d u e t o L e is h m a n ia in f a n t u m

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D e t e c t io n o f s e r u m a n t ib o d ie s a g a in s t L e is h m a n ia 9 4 k D a

ANTIGEN IN VISCERAL AND CUTANEOUS LEISHMANIOSIS

d u e t o L e is h m a n ia in f a n t u m

ROLLAND L.*, BELKAID M.**, SEYE A.***, SCHNEIDER P.**** & GENTILINI M.*

Summary :

Leishmania promastigotes polypeptides are analyzed by immuno- blotting with sera from patients infected with different Leishmania species and presenting visceral or cutaneous infections. These sera recognize Leishmania polypeptides in several molecular masses.

The major findings of this study are as follow. 1) The Leishmania 9 4 kDa antigen, which is specifically recognized by all sera from L. infantum-infected patients with visceral infection, is recognized by some sera from L. infanium-infected patients presenting cuta

neous infection. 2) All patients with cutaneous infections due to i.

tropica, L. amazonensis, or L. guyanensis do not develop anti-94 kDa antibodies, whatever the Leishmania species used as anti

gens. 3) Difference in electrophoretic mobilities is seen between the 9 4 kDa antigen identified by sera from Leishmania infantum infected patients, and the antigen both recognized by the Concavalin A lectin and a rabbit antiserum raised against degly

cosylated Promastigote Surface Protease.

K E Y WORDS :

Leishmania.

leishmaniosis. immunoblot. antibodies, diagnosis.

MOTS CLÉS : Leishmania. le

ishmaniose. immunoempreinte. anticorps, dia

gnostic.

INTRODUCTION

P

rotozoans o f the genus L e is h m a n ia include species causing a wide variety o f diseases in hum ans. Sym ptom s range from self-healin g cutaneous lesions to diffuse cutaneous and mucosal manifestations, or severe visceral involvement o f the s p le e n , liv e r, ly m p h n o d e s an d h o n e m a rro w . V isceral leishm aniosis (VL) is classically cau sed by species o f the L e is h m a n ia d o n o v a n i ( L. d o n o v a n i) com plex : L. c h a g a s i in Latin America, L. d o n o v a n i an d L. i n f a n t u m in A fric a , A sia an d th e M editerranean region. L. c h a g a s i and L. in fa n t u m infections can present in two distinct clinical forms.

The great majority o f cases occur as a VL w hich is

* Laboratoire de P arasitologie, Facu lté de M éd ecin e Pitié-Salpêtrière, Paris, France

** Service de Parasitologie, Institut Pasteur, Alger, Algérie

*** L a b o ra to ire d e P h o to b io lo g ie , F a c u lté d e s S c ie n c e s , R o u en , France - presen t adress : Institut C ochin d e G én étiq u e M oléculaire (ICGM ), 22 rue M échain, 7 5 014, France

**** In stitu t d e B io c h im ie , U n iv ersité d e L au san n e, E p a lin g es, Suisse.

C orresp on d en ce : L. Rolland, 1 rue Lam artine, F-38000 G ren o b le - tél : 7 6 4 7 7 7 80

R ésu m é : Dé t e c t io n d’a n t ic o r p sd ir ig é sc o n t r eu na n t ig è n ed e94

kDad a n slesse r u m sd e p a t ie n t sa t t e in t sd e l e ish m a n io se sv isc é ra l e o u c u t a n é ed u e sà Le is h m a n iain f a n t u m.

Des polypeptides de promastigotes de Leishmania sont analysés par immuno-empreintes avec des sérums de patients infectés par diffé

rentes espèces de Leishmania et présentant des infections viscérale ou cutanée. Ces sérums reconnaissent des polypeptides de Leishmania de plusieurs masses moléculaires. Les principaux résultats de cette étude sont les suivants: 1) L'antigène 94 kDa de Leishma

nia, reconnu spécifiquement par les sérums de patients infectés par L. infantum et ayant une infection viscérale, est reconnu par certains serums de patients infectés par L. infantum et présentant une infec

tion cutanée. 2) Les patients infectés par L. tropica, L. amazonensis, ou L. guyanensis, présentant une leishmaniose cutanée, ne dévelop

pent pas d'anticorps anti-94 kDa. 3) Une différence de mobilité électrophorétique est observée entre la bande de 94 kDa reconnue par les sérums de patients infectés par L. infantum, et celle reconnue à la fois par la lectine concavaline-A et un sérum de lapin dirigé contre la forme déglycosylée de la Protéase de Surface des Promastigotes.

typically characterized by fever, anemia, hepatosple- nomegaly, and w eight loss. A marked hypergamma

globulinemia (Shaw and V oller, 1964) and absence o f detectable cell-m ediated immunity are the principal im m unological features o f the disease (Carvalho et al., 1981; Carvalho et a l., 1985; Sacks et al., 1987;

Cillari et al., 1991; Meller-Melloul et al., 1991). The infection has a high mortality rate if untreated and is often com plicated by secondary infections. The rarer form is a cutaneous leishmaniosis (CL). L. in fa n tu m may cause either a single nodule, a larger ulcer, or a m ucosal lesio n (R iou x et co ll., 1980; B ellazo u g et co ll., 1985; G ram iccia et a l., 1987; G ram iccia and Gradoni, 1989; Rioux and Lanotte, 1990; Gradoni et al., 1991; reviewed by Gradoni and Gramiccia, 1994).

L. c h a g a s i m ay c a u s e CL (O liv e ira e t a l ., 1 986;

Zeledon et al., 1989; Ponce et al., 1991).

Numerous studies have investigated L e is h m a n ia anti

gen expression at the level o f specific antibody reco

gnition. Antigens with potential diagnostic value have been selected by their specific recognition by VL sera in W estern blot assays (D os Santos et al., 1987; Reed et al., 1987; Evans et al., 1989; Hoerauf et al., 1992;

Mary e t a l., 1992). In p reviou s studies, using the W e s te r n b lo t t e c h n iq u e , w e h a v e id e n tifie d a Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1995021013

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ROLLAND L., BELKAID M., SEYE A., SCHNEIDER P. & GENTILINI M.

L eish m a n ia com ponent o f 94 kDa which is recogni

zed by sera from patients with Old and New Word VL, but not by sera from patients with other protozoiases, h e lm in th ia s e s , fu n g al and b a c te r ia l d is e a s e s (L.

Rolland-Burger et al., 1991; L. Rolland et al., 1992; L.

Rolland et al., 1994). The aim o f the present study was to detect anti-94 kDa antibodies in high dilution of sera from patients with VL, and in low dilution of sera from patients with CL due to L. in fan tu m , L. tropica, L.

am a z o n en sis, or L. g u yan en sis. While 94 kDa antigen has been found in many species/strains o f L eish m a n ia (L. Rolland-Burger et al., 1991), a comparison with the Promastigote Surface Protease (PSP) was undertaken.

MATERIALS AND METHODS

Pa r a s it e s a n d c u l t u r e c o n d it io n s

T

he strains used in this study w ere : L. in fa n  tu m L E M 497 (M C A N /G R /82/LEM 497), L.

g u y a n e n s i s P 30 (IUM B/GF/82/CAYP30), L.

a m a z o n e n s i s P 95 (C A Y P 9 5 ), L. t r o p i c a LEM 135 (MHOM/IQ/LV 556), L. m a jo r NIH 1 7 3 (MHOM/IR/83/

NIH173), L. m e x ic a n a LV4 (MNYC/BZ/62/M379), and L. h ra z ilien s is (MHOM/BR/75/M2904).

Promastigotes w ere grown at 28°C in culture medium consisting o f RPMI 1640 containing 10% heat-inactiva- ted fetal b o v in e serum , 2mM L-glutam ine, 25 mM sodium carbonate, 4 mM tricine, 100 IU/ml penicillin, and 100 mg/ml streptomycin.

L. in fa n tu m LEM497 and L. b ra z ilien s is M 2904 strains w ere m ain tain ed b y w eek ly p assag e in vitro. All other strains w ere maintained by successive in vivo inoculations into Balb/c mice; parasites w ere harves

ted from cutaneous lesions, and transformation into prom astigotes was perform ed at 28°C in com p lete culture medium.

Hu m a n se r a

Sera w ere collected from four patients with parasito

logically confirm ed VL. O f these four patients, two w ere from the region o f Athens, G reece, one from Chad, and one from the state o f Pernam bucco, Brazil.

CL sera w ere obtained from three Algerian patients infected with L. in fa n tu m : all patients w ere positive fo r L e i s h m a n i a in b io p sy e x a m in a tio n , but w ere n eg ativ e fo r c la ssica l a n ti-L e i s h m a n i a an tib o d ies assays. O ther CL sera w ere obtained from 3 G reek patients, and 11 from French Guyana : a brief sum

mary of. clinical history and parasitological data for each patient with OW-CL and NW-CL is presented in Table 1. Negative serum was obtained from five heal

thy F ren ch adults w ith no k now n past history o f leishmaniosis.

Pr o t e in d e t e r m in a t io n

P ro tein c o n c e n tr a tio n w as d e term in ed u sin g the Pierce protein assay (Pierce Chemical Co., 111., USA), w ith b o v in e seru m a lb u m in ( fr a c tio n V, Sigm a Chemical Co., M o.) as a relative protein standard.

So d iu m d o d e c y l s u l f a t e-p o l y a c r y l a m id e g e l

ELECTROPHORESIS AND TRANSFER

Betw een 1.5 x 108 and 2.5 x 108 promastigotes, cor

responding to 1.2 mg protein, w ere lysed in sample buffer, boiled for five minutes, and subjected to elec

trophoresis on 10% polyacrylamide gels, according to Laemmli, 1970. Standard markers w ere a mixture o f high- and low -m olecular-m ass proteins (220 kDa to 14.4 kDa; Pharmacia, Uppsala, Sw eden). Electropho

retic profiles o f L e is h m a n ia com ponents w ere elec- trotransfered o n to n itro cellu lo se m em bran es (0 .4 5 m m p o re s iz e , S c h le ic h e r & S c h u e ll, D a s s e l, Germany) (Tow bin et al., 1979). The markers transfe- red w ere stained with India Ink (H ancock and Tsang, 1983).

W e s t e r n b l o t t in g

W estern blotting was perform ed as previously descri

b ed (L. R olland-Burger e t a l., 1991). Briefly, strips w ere coated in 100 mM PBS, 5% skimmed milk, and 0.3% Tw een 20. Sera w ere diluted 1:200 (unless sta

ted otherw ise) in PBS, 0.1% T w een 20 (washing buf

f e r ) , an d in c u b a te d 2 h at 3 7 °C ( u n le s s s ta te d otherw ise). After washing, strips w ere incubated with anti-Ig (Nordic, Thilburg, T he N etherlands) or anti- IgG (Biosys, Compiègne, France) horse radish peroxi

dase (H RP) or alcalin e p h o sp h atase (AP) (B io sy s) diluted conjugates. Enzymatic activities w ere revealed using H2O 2 and 3-3’, 4-4’ diam inobenzidine for HRP congugates, and nitroblu e tetrazolium and brom o- chloro-indolyl phosphate for AP conjugate.

El u t io n o f p o l y p e p t i d e s f r o m p o l y a c r y l a m id e

GELS

Slab gels w ere cut horizontally betw een 62 kDa and 98 kD a, by follow ing prestained m olecu lar w eight standards run in parallel (1 4 ,4 to 200 kDa; G ibco- Bethesda Research Laboratories, Gaithersburg, USA).

The selected bands w ere placed in tubes containing 4% polyacrylamide gels, and w ere electroeluted over

night at 50 mA. The leads w ere reversed and the cur

rent (50 mA) was again applied for 120 seconds. The samples w ere dialyzed against double distilled water, overnight at 4°C. Aliquots o f 50 μg protein w ere lyo- philized and stored at -80°C until use.

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anti-94 kD in L. infantum cutaneous leishmaniosis

patient lesion

n° sex age (year) country (area) duration number size (cm ) ethiological agent

AL1 ND ND Algeria primary infection 1 ND L. in fa n tu m

GR1 M 75 G reece (Zakinthos) relapse 1 ND ND

GR2 M 45 G reece (Zakinthos) primary infection 1 3 x 2 L. trop ica

GR3 M 88 G reece (Zakinthos) primary infection 1 ND ND

FG1 M 41 French Guyana primary infection 1 ND L. a m a z o n e n s is

FG2 M 23 French Guyana primary infection 6 ND L. g u y a n en sis

FG4 M 30 French Guyana relapse 2 ND L. g u y a n en sis

T ab le 1 - B rief d escription o f clin ical history data fo r th e cu tan eo u s leish m an iosis patients analysed in this study. Patients w ere from Algeria (AL), G re e c e (G R ), and F ren ch G uyana (F G ). ND: n ot determ ined

Le c t in Af f in o b l o t t in g

A tw o-step procedure was used with Concanavalin A (C on A) acco rd in g to Faye and C hrisp eels, 1985.

Strips w ere saturated for 1 h at 20°C in TBS (20 mM Tris-HCl pH=7.4, 500 mM NaCl) containing 1% Tw een 20. Strips w ere then incubated in washing buffer (TBS containing 0.1% Tw een 20, 1 mM CaCl2, and 1 mM MnCl2) containing 25 mg/ml o f Con A (Sigma). After 1 h at 20°C, strips w ere w ashed and incubated in washing buffer containing 50 mg/ml HRP (Boehringer Mannheim, Mannheim, Germany). After 1 h at 20°C, the strips w ere w ashed and immersed in the staining solution 0.2 7 ml H2O 2 (110V ) and 0.5 mg 3,3' -4 ,4 ’ tetrachlorhydrate diam inobenzidine (Sigm a) per ml TB S. Controls w ere treated accord in g to the sam e protocol, except for the inclusion o f 200 mM methyl- alpha-D-m annoside (Sigma), or 15 mM methyl-alpha- glucoside (Sigm a) in the buffers used for w ashing, Con A binding, and HRP binding.

A one-step procedure was used with the HRP-labeled lectin Ricinus Communis Agglutinin RCA120 (Sigma) (Faye and Salier, 1989). Strips w ere saturated for 1 h at 20°C in TBS with 0.1 % Tw een 20 (washing buf

fer), th en in cu b ated in w ashing b u ffer co n tain ing 5 Hg/ml HRP-labeled RCA120. After 90 min at 20°C, strips w ere w ash ed and im m ersed in the staining solution consisting o f 0.3 ml H2O 2 (110V ) in 110 ml TBS m ixed with 30 mg 4-ch lo ro -l-n ap h to l (BioRad, Richemond, Ca.) in 10 ml methanol.

LEISHMANIA PROMASTIGOTE SURFACE PROTEASE (P S P ) DETECTION

Strips were coated for 1 h at 20°C in 10 mM Tris-HCl pH 7.5, 140 mM NaCl, 5% skimmed milk, and 0.05%

sodium azide. Strips were then incubated for 16 h at 20°C with a rabbit antiserum directed against the che

mically deglycosylated-gp63 (Bouvier et al., 1985) dilu

ted 1:500 in coating buffer. After 3 washes, strips were incubated 1 h with 0.5 mg/ml HRP-Protein A (Sigma) in coating buffer. After three washes, the peroxidase activity was revealed with 0.01% H20 2 and 0.3% 4- chloro-l-naphtol in methanol : 10 mM TBS (1V:3V). As positive control, 1 mg of PSP was submitted to electro

phoresis, blotted onto nitrocellulose, then incubated with the rabbit anti-PSP serum, in the same conditions.

RESULTS

VL DUE TO L . INFANTUM

H

ere w e report about quantitative aspects of the recognition o f the 94 kDa antigen by VL sera. Serum was collected from a 21 year- old G reek patient w ith classical VL : at this tim e hep atosp lenom egaly w as present, and L e is h m a n ia am astigotes w ere detected in sternal b o n e marrow aspirate. This serum w as tested for IFAT and had antibody titer o f 1600. IgG, IgM, IgA, and IgE reacti

vity to SDS-PAGE separated L. in fa n t u m (LEM 497)

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F ig . 1. - Im m u n o b lo t o f L. in fa n tu m L E M 497 p o ly p e p tid e s r e a c t e d w ith th e seru m o f a G r e e k p atie n t w ith VL (le ft) and w ith a F ren ch negative serum (right).

Sera w ere incu bated at dilutions indicated at th e top o f th e b lots : from 200 to 12,800 for V I serum , and from 50 to 20 0 fo r n eg a

tive serum . H R P -labeled g o at an ti-h um an IgG as se c o n d a n tib o d y (1 :5 0 0 0 ). A rrow indicates th e 9 4 kD a antigen position.

F ig . 3. - I m m u n o b lo t o f p o l y p e p t i d e s fr o m d if f e r e n t Leishm ania sp e cies (L. guyanensis P30, L. m ajor NIH 173, L.

m exican a LV4, L. am azon en sis P 95) reacted w ith L. tropica- infected patient sera. Lanes : 1-3, individual CL G reek patient sera (1 :1 0 0 ); VL1, individual VL G reek patient serum (1 :2 0 0 );

N, p ool o f Fren ch n egativ e sera (1 :1 0 0 ). H R P -labeled an ti

h um an Ig (G , A, M ) as s e c o n d an tib o d y (1 :1 ,0 0 0 ). Arrow ind icates th e 9 4 kD a an tigen position.

F ig . 2 . - I m m u n o b lo t o f L. infantum LEM 497 prom astig o te p o ly p e p tid e s re a c te d w ith L. in fa n t u m -infected p a t ie n t s s e r a . L a n e s : 1 -3 , in d iv id u a l CL A lg e r ia n patient sera (1 :5 0 ); VL2, indi

v id u a l VL T c h a d p a t ie n t s e r u m ( 1 : 2 0 0 ) . A P -la b e le d a n ti-h u m a n Ig G as s e c o n d an tibody (1 :2 5 0 0 ).

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a n t i - 9 4 k D in L. in f a n t u m c u ta n e o u s leish m an xosis

polypeptides blotted onto n itrocellulose w ere then tested for 2 h at 37°C. No IgA and IgE were detected, som e IgM w ere d etected , in particular IgM anti-94 kDa (data not show n). In this serum, IgG anti-94 kDa antibodies w ere detectable at the 12,800 serum dilu

tion (Fig. 1, left, arrow). In contrast, a 200, 100, or 50 fold diluted French negative serum does not detect the 94 kDa antigen (Fig. 1, right).

C l d u e t o L. In fa n tu m

Sera from th r e e A lg e rian p a tie n ts in fe c te d w ith L. in fa n tu m and presenting CL, w ere tested for 2 h at 37°C, for IgG reactivity to L. in fa n tu m LEM497 poly

p e p tid e s (F ig . 2). T h e s e ro re a c tiv ity p a tte rn s o f L. in fa n tu m CL sera w ere distinguishable from those o f L . in fa n tu m VL sera by the few er immunostaining o f Z. in fa n tu m polypeptides. Indeed, in contrast to the antigen-binding pattern observed with the serum o f a VL patient from Chad (lane VL2), the sera from three Algerian CL patients primarily identified anti

gens o f 94, 92, 77, 75, 23 and 19 kDa (lanes 1-3). O f particular interest, two out o f the three CL sera identi

fied the 94 kDa antigen (lanes 1, 2, arrow).

C l DUE TO L. TROPICA, L. AMAZONENSIS, OR L.

GUYANENSIS

Sera from three Greek patients with OW-CL w ere tes

ted for Ig reactivity to OW (L . m a jo r NIH173), as well as NW (Z. g u y a n en sis P30, L. m e x ic a n a LV4, L. a m a - z o n e n s is P95) L e is h m a n ia polypeptides (Fig. 3, lanes 1-3). Sera identified few or no antigens, whatever the L e is h m a n ia species used as antigens for the immuno- blot.

Similarly, sera from 11 French Guyana patients with NW-CL w ere tested for Ig reactivity to OW (Z. tro p ic a LEM135), and NW ( L. g u y a n e n s is P30, L. m e x ic a n a LV4, Z. a m a z o n e n s is P95) L e is h m a n ia polypeptides (Fig. 4, lanes 1-11). Responses are slightly different, depending o f the L e is h m a n ia sp ecies used for the immunoblotting. These 11 NW-CL patient sera prima

rily id entified an tigens o f 55 kDa to 96 kDa with Z. g u y a n en sis P30 blot, and antigens o f 55 kDa to 70 kDa with Z. tro p ic a LEM135, Z. m e x ic a n a LV4, and Z. a m a z o n e n s is P95 blots.

Neither Z. trop ica-infected nor Z. g u y a n en sis-infected p atients sera cou ld reco g n ize the 94 kD a antigen identified by the VL1 G reek patient serum (Fig. 3 and fig. 4. lanes VL1, arrows).

9 4 k D a ANTIGEN AND P SP DETECTIONS

Electroeluted Z. b ra z ilien sis (M 2904) fraction (62 kDa

< Mr < 98 kDa) was re-electrophoresed, then transfe- red onto nitrocellulose m em brane. India ink staining

revealed 9 bands (28 kDa, 40 kDa, 63 kDa, 67 kDa, 70 kDa, 90 kDa, 94 kDa, 96 kDa, and 98 kDa (Fig. 5 : lane 1). Most o f them are recognized by the Brazilian VL serum (lane 2) and by Con A, with a major band at 70 kDa, that reveal the presence o f a-D-mannose and/or glu cose term inal residues (lane 3). Binding with Con A was com p letly blo ck ed by incubation with 200 mM methyl-a-D-mannoside (lane 5), but not with 15 mM methyl-a-glucoside (lane 4). HRP-labeled RCA120 did not bind to the L. b r a z ilie n s is fraction (lane 6).

The reactions o f anti-L. major-PSP rabbit antiserum on Z. b ra z ilie n sis fraction and on purified Z. m a jor-PSP w ere compared. The purified Z. m a jor-PSP was stron

gly stained by the rabbit antiserum (lane PSP), w he

reas the Z. b r a z ilie n s i s fractio n w as very slightly stained by the antiserum (lane 7, arrow b). The PSP band comigrates with the major Con A band (co m pare lane 7 to 3, arrow b). As can be seen, relative m olecular mass o f the 94 kDa (arrow a) and o f PSP (arrow b, around 70 kDa) are different.

DISCUSSION

T

w o forms o f disease can develop in patients infected with Z. in fa n tu m , a limited form o f cutaneous leishm aniasis, or a fatal system ic form termed VL or Kala-azar, in which parasites deve

lop in viscera. D ifferences in antigen expression by natural variants o f Z. in fa n tu m may induce the deve

lopm ent o f lymphocytes with different effector func

tions in patients with the two forms o f disease. In the present study, w e have analysed Z. in fa n tu m LEM497 promastigotes (a viscerotropic strain) by im munoblot

ting with sera from Z. in /a n tu m -infected patients with VL or CL. By comparing som e VL and CL patient anti

b o d y r e s p o n s e s a g a in s t a n tig e n s e x p r e s s e d by Z. in fa n tu m LEM497 parasites, w e found that the anti- gen-binding patterns w ere different. Blot analyses o f L e is h m a n ia lysates with VL sera show ed that they react with the majority o f the polypeptides present in the total Z. in fa n tu m extract. This confirms that high levels o f L eish m a n ia -specific antibodies are produced during VL. In contrast, few Z. in fa n tu m polypeptides are id entified by sera from Z. in fa n t u m in fected - patients presenting cutaneous lesion. O ne possible explanation for this finding is related to differences in the ‘parasite load’ o f the VL and CL patients. The VL patients contain more parasites than do CL patients.

Moreover, the distribution o f parasites in VL patients viscera may induce different recruited B-cell clones than the parasites found in the lesions o f patients with CL.

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Fig. 4. - Im m u n o b lo t o f p o ly p e p tid e s fro m d iffere n t Leishm ania p rom astigotes sp e cie s (L. guyanensis P30, L. tropica LEM 135, L. m ex ican a LV4, L. a m azon en sis P 9 5 ) re a cte d w ith L. am azon en sis- o r L. guyanensis- infected patient sera. Lanes : 1-11, individual CL sera from Fren ch G uyana patients (1 :1 0 0 ); VL1, individual VL G reek patient serum (1 :2 0 0 ); N, p o o l o f Fren ch n egative sera (1 :1 0 0 ). H R P -labeled anti-Ig (G , A, M) as seco n d antibody (1 :1 ,0 0 0 ). Arrow ind icates th e 9 4 kD a antigen position.

Fig. 5. - D ifferential affinity o f Con A, RCA120, and serum anti-PSP for a L. braziliensis (M 2904) fraction (9 8 kD a > Mr > 6 2 kD a) blotted on to n itrocellulose m em brane. Sh eets w ere treated with: lanes: 1, India ink; 2, individual Brazilian VL serum (1 :1 0 0 ); 3, Con A + HRP ; 4, Con A + HRP in th e p re sen ce o f 15 mM m ethyl-oc-glucoside; 5, C on A + HRP in the p re sen ce o f 2 00 mM m eth yl-a-D -m an n osid e; 6, H R P-labeled RCA120; 7.

rabbit an tib od ies raised against d eglycosylated L. m ajor PSP. Arrow (a) ind icates th e 9 4 kD a antigen position ; arrow (b ) ind icates th e 7 0 kDa position.

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The L. in fa n tu m infected-patient sera tested in this study show ed various d eg rees o f h etero g en eity in antibody reactivity to Z. in fa n tu m polypeptides. This was observed in both VL and CL patients. W e obser

ved that in general, each patient responded to only a subset o f antigen expressed by parasites. As sugges

ted by Mengistu et al., 1990, reporting heterogeneity in serum antibody specifities to L. a e tb io p ic a antigens in patients with localized and diffuse CL, it is possible that these nascent reactivities play an influential role in determining the type o f immune response w hich any one individual will m ake after encountering these antigens during the infectious process.

Similarly, CL sera from L. tropica, L. a m a z o n e n s is , or L. b ra z ilien sis-infected patients, reacted in an hetero

geneous m anner with few L e is h m a n ia polypeptides, whatever the L e is h m a n ia strain used. The responses o f L. in fa n tu m -infected CL patients w ere less strong than those o f the L. a m a z o n e n s is and L. b ra z ilien sis CL patients.

In previous studies, in spite o f heterogeneity o f anti

body responses, w e have identified a L eish m a n ia -94 kDa com ponent w hich was recognized by Old World (L. Rolland-Burger et al., 1991) as well as New World (L. Rolland et al., 1994) VL patients sera, but not by other infection sera, am ong them cutaneous leishmaniosis, C hagas’ d isease, m alaria, tu bercu lo sis, and leprosy. T he sp ecific d etection o f th ese antibodies could be important in the serological diagnoses o f VL in areas in w hich Chagas’ disease, tuberculosis, or leprosy are co-endem ic, particularly if these antibo

dies recognizing the 94 kDa antigen are elicited early in the infection. Here, w e report that the 94 kDa anti

gen is recognized by VL sera at high serum dilution.

A minimum o f 5 min for serum incubation, in a stan

dard procedure (1:200 serum dilution, incubation for 2 h at 37°C), has b een necessary to visualize the 94 kDa band (data not shown).

In contrast, two out the three CL sera from Z. in fa n - tum -infected patients identified this antigen. There are stricking differences betw een the pattern o f anti

gens recognized by CL serum 3, com pared with the other two. H ow ever, there are no know n stricking differences betw een clinical and parasitological status o f the CL patient 3, com pared to the other two. Thus, the recognition o f the L e is h m a n ia 94 kDa antigen is not related to the clinical picture o f the Z. in fa n tu m - infected patients. T he 94 kDa com ponent may be a com m on m olecule to natural variants o f Z. in fan tu m . Numerous m olecules have b een characterized in dif

ferent sp ecies o f L e is h m a n ia , in particular surface m olecules, including lipophosphoglycan (LPG), PSP, transporters and ectoenzym es (review ed in Chang et al., 1990; Alexander and Russell, 1992; Schneider et

al., 1992). PSP, with molecular mass o f 63 kDa, is a m em brane m etalloprotease expressed at the promas- tigote surface o f all L e is h m a n ia that have b een ex a

m in ed (B o u v ie r e t a l ., 1 9 8 5 ; E tg es e t a l ., 1 9 8 6 ; Bouvier et al., 1987; Bouvier et al., 1989; Etges et al., 1992). Antigenic and structural relationships betw een PSP o f d ifferent L e is h m a n ia p rom astigote sp ecies have b een reported (Bouvier et al., 1985; Etges et al., 1985; Lemesre et al., 1985; Bouvier et al., 1987). Anti- PSP antibodies have b een detected in sera from CL and VL patients; PSP is thus potentially usefull anti

gen for immunodiagnosis o f leishm aniosis (Reed et al., 1990). The 94 kDa antigen has been identified by VL s e r a , o n d iffe r e n t L e i s h m a n i a p ro m a s tig o te sp e cie s : Z. in fa n t u m , L. d o n o v a n i , L. t r o p ic a , L.

m ajor, L. g u y an en sis, L. m e x ic a n a (Rolland-Burger et al., 1991), Z. a m a z o n e n s is , (Rolland et al., 1994) and L. b r a z ilie n sis (present study). This suggests the exis

tence o f com m on antigenic ep itop e(s) betw een the 94 kDa antigens o f different L e is h m a n ia promastigote sp ecies. Interestingly, w e show ed that the 94 kDa antigen was not identified on L . in fa n tu m blots by 1 :2 0 0 d ilu ted sera from 4 0 CL p a tie n ts (G r e e c e , Bolivia, French Guyana) (Rolland-Burger et al., 1991), nor by 1:50 diluted sera from 26 CL patients on four L e i s h m a n i a s p e c ie s b lo ts (Ira n , B o liv ia , B ra z il) (Rolland et al., 1994). Similarly, in the present study, no anti-94 kDa antibodies have b een identified, on five L e is h m a n ia species blots, in 1:200 diluted sera from 14 patients with CL due to L . tropica, L. a m a z o 

nensis, or L . gu y an en sis. This differential immunoge- n ic ity m ay b e re la te d to d iffe r e n c e s in a n tig e n processing.

W e have electro elu ted the 62-98 kDa region from SD S-PA G E g els o f a Z. b r a z i l i e n s i s p ro m astig o te ly sate. T w o p ie c e s o f e v id e n c e su g g est th at the L e is h m a n ia 94 kDa antigen is not PSP, the major pro

mastigote surface protein o f L eish m a n ia . Firstly, the 94 kDa antigen (stained with VL sera) migrates above of Z. b ra z ilie n sis gp63 (identified by an affinity puri

fied rabbit an tib o d ies d irected against ch em ically deglycosylated Z. m a jo r gp63 (Bouvier et al., 1985).

Secondly, HRP-Con-A does not stain the 94 kDa anti

gen, but stains a band that comigrates with that reco

gnized by the rabbit antibodies.

In conclusion, a L eish m a n ia -94 kDa antigen, different from L e is h m a n ia PSP, was recognized by serum anti

bodies from L. in fa n tu m -infected patients. This anti

g e n is n o t id e n tifie d by seru m a n tib o d ie s from Z. tropica-, L. a m a z o n en sis-, or L . g u y a n en sis-infected patients. Thus, differential im m unogenicity (circu la

ting anti-94 kDa antibodies in L . in fa n tu m -infected patients sera but not in other infection patient sera) makes these antibodies immunological markers o f the

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ROLLAND L., BELKAID M , SKYE A.. SCHNEIDER P. & GENTILINI M.

L. in fa n tu m infection. The observed sensibility o f the detection o f such antibodies (presence in all L. in fa n - tu m-infected VL patients but not in all L. in fan tu m - in fe c te d CL p a tie n ts) c o n fe rs to ou r im m u n o blo t m ethod its value in the serodiagnosis o f the visceral form o f the disease.

ACKNOWLEDGMENTS

W

e th a n k Je a n -P ie r r e D ed et and M aria Hadziandoniou for kindly providing sera from French Guyana and G reek patients .with cutaneous leishmaniosis, respectively.

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Accepté le 28 novembre 1994

d u e t o L e is h m a n ia in f a n t u m

D e t e c t io n o f s e r u m a n t ib o d ie s a g a in s t L e is h m a n ia 9 4 k D a

ANTIGEN IN VISCERAL AND CUTANEOUS LEISHMANIOSIS