Scaml. J. Immunol. 36, 811 821, 1992

Induction of Cellular Immune Reactions by A36, an Antigen Complex of Mycobacterium paratuberculosis: Comparison of A36 and Johnin Components p. GILOT*. M. DE KESEL*, M. C O E N E * t & C. COCITO* *Microbiotogy and Genetics Unit, Institute of Cell Pathology, University of Louvain Medical School, Brussels. Belgium, and tinnogenctics N.V,, Zwijnaarde, Belgium

Gilot P, De Kesel M, Coene M. Cocito C. Induction of Cellular Immune Reactions by A,'^6. an Antigen Complex of Mycotiacieriwn paratuhcreulosis: Comparison of A36 and Johntn Components, Scand J Immunol 1992:36:811-21 Paraluberculosis (johne's disease) is a chronic enterilifi syndrome of ruminants, which is due to infection by Mycohacterium paratuherculo.sis. Culaneous testing with proteins extracted from a mycobacterial culture fluid (johnin-PPD) is currently used to evaluate lhe cellular itiimune status. We have compared lhe components of johnin-PPD wilh those of lhe A36 complex, a thermostable macromolecular aniigen (TMA) presenl in the cytoplasm and associated with (he cell wall of M. paratuhcrculosi.s. The presence in the johnin-PPD of fifteen A36 components has been shown by Western blotting. Moreover, monoclonal anlibodies. which bmd respectively to the 65-kDa M. leprae heal shock protein, the 28-kDa M. leprae superoxide dismulase. and M. tuberculosis iipoarabinomannan, recognized components of the johnin-PPD. The ability of A36 to trigger delayed hypersenstlivity reactions in sensitized rabbits, and to induce the proliferation of T lymphocytes from the lymph nodes of A36-sensitized mice, matched that of johnin-PPD, The homology levels of T epitopes between A36 and Ihe TMA complexes of ,W. phtei. M. bovis. M. tuberculosis and A/, aiiw/H were estimated, in a iymphoproliferalion assay, lo be 51, 52, 59 and 94% respectively, A strong cross-reactivity of A.'^6 wilh an ,'V/, leprae sonicate was also observed by cutaneous testing. The A36 components wilhin the 45,2 26,8-kDa and lhe 21,6-19,H-kDa ranges were proved to Induce the proliferation of T lymphocyles from sensilized mice. This work supports the possible use of the A 36 complex, and of some of iis components, for cutaneous tests and lymphocyle proliferation assays, tn order lo monitor cellular immunity in Johne"s disease. C. Cocito, GEMO-ICP-VCL-744H, Ave Hippocrate 75, B-1200 Brussels. Belgium

Paratuberculosis (Johne's disease), an infectious disease of cattle and other ruminants, is characterized by a chronic enteritis leading to emaciation, cachexy and death. The actiological agent, Mycohacterium paratuherculosis, is transmitted through infected pastures and located in the intestinal tract of ruminants. The disease progresses through three clinical forms: asymptomatic non-exeretory (stage I), asymptomatic excretory (stage II), and symptomatic excretory (stage HI). It is believed that animals at stages I and II, which are difficult to recognize in the absence of evident clinical symptoms, largely outnumber those at stage III, Cattle at stages II or III contribute to propagation of the disease by

shedding large quantities of mycobacteria in the environment [1. 2], Paratuberculosis involves strong immunological reactions of both humoral and cellular types. The overall elinical picture entails two extremes of high and low reactivity of cell-mediated immunity, which correspond respectively to the tuberculoid and lepromatous forms of human leprosy. The reagent used, in cutaneous testing and lymphocyte proliferation tests, is a purified protein derivative (johnin-PPD) similar to the tuberculinPPD preparalion used for the diagnosis of tuberculosis. At stage I these tests yield a positive reaction, which weakens progressively at stage 11 and becomes negative at slage III (anergy) [I, 1]. 81

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Several mycobacterial components are responsible for the immune reactions occurring in the course of infection. Certain large complexes such as the Iipoarabinomannan (LAM) [3, 4] and the thermostable macromolecular antigens (TMA) [5] seem to be particularly important in this respect. The TMA complexes, which contain polysaccharide, protein and lipid moieties, are present in all mycobacteria [6]. The most studied members of this family are A60, of A/, hovis BCG [7], and A7, of M. leprae[^]. They are able to elicit both humoral and cellular type reactions [9, 10]. We have recently purified A36, the TMA complex of M. paratuhercutosis. The protein components of A36 have been fractionated by electrophoresis. Some of these proteins were shown to possess B-cell epitopes specitic with respect to M. avium, M. hovis or M. phlei [ I I ] . Previous studies have shown that A60 (the TMA of M. hovis BCG and M. tuhercuto.iis) is the main component of tubereulin-PPD and the main thermostable antigen of mycobacterial sonicates [12]. By analogy, these facts suggest the presence of A36 in the johnin-PPD. In the present work, the components of A36 and johnin-PPD are compared by the use of antisera directed against the two preparations and of monoclonal antibodies recognizing single A36 components. The ability of A36 to induce cellular immune reactions is explored by in vivo and in vitro experiments: produclion of delayed hypersensitivity and induction of T-lymphocyte proliferation, A36 protein fractions are tested for the induction of T-cell clonal expansion. A hint of the species-specificity of the cellular immune response elicited by A36, with respect to the other TMA complexes, is obtained, of interest for the future development of species-specific reagents for cutaneous testing of paratuberculosis.

M A T E R I A L S AND M E T H O D S Bacteria. The following mycobacteria were used in this work: M. paratuht-rculosi.s strain 2E (from Dr F. Saxegaard, National Veterinary Instilute. Oslo, Norway); M, paratuhercniosis sirain 3 + 5 and M. phlei strain AM76(from Dr Desmecht, National Instilute of Veterinary Researeh. Brussels, Belgium): M. avium D4 and M. tuberculosis (from Dr F, Porlaels, Institute of Tropical Medicine, Antwerp, Belgium): W, hoiis strain BCG (from Dr M Weckx, Pasteur Instilute, Brussels, Belgium), Preparation of TMA antigen conipte.xe.s. Bacterial

suspensions in buffered saline (200 mg wet wetght cells/ ml, 0,15 M NaCl, 0,02 M K ^ H P O J , pH 7.5), containing 10 mM phenylmethylsulphonyl fluoride, were disrupled by sonication (15 min with a 500-W ultrasonic Vibra cell processor from Sonics and Materials Inc, Danbury, CO, USA). Sonic extracts were centrifuged (35,000 g. 10 min, 4 C). and supernatants were submitted to RNAase digestion (10 /jg enzyme, 100 (ig wet weight bacteria, 20 min, 37 C( and fractionated by chromatography on Sepharose 6B columns (Pharmacia, Uppsala, Sweden) previously equilibraled with bulfered saline, as previously detailed [13], TMA complexes were found within the excluded fractions (which contained on average 1 mg soluble proteins/ml as measured with the Folin reagent [14] using bovine scrum albumin as standard). Solutions of TMA were sterilized by ultraviolet irradiation (260 nm) and stored at - 2 0 C, Purity of TMA complexes was checked by twodimensional immunoeleclrophoresis according to reference systems [7, 15], Agarose gels (l%,type 2 agarose from Sigma. St Louis, MO. USA) on glass plates ( 5 x 7 em) were used for this purpose. The top gel contained 200 ^l of antimycobacterial Ig from immunized rabbits, Mycobacterial antigen (10 /il of samples containing 0,5 mg TMA/ml) was applied lo a corner well, and electrophoretic runs were made as previously described [7, t5](l h,8 V/cm. 15 Ctn I st dimension; 3 V/cm. i8h, 15 C in 2nd dimenston). Slants were washed, dried and slained with Coomassie blue. Aniihodies. For produclion of polyclonal antisera, TMA preparations (10 fig proteins/0,5 ml buffered saline, emulsified with an equal volume of incomplete Freund's adjuvant) or johnin-PPD (100 /ig/0,5 ml buHered saline, emulstfied with an equal volume of incomplete Freund's adjuvani) were repeatedly injected subeutaneously (six inoculations at 1-week intervals) in rabbits. Monoclonal anlibodies MC 4220 and MC 5041 directed respectively against the 65-kDa M. leprae heat shock protein and lhe 28-kDa M. teprae superoxide dismutase [16] were provided by the World Health Organization (WHO. Geneva, Switzerland) through the courtesy of Dr H, Van Heuverswyn (Innogenetics, Ghent, Belgium), Monoclonal aniibody C5-35 directed against M. tuberculosis Iipoarabinomannan was donated by Dr D, Chatterjee {Colorado State University, Fort Collins. CO. USA), Johnin-PPD preparation. A culture of M. paratubercutosis 3-1-5 strain cells was steam-sterilized, filtered and acidified with trichloroacctic acid {.A"/., TCA fmal concentration), Afler I 4 h a t 4 C, the sediment obtained by centrifugal ion was washed with a I'-'i- TCA. 5% NaCl mixture, di.ssolved in 22 mM NaOH, 0,1 M Na;HPO42H2O soluiion, and added to an equal volume of a mixture containing 0,5"';) phenol wt/vol, 14% glucose wt/vol, 168 mM Na:HPO42H;O and 110 mM KH;PO4, The preparation was lyophilized and dissolved at the moment of use. It was a gift from Dr M. Desmecht (National Instilute of Veterinary Research, Brussels, Belgium), Electrophoretic fraclionaiion of TMA and johninPPD. The components of A36 and johnin-PPD were fractionated by eleelrophoresis on polyacrylamide gels, in the presence of sodium dodecyl sulphate (SDS

Cellular Immune Reactivity of A 36 PAGE procedure) [17]. Samples (50 {i% soluble polypeptides in a 50 /J1 solution of 0,125 M Tris-HCI, pH 6.8, containing 5"/u wl/vol SDS, 20% vol/vol glyeerol, WA> vol vol /f-mercaptoelhanol and 0,05"'ii wt/vol bromophenol blue) were incubated for 5 min at 100 C and then applied lo verlieal gel slabs. The molecular weight protein markers (Sigma) used were: bovine serum albumin (66 kDa) ovalbumin (45 kDa), glyceraldchyde3-phosphale dehydrogenase (36 kDa), carbonic anhydrase (29 kDa). trypsinogen (24 kDa), trypsin inhibitor (20,1 kDa)and a-lactalbumin (14,2 kDa), Fleclrophoretic runs were made al a constant current of 30 mA at 20 C in a vertical unil (gel 16x18 cm; Hoefer Scientific Instruments, San F-"ranciseo, CA, USA), Fleclrophoresed components were transferred from polyacrylamide gels to nilrocel]u]ose membranes (BA S5, Macherey-Nagel, Germany) by the use of a transblot unit (217 multiphor 2, LKB, Bromma, Sweden). The transfer bufTer contained 20"/i vol/vol meihanol, 0,039 M glycine and 0,048 M Tris base, pH 8,8, Runs were made al KH) mA for 2 h. Transblotted nitrocellulose sheets were first incubated for 30 min, with TBS bulTer {0.5 M NaCl, 0,023 M Tris-HCI, pH 7,5) containing 3'''ii wt/vol gelatin, and then for 3 h with lhe primary anlibodies diluted with TBST bufTer (TBS containing 0,05"/,. vol/vol Tween 20 and l"/ii wl/vol gelatin). After repeated washings with TBST, sheets were incubated for 2 h with peroxidase-labelled protein A or with peroxidase-labelled rabbit anti-mouse IgG (Dako, Copenhagen, Denmark), This was followed by repeated washings with TBST and TBS buffers, A colour reactton was developed by addition of :;i-chloronaphlhol (Bio-Rad Laboratories. Richmond. CA, USA) in lhe presence of hydrogen peroxide. The colour reaction was slopped by washing the sheets wtth distilled water, A similar protocol was used for antigens directly spolted on nitrocellulose membranes (dot-blot analysis). Reference samples of transblotled total proteins were visualized by India ink staining I,^.\"A. vol/vol solution of founi India ink, Pelikan, Germany, in a 0,2 M NaCI, 0,05 M Tris-HCI, pH 7,4 solution, containing 0.3"-;j vol/ vo! Tween 20), for 3(1 min [I8|, or colloidal gold staining, as described by the producer (Aurodye forte, Amersham. UK) |I9|, Elution of antibodies hound to Western blotted JohninPPD componenis. After incubation with polyclonal antisera, iransblotted nitrocellulose membranes, containing eleclrophoresed johnin-PPD componenis. were repealedly washed with TBST buffer (0,5 M NaCl. i).05".A. vol/vol Tween 20 and 0,023 M Tris-HCI pH 7,5), Bound antibodtes were cluled by incubation of membrane segments with 0.2 M glycine-HCI butTer pH 2,5 (2 min. 18 C), Half volumeof a 5"/. solution of fclal calf serum in I M K2HPO4/KH2PO4 bufTer, pH 9,0, was added lo the eluates. Samples were diluted two times with water and final calf serum concentration was brought lo 2O'H. for storage. Preparation of Western blot for lymphoproliferation i;,v.v((i\ The nitrocellulose blot was divided into equal sections (0,4 mm x 0,4 mm) and sterilized using 50 tnM sodium azide. They were then washed thoroughly wilh sterile buffered saline (0,15 M NaCl, 0,02 M K ^ H P O * pH 7,5) prior to use for proliferation assays. The results shown represent tbe mean of triplicate assays per-

813

formed using blot from adjacent identical lanes in the original gel [20], Lymphocyte proliferation assays. Six-week-old female BALB/C mtce were each immunized by a subcutaneous injection of a solution of 5 ^g A36 emulsified with an equal volume of incomplete Freund's adjuvant (Gibco) in the hind footpads. One week later, popliteal lymph nodes were removed and dissociated lo yield single-cell suspensions in RPMI-1640 medium (Gibco) supplemented wilh 2 niM L-glutamtne, 5x 10"^ M 2-mercaptoelhanol. !00 /ig/ml gentamycin and 10% fetal calf serum. Lymph node cell suspensions were distributed in 96-well fiat-bottomed microtitre plates (5x10^ cells/ well, Nunc. Denmark) to which nitrocellulose-bound aniigens, johnin-PPD or an A36 solution (replaced by Concanavalin A (Con A) or bufVer in conlrols) were added. Plates were incubated for I-6 days, at 37 C, ina 5% CO2 incubator, before being pulsed with 1 ^Ci methyl-{'H]-thymidine (5,0 Ci/mmol. Amersham), Nitrocellulose strips were discarded prior to the addition of lhe radioactive precursor. Cells were harvested 16 h later on a glass fibre filler (Whatman GF/C) and counted in a scintillation spectrometer. Determinations were done in triplicate.

RESULTS Pre.sence of A36 components in johnin-PPD To identify the presence of A36 components in johnin-PPD. the lalter was fractionated on a 14% polyacrylamidc gel (SDS-FAGE) and transblotted to a nitrocellulose membrane. Common components between A36 and johnin-PPD were revealed by the use of anti-A36 serum and monoclonals directed against single A36 components. The johnin-PPD Western blot analysis showed a band heavily stained by anti-A36 serum, coresponding to lhe 28-39-kDa region (Fig. I, track 3A), The monoclonal antibodies MC 5041 and 4220, which were previously found lo bind respectively to the 23-kDa and the 65-kDa proteins of A36[l I], were shown to recognize two johnin-PPD polypeptides (Fig. I, tracks 3B and 4B), Moreover, lhe monoclonal C5-35 directed against M. tuherculosis Iipoarabinomannan was shown to recognize Johnin-PPD and A36 by the dot-blot procedure (results not shown). Since some of the A36 components present in johnin-PPD can be degraded (by the johnin-PPD preparation procedure) into low molecular weight components migrating out of the electrophoresis gel, a further experiment was carried out to determine their presence in the A36 complexWe have produced an antiserum against johnin-

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2

3

A

1

2

3 4

B

FIG, I, Identification of A_16 components in johnin-PPD. The components of johnin-PPD were fractionated by polyacrylamide gel elecirophoresis {\A"/., SDS PAGE) and Iransblotlcd lo nil rocci Itt lose tnembrancs, which were either stained with col]oidal go]d (tracks 2A and 2B) or incLibaled with primary anlibodies as follows: iinli-A3(i polyclonal (track 3A). MC 5041 monoclonal (track 3B). MC 4220 monoclonal (track 4B). Primary anlibodies on membranes were revealed cither by peroxidase-labelled protein A (track 3A) or by peroxidase-labelled antimouse rabbit Ig (tracks _"iB and 4B), Moiecuiar weight standards (66,0. 45.0, 36.0, 24,0, 20,1 and 14,2 kDa in tracks ! A and IB) were stained with colloidal gold. Lines on the right side of the figure point to MC 4220-rcvealed polypeptides.

PPD and used it to identify components common with A36. Twelve A36 components of 83.0, 73.3, 69.3, 67.0, 60.5, 55.9. 52,9. 41.2. 39.0, 37.0, 34,0 and 21.2 kDa were recognized (Fig, 2, track 4), The possibility that the 28 39-kDa johninPPD componenis recognized by anti-A36 (Fig. I, track 3A) originated from A36 components of higher molecular weight, which would be degraded in johnin-PPD, was checked as follows. The anli-A36 immunoglobulins bound lo the 2839-kDa fraction of johnin-PPD (Fig, 1, track 3A) were eluted from the Western blot, and tested on lhe A36 proteins (Fig. 2, track 3), Components of similar molecular weights were revealed, indicat-

ing that no degradation occurs (compare Fig. 2, track 3 and Fig, I, track 3A). The broad 28 39-kDa region stained by the anti-A36 serum on the johnin-PPD Western blot (Fig. l,Track3A)andby iheanti-johnin-PPDon the A36 Western blot (Fig. 2) is in part explained by the presence of Iipoarabinomannan in lhe johnin-PPD and in the A36. The monoclonal antibody C5-35 directed against M. tuherculosis Iipoarabinomannan recognizes the same broad 28 39-kDa region on a Western blot of A36 which was previously fractionated on a 12% acrylamide gel (compare Fig, 2, track 3 and Fig. 3, track C).

Cellular Immune Reactivity of A36

1

A

2

B

815

C

kDa kDa

66,066,0 -

45,0 -

20,1

-

20,1 -

FIG. 2. Analysis of the A36 components shared by johnin-PPD, A36 components were fractionated by polyacrylamide gel electrophoresis (I2";i SDS PAGE) and transblotled to nitrocellulose membranes. They were then either stained with colloidal gold (track 2), or Incubated with anti-A36 Ig which were eluted from a Western blot of johnin (track 3), or incubated with antijohnin serum (track 4), Ig were revealed wilh pero,\idase-labelled protein A, Molecular weighl markers of lhe indicated sizes were labelled wilh colloidal gold. Lines on lhe right side of lhc figure poinl lo ihe antijohnin-PPD-recognized poiypeptides of 77,3 and 83,0, weakly stained in track 4.

These data indicate the presence of at least !5 components of the A36 complex in the johninPPD,

In vivo and in vitro induction of cellular immune responses The ability of A36 to trigger delayed hypersensitivity reactions was tested by measuring the diameter of dermal infiltrations in sensitized rabbits 24 and 48 h after challenge. The occur-

FiG, 3, Presence of Iipoarabinomannan in the A36 complex, A36 components were fractionated by polyacrylamide gel electrophoresis (I2'^r, SDS PAGE) and transblotled lo nitrocellulose membranes. They were then eilher stained with colloidal gold (Irack B), or incubated with the C5-35 monoclonal antibody directed against M. tuberculosis Iipoarabinomannan (track C), Primary Ig were revealed with peroxtdase-labelled antimouse rabbit Ig, Molecular weight markers (track A) of the indicated sizes were labelled with colloidal gold.

rence of a typical cellular immune reaction is shown in Table I. No reaction was visible in unsensitized rabbits. An evaluation of the capacity of A36 to induce cellular immune response was obtained in vitro by the lymphocyte proliferation assay. Cells from the popliteal lymph nodes of A36-sensitized mice were incubated in vitro with increasing concentrations of A36. Lymph node cells from unprimed mice were used as controls and underwent no clonal expansion. Lymphocyte proliferation, monitored by ['H]-thymidine incorporation into DNA, was proportional to both A36 concentration and time after restimulation. The immune response peaked at 90 h (Fig. 4),

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P. Gilot etal.

TABLF I. Abiliiy of A36 to induce delayed liyperscnsitiviiy reactions Erylhemalous dermal infiltration (mtn) after inoculation* Revealing antigen

24 h

48 h

SP buffer (control) A 36 0,5 ;(g

0 12 IH

0 15 20

' Rabbits were sensitized wilh six intradermal inoculations of A36 solutions al 1-week intervals (20 /(g proteins emulsified wilh incomplele Freuiid's adjuvant). The skin test was done 1 m o n i h later.

The ability of the A36 components present in johnin-PPD to promote lymphocyte proliferation was checked by comparing the stimulation levels of A36 and johnin-PPD on lymph node cells from A36-sensitized mice. As indicated in Fig. 5, comparable effects were triggered by the tw{) preparations. These experiments demonstrated the induction of cellular immunity reactions by A36 in vitro and in vivo, and show that some of the A36 components present in the johnin-PPD are able to promote cellular immune responses.

Cellular immunity cross-reactions hetween A36 and the TMA complexes from other mycohacteria The A36 complex from M. para tuherculosis belongs to a family of major mycobacterial complexes called TMA (thermostable macromolecular antigens). An indication of the specificity level of A36, with respect to the TMA complexes from other mycobacteria, was gleaned by in vitro experiments. Lymph node cells from A36-sensitized mice were cultured in the presence of the same amount of either A36 or other TMA, The level of lymphocyte proliferation induced by a heterologous TMA, with respect to thai promoted by the homologous (A36) TMA (equal to lOQ'Yn). was taken as a measure of the cross-reactive Tlymphocyte epitopes shared by the compared complexes. As shown in Table II, the TMA of M. paratuherculosis was highly homologous to that of M. avium. whereas the homology between A36 and the TMA of M. hovis. M. tuherculosis and M. phlei was lower, Immunological cross-reactivity (e,g, the binding of heterologous monoclonals) among antigens of M. paratuherculosis and M. leprae has been reported [II]. These observations concern-

200 r-

100

-

180

FIG. 4, Kinetics of the A36-dependenl in vitro lymphocyle proliferation, Lytitphocyles {2,5 x 10'' ceils/ml) from lymph nodes of A3(i-sensiti7ed mice were cultured, in viiro. in lhe presence of dilTerenl concentrations of A36 (5 n^ (—a—J or 50 ^g (- • )), Cultures were pulse-labelled (! ;jCi |-H]-thymidine;5 x 10^ cells, 16 h): incorporated radioactivity was measured by scinlillalion speclromoiry. The average values of triplicate cuUures are expressed in Acpm (cell-bound radioactivity in stimulated sample sublracted from cellbound radioactivity in unslttiiulaled conlrol) and reported, logeiher wilh standard deviattons.

Cellular Immune Reactivity of A36

150 -1

100 -

•5

50 -

(TJ

CC

Antigens FIG. 5, Ability of A36 componenis present in johnin-PPD to activate the cellular immunity, Lymphocyles (2.5 X 10'' cells/ml) from lymph nodes of A36-sensiti7,ed mice were cultured in vitro (37 C, 72 h) in the presence of; (a) 5/jgCon A/mlUb) 50/ig A36,ml; (c) 50/ig johnin/ml: and (d) SP buffer (control). Cells were then pulse-labelled (1 /jCi[-'H]-thymidine/5 x iO"^ eells, 16 h) and incorporated radioactivity was measured by scintillation spectrometry. The average values of triplicate cultures are reported (standard deviations

TABLE il. Relative potency of TMA complexes from different mycobacteria in stimulating in viiro lymphocyte proliferation from A36-sensitized mice* TMAt (50 /ig/ml) M. paraluberculosis 2E M. avium D4 M. phlei M. bovis BCG M. tuberculosis

Lymphocyte proliferationj (cpm)

Homologous T-cell epitopes§ with respect lo A36 {%)

135.304 126,556 69,478 80,336 70,556

100 94 51 59 52

* Mice were sensitized by subcutaneous inoculalion of A36 solution in the foolpad (5 /jg proteins emulsified with tncomplete Freund's adjuvant). t TMA from dilVerent mycobacteria were used to stimulate lymphocytes from the popliteal lymph nodes of A36-sensili7ed mice. X Lymphocytes (2,5 x IO''/ml) were cultured for 4 days and pulse-label led with ['H]Ihymidine (1 /(Ci/5 x 10^ cells) 16 h before the filtration. Incorporated radioactivity was measured by scintillation speclrometry. The average values of three independent determinations are reported (sfandard deviation < \7"A>). § Per cent hotnology of different TMA wilh respect to that of M. paratuherculosis was estimated by: (cpm Mycobaclcrium sp,) x 100/(cpm M. paraluberculosis).

817

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P. Gilot etal.

ing humoral immunity have been extended in lhc present work to cellular immune reactions with TMA complexes. A rabbit was immunized with a wholelysateof A/./£'^rfle( 10 injections with 30//g of proteins emulsified in incomplete Freund's adjuvant) and challenged I month later with A36 (5 /ig of protein) on one side, and with M. leprae lysate (5 /ig of protein) on the other side (A7, the TMA complex of this micro-organism was unavailable). Homologous and helerologous delayed hypersensitivity reactions were respectively observed with M. leprae lysate and A36. Homologous reactions (15 mm infiltrate after 24 and 48 h) were stronger than the heterologous ones (7.5 mm). In conclusion, cellular immunity cross-reactions among A36 and all other TMA eomplexes was evident. Identification of the A36 components inducing cellular immune reactions Since A36 is able to induce cellular immunity

reactions, it was of interest to identify the A36 eomponents responsible for these effects. Upon dissoeiation of the A36 complex, its components were fractionated by polyacrylamide gel eleetrophoresis (12"A, SDS-PAGE) and transferred to nitroeeilulosc membranes, the segments of which were submitted to the lymphocyte proliferation test. For this purpose, the lymph nodes of mice sensitized by footpad injection of A36 were dissected and lymphocytes were cultured, in lhe absence and presence of nitrocellulose transblotted A36 fractions. Cell proliferation was monitored by pH]-thymidine incorporation into DNA. In preliminary trials, similar proliferation kinetics were obtained with soluble and nitrocellulose-bound A36 eomponents. Two regions of the A36 patterns., the 45.2-26.8 kDa and 21.6-19.8 kDa ones, promoted lymphocyte proliferation (Fig. 6), whereas the high molecular weight region was almost ineffective. In conclusion, only certain components of A36 proved to be able to elicit cellular immune reactions in vitro. About one-half of the proteins

30 25 -

-X.

E 20 ^ a.

5" 15

_L

xl\ 14,2-

66.0

45,0 36,0 29.0 24.0

14,2

Molecular weigbt (kDa)

FIG, 6. Ability of different A36 components to stimulate the proliferation of lymphocytes from sensitized mice. Lymphocytes (2,5 x 10*" ceils/ml) from lymph nodes of A36-sensilized tnice were cultured in viiro. The bands of nitrocellulose membranes transblolted wilh A36 componenis fractionated by \2%, SDS PAGE were cut and used to stimulate the cell growth (37 C, 72 h). Cultures were then pulse-labelled (1 /(Ci [-'H]-lhymidinc/5 x 10' cells. 16 h) and incorporated radioactivity was measured by scintillation spectrometry. The average values of Implicate eultures and standard deviations are reported. Response lo nitrocellulose alone ( ) was 8484+ 1548 cpm (measure repeated 10 times). A Student's /-test was used to determine the significance of the differences between responses lo antigenic fractions and nitrocellulose (highly significant values; T, significant values: 0), The left-side insert is the extremity of the transblolted nitrocellulose membrane, India ink staining of the membrane revealed the A36 polypeptides and the molecttlar weighl tnarkers.

Cellular Immune Reactivity of A36 shared by both A36 and johnin-PPD corresponding to the active regions identified by the lymphocyte proliferation test in vitro.

DISCUSSION It was previously shown by Harboe [12] that autoclaving of either whole sonicates or the culture medium of M. hovis BCG brought about the inactivation of most antigens (they failed to react with the corresponding antibodies and to yield precipitinogen lines). Among the surviving antigens, the 60 and 89 components stood out: both of them were present in old tuberculin preparations, but only A60 was found in tuberculin-PPD. The corollary of these findings was that A60 is the main active component of tuberculinPPD, the widely used reagent for cutaneous testing in tuberculosis. Such an inference justifies the present approach, whereby A36 components were compared to those of johnin-PPD (A60 of M. tuherculosis corresponds to A36 of M. paratuherculosis, as tuberculin-PPD does to johninPPD), Data in Figs 1, 2 and 3 indicate the presence of at least 15 components common to both A36 and johnin-PPD. Twelve of these components were identified by allowing a johnin-PPD polyclonal antiserum to react with a Western blot of A36 components (Fig. 2, track 4). Three other common constituents were detected with monoclonal antibodies: an anti-65-kDa heat shock protein (MC 4220) (Fig. 1, track 4B) and an anti-23-kDa superoxide dismutase (MC 5041) (Fig. 1, track 3B) which were previously found to bind to A36 components [II): and an anti-lipoarabinomannan (C5-35) which was found in this work to bind to A36 (Fig, 3, track C) and to johnin-PPD (not shown). Six of these 15 components (23 kDa, Iipoarabinomannan, and the four components between 28 and 39 kDa) have similar molecular weight in A36 and in johnin-PPD while the others are degraded in johnin-PPD, The monoclonal antibody MC 4220 was found to recognize an epitope of the 65-kDa heat shock proteins of some mycobacteria [21-23]. These 65-kDa proteins are present in A7 [16], A36 [11] and A60 (unpublished results), the TMA complexes of M. leprae.. M. paratuherculosis and M. hovis BCG respectively. They are also present in tuberculin-PPD [24] and johnin-PFD (Fig. I). It is worth noting that the 65-kDa protein of

M.gordonae induces delayed hypersensitivity reactions in guinea pigs sensitized with either M. gordonae, M. leprae or M. hovis BCG [25]. In Fig. 1 (track 4B), MC 4220 recognized multiple bands in the 65 55-kDa region of the johninPPD, These bands presumably originated from a carboxyl terminal degradation of the 65-kDa component, as was previously proposed [16, 26]. On the other hand, the monoclonal antibody MC 5041 (SA1D2D) also recognizes a series of proteins which have simiiar but not always identical size in dilTerent mycobacteria. Thus, this monoclonal antibody, which binds to the 23-kDa protein of M. paratuherculosis [II], also recognizes a 23-kDa component of M. tuherculosis and a 28-kDa component of M. leprae. All these proteins thus share cross-reacting epitopes and possibly correspond one to another, as indicated by the fact that the M. tuherculosis 23-kDa protein and the M. leprae 28-kDa protein share 81% homology and superoxide dismutase activity [27-30]. In this work, we show that this protein is also present in the johnin-PPD (Fig, 1, track 4B). This fact can be correlated with the finding that the homologous 23-kDa protein of M. tuherculosis is pres^eni in the culture filtrates of exponentially growing bacteria [30, 31]. The steam-sterilized culture filtrates of M. tuherculosis or M. paratuherculosis are the basic reagents for the preparation of the tuberculin-PPD and the johnin-PPD respectively. Since johnin-PPD is a widely used reagent for cutaneous testing, and at least 15 components of the A36 complex are present in johnin-PPD, the ability of A36 to trigger delayed hypersensitivity reactions could be predicted with confidence. In the present work, evidence is further provided for the ability of A36 to induce the proliferation of lymphocytes from lymph nodes of A36-primed mice in a concentration-dependent manner. This is the key step in the activation process of macrophages, which prevents intracellular mycobacterial proliferation and is the basis of protective immunity. It has been shown in other works that the stimulation of lymphocyte proliferation by the A60 complex was concentration-dependent and antigen-specific, and that A7 was able to stimulate in vitro lymphocytes from patients suffering from the tuberculoid stage ofleprosy [9, 32], A60 was also proved to elicit delayed hypersensitivity reactions in sensitized animals [10. 33]. In conclusion, data presented in Table I, Fig. 5 and previous works on A7 and A60 [9, 10, 12, 32.

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P. Gilot et at.

33] suggest that A36 preparations could replace johnin-PPD to monitor cellular immunity in paratuberculosis. The strong humoral immunity cross-reactivity among TMA complexes from different mycobacteria is well known [11, 34], Such cross-reactivity also applies to cellular immunity. Table II provides information on the degree of homology of dilTerent TMA as far as the lymphocyte stimulation ability was concerned. In praetice, these data suggest the level of chance of finding, within the components of a given TMA complex, T-cell epitopes endowed with species-specificity. Thus, according to the data in Table II. the homology level between A36 and the TMA of M. tuherculosis, M. hovis BCG and M. phlei is relatively low (homology level near to 55%), whereas that from M. avium is high (94% homology). These data are similar, but not identical, to those previously found for humoral immunity (the TMA of M. paratuherculosis has a level of specificity of l"/n towards the TMA of M. avium, 10% towards the TMA of M. hovis and 4S% towards the TMA of M. phlei) [ 11 ]. A comparison of these sets of data suggests that it might be easier to find speciesspecific proteins for cutaneous testing than for serological assay (at least with respect to M. hovis). In this respect, let us mention that, within the whole A36 protein set, only one protein, that of 34 kDa, was found to contain B epitope(s) specific for M. paratuherculosis with respect to M. avium,

M. hovis a n d M. phlei [ I I ] .

Some conclusions on the immunogenicity of lhe A36 components can be drawn by comparing the pictures of electrophoretic patterns stained with colloidal gold (Fig. 2, track 2) or immunostained (Fig. 2, track 4 and Ref. II), and the activity diagram of lymphocyte proliferation assays (Fig. 6). No direct relationship was observed between the concentration of any given component and its immunogenicity. In fact, the components most heavily stained with gold or India ink were not those preferentially revealed by sera of M. paratuherculosis infected bovine or those inducing the strongest lymphocytes responses. The components within the 45 28-kDa range were the most active in eliciting immune reactions of both cellular (Fig. 6) and humoral [11] type. The presence of Iipoarabinomannan in this same range could explain the high antibody affinity for the components of that region. Indeed, Iipoarabinomannan is well known for its high humoral antigenicity [3]. Nevertheless, the

Iipoarabinomannan is not the only factor accounting for the high antigenicity of this region, since other very antigenic components, for example a 34-kDa protein, are also present there [11]. As for cellular immunity, the same 4528-kDa region is a potent activator of lymphocyte proliferation, despite the described inhibitory etfect of the Iipoarabinomannan [35]. This reveals the high activation power of the stimulating componenis of that region. Finally, about half of the components shared by both A36 and johninPPD corresponded to the regions inducing lymphocyte proliferation in vitro. The experiments described in the present work suggest A36 to be a suitable reagent for cutaneous testing in Johne's disease. Although such an assay lacks species-specificity, it indicates that A36 contains a sufficient number of species-specific T-cell epiiopes to justify the search for its components inducing specific delayed hypersensitivity reactions. ACKNOWLEDGMENTS P, Giiot and M. De Kesel were predoctoral fellows supported by the Belgian Foundation IRSIA. Anti-M, leprae monoclonals were provided by WHO (Geneva, Switzerland)/(UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases). We thank Dr D. Chatterjee (Colorado State University, Fort Collins. USA) for the gift of anti-LAM monoclonal, and Dr M. Desmecht (National Veterinary Research Institute, Brussels, Belgium). Dr F, Saxegaard (National Veterinary Institute. Oslo, Norway) and Dr M, Weckx (Pasteur Institute. Brussels, Belgium) for the gift of strains. The senior author thanks H. Maes for reading the manuscript. REFERENCES 1 Chiodini RJ, Van Kruiningen HJ, Merkal RS, Ruminant paraluberculosis (Johne's disease); the current status and future prospects, Cornell Vet 1984:74:218-68, 2 Brugere-Picoux J, Le diagnostic de la paraluberculose chcz les ruminants, Rec Med Vel 1987;I63: 539-46. ? Sugden EA, Samagh BS. Bundle DR. Duncan JR, Lipoarabinomannan and lipid-free arabinomannan antigens of Mycobaclerium paraluberculosis. Infect Immun 1987:55:762 70. 4 Chan J, Fan X, Hunter SW, Brennan PJ, Bloom BR, Lipoarabinomannan, a possible virulcnee factor

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