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618^ International Journal of Leprosy^ 1985

Monoclonal Antibodies and Recombinant DNA Technology:Present and Future Uses in Leprosy and Tuberculosis*

Leprosy (Hansen's disease) and tubercu-losis (TB) are both chronic granulomatousinfections of man caused by related myco-bacteria. They are found worldwide, al-though the most seriously affected areas arethe developing countries of Asia and Africa.Historically, leprosy is possibly a recent dis-ease, whereas tuberculosis has been knownsince 23,000 B.C.'

According to the World Health Organi-zation (WHO), over 10.5 million people areestimated to have leprosy and between 1.2-1.4 billion are exposed to the risk of con-tracting the disease.' There are some 30 mil-lion new cases of tuberculosis per year. 3

However, only 50% of patients with my-cobacterial disease are registered, and only50% again receive regular treatment.' Thisfailure to register and treat patients is largelydue to an inadequate public health infra-structure and poor patient compliance. Asa result of these factors, both antileprosyand antituberculosis drugs have failed tocontrol and eradicate these diseases. In ad-dition, both primary" and secondary 6 • 7

* This review was written in 1984 by Michael J.Seck1,13.Sc., University College, London, while a third-year medical student at University College Hospital.It won First Prize in the annual competition set up bythe British Leprosy Relief Association (LEPRA) foressays on various aspects of leprosy. We take pleasurein publishing this review. Mr. Seckl's address is 30Fairmead Road, London NI9 4DF, England.

' Grange, J. M. Current Topics in Infection Series.I. Alycobacterial Diseases. London: Arnold Press, 1980.

2 WHO report on the sixth meeting of the scientificworking group on the immunology of leprosy. WHOdocument TDR/IMMLEP-SWG 6,1982.

Styblo, K. Recent advances in epidemiological re-search in tuberculosis. Adv. Tuberc. Res. 20 (1980) 1-63.

4 Pearson, J. M. H. and Haile, G. S. Primary dap-sone-resistant leprosy. Lepr. Rev. 48 (1977) 129-132.

Janowiec, M., Zwolska-Kwiek, Z. and Bek, E. Drugresistance in newly discovered untreated tuberculosispatients in Poland 1974-1977. Tubercle 60 (1979) 233-237.

Pettit, J. H. S., Rees, R. J. and Ridley, D. S. Studieson sulfone resistance in leprosy. Int. J. Lepr. 34 (1966)375-390.

7 Nielson, N. J. Primary and secondary resistance ofMycobacterium tuberculosis in Eastern Botswana. Tu-bercle 69 (1980) 239-243.

drug-resistant bacteria have now been doc-umented.

Primary prevention, in the form of vac-cination, continues to be a major area ofresearch. The BCG program for TI3, al-though effective in some areas, has been atotal failure in others!' Rapid diagnosis ishampered by the slow in vitro culture ofAl ycobacterizon tuberculosis and the inabil-ity to grow Al. leprac except in vivo in mice,''armadillos, and monkeys," thus furtherdelaying therapy.

The use of monoclonal antibodies (MABs)and recombinant DNA technology will,hopefully, herald a new era in which thegeneration in bulk of mycobacterial prod-ucts may reveal much about the biochem-istry and surface structure of these organ-isms. Knowledge thus acquired should speeddiagnosis and has obvious roles in therapyand prevention.

This review attempts to collate work donein these areas and to indicate the potentialbenefits to be accrued.

MONOCLONAL ANTIBODIESIn 1959, Burnet'' proposed the theory of

clonal selection which predicted that oneplasma cell would produce only one im-munoglobulin specificity. Confirmationcame from studies of human myeloma pro-teins and from animals with B-cell lympho-proliferate disorders. The discovery of cellhybrids formed by spontaneous fusion oftwo different cells in culture was made in

Bally, G. V. J. Trial of BCG vaccines in South Indiafor tuberculosis prevention. Indian J. Med. Res. 70(1979) 349-363.

Shepard, C. C. The experimental disease that fol-lows the injection of human leprosy bacilli into foot-pads of mice. Br. J. Exp. Med. 112 (1960) 445-454.

'" Kirchheimer, W. F. and Storrs, E. E. Attempts toestablish the armadillo (Dasypus novemcinctus Linn.)as a model for the study of leprosy. Int. J. Lepr. 39(1971) 692-702.

" Meyers, W. M., Walsh, G. P., Brown, H. L., Fu-kunishi, Y., Binford, C. H., Gerone, P. J. and Wolf,R. H. Naturally acquired leprosy in a mangabey mon-key (C'ercocebus sp.). Int. J. Lepr. 48 (1980) 495-496.

12 Burnet, F. M. The Clonal Selection Theory of Ac-quired lnnumitr. Cambridge: University Press, 1959.

53,4^ Editorials^ 619

1960,' 3 but it was not until 1975 that Mil-stein and his colleagues' 4 published a suc-cessful technique for the production of im-mortalized mouse B-cell hybridomassecreting MABs raised to specific antigens.

SynthesisHybridomas are formed by fusing togeth-

er two different cells so that the resultinghybrid cell survives, whereas the nonfusedparent cell populations do not. Lines ofmyeloma cells, preferably not secreting im-munoglobulins, have been established inlong-term culture for several species,'' - ' 7 in-cluding man. By fusing such cells (using in-activated Sendai virus or polyethylene gly-col)' 0 with another population, for example,spleen cells from an immunized mouse, ithas proved possible to produce immuno-globulin-secreting hybrids of which as manyas 10% may secrete a specific antibody. Thenon-myeloma parent cells that do not fusedie naturally in culture, but the same is nottrue of nonfused parent myeloma cells.These have to be eliminated by growth ofthe mixed culture in a selective mediumcontaining hypoxanthine, aminopterin, andthymidine (HAT).''' A selective enzyme de-fect can be induced in the myeloma line (forexample, deficient hypoxanthine guanine ri-bosyl transferase or thymidine kinase) sothat when the cells are grown in HAT, theycannot use hypoxanthine and, since the onlyother route to nucleotide synthesis is blockedby aminopterin, they die. The hybridoma

Ringertz, N. R. and Savage, R. E. In: Cell hybrids.New York: Academic Press, 1976.

14 Köhler, G. and Milstein, C. Continuous culturesof fused cells secreting antibody of predefined specific-ity. Nature 256 (1975) 495-497.

15 GalfrC, G., Milstein, C. and Wright, B. Rat x rathybrid myelomas and a monoclonal anti-Fd portionof mouse 1gG. Nature 277 (1979) 131-133.

1 ' Olsson, L. and Kaplan, H. S. Human-human by-bridomas producing monoclonal antibodies of prede-fined antigenic specificity. Proc. Natl. Acad. Sci. U.S.A.77 (1980) 5429-5431.

' 7 Croce, C. M., Linnenbach, A., Hall, W., Steplew-ski, Z. and Koprowski, H. Production of human by-bridomas secreting antibodies to measles virus. Nature288 (1980) 488-489.

Pontecorvo, G. Production of mammalian so-matic cell hybrids by means of polyethylene glycoltreatment. Somatic Cell Genet. 1 (1975) 397-400.

Szybalski, W., Szybalski, E. H. and Ragni, G. Ge-netic studies with human cell lines. Natl. Cancer Inst.Monogr. 7 (1962) 75-89.

ANTIGEN

adMYELOMA CELL LINE:

GROW IN TC

^

SPLEEN CELLS^DIE IN HAT

FUSION

HYBRID CELLSCONTAMINATED^SPLEEN CELLS NOT IMMORTALWITH UNFUSED^THEREFORE DIEPARENT CELLS

HAT MEDIUM ---0 MYELOMA CELLS DIE

HYBRID CELLS

^1 ^1^1^1^1^1CRUDE SPECIFICITY CHECK

RIA+VE WELLS SELECTEDFOR CLONING VIA

LIMITING DILUTIONSTORED

BELOW -80C.-80MORE SELECTIVE

SPECIFICITY CHECK

STOREDê--- +VE CLONESBELOW -BO .0

Eamu

PROPAGATION VIA

CULTUREÂSCITESIN VITROÎN VIVO

EXACT SPECIFICITY CHECKEDAGAINST A DIVERSE ANTIGEN BANK

FR:. I. Technique for mouse monoclonal antibodysynthesis (much simplified). TC = tissue culture; HAT =hypoxanthine, aminopterin and thymidine; RIA = ra-dioimmunoassay.

survives because of the ability conferred bythe immune cell genome to use hypoxan-thine in nucleotide synthesis.

Hybridization takes less than one hour.The next stages in the production of MABsmay take several months. Briefly, the hybridcells are distributed into wells in a plastictray and by 7-14 days the cell growth issufficient to test for the presence of anti-bodies by immunoassay. The cells from theantibody-positive wells are then cloned bylimiting cell dilution'" as shown in FigureI. Final mass production of the MAB maybe achieved via an ascitic tumor in mice"'or in culture media. MABs do not suffer the

2" Köhler, G. and Milstein, C. Derivation of specificantibody-producing tissue culture and tumour lines bycell fusion. Eur. J. Immunol. 6 (1976) 511-519.

2 ' Wright, P. Production of monoclonal antibody as-citic fluid and sera in mice with hybrid myeloma tu-mors. J. Inst. Animal Technicians 31 (1980) 113-118.


disadvantages of polyclonal antisera, suchas batch variation, low titers, and extensivetissue absorportions required to obtainspecificity. 22-24

Mouse vs human MABsThe use of mouse MABs (mMABs) to

study and to intervene in human disease hasseveral shortcomings: a) mMAl3s used inman are immunologically foreign and mayprovoke immune responses to them by thehost. b) The antigenic determinants selectedmay vary considerably between differentspecies, and less so between different mem-bers of the same species. Thus, immunizinga mouse against antigens A, B, and C mayproduce a response against A and B but notagainst C. The same procedure carried outin man may produce antibodies to B and C.Clearly, this has far-reaching implications,including the hampering of attempts to de-velop serodiagnostic tests and new vaccines(sec Scrodiagnosis). c) There is recent evi-dence to suggest that anti-idiotypes may beimportant in the regulation of immune re-sponses to Al. tuberculosis and Al. lep-rae. 25' Immunomodulation using anti-idi-otypes has therapeutic potential, butmMABs are not easily raised against idi-otypic determinants of human antibodies.d) The raising of mMABs cannot providethe means ofdefining or comparing the rangeof specificities and idiotypes present in theantibody responses of patients and immuneindividuals. Thus, human MABs appear ofgreater scientific and therapeutic interest.

" Strelkauskas, A. J., Schauf, V., Wilson, B. S., Chess,L. and Schlossman, S. F. Isolation and characterizationof naturally occurring subclasses of human peripheralblood T cells with regulatory functions. J. Immunol.120 (1978) 1278-1282.

" Moretta, L., Ferrarini, M., Mingari, M. C., Mo-retti, A. and Webb, S. R. Subpopulations of human Tcells identified by receptors for immunoglobulins andmitogen responsiveness. J. Immunol. 117 (1976) 2171-2174.

Wybran, J. and Fundenberg, H. H. Rosette for-mation, a test for cellular immunity. Trans. Assoc. Am.Physicians 84 (1971) 239-244.

" Campa, M., Benedettini, G., de Libero, G., Mori,L. and Falcone, G. T Suppressor cells as well as anti-hapten and anti-idiotype B lymphocytes regulate con-tact sensitivity to oxazolone in mice injected withpurified protein derivative and Mycobacterium tuber-culosis. Infect. Immun. 45 (1984) 701-707.

Human MABsHuman (hMABs) can be raised in two

different ways: fusion of human B cells witha human myeloma line (human/human hy-brid),'`' and fusion of human B cells with amouse mycloma line (human/mouse hy-brid).'' Both methods have drawbacks. Thehuman/human hybrids have three disad-vantages: a) The current human myelomalines are secretors of immunoglobulin (Ig),so there is contamination with irrelevantantibodies. b) The rate of Ig secretion is low.c) Most authors report a low percentage ofsecreting hybrids.

The main disadvantage of human/mousehybridization is contamination with mousecomponents. The genetic instability prob-lem has been overcome by re-cloning everythree weeks for the first two months. Workis now under way to synthesize these hu-man/mouse hybrids against mycobacterialantigens which will hopefully replace manyof the mMABs used today. Such hybridshave already been generated against humanidiotypes. 25 The dangers of injecting foreignprotein into man to generate B cells forhMAB production must not be forgotten.

EpidemiologyVarious methods have been employed to

classify mycobacteria into species and strains(extensively reviewed elsewhere)."' Themost successful immunological methodshave involved precipitation of soluble an-

" Strike, L. E., Devens, B. H. and Lundak, R. L.Production of human-human hybridomas secretingantibody to sheep erythrocytes after in vitro immuni-zation. J. Immunol. 132 (1984) 1798-1803.

" Butler, J. L., Lane, H. C. and Fauci, A. S. Delin-eation of optimal conditions for producing mouse-hu-man heterohybridomas from human peripheral bloodB cells of immunized subjects. J. Immunol. 130 (1983)165-168.

28 Abrams, P. G., Ochs, J. J., Giardina, S. L., Mor-gan, A. C., Wilburn, S. B., Witt, A. R., Oldham, R. K.and Foon, K. A. Production of large quantities of hu-man immunoglobulin in the ascites of athymic mice:implications for the development of anti-human idio-typic monoclonal antibodies. J. Immunol. 132 (1984)1611-1613.

Jenkins, P. A., Pattyn, S. R. and Portaels, F. Di-agnostic bacteriology. In: The Biology of the Myco-bacteria. Ratledge, C. and Stanford, J. L., eds. London:Academic Press, 1982, vol. I, pp. 441-470, and Stan-ford, J. L. Immunologically important constituents ofmycobacteria: antigens. In: The Biology of the Myco-bacteria, 1983, vol. 2, pp. 85-127.


tigen in gel matrices by double diffusion,immunoelectrophoresis, and crossed im-munoclectrophoresis. Essentially, there aretwo problems with these techniques: a) Theyrequire hyperimmune antisera of very highquality. As already mentioned, MABs haveseveral advantages over polyclonal antisera.b) Even with the best antisera, it has beendifficult to distinguish between differentmembers of the tuberculosis complex (M.tuberculosis, Al. africanum, AI. bolls, andAI. microti) and impossible to detect otherstrains. 3" Nevertheless, several groups havenow produced mMABs to mycobacteria(Table 1).

Tuberculosis taxonomy. M. tuberculosis,Al. africal111111, and M. boris are currentlydistinguished by morphological and bio-chemical criteria and by phage typing (A,B, and 1). 4 ' The report by Coates, et al. in

3" Stanford, J. L. and Grange, J. M. The meaningand structure of species as applied to mycobacteria.Tubercle 55 (1974) 143-152.

31 Coates, A. R. M., Hewitt, J., Allen, B. W., Ivanyi,J. and Mitchison, a A. Antigenic diversity of Myco-bacterium tuberculosis and M•cobacterium boris de-tected by means of monoclonal antibodies. Lancet 2(1981) 167-169.

" Hewitt, J., Coates, A. R. M., Mitchison, D. A. andIvanyi, J. The use of murine monoclonal antibodieswithout purification of antigen in the serodiagnosis oftuberculosis. J. Immunol. Methods 55 (1982) 205-211.

Ivanyi, J., Krambovitis, E. and Keen, M. Evalu-ation of a monoclonal antibody (T1372) based serolog-ical test for tuberculosis. Clin. Exp. Immunol. 54 (1983)337-345.

34 Minden, I'., Kelleher, P. J., Freed, J. 1-I., Nielson,L. I3., Brennan, P. J., McPherson, L. and McClatchy,J. K. Immunological evaluation of a component iso-lated from Mycobacterium boris BCG with a mono-clonal antibody to Al. bolls BCG. Infect. Immun. 46(1984) 519-525.

Gillis, T. P. and Buchanan, T. M. Production andpartial characterization of monoclonal antibodies toMycobacterium leprae. Infect. Immun. 37 (1982) 172-178.

Ivanyi, J., Sinha, S., Aston, R., Cussell, D., Keen,M. and Sengupta, U. Definition of species-specific andcross-reactive antigenic determinants of Mycobacte-rium leprae using monoclonal antibodies. Clin. Exp.Immunol. 52 (1983) 528-536.

Sinha, S., Sengupta, U., Ramu, G. and Ivanyi, J.A serological test for leprosy based on competitive in-hibition of monoclonal antibody binding to the MY2adeterminant of M. leprae. Trans. R. Soc. Trop. Med.Hyg. 77 (1983) 869-871.

" Young, D. B., Khanolkar, S. R., Barg, L. L. andBuchanan, T. M. Generation and characterization ofmonoclonal antibodies to the phenolic glycolipid ofMycobacterium leprae. Infect. Immun. 43 (1984) 183-188.

19813' of mMABs against tuberculosis gavehope for both a better mycobacterial tax-onomy and the possibility of a scrodiagnos-tic test. A variety of whole tuberculosis andBCG strain bacteria were inoculated to gen-erate MABs against intact cell surface an-tigens. The resulting MABs were then testedagainst a bank of mycobacteria for theirspecies and strain specificity. Unfortu-nately, this bank was not extensive enoughand notably did not include AI. africanum,AI. raccae, and Al. leprae, nocardia, or cory-nebacteria. Furthermore, although theMABs were generated against whole bac-teria, they were tested against pressates(broken-down bacteria). Thus, there is apossibility of crossrcaction between exter-nal and internal components of different or-ganisms. Some biochemical analysis of theseantigens has been made (personal commu-nication, Dr. T. Coates). MAB TB68 bindsa four-chain glycoprotein of 120KD andMAB TB72, a two-chain protein of 35KD.The tentative conclusions were that theseMABs were able to detect differences be-tween particular strains of AI. tuberculosis,virulent M. boris, and Al. boris BCG.

Leprosy taxonomy. The development ofmMABS against AI. leprae has recently beenreviewed. 42 Gillis and Buchanan" were thefirst to produce mMABs of which only two(IVD8-1gG, and 111E9-IgG,„) were specificagainst Al. leprae, binding to cytosolic an-tigens. The methods of antigen productionfor immunization and for MAB-specificitytesting were the same, the antigens being amixture of extracellular and intracellularcomponents. For epidemiological purposesthis is a far more sensitive method of iden-tifying differences between strains, sincethese differences may not be confined to the

4--" Mehra, V., Brennan, P. J., Rada, E., Convit, J. and

Bloom, B. R. Lymphocyte suppression in leprosy in-duced by unique M. lepraeglycolinid. Nature 308 (1984)194-196.

4" Bach, M.-A. and Ilolfenbach, A. A. A monoclonalantibody against Mycobacterium lepraenturium whichrecognises a cross-reacting mycobacterial antigen. Ann.Immunol. (Paris) 134C (1983) 301-309.

41 Grange, J. M. and Redmond, W. B. Host-phagerelationship in the genus Mycobacterium and their clin-ical significance. Tubercle 59 (1978) 203-225.

" Ivanyi, J. Application of monoclonal antibodiestowards immunological studies in leprosy. Lepr. Rev.55 (1984) 1-9.


TABLE 1. Summarizing niMilBs against Mycobacterium tuberculosis, BCG, and M.leprae, demonstrating the mut using nomenclature.

Foot-noterefer-once

Example of mMABSproduced or used

Chemical structureand specificity

Molecularweight (KD)and no. of

chains

Localization Crossrcactivity

31 • " T1!68-IgG, Glycoprotein 120 Cell wall Narrow"4 chains

''-" T1372-IgG, Protein or glycoprotein 35 Cell wall Narrow"2 chains

34 BCG SAl2-IgG Protein 10 Cell wall Narrow'" IVD8-IgG, — — Cytoplasm None" 111-19-IgG, Protein 68 Cytoplasm Broad36 ML06/MY l a-IgG, Protein 12 Cytoplasm None

3637 ML04/MY2a-IgG, Protein — Cytoplasm? Marginal36

36

ML02/MY4a-IgG,ML35/MY4b-IgM Polysaccharide 40-50 Cell wall Broad

'a 13G, 13511-1gM 13GL-1-M. /eprae-specilicdisaccharide

? Cell wall Marginal°

PG, B2G-IgM PGL-l-A1. /eprae-specilicdisaccharide

? Cell wall Moderate

39 PG, 46.7-IgG, PGL-l-A1. /eprae-specilicdisaccharide

7 Cell wall Not shown

39 Y,-IgG, Protein 68 7 Not shownSAI D2D-IgG,' Protein 28 Cytoplasm BroadSA1 A1B-IgG,' Protein 28 Cytoplasm Narrow

'" A494-IgM 7 ? Cell wall All mycobacteria

a M. tuberculosis strains H37Ra, H37Rv, 6067, 729, and SI.M. tuberculosis strains and Al. bovis BCG and Vallee.Al. bolls BCG, M. tuberculosis H37Rv (other strains not tested).

° M. terrae and M. nonchromogenicum.Personal communication, Dr. D. B. Young.

bacterial cell wall. In this respect, genomeanalysis will provide the ultimate sensitiv-ity.

In a later report, 38 Buchanan, et al. de-scribed a set of IgM mMABs against the M.leprae-specific trisaccharide (Fig. 2). Confir-mation that this trisaccharide was only foundin Al. leprae has come from structural 43 andserological 44 studies. However, some theMABs which apparently only bound to theintact trisaccharide (i.e., Al. leprae specific)also crossreacted with AI. bovis, Al. non-chromogenicum, and Al. terrae. 38 At leasttwo possibilities therefore exist: a) The tri-saccharide, although unique structurally,antigenically is not, and the previous sero-logical studies were not sufficiently sensi-

4‘ Payne, S. N., Draper, P. and Rees, R. J. W. Se-rological activity of purified glycolipid from Mycobac-terium leprae. Int. J. Lepr. 50 (1982) 220-221.

1 Yanagihara, D. L., Knisley, C. V., Barr, V. L.,Hunter, S. W. and Brennan, P. J. ELISA and the spe-cific glycolipid antigens of mycobacteria. Fed. Proc. 42(1983) 2022.

tive. Indeed, the latest investigations haveshown crossrcactivity with M. bovis and M.kansasii (although this crossreactivity maybe phenol-ring related). 45 b) Nonspecificbinding occurred to the other organisms.Although this occurs with IgM-class anti-bodies, it seems unlikely since only someorganisms were stained. However, theseMABs have already been of great use ininvestigating the T-suppressor-cell activitygenerated by the trisaccharide. 39

One of the disadvantages of trying to useMABs to isolate species-specific antigens is,indeed, their extreme specificity. While suchMABs recognize an epitope on a moleculewhich may be species specific, the other epi-topes may be crossreactive, and vice versa.An example of this has just been reported(personal communication, Dr. D. B. Young).Five mMABs have been synthesized which

Brett, S. J., Payne, S. N., Draper, P. and Gigg, R.Analysis of the major antigenic determinants of thecharacteristic phenolic glycolipid from Mycobacteriumleprae. Clin. Exp. Immunol. 56 (1984) 89-96.

53, 4^ Editorials^ 623

bind to an intracellular antigen of 28KDfound in all mycobacteria. Two of theseMABs are M. leprae specific, one is cross-reactive with all mycobacteria, and two arepartially specific (AI. leprae, Al. trivia/e, andAl. piaci). Interestingly, competition studieshave suggested that the species-specific epi-topes are closely clustered while the cross-reactive epitopes are on another distinct partof the molecule.

MAB taxonomy. A rapidly enlarging ar-ray of MABs exist against mycobacteria(Table 1). A classification system, perhapsthat of Ivanyi, 3 ' should be formed. Work-shop meetings would produce much usefulinterchange. (A good example of this hasbeen the development of a human leukocyteMAB classification scheme. 46 . 47 )

Other epidemiological uses. The use ofMABs in taxonomy is not promising sincemost are crossreactive. Nevertheless, theMABs which are Al. leprae or M. tubercu-losis specific could be used to explore modesof transmission of infection, organism vir-

46 Beverley, P. Monoclonal antibodies and humanleukocyte antigens. Immunol. Today 4 (1983) 61-62.

" Bernard, A., Berstein, 1., Boumsell, L., Dausctt,J., Evans, R., Hansen, J., Haynes, 13., Kersey, J., Knapp,W., McMichael, A., Milstein, C., Reinherz, E., Ritts,R. E. and Schlossman, S. F. Proposed nomenclaturefor human leukocyte differentiation antigens: a correc-tion. Immunol. Today 5 (1984) 280.

ulence, host susceptibility, the epidemiol-ogy of small epidemics, and such interestingquestions as whether or not tuberculosisoriginates by an infection with a single orseveral bacilli.

MABs and immunopathologyTuberculosis, and particularly leprosy,

exist as disease spectra. In leprosy, at oneextreme are patients who mount a vigorouscell-mediated immune (CMI) response,tuberculoid leprosy (TT); at the other ex-treme are patients who have a profound ab-sence ofCMI, lepromatous leprosy (LL). Thefailure of CMI, particularly in tuberculosis,may be fatal for the patient. The exactmechanism(s) involved and why they occuronly in some patients are still unclear. Inorder to understand the possible roles ofMABs, the CMI pathway and its regulationare outlined hereafter and pertinent recentadvances in immunology are highlighted.

CMI pathway (Fig. 3). Mycobacteriapenetrate the body through a variety of sites.For M. tuberculosis the usual portal of entryis the lungs, "wild-type" mycobacteriaprobably enter during their passage alongthe gastrointestinal tract, while AL leprae'sportal is unknown. The bacteria are thenphagocytosed by macrophages and partiallydigested ("antigen processing"), allowingsome bacterial components to be extruded

624^ International Journal of Leprosy^ 1 985

HG. 3. A much-simplified view of the immune pathways in leprosy and tuberculosis. Suppressor mechanisms( ) may therefore include: a) an antigen-presenting cell defect, b) a hole in the Th repertoire, c) IL-1/1L-2and/or PGE,/PGE 2 defect, d) Ts cells, e) anti-idiotypes, 0 a lymphon defect. Ag = antigen; MHC = majorhistocompatibility complex; IL-1 = interleukin-l; PGE, = prostaglandin E i ; IFN = interferon; MIF and MAF =migration inhibition and macrophage activation factor, respectively; Th = helper T lymphocyte; Ts = suppressorT lymphocyte; B = B lymphocyte; BCGF and BCMF = B cell growth and maturation factors, respectively;+ve = positive effect; — ye = negative effect.

onto the cell surface for antigen presenta-tion. 48 Effective presentation can only occurwhen the foreign antigen is combined withclass I (HLA A B C) or class II (HLA DDP, DQ, DR) 4" major histocompatibility(MHC) molecules. Class I MHC moleculesare found on all nucleated cells; whereasclass II MHC molecules only occur on an-tigen-presenting cells (APCs) (usually ofmonocyte origin), some T and B cells, thy-mus epithelium, and a few other cell types. 49

48 Unanue, E. R., Beller, D. I., Lu, C. Y. and Allen,P. M. Antigen presentation: comments on its regula-tion and mechanism. J. Immunol. 132 (1984) 1-5.

"" Bodmer, J. and Bodmer, W. Histocompatibility1984. Immunol. Today 5 (1984) 251-254.

4' Nixon, D. F., Ting, J. P. and Frelinger, J. A. laantigens on nonlymphoid tissues: their origins andfunctions. Immunol. Today 3 (1982) 339-342.

The use of highly purified allo- and het-ero-antisera and MABs have permitted dif-ferentiation and analysis of "functional"T-cell subsets (Table 2). In addition, anti-gen- or mitogen-activated T cells may bedistinguished from resting T cells by theirincreased density of class II antigens'''• 5 ' andFc receptors for IgG. 52

'" Evans, R. L., Faldetta, T. J., Humphreys, R. E.,Pratt, D. M., Yunis, E. J. and Schlossman, S. F. Pe-ripheral human T cells sensitized in mixed leukocyteculture synthesize and express Ia-like antigens. J. Exp.Mcd. 148 (1978) 1440-1445.

Ko, H. S., Fu, S. M., Winchester, R. J., Yu, D. T.Y. and Kunkel, H. G. Ia determinants on stimulatedhuman T cells. Occurrence on mitogen and antigenactivated T cells. J. Exp. Mcd. 150 (1979) 246-255.

Kaszubowski, P. A., Goodwin, J. S. and Williams,R. C., Jr. la antigen on the surface of a subfraction ofT cells that bear Fc receptors for IgG. J. Immunol. 124(1980) 1075-1078.


TABLE 2. Functional T-cell subset identification on mouse and man.

T-cell function^Mouse phenotype^Human phenotype

T helper/inducer (Th/i)^Lyt 1 +/23—"^TH2-5" " OKT4+ 56

T suppressor/cytotoxic (Ts/c)^Lyt 1—/23+"^TH, "' " OKT5 +, OKT8+ 57

The functional purity of these T-cell sub-sets is questionable. For example, mousesuppressor T cells (Ts) have been foundwhich are Lyt-1 +/ 59 or Lyt-1 +/2,3 + .('" Helper T cells (Th) may also expressdifferent Lyt phenotypes." Similar func-tional heterogeneity is known to exist inman.`''

It has been shown that Th cells will onlyrespond to foreign antigen when it has beenprocessed and is properly presented (usually

" Cantor, H. and Boyse, E. A. Functional subclassesof T lymphocytes bearing different Ly antigens. Co-operation between subsets of Ly cells in the generationof killer activity. J. Exp. Med. 141 (1975) 1390-1405.

Reinherz, E. L. and Schlossman, S. F. ConA-in-ducible suppression of MLC: evidence for mediationby the TH,+ cell subset in man. J. Immunol. 122(1979) 1335-1342.

Reinherz, E. L., Kung, P. C., Goldstein, G. andSchlossman, S. F. A monoclonal antibody reactive withthe human cytotoxic/suppressor T cell subset previ-ously defined by a heteroantiserum termed TH 2 . J.Immunol. 124 (1980) 1301-1307.

'" Reinherz, E. L., Kung, P. C., Breard, J. M., Gold-stein, G. and Schlossman, S. F. T cell requirements forgeneration of helper factor(s) in man: analysis of thesubsets involved. J. Immunol. 124 (1980) 1883-1887.

" Reinherz, E. L. and Schlossman, S. F. The differ-entiation and function of human T lymphocytes. Cell19 (1980) 821-827.

Maier, J., Levy, J. G. and Kilburn, D. G. The Lytphenotype of cells involved in the cytotoxic responseto syngenic tumour and of tumor-specific suppressorcells. Cell. Immunol. 56 (1980) 392-399.

Ramshaw, I. A., McKenzie, I. F. C., Bretscher, P.A. and Parish, C. R. Discrimination of suppressor Tcells of humoral and cell mediated immunity by anti-Ly and anti-Ia sera. Cell. Immunol. 31 (1977) 364-369.

Al-Adra, A. R., Pilarski, L. M. and McKenzie, I.F. C. Surface markers on the T cells that regulate cy-totoxic T-cell responses. I. The Ly phenotype of sup-pressor T cells changes as a function of time and isdistinct from that of helper or cytotoxic T cells. Im-munogenet. 10 (1980) 521-533.

61 Thomas, D. B. and Calderon, R. A. T helper cellschange their Lyt 1,2 phenotype during an immune re-sponse. Eur. J. Immunol. 12 (1982) 16-23.

" 2 Lamb, J. R., Eckels, D. D., Lake, P., Woody, J.N. and Green, N. Human T cell clones recognise chem-ically synthesized peptides of influenza hemagglutinin.Nature 300 (1982) 66-69.

in conjunction with self-class II mole-cules). 49 Cytotoxic T cells (Tc) are similarlyrestricted but usually to self-class I anti-gens." Clearly, any defect in macrophageprocessing and antigen presentation may al-ter Th or Tc cell responsiveness to foreignantigen. (Indeed, HLA DR3-negative in-dividuals mount a poor immune responseto M. leprae. 64 )

The binding of Th cells to an antigen plusclass II MHC complex triggers interleukin-1(IL-1) release from the antigen-presentingcell (APC). 48 This, in turn, causes Th cellrelease of various humoral, non-immuno-globulin molecules (lymphokines) whichmediate a variety of biological effects. Atthis point the two arms of the immune re-sponse, CMI and humoral immunity, di-verge. The types of lymphokines secretedare dependent upon the type of antigen pre-sented and, thus, upon the responding Thcell. If the antigen creates a predominantlyT-cell-dependent, B-cell response, then mostof the lymphokines released will activateantigen-bound B cells (for example, B-cellgrowth factor). 65 Conversely, antigens stim-ulating CMI are associated with the secre-tion of macrophage and/or Tc-activatinglymphokines. These include IL-2, alpha-in-terferon (a-I FN), macrophage inhibitionfactor (MIF), and macrophage activatingfactor (MAF)."."" In leprosy and tubercu-

" Zinkernagel, R. M. and Doherty, P. C. H-2 com-patibility requirement for T-cell mediated lysis of tar-get cells infected with lymphocytic choriomeningitisvirus: Different cytotoxic T-cell specificities are asso-ciated with structures coded in H-2K or H-2D. J. Exp.Med. 141 (1975) 1427-1436.

" van Eden, W., de Vries, R. R. P., D'Amaro, J.,Schreuder, G. M. T., Leiker, D. L. and van Rood, J.J. HLA-DR-associated genetic control of the type ofleprosy in a population from Surinam. Human Im-munol. 4 (1982) 343-350.

" Muraguchi, A. and Fauci, A. S. Proliferative re-sponses of normal human B lymphocytes. Develop-ment of an assay system for human B cell growth factor(BCGF). J. Immunol. 129 (1982) 1104-1108.

Howie, S. and McBride, W. H. Cellular interac-tions in thymus-dependent antibody responses. Im-munol. Today 3 (1982) 273-278.


losis, the most important part of the im-mune response is the effective activation ofmacrophages to enable mycobacterial de-struction.

The immune response, like most biolog-ical systems, may be "down" regulated. Thiscan be achieved through a variety of mech-anisms involving suppressor T cells (Ts),excess circulating antibodies, or idiotypicantibodies. Thus, the suppression can beboth cell mediated and humoral. Recently,there has been increasing evidence thatprostaglandins, PGE, and PGE,, producedon the surface of APCs can "down" regulatethe response by inhibiting Th cells'' (prob-ably by prolonged activation of adenylatecyclase; cAMP inhibits lymphocyte trans-formation), activating Ts cells," or "down"regulating the expression of class II MHCantigens on APCs."

Finally, it is important to remember therole of the lymphon, the collective name forthe primary and secondary lymphoid or-gans and their constituent cells. All too oftenthe cellular interactions and functions un-derlying immune responses are consideredout of the context of their physiological en-vironment. The crucial importance of thecyto-architecture (e.g., the role of dendriticreticular cells in germinal center formation)and the concept of lymphocyte traffic, to-gether with its disturbance by antigen,''-needs to be considered when studying de-ficient or abnormal immunology.

Histology. In addition to their use in sub-dividing T cells, MABs have been devel-

`' Staitc, N. D. and Panayi, G. S. Prostaglandin reg-ulation of B-lymphocyte function. Immunol. Today 5(1984) 175-178.

Hansen, J. M., Rumjanek, V. M. and Morley, J.Mediators of cellular immune reactions. Pharmacol.Ther. 17 (1982) 165-198.

Snyder, D. S., Beller, D. 1. and Unanue, E. R.Prostaglandins modulate macrophage la expression.Nature 299 (1982) 163-165.

Palacios, R. and Willer, G. T-cell growth factorabrogates concanavalin A-induced suppressor cellfunction. J. Exp. Med. 153 (1981) 1360-1365.

Urbain, C. and Wilmart, J. Some thoughts on idio-typic networks and immuno-regulation. Immunol. To-day 3 (1982) 88-92,125-127.

72 McConnell, I., Munro, A. and Waldmann, H. TheImmune System. A Course on the Molecular and Cel-lular Basis of Immunity. 2nd ed. Oxford: BlackwellScientific Publications, 1981.

oped against various class II molecules, 73

IL-2, 74 the IL-2 receptor, 75 and subpopu-lations of APCs. 76 Some of these MABs arenow being used in the study of histologicalsections of leprosy granulomas. Initial re-sults have shown that the well-organized ep-ithelioid granulomas of tuberculoid leprosy(TT) lesions contain large numbers ofOKT4 + (helper/inducer T cells, Th/i) lym-phocytes surrounded by a heavy "mantle"of predominantly suppressor/cytotoxic Tcells (Ts/c). The overall Th : Ts ratio is nor-mal (2:1). The diffuse lepromatous lesion,by contrast, has few lymphocytes, and thereis controversy over the existence of a de-creased Th Ts/c ratio.' 77 ' 78 As alreadymentioned, subdividing T cells by their sur-face phenotypes is functionally very mis-leading. In their most recent paper, Modlin,et al. 7' have attempted to overcome thisproblem by MAB analysis of IL-2-produc-ing and IL-2-accepting cells in TT and LLbiopsy specimens. Using double-stainingtechniques, they found that in TT lesionsthere were large numbers of IL-2-producing(Th phenotype) cells in intimate contact with

Beckman, I. Monoclonal antibodies to HLA-DRantigens. Immunol. Today 5 (1984) 29-32.

Gillis, S., Mochizuki, D. Y., Conlon, I'. J., Hcfc-neidcr, S. H., Ramthun, C. A., Gillis, A. E., Frank, M.B., Henney, C. S. and Watson, J. D. Molecular char-acterization of interlcukin 2. Immunol. Rev. 63 (1982)167-209.

Osawa, H. and Diamantstein, T. Studies on T lym-phocyte activation. II. Monoclonal antibody inhibitingthe capacity of rat T lymphoblasts to absorb and torespond to IL-2: an anti-IL-2 receptor antibody? Im-munol. 48 (1983) 617-621.

76 Todd, R. F., 111 and Schlossman, S. F. Utilizationof monoclonal antibodies and characterization ofmonocytc-macrophage differentiation antigens. In:Ref iczdoendothelial System: A Comprehensive Trea-tise: Vol. 6: Immunolo,u. 13ellanti, A. and Herscowitz,H. 13., eds. New York: Plenum Press, 1984, pp. 87-112.

' 7 Narayanan, R. B., Bhutani, L. K., Sharma, A. K.and Nath, I. T-cell subsets in leprosy: in situ charac-terization using monoclonal antibodies. Clin. Exp. Im-munol. 41 (1983) 421-429.

Modlin, R. L., Holman, F. M., Meyer, P. R., Shar-ma, 0. P., Taylor, C. R. and Rea, T. H. In situ dem-onstration of T-Iymphocyte subsets in granulomatousinflammation: leprosy, rhinoscleroma and sarcoidosis.Clin. Exp. Immunol. 51 (1983) 430-438.

7 ' Modlin, R. L., Holman, F. M., Horwitz, D. A.,Husmann, L. A., Gillis, S., Taylor, C. R. and Rea, T.H. In situ identification of cells in human leprosy gran-ulomas with monoclonal antibodies to interleukin 2and its receptor. J. Immunol. 132 (1984) 3085-3090.


macrophages surrounded by a mantle ofmainly IL-2-accepting Th phenotype cells.The IL-2-producing cells did not possessIL-2 receptors. By contrast, the disorgan-ized LL lesions had few I L-2-producing cellsand decreased numbers of IL-2-acceptingcells of both helper and suppressor pheno-types. From these data it is tempting to sug-gest that defective CM I in LL is associatedwith decreased IL-2 production, but thisseems unlikely to be the primary defect. Bi-opsy work with borderline lepromatous,borderline tuberculoid, and indeterminateleprosy patients should now be done in anattempt to clarify the situation.

Unfortunately, similar investigations havenot yet been performed in tuberculosis le-sions. This presumably reflects both the dif-ficulty in obtaining lung biopsies and thecomparative paucity of workers in the field.

Clearly, MABs can play a vital role in theanalysis of cell types and cell products inhistological sections, and this is likely toyield far more relevant data than the in-vestigation of peripheral blood cells.

MABs and erythema nodosum leprosum(ENL). Classically, type II lepra reactions(ENL) have been thought to result from anArthus type of hypersensitivity—circulat-ing immune complexes (CIC) being depos-ited in capillary beds and then activatinginflammatory processes via the comple-ment cascade."" However, there is consid-erable evidence to suggest that the CIC andalterations in complement levels found aresecondary phenomena for the following rea-sons: a) Similar levels of CIC occur in LLpatients with or without ENL."' b) In areaswith endemic leprosy, CIC may be foundthroughout the entire leprosy spectrum, al-though ENL only develops in LL patients.'''c) Thalidomide, used in the treatment of

"" Coda!, T. Immunological aspects of leprosy. Pres-ent status. Frog. Allergy 25 (1978) 211-242.

"1 Moran, C. J., Ryder, G. and Turk, J. C. Evidencefor circulating immune complexes in lepromatous lep-rosy. Lancet 2 (1972) 572-573.

Valentijn, R. M., Faber, W. R., Lai—A—Fat, R. F.,Chan Pin Jie, J. C., Daha, M. R. and van Es, L. A.Immune complexes in leprosy patients from an en-demic and non-endemic area and a longitudinal studyof the relationship between complement breakdownproducts and the clinical activity of erythema nodosumleprosum. Clin. Immunol. Immunopathol. 22 (1982)194-202.

ENL, has no suppressive effect on experi-mental Arthus reactions. d) Local levels ofC3d in lepromatous lesions do not correlatewell with the circulating levels of C3d."

The latter point may suggest that localcomplement production by tissue macro-phages may be more important in ENL.Complement fragments such as C3a are ex-tremely chemotactic for neutrophils. 72 Analternative pathogenesis for ENL reactionscould involve local tissue dissemination ofbacteria, triggering the release of local com-plement and, thus, neutrophil chemotaxistoward multiple sites in the body, wheredegranulation would cause tissue injury. In-deed, neutrophil chemotaxis is depressed inuntreated LL patients but enhanced by dap-sone chemotherapy." Thus, the increasedoccurrence of ENL in LL patients duringtheir first year of dapsone therapy may berelated to increased chemotaxis. However,not all LL patients on chemotherapy de-velop ENL. Although such a hypothesis hasobvious weaknesses, it would be interestingto investigate further the role of macro-phages and neutrophils in ENL reactions,perhaps using MABs to define specific celltypes and products.

SerodiagnosisThe possibility of accurate serodiagnosis

from a drop of blood, urine, or cerebrospi-nal fluid in leprosy or tuberculosis has beenthe dream of many investigators. It wouldbe of particular use in the following: a) caseswith uncertain diagnosis; b) paucibacillarycases with negative immediate staining orlong-term culture; and c) in a case-findingcontrol program together with, or as an al-ternative to, biological examination.

Such a test would have the following re-quirements: a) species-specific antigens; b)evoke an antibody response in all infectedindividuals, including severely ill patients;and c) evoke a response which wanes rap-

" Ridley, M. J. and Russel, D. An immunoperoxi-dase study of immunological factors in high immuneand low resistance granulomas in leprosy. J. Pathol.137 (1982) 149-157.

" Anderson, R., Gatner, E. M., Van Rensburg, C.E., Grabow, G., lmkamp, F. M., Kok, S. R. and VanRensburg, A. J. In vitro and in vivo effects of dapsoneon neutrophil and lymphocyte functions in normal in-dividuals and patients with lepromatous leprosy. An-timicrob. Agents Chemother. 19 (1981) 495-503.


FIG. 4. Principles of an indirect antibody assay toa species-specific antigen.

idly when infection is no longer active orafter effective vaccination.

In principle, the presence of infectionwould be determined either by detection ofcirculating species-specific antigens or bydetection of circulating antibodies gener-ated by an immune response to species-spe-cific antigens. Clearly, the presence of an-tigen is better proof of infection than anantibody assay, but it is not yet clear wheth-er M. tuberculosis releases sufficient antigento make this a realistic possibility. Even inLL, where the bacterial load is enormous,it has proved remarkably difficult to dem-onstrate mycobacterial antigen in the se-rum, partly because much of it is circulatingas immune complexes."' It is conceivablethat MABs will allow the development ofassays of sufficient sensitivity.

To date, all attempts at serodiagnosis haverelied on detecting circulating antibodies tospecies-specific antigens. This has been per-formed by solid- or liquid-phase bindingassays using enzyme or radiolabeled sec-

ond-layer antibodies (Fig. 4). Such assaysare not dependent upon secondary antibodycharacteristics, such as complement fixationor agglutination, and thus, in theory, candetect all antibody classes which bind to theantigen in question. Unfortunately, no an-tigen has yet been found with epitopes spe-cific to a single species. This was previouslyattributed to the physical characteristics ofmycobacteria, which do not break down intosub-units of predictable molecular weightsand properties." However, the recent MABwork has overturned this view and has de-lineated reproducible antigens for both M.leprae and Al. tuberculosis (Table 1). Yeteven these have crossrcacting epitopes (Fig.5).

Two possible solutions exist. Firstly,species-specific epitopes (e.g., the M. lepraedisaccharide) could be synthesized as shownin Figure 4. An alternative and simpler ap-proach would be the use of whole moleculesin an inhibition assay (Fig. 6).3' 11 17

The so-called A/. /eprae-specific, phenolicglycolipid-I (PGL-I), as aforementioned, hasbeen investigated by MABs 38 and serolo-gy."' 45 Both methods have revealed that theterminal saccharide is the most importantepitopc. One group has recently synthesizedthe terminal disaccharide and conjugated itto bovine serum albumin (BSA)." Prelim-inary results in screening sera from healthycontrols, tuberculosis patients, and leprosypatients have, however, shown some cross-reactivity between mycobacterial species.This has been attributed to nonspecific BSAbinding, but this seems unlikely since theMABs also show species crossreactivity. Inaddition to this, there are two other prob-lems with such assays. Firstly, when the im-mune response of an outbred species suchas man is presented with an immenselycomplex antigenic structure such as myco-bacteria, only some of the thousands of epi-

" Daniel, T. M. and Janicki, B. W. Mycobacterialantigens: a review of their isolation, chemistry andimmunological properties. Microbiol. Rev. 42 (1978)84-113.

86 Fujiwara, T., Hunter, S. W., Cho, S.-N., Aspinall,G. D. and Brennan, P. J. Chemical synthesis and se-rology of disaccharides and trisaccharidcs of phenolicglycolipid antigens from the leprosy bacillus and prep-aration of a disaccharide protein conjugate for sero-diagnosis of leprosy. Infect. Immun. 43 (1984) 245-252.


FIG. 5. A single molecule (antigen) with varyingepitope specificities.

topes evoke an antibody response. Whichones arc selected will depend partly on ge-netic factors and partly on the previouspriming of antibody responses by, for ex-ample, "wild type" mycobacteria. Severalreports illustrate this points'• 88 When serafrom tuberculosis patients were tested againsta range of glycolipids isolated from M. tu-berculosis, different individuals were foundto have responded to different selections ofthese molecules." Thus, a diagnostic assaymay require a battery of species-specific,MAB-defined epitopes. Interestingly, a bankof TB-specific MABs 32 was no more suc-cessful than a single TB-specific MAB 33 inserodiagnostic testing. Neither approach wasparticularly encouraging, achieving 74%positive diagnoses in a highly selected pop-ulation of patients. The recent develop-ments of hybrid hybridomas producing bi-specific MABs may reduce the total bank ofMABs necessary for serodiagnosis, provid-ed the species-specific epitopes occur closetogether on the same molecule. Some ten-tative evidence may exist for this (see Lep-rosy taxonomy).

The second problem with any assay ofantibody titer against infecting mycobac-teria has been with the serum antibody con-centration. Severely ill tuberculosis patientswho are not on treatment have low antibody

" Kaplan, M. H. and Chase, M. W. Antibodies tomycobacteria in human tuberculosis: the response tonine defined mycobacterial antigens, with evidence foran antibody common to tuberculosis and lepromatousleprosy. J. Infec. Dis. 142 (1980) 835-843.

8" Reggiardo, Z., Aber, V. R., Mitchison, D. A. andDevi, S. Hemagglutination tests for tuberculosis withmycobacterial glycolipid antigens. Am. Rev. Respir.Dis. 124 (1981) 21-25.

levels.89 This has been attributed to de-pressed immunity. Conversely, LL patientswith severely depressed CMI have highlevels of circulating antibodies, some ofwhich arc M. leprae specific.'" ) . 91 Further-more, the response to M. tuberculosis is oftendelayed, with the highest titers and thegreatest variety of antibodies occurring sev-eral weeks after the disease is initially di-agnosed. 88 Thus, subclinical diagnosis in ep-idemiological studies may not be possiblefor tuberculosis. Abe's fluorescent antibodyabsorption test has successfully detected M.leprae-specific antibodies in subclinical in-fections. 92 . 91 Clearly, leprosy has betterprospects for serodiagnosis than tubercu-losis.

A recent paper has attempted to hastenthe search for new M. leprae-specific MABsby testing LL sera against M. leprae com-ponents of known molecular weights de-rived from M. leprae sonicates run on SDS-PAGE. 94 Six of these were only identified

8° Kaplan, M. H. and Chase, M. W. Antibodies tomycobacteria in human tuberculosis; the developmentof antibodies before and after antimicrobial therapy.J. Infect. Dis. 142 (1980) 825-834.

"O Levinson, A. I., Lisak, R. P. and Zweiman, B.Immunologic aspects of leprosy. Int. J. Dermatol. 16(1977) 103-112.

"' Harboe, M. Significance of antibody studies in lep-rosy and experimental models of the disease. Int. J.Lepr. 50 (1982) 342-350.

92 Abe, M., Minagawa, F., Yoshino, Y., Ozawa, T.,Saikawa, K. and Saito, T. Fluorescent leprosy antibodyabsorption (FLA-ABS) test for detecting subclinical in-fection with Mycobacterium leprae. Int. J. Lcpr. 48(1980) 109-119.

Abe, M., Ozawa, T., Minagawa, F. and Yoshino,Y. Subclinical infection in leprosy—its detection andcontrol by fluorescent antibody absorption (FLA-ABS)test. Lepr. Rev. 52 Suppl. (1981) 263-273.

9" Klatscr, P. R., van Rens, M. M. and Eggelte, T.A. Immunochemical characterization of Mycobacte-rium leprae antigens by the SDS-polyacrylamide gelelectrophoresis immunoperoxidase technique (SGIP)using patients' sera. Din. Exp. Immunology 56 (1984)537-544.

630^ International .Thurnal of Leprosy^ 1985

by sera from leprosy patients and not bysera from normal controls. Obviously, thisstudy needs to be extended to include serafrom the whole spectra of leprosy and tu-berculosis. Such M. leprae-specific antigensmay then be useful for generating MABs forimmunodiagnosis.

Hopefully, the creation of human MAI3swill reveal a species-specific epitope whichwill react with more than 95% of patientsera. Then we may have a truly useful tool.

TreatmentImmunomodulation. Several theories have

been proposed to explain the deficient CM Iresponse to mycobacteria observed in somepatients with TB or leprosy. The evidencefor and against these hypotheses is outlinedbelow.

Ts cells. Bloom, et al., in a series of well-controlled experiments, have shown thatexposure of peripheral blood mononuclearcells from lepromatous (LL) or borderline(BL) patients to Dharmendra lepromin (D.lepromin) in vitro consistently resulted inthe suppression of allogenic lymphocytetransformation in response to concanavalinA (a polyclonal T-cell activator)." The sup-pressor activity was shown to originate frommonocytes and OKT8 phenotype T cells(Ts/c). 96 ' 97 These Ts/c cells also expressedFc and Ia receptors, indicating an active cellpopulation," 7 although this activity may benonspecific. Removal of the OKT8 phe-notype T cells from blood samples of BLand LT patients allowed proliferation ofOKT4 phenotype T cells in response to D.lepromin. In LL patients, removal of the Tscells did not permit proliferation, suggestingextremely low levels of D. lepromin-re-sponsive Th cells. This group's most recentresults have indicated that some of thesuppression may be mediated through Tscell recognition of the PGL-I terminal disac-

'5 Mehra, V., Mason, L. H., Fields, J. P. and Bloom,B. R. Lepromin-induced suppressor cells in patientswith leprosy. J. Immunol. 123 (1979) 1813-1817.

"' Mehra, V., Mason, L. H., Rothman, W., Reinherz,E., Schlossman, S. F. and Bloom, B. R. Delineation ofa human T cell subset responsible for lepromin inducedsuppression in leprosy patients. J. Immunol. 125 (1980)1183-1188.

°' Mehra, V., Convit, J., Rubinstein, A. and Bloom,B. R. Activated suppressor T-cells in leprosy. J. Im-munol. 129 (1982) 1946-1951.

charide.39 MABs specific for this Al. lepraedisaccharide completely inhibited thesuppression induced by PGL-I. In patientswho have improved following vaccinationwith the new Convit mixture of killed M.leprae and live I3CG, there is an almost totalreduction in both D. lepromin-induced andPGL-I-induced suppression." This suggeststhat the Ts cells reactive with PGL-I arcrelevant to the immunological anergy of LLpatients. Suppression has also been foundagainst 13CG and tuberculin, in the casesmediated by MHC-restricted OKT4 phe-notype cells."

In animal models, BCG-activated Ts cellshave been shown to elaborate an as yet un-characterized nonspecific suppressor factorwhich inhibits in vitro DNA synthesis." Thisis associated with a lack of IL-2 produc-tion."' A lack of IL-2 has been reported inLL" and in Al. lepmentu•ium infections.'"'Thus, it is conceivable that Ts cells, in re-sponse to certain mycobacterial epitopes,secrete a suppressor factor (SF) which mayact directly on Th cells or indirectly on APCs(by reducing IL-2 production and secretion;Fig. 7). There is also skin-test evidence forsuch circulating SFs."" The fact that IL-1production appears to be normal in LLpatients"" suggests that SF may not act onthe APC (but, of course, this is not ruledout). Normal IL-1 levels may also imply

"" Mustafa, A. S. and Godal, T. In vitro inductionof human suppressor T-cells by mycobacterial anti-gens. BCG activated OKT4+ cells mediate suppres-sion of antigen induced T-ccll proliferation. Clin. Exp.Immunology 52 (1983) 29-37.

Colizzi, V., Ferluga, J., Garreau, F., Malkovsky,M. and Asherson, G. L. Suppressor cells induced byBCG release non-specific factors in vitro which inhibitDNA synthesis and interleukin-2 production. Immu-nol. 51 (1984) 65-71.

1 "0 Colizzi, V. In vivo and in vitro administration ofinterleukin 2-containing preparation reverses T-cellunresponsiveness in Mycobacterium bovis BCG-in-fected mice. Infect. Immun. 45 (1984) 25-28.

'"' Haregewoin, A., Godal, T., Mustafa, A. S., Be-lchu, A. and Yemaneberhan, T. T-cell conditioned me-dia reverse T-ccll unresponsiveness in lepromatousleprosy. Nature 303 (1983) 342-344.

10 ' Hoffenbach, A., Lagrange, P. H. and Bach, M.-A.Deficit of interleukin 2 production associated with im-paired T-cell proliferative responses in Mycobacteriumlepraenzurium infection. Infect. Immun. 39 (1983) 109-116.

Nath, I. Immunology of human leprosy—currentstatus. Lepr. Rev. 31 Special Issue (1983) 194-196.


normal antigen presentation even in HLADR3-negative individuals.

If this suppressive mechanism exists, thenthe following roles for MABs can be pre-dicted: a) Bloom, et al. have suggested theuse ofOKT8 MAI3s" either as complementactivators or conjugated to the potent planttoxin ricin."" This would involve the de-struction of the entire circulating Ts/c cellpool. Such a drastic measure might lead tounwanted reactions (e.g., autoimmune dis-ease and viral infections). This is clearly un-desirable and, until we can develop humanMABs to Al. /cprac-specilic Ts cells, suchmethods are unlikely to be used. PerhapsTs cells which bind the Al. /eprac-specificdisaccharide could be isolated and then usedto generate specific MABs. An alternativeapproach may involve the use of recombi-nant DNA technology to isolate the DNAcoding for Ts cell receptor diversity. b)hMAI3s against mycobacterial suppressorepitopes; c) hMABs to purify IL-2. In at leastsome cases, IL-2 extracts have been shownto reverse in vitro suppression seen in LL."However, IL-2 has been used in AIDS pa-tients with mixed success.'"' Another dis-advantage is its short half-life.'"" PerhapsIL-2 will be useful in treating downgradingreactions (leprosy patients who rapidly pro-gress toward the LL pole).

Some discrepancies exist in this Ts cellmodel. Nath, et al. have been unable to showTs cell activity in LL.'" Indeed, suppres-sion was seen at the tuberculoid end of thespectrum. This difference could be attrib-uted to the different M. leprae preparationsused. In addition to these antigenic differ-ences, there was considerable variation be-

'"4 Thorpe, P. E., Edwards, D. C., Davies, A. J. S.and Ross, W. C. J. Monoclonal antibody-toxin con-jugates: aiming the magic bullet. In: Monoclonal An-tibodies in Clinical Medicine. McMichael, A. J. andFabre, J. W., eds. London: Academic Press, 1982, pp.167-201.

'" 5 Lifson, J. D., Benike, C., Mark, D. F., Koths, K.and Engleman, E. G. Human recombinant interleu-kin-2 partly reconstitutes deficient in - vitro immune re-sponses of lymphocytes from patients with AIDS. Lan-cet 1 (1984) 698-702.

'"' Nath, I., van Rood, J. J., Mehra, N. K. and Vai-dya, M. C. Natural suppressor cells in human leprosy:the role of HLA-D-identical peripheral lymphocytesand macrophages in the in vivo modulation of lympho-proliferative responses. Clin. Exp. Immunology 42(1980) 203-210.

tween the patient populations and theirtreatment status investigated by these re-search groups. Probably of most relevanceis the antigen overload, particularly ofPGL-I, which occurs in LL.' This is likelyto give secondary immune suppression, 107

and raises the important question of wheth-er or not the T suppression observed byBloom, et al. is a primary or secondary phe-nomenon. Rook, et al. have indicated thatleprosy patients only respond to the species-specific mycobacterial antigens (group iv).This is suggested from delayed-type hyper-sensitivity (DTH) responses to dermal chal-lenge with various mycobacteria. "'t• ")9 LLpatients do not respond to M. lcprae anti-gens but following treatment, many patientsrespond to antigen preparations from othercrossreactive species. If DTH correspondsto CM I, then the Ts cells specific to M. lep-roe determinants may not be relevant, andwe will need to look for the mechanism ofsuppression of common mycobacterial an-tigens. It seems unlikely that both types ofsuppression exist and may be important indiffering circumstances. ' 1 "- ' 13 Again, MABscould be of great use in defining and ana-lyzing the roles of group i and group iv an-

117 Playfair, J. H. L. Workshop report: suppressorcells in infectious disease. Parasite Immunol. 4 (1982)299-310.

1 " Stanford, J. L., Nye, P. M., Rook, G. A. W., Sam-uel, N. and Fairbank, A. A preliminary investigationof the responsiveness or otherwise of patients and staffof a leprosy hospital to groups of shared or species-specific antigens of mycobacteria. Lepr. Rev. 52 (1981)321-327.

"" Rook, G. A. W. and Stanford, J. L. The hetero-geneity of the immune response to mycobacteria andthe relevance of the common antigens to their patho-genicity. Ann. Immunol. (Paris) 132D (1981) 155-164.

"" Nye, P. M., Price, J. E., Ravenkar, C. R., Rook,G. A. W. and Stanford, J. L. The demonstration of twotypes of suppressor mechanism in leprosy patients andtheir contacts by quadruple skin testing with myco-bacterial reagent mixtures. Lepr. Rev. 54 (1983) 9-18.

"' Morton, A., Nye, P. M., Rook, G. A. W., Samuel,N. and Stanford, J. L. A further investigation of skintest responsiveness and suppression in leprosy patientsand healthy school children in Nepal. Lepr. Rev. 55(1984) 273-278.

"2 Nye, P. M., Stanford, J. L., Rook, G. A. W., Law-son, P., MacGregor, M., Kelly, C., Humber, D., Drege,P., Revankar, C. R. and Torres, P. Suppressor mech-anisms and fractionated antigens of Mycobacteriumvaccae investigated in East Africa, India and Spain.(submitted for publication to Lepr. Rev., 1984)

Ralf, M. C. Do antigen presenting cells distinguishself from non-self? Nature 298 (1982) 791-792.


Fui. 7. Possible targets of a T-suppressor factor.T,, = T-suppressor fiictor (see Figure 3 for other ab-breviations).

tigens on slow- and fast-growing mycobac-terial species.

Nlacrophage-Th cell axis. A macrophagedefect may reside in the failure to process,present, destroy, or elaborate factors in lep-rosy or tuberculosis. Little is known aboutthe mechanisms of antigen processing."'MABs can be used in pulse-chase experi-ments to detect what happens to individualepitopes as they are processed. This mayreveal which epitopes are selected for pre-sentation (and how they are modified) andwhich are discarded. Perhaps antigen pro-cessing is not required at all."' It is possiblethat only certain macrophage subpopula-dons are capable of correct presentation.These subpopulations may be analyzed byMABs. 76 MABs generated against sulfa-tides"' may reveal the details by which M.tuberculosis and M. leprae prevent phago-some-lysosome fusion and Al. leprae canescape from phagosomes and exist withinthe cytosol.' ''' Armed with such knowledge,we may hope to develop new therapies, per-haps by encouraging lysosome-phagosomefusion.

Kaye, P. M., Chain, B. M. and Feldman, M. Ac-cessory cell heterogeneity in anti-mycobacterial re-sponses. Immunobiol. 167 (1984) 155-156.

' 1 ' Goren, M. B., D'Arcy Hart, P., Young, M. R. andArmstrong, J. A.Prevention of phagosome lyosome fu-sion in cultured macrophages by sulfatides ofbacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A.73 (1976) 2510-2514.

'''' Lowrie, D. 13. Mononuclear phagocyte-mycobac-terium interaction. In: The Biology of the Mvcobacter-ia. Ratledge, C. and Stanford, J. L., eds. London: Ac-ademic Press, 1983, vol. 1, pp. 235-278.

At present there is no concrete evidenceof a specific macrophage defect."' An as-sociation between HLA DR3-negative in-dividuals and unresponsiveness to Al. lep-rae has been claimed." 4 However, thisgroup's latest results confuse the matter-HLA DR3-negative macrophages presentbetter than FILA DR3-positive macro-phages."` Nath, el al."' and other workers"have described a macrophage-derived sup-pressor factor. If this exists, then MABs di-rected against it would block unwantedsuppression.

A hole in the Th cell repertoire is unlikelyfor several reasons, and it seems improbablethat Th cells are unresponsive to all Al. lep-rae antigens. The development of MABs,T-cell clones, and recombinant DNA tech-nology may all help to solve the unansweredproblem of T-cell restriction and, hence, theongoing immunological argument of deter-minant selection versus hole in repertoire.

Idiotypes. Collizzi, et al. have recentlypublished exciting work demonstrating thepossible role of idiotypes in regulating theimmune response in tuberculosis and lep-rosy."' Mice heavily infected with BCGproduced antibodies which specifically in-hibited DTH to TB purified protein deriv-ative (PPD) in transfer experiments. Therewas some evidence to suggest that these an-tibodies bind to T MABs could beused to define which T-cell subpopulationwas involved and might indicate whetheror not the T-cell receptor or its associatedmolecules are the target of anti-idiotypes inmycobacterial suppression. Daus, et al.' 20

have shown that antibody subclass is im-portant in determining suppression or stim-ulation of T and B cells in immune re-sponses to BCG in guinea pigs. The

' '' Rook, G. A. W. The immunology of leprosy. Tu-bercle 64 (1983) 297-312.

''" van Eden, W., Elferink, II. G., de Vries, R. R. P.,Leiker, D. L. and van Rood, J. J. Low T lymphocyteresponsiveness to Mycobacterium leprae antigens inassociation with HLA-DR3. Clin. Exp. Immunology55 (1984) 140-148.

Ferluga, J., Colizzi, V., Ferrante, A., Colston, M.J. and Holborow, E. J. Hypothesis: possible idiotypesuppression ofcell-mediated immunity in lepromatousleprosy. Lepr. Rev. 55 (1984) 221-227.

"=" Daus, H., Hammer, H. J., Rajki, K. and Mauch,H. Influence of subclass-specific anti-idiotypic anti-bodies on the kinetics of the immune response to I3CG.Immunology 52 (1984) 697-702.


relationship between Gm allotypcs andidiotypes''' may provide another geneticlink with the susceptibility of individuals.MAI3s against Gm allotypcs may help todefine this. Another potential role of idio-types would be to mimic antigen, and thusstimulate a host immune response."' Sim-ilarly, idiotypes resembling the Ts-cell re-ceptor might allow the host to overcomedeleterious suppressor mechanisms.

MARs could, of course, be developed toidiotypes or idiotopes in order to modulatethe immune response in a beneficial way. Itseems likely that several mechanisms ofsuppression are at work in mycobacterialdisease, and MARS will play a major rolein analyzing these. However, for the pres-ent, the use of MARS in the treatment ofpatients cannot be foreseen, not only be-cause of our lack of understanding but alsobecause of the difficulty in transport (refrig-eration) and administration. A vaccine isthe best hope for the Third World.

Prophylaxis. Traditionally, antibodieshave been used to provide passive immu-nity. Clearly, in leprosy and tuberculosis,where the organisms are concealed from cir-culating antibodies, such prophylaxis is use-less. The ideal vaccine for developing coun-tries should have the following properties:a) inexpensive to produce, b) provides fullimmunization after one dose, c) easily dis-tributed and administered (e.g., no refrig-eration required), and d) has no side effects.

MARs in conjunction with recombinantDNA technology are being used to make anew tuberculosis vaccine. The details andprospects for this are outlined in the section,Prophylaxis—new vaccines.

PrognosisMany different factors may relate to the

prognosis of mycobacterial disease. For ex-ample: a) There may be changes in antibodysubpopulations (e.g., circulating idiotypesagainst Th cells) which may affect the prog-nosis." b) Many patients with LL have CICbut do not suffer ENL. MARS may be usedto analyze which CICs correspond withENL. Recent work has indicated that the

1 = 1 13inz, H., Wigzell, H. and Bazin, H. T cell idio-types are linked to immunoglobulin heavy chain genes.Nature 264 (1976) 639-642.

number of mycobacterial constituents inthese complexes is rather restricted and, todate, only one protein antigen (67KD) hasbeen identified,'" possibly that recognizedby MAR 11H9. 35 c) Serum markers may bediscovered (e.g., SF). d) It may become pos-sible to measure the proportion of M. lep-rae - or M. tuberculosis - specific Ts cells.

Thus, MARS will provide both the defi-nition of disease markers and the precisemethods of monitoring changes in theirlevels.

RECOMBINANT DNA TECHNOLOGYRecombinant DNA technology or genetic

engineering is legally defined as "the for-mation of new combinations of heritablematerial by the insertion of nucleic acidmolecules, produced by whatever meansoutside the cell, into any virus, bacterialplasmid, or other vector system so as toallow their incorporation into a host organ-ism in which they do not naturally occurbut in which they are capable of continuedpropagation."

The historical background for genetic en-gineering is difficult to trace because of itsmultiple origins within science. For exam-ple, the discovery of useful restriction en-donucleases came from virologists in1970, 123124 transformation from bacteriol-ogists in 1970,'" and effective DNA sepa-ration by agarose gel electrophoresis frombiochemists in 1977.' 2" Some of the rele-vant principles of this technology are briefly

1 " Chakrabarty, A., Maire, M., Saha, K. and Lam-bert, P.-H. Identification of components of IC purifiedfrom human sera. II. Demonstration of mycobacterialantigens in immune complexes isolated from sera oflepromatous patients. Clin. Exp. Immunology 51 (1983)225-231.

1 " Smith, H. 0. and Wilcox, K. W. A restrictionenzyme from Ilemophilus iMluenzae. I. Purificationand general properties. J. Mol. Biol. 51 (1970) 379—39 I.

12 ' Kelly, T. J. and Smith, H. O. A restriction enzymefrom Ilemophilus MIluenzae. II. Base sequence of therecognition site. J. Mol. Biol. 51 (1970) 393-409.

1 " Mandel, M. and Higa, A. Calcium-dependentbacteriophage DNA infection. J. Mol. Biol. 53 (1970)159-162.

Johnson, P. H. and Grossman, L. I. Electropho-resis of DNA in agarose gels: optimizing separationsof conformational isomers of double-stranded and sin-gle-stranded DNAs. Biochem. 16 (1977) 4217-4225.


TABLE 3. Properties of classes of vectorsfor recombinant DNA technology.

Maximum sizeof foreign Size of vectorVector class^DNA insert DNA (kb)in kilobases

(kb)

Plasmid^15^3-15Phage^25^50Cosmid^50^25

discussed below and more comprehensivereviews may be found elsewhere.'''

The principlesDNA isolated from bacteria must first be

broken into manageable lengths using re-striction endonuclease enzymes. These en-zymes form the basis of our present-dayDNA technology. Over 200 have been de-scribed, and there is now a complicated clas-sification system. The restriction endonu-clease is able to precisely cut DNA at aspecific site and does this reproducibly. Thefragments arc then cloned to form a geno-mic library. Cloning involves the bulk pro-duction ofeach DNA fragment isolated. Thisis achieved by inserting the DNA into vec-tors which are taken up by dividing cells(e.g., Escherichia coli). The DNA fragmentis then copied whenever the cell divides.Three vector classes have been described,and their properties are summarized in Ta-ble 3.

The advantages and disadvantages ofthese vectors have been reviewed.''' In or-der to distinguish between replicating cells(for example, between E. coil containing thevector with the DNA fragment and E. colicontaining the vector alone, or no vector),certain characteristics such as penicillin re-sistance may be pre-incorporated into thevector (Fig. 8). This principle is similar tothe selection of MAB-producing hybrid-omas with HAT medium. Because of thevery rapid growth of E. coli (even if onlyone organism in 10,000 has taken up a re-combinant plasmid), mass cultures consist-ing exclusively of a specific small subpop-ulation can be produced in a matter of days.

'" Old, R. W. and Primrose, S. B. Principles of GeneManipulation. 2nd ed. Oxford: Blackwell ScientificPublications, 1981.

GRoW IA AMPICILLIN —v. NON INFECTED BACTERIA DIE

GROW IN TETRACYCLINE—v. ENSURES GROWTH STASIS OFRECOMBINANT BACTERIA

ADD POISVN FUR^--■ DIVIDING BACTERIA OITHDIVIDING BACTERIA^REPAIRED PLASMID DIE

JRE. CULT WITH kECOMBINANT PLASMID SURVIVES

i. 8. Principles involved in the selection of re-combinant plasmids. A = ampicillin resistance; T =tetracycline resistance.

It is now possible to mix the collection ofrandom fragments of total genomic DNAwith vector DNA (e.g., plasmid) in linearform. Physical conditions are adjusted togive the maximum likelihood of closure ofeach plasmid circle by insertion of just onegenomic fragment. Thus, a heterogeneouspopulation of E. coli will be produced con-taining plasmids with different insertedfragments. Conventional bacteriologicalprocedures, however, allow colonies of E.coli to be grown from single organisms, andif all of the original DNA has been incor-porated, virtually every DNA fragment fromthe original mixture is represented in the setof isolates forming a genomic library. Suchlibraries now exist for some strains of thetuberculosis complex and M. leprae (per-sonal communication, Dr. T. Coates).

The next, rather tedious, step involvesreading the libraries. The vector DNA isseparated from E. coli DNA by centrifu-gation. Recombinant plasmid DNA is thencleaved by the same restriction enzyme usedfor the insertion step and separated fromthe foreign DNA by agarose gel electropho-resis. The phenomenon of hybridization is


then exploited, in which single-strandedDNA (ssDNA) always pairs with its com-plementary DNA sequence (guanine op-posite cytosine, adenine opposite thymi-dine). In this way, biotin or radiolabeledcomplementary DNA (cDNA) probes of 15base pair length constructed based upon,e.g., amino acid sequences of a particularprotein, can be used to locate the protein-producing DNA. The techniques involvedin locating a desired gene from a clone bankinclude Southern blotting (after Dr. E. M.Southern), where cDNA hybridizes withDNA;'" northern blotting, where RNAprobes hybridize with DNA; ' 2 ' and westernblotting, where antibody binds to proteinproduced from the clone bank.'"

EpidemiologyTheoretically, the most sensitive meth-

ods of detecting differences between speciesand strains of mycobacteria would operateat the genomic level. This problem can beapproached in several ways. Imaeda, et al.have used whole genome hybridization toanalyze species homology."' A more sen-sitive method involves DNA fragmentationby restriction endonucleases and separationinto electrophoretic DNA patterns. Eachmycobacterial species would then have itsown DNA "fingerprint." However, differ-ences may occur in non-coding parts of thegenome. Thus, these tests may suffer frompoor specificity.

One group working in New Zealand hasrecently published a preliminary report us-ing the latter method.' 32 While they could

12 " Southern, E. M. Detection of specific sequencesamong DNA fragments separated by gel electropho-resis. J. Mol. Biol. 98 (1975) 503-517.

1211 Alwine, J. C., Kemp, D. J., Parker, B. A., Reiser,J., Renart, J., Stark, G. R. and Wahl, G. M. Detectionof specific RNAs or specific fragments of DNA by frac-tionation in gels and transfer to diazobenzyloxy-methylpaper. Methods Enzymol. 68 (1979) 220-242.

"° Thomas, P. S. Hybridization of denatured RNAand small DNA fragments transferred to nitrocellulose.Proc. Natl. Acad. Sci. U.S.A. 77 (1980) 5201-5205.

" 1 Imaeda, T., Kirchheimer, W. F. and Barksdale,L. DNA isolated from Mycobacterium leprae: genomesize, base ratio and homology with other related bac-teria as determined by optical DNA-RNA reassocia-tion. J. Bacteriol. 150 (1982) 414-417.

'" Collins, D. M. and De Lisle, G. W. DNA restric-tion endonuclease analysis of Mycobacterium tuber-culosis and Mycobacterium bovis BCG. J. Gen. Micro-biol. 130 (1984) 1019-1021.

successfully distinguish M. tuberculosis fromM. boris, they were unable to demonstrateany difference between Al. tuberculosisstrains H37Ra and H37Rv (17 types of re-striction enzymes were tried). If strain dif-ferences reside in single amino acid substi-tutions, then only full DNA sequencing willallow distinctions to be made. Such substi-tutions may determine infectivity andpathogenicity of mycobacteria.

In addition to studying mycobacterialvirulence, recombinant DNA techniques canbe used to determine the genetics and mech-anisms of host susceptibility. Indeed, T-cellreceptor research has been greatly advancedby genetic engineering, and the genes codingfor part of this elusive receptor have beenrevealed.' 33- ' 35 While we are close to un-derstanding the generation of Th-cell recep-tor diversity, we still do not possess the mo-lecular explanation for T-cell restriction orthe nature of the Ts-cell receptor. Such an-swers have far-reaching implications, notonly in terms of host susceptibility but alsofor the development of effective vaccines.' 36

Recombinant DNA technology could beused to analyze interesting phenomena, forexample, a) the relationship between cory-nebacteria and Al. leprae. Preliminary workhas shown great DNA homology betweencorynebacteria isolated in LL patients andM. leprae.' 31 Certainly, corynebacteria andM. leprae ribosomes are closely related."'. '' sb) Why is M. leprae more pathogenic thanM. tuberculosis? Despite numerous claimsof in vitro growth of M. leprac, none has yetbeen substantiated.' 37 Research into thebiochemical pathways ofM. leprae has beenhampered by the small numbers of bacteriaavailable from in vivo growth systems (250mg dry weight bacteria from 50 g infectedtissue) and the impure samples obtainedeven with NaOH extraction.' 37 Genetic en-

1 " Williams, A. F. The T-lymphocyte antigen re-ceptor—elusive no more. Nature 308 (1984) 108-109.

'" Robertson, M. T-cell antigen receptor—the cap-ture of the snark. Nature 312 (1984) 16-17.

1 " Owen, M. J. T-cell receptor companions. Nature312 (1984) 406.

11" Mitchison, N. A. Immunology—rational designof vaccines. Nature 308 (1984) 112-113.

'" Wheeler, P. R. Metabolism in Mycobacteriumleprac: its relation to other research on M. leprac andto aspects of metabolism in other mycobacteria andintracellular parasites. Int. J. Lcpr. 52 (1984) 208-230.

636^ into-natio/7a/ Journal of Leprosy^ 1985

gineering could be used to generate largequantities of pure enzymes ofanalysis. Thus,differences between M. leprae and host met-abolic pathways may be discovered, pavingthe way for the development of new ther-apeutic approaches and convenient culturemedia.

DiagnosisIn diagnosis, as in many other fields of

mycobacterial research, recombinant DNAtechniques have yet to be fully exploited.Perhaps they could be used in conjunctionwith MABs as the basis of serodiagnostictests (an example of this already exists forhepatitis B)." 8 Alternatively, cDNA probesmight be synthesized to detect circulatingbacterial DNA. At present, Stanford, et a!.are attempting to provide evidence for amycobacterial etiology of Crohn's diseaseby using cDNA probes in tissue biopsy spec-imens. However, these methods are notstraightforward and are unlikely to be ap-propriate for the development ofa clinicallyapplicable blood test. If mycobacterial DNAdoes circulate in the blood, it will be difficultto determine the relative importance of thevarious fragments and their specificity indifferent individuals. This is a problem notdissimilar to that encountered with sero-diagnosis.

TreatmentRecombinant DNA technology is a rel-

atively inexpensive method of generatingprotein products in bulk. There are a num-ber of obvious applications for this (for ex-ample, IL-2 or IFNs). The role of IL-2 hasalready been discussed. Kiderlen, et a!. haveprovided some tentative evidence that re-combinant y-IFN induces mouse macro-phage killing of Listeria monocytogencs invivo. 1 39 The immune response to L. mono-cvtogenes and its cellular localization arevery similar to those of M. leprae and M.tuberculosis. It would be interesting to ex-amine the role of y-IFN in mycobacterialdisease.

' '" Murray, K. New routes to drugs, diagnostic agentsand vaccines. Lancet 2 (1984) 1194-1198.

Kiderlen, A. F., Kaufmann, S. 11. E. and Loh-mann-Matthes, M. L. Protection of mice against theintracellular bacterium Listeria monocytogenes by re-combinant immune interferon. Eur. J. Immunol. 14(1984) 964-967.

Foreign DNA transfer into human cellsto promote resistance. The possibility of in-serting useful foreign DNA into the humangenome has excited scientists for many years.The best example of such a technique isprovided by Palmiter's "super mouse."" )

A DNA fragment containing the promotorof the mouse metallothionein I gene fusedto the structural gene of the rat growth hor-mone (GI-I) was micro-injected into thepronuclei of fertilized mouse eggs. Severalof these transgenic mice had extraordinarilyhigh levels of serum GH and grew into giantmice. Second-generation mice had variablegene expression. Thus, the stability of thesegenes in vertical transmission is question-able. It seems very unlikely that there willever be a requirement for embryo therapy,but the possibility of using control se-quences, such as the metallothionein pro-motor, to correct gene disorders or to alterimmune imbalances in isomatic cells of af-fected patients is very exciting. Such cor-rective procedures might be achieved usingviral vectors. For example, the nucleotidesequence of a protein antibiotic capable ofprotection against mycobacterial diseasecould be inserted into viral DNA and usedas a vaccine. Depending on the virus used,the antibiotic coding DNA could be insertedinto the host genome (retro viruses) or rep-licated within the host cytosol (vaccinia). Asingle dose ofrecombinant virus might thenbe able to protect the host for life.

Clearly, there are many problems to besurmounted both technologically and ethi-cally. Until we can precisely insert and re-move DNA from animals"' without oth-erwise causing harm (e.g., neoplasia' 43 ), suchprocedures cannot be performed in man.

Prophylaxis—new vaccines. The devel-opment of an effective, cheap vaccine against

Palmiter, R. D., Brinster, R. L., Hammer, R. E.,Trumbauer, M. E., Rosenfeld, M. G., Birnberg, N. C.and Evans, R. M. Dramatic growth of mice that de-velop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300 (1982) 611—615.

Cooke, A., Lydyard, P. M. and Roitt, 1. M. Au-toimmunity and idiotypes. Lancet 2 (1984) 723-724.

" Lerner, R. A. Synthetic vaccines. Sci. Am. 248(1983) 48-56.

Palmiter, R. D., Wilkie, T. M., Chen, H. Y. andBrinster, R. L. Transmission, distortion and mosai-cism in an unusual transgenic mouse pedigree. Cell 36(1984) 869-877.


mycobacterial disease would have a tre-mendous impact, particularly in South In-dia where BCG has failed to provide ade-quate levels of protection against eithertuberculosis or leprosy.s The reasons for thisfailure are not clearly understood. However,there is increasing evidence that pre-sensi-tization to certain "wild-type" mycobacte-ria may induce suppression which is en-hanced or unchanged with BCG, M.tuberculosis, or AI. leprae. 1 " 8 - " 2 Thissuppression is probably against the com-mon mycobacterial antigens (group i).

Developments in molecular immunolo-gy, immunochemistry, and recombinantDNA technology have provided the foun-dations for the production of a new gener-ation of vaccines. Many of the antigens as-sociated with immune disorders in infectiousdiseases are attributed to protein molecules.Clearly, knowledge of the molecular fea-tures responsible for the antigenicity of pro-tein molecules has at the basis of under-standing, in molecular terms, the cellularevents of the immune response. Localiza-tion and synthesis of appropriate epitopesof mycobacteria should afford valuable syn-thetic vaccines which will, in principle, beexpected to have few or no side effects.

Recombinant DNA technology couldtherefore be used to make new vaccines by:1) synthesis of epitopes for direct injec-tion;' 44 2) synthesis of epitopes to be com-bined with a) an adjuvant. Unfortunately,none of the adjuvants used in animals maybe used in man, largely because of local skinreactions. Recently, a new adjuvant (Iscom)derived from tree bark has been discovered.Initial experiments have shown no side ef-fects in animals.'" b) class II molecules inliposomes; 3) insertion of epitope-produc-ing DNA into a vector, e.g., vaccinia virus(see below).

It is difficult for the "vaccinologists" toisolate a mycobacterial antigen capable ofgenerating a beneficial immune response.

"4 Murray, K., Bruce, S. A., Hinncn, A., Wingfield,P., van Erd, P. M., de Reus, A. and Schellekens, H.Hepatitis B virus antigens made in microbial cells im-munize against viral infection. EMBO J. 3 (1984) 645-650.

16 Morcin, B., Sundquist, B., Hoglund, S., Dals-gaard, K. and Osterhaus, S. Iscom, a novel structurefor antigenic presentation of membrane proteins fromenveloped viruses. Nature 308 (1984) 457-460.

The use of MABs has shown that each an-tigen has many epitopes. Just which epitopewill generate the required immune responsein unknown. It may be important to distin-guish between B-cell and T-cell epitopes.Although B-cell responses can be importantin idiotypic regulation, it is the primaryT-cell response which generates CMI againstmycobacteria. Matthews, et al. have createda set of T-cell clones to the MAB-purifiedTB68 antigen.'" At least three epitopes havebeen defined which are capable of inducingMAF release from the T-cell clones. One isspecies specific; one, partially specific; andone, broadly cross-relative (corroboratingthe antibody work mentioned above). Un-fortunately, all of the clones analyzed wereselected for helper activity. It would havebeen interesting to investigate the suppres-sor/cytotoxic subset for epitopes triggeringsuppression on the TB68 antigen. If no suchsuppressor epitopes exist, then we may havean antigen which could promote a beneficialimmune response.

Coates, et al. (personal communication)are now developing a new TB vaccine usingrecombinant DNA technology. At present,they are trying to amino acid sequence theTB68 and TB72 antigens. Thus, an N-ter-minal, 15-base pair, cDNA probe could besynthesized in an attempt to isolate the genescoding for these antigens. Unfortunately,such oligo-nucleotide probes are affected bydegeneracy of the code (e.g., serine has fourcodons). Thus, all the possible probes mayhave to be synthesized or, alternatively, anamino acid sequence with least degeneracyselected. Furthermore, short probes arelikely to give rise to more false-positives(i.e., binding to irrelevant genes with similarhomology). Despite these failings, thismethod is probably the most rapid to detectthe gene (providing it is present in the clonebank) producing the antigen required. Hav-ing isolated this gene, Coates, et al. intendto insert it into modified vaccinia virus foruse as a live vaccine (Figs. 9 and 10).

Bloom and colleagues have approachedthe problem in a slightly different way (per-sonal communication, Dr. T. Coates) (Fig.

Matthews, R., Scoging, A. and Rees, A. D. M.Mycobacterial antigen-specific human T-cell clones se-creting macrophage activating factors. I mmunology 54(1985) 17-23.


A.R.M. COATIS et 11^ 5.R. BLOOM et al

TB 68 1 7

I2 ANTIGENS^ M. LEPRAE ANTIGEN

N-TERMINAL AMINO ACIDSEQUENCING

Î^ GENERATING MABTO ANTIGEN

TB68^TB72

^

5 AMINO ACIDS-N^5 AMINO ACID-N

IOLIGONUCLEOTIDE

RADIOLABELLED PROBESYNTHESIS

Î^PROBLEMS^WESTERN BLOT WITHWITH^---. EXPRESSED PRODUCTS

15 BASE PAIRS EACHÊXPRESSION^FROM M, LEPRAE GENOMIC

Î ^CLONE BANK (PHAGE X 9t11IN E.^COLT)

SOUTHERN BLOTTING WITHTB GENOMIC CLONE BANK(PLASMID PUN 290 INE.^COLII

IISOLATE PLASMID(S) WITH^ ISOLATE PHAGE(S) WITHDNA CODING FOR ANTIGEN^ DNA CODING FOR ANTIGEN

EXCISE DNA

IINSERT INTO VACCINIA VIRUS IN VIVO

ISELECT FOR RECOMBINANT VACCINIA VIA

GROWTH MEDIA

MASS CULTURE FOR VACCINE PRODUCTION

ITEST PRODUCT IN ANIMALS--, NUMANS

FIG. 9.^Stages involved in vaccine production usingrecombinant vaccinia virus.

infectious but not pathogenic. The advan-tages of such a vaccine include: a) economyof mimulacture (only a few cents per doseduring the smallpox campaign); b) stabilityin dried form without refrigeration, en-abling easy transportation in tropical coun-tries without loss of potency; c) simpleadministration, e.g., jet gun for use by rel-atively medically unskilled field workers; d)large DNA capacity, allowing multiple an-tigen expression and thus polyvalent vac-cines; and e) generation of both CMI andhumoral responses.

To date, vaccinia virus recombinants ex-pressing the hepatitis B virus surface anti-gen,' 4" 4 " influenza virus hemaggluti-nin, 1504 151 herpes simplex virus (HSV)glycoprotein D,' 4" Plastnodiunz knowlesisporozoite antigen' 52 and, most recently, ra-bies virus glycoprotein'" have been con-structed. Moreover, single intradermal vac-cinations with these recombinants haveeither protected or prevented clinical signsof infection with hepatitis B,' 54 influenza, ' 5 °and rabies' 53 in animals. Intraperitoneal in-

'" Smith, G. L. and

vectors have capacity for at least 25,000 base pairs offoreign DNA. Gene 25 (1983) 21-28.

"8 Smith, G. L., Mackett, M. and Moss, B. Infectiousvaccinia virus recombinants that express hepatitis Bvirus surface antigen. Nature 302 (1983) 490-495.

1 " Paoletti, E., Lipinskos, B. R., Samsonotf, C.,Merger, S. and Panicalli, D. Construction of live vac-cines using genetically engineered poxviruses: biolog-ical activity of vaccinia virus recombinants expressingthe hepatitis B virus surface antigen and the herpessimplex virus glycoprotein D. Proc. Natl. Acad. Sci.U.S.A. 81 (1984) 193-197.

1 ' 11 Smith, G. L., Murphy, B. R. and Moss, II. Con-struction and characterization of an infectious vacciniavirus recombinant that expresses the influenza virushemagglutinin gene and that induces resistance to in-fluenza virus infection in hamsters. Proc. Natl. Acad.Sci. U.S.A. 80 (1983) 7155-7159.

151 Panicali, D., Davis, S. W., Weinberg, R. L. andPaoletti, E. Construction of live vaccines using genet-ically engineered poxvirus: biological activity of re-combinant vaccinia virus expressing influenza virushemagglutinin. Proc. Natl. Acad. Sci. U.S.A. 80 (1983)5364-5368.

'" Smith, G. L., Godson, G. N., Nussenzweig, V.,Nussenzweig, R., I3arawell, J. and Moss, B. Plasmo-dium knowlesi sporozoite antigen: expression by aninfectious vaccinia virus recombinant. Science 224(1984) 197-199.

1" Kieny, M. P., Lathe, R., Drillien, R., Spehner,D., Skory, S., Schmitt, D., Wiklor, T., Koprowski, H.and Lecocq, J. R. Expression of rabies virus glycopro-tein from a recombinant vaccinia virus. Nature 312

Moss, B. Infectious poxvirus (1984) 163-166.

8). Working with a M. leprae clone bank (Agt 1 1 vector), they have used MABs in west-ern blots to detect expression of antigen.Unfortunately, they and others (e.g., R. Cur-tiss, III) have suffered expression problems.Failure of expression can occur as a resultof some of the following: a) an incompletegenomic library leading to the absence of arequired product, b) nonfunctioning pro-motors, c) incomplete genes, d) suppressedgenes, e) toxicity of a gene product to thehost cell, and/or f) instability of a novel pep-tide in the host cell environment. Hopeful-ly, these difficulties with gene expression willbe surmounted, allowing analysis of the M.leprae products. Bloom, like Coates, thenintends to construct a recombinant vacciniavaccine.

Vaccinia for vaccines. Following the erad-ication of smallpox, medical interest in thevaccinia virus has dwindled, but a flood ofrecent papers has revealed a major new rolefor this virus in vaccination. RecombinantDNA technology has been used to recon-struct vaccinia into a "DNA-carrier" oflarger than 25,000' 47 base pairs which is


FIG. 10. Construction of vaccinia virus recombi-nants. R = antibiotic resistance; 0 = origin of bacterialreplication; TK = thymidine kinase; P = vaccinia pro-motor.

jection of the herpes recombinant vaccine' 4 'prevented HSV infection, but inoculationof rabbits with the malarial recombinantvaccine produced variable results.' 52 Somerabbits responded with a high antibody ti-ter; others, with a low titer.

The difficulties encountered with recom-binant vaccinia vaccines include: a) variableexpression of foreign genes. This may beovercome by using stronger promotors orby inserting multiple gene copies. b) risk ofcomplications including mutation into amore virulent virus. In the U.S.A., smallpoxvaccination had a serious complication rateof 1:10 5 (and nine deaths from 14.5 millionvaccinations). Clearly, the risks of the pri-mary disease outweigh the risks of vacci-nations in smallpox. Nevertheless, attemptsto attenuate the virus and to improve vac-cine safety would be welcome.

Vaccinia for tuberculosis and leprosy? Therecombinant vaccinia vaccine clearly fillsthree of the four requirements of a vaccineoutlined above. Exactly how successful such

a vaccine would be in improving the exist-ing BCG is not clear. It may well be thatCoates, et al.'s TB68 and TB72 antigenscontain epitopes generating clinically ben-eficial immune responses (as discussedabove). If, however, the epitopes generatesuppression or a deleterious type of T help(or, indeed, Tc-cell activity), then we mayexpect such a vaccine to fail. A perfect il-lustration of our lack of understanding ofmycobacterial vaccines is provided by Con-vit, ct al. who had empirical success in re-versing LL anergy with a mixture of M. Lep-rac and BCG.'" Exactly why this particularmixture of antigens should be effective isobscure. Hopefully, the use of MABs, T-cellclones, and recombinant DNA technologywill pave the way to a clearer understandingof these processes.

CONCLUSIONThe development of MABs and recom-

binant DNA technology has permitted theexploration of vast new areas of research.In 1984, Köhler and Milstein were awardedthe Nobel Prize for their work with MABs.Do these new techniques offer any hope ofa solution to the mycobacterial misery ofthe Third World?

Present usesThe exquisite specificity of MABs clearly

allows a remarkable opportunity to define,purify, and analyze bacteria and immunecells and their structures and products. Anarray of mouse MABs has now been syn-thesized against Al. lcprac and Al. tuber-culosis. The initial results show that theseare of limited use in taxonomy and sero-diagnosis. By contrast, MAB studies of T-cellfunction in histological sections have pro-vided structural evidence of the immuno-pathology present.

Recombinant DNA technology, whilehaving enormous potential uses, has not asyet produced results. Only a limited numberof mycobacterial clone banks have been

1 " Moss, B., Smith, G. L., Gerin, J. L. and Purcell,R. Live recombinant vaccinia virus protects chimpan-zees against hepatitis B. Nature 311 (1984) 67-69.

1 " Convit, J., Aranzazu, N., Ulrich, M., Pinardi, M.E., Reyes, 0. and Alvarado, J. Immunotherapy with amixture of Mycobacterium leprae and BCG in differentforms of leprosy and in Mitsuda-negative contacts. Int.J. Lcpr. 50 (1982) 415-424.


made; thus only a few strains may be ana-lyzed. The Al. leprae clone banks have not,by and large, expressed proteins successful-ly, and attempts at mycobacterial taxonomyusing restriction enzyme patterns havefailed. Nevertheless, these techniques havebeen ofgreat use in analyzing T-cell receptordiversity, although the final solutions toT-cell restriction and the Ts-cell receptorarc still lacking. Such work is of great rel-evance in trying to comprehend mycobac-terial-induced anergy.

The FutureClearly, much of the existing MAB work

needs to be expanded, and a classificationscheme for the mycobacterial MABs shouldbe introduced. In immunopathology, theimportance of common and species-specificantigens (from fast- and slow-growing my-cobacteria) in triggering suppressor circuitsmay be examined with MABs. Further-more, selective inhibition of one suppressormechanism with MABs may allow analysisof the relative effects of other suppressormechanisms. Temporal studies comparinghealthy populations, exposed groups, andthose with disease may help to resolvewhether or not the CMI deficit in myco-bacterial disorders is the primary cause ofthe pathology or a secondary result of otherprocesses. The possibility of immunomod-ulation with MABs is also speculative andwill depend on both a better understandingof the immunopathology and the develop-ment of hMABs (MABs raised from otherspecies may induce graft versus host re-sponses). Of course, any MAB destined forhuman use would undergo extensive testingfor undesirable crossreactions with othertissues.

Recombinant DNA technology will pro-vide for "in bulk" generation of uncontam-inated M. leprae and M. tuberculosis prod-

ucts. In this way, we may be able to derivefurther knowledge of both the external andinternal biology (e.g., metabolic pathways)of these organisms and, hence, develop newtherapeutic approaches and, perhaps, thecorrect conditions for in vitro growth of M.leprae.

Genetic engineering techniques are cur-rently providing a new generation of cheapvaccines ideal for use in developing coun-tries. The attempts to construct a recom-binant vaccinia virus expressing M. tuber-culosis and Al. leprae antigens are veryencouraging. However, since we do not un-derstand the nature of the CMI suppressionseen in mycobacterial disease (and againstwhich epitopes this is directed), the successof these vaccines cannot be predicted.

Hopefully, the combined use of MABs,T-cell clones, and recombinant DNA tech-nology will help to solve the puzzles of my-cobacterial disease, and both leprosy andtuberculosis will dwindle into the historicalannals of medical literature.

—Michael J. Seckl

Acknowledgments. I would like to thank Drs. J. L.Stanford, G. A. W. Rook, T. Coates, A. D. M. Rees,Professor N. A. Mitchison, and Barbara Sargeant fortheir kind help in this project.

Note added in proof. Since typing, Krambovitis, etal. (Lancet 2:1984, 1229-1231 and editorial, p. 1254)have published an encouraging preliminary report onthe rapid diagnosis of tuberculous meningitis, basedon an antigen agglutination test. An absorbed rabbitantiserum was generated against one of the M. tuber-culosis plasma membrane antigens. This effectively di-agnosed 17 of 18 patients (the remaining patient wasonly proved positive at autopsy). The reason for thiscrossreactivity was unclear.

MABs would have obvious advantages over the het-eroantisera used in this study. If these results are sub-stantiated, then we may look forward to earlier treat-ment of a disease which is all too often misdiagnosed.

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