INT1,12NATIONAI. JOURNAL01 I.11.1“)SY^ volume (in. Nuinhci 2l'tinted in the (IS.. I.

(ISSN 01.3S-91()X1

Two-Dimensional Electrophoretic Analysis of HumoralResponses to Culture Filtrate of Mycobacterium bovis

BCG in Patients with Leprosy and Tuberculosis'Mariko Naito, Shinzo lzumi, and Takeshi Yamada'

Leprosy still remains a major chronic in-fectious disease affecting more than1,260,000 patients. There are two polartypes of the diseases. One is lepromatousleprosy (LL), which represents no specificcellular immune response to Mycobac-terium leprac and has a large number ofbacilli within phagocytes in the macro-phage-rich granuloma. The other is tuber-culoid leprosy (TT), which is characterizedby a vigorous cellular immune response andcan result in irreversible nerve destructionin a high proportion of cases. Precise earlydiagnosis is important for the initiation ofefficient chemotherapy. A variety of tech-niques to detect an infection with M.. lepraehave been offered. In prognosis and treat-ment, there are differences between the LLand TT types of the disease. Many studieson the diagnosis of leprosy also have beenreported (4.12).

The antigen 85 (Ag85) complex, Ag85-A= MPB44 C), Ag85-B MPB59 (it anti-gen (5) and Ag85-C MPB45 (), is se-creted by mycobacteria. These antigens arepresented both on the cell surface and in thecytosol of M. /eprac within the infected le-sions of LL patients ("). The Immoral re-sponses to Ag85 complex have been re-ported by different groups ( ''). However,so far no comparisons of the reactions withwhole secreted proteins have been pre-sented.

' Received for publication on 3 November 1997.Accepted l'or publication in revised form on 27February 1998.

:NI. Naito, D.D.S., Ph.D.: T. Yamada, M.D., N.D.,Department of Oral Bacteriology, Nagasaki UniversitySchool of Dentistry, Sakainoto 1-7-1, Nagasaki 852-8588. Japan. S. Iitnni, M.D., Ph.D., National Lep-rosarium Ohshima-Seisyouen, Kagawa, Japan.

Reprint requests to Dr. Yamada at the above ad-dress or FAX 81-95-849-7650; email: yamatake0)net.nagasaki-u.acjp

208

The genes for the culture filtrate (CF)proteins, (Y. antigen (5), MPI351 ("), MPB64(15), MPB70 MPB83 (7), and MP1357(1") were first cloned by us from M. bovisbacillus Calmette-Guerin (BCG). On the ba-sis of this background, comparative analy-ses were carried out on Immoral responsesof patients against whole CF proteins in thehope that these proteins in contain anti-gens useful in predicting the prognosis ofthe disease. Two-dimensional electrophore-sis (2-DE) has great resolving power to sep-arate individual proteins. The CF of BCGwas analyzed by 2-DE. Ag85 complex andMPB51 were immunodominant antigens aspreviously reported (L). In addition, weidentified three new antigens that were rec-ognized by leprosy sent.

MATERIALS AND METHODSSerum. Patients were classified clini-

cally and histopathologically. Serum sam-ples were collected from 3 LL, 3 borderlinelepromatous (BL), 3 mid-borderline (BB), 3borderline tuberculoid (I3T), 2 TT and 4 tu-berculosis (TB) patients (The Table). Allpatients were studied before chemotherapy.The healthy control sent were collectedfrom three persons who had no prior historyof exposure to leprosy.

CE BCG substrain Tokyo was used inthis study. It was cultured on the surface ofSauton liquid medium for 3 weeks. The cul-ture was passed through a membrane filterwith a pore size of 0.45 pm to remove BCGcells. The proteins in the filtrate were con-centrated by solid ammonium sulfate at80% saturation, as described previously C),and the concentrate was dialyzed againstTMNSH buffer (10 mM Tris-HCI buffer,pH 7.8, 10 inM Mg acetate, 60 InM NH 1 C1and 6 mM 2-mercaptoethanol).

2-DE. The proteins in Ihe CF were ana-lyzed by 2-DE according to the method of

66, 2^Naito, et al.: Humoral Immune Responses to BCG CF A gS^209

Tiff, TAitkr. Classification and clinicalhistory of untreated patients".

Clinical/histopathological^Clinical history

diagnosis

I-)

111,WI'

RecurrenceRecurrence

3 II. Recurrence4 TT Primary in5 BL Primary infection6 1.1 Priinary infection7 11 Primary infection8 TI' Primary infection9 IIT Pdmary infection

I (1 BB Primary infectionII BL Primary infection12 BB Primar■,,' infection13 BB Primary infection14 BT Primary infection15 TB Primary infection16 TB Primary infection17 TB Primary infectionIS TB Primary infection

Sera \yen: obtained I'M in Kyoto University I lospitalattached to the School of Medicine, Kyoto (patients1-12): Kitano hospital. Osaka (patient 13): KitasatoUniversity hospital attached to the School of Medicine,Kanagawa (patient 14): Johoku Hospital. Aichi (patient15) and "Ibkyo [hospital of National Sanatorium, Tokyo(patients 16-18).

O'Farrell (I") with a few modifications asdescribed previously ("). In each 2-DE gel,20 pg of CF protein was loaded. The firstdimension was isoelectric focusing in 8 Murea with a 2% ampholyte mixture (Bio-Lyte; Bio-Rad mixture of 3/10:3/5:5/7 =4:1:1) (Bio-Rad Laboratories, Hercules,California, U.S.A.). The second dimensionwas sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE)with a 12.5% polyacrylamide gel. The gelswere processed for Western blotting or pro-teins were visualized by silver staining.

Immtmoblotting. Immunoblotting wascarried out accordinL, to the method of Tow-bin, et al. The blots were probed withI: IOU diluted serum antibodies, and de-tected with 1:1000 diluted peroxidase-con-jugated rabbit antihuman immunoglobulinG (Dako A/S, Glostrup, Denmark).

RESuus2-DE. 2-DE of the CF derived from

BCG is presented in Figure 1. The silver-stained gel showed the well-separated pro-teins. For example, Ag85-A, a antigen,

Ag85-C, MPB51, MPB64, MPB70, the 65-kat protein (groEL/hsp60 analog) ("),MP1357 (groES/hsp10 analog), and BCG45/47-kDa complex (") were defined asclear spots.

Humoral responses of LL and III, pa-tients to CI; proteins. Represented in Fig-ure 2A, all sera showed the strongest Im-moral reactions to Ag85 complex andMPB51 among the CF proteins. A majorityof the sera reacted to BCG 45/47-kDa com-plex (5/6 = 83%). Only a smaller number ofsera recognized the NIP1357 and 65-kDaprotein spots (MPI357: 1/6 = 16.7%, 65-kDa: 3/6 = 50%). These responses wereweaker than those to the Ag85 complex andBCG 45/47-kDa complex. There were alsothree small seroreactive spots, the 29-, 24-and 23-kDa spots, that had not as yet beencharacterized (arrows in Fig. 2). All sera re-acted weakly to the 29- and 24-kDa spots.Concerning the 23-kDa spot, half of thesera gave a positive reaction.

I tumoral responses Of BR, WE and Ti'patients to CI; proteins. Proteins wererecognized relatively less intensely or not atall when probed with BB, BT and TT sera(Fig. 213). All of these sent showed weak re-actions against Ag85 complex and MPB51,but only I out of 3 BB and 1 out of 3 I3Tsent showed strong reactions. Other spotswere recognized with only a few sera(MPB57: 2/8 = 25%; 65-kDa: 2/8 = 25%;BCG 45/47-kDa complex: 1/8 = 12.5%; 29-kDa: 3/8 = 37.5%; 24-kDa: 2/8 = 25%; 23-kDa: 1/8 = 12.5(k). There were no spotsthat were recognized by only the I3T andTT patients.

Humoral responses of TB patients toCF proteins. TB patients showed reactionssimilar to the TT and BT patients. AL*85complex, MPB51 and 29-kDa spots wereweakly recognized by these sent (Fig. 2C).Other spots were recognized with some sent(65-kDa: 3/4 = 75%; 24-kDa: 1/4 = 25%;23-kDa: 1/4 = 25%). MPB57 showed no re-action with any sent. The Immoral re-sponses of healthy control sent showed noreactions to any of the CF proteins (data notshown).

DISCUSSIONOne of the main limitations on the suc-

cessful control of leprosy as a public healthproblem is the lack of methods for its early

Patientno.

210^ International Journal of Leprosy^ 1998

94-

67-45/47

kDacomplex

43-

29kDaA — MPB64

24kDa^4111PAPB70

MPB57

30-MPB51 —

20-

14.4-

•23kDa

basic^ acidicFIG. 1. 2-DE of CF. proteins derived from BCG. The gel was visualized by silver staining. Apparent sizes of

molecular weight standards are indicated in kDa along the left side. 85A = Ag85-A; a = antigen; 85C = Ag85-C; 65 kDa = 65-kDa protein (groEL analog); MPB = major protein secreted from BCG.

diagnosis in subclinical cases. Because ofthe long incubation period of the disease,the liberation and spread of M. leprae intothe environment during that stage constitutethe main sources of infection. We searchedfor antigens that could be applied to the pre-diction of leprosy infection in subclinicalcases.

Separation of proteins in one-dimen-sional electrophoresis is generally not suffi-cient to resolve a large number of con-stituents. 2-DE as described by O'Farrell(w) is very powerful since two differentprinciples of protein separation are appliedin the first and second dimensions, permit-ting a precise definition of individual anti-gens of samples. Proteins secreted bymycobacteria have been suggested as majorimmune targets in the early phase of infec-tion ('). The CF of BCG was utilized to ex-amine reactivities with sera from leprosypatients because cultivation of M. lepraehas not yet succeeded in vitro. A study of

crossreactivities with these proteins thatcontain well-characterized antigens mayhave potential value for finding new im-munogenic antigens. The early cultureswere used to exclude the possible contami-nants with degraded products of bacteria.

In this study, we have defined three newantigens that are recognized by sera fromleprosy and tuberculosis patients, the 29-,24- and 23-kDa antigens (Fig. 2). The 29-and 24-kDa antigens were recognized veryoften in lepromatous patients but rarely intuberculoid or TB patients. The reactions tothese antigens can be good candidates forprediction of the infection, especially inlepromatous patients. Further study of theseantigens may provide a better understand-ing of the pathogenesis.

Immunoblot analysis gave some patternsof reactivities with individual patient sera.There was a clear trend. Flumoral responseintensities against Ag85 complex andMPB51 were well correlated with the types

21166, 2^Naito, et al.: Humoral Immune Responses to BCG CF Ags

A

45/47kDa

complex

94-

^

67-^65kDa —

^

43-^85A 85C •46"

I^I

^

30-^MPB51^Not,

• " I- 29kDa20- 23kDa^24kDa^•

14.4-^ — MPB57

85C85A■• •

•MPB51^a

20-

85A 85CII

.^• •3 0 —^ /

MPB51 — a^29kDa

20-^ •

14.4-

basic^ acidic

PIG. 2. Patterns of antibody responses to CI: proteins separated by 2-DE of three individual patients: A =1.1.,B = Ti' and C = TB. --> = spots with molecular weights of 29-, 24-, and 23-k Da. Apparent sizes of molecularweight standards are indicated in kDa along the left side; other spots are as indicated in Figure I.

94-

67-

43-

30-

14.4-

94-

67-

43-

21 2^ International Journal of Leprosy^ 1 998

of disease. The reactions with sera fromlepromatous patients were stronger thanthose from tuberculoid and TB patients. Fo-cusing on A$5-l3. an ELISA analysis wasperformed and yielded a similar conclusion(data not shown). Ag85 complex has re-cently been shown to be a mycolyl trans-ferase that is one of the important enzymesfor unique mycobacterial cell-wall synthe-sis ('). The At85 complex may play impor-tant roles as the first row antigens secretedby Al. leprae after infection. The antigensare suggested to be the most principal onesfor determining the clinical prognosis afterinfection.

Although relapse of lepromatous leprosyis a serious problem, this clinical changecannot be detected clearly in an early stage.Recently, multidrug-resistant M. leprae hasbeen isolated from relapsing leprosy (').Drug therapy for a long period to avoid re-lapse of the disease raises the possibility ofselecting the drug-resistant Al. frprac. Theantibody responses to spots defined in thisstudy might provide successful predictionfor controlling the disease and for efficienttherapy. Our findings may also contribute tothe understanding of the pathogenesis ofleprosy and the mechanism of the infection.

SUMMARYSera from 3 lepromatous (LL), 3 border-

line lepromatous (BL), 3 mid-borderline(BB), 3 borderline tuberculoid (BT), 2 tu-berculoid (TT) and 4 tuberculosis (TB) pa-tients and 3 healthy individuals were exam-ined for their reactivities against the pro-teins in the culture filtrate of BCG separatedby two-dimensional electrophoresis (2-DE).The sera were obtained from patients whowere untreated. Sera from LL and BL pa-tients reacted strongly with the antigen 85(Ag85) complex and MPB5 I. Sera from LLand BL patients also weakly reacted withthe newly identified 29-, 24- and 23-kDaspots. Sera from the other patients reactedsimilarly, but the levels of reaction were re-markably lower than those from LL and BLpatients. Mrcobacterium leprae antigensthat are analogous to BCG Ag85 andMPB5 I are suggested as the main targetsfor the humoral immunity of untreated pa-tients. The reactivities of sera with newly

identified antigens may provide the poten-tial for predicting the severity and progno-sis of diseases.

RESUNI 1.7,NSc probaron los sueros de 3 pacientes lepromatosos

3 lepromatosos subpolares (ItI.), 3 pacientes in-termedios (BB). 3 tuberculoides subpolares (13T). 2 in-berculoides (17), 4 pacientes con tuberculosis (TB), y3 individuos sanos, para establecer su reactilidad con-tra Its protein:1s secretadas por BCC; fraccionadas poreletroforesis bidimensional (2-DE). Todos los individ-uos lueron cases no tratados. Los sueros de los pa-cientes LI. y 131. reaccionaron fuertemente con el com-plejo antigénico 85 (Ag 85) y con NIP1351. Los suerosde los pacientes II. y 131. también reaccionaronmente con moléculas novelas de 29-, 24- y 23 kDa.Los sueros de los otros pacientes reaccionaron de ma-nera sniitlitr pero los niveles de reacciOn fueron mar-cadamente menores que los de los pacientes LL y 131..Se sugierc que los antigenos de Mycobacterium lepraeque son aniilogos a los antigenos Ag 85 y NIP1151 deBCC, sean los Mimeos principales de la inmunidad Im-moral en los pacientes no tratados. Las reactividadesde los sueros con los nuevos antigenos identilicadospodrian Ser de militia(' potencial para predecir la se-veridad y el prontistico de la enlermcdad.

RÉSUMÉ

Les sirtuns col lectés it partir de patients hanséniensincluant 3 lépromateux (LL), 3 lépromateux border-lines (BL), 3 borderlines (BB). 2 tuberculoides (TT),ainsi que 4 patients tuberculeux (TB) et 3 individus enbonne sank iureitt analyses pour lcur réactivité contreles prokines (+kitties partir de filtrats de cultures de

protéines séparées par électrophrese it 2 dimen-sions (2-DE). Les serums furent °Menus itpartir de pa-tients non traités. Les serums des patients LI, et BL ontfortement réagis contre le complexe antigénique 85(Ag 85) et NIP135 I. Les sérums des patients LL et BLout aussi Rtiblement réagis avec des spots nouvelle-heft identitiés de 29, 24 et 23 kilodaltons (kDit). Lessérums des antics patients (lilt réaL",is de laqon similaire,avec toutelois des niveaux de reaction tres 'tenementinférieurs a ceux observes chez les patients LL et 131_

II est suppose qui; les antigenes de M. leprac quisoft analogues it ceux des antigènes Ag85 et NIP1351du BCC, sont les cibles principales de l'immunité it mé-diation humor:tie des patients non traités. La présenced' tine react ion de ces serums avec des antigénes nou-vellement identifiés pourrait potentiellement permettrede préchre lit sévérik et le pronostic de maladies.

nil ledgment. 'Fhis work was partly supportedby a grant from the !Inman Science Foundation and bya grant-in-aid for Scientific Research ())8771705) fromthe Nlinistry of Fducation. Science and Culture, Japan.

66, 2^Naito, et al.: Humeral Immune Re.spotiscs to BCG CF Ags^213

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