AN OBSCURE BUT fascinating paper ontreated pulmonary tuberculosis (TB)

lesions—provocatively subtitled Thedeath and resurrection—clearly definedone of the central enigmas of tuberculo-sis and challenges to its treatment: theability of the tubercle bacillus to persistdespite prolonged chemotherapy1. Inthis clinical study, samples taken fromopen and active lesions yielded coloniesof tubercle bacilli that were drug-resis-tant and could develop into colonieswithin the usual time frame of 3–8weeks. Samples taken from the samelung, but from lesions that were closedand encapsulated, an environment usu-ally unfavorable for bacterial growth,developed into colonies only after 3–10

months in culture. Interestingly, the‘resurrected’ bacteria retained full drugsensitivity, but their physiological statein the lung was refractory to anti-tuber-culosis drugs. The mechanism of thispersistent state, also known as latency,dormancy or drug tolerance, remains afundamental mystery of the disease. Inthe words of epidemiologist GeorgeComstock, “Following infection, the in-cubation period of tuberculosis rangesfrom a few weeks to a lifetime.”2 This isin contrast to the latent period of mostinfectious diseases, which is measuredin days or weeks. Whether there is a sin-

gle mechanism of persistence or severaldistinct mechanisms, the phenomenonis the reason that treatment of TB takesso long and relapse remains a problem3.Persistence also explains why the epi-demiology of TB is characterized by anepidemic cycle of centuries rather thanthe more usual months or years4,5.

The tenacity of Mycobacterium tubercu-losis poses a formidable obstacle to con-trol strategies based on drug therapy ofinfected individuals (Fig. 1). A vaccinethat could eliminate persistent infec-tion—alone or in combination withchemotherapy—would be a powerfulnew strategy for TB control. A DNA vac-cine based on the 65-kDa heat shockcognate antigen of M. tuberculosis pro-vided partial protection against chal-

The death and resurrection of tuberculosisDNA vaccines may be useful in overcoming persistent tuberculosis infections

BARRY R. BLOOM1 & JOHN D. MCKINNEY2

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rats, but febrile convulsions are rela-tively rare in children. In humans theremay be predisposing factors such as hip-pocampal malformations and hered-ity1,13. Despite these differences, thismodel can give us some insight into thespecific changes that may occur with hy-perthermia-associated seizures.

Although Chen et al. conclude thatfebrile convulsions should not be consid-ered ‘benign’, they are generally consid-ered a separate entity from epilepsyprecisely because of their benign progno-sis, as determined by epidemiologicalstudies. Although approximately 30% ofaffected children have recurrent febrileconvulsions, only 7% with febrile con-vulsions go on to develop unprovokedseizures by the age of 25 years2. For solelyfebrile convulsions lasting 10–29 min-utes and with no focal features, the risk is3%, and even with very prolonged (≥ 30minutes) febrile convulsions with nofocal features the risk is 7% (ref. 2). Thesefigures are much lower than the risk ofunprovoked seizures after other acutesymptomatic seizures; some estimates ofthis risk have been as high as 46% (ref.14). Furthermore, children who hadfebrile seizures show no significant intel-lectual or behavioral deficit by 10 years15.Thus, given that many children withfebrile convulsions may have a predis-posing susceptibility to seizures such ashippocampal malformations, it is some-what surprising that the condition ismore benign than other symptomatic

causes of acute seizures. Perhaps febrileconvulsions confer upon the individualsome protection from further seizures.The upregulation of GABAergic signalingreported by Chen et al.3 may therefore bean effective, compensatory alteration.This possibility has been mostly unex-plored both experimentally and clini-cally, but one tantalizing consequence isthat prior febrile convulsions might pro-tect against the pro-epileptogenic effectsof subsequent acute provoked seizures.

The study by Chen et al. thus presentsus with clinical challenges to discoverhow their findings relate to humanfebrile convulsions. They also present uswith a scientific challenge to determinewhy hyperthermia-triggered seizures inparticular result in pre-synaptic changesin GABA release, and whether thechanges are pro-epileptogenic or com-pensatory.

1. VanLandingham, K.E. Heinz, E.R., Cavazos, J.E. &Lewis, D.V. Magnetic resonance imaging evidenceof hippocampal injury after prolonged focal febrileconvulsions Ann. Neurol. 43, 413–426 (1998)

2. Annegers, J.F., Hauser, W.A., Shirts, S.B. & Kurland,L.T. Factors prognostic of unprovoked seizures afterfebrile convulsions. N. Engl. J. Med. 316,493–498(1987)

3. Chen, K., Baram, T.Z. & Soltesz, T. Febrile seizuresin the developing brain result in persistent modifi-cation of neuronal excitability in limbic circuits.Nature Med. 5, 888–894 (1999)

4. Toth, Z., Yan, X.X., Haftoglou, S., Ribak, C.E. &Baram, T.Z. Seizure-induced neuronal injury: vul-nerability to febrile seizures in an immature ratmodel. J. Neurosci. 18, 4285–4294 (1998).

5. Nusser, Z., Hajos, N., Somogyi, P. & Mody, I.Increased number of synaptic GABA(A) receptorsunderlies potentiation at hippocampal inhibitory

synapses. Nature 395, 172–177 (1998).6. Buhl, E.H., Otis, T.S. & Mody, I. Zinc-induced col-

lapse of augmented inhibition by GABA in a tempo-ral lobe epilepsy model. Science 271, 369–373(1996).

7. Brooks-Kayal, A.R., Shumate, M.D., Jin, H., Rikhter,T.Y. & Coulter, D.A. Selective changes in single cellGABA(A) receptor subunit expression and functionin temporal lobe epilepsy. Nature Med. 4,1166–1172 (1998).

8. Prince, D.A. & Jacobs, K. Inhibitory function in twomodels of chronic epileptogenesis. Epilepsy Res. 32,83–92 (1998)

9. Huang YY. et al. A genetic test of the effects of mu-tations in PKA on mossy fiber LTP and its relation tospatial and contextual learning. Cell 79, 69–79(1994).

10. Nicoll, R.A. & Malenka, R.C. Contrasting propertiesof two forms of long-term potentiation in the hip-pocampus. Nature 377, 115–118 (1995).

11. Chain, D.G., Hegde, A.N., Yamamoto, N., Liu-Marsh, B. & Schwartz, J.H. Persistent activation ofcAMP-dependent protein kinase by regulated pro-teolysis suggests a neuron-specific function of theubiquitin system in Aplysia. J. Neurosci. 15,7592–7603 (1995).

12. Cobb, S.R., Buhl, E.H., Halasy, K., Paulsen, O. &Somogyi, P. Synchronization of neuronal activity inhippocampus by individual GABAergic interneu-rons. Nature 378, 75–78 (1995).

13. Fernandez, G. et al. Hippocampal malformation asa cause of familial febrile convulsions and subse-quent hippocampal sclerosis. Neurology 50, 909–17(1998)

14. Hart, Y.M., Sander, J.W., Johnson, A.L. & Shorvon,S.D. National General Practice Study of Epilepsy: re-currence after a first seizure. Lancet 336,1271–1274 (1990)

15. Verity, C.M., Greenwood, R. & Golding, J. Long-term intellectual and behavioral outcomes of chil-dren with febrile convulsions. N. Engl. J. Med. 338,1723–1728 (1998).

University Department of Clinical Neurology,Institute of Neurology, UCL,Queen Square,London WC1N 3BG,United Kingdom

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lenge with virulent M. tuberculosis in amurine model6. DNA vaccines express-ing other antigens have shown similarprophylactic protection in the mousemodel7. But there has been no prospectfor development of a therapeutic or‘post-exposure’ vaccine...until now.

In the 16 July 1999 issue of Nature,Lowrie et al. reported that the hsp65DNA vaccine was also protective whenapplied therapeutically in two distinctmodels of persistent infection in mice8.In a model of chronic TB initiated by in-fection with a sublethal dose of bacilli,bacterial numbers increased for severalweeks and then plateaued as a result ofthe emergence of bacteriostatic immu-nity. A stalemate was achieved in whichthe host was unable to eradicate thebacteria, yet suppressed their furthermultiplication9, resulting in a chronicinfection in which both the bacteriumand the host survived. Remarkably, thisstalemate was broken when chronicallyinfected animals were vaccinated withthe hsp65 DNA vaccine: the numbers ofviable bacteria actually decreased in the

vaccinated animals. Equally impressivewas the demonstration that vaccinationcould suppress bacteriologic relapsewhen applied to mice that had been in-fected and treated for 12 weeks withanti-tuberculosis drugs. Vaccinationprevented relapse even when the ani-mals were later immunosuppressed withcorticosteroids, suggesting that in somemice the infection was completely elim-inated. This is a remarkable achieve-ment, because infection in the mouse isnot eradicated by chemotherapy alone10

or by immunization with the live atten-uated bacille Calmette-Guérin vaccine7.Apparently, a more effective host im-mune response is elicited by the hsp65DNA vaccine than by exposure to livingbacilli, whether virulent or attenuated.Although the protective mechanismwas not identified, DNA vaccines areparticularly effective inducers of cyto-toxic T lymphocytes in mice, and it istempting to speculate that cytotoxic Tlymphocytes, as well as interferon-γ andmacrophage activation, might be in-volved.

The potential for a therapeutic vac-cine is an important step for TB therapy.Current anti-tuberculosis regimens re-quire administration of multiple drugsfor a minimum of 6–9 months. Withoutdirectly observed therapy, patient ad-herence to such lengthy and complexregimens is poor, resulting in high ratesof treatment failure, relapse, and devel-opment of bacterial drug resistance (seeFig. 1). Directly observed therapy, inwhich each dose of drugs is adminis-tered by a health care provider or socialworker, dramatically improves patientadherence. At present, however, only15% of patients worldwide receive thedirectly observed therapy regimen rec-ommended by the World HealthOrganization.

Why is TB so difficult to treat? Thedrugs themselves are not at fault: Thesame compounds that are so poorly ef-fective in vivo will sterilize a culture ofM. tuberculosis in vitro within days. Thepossibility of suboptimal penetration ofdrugs into tuberculous tissue has alsobeen eliminated by analysis of resectedlung tissue from patients who were in-jected with a radiolabeled drug prior tosurgery11. Again, the mysterious physio-logical state of the persistent organismsmay hold the key to the problem, be-cause conventional drugs are ineffectiveagainst bacteria that have exited the celldivision cycle and become stationary3.

Despite the promise of the findings ofLowrie et al.8, many important ques-tions still remain. How closely do themouse models of TB reflect persistencein humans? If the mechanisms of persis-tence differ between mice and man, astrategy that works in mice may notgeneralize to humans. Will DNA vac-cines, which elicit powerful immune re-sponses in the mouse, prove equallyeffective in humans? Early clinical stud-ies of DNA vaccines in humans havebeen disappointing so far. Can a combi-nation of drugs and vaccination achievethe total eradication of viable tuberclebacilli from the tissues, in mice or hu-mans? Could such a strategy eliminateviable M. tuberculosis in HIV-infected in-dividuals, a third of whom, in sub-Saharan Africa, succumb to TB?

If we learn the answers to those ques-tions, a post-exposure vaccine wouldprovide the basis for a totally novelglobal TB control strategy. Current esti-mates place the number of individualsinfected with M. tuberculosis at 1–2 bil-lion globally. Approximately 16 millionof these individuals have active disease,and the rest are presumed to harbor theinfection in a latent form. As the risk ofreactivation is 5–10% in a lifetime, itfollows that 50–200 million of these la-tently infected individuals will eventu-ally develop full-blown TB. At present,nothing is being done to prevent theemergence of disease in these individu-als. Chemoprophylaxis of latent infec-tions can reduce the risk of reactivation,but it is not widely used because it isdeemed too costly and too difficult toadminister. Elimination of TB as a pub-lic health problem will not be easilyachieved without an effective and af-fordable intervention targeting this vastreservoir of contagion. The presentwork gives us hope that application of apost-exposure vaccine to individualswho are positive by the tuberculin skintest could accelerate the treatment ofchronic TB and block the reactivation oflatent infections. This is not only ourbest hope, but may be the only feasiblestrategy for TB control on a global scale.If a therapeutic vaccine is found to beeffective in humans, it could save50–200 million lives, and reduce theepidemic cycle of TB from centuries towithin our lifetime.

1. Vandiviere, H.M., Loring, W.E., Melvin, I. & Willis,S. The treated pulmonary lesion and its tuberclebacillus. II. The death and resurrection. Am. J.Med. Sci. 232, 30–37 (1956).

Fig.1 Persistence and pathogenesis: Activepulmonary tuberculosis with cavitation of 41years’ duration12. This chest X-ray of a 70-year-old woman taken shortly before her deathfrom chronic TB shows a collapsed left lungand a huge tuberculous cavity in the rightapex. The patient was first hospitalized withactive cavitary TB in 1927 at the age of 29. Herinfection became latent after thoracic surgeryin 1945, but reactivated after a traumatic acci-dent in 1955. Chemotherapy wasadministered until 1966, but the patient wasnoncompliant. Bacterial resistance developedto all seven of the available anti-tuberculosisdrugs and chemotherapy was discontinued.The patient eventually died in 1967.

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2. Comstock, G.W., Livesay, V.T., and Woolpert, S.F.The prognosis of a positive tuberculin reaction inchildhood and adolescence. Am. J. Epidemiol. 99,131–138 (1974).

3. McKinney, J.D., Jacobs, W.R. & Bloom, B.R. inEmerging Infections (eds. Krause, R., Gallin, J.I. &Fauci, A.S.) 51–146 (Academic, New York, 1998).

4. Grigg, E.R.N. The arcana of tuberculosis, with abrief epidemiologic history of the disease in theUSA. Am. Rev. Tuberc. Pulm. Dis. 78, 151–172;426–453; 583–603 (1958).

5. Blower, S.M. et al. The intrinsic transmission dy-namics of tuberculosis epidemics. Nature Med. 1,815–821 (1995).

6. Tascon, R.E. et al. Vaccination against tuberculo-sis by DNA injection. Nature Med. 2, 888–892(1996).

7. Huygen, K. et al. Immunogenicity and protectiveefficacy of a tuberculosis DNA vaccine. Nature

Med. 2, 893–898 (1996).8. Lowrie, D.B. et al. Therapy of tuberculosis in mice

by DNA vaccination. Nature 400, 269–271(1999).

9. Rees, R.J.W. & Hart, P.D. Analysis of the host-par-asite equilibrium in chronic murine tuberculosisby total and viable bacillary counts. Br. J. Exp.Pathol. 42, 83–88 (1961).

10. McCune, R.M. & Tompsett, R. Fate ofMycobacterium tuberculosis in mouse tissues asdetermined by the microbial enumeration tech-nique. I. The persistence of drug-susceptible tu-bercle bacilli in the tissues despite prolongedantimicrobial therapy. II. The conversion of tuber-culous infection to the latent state by the admin-istration of pyrazinamide and a companion drug.J. Exp. Med. 104, 737–801 (1956).

11. Barclay, W.R., Ebert, R.H., Le Roy, G.V., Manthei,R.W. & Roth, L.J. Distribution and excretion of ra-

dioactive isoniazid in tuberculosis patients. J. Am.Med. Assn. 151, 1384–1388 (1953).

12. Edwards, W.M., Cox, R.S., Jr., Cooney, J.P. &Crone, R.I. Active pulmonary tuberculosis withcavitation of forty-one years’ duration. Am. Rev.Respir. Dis. 102, 448–455 (1970).

1Harvard School of Public Health667 Huntington AvenueBoston, MA 02115-6096email: bloom@hsph.harvard.edu2The Rockefeller University1230 York Avenue, Box 21New York, NY 10021-6399email: mckinney@mail.rockefeller.edu

VIRUSES HAVE DEVELOPED many well-stud-ied mechanisms to ‘hijack’ their

hosts’ metabolic pathways for their ownbenefit and to evade immune attack.Similarly, tumor cells are under selectivepressure to manipulate their environ-ment and to escape the growth-restrict-ing mechanisms that act on the rest ofthe organism. For example, stromal cellscan provide cancer cells with nutrientsand soluble or membrane-anchoredgrowth-promoting factors such as cy-tokines or integrins (Fig. 1). Tumor cellscan also produce factors that modify theenvironment to their advantage, for ex-

ample, by promoting blood vesselgrowth (neovascularization) to meettheir increasing demand for oxygen andnutrients. In normal conditions, tissuegrowth is limited by inhibitory signalsfrom neighboring cells. Mutations inoncogenes or tumor suppressor genes,however, allow the tumor to overcomethe normal growth constraints. Tumorsdevelop independence from externalgrowth signals and become refractory to

cellular senescenceand growth-in-hibitory or apopto-sis-inducing stimuli.

Certain types of

cancer cells have also developed strate-gies to evade or counterattack the im-mune system (Fig. 1). Macrophages andgranulocytes can kill cancer cells in anantigen- and major histocompatibilitycomplex (MHC)-independent manner.In contrast, cytotoxic T lymphocytes(CTLs) must recognize antigen-derivedpeptides presented by MHC class I mole-cules with their T-cell receptor (TCR) tobecome activated, whereas natural killer(NK) cells are triggered by cells lackingMHC class I molecules. Mutation-in-duced resistance to immune attack maytherefore contribute to neoplastic trans-formation. Reduced MHC class I expres-sion or impaired antigen processingprevent recognition of tumor cells byCTLs, but may target them for NK cellattack1. Certain viruses overcome this

The great escape: Is immune evasion required for tumorprogression?

A study on page 938 reports the identification of an ovarian and uterine tumor-associated ligand, RCAS1, whichinhibits growth of activated T lymphocytes.

ANDREAS VILLUNGER & ANDREAS STRASSER

Fig. 1 Upper panel, complex three-way interactions between tumorcells, their microenvironment and the immune system. Cancer cellscan receive nutrients and growth-stimulatory signals from neighboringstromal cells and stimulate neovascularization. Cells of both the innateand adaptive immune systems may attack cancer cells. Myeloid cellscan attack tumor cells in an antigen- and MHC-independent way.Cytotoxic T lymphocytes need to be activated by antigen-derived pep-tides presented by MHC molecules, whereas NK cells are triggered bycells lacking MHC class I molecules. Lower panel, different mechanismsmay lead to evasion or counter-attack of cancer cells towards theimmune system: impaired antigen presentation with limited CTLactivation; expression of decoy receptor DcR3 on cancer cells and neu-tralization of Fas ligand produced by CTL and NK cells; expression ofFas ligand may kill tumor-infiltrating CTL, NK cells, granulocytes ormacrophages; and finally mucins, such as DF3/MUC1, or the newligand RCAS1, described here, block T-cell proliferation, adding to thearsenal of weapons that tumors may use to evade control by theimmune system.

Immune system Stromal cells

IntegrinsGrowth factors

Cytokines

Immune escapeClonal expansionVascularization

Myeloid cells,NK cells,

Activated lymphocytes

Cancer cells

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B7-1/2

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