CLINICAL MICROBIOLOGY REVIEWS, JUly 1988, p. 330-348 Vol. 1, No. 30893-8512/88/030330-19$02.00/0Copyright X 1988, American Society for Microbiology

LeprosyROBERT C. HASTINGS,* THOMAS P. GILLIS, JAMES L. KRAHENBUHL, AND SCOTT G. FRANZBLAU

Laboratory Research Branch, Gillis W. Long Hansen's Disease Center, U.S. Public Health Service,Carville, Louisiana 70721

OVERVIEW OF LEPROSY ................................................................ 330Epidemiology ................................................................ 330Clinical Aspects ................................................................ 331

Indeterminate leprosy ................................................................ 331LL ................................................................ 331TT ................................................................ 331Borderline leprosy ................................................................ 331

Reactions................................................................ 332Patient Management ................................................................ 332

CELL WALL STRUCTURE AND ASSOCIATED ANTIGENS OF M. LEPRAE ................................333Cell Wall of M. leprae ............................................................. 333Protein Antigens of M. leprae ................................................................ 333Molecular Biology of M. leprae ................................................................ 334

IMMUNOLOGY ................................................................ 335Immunologic Research ................................................................ 335

Goals ................................................................ 335Obstacles to research ................................................................ 335Major breakthroughs ................................................................ 335

Clinical Immunology ................................................................ 336Lepromin test................................................................ 336Humoral immunity and serodiagnosis in leprosy ................................................................ 336

CMI in Leprosy ................................................................ 336Mechanisms of Specific Anergy in LL................................................................ 337

Genetic control of CMI in leprosy................................................................ 337Role of the lymphocyte in specific anergy in leprosy ...............................................................337

The Macrophage in Host Resistance to Leprosy ................................................................ 337Leprosy Vaccine ................................................................ 338

MICROBIOLOGY ............................................................. 339Metabolism ............................................................. 339

Catabolic activity ............................................................. 339Amino acid metabolism ............................................................ 340Nucleic acid metabolism ............................................................ 340Lipid metabolism ............................................................ 340Iron ............................................................. 340Biophysical parameters............................................................. 340

In Vivo Drug Testing ............................................................. 340In Vitro Drug Testing ............................................................. 341

ACKNOWLEDGMENTS ............................................................ 342LITERATURE CITED ............................................................. 342

OVERVIEW OF LEPROSY person. There is evidence that transmission of leprosy canoccur through (i) intact skin, (ii) inhalation and deposition

Leprosy or Hansen's disease is a chronic infectious dis- of bacilli onto intact nasal mucosa, and (iii) penetratingease caused by Mycobacterium leprae. wounds, such as thorns or biting arthropods. Which of these

mechanisms is most common is unknown. Perhaps the mostEpidemiology popular view is that the leprosy bacilli are expelled from the

The total number of leprosy patients in the world has been nose of a patient with active disease and impact on the nasalestimated to be about 10.6 million (199). Of these, about 62% mucosa of another individual.are in Asia and 34% are in Africa (170). Expressed in terms Although the disease is predominantly one of humans,of the intensity of the disease in a population, i.e., as mean since 1975 it has been demonstrated to be a natural infectionprevalence, the disease is about three times as intense in of wild armadillos (Dasypus novemcinctus) in Louisiana andAfrica as it is in Asia. Texas (236, 237). Spontaneous cases of leprosy have alsoTransmission of leprosy is thought to be from person to been described in two mangabey monkeys (78), and experi-

mental leprosy has been transmitted to the rhesus monkey* Corresponding author. (10). The disease has been present in wild armadillos since at

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least 1961 (232). The relationship between leprosy in wildarmadillos and the disease in humans is not clear. Therehave been antecdotal reports of leprosy in humans followingcontact with armadillos (132, 230). On the other hand, mosthuman leprosy occurs in areas (e.g., India) in which arma-dillos do not exist. If a relationship exists between leprosy inwild armadillos and that in humans, it seems to be quantita-tively minor.

Clinical Aspects

Leprosy predominantly affects the skin, peripheralnerves, and mucous membranes. The clinical features of thedisease can be grouped into three parts depending on mech-anism: (i) features due to bacterial proliferation, (ii) featuresdue to the immunologic responses of the host to the leprosybacilli, and (iii) features due to the peripheral neuritis causedby the first two processes (179). Leprosy always involvesperipheral nerves, almost always involves skin, and fre-quently involves mucous membranes. The three cardinalsigns of the disease are skin lesion(s), skin anesthesia(s), andenlarged peripheral nerve(s).The majority of people effectively resist infection with M.

leprae even in highly endemic areas. It is now thought that as

many as 200 individuals become infected with M. leprae foreach overt case which develops and is detected (1).

Indeterminate leprosy. For those individuals unable toresist infection with M. leprae, the incubation period varies,but it is usually in the range of 2 to 4 years. The earliest signof the disease is usually one or a few hypopigmented skinmacules with minimal sensory loss confined to the lesion.The histopathology of such a lesion may be only that of a

nonspecific, chronic dermatitis with scattered round cellinfiltrates of the dermis. The presence of acid-fast bacilli oran infiltrate selectively in a nerve bundle in the dermis isdiagnostic. Indeterminate leprosy has a variable course. Inapproximately three-fourths of such patients, the diseaseheals spontaneously; some cases remain indeterminate for a

prolonged period of time, and some progress to one of theestablished forms of the disease.

Established leprosy illustrates a continuous spectrum ofdisease from a localized, self-healing, granulomatous diseasewith very few demonstrable leprosy bacilli to a widespread,progressive, anergic disease with massive numbers of M.leprae. To describe the position of a leprosy patient on thisspectrum, most centers use the classification of Ridley andJopling for research purposes (192). The so-called polartypes of leprosy are stable clinically. The so-called border-line types of leprosy are characteristically unstable clinicallyand form a continuous spectrum between the two polarforms.LL. Polar lepromatous leprosy (LL) is the widespread,

anergic form of the disease. Proliferation ofM. leprae resultsin skin lesions of a variety of types ranging from diffusegeneralized skin involvement to nodules (called lepromas) ina widespread, usually symmetrical distribution. These skinlepromas in advanced LL may contain 1010 M. leprae per gof tissue. Characteristically, lepromatous skin lesions in-volve cooler parts of the body surface. This is thought to bedue to preferential growth of M. leprae at temperaturescooler than core body temperature (91). With this tempera-ture preference of M. leprae, the anterior third of the eye(but not the warm, posterior two-thirds), the nasal mucosa(but not the oral mucosa in a nose breather because the oralmucosa is not cooled by inspired air), and peripheral nervetrunks as they course superficially (the ulnars at the elbow,

medians at the wrist, common peroneals at the knee, andposterior tibials at the ankle) are affected in bacteriologicallyprogressive disease. There is a characteristic pattern ofsensory loss due to dermal nerve fiber involvement inadvanced lepromatous leprosy which affects cooler areas ofthe body surface (194). Despite the widespread involvementin bacteriologically progressive LL, the patient has remark-ably few symptoms other than those caused by the bacterialmass and the accumulations of macrophages required tocontain them. Histopathologically, lepromatous lesions arecharacterized by massive collections of macrophages con-taining large numbers of acid-fast bacilli and often containinglarge amounts of lipids which create a foamy appearance onhematoxylin and eosin staining. These foamy macrophagesmay occupy 90% of the dermis. The dermal foam cellaccumulations are separated from the epidermis by a clearzone.TT. Polar tuberculoid leprosy (TT) is the localized form of

the disease. In contrast to uncomplicated LL in whichbacterial proliferation results in signs and symptoms, muchof the clinical picture in TT is due to a combination ofbacterial proliferation and the immunologic responses of thehost to the bacilli. Characteristically, TT consists of one or,at most, a few well-circumscribed skin lesions with profoundanesthesia of the skin lesion itself. There may be an enlargedperipheral nerve in the vicinity of the skin lesion(s). Histo-pathologically, there are very few, if any, demonstrableacid-fast bacilli. There is a dense, well-organized granulomaconsisting of epithelioid cells, surrounded by lymphocytes,and containing multinucleated Langhans giant cells. Thegranuloma involves the basal layer of the epidermis in polarTT.

Borderline leprosy. Borderline leprosy encompasses thosetypes of the disease between LL and TT. Mid-borderline israre because it is very unstable. A patient with borderlineleprosy can develop clinical, bacteriological, and histopatho-logic features of more tuberculoid disease with time, and thisis called upgrading. Conversely, developing more leproma-tous disease with time is called downgrading. As in TT, thesigns and symptoms of borderline leprosy tend to be due toa mixture of bacterial proliferation and the immunologicresponse of the host to them. Borderline tuberculoid leprosy(BT) resembles tuberculoid disease except that the numberof skin lesions is usually greater, the edges of the skin lesionsare less well defined, there is a tendency for satellite lesionsto develop near the edges of the larger lesions, and individuallesions tend to be larger. Damage to peripheral nerves tendsto be more widespread and more severe in BT than in TT.This nerve damage is largely on the basis of the immuneresponse of the host to the bacilli. The histopathology of BTskin lesions resembles that of TT except that the granulomadoes not extend up to involve the basal layer of the epider-mis. The numbers of acid-fast bacilli in lesions vary fromundetectable to 1 in every 10 to 100 oil immersion micro-scopic fields. Borderline lepromatous (BL) leprosy resem-bles lepromatous disease except that at least some of theskin lesions are selectively anesthetic and show varyingdegrees of distinctness in their borders. Peripheral nervetrunk involvement (due to the immune response of the host)is more widespread than in LL, but mucous membraneinvolvement (due to bacterial proliferation) is less than inLL. Skin lesions of BL leprosy contain predominantlymacrophages with relatively large numbers of lymphocytes.The numbers of acid-fast bacilli are usually somewhat lessthan those in an LL lesion of comparable duration, butsubstantially more than in a BT lesion. BT disease can

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develop from indeterminate leprosy; it can also develop fromBL disease by upgrading (by increasing the immune re-sponse of the host). Similarly, BL leprosy can develop fromindeterminate leprosy or by downgrading from BT.

ReactionsSo-called reactions in leprosy are clinically apparent,

immunologically mediated inflammatory conditions occur-ring during the course of the disease in about 50% ofpatients. These manifestations of leprosy are due to theimmunologic response of the host to the bacilli. They arebasically of two types. Type 1 or reversal reactions aregenerally agreed to be a result of delayed hypersensitivityand affect patients with borderline to tuberculoid leprosy.They are characterized by edema and erythema of preexist-ing lesions and a tendency for the overall disease classifica-tion to upgrade. Type 2 or erythema nodosum leprosumlesions have long been thought to be manifestations of anArthus type of hypersensitivity reaction. They are seen inBL and LL patients and are characterized by the develop-ment of crops of tender, erythematous skin nodules andfever. Both types of reaction can involve peripheral nerves,but type 1 reactions more characteristically do so. Type 2reactions can involve any tissue containing antigens of theleprosy bacillus; therefore, lesions of erythema nodosumleprosum are not confined to the skin, but can involve theeye, joints, nasal mucosa, etc.Type 1 reactions histologically consist of edema on a BT

or TT background, initially. If the outcome of the type 1reaction is upgrading, there may be an early increase in thenumber of lymphocytes. In severe type 1 reactions, case-ation necrosis may occur. Type 2 reactions are characterizedby an influx of neutrophils on a BL or LL background. Avasculitis involving arterioles or venules is demonstrable inabout half of the cases in type 2 reactions.

Patient ManagementSuccessful management of the leprosy patient consists of

controlling the three mechanisms by which the diseasecauses symptoms, i.e., (i) bacterial proliferation, (ii) theimmunologic response of the host to the leprosy bacilli, and(iii) peripheral neuritis caused by the first two processes.

Antibacterial chemotherapy for leprosy, in general, hasbeen both adequate and available since the early 1940s (57).The problems in antibacterial chemotherapy have come frominadequacies in health care delivery systems in leprosy-endemic areas, inadequacies in patient compliance for vari-ous reasons, and the potential for the development ofdrug-resistant M. leprae on a large scale. At present, fourdrugs are used widely: dapsone, rifampin, clofazimine, and athioamide, either ethionamide or prothionamide. A varietyof drug combinations are used in various parts of the world.Some centers use monotherapy with dapsone. Many centersutilize the multidrug regimens recommended by the WorldHealth Organization (253). For indeterminate, TT, and BTleprosy, for example, daily dapsone is recommended plusrifampin once monthly. The total duration of treatmentrecommended in these so-called paucibacillary cases is 6months. For mid-borderline, BL, and LL disease, the WorldHealth Organization recommends daily dapsone, plus dailyclofazimine, with additional clofazimine given once monthlytogether with once-monthly rifampin. The total duration oftherapy in these so-called multibacillary cases is at least 2years and preferably until bacilli are no longer demonstrableby the usual slit-skin smear techniques.

Current U.S. recommendations for therapy are 6 monthsof rifampin plus dapsone daily and then dapsone alone until3 years after disease inactivity for indeterminate and TTpatients. BT patients are treated the same, except dapsone iscontinued until 5 years beyond disease inactivity. BL andLL patients are treated with rifampin plus dapsone daily forat least 3 years, and dapsone is then given alone for theremainder of the patient's life.The rate of clearance of bacilli from a patient on effective

chemotherapy is a function of the host's immune response.It is 0.5 to 1.0 log per year of effective chemotherapy in LLand increases progressively as the disease is more tubercu-loid. The nature of the chemotherapy, e.g., bactericidal or

bacteriostatic, does not affect the rates of clearance ofbacilli.

In approximately 50% of leprosy patients, managementmust also include controlling reactions caused by the host'simmunologic response to the leprosy bacilli. The aim of suchanti-inflammatory drug treatment is to prevent or minimizepermanent disability and is directed predominantly at con-

trolling peripheral nerve damage and irreversible eye dam-age. Inflammatory eye changes occur predominantly inlepromatous patients and can frequently be managed withtopical corticosteroids. Peripheral nerve damage from reac-

tional leprosy can occur throughout the spectrum, but isparticularly prominent in BT disease. In general terms, acutelosses in peripheral nerve function are treated with cortico-steroids. More subacute or chronic reactional states can betreated with limited courses of relatively high doses ofclofazimine, an antileprosy drug with both antibacterial andanti-inflammatory properties. Erythema nodosum leprosumis usually completely suppressed with thalidomide, a terato-genic sedative-hypnotic with remarkable immunosuppres-sive/anti-inflammatory activity in this condition (92).

In virtually all leprosy patients, there is some degree ofirreversible peripheral nerve damage caused by bacterialproliferation or the immunologic response of the host tothese bacilli or both. In some patients, e.g., an indeterminatecase with minor sensory impairment confined to a small skinlesion on the trunk, this is inconsequential. In others,widespread destruction of mixed peripheral nerve trunks can

result in widespread skin anesthesia and widespread perma-nent muscle paralysis involving the face, hands, and feet.For patients with muscle paralysis due to irreversible nerve

damage due to leprosy, there are a variety of reconstructivesurgery techniques which can frequently restore reasonablemotor function (17). It should be pointed out that most of thedeformities attributed to leprosy are not caused by thedisease itself. Leprosy removes the sensation of pain. Thelack of feedback provided by pain allows the leprosy patientto damage and deform himself (as it does any patient lackingthe feedback of pain). This secondary damage is the mostdisabling, and all of the secondary damage and deformity are

preventable. A variety of techniques and principles apply towound prevention in anesthetic extremities. Perhaps, themost important principle relates to the care of wounds afterthey are sustained. Lacking sensation, individuals will con-

tinue to use a wounded and infected extremity and subject itto stress in spite of the infection. More than any other cause,this accounts for the destruction of hands and feet in leprosywhich, to a large extent, accounts for the stigma of thedisease. The continued use of an infected extremity leads to

osteomyelitis and septic destruction of bones, resulting inpermanent secondary deformities. These can often be pre-vented by simply splinting wounded extremities to preventtheir use until the wound has healed (17).

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CELL WALL STRUCTURE AND ASSOCIATEDANTIGENS OF M. LEPRAE

All pathogenic microorganisms have evolved characteris-tics which provide survival advantages in potential hosts.These may be as overt as the production of a potent,tissue-destroying exotoxin or as subtle as modulating theimmune response elicited in the host against the invadingpathogen. Intracellular bacteria, such as M. leprae, gener-ally fall into the latter category, since their survival dependsupon maintaining a stable environment in phagocytic cells(particularly macrophages) of the infected host. Two majorareas of study central to the understanding of host-parasiteinteractions are bacterial metabolism and physiology, in-cluding cell wall structure-function relationships. Also, re-lated antigenic analysis of the pathogen is helpful in definingthose structures of the bacterium involved in the immuneresponse of the host during infection. Since metabolic pro-cesses of M. leprae are covered below, we summarize herethe biochemical data related to the major cell wall structuresof M. leprae, and, when appropriate, indicate those mole-cules which have been shown to induce immune responses ineither humans or experimental animals.

Cell Wall of M. keprae

Extensive chemical analysis has shown mycobacterial cellwalls to be highly complex, lipid-rich, macromolecular struc-tures. While many cell components appear to be unique tothe mycobacteria, the common bacterial structure, peptido-glycan, is present in all mycobacteria with only a few minorvariations. For example, meso-diaminopimelic acid is foundin the tetrapeptide allowing cross-linking of the peptidogly-can through adjacent meso-diaminopimelic acid residues or

through meso-diaminopimelic acid-D-alanine residues (9,125, 202). In addition, M. leprae peptidoglycan, unlike thatin other mycobacteria, contains the unusual substitution ofglycine for alanine in the tetrapeptide (49-51). While thesesubstitutions may provide some advantage in pathogenicityfor M. leprae (e.g., resistance to degradation), it is unlikelythat they affect the adjuvant properties of the muramyldipeptide region found in all bacterial peptidoglycans (238),including that of M. leprae.

Covalently linked to the peptidoglycan is an arabinogalac-tan-mycolic acid complex, which constitutes approximately70% of the cell wall mass and forms a major amphipathicregion external to the peptidoglycan. The arabinogalactanpolymers consist of linear arrays of D-arabinose-D-galactosewith appendages of D-arabinose extending laterally (2, 5, 7).The absence of the more common L-isomer of arabinose inmycobacterial cell walls suggests a heightened resistance todegradation by host enzymes, possible contributing to per-sistence of cell wall material in host cells upon death of themycobacterial cell. Esterified to the terminal arabinose moi-eties are mycolic acids which contribute significantly to thehydrophobicity of mycobacterial cell walls (8). In M. lepraethese long-chain fatty acids form two groups, the alpha-mycolates and beta-mycolates, and can be used for taxo-nomic purposes to differentiate M. leprae from other myco-bacteria (77, 103, 142).While peptidoglycan, arabinogalactan, and mycolates con-

tribute significantly to the structural integrity of the cell wallof M. leprae and other mycobacteria, they do not appear toconstitute major immunogens of the bacteria. Instead, otherglycolipids (20, 94), glycopeptidolipids (19, 21), and treha-lose containing lipooligosaccharides (97, 98) have been

found to be active antigenic components of mycobacteria.The most notable of the cell wall-associated glycolipidmolecules of M. leprae is phenolic glycolipid I (PGL-I)which has been shown to be species specific and immuno-genic during infections with M. leprae (20, 255). Brennanand co-workers established the chemical structure and im-munologic specificity of PGL-I through a series of elegantstudies which have been reviewed in detail elsewhere (18,69). The general structure of PGL-I can be described as atrisaccharide moiety composed of 3,6-dimethyl-p-D-glucose(1- 4) 2,3-dimethyl-a-L-rhamnose (1-*2) 3-methyl-a-L-rhamnose linked to a phthiocerol lipid core through a phe-nolic group. The terminal sugar, 3,6-dimethyl-p-D-glucose,constitutes the major immunodominant region of the trisac-charide, with the penultimate 2,3-dimethyl-a-L-rhamnosecompleting the composite native epitope (96). PGL-I appearsto be associated with the outer surface of M. Ieprae (258) andhas been isolated from purified bacteria and M. leprae-infected tissues in relatively high concentrations (94). Takentogether, these characteristics suggest that PGL-I may rep-resent the M. leprae "capsule" which could function as avirulence factor providing an important interface betweenparasite and host, critical for maintenance of the parasiticrelationship. The immunogenicity of PGL-I during M. lepraeinfection in humans and various other animals has beenfirmly established, but further studies need to be performedon potentially important immune and nonimmune regulatoryfunctions of PGL-I and related extracellular glycolipids inrelation to intracellular parasitism.

Recently, Brennan and co-workers isolated and character-ized a group of arabinose and mannose-containing phospho-rylated lipopolysaccharides from M. leprae (97). Earlierwork on similar carbohydrate-rich fractions from mycobac-teria (204) and M. leprae (141) had established the serologicreactivity of the arabinomannan component but not thecellular location of the component. Detailed studies byHunter et al. (97) established that the serologically activecomponent, lipoarabinomannan B (LAM-B), is acylated,contains substituents of phosphatidylinositol, and may bemembrane associated. LAM-B has proven to be highlyimmunogenic in most mycobacterial infections, inducingstrong humoral antibody responses. Since LAM-B is acommon antigen among mycobacteria, it cannot be usedeffectively for serologic tests to detect early infection withM. leprae. However, there are data to suggest that highlevels of anti-LAM-B are correlated with high bacterial loadsin leprosy (129). This may make possible the estimation ofbacterial clearance during antimicrobial chemotherapy bymonitoring serum antibody levels to LAM-B.

Protein Antigens of M. lepraeImmunochemical information combined with detailed

fine-structure analyses ofM. leprae cell walls and associatedmolecules has led to our understanding of a cell wall essen-tially devoid of proteins and polypeptides. Since proteins aremajor structural and functional molecules in all biologicalsystems, the need to elucidate their role in M. leprae hasbeen of paramount importance. The major impediment tostudying the proteins ofM. leprae stems from the inability toculture the bacteria in vitro, limiting workers to bacilliobtained from tissues of experimentally infected mice, rats,or armadillos. These relatively low numbers of organismsprovide only small quantities of purified proteins, severelylimiting detailed analysis.

Prior to the application of monoclonal antibodies (MAb)and recombinant deoxyribonucleic acid (DNA) techniques,

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immunochemical approaches for studying proteins of M.leprae proved tedious and highly complex. Most of the earlywork was performed by immunoprecipitation of protein andother antigens with polyclonal antisera in agarose gels bytwo-dimensional immunoelectrophoresis. A sophisticatedscheme for analysis of these profiles was developed, butlittle has been learned about the chemical nature of theantigens contributing to the various immunoprecipitates. Adetailed description of this approach is beyond the scope ofthis review, but was reviewed recently by Harboe (85).More recent developments on proteins of M. leprae have

come from studies with murine MAb as monospecific probesto identify and characterize the eliciting antigens. The firstreported murine MAb to M. Ieprae recognized a 65-kilo-dalton (kDa) protein which was subsequently found to beassociated with the cell wall of the bacteria (73, 74). Four-teen nonoverlapping antigenic epitopes have been defined onthe 65-kDa protein by competitive inhibition studies withmurine MAb (25). The majority of these epitopes werecross-reactive and present on homologous 65-kDa proteinsof other mycobacteria, while one epitope, defined by MAbIIIE9 and IVD8, was specific for M. leprae within thecontext of the 23 mycobacterial species studied. Serologictests in humans for antibody to this species-specific epitopehave revealed antibody primarily in patients with LL, limit-ing the usefulness of this antigen for detecting early infectionwith M. leprae (128).The general scheme of multiepitopic proteins of M. leprae,

containing both species-specific and cross-reactive B-celldeterminants, has been reported by other workers, usingmurine MAb (101, 119). A collaborative study for the com-parison of 20 MAb to M. leprae was recently reported by theWorld Health Organization in which M. leprae-specific andcross-reactive epitopes were found on proteins with molec-ular weights of 12,000, 18,000, 28,000, 36,000, 55,000 to65,000, and 200,000 (56). Antigenic analysis with Mab is onlya first approximation of the extent and complexity of M.leprae protein antigens. Moreover, the relevance of theseproteins to the human immune response during infectionwith M. leprae remains unanswered. Obviously, immuno-genic proteins might provide suitable components for devel-opment of vaccines against leprosy, but detailed studies areneeded to define the B- and T-cell stimulatory capacity ofthese molecules. This work will require relatively largeamounts of purified proteins which are not attainable fromnative purified bacilli.Major accomplishments have been realized in the area of

mycobacterial cell wall chemistry and structure. These ad-vances have not been accompanied by parallel advances inour understanding of the biological significance of thesesame components. In particular, our knowledge concerningthe role of M. leprae cell wall and cell wall-associatedconstituents, as they relate to virulence factors of M. leprae,remains speculative at best. Further research aimed atdefining structure-function relationships, using highly de-fined and purified components of M. leprae and othermycobacteria, should lead to a more complete understandingof the pathogenic mechanisms of this group of highly suc-

cessful pathogens.

Molecular Biology of M. lepraeAnalysis of genome size and guanine-plus-cytosine con-

tent are two important characteristics of bacteria used incomparing relatedness among bacterial species. Molecularanalysis of purified M. leprae DNA by guanine-plus-cytosine

content and by molecular size of the genome indicated thatM. leprae was significantly different from other mycobacte-rial species (16, 36, 99). Guanine-plus-cytosine content hasnow been established at approximately 56%, with most othermycobacteria exhibiting guanine-plus-cytosine contents of>60%. Genome size for M. leprae (2.2 x 109 daltons)appears to be smaller than that for most other mycobacteria(2.8 x 109 to 4.5 x 109 daltons) with the exception of M.tuberculosis H37Ra, which has been reported to be between2.0 x 109 and 2.5 x 109 daltons (16, 36).

Until recent developments in recombinant DNA tech-niques, major areas of M. leprae genetics and physiologyremained unexplored. Since M. leprae has not been culti-vated in vitro, recombinant DNA methods provide a power-ful, indirect approach for studying genes and gene productsof the organism. Application of this approach has fosteredresearch in areas as diverse as analysis of metabolic path-ways (36, 102), immunochemical analysis of antigenic pro-teins (25), and taxonomic studies measuring genotypic relat-edness of phenotypically similar bacterial species (J. E.Clark-Curtiss, Abstr. Annu. Meet. Am. Soc. Microbiol.1987, U-172, p. 117).The initiation of recombinant DNA studies with M. leprae

required two major breakthroughs. The first came with thesuccessful propagation of M. leprae in the armadillo (113),providing large quantities of bacteria from which purifiedDNA could be obtained ahd analyzed. The second break-through came with the establishment of M. leprae genomiclibraries which could be manipulated in cultivable bacteria,such as Escherichia coli, for subsequent analyses of M.leprae genes and gene products. Libraries of this type are

composed of small segments of M. leprae DNA (4 to 20kilobases) present in independent recombinant moleculeswhich can be manipulated to meet experimental needs. Forexample, a small segment ofDNA from M. leprae, localizedin a plasmid or bacteriophage vector, can be directed by thevector to be transcribed. Subsequent translation of theresultant messenger ribonucleic acid should result in an M.leprae protein(s), the gene(s) for which is encoded in thecloned segment of M. leprae DNA. Should an intact proteinor partial peptide result, the enzymatic or antigenic nature ofthe molecule can be studied. Theoretically, successful appli-cation of this approach should make possible the elucidationof enzymatic pathways important for M. leprae growth andmetabolism and, thereby, suggest strategies for in vitrocultivation studies and indicate potential sites for chemo-therapeutic agents with activity against M. leprae. In addi-tion, potentially important immunogenic proteins of M.leprae may be produced and tested as vaccines.Genomic libraries of M. leprae have been established in

the cosmid vector pHC79 and the plasmid vector pYA626 bythe Clark-Curtiss group (36, 102). Initially, clones from thelibraries were used to explore the ability of M. leprae genesto complement known enzymatic pathways in auxotrophicmutants of E. coli. Complementation with M. leprae DNAwas observed for the gltA (citrate synthase) and aroB(dehydroquinate synthetase) mutations in E. coli (102). Com-plementation of three gltA mutants was found only when M.leprae DNA was linked to a strong promoter from Strepto-coccus mutans, suggesting that mycobacterial promoters are

utilized only weekly if at all in E. coli. The complementingDNA fragment specified a 46-kDa polypeptide and probablyrepresented the citrate synthase gene ofM. leprae. Other M.leprae DNA sequences capable of complementation of E.coli mutants have not been reported, reflecting possibleinherent difficulties with this approach due to basic genetic

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and physiologic differences between E. coli and M. lepare. Asimilar approach using other host bacteria, such as Bacillussubtilis and appropriate cloning vectors, could be tried totest this hypothesis. Unfortunately, all other potential hostsand related cloning vectors are less well characterizedcompared with E. coli, limiting their usefulness in theimmediate future. Nevertheless, this general approach re-mains a potentially powerful tool for studying basic physio-logical mechanisms operative in a noncultivable microorgan-ism, such as M. leprae.The other significant work in this area was the construc-

tion of a genomic library in bacteriophage X gtll by Young'sgroup, which was designed for regulated expression of M.leprae proteins in E. coli (260). So far, five protein antigensof M. leprae have been expressed in E. coli and analyzed indetail (140, 260). Cloned segments of M. leprae DNA fromthe X gtll library have provided DNA for sequencing of atleast two M. leprae proteins (18 and 65 kDa), both of whichhave been shown to be immunogenic subsequent to vacci-nation with M. leprae (158, 159). Sequence data of this typefor the 65-kDa proteins of M. leprae, M. bovis BCG, and M.tuberculosis have provided detailed amino acid sequenceinformation concerning cross-reactive and species-specificantibody-reactive epitopes (219). Similar work with T-cellclones should provide information related to T-cell recogni-tion regions on these and other proteins, allowing for de-tailed immunochemical analysis of M. leprae proteins.Moreover, DNA-derived peptide sequences and subsequentsynthesis of these moieties will provide reagents needed toanalyze the complex interactions of antigenic peptides andclass II major histocompatibility complex molecules onantigen-presenting cells. Studies on this type may helpelucidate the molecular aspects of triggering M. leprae-reactive T cells, important in mediating protective cell-mediated immunity (CMI) against M. leprae.

Finally, application of molecular techniques for analysis ofgenomic DNA ultimately may provide new methods fortaxonomic purposes and new insight into our thinking aboutM. leprae and related bacterial species. Traditionally, genus,species, and strain differentiation of microorganisms hasbeen based on phenotypic differences observed betweenbacteria which ultimately are the result of, and consistentwith, the genetic constitution of those organisms. However,it has been estimated that only 20% of the genomic capabilityof a microorganism is represented when applying detailedphenotypic analyses (22). Accordingly, major areas of thegenome, possibly encoding important functional or regula-tory genes not demonstrable as quantifiable phenotypiccharacteristics, may go undetected.

Obviously, new approaches are needed for studying largerareas of all bacterial genomes and particularly noncultivablebacteria, such as M. leprae. Analysis of DNA restrictionfragments after restriction endonuclease digestion of ge-nomic DNA (38, 175) and the related approach of restrictionfragment length polymorphism analysis hold promise in thisarea. As we obtain a more complete understanding of the M.leprae genome and its related functional capacity, it isanticipated that most areas of study related to the leprosybacillus will be enriched and, thereby, advance our under-standing of M. leprae and its basis for pathogenicity.

IMMUNOLOGY

Active infection with M. leprae is characterized by abroad spectrum of host response, with great variability inhistopathology and clinical course of infection. There is,

likewise, a spectrum in the host humoral or CMI response toinfection. Individuals with TT manifest a strong delayed-type hypersensitivity (DTH) to M. leprae antigens but pro-duce relatively low levels of antibody. At the opposite end ofthe clinical spectrum (LL), there is a potent humoral anti-body response but a progressive anergy in CMI to M. lepraeantigens.

Immunologic Research

Goals. As with the study of any infectious disease, thegeneral goals of an immunologist working in the leprosy fieldare clearly defined. As a practical measure, tests should bedeveloped to aid in the diagnosis of subclinical disease.These tests should also find application in the monitoring ofthe course of control measures in endemic areas and inepidemiological studies. More basic research can be focusedon the immunological mechanisms that contribute to theprotective as well as pathologic aspects of host responses toleprosy. Ultimately, exploitation of fundamental knowledgeof the immune response to leprosy should allow develop-ment of an effective vaccine.

Obstacles to research. Although the leprosy bacillus wasidentified as a human bacterial pathogen in 1874, untilrecently workers in the leprosy field have made few contri-butions to the field of immunology and have adopted little inthe way of immunological know-how to their studies. Thislack of progress has not been due to a lack of effort,expertise, or dedication by researchers. At virtually everyturn in investigating this disease seemingly insurmountableobstacles arose that stymied progress. Even 115 years afterits discovery, M. leprae remains uncultivable. Until re-cently, human biopsies served as the sole source of leprosybacilli. These crude preparations yielded inadequate num-bers of organisms, and the lack of reliable methods forquantitating viable organisms prevented standardization ofdifferent preparations.Major breakthroughs. Three major discoveries in the past

25 years have allowed leprosy workers in general andimmunologists in particular to put leprosy research in pacewith other infectious diseases. (i) The mouse footpad modelfor M. leprae infection, developed by Shepard in 1960 (206)offered a means of quantitating the growth of the leprosybacillus. Although only a relatively few organisms could beobtained, this model allowed evaluation of chemotherapeu-tic regimens (209) and serves as the basic model for testingthe efficacy of potential leprosy vaccine preparations. Theuse of athymic (nu/nu) mice (33, 39, 117) and rats (60) definedthe importance of CMI in host resistance to leprosy andprovided models that more closely mimic LL. (ii) In 1971,Kirchheimer and Storrs (113) identified the nine-bandedarmadillo as a susceptible host for the growth of M. leprae.Armadillos in Louisiana and Texas have subsequently beenshown to harbor a naturally acquired M. leprae infection(226, 231, 237). The armadillo model provided for the firsttime large quantities of leprosy bacilli for immunologic,immunoprophylactic, immunotherapeutic, and immuno-chemical studies. (iii) Using armadillo-derived M. leprae,Brennan and his colleagues in Colorado (18, 20, 94, 96, 98)were able to identify in preparations of the leprosy bacillus aphenolic glycolipid that possesses a unique trisaccharidestructure that is immunologically specific for M. leprae.Immunochemical studies and electron microscopy indicatethat PGL-I is located on the surface of the organism and thatit may accumulate in the tissues of the armadillo in quantitiesequal to half the total weight of the leprosy bacilli present

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(94, 96). The "foam" seen in heavily infected macrophagescharacteristic of the lepromatous granuloma is thought tocontain PGL-I (67, 84). The ability to produce purifiedpreparations of specific M. leprae antigens allowed immu-nologists to use state-of-the-art techniques to further explorethe immunology of leprosy.

Clinical Immunology

Lepromin test. The practical clinical immunology of lep-rosy is at present based almost entirely on the reactivity tointradermal skin tests with lepromin, a heat-killed suspen-sion of M. leprae obtained originally from homogenizedhuman leproma (48, 143) but now prepared from infectedarmadillo tissue. Reactivity to lepromin has no real diagnos-tic value but does establish the immune status of the indi-vidual to M. leprae and thus is of prognostic value (189).

Typically, a positive reaction is biphasic. An early (24 to48-h) Fernandez reaction (58) is a DTH reaction (probably tosoluble protein antigens in the preparation) and occurs in TTpatients as well as contacts or healthy individuals sensitizedto M. leprae or cross-reacting antigens from other mycobac-teria. A major goal of recombinant DNA research in leprosyis the production of M. leprae-specific protein antigens thatcan serve as a reagent for detection of specific DTH inleprosy.The late (Mitsuda) reaction to lepromin is measured at 21

days and reflects the induction of acquired CMI to M.leprae, manifested by formation of an organized epithelioidcell granuloma (143, 189). Positive Mitsuda reactions areseen in the vast majority of contacts and unexposed individ-uals as well as in persons with TT. Weakly positive reactionsaid in classification of borderline disease. In LL, no responseis seen, indicating the absence of CMI to M. leprae. Evenafter many years of chemotherapy, the lepromin test remainsnegative in LL (156, 232).Humoral immunity and serodiagnosis in leprosy. Because

of the intracellular nature of M. leprae, humoral antibody isprobably not important in resistance but could play a role inthe pathogenesis of erythema nodosum leprosum reactionsby formation of antigen-antibody complexes (14). LL haslong been associated with a state of hypergammaglobuline-mia (27, 205), and there is, in general, an inverse correlationbetween the anti-M. leprae antibody titer of a patient and thepotency of his CMI response to the leprosy bacillus, i.e., adirect correlation between antibody level and bacillary load.Demonstration of antibody to specific M. leprae antigens isnot as straightforward. Prior to the availability of armadillo-derived M. leprae, organisms extracted from human tissuewere used in indirect fluorescent antibody studies, radioim-munoassays, and crossed immunoelectrophoresis assays (1,37, 86, 87), but M. leprae-specific antigens could not bedemonstrated.The discovery that PGL-I is biochemically unique (94, 95)

and immunologically specific (20, 177) for M. leprae reawak-ened interest in the serodiagnosis of subclinical leprosy. Adetailed summary of leprosy serodiagnosis can be found inthe recent review by Gaylord and Brennan (69). The major-ity of human antibody to PGL-I is of the immunoglobulin Msubclass (35, 256), and virtually all MAb generated againstPGL-I in mice are immunoglobulin M (258). Enzyme-linkedimmunosorbent assay technology that uses the highly hydro-phobic native PGL-I (35) has been worked out, and attemptshave been made to standardize (177, 198) and simplify theprocedures (257). Alternatively, hydrophilic synthetic neo-glycoprotein antigens bearing the immunologically distinct

moiety specific for M. leprae, and linked to a protein carrier(31, 32, 34, 35, 65, 66), have found widespread application.Anti-PGL-I antibody is found in virtually all LL patients (23,35, 255). However, although the number of false-positiveresponses is very low, the response of TT patients andcontacts is also disappointingly low, thus limiting the use ofthis test for epidemiological purposes or for identification ofpatients with subclinical infection. There is some evidencethat there is a correlation between decreased bacillary loadand anti-PGL-I antibody levels after chemotherapy in LL(129). Because PGL-I is a major component ofM. leprae andis present in abundance in infected tissue and the blood ofLL patients, detection of this antigen by serological meanscould identify infected individuals (254), although this ap-proach may not be feasible at the TT end of the spectrum orin subclinical infections.

Sera from individuals with leprosy, including TT patients,react strongly with the highly immunogenic LAM isolatedfrom the cell wall of M. Ieprae (130). In general, theseantibodies cross-react with LAM from M. tuberculosis,although specific epitopes for M. leprae LAM are demon-strable with MAb (70). Distinct chemical features for arabi-nogalactan and peptidoglycan of M. leprae cell wall havebeen demonstrated (70, 97). Further work is necessary todetermine whether a routine assay for specific M. lepraeantibodies to these cell wall subunits can be developed.MAb technology has focused attention on at least five

major protein antigens of M. leprae: those of 65, 36, 28, 18,and 12 kDa. Of these, the 65-kDa antigen is the most widelystudied, including complete expression in the X gtll recom-binant DNA system (259, 260). The 65-kDa protein has beenshown to possess epitopes that cross-react with those ofother mycobacteria (73), but M. leprae-specific epitopeshave been found as well (25, 74). With MAb, M. leprae-specific epitopes have also been found on the 36- and 12-kDaproteins (101, 115, 119). These specific MAb lend themselvesto use in competitive inhibition assays for the serodiagnosisof leprosy (114, 225) and may detect a specific antibodyresponse in TT patients.

CMI in Leprosy

Early pathologists recognized the importance of CMI,manifested by the epithelioid cell granuloma, in conferringresistance in TT. In addition, DTH appears to underlie muchof the host tissue damage observed in reactional episodes inBT disease (170, 252). As discussed above, the lepromin testis uniformly negative in LL patients and remains negativeeven after years of successful chemotherapy. An in vitromanifestation of anergy in LL is demonstrated by the lack oflymphocyte blast transformation in the presence of M.leprae antigen (75, 160). Early indications that LL wasassociated with a general suppression of CMI (83, 188, 205)have not held up. The T4/T8 (helper-inducer/suppressor-cytotoxic) ratio of T lymphocytes in the peripheral blood ofLL patients ranges near the normal value of 2:1 (187). In LL,there is no evidence for an increased incidence of cancer orinfection with the opportunistic pathogens commonly asso-ciated with the immunocompromised host. For example, theclinical course of pulmonary tuberculosis in an LL patient isidentical to that in an otherwise healthy individual. Theabsence of specific CMI to antigens of M. leprae in LL hasattracted widespread attention by immunologists interestedin exploring immunoregulatory mechanisms in a model ofnonfatal immunodeficiency disease in humans.

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Mechanisms of Specific Anergy in LL

Several hypotheses are under investigation to explain thespecific anergy in CMI to M. leprae antigens in LL, includ-ing a genetic predisposition for LL, clonal deletion of M.leprae-specific T cells, and specific suppression of T-cellfunction.

Genetic control of CMI in leprosy. Studies of leprosy typein homozygous twins showed neither the random concor-dance expected if there was no predisposition for type ofdisease nor complete concordance for disease at the TT orLL ends of the spectrum, expected if precise genetic controlwas operating (29, 154). Other attempts have been made toexplore genetic restriction in leprosy, and evidence is focus-ing on the importance of the major histocompatibility com-

plex class II genes. Individuals with the HLA-DR2 orHLA-DR3 haplotype or both may be predisposed to the TTform of leprosy (173, 233, 234), while the incidence ofDR2-DQW1 may be increased in individuals with LL (201).Genetic predisposition and as yet unclarified exogenousfactors such as environment, mode of transmission, andinfecting dose should be important considerations in anywidespread vaccine trials for prevention of leprosy.

Role of the lymphocyte in specific anergy in leprosy. At thelymphocyte level, the presence of T-helper cells specific forantigens of the leprosy bacillus is a key characteristic of theTT end of the clinical spectrum of leprosy. The reason fortheir absence or lack of function in LL remains unclear.Haregewoin et al. (88, 89) appeared to restore T-helper cellresponse to M. leprae in vitro by supplementing the cellculture media with the T-cell-amplifying cytokine interleu-kin-2 (IL-2). However, others have been unable to confirmthese findings (105, 107, 163) in totally anergic LL patients.Mohagheghpour et al. (153) suggested that lack of specificT-cell response in LL may be due to absence of the IL-2receptor on these cells. On the other hand, Modlin et al.(146, 147) have shown T cells bearing IL-2 receptors in theLL lesions themselves, although there were far fewer IL-2-secreting T cells in LL compared with TT lesions. Mohag-heghpour et al. (152) have recently reported that M. leprae-specific T4 cells are present and respond to M. Iepraeantigen, but only after a 48-h "rest" in culture.Although specific reactivity in TT or specific anergy in LL

is routinely demonstrated in vitro with peripheral bloodlymphocytes, the reactivity of the cells in the leprosy le-sions, not the blood, is probably more relevant to the CMIresponse of the host to M. leprae. In a series of studies,Modlin et al. have investigated the cellular makeup of the TTand LL lesion, using immunopathological techniques (149-151). Briefly, their findings indicate that, in contrast to thenear-normal (2:1) T4/T8 ratio in the blood in LL, the relativenumber of helper T cells is markedly lower (0.5:1) in thelepromatous lesions. The T4/T8 ratio remains approximately2:1 in the lesions in TT leprosy, but more importantly, thecells are arranged in a distinct architecture within the lesion:T4 cells in the centers of the epithelioid granulomas and T8suppressor cells in the margins.

In a series of studies by Mehra et al. (136, 138, 139), M.leprae-specific suppressor T cells were demonstrated in theperipheral blood of LL patients. Immunologically specifictriggering of the suppressor cells was shown to be elicited bythe unique PGL-I antigen of M. leprae (136). The demon-strated effects of these suppresor cells in vitro involvenonspecific suppression of helper T-cell function as mea-sured by a concanavalin A mitogenic response. Suppressionof a CMI response as potent as that of concanavalin A

mitogenicity might also suggest that more vital T4 lympho-cyte-antigen interactions could also be blocked, a conceptnot consistent with CMI anergy in LL being specific for M.leprae. However, in the LL lesion, the random distributionof T4 and T8 cells at a 0.5:1 ratio (148) may favor localsuppression of helper cell function and not result in ageneralized immunocompromised state. Recently, clones ofT8 lymphocytes isolated from LL lesions were shown tomediate major histocompatibility complex class II antigen-restricted suppression of T4 clones after interacting specifi-cally with lepromin (149, 169).The suppressor cell hypothesis in LL is controversial.

Nath (161) concluded that T-cell suppression in leprosyregulates the CMI response in the TT but not LL stage.Moreover, although the suppressor cell hypothesis may belinked to the specific anergy characteristic of LL, it does notas yet address the actual mechanisms whereby sensitizedhelper T cells fail to respond to antigen. Nor does thishypothesis address the argument of how and why onlycertain individuals develop a T suppressor cell responseupon challenge with the leprosy bacillus and subsequentlyprogress to the lepromatous form of the disease.Other groups have developed T-cell clones from the

peripheral blood lymphocytes of leprosy patients by usingautologous antigen-presenting cells to define their reactivityto M. leprae antigen. Mostly, antigen-reactive clones, in-cluding clones from M. Ieprae-vaccinated volunteers, havebeen T4 in phenotype (158), some of which proliferated inresponse to the 18-kDa M. leprae recombinant protein (260).Some T4 clones from a TT patient were shown to reactspecifically with M. leprae, while others responded to othermycobacterial antigens (55). As an alternative to autologousblood monocytes as antigen-presenting cells, some workersused Epstein-Barr virus-transformed autologous B cells tosupport T-cell clones from TT and BL patients (80, 172, 174).Clones from the TT patient were all T4, and, although somecross-reacted with other mycobacteria, others appeared toproliferate specifically in the presence of M. leprae 36-kDaprotein (174). Interestingly, clones of the T8 suppressorphenotype from a BL patient suppressed autologous T4lymphocyte response to M. leprae (172).

The Macrophage in Host Resistance to Leprosy

Macrophages participate in the immune response in bothafferent and efferent roles. In their afferent role they assist inregulation of the immune response (4, 131) by interactingwith T cells in the presentation of antigens or by secretion ofsoluble mediators. Macrophages have been shown to sup-press T-cell responses in leprosy (164, 196) and may bedefective in their ability to present M. Ieprae antigens tosensitized T cells (93). Studies have also revealed (191, 239)that, in LL, macrophages may be defective in the productionof IL-1, the cytokine that can amplify the production of IL-2by T cells.

Regardless of the lack of a clear understanding of theunderlying mechanism of defective CMI in LL and thepossible role of macrophages in anergy, the failure of themacrophage to kill or inhibit M. leprae is a conspicuouscharacteristic of the lepromatous form of the disease. Itsinability to cope with M. leprae is an issue central tounderstanding the mechanisms of host resistance to theleprosy bacillus.Macrophages, activated by lymphokines (gamma inter-

feron [IFN--y], especially) released from sensitized helper Tcells responding specifically to antigen, are thought to play a

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major role in resistance to a wide variety of obligate andfacultative intracellular pathogens (122, 166, 167). Theevents leading to formation of an epithelioid cell granulomaand the subsequent limitation and elimination of the localbacilli in TT leprosy or a positive lepromin reaction are likelythe consequences of such a scenario. Activated macro-phages are distinct from normal macrophages by numerousmorphological and metabolic criteria (108, 171), includingenhanced production of oxidative metabolites such as super-oxide and hydrogen peroxide which may underlie theirenhanced microbicidal capacity (167, 168).A fundamental problem plaguing studies of the role of the

macrophage in resistance to leprosy is the inability toroutinely quantitate the viability of the uncultivable leprosybacillus. Thus, macrophage effector function in leprosy hasbeen addressed indirectly. Although there is some evidencethat macrophages from LL patients are deficient in theirability to digest M. leprae (12), not all workers have foundsuch deficiencies (52, 197). Our own studies showed thatmice with potent populations of activated macrophages weremarkedly resistant to footpad infection with M. Ieprae (120,121). In other indirect studies, oxidative metabolism ofpatient phagocytes was tested and found to be normal orabove normal (68, 76, 133). On the other hand, Nathan et al.(165) and Kaplan et al. (106) showed not only that monocytesfrom LL patients were deficient in hydrogen peroxide pro-duction (and presumably microbicidal capacity as well), butalso that this defect was reversed by treatment with IFN-y.These findings are not consistent with studies that showedno defect in the innate microbicidal capacity towards otherpathogens of macrophages from LL patients (52). We haverecently demonstrated that activated mouse macrophageshave a deleterious, probably microbicidal effect on M. Iepraeas shown by electron microscopy (220) and marked inhibi-tion of adenosine 5'-triphosphate (ATP) content and synthe-sis of PGL-I (186, 224).Because of the T-cell anergy associated with LL, the

signals are not produced that would activate the microbicidalcapacity of the infected macrophages in the lesion. Regard-less, our recent studies (222, 223) show that the M. leprae-gorged macrophages from the footpad lesions of infectednude (nulnu) mice, a model of experimental LL (33), arerefractory to IFN--y in vitro as measured by four parametersof activation: microbicidal capacity, cytotoxicity for neo-plastic cells, superoxide anion production, and expression ofmajor histocompatibility complex class II (Ia) antigen. Peri-toneal macrophages from these same mice were fully respon-sive to IFN--y.

Interestingly, a similar refractory response to IFN--y couldbe induced in vitro in mouse peritoneal macrophages heavilyinfected with live M. leprae (L. D. Sibley and J. L. Krahen-buhl, Infect. Immun., in press). The development of defec-tive activation was time and dose related, requiring 48 to 72h of incubation with large numbers of bacilli, and was clearlyparalleled by an increase in macrophage production ofprostaglandin E2 (PGE2). PGE2 is a potent immune modula-tor that suppresses macrophage Ia induction by lymphokines(227), macrophage tumoricidal capacity (228), and lympho-cyte blast transformation (54). Immunosuppressive levels ofPGE2 have been demonstrated to be produced in response tomycobacterial products (116) or by infection of mice with M.intracellulare (53). In support of this hypothesis, Ridel et al.(191) reported elevated production of PGE2 by human mono-cytes from LL patients. Moreover, we have recently dem-onstrated that skin biopsy tissue from LL patients thatproduced high levels of a 235-kDa inhibitor of monocyte

chemotaxis (28) also produced high levels of PGE2 in vitro(Sibley and Krahenbuhl, unpublished results).Although indomethacin blocked production of PGE2 and

reversed the defective response to IFN-y in our in vitrostudies (Sibley and Krahenbuhl, in press), it did not reversethe defective response of footpad granuloma macrophages(Sibley and Krahenbuhl, in press), suggesting that prosta-noid production is not the sole mechanism inhibiting macro-phage function in vivo in the lepromatous lesion. Of interest,a mycobacterial component, LAM, not only blocks T-cellproliferation (105) but also induces a refractory response toIFN--y in vitro in mouse macrophages (221) as well as inhuman monocyte-derived macrophages (Sibley and Krahen-buhl, submitted for publication). Defective macrophage re-sponse induced by LAM was not associated with PGE2production. These findings again emphasize the probabilitythat immunological events occurring in the lepromatouslesion do not necessarily represent similar events in otheranatomical compartments.Two recent reports stemming from clinical trials of LL

patients treated with IFN--y address local changes at the siteof injection (106, 165). Local changes included indurationdue to accumulation of T4 lymphocytes and monocytes andenhanced keratinocyte proliferation and la expression, butthere was no obvious evidence of clearance of bacilli. Incontrast, the reports of Convit and his associates (41, 44)suggest that local injection of lepromatous lesions with BCGresults in clearance of leprosy bacilli. These findings serve,in part, as the basis for including BCG with a killed M. lepraevaccine to exploit this immunotherapeutic effect (42).

Collectively, our in vivo and in vitro studies suggest thatany clearance of bacilli from lepromatous lesions as aconsequence of local immunotherapeutic measures (41, 44,106, 165) or chemotherapy (190) likely depends on the influxof new mononuclear phagocytes into the local lesion, ratherthan activation of the resident lepromatous macrophages.Even then, these newly arrived macrophages encounterlocal conditions that rapidly restrict their responsiveness toIFN--y. These findings emphasize that defects in CMI in LLlikely extend beyond the level of the T cell to includelocalized restriction of macrophage-afferent and -efferentfunctions influenced by lymphokines.

Leprosy Vaccine

Perhaps the main goal of immunological investigations inleprosy is the development of a safe, effective, low-costvaccine that can be used in conjunction with other controlmeasures in endemic areas, resulting ultimately in the erad-ication of leprosy. Vaccine research must, of course, takeinto consideration progress made in defining environmentaland genetic factors that determine susceptibility to leprosy.Two broad approaches are currently being explored, and athird approach involves the genetic engineering of a leprosyvaccine.The first approach depends on cross-reactivity between

M. leprae and other mycobacteria. BCG seems to be theonly other mycobacterium that affords protection against M.leprae challenge in mice (215). In large field trials, BCGvaccination protected against leprosy in Uganda (24) but wasmuch less effective in Burma (11). Other cross-reactivemycobacteria are being actively investigated as leprosyvaccine candidates, including the ICRC bacillus (47) and"Mycobacterium w." (229).The availability of large amounts of armadillo-derived,

purified M. leprae provides the basis for using killed M.

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TABLE 1. Sources of M. leprae

Host Tissue Incubation Yielda Disadvantagestime (mo)

Armadillo Liver, spleen 18 101o-1013 Expensive; requires specialized facility; highly variable yield;unused tissue must be frozen; often contaminated with otherslow-growing mycobacteria (180, 181)

Athymic "nude" mouse Footpad 12-18 101o-1011 Requires special isolator, sterile water, feed, and beddingHuman Skin nodule 107-109 Often contaminated with other skin-borne bacteria; low viabilityMouse Footpad 6-12 106 Yield too low for physiological studies

a From Wheeler (242).

leprae as a vaccine preparation. Killed M. leprae affordprotection against experimental challenge with viable bacilliin the mouse (216, 217) and armadillo (112) and induction ofDTH in guinea pigs (135). Human volunteers vaccinatedwith killed M. leprae have been shown to develop a DTHresponse (72).A novel approach to vaccination combines killed M.

leprae with viable BCG and seems to exploit specific, butundefined, immunity to M. Ieprae, with relevant cross-reac-tive immunity to BCG. Convit and his colleagues devisedthis immunoprophylactic-immunotherapeutic approachbased on their observations that local injection of thismixture into lesions of BL and LL patients induced conver-sion to positive lepromin skin test reactivity, clearance ofbacilli, histopathological upgrading, and clinical improve-ment and decreased suppressor cell activity in some patients(42, 43, 137). Long-term trials of the BCG-M. leprae vaccineare under way or are being planned in Africa, South Amer-ica, and southern India.

Finally, propagation of M. leprae genes in E. coli (260)allows potentially unlimited production ofM. leprae proteinsfor immunologic analysis and for use as diagnostic skin testreagents. The contribution of any of the M. leprae-definedproteins to a protective response against the invading bacilliis difficult to assess. However, recombinant proteins of M.leprae have been used to provide evidence that human T-cellclones recognize the 18-kDa (158) and 36-kDa (174) proteinsof M. leprae. This suggests that both proteins may berelevant to protection against leprosy.However, even highly M. leprae-specific pure proteins or

peptides will probably require incorporation in an adjuvantto induce long-term CMI and protection in humans. Few, ifany, potential adjuvants suitable for human use are avail-able. Ingenious plans are under way (15) to engineer a safe,potent leprosy vaccine consisting of highly immunogenicBCG containing the appropriate genes of M. leprae, whichmay allow these organisms to express those M. lepraeantigens associated with a protective immune response.

MICROBIOLOGY

Metabolism

The relative difficulty in studying metabolism in a bacte-rium which can be propagated only in vivo cannot beoveremphasized. The sources of M. leprae listed in Table 1all have drawbacks for research. In practice, the armadillohas served as the source of bacilli for most physiologicalstudies. However, there are only a few armadillo farms inthe world, which severely limits the number of availablebacilli. This problem is further exacerbated by the extremelyslow growth rate (generation time, approximately 12 days)and apparently low overall viability ofM. leprae populations

found in animal tissue. The need to purify the bacterialsuspensions to rid them of contaminating host tissue debrisfurther reduces bacillary yield.Another major problem in studying the metabolism of

host-derived microorganisms is that of differentiating be-tween host- and bacillary-derived activities. Since host-derived enzymes may be adsorbed to the surface of intracel-lular bacilli during tissue homogenization, mere removal ofmicroscopically detectable tissue debris is not sufficient toestablish bacterial activity. Surface treatments, usually in-volving washing with NaOH, have been shown to abolishhost-derived activities but may also destroy bacterial surfaceenzymes (249). Host and bacterial activities can often bedifferentiated via electrophoretic migration, substrate spec-ificity, serology, or the use of differential enzyme inhibitors(242).

Considering its uniqueness at a molecular level (guanine-plus-cytosine percentage and DNA homology) (6, 36, 45),one might expect to find a number of unusual or uniquemetabolic activities in M. leprae compared with other my-cobacteria. Unfortunately, some comparative studies haveused cultivable mycobacteria which were propagated in vitroand thus have compared an in vivo-grown organism (M.leprae) with in vitro-grown bacilli. Since phenotypic expres-sion may be strongly influenced by the growth environment(203), the results of such experiments do not necessarilyclarify the phenotypic relationship between M. leprae andother mycobacteria.

Catabolic activity. M. leprae appears to be metabolicallycompetent with regard to catabolic pathways for energygeneration. Glucose is actively transported (109) and, as inmost mycobacteria, is oxidized to carbon dioxide (240, 241),although rather slowly. Also, as with other mycobacteria,oxidation appears to occur primarily through the Embden-Meyerhof pathway and, to a lesser extent, via the hexosemonophosphate pathway (240). Strongly supporting the ex-istence of these pathways was Wheeler's demonstration ofthe component enzyme activities (241), although two Emb-den-Meyerhof pathway enzymes could not be detected inanother laboratory (155). Also, as with other mycobacteria,glycerol is oxidized through these pathways. What appearsunique to M. leprae is its extremely high level of 6-phospho-gluconate dehydrogenase production (241), over 100 timesthe level of the first hexose monophosphate pathway en-zyme, glucose 6-phosphate dehydrogenase. In addition, theability of the bacillus to oxidize 6-phosphogluconate, but notgluconate, suggests that M. leprae may actively scavenge6-phosphogluconate in vivo for use as an energy source(245).

Pyruvate produced via glycolosis may be converted tolactate by lactate dehydrogenase (250) or cycled through thetricarboxylic acid cycle (243). The existence of an intacttricarboxylic acid cycle in M. leprae was suggested by the

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ability of whole cells to oxidize pyruvate, citrate (243),malate (248), and succinate (240) to carbon dioxide, stimu-lation of pyruvate oxidation by citrate (243), and finally,demonstration of a full complement of tricarboxylic acidcycle enzymes (243). Of interest was the apparent proteo-lytic inactivation of fumarase in the M. leprae crude ex-tracts, an activity which could play a regulatory role. Thepresence of the glyoxylate cycle enzymes also suggests thecapacity to regulate tricarboxylic acid cycle activity (245).M. leprae appears to possess an electron transport system

as suggested by reduced nicotinamide adenine dinucleotideoxidase activity (100) and the presence of cytochromes (100,155). In addition, it is conceivable that diphenoloxidase mayparticipate in electron transport by formation of a quinonewhich can undergo reversible oxidation-reduction (155, 185).

Unlike chlamydiae, M. leprae is capable of generating itsown ATP, as demonstrated by the presence of adenylatekinase (127), incorporation of exogenous phosphate intoATP (126), and short-term 2,4-dinitrophenol-sensitive in-creases in ATP pools immediately following harvest fromarmadillo tissue (124). In addition, extended maintenance ofintracellular pools of such a labile compound under appro-priate conditions (62a) indirectly suggests that some activesynthesis is occurring.M. leprae contains a manganese-dependent superoxide

dismutase (123, 244, 251) but appears to be devoid ofcatalase (155, 251), thus suggesting a microaerophilic dispo-sition. This is supported by optimal maintenance of intracel-lular ATP at reduced oxygen concentrations, as mentionedbelow.Amino acid metabolism. M. leprae is capable of amino acid

uptake (245) and incorporation into protein (109), althoughthere is a lack of data concerning most individual aminoacids. 3,4-Dihydroxyphenylalanine is taken up (3) and oxi-dized (185), although the nature, uniqueness, and signifi-cance of this activity continue to be a matter of controversy(183, 242). 3,4-Dihydroxyphenylalanine oxidase activity isaccepted by many as a specific taxonomic marker of M.leprae. Other enzymes of amino acid metabolism which havebeen detected are glutamate decarboxylase (184) and gam-ma-glutamyl transpeptidase (218).

Nucleic acid metabolism. Thymidine is taken up by M.leprae residing in macrophage cell cultures (162) or sus-pended in axenic culture media (111). Uracil is also taken upin axenic medium, as is the pyrimidine precursor orotic acid.In general, purine bases are taken up and incorporated intotrichloroacetic acid-insoluble material at higher rates thanare pyrimidipes. M. leprae appears to be incapable of denovo purine synthesis, as evidenced by lack of incorporationof [14C]serine or ['4C]glycine into the purine fraction, andmay obtain purines primarily via scavenging mechanisms(246, 247). Enzymes for interconversion of purine baseshave been detected in cell-free extracts, and rates of incor-poration of purines could be accounted for by the levels ofphosphoribosyltransferases in cell-free extracts. Of interestis the relatively high level of adenosine kinase in M. lepraecompared with other host-grown mycobacteria.

Lipid metabolism. M. leprae residing within Schwann cellsreportedly incorporate acetate into the PGL-I fraction (157).Similarly, bacilli residing within macrophages have beenshown to rapidly incorporate palmitic acid into PGL-I (186).The latter also occurs in M. leprae residing extracellularly inan axenic medium (63). Other precursors, including acetate,were not incorporated into PGL-I in this system. Incorpora-tion of 32P into the phospholipid fraction has been reported

(46). The extracellular bacilli can also rapidly oxidize palmi-tic acid to carbon dioxide (62).

Iron. Exochelins and mycobactins, the iron transportcompounds produced by cultivable mycobacteria, have yetto be detected in M. leprae, probably due to our inability tocultivate the bacillus. However, exochelins isolated from M.neoaurum and from an armadillo-derived mycobacteriumhave been shown to mediate iron uptake in M. leprae,possibly by facilitated diffusion (81, 82). Since iron transportwas found to be the critical factor in the in vitro cultivationof M. paratuberculosis, there is an obvious interest in thepossibility of a similar situation with regard to cultivation ofM. leprae.

Biophysical parameters. As mentioned above, the prefer-ence of M. leprae for temperatures below 370C was firstsuggested by the observation that the organism was found incooler regions of the body. In the mouse footpad, M. lepraeshowed a reduced growth rate when mice were housed in theconditions where the average footpad temperature was 360Cas compared with conditions maintaining footpad tempera-tures of 27 to 30'C (207). These in vivo observations havebeen corroborated by the observation that the optimumtemperature for in vitro oxidation of palmitic acid by M.leprae is 330C (62). This activity ceases within 3 days at 370Cin contrast to a linear response for .1 week at 330C. Inaddition, M. leprae reportedly takes up 3,4-dihydroxyphen-ylalanine optimally at 34WC (110). Consistent with an optimalpH of 5.8 to 6.5 for slowly growing mycobacteria in general(182), M. leprae reportedly has a pH optimum of 5.8 forrespiration (100). As mentioned above, most studies suggestthat M. leprae utilizes free oxygen in its energy-generatingprocesses. A recent study on ATP maintenance and PGL-Isynthesis indicated biophysical optima of 330C and pH 5.6and a reduced but maintained oxygen concentration (62a).

In Vivo Drug TestingPrior to the demonstration in 1960 of limited multiplication

of M. leprae in the footpads of immunologically intact mice(206), therapeutic agents for leprosy were selected solely onthe basis of empirical results in clinical trials. Treatment withchaulmoogra oil, based on Burmese folk medicine, was onlypartially effective and was used until the sulfones were foundto be highly efficacious. Until the development of the mousefootpad model, activity against M. tuberculosis had beenconsidered a valid criterion for initiation of clinical trials inleprosy.

In the immunologically intact mouse, M. leprae can attainonly a density of approximately 106 bacilli per footpad,regardless of the inoculum dosage (206). This, however, issufficient for detection of growth-inhibitory compoundswhen low inocula (5 x 103 to 1 x 104) are used. Bacilli,obtained either from patient biopsies or by animal passage,are inoculated into one or both hind footpads in a volume ofapproximately 0.03 ml. Test compounds are usually pow-dered and mixed in the diet at concentrations ranging from0.0001 to 0.1% (wt/wt). Alternatively, compounds may beadministered by gavage or parenterally, although the repeti-tion of such procedures over the course of several months islaborious. All of the presently used antileprosy drugs can beadministered in the feed, and ultimately any new usefuldrugs should be active orally in humans. Bacillary growth isevaluated by direct microscopy (Ziehl-Neelsen staining)(213) of footpad homogenates following incubation periodsof several months to 1 year.Four techniques have been described and utilized in the

evaluation of potential antileprosy compounds. The earli-

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est such technique used continuous drug administrationthroughout the experiment (211). While a number of com-

pounds are active in this system, bacteriostatic and bacteri-cidal activity cannot be differentiated. This method has theadvantage of requiring the lowest number of mice and theleast labor.

In the kinetic method (208), drug administration is delayeduntil the bacteria have reached the early logarithmic phase ofgrowth, 60 to 70 days postinoculation, and is continued forapproximately 50 to 60 days. Bacillary growth is determinedat selected intervals during drug administration and thereaf-ter. Bacteriostatic drugs cause a growth delay equal to theperiod of drug administration, while bactericidal compoundseffect a growth delay greater than the period of drug admin-istration. In this method, the potential exists for falselyattributing bactericidal activity to drugs with tissue-reposi-tory or bacteriopausal activity.The proportional bactericidal method (40) involves inocu-

lation of groups of mice with 10-fold dilutions of M. lepraeand administration of test compounds for 2 months. Growthof bacilli is determined after 12 months, using most-proba-ble-number calculations. Only bactericidal agents appear

active in this system, while inactive and bacteriostatic agentscannot be differentiated.The rapidity of onset of bactericidal activity can be

determined by serial inoculation of mice with patient skinbiopsy material at time intervals following the initiation ofdrug therapy (210).Only a relatively small number of compounds have dem-

onstrated bactericidal activity in these systems, including allof the clinically useful antileprosy agents: dapsone (weak),rifampin and related compounds, and clofazimine, with onlyrifampin being rapidly bactericidal. Ethionamide and pro-

thionamide (212), thiacetazone, some of the fluoroquino-lones (79, 176, 195), and certain cephalosporins (214) havealso shown bactericidal-like activity. An effective therapeu-tic regimen should include at least one bactericidal drug ifthat drug is to be used intermittently, e.g., once monthly,and therefore such drugs are actively sought.

Drug-resistant leprosy bacilli can be detected by thecontinuous-feed technique (178). High-, medium-, and low-level resistance to dapsone are defined as growth in thepresence of 0.01, 0.001, and 0.0001% drug, respectively (90).Primary resistance is usually low level, and patients harbor-ing such bacilli would normally be expected to respond to

full-dosage dapsone. Testing for secondary dapsone resis-tance enables the physician to determine whether the failureto respond to- therapy is due to true drug resistance of thebacilli or to a lack of compliance by the patient (104).The armadillo (113), athymic (nude) mouse (33), and

neonatally thymectomized Lewis rat (61) all support exten-

sive multiplication of M. leprae. Athymic rodents have beenused as models for treatment of LL, in which initial bacillaryloads may be quite high (118). In addition, their use enableslarger inocula to be used when attempting to detect thepresence of resistant or "persister" bacilli in treated pa-

tients.

In Vitro Drug Testing

Although the development of the mouse model has madepossible the screening of potential antileprosy compoundsprior to clinical evaluation, the long incubation time, highcost, requirement that the test compound have favorablepharmacokinetics in the mice, and requirement for gram

quantities of test compounds have undoubtedly limited the

number of agents which can be evaluated. Therefore, anumber of attempts have been made in the last decade totake advantage of the ability to rapidly quantitate temporalmetabolic activity of M. leprae (as described above), con-sidering the inhibition of such activities to be indicative ofdrug activity.These systems have used intact bacilli suspended in either

simple axenic media or macrophage cell cultures, require 1to 3 weeks of incubation, and rely upon drug action at a locusnot necessarily directly involved in the metabolic activitybeing assayed. Relatively simple systems having bacillaryrequirements of 106 to 107 per assay might be candidates forclinical detection of secondary drug resistance, using skinbiopsy-derived inocula. Detection of primary drug resistancein which only a small percentage of the bacterial populationmay be resistant would not be possible in these systems andwill await the development of a growth-supporting method-ology.

Incorporation of tritiated thymidine into the trichloroace-tic acid-insoluble fraction of phagocytized M. leprae isinhibited by clinical antileprosy agents (162) and has beenused to evaluate a number of phenazines (145). Results inthis system have been found to correlate well with the mousefootpad technique in evaluating secondary drug resistance inclinical isolates (200). A scaled-down version of this tech-nique (144) reduces the requirement for both bacilli andmacrophages, making its clinical use more practical. Whilethymidine is also taken up by extracellular M. leprae, othernucleic acid precursors such as hypoxanthine are taken upmore rapidly and are also sensitive to the action of antile-prosy agents (111). Thus, these compounds may ultimatelyprove to be superior substrates when nucleic acid synthesisis used in a drug-screening system.Our laboratory has recently developed three distinct sys-

tems for large-scale screening of potential antileprosyagents. The incorporation of [14C]palmitic acid into PGL-I ofM. leprae has been shown to be sensitive to clinical antile-prosy drugs as well as a number of other compounds in bothintracellular (186) and extracellular (63) M. leprae. The useof these systems in parallel has the potential for evaluatingthe effect of intracellular residence on bacillary metabolicstability and drug susceptibility. Over 25 antimicrobialagents have been evaluated by their ability to effect anaccelerated rate of ATP decay in extracellular M. lepraesuspended in axenic media (64). Finally, palmitate oxidationto carbon dioxide, measured by radiorespirometry, wasfound to be sensitive to antileprosy agents (62). The use of anautomatic Buddemeyer-type counting system (26) makesthis system the simplest described to date for evaluatingantileprosy agents. The ability to readily detect activity withapproximately 106 bacilli and the precedence for using thisactivity in the rapid drug susceptibility testing of cultivablemycobacteria (134, 193, 235) make this assay a strongcandidate for use in the clinical detection of secondary drugresistance pending further studies.These systems have identified fluoroquinolines, minocy-

cline, and some phenazines as having anti-M. leprae activ-ity, findings consistent with the activity of these compoundsin the mouse footpad (71, 79, 176, 195). Perhaps mostinteresting is the potent activity in these systems of eryth-romycin and two new semisynthetic macrolides (S. G.Franzblau, N. Ramasesh, E. B. Harris, and R. C. Hastings,Program Abstr. 27th Intersci. Conf. Antimicrob. AgentsChemother., abstr. no. 1368, 1987), Roxithromycin (RU 965;Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J.)(30) and Clarithromycin (TE-031, A-56268; Abbott Labora-

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342 HASTINGS ET AL.

tories, North Chicago, Ill.) (59). The semisynthetic macro-lides have demonstrated markedly superior pharmacokineticproperties. Recent (unpublished) studies in our laboratoryhave shown that, when administered in the feed of mice at0.01% (wt/wt), erythromycin ethylsuccinate and Roxi-thromycin are unable to inhibit multiplication of M. leprae inthe footpad, while Clarithromycin fully suppresses growth ofthe bacillus. As thermostable, relatively inexpensive com-pounds acting at a locus not currently exploited in leprosychemotherapy and with proven activity against a variety ofintracellular pathogens, including mycobacteria (13), macro-lides possess many desirable features for inclusion in multi-drug therapy against leprosy.

In summary, the currently uncultivable M. leprae contin-ues to present enormous challenges in all aspects of leprosyresearch. The bacterium has no readily apparent biochemi-cal lesions with regard to energy generation. However,further studies are required to determine the complete ana-bolic requirements and biophysical optima which shouldfacilitate the development of an in vitro growth-supportingmethodology. While well-established in vivo systems forassessing antileprosy drug activity exist, the more recentlydeveloped rapid metabolic assays should soon find accep-tance in large-scale preliminary drug screening and in theclinical detection of secondary drug resistance.

ACKNOWLEDGMENTS

The research reported in this review was supported in part byPublic Health Service grants from the National Institutes of Health(AI22492, AI22442, A122007, and NIAID Interagency AgreementY01-AI-50001) and by grants from the Victor Heiser Foundation,The Baton Rouge Area Foundation, and the Hansen's DiseaseFoundation.We are deeply grateful to Rened Painter, Rosie Hauge, and Penne

Cason for their help in the literature search and in preparation of themanuscript.

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