CLINICAL MICROBIOLOGY REVIEWS, JUly 1988, p. 330-348 Vol. 1, No. 3 0893-8512/88/030330-19$02.00/0 Copyright X 1988, American Society for Microbiology ROBERT C. HASTINGS,* THOMAS P. GILLIS, JAMES L. KRAHENBUHL, AND SCOTT G. FRANZBLAU Laboratory Research Branch, Gillis W. Long Hansen's Disease Center, U.S. Service, Carville, Louisiana 70721

OVERVIEW OF LEPROSY...... 330 Epidemiology ...... 330 Clinical Aspects ...... 331 Indeterminate leprosy ...... 331 LL ...... 331 TT ...... 331 Borderline leprosy ...... 331 Reactions...... 332 Patient Management ...... 332 CELL WALL STRUCTURE AND ASSOCIATED OF M. LEPRAE ...... 333 Cell Wall of M. leprae ...... 333 Protein Antigens of M. leprae ...... 333 Molecular Biology of M. leprae ...... 334 ...... 335 Immunologic Research ...... 335 Goals...... 335 Obstacles to research ...... 335 Major breakthroughs ...... 335 Clinical Immunology ...... 336 test...... 336 and serodiagnosis in leprosy ...... 336 CMI in Leprosy ...... 336 Mechanisms of Specific Anergy in LL...... 337 Genetic control of CMI in leprosy...... 337 Role of the lymphocyte in specific anergy in leprosy ...... 337 The Macrophage in Host Resistance to Leprosy ...... 337 Leprosy Vaccine ...... 338 MICROBIOLOGY ...... 339 Metabolism ...... 339 Catabolic activity ...... 339 Amino acid metabolism ...... 340 Nucleic acid metabolism ...... 340 Lipid metabolism ...... 340 Iron ...... 340 Biophysical parameters...... 340 In Vivo Drug Testing ...... 340 In Vitro Drug Testing ...... 341 ACKNOWLEDGMENTS ...... 342 LITERATURE CITED...... 342

OVERVIEW OF LEPROSY person. There is evidence that transmission of leprosy can occur 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) penetrating ease caused by leprae. wounds, such as thorns or biting arthropods. Which of these mechanisms is most common is unknown. Perhaps the most Epidemiology 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 nasal estimated 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 infection prevalence, the disease is about three times as intense in of wild armadillos (Dasypus novemcinctus) in Louisiana and Africa as it is in Asia. Texas (236, 237). Spontaneous cases of leprosy have also Transmission 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 330 VOL. 1, 1988 LEPROSY 331

least 1961 (232). The relationship between leprosy in wild medians at the wrist, common peroneals at the knee, and armadillos and the disease in humans is not clear. There posterior tibials at the ankle) are affected in bacteriologically have been antecdotal reports of leprosy in humans following progressive disease. There is a characteristic pattern of contact with armadillos (132, 230). On the other hand, most sensory loss due to dermal nerve fiber involvement in human leprosy occurs in areas (e.g., India) in which arma- advanced which affects cooler areas of dillos do not exist. If a relationship exists between leprosy in the body surface (194). Despite the widespread involvement wild armadillos and that in humans, it seems to be quantita- in bacteriologically progressive LL, the patient has remark- tively minor. ably few symptoms other than those caused by the bacterial mass and the accumulations of macrophages required to Clinical Aspects contain them. Histopathologically, lepromatous lesions are characterized by massive collections of macrophages con- Leprosy predominantly affects the skin, peripheral taining large numbers of acid-fast bacilli and often containing nerves, and mucous membranes. The clinical features of the large amounts of lipids which create a foamy appearance on disease can be grouped into three parts depending on mech- hematoxylin and eosin staining. These foamy macrophages anism: (i) features due to bacterial proliferation, (ii) features may occupy 90% of the dermis. The dermal foam cell due to the immunologic responses of the host to the leprosy accumulations are separated from the epidermis by a clear bacilli, and (iii) features due to the peripheral neuritis caused zone. by the first two processes (179). Leprosy always involves TT. Polar (TT) is the localized form of peripheral nerves, almost always involves skin, and fre- the disease. In contrast to uncomplicated LL in which quently involves mucous membranes. The three cardinal bacterial proliferation results in signs and symptoms, much signs of the disease are skin lesion(s), skin anesthesia(s), and of the clinical picture in TT is due to a combination of enlarged peripheral nerve(s). bacterial proliferation and the immunologic responses of the The majority of people effectively resist infection with M. host to the bacilli. Characteristically, TT consists of one or, leprae even in highly endemic areas. It is now thought that as at most, a few well-circumscribed skin lesions with profound many as 200 individuals become infected with M. leprae for anesthesia of the skin lesion itself. There may be an enlarged each overt case which develops and is detected (1). peripheral nerve in the vicinity of the skin lesion(s). Histo- Indeterminate leprosy. For those individuals unable to pathologically, there are very few, if any, demonstrable resist infection with M. leprae, the incubation period varies, acid-fast bacilli. There is a dense, well-organized granuloma but it is usually in the range of 2 to 4 years. The earliest sign consisting of epithelioid cells, surrounded by lymphocytes, of the disease is usually one or a few hypopigmented skin and containing multinucleated Langhans giant cells. The macules with minimal sensory loss confined to the lesion. granuloma involves the basal layer of the epidermis in polar The histopathology of such a lesion may be only that of a TT. nonspecific, chronic dermatitis with scattered round cell Borderline leprosy. Borderline leprosy encompasses those infiltrates of the dermis. The presence of acid-fast bacilli or types of the disease between LL and TT. Mid-borderline is an infiltrate selectively in a nerve bundle in the dermis is rare because it is very unstable. A patient with borderline diagnostic. Indeterminate leprosy has a variable course. In leprosy can develop clinical, bacteriological, and histopatho- approximately three-fourths of such patients, the disease logic features of more tuberculoid disease with time, and this heals spontaneously; some cases remain indeterminate for a is called upgrading. Conversely, developing more leproma- prolonged period of time, and some progress to one of the tous disease with time is called downgrading. As in TT, the established forms of the disease. signs and symptoms of borderline leprosy tend to be due to Established leprosy illustrates a continuous spectrum of a mixture of bacterial proliferation and the immunologic disease from a localized, self-healing, granulomatous disease response of the host to them. Borderline tuberculoid leprosy with very few demonstrable leprosy bacilli to a widespread, (BT) resembles tuberculoid disease except that the number progressive, anergic disease with massive numbers of M. of skin lesions is usually greater, the edges of the skin lesions leprae. To describe the position of a leprosy patient on this are less well defined, there is a tendency for satellite lesions spectrum, most centers use the classification of Ridley and to develop near the edges of the larger lesions, and individual Jopling for research purposes (192). The so-called polar lesions tend to be larger. Damage to peripheral nerves tends types of leprosy are stable clinically. The so-called border- to be more widespread and more severe in BT than in TT. line types of leprosy are characteristically unstable clinically This nerve damage is largely on the basis of the immune and form a continuous spectrum between the two polar response of the host to the bacilli. The histopathology of BT forms. skin lesions resembles that of TT except that the granuloma LL. Polar lepromatous leprosy (LL) is the widespread, does not extend up to involve the basal layer of the epider- anergic form of the disease. Proliferation of M. leprae results mis. The numbers of acid-fast bacilli in lesions vary from in skin lesions of a variety of types ranging from diffuse undetectable to 1 in every 10 to 100 oil immersion micro- generalized skin involvement to nodules (called lepromas) in scopic fields. Borderline lepromatous (BL) leprosy resem- a widespread, usually symmetrical distribution. These skin bles lepromatous disease except that at least some of the lepromas in advanced LL may contain 1010 M. leprae per g skin lesions are selectively anesthetic and show varying of tissue. Characteristically, lepromatous skin lesions in- degrees of distinctness in their borders. Peripheral nerve volve cooler parts of the body surface. This is thought to be trunk involvement (due to the immune response of the host) due to preferential growth of M. leprae at temperatures is more widespread than in LL, but mucous membrane cooler than core body temperature (91). With this tempera- involvement (due to bacterial proliferation) is less than in ture preference of M. leprae, the anterior third of the eye LL. Skin lesions of BL leprosy contain predominantly (but not the warm, posterior two-thirds), the nasal mucosa macrophages with relatively large numbers of lymphocytes. (but not the oral mucosa in a nose breather because the oral The numbers of acid-fast bacilli are usually somewhat less mucosa is not cooled by inspired air), and peripheral nerve than those in an LL lesion of comparable duration, but trunks as they course superficially (the ulnars at the elbow, substantially more than in a BT lesion. BT disease can 332 HASTINGS ET AL. CLIN. MICROBIOL. REV. develop from indeterminate leprosy; it can also develop from Current U.S. recommendations for are 6 months BL disease by upgrading (by increasing the immune re- of rifampin plus dapsone daily and then dapsone alone until sponse of the host). Similarly, BL leprosy can develop from 3 years after disease inactivity for indeterminate and TT indeterminate leprosy or by downgrading from BT. patients. BT patients are treated the same, except dapsone is continued until 5 years beyond disease inactivity. BL and Reactions LL patients are treated with rifampin plus dapsone daily for So-called reactions in leprosy are clinically apparent, at least 3 years, and dapsone is then given alone for the immunologically mediated inflammatory conditions occur- remainder of the patient's life. ring during the course of the disease in about 50% of The rate of clearance of bacilli from a patient on effective patients. These manifestations of leprosy are due to the chemotherapy is a function of the host's immune response. immunologic response of the host to the bacilli. They are It is 0.5 to 1.0 log per year of effective chemotherapy in LL basically of two types. Type 1 or reversal reactions are and increases progressively as the disease is more tubercu- generally agreed to be a result of delayed hypersensitivity loid. The nature of the chemotherapy, e.g., bactericidal or and affect patients with borderline to tuberculoid leprosy. bacteriostatic, does not affect the rates of clearance of They are characterized by edema and erythema of preexist- bacilli. ing lesions and a tendency for the overall disease classifica- In approximately 50% of leprosy patients, management tion to upgrade. Type 2 or erythema nodosum leprosum must also include controlling reactions caused by the host's lesions have long been thought to be manifestations of an immunologic response to the leprosy bacilli. The aim of such Arthus type of hypersensitivity reaction. They are seen in anti-inflammatory drug treatment is to prevent or minimize BL and LL patients and are characterized by the develop- permanent disability and is directed predominantly at con- ment of crops of tender, erythematous skin nodules and trolling peripheral nerve damage and irreversible eye dam- age. Inflammatory eye changes occur predominantly in fever. Both types of reaction can involve peripheral nerves, can with but type 1 reactions more characteristically do so. Type 2 lepromatous patients and frequently be managed the topical corticosteroids. Peripheral nerve damage from reac- reactions can involve any tissue containing antigens of occur but is leprosy bacillus; therefore, lesions of erythema nodosum tional leprosy can throughout the spectrum, leprosum are not confined to but involve the particularly prominent in BT disease. In general terms, acute the skin, can nerve are treated with cortico- eye, joints, nasal mucosa, etc. losses in peripheral function or reactional states can be Type 1 reactions of edema on a BT steroids. More subacute chronic histologically consist with limited courses of doses of or TT background, initially. If the outcome of the type 1 treated relatively high clofazimine, an antileprosy drug with both antibacterial and reaction is upgrading, there may be an early increase in the nodosum number of lymphocytes. In severe type 1 reactions, case- anti-inflammatory properties. Erythema leprosum is usually completely suppressed with thalidomide, a terato- ation necrosis may occur. Type 2 reactions are characterized genic sedative-hypnotic with remarkable immunosuppres- by an influx of neutrophils on a BL or LL background. A in this condition vasculitis involving arterioles is in sive/anti-inflammatory activity (92). or venules demonstrable all there is some of about half of the cases in type 2 reactions. In virtually leprosy patients, degree irreversible peripheral nerve damage caused by bacterial or the of the host to Patient Management proliferation immunologic response these bacilli or both. In some patients, e.g., an indeterminate Successful management of the leprosy patient consists of case with minor sensory impairment confined to a small skin controlling the three mechanisms by which the disease lesion on the trunk, this is inconsequential. In others, causes symptoms, i.e., (i) bacterial proliferation, (ii) the widespread destruction of mixed peripheral nerve trunks can immunologic response of the host to the leprosy bacilli, and result in widespread skin anesthesia and widespread perma- (iii) peripheral neuritis caused by the first two processes. nent muscle paralysis involving the face, hands, and feet. Antibacterial chemotherapy for leprosy, in general, has For patients with muscle paralysis due to irreversible nerve been both adequate and available since the early 1940s (57). damage due to leprosy, there are a variety of reconstructive The problems in antibacterial chemotherapy have come from techniques which can frequently restore reasonable inadequacies in health care delivery systems in leprosy- motor function (17). It should be pointed out that most of the endemic areas, inadequacies in patient compliance for vari- deformities attributed to leprosy are not caused by the ous reasons, and the potential for the development of disease itself. Leprosy removes the sensation of pain. The drug-resistant M. leprae on a large scale. At present, four lack of feedback provided by pain allows the leprosy patient drugs are used widely: dapsone, rifampin, clofazimine, and a to damage and deform himself (as it does any patient lacking thioamide, either ethionamide or prothionamide. A variety the feedback of pain). This secondary damage is the most of drug combinations are used in various parts of the world. disabling, and all of the secondary damage and deformity are Some centers use monotherapy with dapsone. Many centers preventable. A variety of techniques and principles apply to utilize the multidrug regimens recommended by the World wound prevention in anesthetic extremities. Perhaps, the Health Organization (253). For indeterminate, TT, and BT most important principle relates to the care of wounds after leprosy, for example, daily dapsone is recommended plus they are sustained. Lacking sensation, individuals will con- rifampin once monthly. The total duration of treatment tinue to use a wounded and infected extremity and subject it recommended in these so-called paucibacillary cases is 6 to stress in spite of the infection. More than any other cause, months. For mid-borderline, BL, and LL disease, the World this accounts for the destruction of hands and feet in leprosy Health Organization recommends daily dapsone, plus daily which, to a large extent, accounts for the stigma of the clofazimine, with additional clofazimine given once monthly disease. The continued use of an infected extremity leads to together with once-monthly rifampin. The total duration of osteomyelitis and septic destruction of bones, resulting in therapy in these so-called multibacillary cases is at least 2 permanent secondary deformities. These can often be pre- years and preferably until bacilli are no longer demonstrable vented by simply splinting wounded extremities to prevent by the usual slit-skin smear techniques. their use until the wound has healed (17). VOL. 1, 1988 LEPROSY 333

CELL WALL STRUCTURE AND ASSOCIATED found to be active antigenic components of mycobacteria. ANTIGENS OF M. LEPRAE The most notable of the cell wall-associated glycolipid molecules of M. leprae is phenolic glycolipid I (PGL-I) All pathogenic microorganisms have evolved characteris- which has been shown to be species specific and immuno- tics which provide survival advantages in potential hosts. genic during infections with M. leprae (20, 255). Brennan These may be as overt as the production of a potent, and co-workers established the chemical structure and im- tissue-destroying exotoxin or as subtle as modulating the munologic specificity of PGL-I through a series of elegant immune response elicited in the host against the invading studies which have been reviewed in detail elsewhere (18, pathogen. Intracellular bacteria, such as M. leprae, gener- 69). The general structure of PGL-I can be described as a ally fall into the latter category, since their survival depends trisaccharide moiety composed of 3,6-dimethyl-p-D-glucose upon maintaining a stable environment in phagocytic cells (1- 4) 2,3-dimethyl-a-L-rhamnose (1-*2) 3-methyl-a-L- (particularly macrophages) of the infected host. Two major rhamnose linked to a phthiocerol lipid core through a phe- areas of study central to the understanding of host-parasite nolic group. The terminal sugar, 3,6-dimethyl-p-D-glucose, interactions are bacterial metabolism and physiology, in- constitutes the major immunodominant region of the trisac- cluding cell wall structure-function relationships. Also, re- charide, with the penultimate 2,3-dimethyl-a-L-rhamnose lated antigenic analysis of the pathogen is helpful in defining completing the composite native epitope (96). PGL-I appears those structures of the bacterium involved in the immune to be associated with the outer surface of M. Ieprae (258) and response of the host during infection. Since metabolic pro- has been isolated from purified bacteria and M. leprae- cesses of M. leprae are covered below, we summarize here infected tissues in relatively high concentrations (94). Taken the biochemical data related to the major cell wall structures together, these characteristics suggest that PGL-I may rep- of M. leprae, and, when appropriate, indicate those mole- resent the M. leprae "capsule" which could function as a cules which have been shown to induce immune responses in virulence factor providing an important interface between either humans or experimental animals. parasite and host, critical for maintenance of the parasitic relationship. The immunogenicity of PGL-I during M. leprae Cell Wall of M. infection in humans and various other animals has been keprae firmly established, but further studies need to be performed Extensive chemical analysis has shown mycobacterial cell on potentially important immune and nonimmune regulatory walls to be highly complex, lipid-rich, macromolecular struc- functions of PGL-I and related extracellular glycolipids in tures. While many cell components appear to be unique to relation to intracellular parasitism. the mycobacteria, the common bacterial structure, peptido- Recently, Brennan and co-workers isolated and character- glycan, is present in all mycobacteria with only a few minor ized a group of arabinose and mannose-containing phospho- variations. For example, meso-diaminopimelic acid is found rylated lipopolysaccharides from M. leprae (97). Earlier in the tetrapeptide allowing cross-linking of the peptidogly- work on similar carbohydrate-rich fractions from mycobac- can through adjacent meso-diaminopimelic acid residues or teria (204) and M. leprae (141) had established the serologic through meso-diaminopimelic acid-D-alanine residues (9, reactivity of the arabinomannan component but not the 125, 202). In addition, M. leprae peptidoglycan, unlike that cellular location of the component. Detailed studies by in other mycobacteria, contains the unusual substitution of Hunter et al. (97) established that the serologically active glycine for alanine in the tetrapeptide (49-51). While these component, lipoarabinomannan B (LAM-B), is acylated, substitutions may provide some advantage in pathogenicity contains substituents of phosphatidylinositol, and may be for M. leprae (e.g., resistance to degradation), it is unlikely membrane associated. LAM-B has proven to be highly that they affect the adjuvant properties of the muramyl immunogenic in most mycobacterial infections, inducing dipeptide region found in all bacterial peptidoglycans (238), strong humoral responses. Since LAM-B is a including that of M. leprae. common among mycobacteria, it cannot be used Covalently linked to the peptidoglycan is an arabinogalac- effectively for serologic tests to detect early infection with tan-mycolic acid complex, which constitutes approximately M. leprae. However, there are data to suggest that high 70% of the cell wall mass and forms a major amphipathic levels of anti-LAM-B are correlated with high bacterial loads region external to the peptidoglycan. The arabinogalactan in leprosy (129). This may make possible the estimation of polymers consist of linear arrays of D-arabinose-D-galactose bacterial clearance during antimicrobial chemotherapy by with appendages of D-arabinose extending laterally (2, 5, 7). monitoring serum antibody levels to LAM-B. The absence of the more common L-isomer of arabinose in mycobacterial cell walls suggests a heightened resistance to Protein Antigens of M. leprae degradation by host enzymes, possible contributing to per- Immunochemical information combined with detailed sistence of cell wall material in host cells upon death of the fine-structure analyses of M. leprae cell walls and associated mycobacterial cell. Esterified to the terminal arabinose moi- molecules has led to our understanding of a cell wall essen- eties are mycolic acids which contribute significantly to the tially devoid of proteins and polypeptides. Since proteins are hydrophobicity of mycobacterial cell walls (8). In M. leprae major structural and functional molecules in all biological these long-chain fatty acids form two groups, the alpha- systems, the need to elucidate their role in M. leprae has mycolates and beta-mycolates, and can be used for taxo- been of paramount importance. The major impediment to nomic purposes to differentiate M. leprae from other myco- studying the proteins of M. leprae stems from the inability to bacteria (77, 103, 142). culture the bacteria in vitro, limiting workers to bacilli While peptidoglycan, arabinogalactan, and mycolates con- obtained from tissues of experimentally infected mice, rats, tribute significantly to the structural integrity of the cell wall or armadillos. These relatively low numbers of organisms of M. leprae and other mycobacteria, they do not appear to provide only small quantities of purified proteins, severely constitute major immunogens of the bacteria. Instead, other limiting detailed analysis. glycolipids (20, 94), glycopeptidolipids (19, 21), and treha- Prior to the application of monoclonal (MAb) lose containing lipooligosaccharides (97, 98) have been and recombinant deoxyribonucleic acid (DNA) techniques, 334 HASTINGS ET AL. CLIN. MICROBIOL. REV. immunochemical approaches for studying proteins of M. content and by molecular size of the genome indicated that leprae proved tedious and highly complex. Most of the early M. leprae was significantly different from other mycobacte- work was performed by immunoprecipitation of protein and rial species (16, 36, 99). Guanine-plus-cytosine content has other antigens with polyclonal antisera in agarose gels by now been established at approximately 56%, with most other two-dimensional immunoelectrophoresis. A sophisticated mycobacteria exhibiting guanine-plus-cytosine contents of scheme for analysis of these profiles was developed, but >60%. Genome size for M. leprae (2.2 x 109 daltons) little has been learned about the chemical nature of the appears to be smaller than that for most other mycobacteria antigens contributing to the various immunoprecipitates. A (2.8 x 109 to 4.5 x 109 daltons) with the exception of M. detailed description of this approach is beyond the scope of H37Ra, which has been reported to be between this review, but was reviewed recently by Harboe (85). 2.0 x 109 and 2.5 x 109 daltons (16, 36). More recent developments on proteins of M. leprae have Until recent developments in recombinant DNA tech- come from studies with murine MAb as monospecific probes niques, major areas of M. leprae genetics and physiology to identify and characterize the eliciting antigens. The first remained unexplored. Since M. leprae has not been culti- reported murine MAb to M. Ieprae recognized a 65-kilo- vated in vitro, recombinant DNA methods provide a power- dalton (kDa) protein which was subsequently found to be ful, indirect approach for studying genes and gene products associated with the cell wall of the bacteria (73, 74). Four- of the organism. Application of this approach has fostered teen nonoverlapping antigenic epitopes have been defined on research in areas as diverse as analysis of metabolic path- the 65-kDa protein by competitive inhibition studies with ways (36, 102), immunochemical analysis of antigenic pro- murine MAb (25). The majority of these epitopes were teins (25), and taxonomic studies measuring genotypic relat- cross-reactive and present on homologous 65-kDa proteins edness of phenotypically similar bacterial species (J. E. of other mycobacteria, while one epitope, defined by MAb Clark-Curtiss, Abstr. Annu. Meet. Am. Soc. Microbiol. IIIE9 and IVD8, was specific for M. leprae within the 1987, U-172, p. 117). context of the 23 mycobacterial species studied. Serologic The initiation of recombinant DNA studies with M. leprae tests in humans for antibody to this species-specific epitope required two major breakthroughs. The first came with the have revealed antibody primarily in patients with LL, limit- successful propagation of M. leprae in the armadillo (113), ing the usefulness of this antigen for detecting early infection providing large quantities of bacteria from which purified with M. leprae (128). DNA could be obtained ahd analyzed. The second break- The general scheme of multiepitopic proteins of M. leprae, through came with the establishment of M. leprae genomic containing both species-specific and cross-reactive B-cell libraries which could be manipulated in cultivable bacteria, determinants, has been reported by other workers, using such as Escherichia coli, for subsequent analyses of M. murine MAb (101, 119). A collaborative study for the com- leprae genes and gene products. Libraries of this type are parison of 20 MAb to M. leprae was recently reported by the composed of small segments of M. leprae DNA (4 to 20 World Health Organization in which M. leprae-specific and kilobases) present in independent recombinant molecules cross-reactive epitopes were found on proteins with molec- which can be manipulated to meet experimental needs. For ular weights of 12,000, 18,000, 28,000, 36,000, 55,000 to example, a small segment of DNA from M. leprae, localized 65,000, and 200,000 (56). Antigenic analysis with Mab is only in a plasmid or bacteriophage vector, can be directed by the a first approximation of the extent and complexity of M. vector to be transcribed. Subsequent translation of the leprae protein antigens. Moreover, the relevance of these resultant messenger ribonucleic acid should result in an M. proteins to the human immune response during infection leprae protein(s), the gene(s) for which is encoded in the with M. leprae remains unanswered. Obviously, immuno- cloned segment of M. leprae DNA. Should an intact protein genic proteins might provide suitable components for devel- or partial peptide result, the enzymatic or antigenic nature of opment of vaccines against leprosy, but detailed studies are the molecule can be studied. Theoretically, successful appli- needed to define the B- and T-cell stimulatory capacity of cation of this approach should make possible the elucidation these molecules. This work will require relatively large of enzymatic pathways important for M. leprae growth and amounts of purified proteins which are not attainable from metabolism and, thereby, suggest strategies for in vitro native purified bacilli. cultivation studies and indicate potential sites for chemo- Major accomplishments have been realized in the area of therapeutic agents with activity against M. leprae. In addi- mycobacterial cell wall chemistry and structure. These ad- tion, potentially important immunogenic proteins of M. vances have not been accompanied by parallel advances in leprae may be produced and tested as vaccines. our understanding of the biological significance of these Genomic libraries of M. leprae have been established in same components. In particular, our knowledge concerning the cosmid vector pHC79 and the plasmid vector pYA626 by the role of M. leprae cell wall and cell wall-associated the Clark-Curtiss group (36, 102). Initially, clones from the constituents, as they relate to virulence factors of M. leprae, libraries were used to explore the ability of M. leprae genes remains speculative at best. Further research aimed at to complement known enzymatic pathways in auxotrophic defining structure-function relationships, using highly de- mutants of E. coli. Complementation with M. leprae DNA fined and purified components of M. leprae and other was observed for the gltA (citrate synthase) and aroB mycobacteria, should lead to a more complete understanding (dehydroquinate synthetase) mutations in E. coli (102). Com- of the pathogenic mechanisms of this group of highly suc- plementation of three gltA mutants was found only when M. cessful pathogens. leprae DNA was linked to a strong promoter from Strepto- coccus mutans, suggesting that mycobacterial promoters are utilized if at all in E. The Molecular of M. only weekly coli. complementing Biology leprae DNA fragment specified a 46-kDa polypeptide and probably Analysis of genome size and guanine-plus-cytosine con- represented the citrate synthase gene of M. leprae. Other M. tent are two important characteristics of bacteria used in leprae DNA sequences capable of complementation of E. comparing relatedness among bacterial species. Molecular coli mutants have not been reported, reflecting possible analysis of purified M. leprae DNA by guanine-plus-cytosine inherent difficulties with this approach due to basic genetic VOL. 1, 1988 LEPROSY 335 and physiologic differences between E. coli and M. lepare. A likewise, a spectrum in the host humoral or CMI response to similar approach using other host bacteria, such as Bacillus infection. Individuals with TT manifest a strong delayed- subtilis and appropriate cloning vectors, could be tried to type hypersensitivity (DTH) to M. leprae antigens but pro- test this hypothesis. Unfortunately, all other potential hosts duce relatively low levels of antibody. At the opposite end of and related cloning vectors are less well characterized the clinical spectrum (LL), there is a potent humoral anti- compared with E. coli, limiting their usefulness in the body response but a progressive anergy in CMI to M. leprae immediate future. Nevertheless, this general approach re- antigens. mains a potentially powerful tool for studying basic physio- logical mechanisms operative in a noncultivable microorgan- Immunologic Research ism, such as M. leprae. The other significant work in this area was the construc- Goals. As with the study of any infectious disease, the tion of a genomic library in bacteriophage X gtll by Young's general goals of an immunologist working in the leprosy field group, which was designed for regulated expression of M. are clearly defined. As a practical measure, tests should be leprae proteins in E. coli (260). So far, five protein antigens developed to aid in the diagnosis of subclinical disease. of M. leprae have been expressed in E. coli and analyzed in These tests should also find application in the monitoring of detail (140, 260). Cloned segments of M. leprae DNA from the course of control measures in endemic areas and in the X gtll library have provided DNA for sequencing of at epidemiological studies. More basic research can be focused least two M. leprae proteins (18 and 65 kDa), both of which on the immunological mechanisms that contribute to the have been shown to be immunogenic subsequent to vacci- protective as well as pathologic aspects of host responses to nation with M. leprae (158, 159). Sequence data of this type leprosy. Ultimately, exploitation of fundamental knowledge for the 65-kDa proteins of M. leprae, M. bovis BCG, and M. of the immune response to leprosy should allow develop- tuberculosis have provided detailed amino acid sequence ment of an effective vaccine. information concerning cross-reactive and species-specific Obstacles to research. Although the leprosy bacillus was antibody-reactive epitopes (219). Similar work with T-cell identified as a human bacterial pathogen in 1874, until clones should provide information related to T-cell recogni- recently workers in the leprosy field have made few contri- tion regions on these and other proteins, allowing for de- butions to the field of immunology and have adopted little in tailed immunochemical analysis of M. leprae proteins. the way of immunological know-how to their studies. This Moreover, DNA-derived peptide sequences and subsequent lack of progress has not been due to a lack of effort, synthesis of these moieties will provide reagents needed to expertise, or dedication by researchers. At virtually every analyze the complex interactions of antigenic peptides and turn in investigating this disease seemingly insurmountable class II major histocompatibility complex molecules on obstacles arose that stymied progress. Even 115 years after antigen-presenting cells. Studies on this type may help its discovery, M. leprae remains uncultivable. Until re- elucidate the molecular aspects of triggering M. leprae- cently, human biopsies served as the sole source of leprosy reactive T cells, important in mediating protective cell- bacilli. These crude preparations yielded inadequate num- mediated immunity (CMI) against M. leprae. bers of organisms, and the lack of reliable methods for Finally, application of molecular techniques for analysis of quantitating viable organisms prevented standardization of genomic DNA ultimately may provide new methods for different preparations. taxonomic purposes and new insight into our thinking about Major breakthroughs. Three major discoveries in the past M. leprae and related bacterial species. Traditionally, genus, 25 years have allowed leprosy workers in general and species, and strain differentiation of microorganisms has immunologists in particular to put leprosy research in pace been based on phenotypic differences observed between with other infectious diseases. (i) The mouse footpad model bacteria which ultimately are the result of, and consistent for M. leprae infection, developed by Shepard in 1960 (206) with, the genetic constitution of those organisms. However, offered a means of quantitating the growth of the leprosy it has been estimated that only 20% of the genomic capability bacillus. Although only a relatively few organisms could be of a microorganism is represented when applying detailed obtained, this model allowed evaluation of chemotherapeu- phenotypic analyses (22). Accordingly, major areas of the tic regimens (209) and serves as the basic model for testing genome, possibly encoding important functional or regula- the efficacy of potential leprosy vaccine preparations. The tory genes not demonstrable as quantifiable phenotypic use of athymic (nu/nu) mice (33, 39, 117) and rats (60) defined characteristics, may go undetected. the importance of CMI in host resistance to leprosy and Obviously, new approaches are needed for studying larger provided models that more closely mimic LL. (ii) In 1971, areas of all bacterial genomes and particularly noncultivable Kirchheimer and Storrs (113) identified the nine-banded bacteria, such as M. leprae. Analysis of DNA restriction armadillo as a susceptible host for the growth of M. leprae. fragments after restriction endonuclease digestion of ge- Armadillos in Louisiana and Texas have subsequently been nomic DNA (38, 175) and the related approach of restriction shown to harbor a naturally acquired M. leprae infection fragment length polymorphism analysis hold promise in this (226, 231, 237). The armadillo model provided for the first area. As we obtain a more complete understanding of the M. time large quantities of leprosy bacilli for immunologic, leprae genome and its related functional capacity, it is immunoprophylactic, immunotherapeutic, and immuno- anticipated that most areas of study related to the leprosy chemical studies. (iii) Using armadillo-derived M. leprae, bacillus will be enriched and, thereby, advance our under- Brennan and his colleagues in Colorado (18, 20, 94, 96, 98) standing of M. leprae and its basis for pathogenicity. were able to identify in preparations of the leprosy bacillus a phenolic glycolipid that possesses a unique trisaccharide IMMUNOLOGY structure that is immunologically specific for M. leprae. Immunochemical studies and electron microscopy indicate Active infection with M. leprae is characterized by a that PGL-I is located on the surface of the organism and that broad spectrum of host response, with great variability in it may accumulate in the tissues of the armadillo in quantities histopathology and clinical course of infection. There is, equal to half the total weight of the leprosy bacilli present 336 HASTINGS ET AL. CLIN. MICROBIOL. REV.

(94, 96). The "foam" seen in heavily infected macrophages moiety specific for M. leprae, and linked to a protein carrier characteristic of the lepromatous granuloma is thought to (31, 32, 34, 35, 65, 66), have found widespread application. contain PGL-I (67, 84). The ability to produce purified Anti-PGL-I antibody is found in virtually all LL patients (23, preparations of specific M. leprae antigens allowed immu- 35, 255). However, although the number of false-positive nologists to use state-of-the-art techniques to further explore responses is very low, the response of TT patients and the immunology of leprosy. contacts is also disappointingly low, thus limiting the use of this test for epidemiological purposes or for identification of Clinical Immunology patients with subclinical infection. There is some evidence that there is a correlation between decreased bacillary load Lepromin test. The practical clinical immunology of lep- and anti-PGL-I antibody levels after chemotherapy in LL rosy is at present based almost entirely on the reactivity to (129). Because PGL-I is a major component of M. leprae and intradermal skin tests with lepromin, a heat-killed suspen- is present in abundance in infected tissue and the blood of sion of M. leprae obtained originally from homogenized LL patients, detection of this antigen by serological means human leproma (48, 143) but now prepared from infected could identify infected individuals (254), although this ap- armadillo tissue. Reactivity to lepromin has no real diagnos- proach may not be feasible at the TT end of the spectrum or tic value but does establish the immune status of the indi- in subclinical infections. vidual to M. leprae and thus is of prognostic value (189). Sera from individuals with leprosy, including TT patients, Typically, a positive reaction is biphasic. An early (24 to react strongly with the highly immunogenic LAM isolated 48-h) (58) is a DTH reaction (probably to from the cell wall of M. Ieprae (130). In general, these soluble protein antigens in the preparation) and occurs in TT antibodies cross-react with LAM from M. tuberculosis, patients as well as contacts or healthy individuals sensitized although specific epitopes for M. leprae LAM are demon- to M. leprae or cross-reacting antigens from other mycobac- strable with MAb (70). Distinct chemical features for arabi- teria. A major goal of recombinant DNA research in leprosy nogalactan and peptidoglycan of M. leprae cell wall have is the production of M. leprae-specific protein antigens that been demonstrated (70, 97). Further work is necessary to can serve as a reagent for detection of specific DTH in determine whether a routine assay for specific M. leprae leprosy. antibodies to these cell wall subunits can be developed. The late (Mitsuda) reaction to lepromin is measured at 21 MAb technology has focused attention on at least five days and reflects the induction of acquired CMI to M. major protein antigens of M. leprae: those of 65, 36, 28, 18, leprae, manifested by formation of an organized epithelioid and 12 kDa. Of these, the 65-kDa antigen is the most widely cell granuloma (143, 189). Positive Mitsuda reactions are studied, including complete expression in the X gtll recom- seen in the vast majority of contacts and unexposed individ- binant DNA system (259, 260). The 65-kDa protein has been uals as well as in persons with TT. Weakly positive reactions shown to possess epitopes that cross-react with those of aid in classification of borderline disease. In LL, no response other mycobacteria (73), but M. leprae-specific epitopes is seen, indicating the absence of CMI to M. leprae. Even have been found as well (25, 74). With MAb, M. leprae- after many years of chemotherapy, the lepromin test remains specific epitopes have also been found on the 36- and 12-kDa negative in LL (156, 232). proteins (101, 115, 119). These specific MAb lend themselves Humoral immunity and serodiagnosis in leprosy. Because to use in competitive inhibition assays for the serodiagnosis of the intracellular nature of M. leprae, humoral antibody is of leprosy (114, 225) and may detect a specific antibody probably not important in resistance but could play a role in response in TT patients. the pathogenesis of erythema nodosum leprosum reactions by formation of antigen-antibody complexes (14). LL has long been associated with a state of hypergammaglobuline- CMI in Leprosy mia (27, 205), and there is, in general, an inverse correlation between the anti-M. leprae antibody titer of a patient and the Early pathologists recognized the importance of CMI, potency of his CMI response to the leprosy bacillus, i.e., a manifested by the epithelioid cell granuloma, in conferring direct correlation between antibody level and bacillary load. resistance in TT. In addition, DTH appears to underlie much Demonstration of antibody to specific M. leprae antigens is of the host tissue damage observed in reactional episodes in not as straightforward. Prior to the availability of armadillo- BT disease (170, 252). As discussed above, the lepromin test derived M. leprae, organisms extracted from human tissue is uniformly negative in LL patients and remains negative were used in indirect fluorescent antibody studies, radioim- even after years of successful chemotherapy. An in vitro munoassays, and crossed immunoelectrophoresis assays (1, manifestation of anergy in LL is demonstrated by the lack of 37, 86, 87), but M. leprae-specific antigens could not be lymphocyte blast transformation in the presence of M. demonstrated. leprae antigen (75, 160). Early indications that LL was The discovery that PGL-I is biochemically unique (94, 95) associated with a general suppression of CMI (83, 188, 205) and immunologically specific (20, 177) for M. leprae reawak- have not held up. The T4/T8 (helper-inducer/suppressor- ened interest in the serodiagnosis of subclinical leprosy. A cytotoxic) ratio of T lymphocytes in the peripheral blood of detailed summary of leprosy serodiagnosis can be found in LL patients ranges near the normal value of 2:1 (187). In LL, the recent review by Gaylord and Brennan (69). The major- there is no evidence for an increased incidence of cancer or ity of human antibody to PGL-I is of the immunoglobulin M infection with the opportunistic pathogens commonly asso- subclass (35, 256), and virtually all MAb generated against ciated with the immunocompromised host. For example, the PGL-I in mice are immunoglobulin M (258). Enzyme-linked clinical course of pulmonary tuberculosis in an LL patient is immunosorbent assay technology that uses the highly hydro- identical to that in an otherwise healthy individual. The phobic native PGL-I (35) has been worked out, and attempts absence of specific CMI to antigens of M. leprae in LL has have been made to standardize (177, 198) and simplify the attracted widespread attention by immunologists interested procedures (257). Alternatively, hydrophilic synthetic neo- in exploring immunoregulatory mechanisms in a model of glycoprotein antigens bearing the immunologically distinct nonfatal immunodeficiency disease in humans. VOL. 1, 1988 LEPROSY 337

Mechanisms of Specific Anergy in LL mitogenicity might also suggest that more vital T4 lympho- cyte-antigen interactions could also be blocked, a concept Several hypotheses are under investigation to explain the not consistent with CMI anergy in LL being specific for M. specific anergy in CMI to M. leprae antigens in LL, includ- leprae. However, in the LL lesion, the random distribution ing a genetic predisposition for LL, clonal deletion of M. of T4 and T8 cells at a 0.5:1 ratio (148) may favor local leprae-specific T cells, and specific suppression of T-cell suppression of helper cell function and not result in a function. generalized immunocompromised state. Recently, clones of Genetic control of CMI in leprosy. Studies of leprosy type T8 lymphocytes isolated from LL lesions were shown to in homozygous twins showed neither the random concor- mediate major histocompatibility complex class II antigen- dance expected if there was no predisposition for type of restricted suppression of T4 clones after interacting specifi- disease nor complete concordance for disease at the TT or cally with lepromin (149, 169). LL ends of the spectrum, expected if precise genetic control The suppressor cell hypothesis in LL is controversial. was operating (29, 154). Other attempts have been made to Nath (161) concluded that T-cell suppression in leprosy explore genetic restriction in leprosy, and evidence is focus- regulates the CMI response in the TT but not LL stage. ing on the importance of the major histocompatibility com- Moreover, although the suppressor cell hypothesis may be plex class II genes. Individuals with the HLA-DR2 or linked to the specific anergy characteristic of LL, it does not HLA-DR3 haplotype or both may be predisposed to the TT as yet address the actual mechanisms whereby sensitized form of leprosy (173, 233, 234), while the incidence of helper T cells fail to respond to antigen. Nor does this DR2-DQW1 may be increased in individuals with LL (201). hypothesis address the argument of how and why only Genetic predisposition and as yet unclarified exogenous certain individuals develop a T suppressor cell response factors such as environment, mode of transmission, and upon challenge with the leprosy bacillus and subsequently infecting dose should be important considerations in any progress to the lepromatous form of the disease. widespread vaccine trials for prevention of leprosy. Other groups have developed T-cell clones from the Role of the lymphocyte in specific anergy in leprosy. At the peripheral blood lymphocytes of leprosy patients by using lymphocyte level, the presence of T-helper cells specific for autologous antigen-presenting cells to define their reactivity antigens of the leprosy bacillus is a key characteristic of the to M. leprae antigen. Mostly, antigen-reactive clones, in- TT end of the clinical spectrum of leprosy. The reason for cluding clones from M. Ieprae-vaccinated volunteers, have their absence or lack of function in LL remains unclear. been T4 in phenotype (158), some of which proliferated in Haregewoin et al. (88, 89) appeared to restore T-helper cell response to the 18-kDa M. leprae recombinant protein (260). response to M. leprae in vitro by supplementing the cell Some T4 clones from a TT patient were shown to react culture media with the T-cell-amplifying cytokine interleu- specifically with M. leprae, while others responded to other kin-2 (IL-2). However, others have been unable to confirm mycobacterial antigens (55). As an alternative to autologous these findings (105, 107, 163) in totally anergic LL patients. blood monocytes as antigen-presenting cells, some workers Mohagheghpour et al. (153) suggested that lack of specific used Epstein-Barr virus-transformed autologous B cells to T-cell response in LL may be due to absence of the IL-2 support T-cell clones from TT and BL patients (80, 172, 174). receptor on these cells. On the other hand, Modlin et al. Clones from the TT patient were all T4, and, although some (146, 147) have shown T cells bearing IL-2 receptors in the cross-reacted with other mycobacteria, others appeared to LL lesions themselves, although there were far fewer IL-2- proliferate specifically in the presence of M. leprae 36-kDa secreting T cells in LL compared with TT lesions. Mohag- protein (174). Interestingly, clones of the T8 suppressor heghpour et al. (152) have recently reported that M. leprae- phenotype from a BL patient suppressed autologous T4 specific T4 cells are present and respond to M. Ieprae lymphocyte response to M. leprae (172). antigen, but only after a 48-h "rest" in culture. Although specific reactivity in TT or specific anergy in LL The Macrophage in Host Resistance to Leprosy is routinely demonstrated in vitro with peripheral blood lymphocytes, the reactivity of the cells in the leprosy le- Macrophages participate in the immune response in both sions, not the blood, is probably more relevant to the CMI afferent and efferent roles. In their afferent role they assist in response of the host to M. leprae. In a series of studies, regulation of the immune response (4, 131) by interacting Modlin et al. have investigated the cellular makeup of the TT with T cells in the presentation of antigens or by secretion of and LL lesion, using immunopathological techniques (149- soluble mediators. Macrophages have been shown to sup- 151). Briefly, their findings indicate that, in contrast to the press T-cell responses in leprosy (164, 196) and may be near-normal (2:1) T4/T8 ratio in the blood in LL, the relative defective in their ability to present M. Ieprae antigens to number of helper T cells is markedly lower (0.5:1) in the sensitized T cells (93). Studies have also revealed (191, 239) lepromatous lesions. The T4/T8 ratio remains approximately that, in LL, macrophages may be defective in the production 2:1 in the lesions in TT leprosy, but more importantly, the of IL-1, the cytokine that can amplify the production of IL-2 cells are arranged in a distinct architecture within the lesion: by T cells. T4 cells in the centers of the epithelioid granulomas and T8 Regardless of the lack of a clear understanding of the suppressor cells in the margins. underlying mechanism of defective CMI in LL and the In a series of studies by Mehra et al. (136, 138, 139), M. possible role of macrophages in anergy, the failure of the leprae-specific suppressor T cells were demonstrated in the macrophage to kill or inhibit M. leprae is a conspicuous peripheral blood of LL patients. Immunologically specific characteristic of the lepromatous form of the disease. Its triggering of the suppressor cells was shown to be elicited by inability to cope with M. leprae is an issue central to the unique PGL-I antigen of M. leprae (136). The demon- understanding the mechanisms of host resistance to the strated effects of these suppresor cells in vitro involve leprosy bacillus. nonspecific suppression of helper T-cell function as mea- Macrophages, activated by lymphokines (gamma inter- sured by a concanavalin A mitogenic response. Suppression feron [IFN--y], especially) released from sensitized helper T of a CMI response as potent as that of concanavalin A cells responding specifically to antigen, are thought to play a 338 HASTINGS ET AL. CLIN. MICROBIOL. REV. major role in resistance to a wide variety of obligate and chemotaxis (28) also produced high levels of PGE2 in vitro facultative intracellular pathogens (122, 166, 167). The (Sibley and Krahenbuhl, unpublished results). events leading to formation of an epithelioid cell granuloma Although indomethacin blocked production of PGE2 and and the subsequent limitation and elimination of the local reversed the defective response to IFN-y in our in vitro bacilli in TT leprosy or a positive lepromin reaction are likely studies (Sibley and Krahenbuhl, in press), it did not reverse the consequences of such a scenario. Activated macro- the defective response of footpad granuloma macrophages phages are distinct from normal macrophages by numerous (Sibley and Krahenbuhl, in press), suggesting that prosta- morphological and metabolic criteria (108, 171), including noid production is not the sole mechanism inhibiting macro- enhanced production of oxidative metabolites such as super- phage function in vivo in the lepromatous lesion. Of interest, oxide and hydrogen peroxide which may underlie their a mycobacterial component, LAM, not only blocks T-cell enhanced microbicidal capacity (167, 168). proliferation (105) but also induces a refractory response to A fundamental problem plaguing studies of the role of the IFN--y in vitro in mouse macrophages (221) as well as in macrophage in resistance to leprosy is the inability to human monocyte-derived macrophages (Sibley and Krahen- routinely quantitate the viability of the uncultivable leprosy buhl, submitted for publication). Defective macrophage re- bacillus. Thus, macrophage effector function in leprosy has sponse induced by LAM was not associated with PGE2 been addressed indirectly. Although there is some evidence production. These findings again emphasize the probability that macrophages from LL patients are deficient in their that immunological events occurring in the lepromatous ability to digest M. leprae (12), not all workers have found lesion do not necessarily represent similar events in other such deficiencies (52, 197). Our own studies showed that anatomical compartments. mice with potent populations of activated macrophages were Two recent reports stemming from clinical trials of LL markedly resistant to footpad infection with M. Ieprae (120, patients treated with IFN--y address local changes at the site 121). In other indirect studies, oxidative metabolism of of injection (106, 165). Local changes included induration patient phagocytes was tested and found to be normal or due to accumulation of T4 lymphocytes and monocytes and above normal (68, 76, 133). On the other hand, Nathan et al. enhanced keratinocyte proliferation and la expression, but (165) and Kaplan et al. (106) showed not only that monocytes there was no obvious evidence of clearance of bacilli. In from LL patients were deficient in hydrogen peroxide pro- contrast, the reports of Convit and his associates (41, 44) duction (and presumably microbicidal capacity as well), but suggest that local injection of lepromatous lesions with BCG also that this defect was reversed by treatment with IFN-y. results in clearance of leprosy bacilli. These findings serve, These findings are not consistent with studies that showed in part, as the basis for including BCG with a killed M. leprae no defect in the innate microbicidal capacity towards other vaccine to exploit this immunotherapeutic effect (42). pathogens of macrophages from LL patients (52). We have Collectively, our in vivo and in vitro studies suggest that recently demonstrated that activated mouse macrophages any clearance of bacilli from lepromatous lesions as a have a deleterious, probably microbicidal effect on M. Ieprae consequence of local immunotherapeutic measures (41, 44, as shown by electron microscopy (220) and marked inhibi- 106, 165) or chemotherapy (190) likely depends on the influx tion of adenosine 5'-triphosphate (ATP) content and synthe- of new mononuclear phagocytes into the local lesion, rather sis of PGL-I (186, 224). than activation of the resident lepromatous macrophages. Because of the T-cell anergy associated with LL, the Even then, these newly arrived macrophages encounter signals are not produced that would activate the microbicidal local conditions that rapidly restrict their responsiveness to capacity of the infected macrophages in the lesion. Regard- IFN--y. These findings emphasize that defects in CMI in LL less, our recent studies (222, 223) show that the M. leprae- likely extend beyond the level of the to include gorged macrophages from the footpad lesions of infected localized restriction of macrophage-afferent and -efferent nude (nulnu) mice, a model of experimental LL (33), are functions influenced by lymphokines. refractory to IFN--y in vitro as measured by four parameters of activation: microbicidal capacity, cytotoxicity for neo- Leprosy Vaccine plastic cells, superoxide anion production, and expression of major histocompatibility complex class II (Ia) antigen. Peri- Perhaps the main goal of immunological investigations in toneal macrophages from these same mice were fully respon- leprosy is the development of a safe, effective, low-cost sive to IFN--y. vaccine that can be used in conjunction with other control Interestingly, a similar refractory response to IFN--y could measures in endemic areas, resulting ultimately in the erad- be induced in vitro in mouse peritoneal macrophages heavily ication of leprosy. Vaccine research must, of course, take infected with live M. leprae (L. D. Sibley and J. L. Krahen- into consideration progress made in defining environmental buhl, Infect. Immun., in press). The development of defec- and genetic factors that determine susceptibility to leprosy. tive activation was time and dose related, requiring 48 to 72 Two broad approaches are currently being explored, and a h of incubation with large numbers of bacilli, and was clearly third approach involves the genetic engineering of a leprosy paralleled by an increase in macrophage production of vaccine. prostaglandin E2 (PGE2). PGE2 is a potent immune modula- The first approach depends on cross-reactivity between tor that suppresses macrophage Ia induction by lymphokines M. leprae and other mycobacteria. BCG seems to be the (227), macrophage tumoricidal capacity (228), and lympho- only other mycobacterium that affords protection against M. cyte blast transformation (54). Immunosuppressive levels of leprae challenge in mice (215). In large field trials, BCG PGE2 have been demonstrated to be produced in response to vaccination protected against leprosy in Uganda (24) but was mycobacterial products (116) or by infection of mice with M. much less effective in Burma (11). Other cross-reactive intracellulare (53). In support of this hypothesis, Ridel et al. mycobacteria are being actively investigated as leprosy (191) reported elevated production of PGE2 by human mono- vaccine candidates, including the ICRC bacillus (47) and cytes from LL patients. Moreover, we have recently dem- "Mycobacterium w." (229). onstrated that skin biopsy tissue from LL patients that The availability of large amounts of armadillo-derived, produced high levels of a 235-kDa inhibitor of monocyte purified M. leprae provides the basis for using killed M. VOL. 1, 1988 LEPROSY 339

TABLE 1. Sources of M. leprae

Host Tissue Incubationtime (mo) Yielda Disadvantages Armadillo Liver, spleen 18 101o-1013 Expensive; requires specialized facility; highly variable yield; unused tissue must be frozen; often contaminated with other slow-growing mycobacteria (180, 181) Athymic "nude" mouse Footpad 12-18 101o-1011 Requires special isolator, sterile water, feed, and bedding Human Skin nodule 107-109 Often contaminated with other skin-borne bacteria; low viability Mouse Footpad 6-12 106 Yield too low for physiological studies a From Wheeler (242). leprae as a vaccine preparation. Killed M. leprae afford found in animal tissue. The need to purify the bacterial protection against experimental challenge with viable bacilli suspensions to rid them of contaminating host tissue debris in the mouse (216, 217) and armadillo (112) and induction of further reduces bacillary yield. DTH in guinea pigs (135). Human volunteers vaccinated Another major problem in studying the metabolism of with killed M. leprae have been shown to develop a DTH host-derived microorganisms is that of differentiating be- response (72). tween host- and bacillary-derived activities. Since host- A novel approach to vaccination combines killed M. derived enzymes may be adsorbed to the surface of intracel- leprae with viable BCG and seems to exploit specific, but lular bacilli during tissue homogenization, mere removal of undefined, immunity to M. Ieprae, with relevant cross-reac- microscopically detectable tissue debris is not sufficient to tive immunity to BCG. Convit and his colleagues devised establish bacterial activity. Surface treatments, usually in- this immunoprophylactic-immunotherapeutic approach volving washing with NaOH, have been shown to abolish based on their observations that local injection of this host-derived activities but may also destroy bacterial surface mixture into lesions of BL and LL patients induced conver- enzymes (249). Host and bacterial activities can often be sion to positive lepromin skin test reactivity, clearance of differentiated via electrophoretic migration, substrate spec- bacilli, histopathological upgrading, and clinical improve- ificity, serology, or the use of differential enzyme inhibitors ment and decreased suppressor cell activity in some patients (242). (42, 43, 137). Long-term trials of the BCG-M. leprae vaccine Considering its uniqueness at a molecular level (guanine- are under way or are being planned in Africa, South Amer- plus-cytosine percentage and DNA homology) (6, 36, 45), ica, and southern India. one might expect to find a number of unusual or unique Finally, propagation of M. leprae genes in E. coli (260) metabolic activities in M. leprae compared with other my- allows potentially unlimited production of M. leprae proteins cobacteria. Unfortunately, some comparative studies have for immunologic analysis and for use as diagnostic skin test used cultivable mycobacteria which were propagated in vitro reagents. The contribution of any of the M. leprae-defined and thus have compared an in vivo-grown organism (M. proteins to a protective response against the invading bacilli leprae) with in vitro-grown bacilli. Since phenotypic expres- is difficult to assess. However, recombinant proteins of M. sion may be strongly influenced by the growth environment leprae have been used to provide evidence that human T-cell (203), the results of such experiments do not necessarily clones recognize the 18-kDa (158) and 36-kDa (174) proteins clarify the phenotypic relationship between M. leprae and of M. leprae. This suggests that both proteins may be other mycobacteria. relevant to protection against leprosy. Catabolic activity. M. leprae appears to be metabolically However, even highly M. leprae-specific pure proteins or competent with regard to catabolic pathways for energy peptides will probably require incorporation in an adjuvant generation. Glucose is actively transported (109) and, as in to induce long-term CMI and protection in humans. Few, if most mycobacteria, is oxidized to carbon dioxide (240, 241), any, potential adjuvants suitable for human use are avail- although rather slowly. Also, as with other mycobacteria, able. Ingenious plans are under way (15) to engineer a safe, oxidation appears to occur primarily through the Embden- potent leprosy vaccine consisting of highly immunogenic Meyerhof pathway and, to a lesser extent, via the hexose BCG containing the appropriate genes of M. leprae, which monophosphate pathway (240). Strongly supporting the ex- may allow these organisms to express those M. leprae istence of these pathways was Wheeler's demonstration of antigens associated with a protective immune response. the component enzyme activities (241), although two Emb- den-Meyerhof pathway enzymes could not be detected in MICROBIOLOGY another laboratory (155). Also, as with other mycobacteria, glycerol is oxidized through these pathways. What appears Metabolism unique to M. leprae is its extremely high level of 6-phospho- gluconate dehydrogenase production (241), over 100 times The relative difficulty in studying metabolism in a bacte- the level of the first hexose monophosphate pathway en- rium which can be propagated only in vivo cannot be zyme, glucose 6-phosphate dehydrogenase. In addition, the overemphasized. The sources of M. leprae listed in Table 1 ability of the bacillus to oxidize 6-phosphogluconate, but not all have drawbacks for research. In practice, the armadillo gluconate, suggests that M. leprae may actively scavenge has served as the source of bacilli for most physiological 6-phosphogluconate in vivo for use as an energy source studies. However, there are only a few armadillo farms in (245). the world, which severely limits the number of available Pyruvate produced via glycolosis may be converted to bacilli. This problem is further exacerbated by the extremely lactate by lactate dehydrogenase (250) or cycled through the slow growth rate (generation time, approximately 12 days) tricarboxylic acid cycle (243). The existence of an intact and apparently low overall viability of M. leprae populations tricarboxylic acid cycle in M. leprae was suggested by the 340 HASTINGS ET AL. CLIN. MICROBIOL. REV. ability of whole cells to oxidize pyruvate, citrate (243), (46). The extracellular bacilli can also rapidly oxidize palmi- malate (248), and succinate (240) to carbon dioxide, stimu- tic acid to carbon dioxide (62). lation of pyruvate oxidation by citrate (243), and finally, Iron. Exochelins and mycobactins, the iron transport demonstration of a full complement of tricarboxylic acid compounds produced by cultivable mycobacteria, have yet cycle enzymes (243). Of interest was the apparent proteo- to be detected in M. leprae, probably due to our inability to lytic inactivation of fumarase in the M. leprae crude ex- cultivate the bacillus. However, exochelins isolated from M. tracts, an activity which could play a regulatory role. The neoaurum and from an armadillo-derived mycobacterium presence of the glyoxylate cycle enzymes also suggests the have been shown to mediate iron uptake in M. leprae, capacity to regulate tricarboxylic acid cycle activity (245). possibly by facilitated diffusion (81, 82). Since iron transport M. leprae appears to possess an electron transport system was found to be the critical factor in the in vitro cultivation as suggested by reduced nicotinamide adenine dinucleotide of M. , there is an obvious interest in the oxidase activity (100) and the presence of cytochromes (100, possibility of a similar situation with regard to cultivation of 155). In addition, it is conceivable that diphenoloxidase may M. leprae. participate in electron transport by formation of a quinone Biophysical parameters. As mentioned above, the prefer- which can undergo reversible oxidation-reduction (155, 185). ence of M. leprae for temperatures below 370C was first Unlike chlamydiae, M. leprae is capable of generating its suggested by the observation that the organism was found in own ATP, as demonstrated by the presence of adenylate cooler regions of the body. In the mouse footpad, M. leprae kinase (127), incorporation of exogenous phosphate into showed a reduced growth rate when mice were housed in the ATP (126), and short-term 2,4-dinitrophenol-sensitive in- conditions where the average footpad temperature was 360C creases in ATP pools immediately following harvest from as compared with conditions maintaining footpad tempera- armadillo tissue (124). In addition, extended maintenance of tures of 27 to 30'C (207). These in vivo observations have intracellular pools of such a labile compound under appro- been corroborated by the observation that the optimum priate conditions (62a) indirectly suggests that some temperature for in vitro oxidation of palmitic acid by M. active leprae is 330C (62). This activity ceases within 3 days at 370C synthesis is occurring. in contrast to a linear response for .1 week at 330C. In M. leprae contains a manganese-dependent superoxide addition, M. leprae reportedly takes up 3,4-dihydroxyphen- dismutase (123, 244, 251) but appears to be devoid of ylalanine optimally at 34WC (110). Consistent with an optimal catalase (155, 251), thus suggesting a microaerophilic dispo- pH of 5.8 to 6.5 for slowly growing mycobacteria in general sition. This is supported by optimal maintenance of intracel- (182), M. leprae reportedly has a pH optimum of 5.8 for lular ATP at reduced oxygen concentrations, as mentioned respiration (100). As mentioned above, most studies suggest below. that M. leprae utilizes free oxygen in its energy-generating Amino acid metabolism. M. leprae is capable of amino acid processes. A recent study on ATP maintenance and PGL-I uptake (245) and incorporation into protein (109), although synthesis indicated biophysical optima of 330C and pH 5.6 there is a lack of data concerning most individual amino and a reduced but maintained oxygen concentration (62a). acids. 3,4-Dihydroxyphenylalanine is taken up (3) and oxi- dized (185), although the nature, uniqueness, and signifi- In Vivo Drug Testing cance of this activity continue to be a matter of controversy Prior to the demonstration in 1960 of limited multiplication (183, 242). 3,4-Dihydroxyphenylalanine oxidase activity is of M. leprae in the footpads of immunologically intact mice accepted by many as a specific taxonomic marker of M. (206), therapeutic agents for leprosy were selected solely on leprae. Other enzymes of amino acid metabolism which have the basis of empirical results in clinical trials. Treatment with been detected are glutamate decarboxylase (184) and gam- chaulmoogra oil, based on Burmese folk , was only ma-glutamyl transpeptidase (218). partially effective and was used until the sulfones were found Nucleic acid metabolism. Thymidine is taken up by M. to be highly efficacious. Until the development of the mouse leprae residing in macrophage cell cultures (162) or sus- footpad model, activity against M. tuberculosis had been pended in axenic culture media (111). Uracil is also taken up considered a valid criterion for initiation of clinical trials in in axenic medium, as is the pyrimidine precursor orotic acid. leprosy. In general, purine bases are taken up and incorporated into In the immunologically intact mouse, M. leprae can attain trichloroacetic acid-insoluble material at higher rates than only a density of approximately 106 bacilli per footpad, are pyrimidipes. M. leprae appears to be incapable of de regardless of the inoculum dosage (206). This, however, is novo purine synthesis, as evidenced by lack of incorporation sufficient for detection of growth-inhibitory compounds of [14C]serine or ['4C]glycine into the purine fraction, and when low inocula (5 x 103 to 1 x 104) are used. Bacilli, may obtain purines primarily via scavenging mechanisms obtained either from patient biopsies or by animal passage, (246, 247). Enzymes for interconversion of purine bases are inoculated into one or both hind footpads in a volume of have been detected in cell-free extracts, and rates of incor- approximately 0.03 ml. Test compounds are usually pow- poration of purines could be accounted for by the levels of dered and mixed in the diet at concentrations ranging from phosphoribosyltransferases in cell-free extracts. Of interest 0.0001 to 0.1% (wt/wt). Alternatively, compounds may be is the relatively high level of adenosine kinase in M. leprae administered by gavage or parenterally, although the repeti- compared with other host-grown mycobacteria. tion of such procedures over the course of several months is Lipid metabolism. M. leprae residing within Schwann cells laborious. All of the presently used antileprosy drugs can be reportedly incorporate acetate into the PGL-I fraction (157). administered in the feed, and ultimately any new useful Similarly, bacilli residing within macrophages have been drugs should be active orally in humans. Bacillary growth is shown to rapidly incorporate palmitic acid into PGL-I (186). evaluated by direct microscopy (Ziehl-Neelsen staining) The latter also occurs in M. leprae residing extracellularly in (213) of footpad homogenates following incubation periods an axenic medium (63). Other precursors, including acetate, of several months to 1 year. were not incorporated into PGL-I in this system. Incorpora- Four techniques have been described and utilized in the tion of 32P into the phospholipid fraction has been reported evaluation of potential antileprosy compounds. The earli- VOL. 1, 1988 LEPROSY 341 est such technique used continuous drug administration number of agents which can be evaluated. Therefore, a throughout the experiment (211). While a number of com- number of attempts have been made in the last decade to pounds are active in this system, bacteriostatic and bacteri- take advantage of the ability to rapidly quantitate temporal cidal activity cannot be differentiated. This method has the metabolic activity of M. leprae (as described above), con- advantage of requiring the lowest number of mice and the sidering the inhibition of such activities to be indicative of least labor. drug activity. In the kinetic method (208), drug administration is delayed These systems have used intact bacilli suspended in either until the bacteria have reached the early logarithmic phase of simple axenic media or macrophage cell cultures, require 1 growth, 60 to 70 days postinoculation, and is continued for to 3 weeks of incubation, and rely upon drug action at a locus approximately 50 to 60 days. Bacillary growth is determined not necessarily directly involved in the metabolic activity at selected intervals during drug administration and thereaf- being assayed. Relatively simple systems having bacillary ter. Bacteriostatic drugs cause a growth delay equal to the requirements of 106 to 107 per assay might be candidates for period of drug administration, while bactericidal compounds clinical detection of secondary drug resistance, using skin effect a growth delay greater than the period of drug admin- biopsy-derived inocula. Detection of primary drug resistance istration. In this method, the potential exists for falsely in which only a small percentage of the bacterial population attributing bactericidal activity to drugs with tissue-reposi- may be resistant would not be possible in these systems and tory or bacteriopausal activity. will await the development of a growth-supporting method- The proportional bactericidal method (40) involves inocu- ology. lation of groups of mice with 10-fold dilutions of M. leprae Incorporation of tritiated thymidine into the trichloroace- and administration of test compounds for 2 months. Growth tic acid-insoluble fraction of phagocytized M. leprae is of bacilli is determined after 12 months, using most-proba- inhibited by clinical antileprosy agents (162) and has been ble-number calculations. Only bactericidal agents appear used to evaluate a number of phenazines (145). Results in active in this system, while inactive and bacteriostatic agents this system have been found to correlate well with the mouse cannot be differentiated. footpad technique in evaluating secondary drug resistance in The rapidity of onset of bactericidal activity can be clinical isolates (200). A scaled-down version of this tech- determined by serial inoculation of mice with patient skin nique (144) reduces the requirement for both bacilli and biopsy material at time intervals following the initiation of macrophages, making its clinical use more practical. While drug therapy (210). thymidine is also taken up by extracellular M. leprae, other Only a relatively small number of compounds have dem- nucleic acid precursors such as hypoxanthine are taken up onstrated bactericidal activity in these systems, including all more rapidly and are also sensitive to the action of antile- of the clinically useful antileprosy agents: dapsone (weak), prosy agents (111). Thus, these compounds may ultimately rifampin and related compounds, and clofazimine, with only prove to be superior substrates when nucleic acid synthesis rifampin being rapidly bactericidal. Ethionamide and pro- is used in a drug-screening system. thionamide (212), thiacetazone, some of the fluoroquino- Our laboratory has recently developed three distinct sys- lones (79, 176, 195), and certain cephalosporins (214) have tems for large-scale screening of potential antileprosy also shown bactericidal-like activity. An effective therapeu- agents. The incorporation of [14C]palmitic acid into PGL-I of tic regimen should include at least one bactericidal drug if M. leprae has been shown to be sensitive to clinical antile- that drug is to be used intermittently, e.g., once monthly, prosy drugs as well as a number of other compounds in both and therefore such drugs are actively sought. intracellular (186) and extracellular (63) M. leprae. The use Drug-resistant leprosy bacilli can be detected by the of these systems in parallel has the potential for evaluating continuous-feed technique (178). High-, medium-, and low- the effect of intracellular residence on bacillary metabolic level resistance to dapsone are defined as growth in the stability and drug susceptibility. Over 25 antimicrobial presence of 0.01, 0.001, and 0.0001% drug, respectively (90). agents have been evaluated by their ability to effect an Primary resistance is usually low level, and patients harbor- accelerated rate of ATP decay in extracellular M. leprae ing such bacilli would normally be expected to respond to suspended in axenic media (64). Finally, palmitate oxidation full-dosage dapsone. Testing for secondary dapsone resis- to carbon dioxide, measured by radiorespirometry, was tance enables the to determine whether the failure found to be sensitive to antileprosy agents (62). The use of an to respond to- therapy is due to true drug resistance of the automatic Buddemeyer-type counting system (26) makes bacilli or to a lack of compliance by the patient (104). this system the simplest described to date for evaluating The armadillo (113), athymic (nude) mouse (33), and antileprosy agents. The ability to readily detect activity with neonatally thymectomized Lewis rat (61) all support exten- approximately 106 bacilli and the precedence for using this sive multiplication of M. leprae. Athymic rodents have been activity in the rapid drug susceptibility testing of cultivable used as models for treatment of LL, in which initial bacillary mycobacteria (134, 193, 235) make this assay a strong loads may be quite high (118). In addition, their use enables candidate for use in the clinical detection of secondary drug larger inocula to be used when attempting to detect the resistance pending further studies. presence of resistant or "persister" bacilli in treated pa- These systems have identified fluoroquinolines, minocy- tients. cline, and some phenazines as having anti-M. leprae activ- ity, findings consistent with the activity of these compounds In Vitro Drug Testing in the mouse footpad (71, 79, 176, 195). Perhaps most interesting is the potent activity in these systems of eryth- Although the development of the mouse model has made romycin and two new semisynthetic macrolides (S. G. possible the screening of potential antileprosy compounds Franzblau, N. Ramasesh, E. B. Harris, and R. C. Hastings, prior to clinical evaluation, the long incubation time, high Program Abstr. 27th Intersci. Conf. Antimicrob. Agents cost, requirement that the test compound have favorable Chemother., abstr. no. 1368, 1987), Roxithromycin (RU 965; pharmacokinetics in the mice, and requirement for gram Hoechst-Roussel Pharmaceuticals Inc., Somerville, N.J.) quantities of test compounds have undoubtedly limited the (30) and Clarithromycin (TE-031, A-56268; Abbott Labora- 342 HASTINGS ET AL. CLIN. MICROBIOL. REV.

tories, North Chicago, Ill.) (59). The semisynthetic macro- 9. Barksdale, L., and K. S. Kim. 1977. Mycobacterium. Bacteriol. lides have demonstrated markedly superior pharmacokinetic Rev. 41:217-372. properties. Recent (unpublished) studies in our laboratory 10. Baskin, G. B., B. J. Gormus, L. N. Martin, R. H. Wolf, M. have shown that, when administered in the feed of mice at Murphey-Corb, G. P. Walsh, C. H. Binford, W. M. Meyers, 0.01% (wt/wt), erythromycin ethylsuccinate and Roxi- and R. Malaty. 1987. Experimental leprosy in a Rhesus mon- thromycin are unable to inhibit multiplication of M. key: necropsy findings. Int. J. Lepr. 55:109-115. leprae in 11. Bechelli, L. M., P. G. Garbajosa, M. M. Gyi, K. Uemura, T. the footpad, while Clarithromycin fully suppresses growth of Sundaresan, W. M. Dominguez, M. Matejka, C. Tamondong, the bacillus. As thermostable, relatively inexpensive com- R. Quagliato, V. Engler, and M. Altmann. 1973. BCG vaccina- pounds acting at a locus not currently exploited in leprosy tion of children against leprosy: seven-year findings of the chemotherapy and with proven activity against a variety of controlled WHO trial in Burma. Bull. W.H.O. 48:323-334. intracellular pathogens, including mycobacteria (13), macro- 12. Beiguelman, B. 1967. Leprosy and genetics. A review of past lides possess many desirable features for inclusion in multi- research with remarks concerning future investigations. Bull. drug therapy against leprosy. W.H.O. 37:461-476. In summary, the currently uncultivable M. leprae contin- 13. Berlin, 0. G. W., L. S. Young, S. A. Floyd-Reising, and D. A. ues to present enormous challenges in all aspects of Bruckner. 1987. Comparative in vitro activity of the new leprosy macrolide A-56268 against mycobacteria. Eur. J. Clin. Micro- research. The bacterium has no readily apparent biochemi- biol. 6:486-487. cal lesions with regard to energy generation. However, 14. Bjorvatn, B., R. S. Barnetson, G. Kronvall, R. Zubler, and further studies are required to determine the complete ana- P. H. Lambert. 1976. Immune complexes and complement bolic requirements and biophysical optima which should hypercatabolism in patients with leprosy. Clin. Exp. Immunol. facilitate the development of an in vitro growth-supporting 26:388-396. methodology. While well-established in vivo systems for 15. Bloom, B. R. 1986. Learning from leprosy: a perspective on assessing antileprosy drug activity exist, the more recently immunology and the Third World. J. Immunol. 137:i-x. developed rapid metabolic assays should soon find accep- 16. Bradley, S. G. 1973. Relationships among mycobacteria and nocardia based upon deoxyribonucleic acid reassociation. J. tance in large-scale preliminary drug screening and in the Bacteriol. 113:645-651. clinical detection of secondary drug resistance. 17. Brand, P. W., and E. P. Fritschi. 1985. Rehabilitation in leprosy, p. 287-319. In R. C. Hastings (ed.), Leprosy. Church- ACKNOWLEDGMENTS ill Livingstone, Ltd., Edinburgh. 18. Brennan, P. J. 1983. The phthiocerol-containing surface lipids The research reported in this review was supported in part by of M. leprae-a perspective of past and present work. Int. J. Public Health Service grants from the National Institutes of Health Lepr. 51:387-396. (AI22492, AI22442, A122007, and NIAID Interagency Agreement 19. Brennan, P. J., G. 0. Aspinall, and J. E. Nam Shin. 1981. Y01-AI-50001) and by grants from the Victor Heiser Foundation, Structure of the specific oligosaccharides from the glycopepti- The Baton Rouge Area Foundation, and the Hansen's Disease dolipid antigens of serovars in the Mycobacterium avium- Foundation. Mycobacterium intracellulare-Mycobacterium-scrofulaceum We are deeply grateful to Rened Painter, Rosie Hauge, and Penne serocomplex. J. Biol. Chem. 256:6817-6822. Cason for their help in the literature search and in preparation of the 20. Brennan, P. J., and W. W. 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