Review. Immunotherapy for TB. T Mark Doherty

Transcription

1 Immunoterapy TB Keywds: bacterial n immunoterapy n latency n TB M. tuberculosis was one o te irst uman patogens to be identiied as te cause o a speciic disease TB. TB was also one o te irst speciic diseases wic immunoterapy was attempted. In me tan a century since, multiple dierent immunoterapies ave been attempted, alongside vaccination and antibiotic treatment, wit varying degrees o success. Despite tis, TB remains a maj wldwide ealt problem tat causes nearly 2 million deats annually and as inected an estimated 2 billion people. maj reason tis is tat M. tuberculosis is an ancient uman patogen tat as evolved complex strategies persistence in te uman ost. It as tus been long understd tat, to eectively control TB, we will need to address te ability o te patogen to establis a persistent, latent inection in most inected individuals. Tis review discusses wat is presently known abo te interaction o M. tuberculosis wit te immune system, and ow tis knowledge as been used to design immunoterapeic strategies. to understanding te callenges tat immuno terapy aces and some o te dierent approaces tat ave been tried. Controlling TB: social intervention, antibiotics, vaccination & immunoterapy Over te course o te 20t century, te incidence o TB ell dramatically in most industrialized nations [9], initially as a result o improvement in living conditions, public ealt education campaigns and te widespread use o sanatia to seclude inectious TB cases. Furter tecnical advances te use o tuberculin to diagnose latent inection and te introduction o widespread x-ray screening urter elped to identiy TB cases treatment containment. Te introduction o te bacillus Calmette Guérin (BCG) vaccine gave us, te irst time, an eective tl to prevent disease rater tan treat it ater te act, and BCG vaccination campaigns across Europe sowed a clear decrease in TB incidence in te vaccinated cots [10,11]. Te culmination o tis al-century o progress te identiication o eective antibiotics and eective al cemoterapy led many to pronounce tat TB would sn be a ting o te past. Clearly, owever, tat did not appen. In developed countries, TB incidence is restrained by continuing surveillance and treatment; any reduction in vigilance leads to rapid resurgence /IMT Fure Medicine Ltd Immunoterapy (2012) 4(6), Medical airs, GlaxoSmitKline, Brøndby, DK-2605, Copenagen, Denmark Tel.: mark.x.doerty@gsk.com Mycobacterium tuberculosis, te causative agent o TB, is one o te wld s most devastating uman patogens. In 2010, tere were an estimated 8.8 million incident cases o TB and approximately 1.5 million deats [1]. Central to te success o M. tuberculosis as a patogen is its ability to persist witin umans long periods in a clinically latent state; approximately 95% o te people wo become inected develop a latent inection. Te magnitude o tis disease reservoir is estimated to be approximately 2 billion people approximately a tird o te global population [2]. Te global burden o disease as been wsened by te spread o HIV [3,4] : te two diseases m a vicious cycle wit eac wsening te ocome o te oter [5 7]. Finally, te spread o multidrug-resistant TB (MDR TB; deined as a strain o M. tuberculosis tat is resistant to at least isoniazid and riampin, te two drugs tat ave been te backbone o most treatment regimens) and extremely drugresistant TB (XDR TB; deined as resistance to isoniazid and riampin plus resistance to imptant second-line medications: te luoquinolones and at least one o te aminoglycosides, suc as amikacin, kanamycin capreomycin) as sarply raised interest in better terapies TB [8]. ll o tis continues to make TB control a priity issue around te globe. Tis review introduces te disease, particularly te complex interaction o M. tuberculosis wit te ost s immune system, as background T Mark Doerty part o ISSN X 629

2 Doerty must understand te way tat M. tuberculosis itsel aects immune responses. M. tuberculosis survival strategies Inibiting pagocyte unction Te nmal roe o inection M. tuberculosis is te mucosal surace, eiter troug te lung via inalation o airbne spum droplets rom an inected person s coug troug te g ater ingestion o inected material (te latter roe is te way tat anoter TB complex member, Mycobacterium bovis, typically inects umans). Te mucosal surace is monited by pagocytic cells, wic take up and kill invading patogens. It is known tat some eavily M. tuberculosis-exposed individuals remain ealty and wito signs o inection. It is tempting to speculate tat, in tese individuals, te innate immune response as eliminated te patogen bee it can initiate a productive inection. In most cases, owever, te patogen is able to resist killing by te pagocyte. One o te reasons M. tuberculosis as been so successul as a patogen is its ability to persist witin ost cells, including immune cells suc as macro pages and monocytes, wic are responsible bot killing bacteria directly by pagocytosis and priming immune responses by antigen presentation. M. tuberculosis does tis initially by interering wit pagocytosis, a process tat starts wit te irst contact between bacterial cell wall components and te cell s recepts (Figure 1). Pagosome maturation te merging o te pagosome wit oter vesicles carrying a variety o antimicrobial payloads (e.g., superoxides, catelicidins, deensins and ubiquitin) creates an intracellular compartment wit an acidic, ydrolytic environment and M. tuberculosis as evolved multiple mecanisms to block tis process [21], wic would oterwise be atal te patogen, including te EST-6/CFP10 and Sec1/2 proteins [22 24]. In addition, inibition o TPase and GTPase accumulation in te vacuole intereres wit te cell s ability to monit pagosome unctions suc as acidiication [25], wile inibition o calmodulin-dependent Ca 2+ lux and expression o molecules suc as LRG 47 [26,27] intereres wit vacuole tracking and usion [28 30]. However, usion wit oter vesicles and membrane remodeling still occurs to some extent, allowing M. tuberculosis to acquire necessary nrients and cations (especially iron) and expt its own proteins [26,29,31]. Immediately ater pagocytosis, M. tuberculosis activates numerous o te disease [12]. In te less-developed wld, new TB epidemics arose in switly growing cities, me tan compensating te decline elsewere. In some igly TB-endemic regions, suc as sub-saaran rica, TB reaces incidences well above 500 cases per 100,000 individuals in te year-old age group; comparable to te epidemic in te industrializing cities o 19t century Europe and te US [1,9]. In te longer term, vaccination as proved to be only partially eective. ltoug BCG is eective in inants in mycobacteria-naive adults [10,11,13], in individuals presensitized to mycobacteria by, example, exposure to environmental mycobacteria, pri BCG vaccination M. tuberculosis inection, it does not appear to oer any additional protection [14,15]. BCG as never sown any terapeic eicacy against M. tuberculosis, altoug its immunostimulaty properties mean tat it is widely used as an immunoterapeic various ms o cancer [16]. ntibiotics ave likewise proven to only be a stopgap measure. Te diiculty o timely diagnosis, particularly in TB-endemic areas [17], means tat many cases are only treated long ater tey become inectious, so tat te cycle o inection is merely reduced, not interrupted. ltoug drugs are ceap and eective, treatment requires many monts. Te diiculty o maintaining compliance over long treatment regimes reduces successul treatment rates and provides opptunities drug resistance to emerge [18]. Treatment o MDR TB and XDR TB is expensive and time-consuming, and ocomes especially patients wit XDR TB remain po [19]. Tese acts ave revived an interest in immunoterapy M. tuberculosis, bot te development o vaccines wit postexposure eicacy and as adjuncts to terapy. Te potential o immunoterapy as long been recognized; sn ater te discovery o M. tuberculosis, Robert Koc attempted to treat patients wit a bacterial extract tat e called tuberculin. las Robert Koc s repation and immunoterapy generally te treatment was not successul [20]. It did, in a ew individuals, apparently lead to cure. In oters, it led to severe reactions, and may ave even caused teir deats. In te majity, it was painul and ineective. Immunoterapy TB labed under te cloud o tis ailure decades. Today, we understand tat generating a strong immune response is not, by itsel, suicient and recognize tat to manipulate te ost s immune response to M. tuberculosis, we 630 Immunoterapy (2012) 4(6) ure science group

3 Immunoterapy TB Host process: PMP recept ligation (activates immune response) Ferritin (sequester iron) o-apoptotic molecules (promote apoptotic cell deat) oteinases and lipases (lyze bacterial components) V-TPases (lower ph) NPDH complex (generates superoxide burst) Caperonins (sample vesicle antigens to present) ntimicrobial peptides and proteins (disrupt bacterial cell wall and membrane proteins) Endosome Pagosome Pagocytosis Early pagosome Lysosome Vesicles rom Golgi and ER Membrane proteins (disrupt pagosome membrane integrity, interere wit ost vacuole signaling/traicking) Patogen response: Lipoproteins to disrupt PMP signaling (inibit immune activation) Iron scavengers (acquire iron) Superoxide dismase and cell wall lipidoglycans (inibit superoxide burst) Decoy proteins (to misdirect antigen-speciic response) Dimycolates and lipoarabinomannans (inibit antigen uptake) ntiapoptotic molecules (promote necrotic cell deat) Lysosome Figure 1. Patogen intererence wit pagosome maturation. Te maturation o te pagosome, altoug ere depicted as a simple linear process leading to te killing o te patogen, is actually a dynamic process wit many potential end points: ow te pagosome is andled by te cell and ow it develops depends on te signaling markers inserted into, bound to, its surace. Mycobacterium tuberculosis survives by being able to divert te traicking o vesicles carrying potentially armul molecules so tat tey do not use wit te pagosome; instead it recruits vesicles carrying te resources it needs. Tis process starts immediately, wit binding o pagocyte recepts wit ligands on te patogen s surace. Tis triggers te remodeling o te pagocyte s surace membrane and a part o te membrane is pinced o to m a vacuole: te early pagosome. Interactions o patogen surace molecules and ost recepts suc as DC-SIGN, CRD, MINCLE and members o te Toll-like recept amily start to induce cytokine production, modiying te pagocyte s own beavi and sending signals to oter immune cells, promoting ( inibiting) teir recruitment and activation. Te rapid recruitment o te NDPH oxidase complex and TPases (proton pumps) drives te acidiication o te vacuole a low ph (~4.5) is imptant activation o lytic molecules delivered into te maturing pagosome by lysosomes. s it matures, te vacuole acquires a variety o membrane proteins delivered by usion wit oter vesicles rom te recycling endosomal system, te ER, te aopagosome and te Golgi apparatus, wic guide te delivery o speciic ost molecules and vesicles to te pagosome, wit te purpose o eradicating te patogen inside. t te same time, te patogen inside te vacuole inserts its own proteins into te membrane, wic inder tis process and allow te patogen to scavenge te necessary cations and nrients needed survival. ER: Endoplasmic reticulum; PMP: Patogen-associated molecular pattern; V-TPase: Vacuolar TPase. genes, including transpters, iron scavengers and genes involved in lipid accumulation and metabolism [28,29,32 35]. Surace molecules suc as trealose dimycolate and mannose-capped lipoarabinomannan prevent vacuole expression o MHC molecules and costimulats, inibiting antigen presentation. Te seer number o genes involved in interering wit pagosome unction ure science group indicates te imptance o tis approac te survival o M. tuberculosis [26,29 31,33]. However, te ost is able to respond in kind. ctivated pagocytes are less susceptible to tese M. tuberculosis-induced inibity eects [36 38] and invasion o pagocytes tat are not yet activated may be imptant te bacteria s ability to invade te ost. Exposure o macropages to 631

4 Doerty IFN g and/ TNF-a and induction o reactive oxygen and nitrogen intermediates pri to inection reduces teir susceptibility to M. tuberculosis-induced deiciencies in pagosome maturation and unction [26,36,39 42] ; activation o te inlammaty patways tat produce tese molecules as teree played a central role in immunoterapy (discussed in detail below). Despite M. tuberculosis s ability to interere wit traicking, te majity o exposed individuals are able to control te inection and te generation o antigen-speciic immune responses sows tat te PCs are able to present antigens rom te killed bacteria. Inibiting inlammaty immune responses It turns o tat Saw was rigt all along [43] : tere is undamentally only one genuinely scientiic treatment all diseases, and tat is to stimulate te pagocytes. Induction o IFN g is a crucial irst step in tat direction. It as been suggested tat unconventional T cell subsets (gd, NK-T and CD1-restricted cells [44,45]) serve as te initial source o cytokine production [46,47] by responding to targets suc as lipids and glycoproteins ound on te surace o bacterial patogens [48]. Tis noncognate cytokine production may be crucial bsting PC unction in te early pases o inection [49,50], especially te interleukins (IL-12, IL-18 and IL-23) needed to initiate cognate immunity and te development o immune memy. IL-23 drives te maturation and expansion o a subset o T elper cells, called T17 cells, wic are caracterized by production o IL-17, wic recruits and activates neropils, monocytes and IFN g-producing CD4 + T cells. It plays a crucial role in te initial development o te granuloma [51] b, i not downregulated, it can be immunopatological [52]. Genetic deicits in te IL-12/IFN g axis are associated wit extreme susceptibility to mycobacterial disease [53,54], wile animals patients deective in CD4 + T cells IFN g production are igly susceptible to M. tuberculosis inection [55 59], indicating te essential nature o tese acts in immunity to M. tuberculosis. In addition, CD8 + T cells appear to play a role troug cytotoxic activity and IFN g production, altoug tis response appears to take longer to develop [60 62]. Given te imptance o te T1/inlammaty response control o M. tuberculosis inection, it is no surprise tat te patogen counters te development o adaptive immunity (Figure 2). Molecules ound on in te M. tuberculosis cell wall can inibit some o te eects o IFN g [63 65], reduce its production, leading to te suppression o IFN g and T cell responses associated wit progressive TB disease in patients [66]. Tis may be in part due to a bias toward T2 cytokine production. Elevated IL-4 gene expression crelates wit disease severity in TB patients [67 69] and risk o subsequent disease in ealty b TB-exposed individuals [70 72]. Conversely, te ratio o IFN g ( te IL-4 antagonist IL-4d2) to IL-4 tends to increase in patients during terapy, b decreases in TB contacts tat become ill [71]. Similar observations ave also been made in animal models o TB [73,74]. M. tuberculosis cell wall components suc as pospoglycolipids te 19 kda antigen tat induce IL-4 and IL-13 may tus be imptant virulence [32,75,76], wile production o IFN g, TNF-a IL-4 antagonists is essential protection. low IFN g:il-10 ratio is also associated wit a iger risk o progressive disease [71,77,78] and, consistent wit tis, recent studies ave ound tat expression o IL-10 is signiicantly elevated in TB patients wit active disease [79 81]. IL-10 is induced in ost cells by M. tuberculosis cell wall components suc as lipoarabinomannan. Lipoarabinomannan binding to te DC-SIGN recept o dendritic cells inibits teir maturation, MHC expression, antigen presentation and expression o costimulaty recepts, reduces IL-12 production and induces IL-10 secretion instead [82,83]. ltering te balance between IFN g and IL-4 IL-10 production and unction tus seems to be a second maj survival strategy M. tuberculosis. Te second inlammaty cytokine unequivocally associated wit immune protection against M. tuberculosis is TNF-a [84]. Individuals wit latent M. tuberculosis inection, wo are oterwise controlling te patogen, rapidly progress to disease wen treated wit TNF-a recept antagonists [85,86]. TNF-a production is also essential protection in animal models [87,88] b TNF-a is a double-edged swd: in some circumstances, it appears to lead to immuno p atology rater tan protection, and overexpression o IL-4 may promote tis [89,90]. M. tuberculosis (b not avirulent myco bacteria) is able to desensitize ost pagocytes to TNF-a by inducing inected cells to sed teir TNF-a recepts [91 93], turning tem into soluble antago nists; a pattern consistent wit tat seen in TB patients [94]. TNF-a blockade seems to ave a negative eect on pagosome 632 Immunoterapy (2012) 4(6) ure science group

5 Immunoterapy TB Host process: Induce IFN-γ (activate T cells and PC) Induce TNF-α (activate inlammaty and apoptotic cascades) Induce IL-12 and IL-18 (promote T1 cell activation and maturation) oteinases and lipases (lyze bacterial components) Induce IL-23 (promote T17 cell activation and maturation) Induce IL-2 (promote T cell replication) Caperonins (sample vesicle antigens to present) CD8 CD8 CD8 CD4 CD8 CD4 ntigen presentation T1/T17 activation T-cell memy and PC activation CD4 CD4 Induce sedding o TNF-α recepts on inected PC (inibit TNF-α eects on PC) Dimycolates and lipoarabinomannans (inibit antigen uptake) Decoy antigens (induce immune responses tat promote patology, not protection) Pospogycolipids induce IL-4, IL-10 and IL-13 (inibit IFN-γ eect, promote T2 maturation and drive TNF-α dependant patology) Bacterial proteins activate SOCS and IRK-M patways (inibit TLR signaling and downregulate inlammaty responses) Cell wall components induce IL-10 and TGF-β (inibit inlammaty responses) Patogen response: Figure 2. Patogen intererence wit te generation o adaptive immunity. Te intracellular environment in Figure 1 is also aected by te microenvironment tat te pagocyte inds itsel in, most crucially te response o adaptive immunity to TB inection. otection requires te activation o T1/T17 cells and te generation o a strong antigen-speciic memy response. Tis is initiated by te secretion o cytokines rom inected pagocytes most prominently including IL-12, IL-18, IL-23 and TNF-a and presentation o antigens to CD4 + and CD8 + T cells. Nonconventional CD1-restricted T cells and gd T cells, wic respond to conserved nonprotein antigens rom te patogen s cell wall, may play an imptant early role in augmenting responses, b it appears to be te conventional MHC class I and class II memy response and te production o antigen-speciic IFN g, TNF-a and IL-17 tat are most imptant eventual protection. Te patogen can counter te development o protective T1 immunity by promoting te expansion o T2 cells, inibit it by downregulating inlammaty responses subvert it by producing decoy antigens, responses against wic may lead to immunopatology, by interering wit te signaling patways tat respond to IFN g and TNF-a. bsent rom tis simple overview are te various subsets o Tregs. Tese expand ater Mycobacterium tuberculosis inection, b it is not clear yet weter tey represent a potential treat (by inibiting te development o protective immunity) a beneit (by inibiting immunopatology). It is possible, even likely, tat tey can do neiter, depending on te state o te immune response and inection. TLR: Toll-like recept. maturation [95], peraps owing to te role it plays in te induction o reactive nitrogen products. It teree appears tat M. tuberculosis inibits IFN g and TNF-a unction and production by multiple roes, allowing it to modulate te development o te bactericidal pagosome and te expansion o an eective adaptive immune response. Interering wit ure science group TNF-a unction also plays a role in cell deat and tissue destruction in TB. Modulating ost cell deat s a last rest, te ost can remove inected pagocytes by inducing teir deat, eiter by apoptosis necrosis. Cells tat die by apoptosis do not generally activate strong inlammaty 633

6 Doerty granuloma, in wic a small number o inected pagocytes are surrounded by successive zones o activated monocyte/macropages and activated lympocytes [119]. Successul containment o inection at tis stage may leave a small scar calciication and a memy response to M. tuberculosis-derived antigens. However, i bacterial replication is not controlled, te granuloma can increase in size and cellularity and cell deat (as discussed above) may lead to necrosis at te center o te granuloma. Were te tissue destruction caused by necrosis aects te surace o te bronci, te granuloma s contents (including M. tuberculosis bacilli) will drain into te lumen o te lung, giving rise to a cavity and te prototypic symptom o TB a persistent coug, wit bld pus in te spum. t tis point, te bacteria can spread to oter sites in te lung and to oter osts, wo are exposed to bacteria in te spum o te couging patient. Tis process o inlammation and tissue destruction is essential to te survival and spread o M. tuberculosis. Inection wito cavitation is an evolionary dead end, as te bacteria would be unable to spread to new osts. Te cavitation and patology seen in TB is not due to cytotoxic activities o te patogen; it is immunopatological in nature. Tus, M. tuberculosis as evolved to subvert te inlammaty response tat te ost relies on eradicating bacterial inections to promote its own dissemination. Some o te speciic patways ave now been identiied and tey appear to involve subversion o te mecanisms nmally associated wit protective immunity. s an example, te 19 kda lipoprotein o M. tuberculosis appears to be a virulence act [120] tat reduces overall immunity to te patogen [121], even toug it binds to te TLR2 recept [122,123], wic is nmally a irst step in initiating proinlammaty responses [124,125]. Instead, 19 kda binding reduces expression o many IFN g-activated genes [126], and inibits antigen processing and MHC II expression [ ]. noter virulence act, EST-6, also binds to TLR-2 and as a similar inibity eect [129], as do glycoproteins rom some epidemic strains o M. tuberculosis [75,76]. It tus appears tat intererence in IFN g signaling ater TLR2/4 ligation is an imptant mecanism o virulence [130]. Meover, even wen IFN g responses are initiated, M. tuberculosis appears to be able to evade immunity by expressing decoy molecules. Te 27 kda lipoprotein o M. tuberculosis is strongly immunogenic, b vaccination wit responses, allowing inected cells to be removed [96,97] wito inducing damage in te surrounding tissue [98]. Meover, apoptotic cells tend to srink, b remain intact, so tat intracellular patogens are removed along wit te dying cell. By contrast, necrosis leads to lysis o te dying cell, allowing release o te patogen and damaging te surrounding tissue [98]. Resolving granulomas are igly enriced apoptotic cells and reduced apoptotic capacity is asso ciated wit enanced patology in response to M. tuberculosis inection [99], wile unresolved granulomas oten ave necrotic centers. Tese necrotic granulomas are a classic eature o evolving TB. In vitro and in vivo studies bot demonstrate tat living, virulent M. tuberculosis (b not dead avirulent mycobacteria) can inibit apoptosis. It as been suggested tat tis is a patogen survival strategy [ ], allowing it to escape to inect new ost cells by inducing necrosis instead [ ]. Consistent wit tis, elevated levels o necrosis in te granuloma crelate wit genetic susceptibility to M. tuberculosis in mice [112] virulence o uman-derived clinical isolates [113]. TNF-a is a potent inducer o cell deat by apoptosis [114] and induction o apoptosis by te CD43 TNF-a inlammaty patway is asso ciated wit control o M. tuberculosis [115]. In addition, it appears tat IL-4 also plays a role ere by promoting te expression o antiapoptotic genes and antagonizing te eect o TNF-a [93]. In summary, M. tuberculosis is able to interere wit te ost s immune response at multiple stages. However, tere is one me aspect to consider tat is igly relevant to immunoterapy. Paradoxical as it migt seem, M. tuberculosis is not only able to turn o te ost s inlammaty immune response, b as also evolved a ost o mecanisms to turn it on. M. tuberculosis escape strategies Tissue damage & cavitation Despite te immune evasion/modulation response strategies tat M. tuberculosis can induce, te initial response to inection is virtually always a strong inlammaty response. Tis is because te cell wall o M. tuberculosis includes many long-cain atty acids and carboydrates [32, ], wic bind to te ost s patogenassociated molecular pattern recepts, suc as te Toll-like recept (TLR) amily [117], strongly stimulating ost inlammaty responses. Tis response switly recruits and activates immune cells. In turn, tis leads to mation o a 634 Immunoterapy (2012) 4(6) ure science group

7 Immunoterapy TB Supplying immune modulats suc as cytokines vitamins Bot approaces ave been tried alone, togeter as an adjunct to cemoterapy, wit varying degrees o success. Eac approac is discussed in me detail below, ocusing primarily on tose approaces tat ave been tested in umans, wic tere is a substantial body o literature, since te number o candidates tat ave been suggested, tried only in animal models, is t large to cover in a single review (a broader overview is provided by Grange and colleagues [140]). Lessons immunoterapy rom te biology o M. tuberculosis Understanding te basic biology o M. tuberculosis inection allows us in indsigt to understand wy early attempts at immuno terapy ultimately ailed. It also allows us to appreciate te callenge o te task wen it comes to developing new immunoterapies. Many o te evasion strategies discussed above can only be demonstrated wit live M. tuberculosis: tey presumably rely on te patogen responding to te microenvironment induced by te ost s attempts to eradicate it. So, wen Robert Koc and oter docts attempted to treat TB by injecting large quantities o proteins extracted rom killed M. tuberculosis, tey generated as intended a potent inlammaty response. However, wat tey did not appreciate initially is tat, i not appropriately controlled, severe inlammaty responses can be atal te patient, wile M. tuberculosis itsel as evolved to survive suc responses. Meover, TB patients ave by deinition already developed a memy immune response tat as proven unable to contain te patogen. Oten tis response is already quite strong, so stimulating it urter is unlikely to elp very muc. F immunoterapy to be eective against M. tuberculosis, teree, it needs to be able to meet speciic criteria. First, te response needs to be appropriately balanced; stimulating ig levels o TNF-a in a ost in wic TNF-a recepts are sed by inected cells is me likely to be patology-generating tan protective. Likewise, generating ig levels o antigen-speciic IFN g is unlikely to elp i downstream signaling te response to IFN g is being actively blocked. However, increasing inlammaty responses may be appropriate in patients wose response is low (e.g., in latent TB). Second, te immune response generated by immunoterapy needs to be targeted to speciic molecules ( be broad enoug) so tat te patogen cannot simply reduce replace te target molecule, allowing it to evade te memy response. Wit tese limitations in mind, tere is increasing interest in immunoterapy, eiter alone, as an adjunct to cemoterapy as a strategy te design o vaccines wit terapeic postexposure eicacy. Te approaces tat ave been tried all into two basic categies: Stimulation o te immune system wit antigens (eiter bacterial extracts speciic antigens) te lipo protein promotes bacterial growt in vaccinated animals, despite te IFN g induced [131,132]. TB patients oten express ig levels o IFN g in response to members o te igly polympic PE-PGRS and PPE MPTR gene amilies, altoug tey are patently unable to control te inection, leading to te suggestion tat tese are also decoy molecules [133,134]. It as even been suggested tat, by turning gene transcription on o, M. tuberculosis may evade immune surveillance during te latent pase o inection [126,128,135], wile still allowing te initiation o inlammaty immune responses leading to tissue destruction and cavitation during ace inection reactivation. Tis ability to modulate te ost s inlammaty immune response explains wy many inected individuals develop a latent inection tat can persist decades in te ace o demonstrable cognate immunity [ ]. ure science group Immunoterapy wit mycobacterial extracts killed bacteria Tuberculin s already noted in te introduction, te irst attempt at immunoterapy TB was injection wit tuberculin made ater Koc s recipe. In essence, all tese preparations were based on iltering a weak ganic extraction o eatkilled bacterial cultures grown in glycerol-based media to obtain a soluble, protein-ric extract [141]. It was known at te time tat te response derived was speciic TB and immunological in nature, wit Koc noting tat Te most imptant eature o tis remedy is its speciic action on tuberculous processes o any kind; ealty individuals wito TB inection did not react to it [20]. However, te precise nature o te response was beyond te understanding o te science o te time; it was not until von Pirquet s wk tat te delayed-type yper sensitivity response was clearly deined, and in te absence o tat understanding, te necrosis 635

8 Doerty tuberculin terapy ad all b vanised by te mid-20t century. Mycobacterium vaccae Immunoterapy M. tuberculosis began to see renewed interest as concerns abo te development o resistance to current drug treatment regimens and te general inadequacy o te prolonged drug treatments required cure increased. rept rom Jon Stand and colleagues described a small trial in Kuwait in wic patients undergoing cemoterapy received a single dose o te sapropytic M. vaccae, killed by irradiation, as adjunct terapy [143]. ltoug most clinical parameters assessed at te time did not cange signiicantly, patients receiving te adjunct terapy did sow signiicantly improved weigt gain, plus increased lympocyte prolieration and antibody responses to a panel o common mycobacterial antigens. Somewat unexpectedly, tey also demonstrated reduced skin test responses to tuberculin, indicating tat some inlammaty immune responses migt be downregulated. In te intervening years, tere ave been dozens o urter clinical trials testing M. vaccae as an adjunct immunoterapy, many in Cina [144], were M. vaccae is now registered as an approved adjunct terapy TB treatment. Most o tese trials ave been quite small, and many ave used dierent protocols, bot o wic acts ave probably contribed to te decidedly mixed results seen. Wit regard to primary end points (deat spum conversion at 2 monts and at end o terapy), no signiicant eects ave been seen rom trials rated as ig quality [145]. Taken in total, owever, te trials rept quite eterogeneous results, wit a number o trials suggesting a weak increase in te number o individuals wo converted to spum negativity ater 2 monts o cemoterapy [144,145]. ltoug te adjunct terapy generated signiicantly me side eects tan te standard treatment [145], in general, tese eects were transient and not serious. Te exception to te comment abo small trials wit M. vaccae is te Dardar trial, conducted to determine weter a multiple-dose series o M. vaccae, in association wit isoniazid cemoterapy, could prevent HIV-associated TB rom developing [146]. In tis study, 2013 BCG-vaccinated, HIV-inected patients wit CD4 + cell counts o at least 200 cells/µl were randomized to receive eiter ive intradermal doses o M. vaccae, a placebo, plus isoniazid i teir tuberculin skin test response was greater tan 5 mm. Te approac ere was dierent induced by treatment was initially seen as a positive eect te destruction o te tissue werein te patogen resided. Indeed, te precise details are still being wked o at te molecular level [142]. Te treatment itsel was simple: repeated intradermal, subcaneous intramuscular injection o te partially puriied bacterial extract. Untunately, te initial eupia over te discovery o a potential cure TB, and te wave o scientists trying te new tecnique, rapidly evapated amidst repts o severe side eects. Witin 2 years, a rept rom Germany summarizing data rom nearly 1800 patients wo ad been treated wit tuberculin concluded tat te new treatment ad no value treating TB [20]. Not only ad almost all patients suered dramatic eects among tem ever, convulsions and local necrosis; symptoms tat became known as te Koc reaction b te overall ocomes were no better tan tose seen in untreated patients. It was not, owever, te end o attempts to generate a protective response wit bacterial extracts. In te decades ollowing te tuberculin iasco, Friedric Friedmann isolated Mycobacterium celonae rom turtles and tested it as bot a terapeic and propylactic vaccine, by intramuscular injection o bacterial extracts wole, killed bacteria [141]. By tat stage it was known tat te wst Koc reactions were in response to ig doses o antigen. Friedmann reduced is doses and spaced is injections ar enoug apart tat te most severe reactions were avoided. In addition, e oped tat, as M. celonae was tougt to be nonpatogenic in man, te reactions it induced would be milder tan preparations derived rom M. tuberculosis. He treated over 1000 individuals and claimed some success te treatment. He was not alone; in te UK Sir lmrot Wrigt designed a regimen o repeated small doses o tuberculin tat, i noting else, eliminated te wst o te side eects and in te irst ew decades o te 20t century many variants on tuberculin treatment remained widely used [141]. In retrospect, it is impossible to determine weter any o tese were beneicial, weter tey simply persisted in te absence o anyting better. Treatments varied so wildly (in te material used; in te scedule, many o wic were nonstandard, being derived eac patient by titration; and in te assessment o patients) tat any systematic comparison is impossible. It is clear, owever, tat beneicial eects i any were small. Wit te advent o BCG as a propylactic vaccine, better surgical options and, inally, antibiotics treatment, 636 Immunoterapy (2012) 4(6) ure science group

9 Immunoterapy TB b some eect against colony-ming units in te st term being repted wen RUTI was given stly ater primary inection [153]. Similar to BCG, M. vaccae and Mycobacterium microti, M. tuberculosis culture iltrate given as adjuvant, RUTI as been observed to oer some eicacy in animal models wen given pri to inection wit M. tuberculosis [153,154], and it generates a strong T1 response, wit elevated levels o IFN g. It remains to be seen, owever, weter RUTI will perm dierently rom oter mycobacterial preparations wit similar T1-stimulating properties. Immunoterapy wit deined mycobacterial antigens delivered as DN Hsp65 Hsp65 is one o a amily o igly conserved proteins tat stabilize molecular unctions against stress. Te Hsp designation reers to eat sock protein and comes rom te iginal observation tat expression o tis amily o genes was elevated by eat sock (altoug tey are now known to be induced by many dierent ms o stress, including inection). Interest in tis antigen as a vaccine candidate was raised by te initial observation tat DN vaccination wit te gene encoding Hsp65 rom Mycobacterium leprae could give protection against subsequent M. tuberculosis callenge in mice [155]. ltoug it was later sown tat Hsp65 protein was also immunogenic and protective as a vaccine candidate wen given wit an eective adjuvant [156], muc o te researc ocused on DN vaccination and in 2005 it was sown tat Hsp65 DN vaccination given postinection could augment te eicacy o cemoterapy in an animal model [157]. Te immune response generated by DN Hsp65 vaccination appeared to be strongly polarized towards IFN g, wit a relatively low IL-4 component compared wit BCG vaccination [158], and contain an augmented cytotoxic response [159]. PCs taking up Hsp65 ave been sown to be selectively activated: macropages produced ig levels o TNF-a, IL-6 and IL-10, and sowed enanced bacterial killing, wile dendritic cells produced ig levels o IL-12 and low levels o TNF-a, IL-6 and IL-10. Te inlammation-promoting eect o Hsp65 appears to be somewat nonspeciic (possibly due te adjuvant eect o naked DN [160]), wit Hsp65-containing constructs being used to modulate immune responses to oter diseases in animal models, including leismaniasis, diabetes and artritis [ ]. Tis led to concerns rom most pri trials in tat te patients did not ave active TB disease (altoug it can be assumed tat many, i not most, o tem were latently inected): te goal was to prevent disease rom developing, rater tan treat existing disease. Tis study repted a nonsigniicant beneit against disseminated TB, and a weak b signiicant beneit against conirmed pulmonary TB, suggesting tat, in a population were immune responsiveness migt be impaired (HIV-positive individuals), immunoterapy in individuals wito active TB disease may elp reduce subsequent disease [146]. It remains unclear, owever, ow muc o tis beneit is immunoterapeic (i.e., due to an eect on existing latent inection) and ow muc is propylactic (by bsting an existing immune response, wic would protect against subsequent primary inection). Te exact mecanism involved in M. vaccae immuno terapy also remains unclear, despite te many trials and a large number o animal experiments [147]. Wat is clear is tat vaccination wit tis ganism induces a T1 response dominated by IFN g, IL-2 and IL-12, as is seen wit oter mycobacteria. M. vaccae treatment also elicits a strong IL-10 response (again, a common eature o mycobacterial inection/vaccination). Studies using animal models suggest tat tis may come rom induction o IL-10-producing Tregs [148] rater tan activated monocytes. In addition, in vitro experiments suggest tat M. vaccaeprimed dendritic cells enance T cell activation and induce T1 maturation, wile attenuating maturation o T2 cells [Rk G, Pers. Comm.]. s yet, owever, it remains unclear weter te response to M. vaccae is qualitatively dierent rom tat seen wit oter killed mycobacteria mycobacterial extracts. RUTI noter mycobacterial-derived terapeic is RUTI (rcivel Farma, Barcelona, Spain), a mixture o detoxiied cell ragments rom M. tuberculosis delivered in liposomes. Te precise nature o te antigenic component is proprietary. RUTI is also an adjunct terapy, designed to be administered along wit conventional cemoterapy against latent TB inection, in te ope o decreasing te duration and improving te eicacy o treatment [149,150]. RUTI as started clinical trials [151], and appears to be reasonably well tolerated, b clinical eicacy data in umans are still some years away. Trials in animals ave been equivocal, wit no signiicant advantage over isoniazid terapy being observed in a trial in goats [152], ure science group 637

10 Doerty immunity, giving rise to elevated levels o IFN g TNF a, b wito te establisment o te long-term protective memy response needed continued control o inection. I tis were te case, ten it would explain te similarity o te results to tose seen wit cytokine terapy [181] (and discussed below). Noneteless, te seer number o repts lends credence to te idea tat it is possible to improve current cemoterapeic approaces wit adjunct immuno terapy based on mycobacterial antigens, and wk is proceeding in tis area. Cytokine immunoterapy Intererons & IL-12 I adjuvant (and possibly antigen-based) immuno terapy exert teir eects by stimulating te induction o inlammaty proinlammaty cytokines, tere is a certain logic to simply testing te potential o tose cytokines directly. Te obvious irst target was IFN g, given its central role in immunity to M. tuberculosis. Te irst suc attempts, against reracty nontuberculous mycobacterial inections [182,183], sowed promising initial results and wk on M. tuberculosis switly ollowed [184]. In te study by Condos et al., patients wit reracty MDR TB wo ad remained spum positive despite aderence to treatment, received 500 µg o aerosolized IFN g tree-times a week 1 mont [184]. ll patients receiving treatment gained weigt, converted to smear negativity, sowed a small increase in time to culture positivity and sowed radiological improvement. Untunately, in all cases, te eects seen were transient, wit reversion to spum positivity over time. In total, tere ave been nearly a dozen trials wit IFN g, wit consistent b disappointing data; an early improvement, ollowed by relapse wen treatment was paused [185]. None o tese studies ave sown signiicant eects on mtality (altoug, to be air, tey ave been so small tat tey were not powered to do so). It as, owever, been sown tat IFN g used as an adjunct to cemoterapy can improve ocomes [186], and oter groups ave been able to sow similar eects wit IFN a [187]. So ar, owever, given te expense and side eects associated wit intereron treatment, te minimal gains acieved do not seem to warrant te cost. IL-12 a crucial act in te development o IFN g-producing CD4 + T cells is also essential protection against M. tuberculosis inection in umans (as indicated by te susceptibility o patients wit deects in te IL-12 signaling patway [188,189]), b te literature contains only a abo possible immunopatogenic eects [165,166], wic retarded progress to clinical trials in umans, b so ar no convincing data tat Hsp65 is intrinsically me dangerous tan oter mycobacterial antigens ave been presented. Hsp65 as also been integrated into multiple oter immunoterapeic regimens tested in animal models. Te best documented o tese is combination o te Hsp65 gene and te gene uman IL-12 in eiter a viral membrane-derived liposome-like construct a DN vect, wic ave bot propylactic [167,168] and terapeic [169] eicacy in animal models, including nonuman primates. In tis study, te eect o tis terapeic intervention in nonuman primates is best crelated wit IL-2 [170], suggesting a T cell-mediated eect rater tan simply a noncognate immunostimulaty eect. Given tis, it is interesting to note tat a Hsp65 IL-2 DN usion vaccine using te uman IL-2 gene also appeared to ave a terapeic eect in te mouse model o TB, wen given ater inection [171]. ntigen 85 (& oter genes) Te todoxy regarding vaccination against M. tuberculosis inection as been tat antigens expressed in te early pases o inection are likely to be ineective at generating responses protective against establised inection [172], because o te ability o M. tuberculosis to manipulate its genotype, as discussed above. Tat may be crect, b it is also possible tat early ailures o propylactic vaccination migt ave been a ailure o tecnique, rater tan target. Over te last several years tere ave been multiple publications testing a variety o antigens rom M. tuberculosis in terapeic animal models, eiter alone as an adjunct to cemoterapy. ntigen 85 (and its close omolog, antigen 85B) are immuno dominant antigens used in multiple propylactic vaccines currently in clinical trials [173] and tese antigens ave been eective as propylactic vaccines in animal models. However, antigen 85 as also been consistently active in postexposure models, particularly wen used as an adjunct to cemoterapy [ ]. It is t early to write o te todoxy as yet; te experiments publised so ar are all rom animal models and primarily rom studies in te mouse, wic is a relatively po model uman cronic latent disease. In addition, te results seen ave typically been transient assessed st periods o time [177]. It is teree possible tat te responses migt relect stterm bsting o existing antimycobacterial 638 Immunoterapy (2012) 4(6) ure science group

11 Immunoterapy TB TNF-a & steroid terapy TNF-a is anoter cytokine o undoubted imptance in te control o TB [84,87,88] and it is known to be crucial te control o latent TB, as indicated by te increased risk o reactivation o latent TB in patients treated wit TNF-a recept antagonists [85,86]. However, TNF-a is also te cytokine most consistently associated wit patology in TB, and may ave been a primary act in te Koc reaction [197]. Peraps in consideration o te potential risks, no clinical trials wit exogenous TNF-a ave been initiated. However, TNF-a is also asso ciated wit accelerated HIV disease progression in individuals coinected wit M. tuberculosis, and ig levels o serum TNF-a were associated wit a poer response to treatment [198], so inibiting TNF-a in association wit TB treatment as been tested. In a study were HIV M. tuberculosis coinected patients received te soluble TNF-a recept etanercept (a TNF-a antagonist) wile receiving anti-tb terapy, signiicant gains were seen in body mass, radiologic sces and time to spum culture conversion [199]. Tis was accompanied by a signiicant increase in CD4 + T cell count, attribed to a possible decrease in TNF-a driven apoptosis [199]. Tese indings are consistent wit te knowledge tat inibiting inlammaty processes can improve TB cemoterapy. In a study rom te 1950s in wic prednisolone was irst used as an adjunct to TB cemoterapy, a similar astening o spum clearance and improved radiology was seen [200], b it did not aect ocomes in te longer term. In addition, in tese studies a rebound eect on radiology was noted on cessation o prednisolone treatment, consistent wit te concept tat underlying patologic IL-2 IL-2 anoter prototypical T1 cytokine is produced by activated T lympocytes and plays a critical role in te expansion, activation and maturation o T cells. In animal models o mycobacterial inection, IL-2 as been sown to inibit mycobacterial replication [191], possibly by augmenting te eect o TNF-a [192] simply by enancing T cell expansion and maturation. nimal experiments wit IL-2, eiter alone togeter wit TNF-a, sowed weak inibity eects on mycobacterial growt [191,192] and te observation o decreased IL-2 expression and responsiveness in umans wit TB [193] suggested tat IL-2 immunoterapy migt be elpul. Initial results in patients at dierent stages o TB treatment (including some wit MDR TB) wo received ril-2 30 consecive days, as an adjunct to cemoterapy, indicated some improvement [194]. Te numbers o CD25 + and CD56 + cells increased in te periperal bld o patients receiving ril-2 terapy, compared wit placebo, and bacterial load in spum decreased, as did time to spum conversion, wile radiological assessment improved in te majity o (b not all) cases [194,195]. Te treatment was, on te wole, well tolerated. Subsequently, a randomized, doubleblinded, placebo-controlled Pase II clinical trial was conducted in 110 HIV-negative TB patients wit drug-sensitive M. tuberculosis inections, wit 1 year o ollow-up [196]. ltoug it conirmed some o te indings o te earlier studies, te study did not ind any clinical improvement. Te researcers noted tat te increase in te percentage o CD25 + T cells was speciic CD4 + cells (tey speculated tat te inability o te treatment to activate CD8 + cells migt account its ailure) and tey also documented increased levels o soluble IL-2 recept and IL-2 in serum. However, te immunological eects were transient and no signiicant dierences were seen between te groups wit regard to weigt gain, symptoms, relapse rates bacillary clearance [196]. It is possible tat te dierence in ocomes was due to te inclusion o MDR TB cases in te initial studies: a poer response to cemoterapy migt ave increased te window in wic a positive eect o te cytokine adjunct terapy could be seen. However, te modest nature o any beneit, combined wit te cost o te treatment, as meant tat ril-2 terapy TB as not been urter pursued. single reerence to its use as an eective adjunct terapy TB [190]. s IFN g, any enancement o immunity generated by te treatment does not appear to persist. In retrospect, given te discussion above on M. tuberculosis s ability to modulate te ost s immune response, it is not surprising tat te response to exogenous cytokine treatment is transient. Host unctions dependent on IL-12 IFN g may be augmented by exogenous cytokine, b te underlying mecanisms o immunity are unlikely to be canged, given tat bot IL-12 and IFN g are produced in abundance by te inected uman ost and are downregulated as disease becomes progressive. It appears tat augmenting inlammaty responses is ineective in te longer term, as long as te underlying deects in immunity tat allowed disease to develop in te irst place are still present. ure science group 639

12 Doerty ten converts ydroxylated vitamin D25 (te m nmally ound in serum) to te bioactive 1,25-diydroxyvitamin D, wic can potentiate aopagy, pagolysosomal usion and te release o microbial peptides suc as catelicidin [206,207]. Te use o vitamin D as an adjunct to cemoterapy TB as teree been tested in several clinical studies. Some o tese ave ound some beneicial eects b two recent studies, te only moderate-sized, randomized, placebo-controlled trials so ar conducted (one o wic was also double-blinded), ave sown tat te eects i any appear to be restricted to individuals wit severe vitamin D deiciency [205,208]. Vitamin D supplementation did not improve clinical scing TB symptoms, spum smear conversion rates (except in a subgroup wit a deined TaqI vitamin D recept polympism) mtality. Tese indings are not wolly conclusive it is possible tat iger dosages oter canges to te regimen migt improve te eect b tey do not, at any rate, indicate a strong eect o vitamin D supplementation. s as been te case many o te oter acts tested as adjunct immunoterapies, it may be tat a deiciency in a speciic act can be a contribing act to te development o TB, b tat supplementing it above baseline levels does not lead to noticeable improvement. lternatively, i te problem is ailure to convert existing vitamin D to te biologically active m, due to M. tuberculosis-driven intererence wit TLR ligation-activated signaling and IFN g production inibiting CYP27b1 induction, ten adding me vitamin D may not be elpul. Tis would explain te beneicial eect being seen only in tose wo were vitamin D deicient. Last o all, tere ave been a series o recent repts rom Ukraine on te beneicial eects o te erbal extract Dzerelo adjunct terapy alongside conventional cemoterapy. Dzerelo is one o a amily o intereron activats, developed and widely used in te countries o te mer Soviet Union [209], wic act as immunostimulants. Tey are derived rom plant extracts and include a number o as-yet poly deined ig-molecular-mass lipids, proteins and carboydrates [209], possibly similar to neem lea extract, wic as been tested in India [210]. Teir precise makeup and active ingredients are unknown, b te eects publised indicate elevated IL-2 and IFN g, in HIVpositive TB patients receiving Dzerelo adjunct terapy alongside cemoterapy, wit concurrent decreases in IL-6 and TNF-a [211]. Several publications rom te same group indicate tat mecanisms ad been suppressed, not removed [200]. Newer studies, using ig-dose metylprednisolone, ave linked te eect o cticosteroids to reduced TNF-a production. Te most recent o tese a well-controlled Pase II study in HIV M. tuberculosis-coinected patients wit CD4 + T cell counts above 200/µl sowed a transient b substantial decrease in median levels o TNF-a, TNF-a recept type II and neopterin in te prednisolone-treated arm, togeter wit an increase in CD4 + T cell counts and a me rapid clearance o M. tuberculosis rom te spum [201]. Untunately, in a pattern wic is all t amiliar in TB immunoterapy, tese beneits were transient, suggesting again tat te treatment ameliated overproduction o inlammaty mediats tat migt be driving patology (and symptoms) in te patients, b tat treatment did not address te underlying deect in immunity tat ad allowed TB patology to develop in te irst place. Tus, despite trials stretcing back over al a century, we remain unclear on precisely wat mecanisms are involved in tese interventions. Te improvements observed in tese studies, in wic TNF-a and inlammaty patways are inibited, can be ascribed to a reduction in tissue destruction and bacterial escape mediated by TNF-a as easily as to improved control o te disease. It sould be noted tat early studies wit cticosteroids in TB wito concurrent cemo t erapy to kill te patogen demon strated tat tey wsened TB (as etanercept does in latently inected TB patients), illustrating te double role tat TNF-a and inlammaty processes play in TB. Dietary supplements: vitamin D & Dzerelo Po nrition as long been associated wit an increased risk TB (altoug it is oten ard to dissociate po nrition rom oter povertyassociated acts, suc as overcrowding and stress, wic may also contribe to disease) so improved diet as been a part o TB treatment since te days o sanatia. Vitamin D, in particular, was used to treat TB in te preantibiotic era [202] and multiple studies indicated tat it migt play a role in protection against TB (reviewed in [203]). ltoug te mecanism remains speculative, tere is general agreement tat low levels o vitamin D are associated wit a iger risk o active TB [204], particularly in HIV-positive individuals [205]. It as been suggested tat, in an eective immune response, TLR ligation and IFN g production upregulate CYP27b1, wic 640 Immunoterapy (2012) 4(6) ure science group