hypothetical proteins (HP), with as yetstill unknown products and functions. Only a few geneshave been designated a function that vary from possibletransmembrane proteins to probable phosphofructo-kinases and probable ferredoxin A proteins (Table 1).

Previous work on BCG had already demonstrated thatRv3133c was up-regulated in a similar dormancy likeresponse (Boon et al., 2001). Besides Rv3133c, twoother dosR regulon proteins (Rv2623 and Rv2626c) alsodisplayed increased expression during conditions simu-lating dormancy. The response regulator Rv3133c wasnamed dosR for ‘dormancy survival regulator’, as disrup-tion of the gene disabled BCG in adapting to hypoxia andcaused loss of induction of the three dormancy proteins,HspX, Rv2623 and Rv2626c (Boon and Dick, 2002).





Table 1 Genes of the 48-gene dosR regulon of Mycobacterium tuberculosisa.

Rv number Gene name Gene product References

Rv0079 HPRv0080 CHPRv0081 Probable transcriptional regulatory protein

(ArsR family)Rv0569 CHP Rosenkrands et al., 2002Rv0570 ndrZ Probable ribonucleoside-diphosphate

reductase (large subunit)NrdZ (ribonucleotide reductase)

Rv0571c CHPRv0572c HPRv0573c CHPRv0574c CHPRv1733c Probable conserved transmembrane protein Leyten et al., 2006; Roupie et al., 2007;

Lin et al., 2007Rv1734c CHPRv1735c Hypothetical membrane proteinRv1736c narX Probable fused nitrate reductase NarX Sohaskey and Wayne, 2003;

Fenhalls et al., 2002Rv1737c narK2 Probable nitrate/nitrite transporter NarK2 Shi et al., 2005; Sohaskey, 2005Rv1738 CHP Leyten et al., 2006; Roupie et al., 2007;

Lin et al., 2007Rv1812c Probable dehydrogenaseRv1813c CHPRv1996 CHPRv1997 ctpF Probable metal cation transporter P-type

ATPase A CtpFRv1998 CHPRv2003c CHPRv2004c CHP Forero et al., 2005Rv2005c CHP Starck et al., 2004Rv2006 otsB1 Probable trehalose-6-phosphate phosphatase Edavana et al., 2004

OtsB1 (trehalose phosphatase) (tpp)Rv2007c fdxA Probable ferredoxin FdxARv2028c CHPRv2029c pfkB Probable phosphofructokinase PfkB Leyten et al., 2006; Roupie et al., 2007;

(phosphohexokinase) Lin et al., 2007Rv2030c CHPRv2031c hspX, acr Heat shock protein HspX (a-crystallin Yuan et al., 1998; Demissie et al., 2006;

homologue) (14 kDa antigen) Geluk et al., 2007Rv2032 acg CHP Acg Purkayastha et al., 2002; Florczyk et al., 2003;

Roupie et al., 2007Rv2623 TB31.7 CHP TB31.7 Florczyk et al., 2001; Boon et al., 2001;

Shi et al., 2003Rv2624c CHPRv2625c Probable conserved transmembrane alanine

and leucine rich proteinRv2626c CHP Shi et al., 2003; Davidow et al., 2005;

Roupie et al., 2007Rv2627c CHP Leyten et al., 2006; Roupie et al., 2007;

Lin et al., 2007Rv2628 HP Leyten et al., 2006; Roupie et al., 2007;

Lin et al., 2007Rv2629 CHP Starck et al., 2004Rv2630 HPRv2631 CHPRv3126c HPRv3127 CHPRv3128c CHPRv3129 CHPRv3130c tgs CHP (triacylglycerol synthase) tgs Daniel et al., 2004; Sirakova et al., 2006;

Reed et al., 2007Rv3131 CHPRv3132c devS, DosS Two component sensor histidine kinase Sherman et al., 2001; Saini et al., 2004a,b;

DevS/DosS Kumar et al., 2007





Table 1 (Continued)

Rv number Gene name Gene product References

Rv3133c devR, DosR Two-component transcriptional regulatory Sherman et al., 2001; Park et al., 2003;protein DevR/DosR Parish et al., 2003; Voskuil et al., 2003;

Saini et al., 2004a,b; Reed et al., 2007Rv3134c CHP Sherman et al., 2001; Saini et al., 2004a/b;

Bagchi et al., 2005a48 Dormancy genes of Mycobacterium tuberculosis as described by Voskuil et al. (2003).Annotations are from: http://genolist.pasteur.fr/TubercuList/, Murphy and Brown (2007), supplementary files: supplementary file 2;http://www.biomedcentral.com/content/supplementary/1471-2334-7-84-s2.xls and supplementary file 4; http://www.biomedcentral.com/content/supplementary/1471-2334-7-84-s4.doc.HP, hypothetical protein; CHP, conserved hypothetical protein.

Rv3133c is a member of a two-component system andis a transcription factor that mediates the hypoxicresponse of M. tuberculosis and its metabolic shift-downto NRP (Park et al., 2003). Two-component systems usu-ally consist of a response regulator (here Rv3133c) anda histidine sensor kinase, in this case Rv3132c (devR,DosS). Bacteria use two-component systems in theiradaptation to the environment. Signal transductionthrough two-component systems is attained by transientphosphorylation of both components (Saini et al., 2004a).Rv3133c and Rv3132c are co-transcribed and conservedin M. tuberculosis and BCG (Dasgupta et al., 2000), andRv3132c is able to phosphorylate Rv3133c in vitro. Asecond gene, Rv2027c (dosT) which also encodes asensor kinase, can also phosphorylate the Rv3133c/Rv3132c two-component system (Roberts et al., 2004;Saini et al., 2004a). Rv2027c bears strong homology withRv3132c, yet it is not known to be a part of an existingtwo-component system. However, a major differencebetween both histidine sensor kinases is that expressionof Rv2027c is not hypoxia regulated, whereas that ofRv3132c is (Saini et al., 2004a).

Deletion of Rv3133c was shown to increase M. tuber-culosis virulence in one study (Parish et al., 2003). Para-doxically, two other studies concluded otherwise: guineapigs infected with a Rv3133c-disrupted M. tuberculosismutant had decreased bacterial loads and significantlydecreased lesions in lung, liver and spleen (Malhotra etal., 2004). A possible role for dosR as a virulence factorwas also suggested when analysing virulent W/Beijinglineages of M. tuberculosis, which display enhanced epi-demic spread. These strains had constitutive up-regula-tion of dosR regulon genes compared to non-Beijingstrains (Reed et al., 2007), and accumulated high levelsof triacylglycerides, likely due to the 10-fold up-regulationof the dosR regulon gene Rv3130c, which encodes atriacylglycerides synthase. Indeed, Rv3130c deficientM. tuberculosis strains fail to accumulate triacylglyce-rides under hypoxic conditions (Daniel et al., 2004).

The dosR regulon: a source of novelphase/stage-specific M. tuberculosis antigens?

The question remains whether M. tuberculosis dosRregulon genes that are regulated in vitro or in animalmodels of TB are in fact expressed during naturalM. tuberculosis infection in humans; and if so, whethertheir products would prove relevant for the humanimmune system in recognition, and perhaps even con-trolling of M. tuberculosis infection.

HspX, the archetypal dosR regulon gene, is a smallheat-shock protein that has been studied intensely. Cel-lular immune responses to HspX have been observed inlatently infected individuals, while antibodies to this anti-gen were predominantly found in individuals with activeTB disease (Wilkinson et al., 1998). This antigen is amajor target for the human immune system and is wellrecognised by CD4q (Wilkinson et al., 1998; Caccamo etal., 2003; Geluk et al., 2007) and CD8q T-cells (Caccamoet al., 2002; Geluk et al., 2007).

HspX (acr) is required for mycobacterial persistencewithin the macrophage and is dominantly expressed inbacterial stationary phase and under reduced oxygenlevels (Yuan et al., 1998). Clues about its role in hostpathogenesis and mycobacterial persistence came fromstudies in which the acr gene was deleted from M. tuber-culosis; in vivo bacterial growth was increased in (restingand activated) macrophages from Balb/c mice infectedwith a Dacr mutant (Hu et al., 2006). Similarly, infectionof C57/B6 mice with an Dacr mutant of M. tuberculosisdemonstrated a 1–2 log higher bacillary load in the lungsin comparison to mice infected with the parentalM. tuberculosis H37Rv (Stewart et al., 2006).

Human immune responses to dosR regulon encodedgenes other than HspX have not been studied untilrecently. Leyten et al. (2006) studied human T-cellresponses to a set of the 48 dosR encoded proteins. Atotal of 25 genes with the highest fold-expression levelswere selected for analysis. First, human M. tuberculosiseducated T-cell lines were used as sensitive probes forimmune recognition, followed later by direct ex vivo iso-lated PBMC assays. Cells were derived from differenthuman donors, including TB patients, latently M. tuber-culosis infected individuals (TSTq) and non-infectedhealthy controls. The results provided first support for theimmunogenicity of dosR regulon encoded genes: all 25selected M. tuberculosis proteins were recognised byone or more of the CD4q T-cell lines. Several of thesedosR regulon proteins were also found immunogenic inPBMC stimulation assays from the mentionedindividuals.

The recognition pattern of the antigens showed signif-icant inter-individual variation. However, when the cumu-lative IFNg response to the set of antigens was used tocompare overall immune responses to the dosR antigensin TB patients vs. TSTq individuals, the TSTq groupshowed significantly higher cumulative IFNg responsesthan the TB patient group. Moreover, latently infectedindividuals tended to recognise also higher numbers of





these late stage dormancy antigens compared to individ-uals with TB disease. In contrast, the early secretedprotein CFP-10 was predominantly recognised by TBpatients. In addition, there was no significant differencein the response to whole M. tuberculosis lysate (which isa mixture of a large variety of antigens) between thesetwo groups.

Dormancy antigens, Rv1733c, Rv2029c, Rv2627c andRv2628, were identified as the most frequently recognis-ed antigens in latently infected individuals in this cohort(Leyten et al., 2006). Demissie et al. (2006) confirmed andextended the observation of preferential recognition ofdormancy antigens by TSTq individuals compared to TBpatients: in their study, HspX was preferentially recog-nised by latently infected individuals, whereas the earlysecreted M. tuberculosis protein ESAT-6 was shown tobe most responsive in TB patients. Although it remainsto be seen whether dosR regulon encoded antigens areassociated with immunity and protection from latent TBdisease, the above studies provide the first evidence for– preferential – recognition in TSTq individuals.

Another study addressed the immune responses tothese antigens in murine models of chronic TB infection.Roupie et al. (2007) showed that eight dosR encodedproteins (Rv1733c, Rv1738, Rv2029c, Rv2031c, Rv2032,Rv2626c, Rv2627c and Rv2628) were immunogenic fol-lowing DNA vaccination of both Balb/c and C57/B6 mice,as they induced antigen specific T-cells producing highlevels of IFNg. Using overlapping sets of peptides, CD4q

and CD8q T-cell epitopes could be identified forRv2029c, Rv2031c, Rv2626c, Rv2627c and Rv2628. Inaddition, strong cellular immunity to dormancy antigenswas associated with chronically persistent M. tubercu-losis infection in the mice: dosR regulon antigen specificresponses were much stronger in Balb/c mice with per-sistent M. tuberculosis infection compared to acutelyinfected animals. These results correspond with thehuman data, in which immunity to dosR antigens wasassociated with latent TB infection (Demissie et al., 2006;Leyten et al., 2006).

BCG, dosR and lack of induction of immunityagainst latency antigens

As mentioned before, several dosR genes have beenidentified in BCG. Microarray expression profiles in BCGand M. tuberculosis under low NO and hypoxia showedsimilar expression profiles of the dosR regulon. Thus,BCG clearly possesses a functional dosR regulon. Insilico analysis of the dosR regulon in BCG and M. tuber-culosis showed at least 97% homology in nucleotidesequences, with 41 out of 48 genes being identical (Linet al., 2007).

An important observation was that BCG vaccinatedinfants in the Gambia showed no detectable immuneresponses to HspX. Yet, these infants did generateimmunity to non-dosR encoded antigens, such asRv1174, Rv2376c, Rv1196, Rv1793 and Rv0125 (Veke-mans et al., 2004). This apparent and unexpected lackof immune responses to HspX following BCG vaccinationwas supported later by Geluk et al. (2007), who dem-onstrated that T-cells from BCG vaccinated individualsdid not respond to HspX if they had not been exposed

to M. tuberculosis; by contrast, significant IFNg produc-tion to HspX was found in BCG vaccinees that had apositive in vitro ESAT-6/CFP-10 response, compatiblewith exposure to M. tuberculosis. These results were fur-ther corroborated in mice: immune responses to HspXwere not induced by BCG vaccination, even though BCGinduced immunity to non-dosR antigens, such as Ag85Band Hsp65. In these animals, HspX was clearly immu-nogenic as demonstrated by protein and DNA immu-nisation, as well as epitope fine mapping for CD4q andCD8 Tq cells.

These observations are not confined to HspX only, butalso extend to additional dosR encoded antigens. Recentwork by our group shows that the above-mentioned BCGvaccinated subjects that had had no known exposureto M. tuberculosis also had no detectable immuneresponses to other tested dosR antigens. Similar resultswere found again in BCG vaccinated (BALB/c) mice. Bycontrast, immune responses to these same dosR anti-gens were found in BCG vaccinated individuals with sus-pected exposure to M. tuberculosis, as well as in micethat had been immunised with plasmid DNA encodingthe selected dormancy antigens (Lin et al., 2007).

The deficient induction of immunity of BCG againstdormancy antigens might relate to its incomplete induc-tion of protection against TB reactivation in the adult.After administration, BCG is not expected to persist inimmunocompetent individuals. The studies above arecompatible with the view that BCG, when administeredin normal skin, is mostly cleared by the host and may notinduce sufficient expression of its dosR regulon to facil-itate persistence, as well as induction of immunityagainst dosR regulon encoded antigens. In any case, ourstudies suggest that BCG immunisation fails to induceadequate immune responses to M. tuberculosis dorman-cy antigens.

Non-tuberculous mycobacteria and the dosRregulon

DosR-dosS expression has also been observed in non-pathogenic M. smegmatis in response to hypoxia (Mayuriet al., 2002). DosR genes are therefore likely to beexpressed in mycobacterial species other than M. tuber-culosis. The dosR regulon gene Rv2029c encodes aphosphofructokinase, an enzyme involved in glycolysiswhich is probably ubiquitously expressed amongst most(myco)bacteria. Cross-reactivity of dosR encoded anti-gens at the immunologic level between M. tuberculosisand non-tuberculous mycobacterias (NTMs) would there-fore be expected rather than surprising. Many mycobac-terial species, including M. avium, M. marinum andM. microti, show a moderate to high degree of homologyin nucleotide sequences encoded by the different dosRregulon genes (M.R. Klein, unpublished data).

Interestingly, Leyten et al. (2006) showed that a pro-portion of healthy blood donors also showed antigenspecific IFNg production to M. tuberculosis dormancyantigens, despite having had no known M. tuberculosisexposure, as confirmed by absent in vitro T-cellresponses to ESAT-6 and CFP-10. These fortuitouslyobserved responses to dormancy antigens are likely dueto previous exposure to NTM, which are present in high





abundance in the environment. Over half of the healthydonors responded also to M. tuberculosis lysate, in linewith prior exposure to NTM; indeed, responses toM. tuberculosis dormancy antigens were mostly confinedto this group (Leyten et al., 2006).

It remains unknown whether NTM induced cross-reac-tive responses against dosR encoded antigens contrib-ute to natural protection to M. tuberculosis. Likewise, itremains unknown whether these responses may interferewith or mask the efficacy of BCG in protection of tuber-culosis. Future research will need to provide betterinsights into the distribution of DosR encoded genesamongst NTM and their influences on immunity to bothM. tuberculosis and BCG.

Prospects of dosR encoded antigens in thecontrol of latent tuberculosis

M. tuberculosis clearly changes its gene expression phe-notype during the course of infection, a phenomenonthat only recently has been appreciated. Amongst others,this implies that the immune system of the host is facedwith different sets of antigens during the different phasesof infection, including latent M. tuberculosis infection.The identification of the first possible M. tuberculosisstage specific genes has been greatly aided by the devel-opment of genome wide expression arrays. The dosRregulon was up-regulated during conditions which areencountered by intracellular mycobacteria, and may thusrepresent a first example of M. tuberculosis stage spe-cific genes (Sherman et al., 2001; Voskuil et al., 2003).

Many of the dosR encoded antigens were immuno-genic, as discussed above. Significant recognition ofthese antigens was observed in individuals with latentTB, and cumulative responses to dosR antigens werehigher in latently infected individuals compared to TBpatients (Leyten et al., 2006). DosR antigens were alsoimmunogenic in mice, and higher responses were asso-ciated with persistent M. tuberculosis infection, confirm-ing human data (Roupie et al., 2007). Remarkably, BCGvaccination did not induce immunity to dormancy anti-gens, neither in humans nor in mice (Lin et al., 2007).

DosR regulon genes that are essential to bacterialdormancy have unexplored potential: they may providenew potential drug targets, as currently used drugs most-ly target actively metabolising bacteria and are mostlikely ineffective against dormant bacteria. Additionally,they may be explored for diagnostics and vaccinedevelopment.

Most newly developed TB vaccines are based on anti-gens that are expressed during early (growth) phase ofM. tuberculosis infection (e.g., ESAT-6, Ag85B) and aretypically designed for prophylactic purposes. Latent TB,however, is unlikely to be targeted by prophylactic vac-cines, as the latter may not be effective in the alreadyinfected host during the late stage infection, in whichimmunity to other antigens may be more relevant. Onestrategy is to develop post-exposure/therapeutic vac-cines that adequately activate T-cell responses to latestage infection associated antigens. Such vaccines maytarget dormant M. tuberculosis and help eliminate the

pathogen during its dormant or slowly replicating phaseof infection, thereby contributing to prevention from laterreactivation from latency. Sub-unit vaccines based ondormancy antigens might also be applied in combinationwith sub-unit vaccines containing early stage antigens toform multi-phase vaccines (Andersen, 2007b). Such vac-cines would offer the advantage of immune protectionagainst both early and late stages of infection. Alterna-tively, dormancy antigens could be used to complementand improve BCG by developing recombinant BCGstrains that constitutively express selected dosR genes.

Post-exposure TB vaccines ought to be administratedto people on different geographical sites that harbourinfections with different strains of M. tuberculosis. Differ-ences in strains might translate into phenotypically dif-ferent physiology and host immune response profilesduring latency. M. tuberculosis can be classified into sixdifferent phylogeographical lineages, each with specificsympatric human populations (Gagneux and Small,2007). Knowledge about the distribution and expressionof dosR encoded genes amongst different M. tubercu-losis strains is lacking, but possible strain heterogeneitytogether with host-specific pathogen adaptationwill need to be considered in the development of vac-cines, including post-exposure vaccines against M.tuberculosis.

Clearly, genes associated with latent M. tuberculosisinfection require further characterisation, as does thehost immune response during latent infection. Besidesimproved TB vaccines, such knowledge will also help toidentify better correlates of protection in (latent) TB.Future studies need to explore these questions and toevaluate the potential of stage specific antigens aspotential TB vaccine candidates, either alone or in com-bination with BCG.

Acknowledgements

This work was supported by a grant from the Foundation Micro-biology Leiden, the European Commission within the 6th Frame-work Programme, contract no. LSHP-CT-2003-503367 (the textrepresents the authors’ views and does not necessarily repre-sent a position of the Commission who will not be liable for theuse of such information) and the Bill and Melinda Gates Foun-dation, Grand Challenges in Global Health GC6�74, GC12�82.

Note added in proof

After submission of this paper, a publication appeared discuss-ing the role of other potential genes in controlling the responseto hypoxia in M. tuberculosis, next to the dosR regulon that wehave discussed here wT.R. Rustad, M.I. Harrell, R. Liao and D.R.Sherman (2008). The enduring hypoxic response of Mycobac-terium tuberculosis. PLoS ONE 3, e1502. DOI: 10.1371/jour-nal.pone.0001502x. Likely, the conclusions we draw regardingthe dosR regulon genes also apply to the new M. tuberculosisgenes.

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