INFECTION AND IMMUNITY, Mar. 1979, p. 787-7940019-9567/79/03-0787/08$02.00/0

Vol. 23, No. 3

Induction of Cell-Mediated Immunity to Mycobacteriumleprae in Guinea Pigs

VIJAY MEHRA AND BARRY R. BLOOM*

Department ofMicrobiology and Immunology, Albert Einstein College ofMedicine, Bronx, New York 10461

Received for publication 12 September 1978

Guinea pigs immunized with intact or disrupted armadillo-grown human Myco-bacterium leprae administered in aqueous or oil vehicles were tested with variousdilutions of M. leprae suspended in saline, water-soluble M. leprae extract,purified protein derivative, and a water-soluble extract of normal armadillo tissue.The results demonstrated the following. (i) Under no conditions was any skin testreactivity found to normal armadillo tissue extract. (ii) Positive sensitization toboth M. leprae and its water-soluble extract was achieved by sensitizing guineapigs with M. Ieprae suspended in Hanks solution or saline. Autoclaved M. lepraein Hanks solution or saline inoculated intradermally was an effective immunogen.Oil suspensions or emulsions were effective at sensitization, but appeared to beno better and, in general, slightly weaker, than simple inoculation in aqueoussuspension. (iii) Living BCG failed to reveal a significant adjuvant effect onsensitization to M. leprae. However, cord factor appeared to potentiate slightlythe sensitization to M. leprae in aqueous suspension. (iv) The minimum doserequired for sensitization with M. leprae in aqueous suspension was 55 Ag ofpurified bacilli. (v) Animals inoculated with M. leprae in saline or with M. lepraetogether with BCG showed positive skin test reactivity to the first skin testapplication made fully 1 year after the initial sensitization. The efficacy ofautoclaved, irradiated M. leprae in aqueous, oil-free medium suggests a relativelysafe approach to human vaccination studies.

The possibility for developing a vaccineagainst leprosy has become a feasible one due totwo significant recent findings: first, the availa-bility of significant amounts of human leprabacilli from the armadillo and, second, methodsfor their purification from leprous tissues. Toevaluate the potential usefulness of such purifiedarmadillo-grown human Mycobacterium leprae,it is first necessary to demonstrate that it retainsthe capability of inducing delayed-type hyper-sensitivity in experimental animals under appro-priate conditions which could be used in humansand that such M. leprae preparations fail tosensitize to tissue components. As part of theIMMLEP Program of the -Special Program forResearch and Training in Tropical Diseases ofthe World Health Organization, we have beenable to study the conditions required to fulfillthese requirements in guinea pigs. A variety ofregimens in terms of dose, route of administra-tion, adjuvants, and specificity were studied, andthe results indicate that armadillo-grown andpurified M. leprae have an ability to engenderhigh levels of delayed-type hypersensitivity innormal guinea pigs in the absence of oil adju-

vants under conditions in which no sensitizationto armadillo tissues can be found.

MATERIALS AND METHODSAnimals. Albino guinea pigs weighing 450 to 600 g

were sensitized in groups of four to six animals.Antigens. Soluble extract prepared from normal

armadillo liver, purified M. leprae obtained from ar-madillo tissue, and soluble M. leprae antigen (lots A14,AB19, and 22) were supplied by P. Draper and R. J.W. Rees. The method used for the extraction andpurification of M. leprae is described in the report ofthe Second IMMLEP Task Force meeting (9). BCGwas obtained from the Trudeau Institute (2 x 108viable bacilli per ml). The source of purified proteinderivative (PPD) was the Ministry of Food, Fisheryand Agriculture, Weybridge, Surrey, England. Integrallepromin, at a concentration of 1.4 x 108 bacilli per ml,was obtained from A. Dhople and J. Hanks (JohnsHopkins School of Hygiene and Public Health, Balti-more, Md.), and Mycobacterium vaccae was from J.L. Stanford (Middlesex Hospital Medical School, Lon-don, England).

Vehicles. The various vehicles used for immunizingguinea pigs were Hanks solution, saline, and incom-plete Freund adjuvant (IFA; Difco Laboratories; ba-cilli suspended directly in oil or suspended in saline

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788 MEHRA AND BLOOM

and emulsified in oil). Other adjuvants used werebiodegradable adjuvant (adjuvant 65) with peanut oil(prepared by the method of Peck et al. [6]), cordfactor, and muramyldipeptide (MDP) obtained fromE. Lederer (Centre National de la Recherche Scienti-fique, Institut de Chimie des Substances Naturelles,Gif-Sur-Yvette, France).

Animals received 0.5 mg of M. leprae either in fourfootpads (oil suspensions) or intradermally (0.1 ml/site) in five sites on the flank above the foreleg.

Skin tests. The animals were tested with 6, 0.6,and 0.06 tig of soluble extract from armadillo tissue,purified M. leprae, soluble M. leprae antigen (A14,AB19, or 22), and PPD injected intradermally on theflank per 0.1 ml. Diameters of induration of the testsites were measured at 2, 24, and 48 h, and unlessotherwise indicated, 24-h data are shown. Thicknesswas graded by the following scale: 0, ±, +, ++, +++,where + was considered a positive reaction (3).

In vitro stimulation of peripheral blood lym-phocytes with mitogens and antigens. Mononu-clear cells were isolated from heparinized blood of M.leprae-immunized guinea pigs over Ficoll-Hypaquegradients and cultured in 10% guinea pig serum at adensity of 2 x 105 cells per 0.2 ml in microtiter platesin the presence and absence of optimal doses of con-canavalin A (20 ug/ml), phytohemagglutinin (20 ytg/ml), M. leprae (10 jig/ml), PPD (20,ug/ml), and le-promin (1:10). Plates were cultured for 3 and 4 days,and 18 h before harvest, 1 ttCi of [3H]thymidine wasadded.

Results are expressed as a stimulation index (exper-imental counts per minute/control counts per minute).Enumeration of antigen-sensitive cells. Enu-

meration of antigen-sensitive T-cells was carried outby the virus plaque assay (2). This technique measuresthe ability of antigen-sensitive T-cells to permit rep-lication of vesicular stomatitis virus after they areactivated by the specific antigen. In brief, after periph-eral blood mononuclear cells had been cultured in thepresence and absence of mitogens or antigens in cul-ture tubes for 3 days, they were infected with vesicularstomatitis virus at a multiplicity of 10 plaque-formingunits per cell for 2 h. The excess virus was removedand neutralized with anti-vesicular stomatitis virus.Each sample was plated in three dilutions on L-cells.Plates were overlaid with 1% agar in minimal essentialmedium containing 6% fetal calf serum and incubatedfor 2 days. At the end of the incubation period theplates were fixed and stained to count plaques. Theresults are expressed as plaque-forming cells per 103cells in stimulated cultures above the background inunstimulated control cultures.

RESULTSComparison of sensitization of guinea

pigs with intact or sonically treated M. lep-rae in aqueous or oil vehicles. The initialseries of studies compared the ability of a con-stant amount of M. leprae (0.5 mg) to inducedelayed-type hypersensitivity in normal guineapigs. To assess the degree and specificity ofsensitization, animals so immunized were testedintradermally approximately 1 month after sen-

INFECT. IMMUN.

sitization with intact bacilli, a water-soluble M.leprae extract (A14), tuberculin PPD, and asoluble extract of armadillo liver. In the initialseries, each guinea pig was tested with 6, 0.6,and 0.06 jig intradermally, and reactions weremeasured at 2, 24, and 48 h. As Fig. 1 and 2show, no skin test reactivity was detectable inthis or any subsequent experiments to solublearmadillo tissue extract, indicating that, eventhough M. leprae is likely to be as effective anadjuvant as Mycobacterium tuberculosis, thebacilli are sufficiently pure that too little arma-dillo antigen remains to engender detectablesensitization. When intact bacilli were used forsensitization, significant sensitization after a sin-gle immunization was achieved both to M. lep-rae and to the soluble A14 M. leprae extract at6 and 0.6 tig when the immunizing antigen wasgiven in the absence of an oil vehicle or in an oilemulsion. These results were confirmed in fourindependent experiments. In addition, it is im-portant to note that sensitization to armadillo-

nML 6,ug C PPD 6ugEU A14 6/ig E ARMADILLO 6j"g

SENSITIZATION TEST DIAMETER

Ml/HANKS

TEST THICKNESS

ML / IFAL

SONIC Ml/HANKS r

SONIC Ml/IFA

ML /Adjuv.65

F I

0 4 8 12 16 0 . .....

mm

FIG. 1. Sensitization ofguinea pigs with intact orsonically treated M. leprae (ML) in aqueous or oilvehicles. Each guinea pig received 0.5 mg of intact ordisrupted (sonically treated for 5 min at 30-s inter-vals) M. leprae in four footpads. The first three groupswere immunized with M. leprae suspended in Hankssolution or IFA. The other two groups received M.leprae emulsified in IFA or adjuvant 65. Animalswere tested intradermally with 6 jig of intact M.leprae per 0.1 ml, water-soluble M. leprae extract(A14), PPD, or soluble extract of armadillo liver Imonth after immunization, and reactions were meas-ured at 24 h. Each bar represents the mean of reac-tions obtained with four to six guinea pigs.

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CELL-MEDIATED IMMUNITY TO M. LEPRAE 789

SENSITIZATION

ML /HANKSAUTOCLAVED- d

MMITSUDA ML

TE ST DIAME TERS

ML /SALIN"E _EMULSIFIED c

IFA-fp

ML / FA - p

ML/HANKS- 'dBOOSTED - 2 WEEKS

ML/ HANKS -, d

0 4 8 12mm

FIG. 2. Sensitization ofguine(ML) in aqueous or oil vehicle,munized with 0.5 mg ofintact M.Hanks solution or IFA or with aM. keprae emulsified with IFA.pigs was also immunized with Aat 15 Ib/in2 for 15 min. Each gumg ofM. leprae either in four fcdermally (id) in five sites (5 intro0.1 ml each) on the anterior fai,

=AB19 ZPPD ing on the ability ofM. leprae to sensitize guineaTEST THICKNESS pigs was examined. The results clearly indicated

that autoclaved bacilli, again in contrast to ex-perience with other mycobacteria, were as goodor better sensitizers than bacilli not subjected toautoclaving, as Shepard et al. have found in mice(8).As a result of these experiments, we infer that

the optimal procedure for sensitization of guineapigs to M. leprae is intradermal inoculation ofautoclaved intact bacilli suspended in aqueousmedia in multiple sites.Comparison of route of immunization

and the effect of multiple sensitization in-oculations. As Fig. 3 shows, comparison of theintradermal route with the footpad inoculationroute for sensitizing guinea pigs with intact ba-cilli suspended in aqueous suspension indicatedthat, although both were effective, the intrader-

I mal route appeared to be somewhat more effec-16 *C 0 t + +'--- tive. When animals sensitized to 0.5 mg were

!a pigs with M. leprae boosted at intervals of 1 week to 1 month after-s. Animals were im- initial sensitization, only a very slight increase'leprae suspended in in degree of skin test reactivity was observed,saline suspension of suggesting that a single inoculation was effective

. A group of guinea in inducing delayed-type hypersensitivity inM. leprae autoclaved guinea pigs.,inea pig received 0.5 Use of living BCG and other mycobacte-)otpads (fp) or intra- rial products as adjuvants. Because of theEdermal injections of well-known sensitizing and adjuvant propertiesnkL (Phnne, theo fnrn.,1n

In addition to skin testing with M. leprae, solubleantigen AB19, and PPD, animals in two groups wereskin tested with 0.1 ml of human Mitsuda lepromin(1.4 x 108 bacilli per ml).

grown M. leprae in Hanks solution induced pos-itive delayed-type reactivity not only to arma-dillo-grown bacilli, but also to human integrallepromin (Fig. 2). In contrast to virtually allother mycobacteria previously studied, it ap-pears that M. leprae suspended in Hanks solu-tion gave stronger sensitization than M. lepraesuspended in an oil or oil and water emulsion.

Sonically disrupted bacilli emulsified withIFA were ineffective in sensitizing guinea pigs toany of the antigens, whereas a suspension ofdisrupted bacilli in Hanks solution engenderedsome degree of sensitization to M. leprae. Whena variety of adjuvant protocols were compared,there was little difference in the sensitizationwith M. leprae suspended in IFA or emulsifiedin biodegradable adjuvant 65. In any case, sen-sitization with bacilli suspended either in Hankssolution or saline was invariably as good andgenerally slightly stronger.

Last, although the bacilli are killed by radia-tion, for any human vaccine it is likely to bepreferable to have the bacilli sterilized at a latertime by autoclaving, and the effect of autoclav-

_ML 6ug F PPD 6,gM A14 6M9g EM VACCAE 6ag

SENSITIZATION TEST DIAMETER

MUL HAN KSfp-1X

TEST THICKNESS

Ml/HANKSfp-2X

ML/HANKS.d - 1 X

ML/HANKSi.d.-2X

0 4 8 12 16mm

FIG. 3. Comparison of single or double immuni-zation by the footpad (fp) or intradermal (i.d) route.Guinea pigs immunized on two occasions receivedthe second injection 1 week after the first. ML, M.leprae.

0 + .. ...

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790 MEHRA AND BLOOM

of living BCG and the possibility that an admix-ture of living BCG and killed M. leprae mightbe an adjuvant which could be used in humans,a study was made of the ability of M. lepraetogether with BCG to sensitize guinea pigs. AsFig. 4 shows, guinea pigs immunized with M.leprae together with BCG (either 106 or 107viable organisms) showed a significant degree ofspecific sensitization, but the levels were no bet-ter than those of animals immunized with M.leprae inoculated intradermally in aqueous me-dium alone. Indeed, in other experiments notshown here, there was some evidence of de-creased sensitization when BCG was given to-gether with M. leprae. Thus, in guinea pigs, M.leprae appears to be a significantly strong sen-sitizer such that addition ofBCG fails to enhancesignificantly the degree of sensitization achieved,at least when tests are made at 1 month aftersensitization.

It is clear that animals immunized with M.leprae alone showed marked cross-sensitizationto tubercle antigens and to PPD, as well as toM. vaccae (Fig. 3 and 4). Thus, it must beconcluded that M. leprae shares sufficient cross-reactive antigens with certain other mycobacte-ria that specific sensitization was not found. Inthis regard, it was quite remarkable that thesoluble M. leprae antigen preparations were M.leprae specific in skin tests, demonstrating no

M ML 6,4g m PPD 6ougUA 14 6Eug EMM.VACCAE 6,g

SENSITIZATION TEST DIAMETER

ML/HANKS- .d.

ML+BCG (107)fp

BCG (107)fP i555555M

BCG(106),30dML fp

BCG (106)fp _///

0 4 8 12 16 20 0 + +++mm

FIG. 4. Test of the adjuvant activity of living BCGinjected together with killed M. leprae (ML). Guineapigs were immunized with 106 or 107 BCG and 0.5 mgof M. leprae (either admixed with BCG or given 30days later) by footpad (fp) inoculation. i.d., Intrader-mal.

TEST THICKNESS

skin reactivity in guinea pigs immunized withBCG.Cord factor and MDP previously have been

demonstrated by Lederer and his associates tobe effective adjuvants (1, 4). Consequently,guinea pigs were sensitized with mixtures of M.leprae and cord factor or MDP. The results (Fig.5) indicate that cord factor enhanced sensitiza-tion to M. leprae, as detected by reactivity tothe soluble skin test antigen AB19, while MDPhad no effect on sensitizing ability of M. leprae.In addition, neither adjuvant induced sensitiza-tion to itself. In any case, these results indicatethat cord factor might be a useful adjuvant forsensitization to M. leprae.From these studies with guinea pigs, optimal

sensitization would appear to be achieved byintradermal inoculation of autoclaved M. lepraesuspended in aqueous medium in multiple sites,possibly in the presence of cord factor as anadjuvant.

In addition, since M. leprae fails to grow inguinea pigs, it is not possible to test the impor-tant question of the correlation between de-layed-type hypersensitivity and protectionagainst infection in guinea pigs, and such resultsin animal models will have to derive from eithermice or armadillos. With these reservations inmind, however, the studies with guinea pigs haveindicated that optimal conditions for achieving

C; ML 6,y.g =AB 19 6HR

SENSITIZATION TES- DIAMETER

CORD FACTORI2Mg) -id

OPPD 6jg

TEST THICKNESS

,...... _ML+CORD FACTOR ___............__

2Hgl d

MDP (20g1-id

ML+MDPI2 gL- d

0.5 mg ML -id

0 4 8 12 16 0 + ++mm

FIG. 5. Comparison of selected adjuvants, cordfactor, and MDP as vehicles. Animals were immu-nized intradermally (id) with 0.5 mg of M. leprae(ML) in 2 ug of cord factor or MDP per 0.5 ml andskin tested 1 month later with M. leprae, AB19, andPPD.

INFECT. IMMUN.

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CELL-MEDIATED IMMUNITY TO M. LEPRAE 791

delayed-type hypersensitivity to M. leprae-spe-cific antigens appear to be those conditionswhich could be considered for use in a normalhuman population.Dose response relationship. Guinea pigs

were sensitized by the intradermal inoculationin saline of six different doses of purified M.leprae, and skin tests were performed 2 monthslater with 6 and 0.6 ,g of M. leprae, the solubleAB22 extract of purified M. leprae, and PPD.The results indicate that as little as 55 Isg of M.leprae induced positive reactions to both 6 and0.6 [Lg (data not shown), with clear-cut indura-tion in essentially all animals. Lower doses wereessentially ineffective (Fig. 6).Duration of sensitization. Groups of six

animals were immunized with M. leprae inHanks solution, adjuvant 65, or IFA or M. lepraetogether with BCG. The animals were main-tained without further contact with M. lepraeantigens for a period of 1 year and then skintested with the above battery of antigens. Theresults (Fig. 7) show that at the first skin test 1year after sensitization there was positive reac-:, . Xj

_~ avtA;..=_3 'iPA_&wtivity to Mvi. leprae ana tmat soiuoiparticularly strong in groups inoculeprae suspended in Hanks solutioanimals inoculated with M. lepraeBCG. Immunization with 0.5 mg oHanks solution given on only

=ML 6M8g _AB22 6,ig

ML/HANKS TEST DIAMETER.................lmg ::::------..-. ..

500 Hg

............. .......... .... ........ ...

166, ,,,...55 klg

18 9

69-g

0 4 8 12mm

116 2C

FIG. 6. Effect of immunization do(ML) in saline on sensitization. Skinplied 2 months after sensitization.

JML 6,4g _ AB22 6M8g =PPD 6bug

SENSITIZATION TEST DIAMETER TEST THICKNESS

ML/Hanks-,.

Boosted 15 d

ML/Adj 65

f P.

ML/IFA ...........;

ML+BCGf.1P.

BCG1106) L

0 4 8 12 16 20 0 + +++mm

FIG. 7. Duration of sensitization to M. leprae

(ML). Reactions of the first skin test applied 1 year

after sensitization are shown. i.d., Intradermal; f.p.,footpad.

.e antigen was showed comparable, indeed slightly better sen-d*atedwith M. sitization, when tested for the first time at 1 yearIn and in those (18.5-mm induration to M. leprae; 17-mm indur-together with ation to AB22). Animals inoculated with BCGof M. leprae in alone or with M. leprae in IFA showed weakone occasion erythematous reactions without induration.

These results indicate that intradermal inocula-OPPD 6AL9 tion of M. leprae in aqueous suspension is ca-

pable of inducing long-lasting sensitization toTEST THICKNESS antigens of the lepra bacillus.

r7777777M Histology of the skin test sites. Figure 8shows the histology of representative skin testsites of unimmunized guinea pigs and animals

177777777immunized with M. leprae intradermally andskin tested for the first time 1 year later with 6,ug of purified M. leprae, soluble M. leprae anti-gen (AB22), and PPD.The subepidermal region in immunized ani-

mals showed marked cellular infiltration of pre-dominantly mononuclear cells at the M. leprae

CL and PPD test sites, although some polymorpho-nuclear cells were present, particularly in thePPD site. Cellular infiltrate at the AB22 test sitewas less intense but almost entirely mononuclearin composition. Clearly the histological pictureis consistent with classical delayed-type hyper-sensitivity reactions.In vitro assay of antigen responsiveness.

Antigen-sensitive cells in the peripheral blood of) O+ti-+- guinea pigs immunized with M. leprae were

0 + ++ +++

assessed by: (i) incorporation of [3H]thymidinese of M. leprae by the stimulated lymphocytes and (ii) enumer-i tests were ap- ation of antigen-sensitive cells by the virus

plaque assay. Table 1 shows the means of stim-

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INFECT. IMMUN.792 MEHRA AND BLOOM

:

*1'~~~~~~~~~~~~~~~~o

A| *T

: _

I '

I

tX0

C ?t.N,,,

*~ ps L *

v ~~~* v' 4 , A

F~~ ~~ Ce*a r .

- Z b S Or,-;AN fw -'J

F , L **r..

.'V set

* V4 '- *S.

p ~~~ *

. ,,.. I'

,o to ,* IJ~ *. I *t.st

FIG. 8. Histological appearance of skin test sites of M. leprae-sensitized guinea pigs tested for the firsttime at 1 year. The immunized animals received 0.5 mg ofM. leprae in saline at six intradermal sites. The 24-h reaction skin sections represent: (A and E) normal guinea pig skin; (B) M. leprae (6 pug); (C and F) M. lepraesoluble antigen (6 pg); and (D) PPD (6 pig). The M. leprae and PPD sites are infiltrated predominantly withmononuclear cells and with some polymorphonuclear leukocytes, and the soluble antigen sites consistexclusively ofmononuclear cells. A through D, x122; E and F, x300.

ulation indexes obtained in 5 to 10 guinea pigsafter. stimulation with concanavalin A, phyto-hemagglutinin, M. leprae, PPD, and lepromin.M. leprae proved to be an exceptionally strongeliciting antigen, more active than PPD andcomparable to concanavalin A in the virusplaque assay, with 1.4% of the cells responding.

DISCUSSIONThis study was undertaken to explore the

possibility of developing a vaccine against lep-rosy by using purified armadillo-grown killedhuman M. leprae. A variety of immunization

protocols were designed to determine conditionswhich could be used in humans for optimalsensitization to the soluble M. leprae skin testantigen and purified M. leprae in guinea pigs.The results indicate that intact bacilli pro-

duced significant sensitization to both M. lepraeand its soluble antigen when given in the absenceof an oil vehicle or emulsion. Since these bacilliare obtained from armadillos, the contributionof contaminating armadillo tissue antigens to-ward skin reactivity was a theoretical concern,but no skin test reactivity was detectable in anyof our experiments to soluble armadillo extract.Comparison of various adjuvants showed only

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CELL-MEDIATED IMMUNITY TO M. LEPRAE 793

TABLE 1. Stimulation ofperipheral bloodlymphocytes by mitogens and antigens

Virus plaque-Stimulation in- forming cellsMitogen/antigen dexa (AV-PFU/103

cells)bConcanavalin A 9.03 ± 2.74c 13.16 ± 2.80cPhytohemagglutinin 9.67 ± 4.65 8.95 ± 1.05M. leprae 4.37 ± 0.67 14.88 ± 3.76PPD 2.81 ± 0.56 9.40 ± 2.88Dharmendra lepro- 3.00 ± 0.42 NDdmin

a [3H]thymidine incorporated counts in the control culturesranged from 306 to 1,806 (mean ± standard error, 949 ±104.84).

bAV-PFU, Virus plaque-forming units above the back-ground. Background in unstimulated control cultures was 4.27± 1.3 plaque-forming units per 103 cells (mean ± standarderror).

c Mean ± standard error of responses obtained in 5 to 10guinea pigs immunized with M. leprae.

d ND, Not determined.

small differences among the sensitization withM. leprae suspended in IFA, M. leprae sus-pended in saline and emulsified in IFA, or M.leprae emulsified in adjuvant 65. They were alleffective in sensitization but, in fact, were nobetter than bacilli suspended in Hanks solutionor saline. Other studies also showed that irradi-ated M. leprae in aqueous suspension producedgreater delayed-type hypersensitivity responsethan did a water-in-oil emulsion in mice (5).The possibility of using living BCG as an

adjuvant was also explored. Guinea pigs immu-nized with M. leprae together with BCG (106 or107 viable organisms) showed significant sensi-tization, but again the level was no better thanin guinea pigs immunized intradermally with M.leprae alone. Thus, M. leprae appears to be aremarkably strong immunogen for cell-mediatedimmunity, and addition of BCG fails to enhancethe sensitization significantly. Similarly, the ab-sence of an adjuvant effect of BCG on M. lepraein immunization has been observed in mice (7).Animals immunized with M. leprae alone

showed quite strong reactivity to PPD, indicat-ing marked cross-sensitization to other myco-bacterial antigens. Numerous attempts to estab-lish M. leprae specificity by carrying out a seriesof skin test antigen dilution studies failed; therewere no dilutions of antigen at which PPD reac-tivity was absent and reactivity to M. leprae orthe soluble antigen was retained. Thus, it mustbe concluded that M. leprae has sufficient anti-genic cross-reactivity that sensitization tounique antigens of M. leprae and cross-reactingantigens shared with M. tuberculosis occurs si-multaneously. On the other hand, the soluble M.leprae skin test antigen of Draper and Rees wasremarkably M. leprae specific and demon-strated no skin reactivity in guinea pigs immu-

nized with BCG alone. This specificity of thesoluble M. leprae extract for sensitization withM. leprae suggests that this may well be a usefultest antigen for M. leprae sensitization in hu-mans.

In vitro studies on lymphocyte transformationand the virus plaque assay for enumerating ac-tivated T-cells indicated that M. leprae in salinewas an excellent sensitizer and an effective elic-iting antigen. Of interest is the fact that thenumber of antigen-reactive cells found in periph-eral blood (1.4%) is comparable to that found inguinea pig lymph nodes sensitized with M. tu-berculosis in adjuvant (2).Even though the bacilli we received were

killed by irradiation, it might be preferable touse bacilli sterilized by autoclaving before use,and our results indicate that immunization withautoclaved M. leprae was, surprisingly, as goodor better than that with bacilli not subjected toautoclaving, a phenomenon also observed byShepard et al. in mice (8).

Last, animals immunized with M. leprae invarious vehicles and skin tested 1 year later forthe first time demonstrated positive reactivityto M. leprae and soluble antigen. Reactions wereparticularly strong in guinea pigs inoculatedwith M. leprae suspended in Hanks solution andin those inoculated with M. leprae along withBCG. Thus, intradermal inoculation of M. lep-rae in aqueous suspension is not only capable ofproducing high levels of reactivity but also in-duces sensitization that persists for long periodsof time.

ACKNOWLEDGMENTS

This work was supported by a grant from the IMMLEPProgram of the World Health Organization Special Programon Research and Training in Tropical Diseases. V.M. wassupported by a Victor Heiser Fellowship for Leprosy Research.We thank P. Draper and R. J. W. Rees for supplying the

purified M. leprae, soluble M. leprae antigen, and solubleextract prepared from armadillo liver. Cord factor and MDPwere kindly supplied by E. Lederer.

LITERATURE CITED

1. Bekierkunst, A., I. S. Levij, E. Yarkoni, E. Vilkas,and E. Lederer. 1971. Cellular reaction in the footpadand draining lymph nodes of mice induced by mycobac-terial fractions and BCG bacilli. Infect. Immun. 4:245-255.

2. Bloom, B. R., L. Jimenez, and P. I. Marcus. 1970. Aplaque assay for enumerating antigen-sensitive cells indelayed-type hypersensitivity. J. Exp. Med. 132:16-30.

3. Chase, M. W. 1954. Experimental sensitization with par-ticular reference to picryl chloride. Int. Arch. AllergyApple. Immunol. 5:163-191.

4. Lederer, E. 1976. Cord factor and related trehalose esters.Chem. Phys. Lipids 16:91-106.

5. Patel, P. J., and M. J. Lefford. 1978. Induction of cell-mediated immunity to Mycobacterium leprae in mice.Infect. Immun. 19:87-93.

6. Peck, H. M., A. F. Woodhour, and M. R. Hilleman.1968. New metabolizable immunologic adjuvant for hu-

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man use. Chronic toxicity and teratogenic tests. Proc.Soc. Exp. Biol. Med. 128:699-708.

7. Shepard, C. C., R. V. Landingham, and L. L. Walker.1976. Immunity to Mycobacterium leprae infections inmice stimulated by M. leprae, BCG, and graft-versus-host reactions. Infect. Immun. 14:919-928.

INFECT. IMMUN.

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