SECRETED ANTIGENS OF THE AMASTIGOTE AND PROMASTIGOTE FORMS OF LEISHMANIA INFANTUM INDUCING A HUMORAL RESPONSE

SECRETED ANTIGENS OF THE AMASTIGOTE AND PROMASTIGOTE FORMS OF LEISHMANIA INFANTUM INDUCING A HUMORAL RESPONSE

IN HUMANS AND DOGS

CIBRELUS P.*, PRÉCIGOUT E.*, SERENO D.**, CARCY В.*, LEMESRE J.L.** & GORENFLOT Α.*

S u m m a r y :

To study the antigens secteted by promastigote and amastigote forms of Leishmania infantum which are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitro cultured parasites, metabolically labelled with [3 5S ] methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitro culture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum.

Nevertheless, the humoral response directed against the excreted- secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secteted antigens of the amastigote forms are immunoprecipitated with eithet human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion- secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitro life cycle.

KEY WORDS : Leishmania infantum, excrefed-secreted antigens, humoral immunity, amastigote, promastigote.

A B B R E V I A T I O N S : VL: visceral leishmaniasis; AMES-antigens: amastigote excreted-secreted antigens; PMES-antigens: promastigote excreted-secreted antigens.

Résumé : ANTIGÈNES SÉCRÉTES PAR LES FORMES AMASTIGOTES ET PROMASTIGOTES DE LEISMANIA INFANTUM ET INDUISANT UNE RÉPONSE HUMORALE CHEZ L'HOMME ET LE CHIEN

Afin d'étudier les antigènes sécrétés par les formes promastigotes et amastigotes de Leishmania infantum capable d'induire une réponse humorale nous avons immunoprécipité différents surnageants de culture in vitro, marqués à la méthionine[S35], à l'aide de sérums de malades ou de chiens. De plus, certains marquages métaboliques ont été effectués chaque jour et ceci durant un cycle complet de développement in vitro de L. infantum afin d'avoir un suivi temporel de l'excrétion-sécrétion des antigènes parasitaires. Les résultats obtenus ont montré que les deux différents hôtes développaient une réponse humorale contre les antigènes sécrétés par chacun des deux stades parasitaires de L. infantum. Néanmoins, la réponse humorale dirigée contre les antigènes d'excrétion-sécrétion du stade promastigote est qualitativement et quantitativement différente si l'on compare la réponse humorale humaine et canine. Par contre, que l'on immunoprécipite avec un sérum humain ou canin, la réponse humorale dirigée contre les antigènes d'excrétion-sécrétion du stade amastigote est similaire. De plus, l'étude temporelle a montré que l'excrétion-sécrétion des antigènes était qualitativement et quantitativement modulée au cours du cycle parasitaire in vitro de L. infantum.

MOTS CLÉS : Leishmania infantum, antigène d'excrétion-sécrétion, immunité humorale, forme amastigote, forme promastigote.

INTRODUCTION

Z

eishmania parasites, m e m b e r s o f the family o f Trypanosomatidae, are protozoan parasites w h i c h 'infect m a c r o p h a g e s o f m a n a n d o t h e r vertebrate hosts like t h o s e o f the Canidae family. T h e y c a u s e a variety o f h u m a n d i s e a s e s ranging from localized self- healing c u t a n e o u s l e s i o n s to fatal visceral infections.

T h e parasites o f t h e Leishmania donovani c o m p l e x (L. donovani a n d L. infantum in the O l d W o r l d and

* EA MENRT 2413 : Immunothérapie et Chimiothérapie des Parasi- toses et des Mycoses, Laboratoire de Biologie Cellulaire et Molécu- laire, UFR des sciences Pharmaceutiques et Biologiques, 15, avenue Charles Flahault, 34060 Montpellier Cedex 1, France.

** Laboratoire de Biologie Parasitaire, ORSTOM, BP 5045, 34032 Montpellier Cedex 1, France.

Correspondenc e: André Gorenflot.

Tel : 33 (0)4 67 54 64 81 - Fax : 33 (0)4 67 54 66 21.

E-mail: agorenflot@ww3.pharma.univ-montp1.fr

L. chagasi in the New W o r l d ) are r e s p o n s i b l e for the visceral leishmaniasis w h i c h is c h a r a c t e r i z e d , in its active form, by a l a c k o f impaired T - c e l l - m e d i a t e d i m m u n e r e s p o n s e s a n d a m a r k e d h y p e r g a m m a g l o b u - linaemia (Carvalho et al., 1 9 8 1 ; Galvão-Castro et al.,

1 9 8 4 ) . Leishmania presents with t w o m o r p h o l o g i c a l l y distinct forms: i) the flagellated promastigote forms, transmitted to the vertebrate host b y the bite o f an infected sandfly, are found in the alimentary tract o f the v e c t o r and, ii) in the m a m m a l i a n host, Leishmania replicates e x c l u s i v e l y as the aflagellated amastigote form inside the m a c r o p h a g e w h e r e they i n d u c e strong c h a n g e s in t h e cell f u n c t i o n s a n d m o r p h o l o g y o f m a c r o p h a g e s such as the formation o f a p h a g o l y s o - s o m e o r the d o w n regulation o f class II MHC (for a review s e e : A l e x a n d e r & Russell, 1 9 9 2 ) .

During its life cycle, Leishmania organisms s e c r e t e various antigens that c o u l d b e directly implicated in m a n y i m m u n o l o g i c a l or b i o c h e m i c a l alterations o f the Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999062121

C I B R E I X I S P., P R É C I G O U T E . , S E R E N O D . ET AL.

host cell. Different glycoconjugates or glycoproteins such as l i p o p h o s p h o g l y c a n ( H a n d m a n et al., 1984;

King et al, 1987) or acid p h o s p h a t a s e ( B a t e s et al, 1990; Ilg et al, 1 9 9 1 ) have b e e n s h o w n to b e secreted from the promastigote forms o f Leishmania. Similarly, other studies have demonstrated i) the secretion o f amastigote antigens into the p h a g o l y s o s o m a l c o m ­ partment via the flagellar p o c k e t o f the parasite (for review: Chakraborty & B a s u , 1997), ii) the p r e s e n c e o f Leishmania-derived antigens o n the surface o f the parasitized m a c r o p h a g e s ( B e r m a n & D w y e r , 1 9 8 1 ; Williams et al, 1986). A m o n g all the described amas­

tigote antigens o f Leishmania species, s o m e o f them are well characterized: an acid p h o s p h a t a s e ( B a t e s et al, 1 9 8 9 ; D o y l e & Dwyer, 1993), a p r o t e o p h o s p h o - glycan, (Ilg et al, 1995), a sucrase ( B l u m & Opperdoes, 1994), a g l u t a t h i o n e b i n d i n g 66 k D a p o l y p e p t i d e ( Y a h i a o u i et al, 1 9 9 3 ) and a protective LACK antigen (Prina et al, 1996).

T h e recent d e v e l o p m e n t o f a x e n i c cultures o f amasti­

gote forms from different Leishmania s p e c i e s ( B a t e s et al, 1992; Lemesre et al, 1997) have provided n e w opportunities to characterize the antigens e x c r e t e d by the mammalian stage o f the parasite. In this paper, w e studied the humoral response o f dogs and m e n against e x c r e t e d - s e c r e t e d antigens from Leishmania infantum amastigotes and promastigotes forms.

MATERIALS AND METHODS

L. INFANTUM LN VITRO C U L T U R E S

The amastigote and promastigote forms o f L. infan- tum M H O W M A ( B E ) / 6 7 / I T M A P - 2 6 3 clone 2 were

used in the present study.

Axenically grown amastigotes w e r e maintained at 37 + / - 1oС under 5 % C O2 a t m o s p h e r e by subpassaging every five days in cell-free medium called MAA/20.

MAA/20 consisted o f modified medium 199 (Life T e c h ­ nologies, F r a n c e ) b a l a n c e d with Hanks'salts, supple­

m e n t e d with 0.01 mM b a t h o c u p r o i n e disulfonic acid, 3 mM L-cysteine, 5 mM L-glutamine, 0 . 0 2 3 mM hemin, 5 mM D-glucose, 4 mM N a H C O3, 0.5 % soya trypto- casein and 25 mM H e p e s . T h e pH was adjusted at 6.5 and 2 0 % heat-inactivated fetal calf serum ( F C S ) w a s added ( L e m e s r e et al, 1 9 9 7 ) . T h e starting inoculum was 5 X 1 01 amastigotes/ml and 1 07 to 5 x 1 07 para­

sites w e r e regularly o b t a i n e d b y day 5.

Promastigote cultures w e r e derived from axenically cul­

tured amastigote stages and w e r e maintained at 25 + / - 1°C by weekly subculturing in medium RPMI 1640 (Life Technologies, France) buffered with 25 mM HEPES and 2 mM N a H C O3, pH 7.2, s u p p l e m e n t e d with 10 % heat- inactived FCS. T h e starting inoculum was 5 x 105 pro- mastigotes/ml.

H U M A N A N D D O G S E R U M S A M P L E S

Human sera w e r e obtained from five South-American patients infected with Leishmania chagasi (acute leish­

maniasis), the causative agent o f American visceral leishmaniasis (VL). T h e VL-infected h u m a n sera w e r e : HIM-A1, HIM-A3, HIM-C1, HIM-V1 and HIM-L1. T h e VL-infected c a n i n e sera w e r e o b t a i n e d from four dogs (acute infection) living in the mediterranean e n d e m i c area and infected with Leishmania infantum. T h e c a n i n e sera were: CIS-14, CIS-15, CIS-16 a n d CIS-18.

Sera from healthy subjects ( h u m a n or d o g ) w e r e u s e d as negative controls.

M E T A B O L I C R A D I O L A B E L L I N G

T w o different procedures o f m e t a b o l i c radiolabelling w e r e performed. In a first experiment, the Leishmania parasites w e r e labelled during a c o m p l e t e in vitro life c y c l e . W h e n t h e in vitro c u l t u r e s w e r e initiated, 100 µCi/ml o f [3 5S]-methionine w e r e added to MAA/20 medium and the labelled parasites w e r e c o l l e c t e d after five days for the amastigote forms and s e v e n days for the promastigote forms. T h e s e labelled antigens w e r e called "one cycle antigens".

In a s e c o n d experiment, promastigotes and amastigotes w e r e cultured in RPMI 1 6 4 0 with 10 % FCS and in MAA/20 with 2 0 % FCS respectively, and supplemented with [3 5S]-methionine ( 1 0 0 µ C i / m l ) for 24 hours. T h e s a m e p r o c e d u r e was also utilized for the promastigote forms, e x c e p t that it was carried out during seven days.

O n day 0, five culture flasks w e r e initiated with the amastigotes and s e v e n with the promastigotes ( o n e flask for e a c h day o f in vitro culture) and, for e a c h parasitic form, [3 5S]-methionine was only a d d e d to o n e culture flask. O n day 1, the labelled in vitro culture supernatants and parasites w e r e collected. T h e culture supernatants w e r e separated from the parasite pellet by centrifugation at 2,500 g for five minutes. T h e super­

natants w e r e then centrifugated at 15,000 g for 2 0 min and filtered through a diam. 0.2 µm m e m b r a n e (Acro- disc, G e l m a n S c i e n c e ) . T h e parasitic pellets and the supernatants w e r e stored at - 80°C until used. T h e s a m e day (day 1 ) , [3 5S]methionine was added to a s e c o n d flask o f in vitro culture o f promastigotes and amastigotes; after 24 hours labelling (day 2 ) , the super­

natants and the parasites w e r e c o l l e c t e d as described a b o v e . This p r o c e d u r e was repeated every day during the c o m p l e t e in vitro culture life cycle (five days for amastigote forms and s e v e n days for promastigotes forms). Thus, culture supernatants and parasites w e r e e a c h c o l l e c t e d o n e day after labelling.

I M M U N O P R E C I P I T A T I O N A S S A Y S

T h e radiolabelled cells w e r e extensively w a s h e d by centrifugation at 2,500 g in culture medium without

LEISHMANIA INFANTUM S E C R E T E D A N T I G E N S

serum and then incubated in lysis buffer (2 % Triton X - 1 0 0 , 0.6 M KC1, 5 mM EDTA (ethylenediamine-tetra- acetic acid), 3 mM phenylmethylsulfonyl fluoride, 1 % aprotinin, 2.5 % i o d o a c e t a m i d e in Tris-buffered saline [TBS: 50 mM Tris, 100 mM NaCl, pH 7.8]). T h e lysate w a s kept o n ice for o n e hour and then centrifuged at 1 5 , 0 0 0 g for 10 min at 4 ° C . T h e Triton X - 1 0 0 insoluble fraction was discarded and the supernatant (radiola­

belled L. infantum lysate) was used immediatly or kept at - 8 0 ° C . T h e radiolabelled supernatants were c o n c e n ­ trated by lyophilization.

T h e radiolabelled L. infantum lysate ( 1 06 c p m ) or 100 μl o f c o n c e n t r a t e d L. infantum supernatant (2 ml equivalent o f labelled supernatant) w e r e e a c h m i x e d with 2 μl 1 o f human o r dog i m m u n e serum and incu­

bated overnight at 4 ° C . T h e n , the antigen-antibody c o m p l e x e s w e r e precipitated b y adding 7 5 μl protein A-Sepharose CL4B b e a d s (Pharmacia; Uppsala, Swe­

d e n ) diluted 1: 1 in T B S , pH 7.2, with constant stir­

ring at 37° С for o n e hour. After incubation, the c o m ­ p l e x e s w e r e w a s h e d : twice with TEN buffer 1 ( 2 0 mM Tris, 5 mM EDTA, 100 mM NaCl, pH 7 . 5 ) supplemented with 1 % NP-40 and 2.5 % b o v i n e serum albumin, four times with TEN buffer 2 ( 2 0 mM Tris, 5 mM EDTA, 2 M NaCl, pH 7 . 5 ) containing 1 % NP-40 and twice with TEN buffer 1 containing 1 % NP-40. T h e e x c e s s buffer was removed, and the immunoprecipitated anti­

g e n s w e r e prepared for SDS-PAGE and run o n 12.5 % polyacrylamide gels under reducing conditions (Laemmli,

1 9 7 0 ) . T h e gels w e r e dried, treated by immersion in Amplify (Amersham, France), and e x p o s e d to X-OMAT XAR-5 films (Eastman Kodak Co., Rochester, N.Y.). T h e m o l e c u l a r m a s s standards (^{1 4}C - m e t h y l a t e d protein marker) w e r e o b t a i n e d from Amersham.

RESULTS

O N E CYCLE L. INFANTUM PROMASTIGOTE ANTIGENS RECOGNIZED B Y HUMAN AND CANINE IMMUNE SERA

F

ive human sera (Fig. 1A) and four c a n i n e sera (Fig. 1B) have b e e n respectively used to imrau- noprecipitate o n e cycle promastigote antigens.

W h e n the promastigote antigens w e r e immunopreci­

p i t a t e d b y t h e different V L - i n f e c t e d h u m a n s e r a (Fig. 1A, lanes 2 - 6 ) , numerous c o m m o n bands w e r e found. T h e major antigens c o r r e s p o n d e d to molecular weights o f 155, 1 2 0 - 1 1 8 doublet, 97, 7 8 , 72, 67, 6 3 , 5 0 , 46, 4 4 , 3 8 , 34 and 26 kDa. Nevertheless, HIM-C1 (Fig.

1A, lane 4 ) and HIM-V1 (Fig. 1A, lane 5 ) sera strongly immunoprecipitated a 6 3 kDa antigen. W h e n the same a n t i g e n s w e r e i m m u n o p r e c i p i t a t e d b y VL-infected c a n i n e sera (Fig. I B , lanes 2 - 5 ) , the major c o m m o n b a n d s w e r e 7 2 , 7 0 , 50, 4 8 , 4 6 , 34-32 doublet and 22 kDa. Nevertheless, on a general point o f view, there w e r e numerous qualitative a n d / o r quantitative diffe­

r e n c e s b e t w e e n L. infantum promastigote antigens

Fig. 1. - [mmunoprecipitation of one cycle antigens of L. infantum promastigotes with VL-infected sera. (A) VL-infected human sera: lane 1, healthy human serum: lane 2, HIM-A1 ; lane 3. HIM-A3; lane 4, HIM-C1; lane 5, HLM-V1 and lane 6. HIM-L1. (B) VL-infected canine sera: lane 1, healthy dog serum: lane 2, CIS-18; lane 3, CIS-Ιό; lane 4, CLS-14 and lane 5. CIS-15.

CIBRELUS P., PRÉCIGOUT E., SERENO D. ET AL.

immunoprecipited b y each canine serum. For example, serum CIS-15 (Fig. I B , lane 5 ) stongly reacted with a 6 3 k D a antigen.

O N E CYCLE L. INFANTUM AMASTIGOTE ANTIGENS RECOGNIZED B Y HUMAN AND CANINE IMMUNE SERA Similarly, the human sera (Fig. 2A) and c a n i n e sera (Fig. 2 B ) have b e e n used to immunoprecipitate o n e c y c l e a m a s t i g o t e a n t i g e n s . E l e v e n m a j o r c o m m o n bands, corresponding to proteins o f 155, 9 6 , 7 8 , 7 2 , 6 6 , 5 0 , 4 6 , 3 8 , 3 4 , 32 and 2 6 kDa w e r e immunopreci- pitated with b o t h human and c a n i n e sera. As d e m o n s ­ trated o n the o n e cycle L. infantum promastigote anti­

g e n s , HIM-V1 (Fig. 2A, lane 5 ) e x h i b i t e d the strongest humoral r e s p o n s e s against the o n e cycle L. infantum amastigote antigens. B a s e d on the results obtained with the immunoprecipitations o f o n e c y c l e L. infantum antigens, HIM-V1 human serum and CIS-15 canine- serum w e r e c h o s e n to immunoprecipitate e x c r e t e d - s e c r e t e d antigens o f L. infantum promastigote and amastigote forms.

PROMASTIGOTE EXCRETED-SECRETED ANTIGENS RECOGNIZED B Y HUMAN AND CANINE IMMUNE SERA T h e labelled excreted-secreted antigens collected every day o f the in vitro life cycle (from day 1 to day 7 ) were immunoprecipitated with human ( H I M - V l ) and canine

(CIS-15) serum (Figs. ЗА and 3 B ) . All along the in vitro L. infantum life cycle, the major promastigote excreted- secreted antigens (PMES-antigens) inducing humoral response in human w e r e a 63-65 k D a doublet. O n day 1, the major immunoprecipitated antigen had a m o l e ­ cular weight o f 6 3 kDa and three other minor antigens could b e detected ( 7 8 , 7 0 and 6 5 k D a ) . T h e e x c r e t i o n o f the 6 5 k D a antigen increased to reach a m a x i m u m at day 4 o f culture, w h e r e a s , t h e e x c r e t i o n o f the 7 8 and 7 0 k D a antigens was quantitatively stable until day 4. O n day 4, the PMES-antigens that c o u l d b e immunoprecipitated w e r e 9 5 , 7 8 , 7 0 , 6 3 - 6 5 doublet, 5 0 and 4 6 kDa. O n day 5 to 7, the patterns o f i m m u n o ­ precipitated antigens l o o k e d like t h o s e e v i d e n c e d o n day 1 to 3, e x c e p t e d that there was n o qualitative dif­

ference b e t w e e n the antigens o f 6 3 and 6 5 kDa.

T h e i m m u n o p r e c i p i t a t i o n s carried out with c a n i n e serum o n the s a m e labelled antigens, confirmed the timing o f antigen excretion-secretion d e s c r i b e d a b o v e , i.e, an increase from day 1 to day 3, a m a x i m u m o n day 4 and d e c r e a s e from day 5 to 7 o f culture. Never­

theless, CIS-15 c a n i n e serum w a s able to i m m u n o p r e ­ cipitate m o r e e x c r e t e d - s e c r e t e d antigens than HIM-Vl human serum. T h e major antigens ( d e t e c t e d o n day 4 ) displayed a molecular mass o f 120, 7 8 , 7 4 , 7 2 , 6 5 , 6 3 , 50, 4 4 , 4 0 , 3 8 and 32 kDa. T h e s e results s h o w e d that the day 4 o f in vitro culture (logarithmic growth p h a s e ) c o r r e s p o n d e d , quantitatively, to the most important

Fig. 2. - Immunoprecipitation of one cycle antigens of L. infantum amastigotes with VL-infected sera. (A) VL-infected human sera : lane 1, healthy human serum; lane 2, HIM-A1; lane 3. HIM-A3; lane 4, HIM-C1; lane 5, HIM-Vl and lane 6, HIM-L1. (B) VL-infected canine sera : lane 1, healthy dog serum; lane 2, CIS-18; lane 3, CIS-16; lane 4, CIS-14 and lane 5, CIS-15.

LEISHMANIA INFANTUM SECRETED ANTIGENS

Fig. 3. — Analysis of the humoral response of man (A) and dog (B) against promastigote excreted-secreted antigens, Immunoprecipitations were performed with HIM-V1 and CIS-15 sera on radiolabelled culture supernatants collected every day of the in vitro life cycle. Lanes 1 to 7 correspond to each day of the in vitro culture life cycle.

period for L. infantum promastigote antigen excretion- secretion.

T h e human humoral r e s p o n s e against PMES-antigens w a s p r e d o m i n a n t l y d i r e c t e d against a 6 3 - 6 5 k D a e x c r e t e d - s e c r e t e d antigen doublet, w h e r e a s the c a n i n e humoral r e s p o n s e was m o r e c o m p l e x and directed against a greater n u m b e r o f excreted-secreted antigens o f L. infantum promastigotes.

A M A S T I G O T E E X C R E T E D - S E C R E T E D A N T I G E N S

R E C O G N I Z E D B Y H U M A N A N D C A N I N E I M M U N E S E R A

T h e s a m e immunoprecipitation protocol was applied o n radiolabelled amastigote excreted-secreted antigens (AMES-antigens) collected from day 1 to day 5 o f in vitro culture (Figs. 4A and 4 B ) . In contrast to PMES- antigens, a similar pattern was o b s e r v e d with human (Fig. 4 A ) and c a n i n e serum (fig. 4 B ) . T h e major AMES- antigens inducing a humoral response had a molecular mass o f 9 0 , 67, 6 5 and 6 3 kDa. However, other minor a n t i g e n s ( 9 8 , 8 1 , 7 2 , 4 6 a n d 4 4 k D a ) w e r e a l s o detected. T h e maximum o f excretion-secretion antigens was observed on day 2 to 3 (logarithmic growth p h a s e ) o f a x e n i c in vitro cultures o f L. infantum amastigotes.

T h e c h r o n o l o g i c a l a p p e a r a n c e o f the antigens s h o w e d that o n day 1 (latent growth p h a s e ) , the 67 k D a antigen was the major s e c r e t e d antigen. From the beginning o f the logarithmic growth phase (day 2 ) , to the e n d o f the logarithmic growth p h a s e (day 4 ) , a strong secretion-excretion o f the 67 k D a antigen was o b s e r v e d and an increase in the e x c r e t i o n o f the 6 5 and 6 3 kDa antigens was e v i d e n c e d . During the s a m e time lapse (from day 2 to 4 ) , there was a qualitative

decrease o f all the others immunoprecipitated antigens.

During the stationary growth phase (day 5 ) , the secre­

tion-excretion o f the 67 kDa antigen was residual and all the others e x c r e t e d - s e c r e t e d antigens had almost disappeared.

DISCUSSION

The excreted-secreted antigens o f Leishmania are implicated to have a role all along the life cycle o f the parasite. In the sandfly vector, at least three e n z y m e s are k n o w n to b e secreted by promas­

tigotes: chitinase, N-acetylglucasaminidase and sucrase.

Chitinase and N-acetylglucasaminidase intervene in the lysis o f the chitin framework o f the peritrophic m e m b r a n e which permits the forward migration o f the p r o m a s t i g o t e s ( S c h l e i n et al., 1 9 9 1 ) , t h e s e c r e t e d sucrase plays an important role in the nutrition and d e v e l o p m e n t o f promastigotes in the insect gut ( B l u m

& O p p e r d o e s , 1 9 9 4 ) . Early after the infection o f the mammalian host, lipophosphoglycan released by the promastigotes, p r o m o t e s intracellular survival b y pro­

tecting Leishmania promastigote against early intraly- s o s o m a l degradation (Mauël, 1 9 9 6 ) and on a general point o f view, the e x c r e t e d - s e c r e t e d antigens o f the promastigote stages have also b e e n implicated in faci­

litating m a c r o p h a g e infection by the parasite (Mukerji et al., 1 9 8 6 ) , and in decreasing m o n o c y t e and lym­

p h o c y t e differentiation (El-On et al., 1980; Londner et al., 1 9 8 3 ) . More recently, Peters et al. ( 1 9 9 7 ) , have s h o w n that the secretion o f p r o t e o p h o s p h o g l y c a n by

C I B R E X U S P., P R É C I G O U T E. S E R E N O D . ET AL.

Fig. 4. - Analysis of the humoral response of man (A) and dog (B) against amastigote excreted-secreted antigens. Immunoprecipitations were performed with HIM-V1 and CIS-15 sera on radiolabelled culture supernatants collected every day of the in vitro life cycle. Lanes 1 to 5 correspond to each day of the in vitro culture life cycle.

L. mexicana amastigotes c a u s e d vacuolization o f per­

itoneal m a c r o p h a g e s in vitro.

In an attempt to define m o l e c u l e s for immunodia- gnostic or vaccination purposes, several authors have studied the humoral r e s p o n s e to Leishmania promas- tigote antigens by using immunoblotting t e c h n i q u e s with sera from patients affected with leishmaniasis (Rolland-Burger et al., 1 9 9 1 ; Mary et al, 1 9 9 2 ) , and patients cured o f visceral leishmaniasis or with an asymptomatic infection (White et al., 1 9 9 2 ) . However, as far as w e k n o w , n o studies have b e e n carried out o n the L. infantum e x c r e t e d - s e c r e t e d antigens, which provide e v i d e n c e a b o u t the humoral i m m u n e res­

p o n s e s in patients and dogs having visceral leishma­

niasis. Our studies illustrate that, regardless the host serum utilized, the e x c r e t e d - s e c r e t e d antigens o f the promas'tigote forms or o f the intramacrophagic amas­

tigotes are able to induce an humoral r e s p o n s e . T h e major PMES-antigens able to i n d u c e a strong humoral r e s p o n s e in man have an apparent molecular weight o f 63 and 65 kDa. W e have o b s e r v e d that in the promastigote stage o f L. infantum, the 65 kDa e x c r e t e d - s e c r e t e d a n t i g e n is first, t h o u g h slightly, secreted during the latent phase, while the secretion strongly increases o n day 4 o f the in vitro culture (log p h a s e ) . During the m e t a c y c l o g e n e s i s p h a s e (days 6 to 7 ) the e x c r e t i o n o f this e x c r e t e d - s e c r e t e d antigen r e m a i n s p r e d o m i n a n t . In c o n t r a s t to t h e 65 k D a a n t i g e n , t h e 63 k D a a n t i g e n is m o r e a b u n d a n t l y

e x p r e s s e d at the beginning than at the e n d o f the in vitro culture. Thus, the immunoprecipitation assays s e e m to demonstrate two different roles, for the 63 kDa and 65 k D a e x c r e t e d - s e c r e t e d antigens, during the promastigote life cycle. Differences in the e x p r e s s i o n o f these two excreted-secreted antigens o f L. infantum promastigotes might have a m e a n i n g in terms of para­

site virulence a n d / o r infectivity. T h e 63 k D a antigen c o u l d b e implicated in the establishment o f the

para-

s i t a e m i a , w h e r e a s t h e

65

k D a

antigen

c o u l d b e involved in the differentiation o f

promastigote

forms to metacyclic forms. T h e latter hypotheses

are

in

agree­

ment with the studies o f K w e i d e r et al. ( 1 9 8 9 ) w h o have demonstrated that the d e v e l o p m e n t o f metacy­

clic promastigotes o f L. braziliensis is associated with the increasing e x p r e s s i o n o f

gp65.

Immunoprecipitations o f L. infantum promastigotes excreted-secreted antigens with canine infection serum has s h o w n a humoral r e s p o n s e directed against a greater n u m b e r o f antigens than that with h u m a n serum. In addition to the 63 k D a and 65 k D a excreted- secreted antigens majoritarely r e c o g n i z e d b y human serum, the canine serum allowed the detection o f anti­

g e n s in the 7 0 - 1 2 0 k D a range and in the l o w m o l e ­ cular m a s s range. O u r data demonstrate that the pro­

mastigotes p r e d o m i n a n t l y r e l e a s e e x c r e t e d - s e c r e t e d antigens o n day 4, during the log p h a s e o f the in vitro culture. This stronger humoral response of dogs against leishmanial excreted-secreted antigens is in a c c o r d a n c e

LEISHMANIA INFANTUM SECRETED ANTIGENS

with the studies o f different authors (Abranches et al, 1991; Rolland et al, 1 9 9 4 ; Correia et al., 1 9 9 6 ) . In this case, the authors studied the humoral r e s p o n s e o f infected dogs against total antigenic extracts o f the pro­

mastigote forms and, b y western blotting, they have s h o w n that t h e s t r o n g e s t h u m o r a l r e s p o n s e w a s directed against the 3 0 , 4 5 k D a , the 58-84 k D a and the 9 4 k D a regions. If w e c o m p a r e the human and the c a n i n e humoral r e s p o n s e s , the hypothesis that the c a n i n e r e s p o n s e could b e directed against antigens o f lyzed promastigotes must b e discarded. As a matter o f fact, if the immunoprecipitation samples had contained mainly lysis antigens, the immunoprecipitation pat­

terns o f promastigote e x c r e t e d - s e c r e t e d antigens with h u m a n or c a n i n e sera should have b e e n similar to that o f the immunoprecipitation patterns o b s e r v e d w h e n using the total promastigote antigens. Indeed, w e can o b s e r v e that the human antibody r e s p o n s e is c o m ­ pletely different w h e n using total or e x c r e t e d - s e c r e t e d antigens.

T h e s u c c e s s o f having axenically grown amastigotes with b i o c h e m i c a l and m o l e c u l a r characteristics similar to t h o s e o f m a c r o p h a g e - d e r i v e d a m a s t i g o t e s h a s a l l o w e d n e w d e v e l o p m e n t s in the studies o f e x c r e t e d - secreted antigens from the amastigote stage. In contrast to the data obtained with the immunoprecipitations o n the e x c r e t e d - s e c r e t e d antigens o f the promastigote form o f L. infantum, the pattern o f immunoprecipitated amastigote e x c r e t e d - s e c r e t e d antigens and their s e c r e ­ tion timing w e r e quite c o m p a r a b l e w h e n h u m a n or canine sera w e r e used. Working with axenically grown amastigotes implicates that at least three different types o f e x c r e t e d - s e c r e t e d antigens may b e observed. T h e e x c r e t e d - s e c r e t e d antigens present i n / o n the parasito- p h o r o u s vacuole, i n / o n the m a c r o p h a g e , and outside the host-cell. Even if the results o b t a i n e d d o not allow for discrimination b e t w e e n these different e x c r e t e d - secreted antigens, it can b e assumed that the e x c r e t e d - s e c r e t e d antigens immunoprecipitated during the first t w o days o f in vitro culture could b e directly involved in the parasite establishment inside the parasitophorous vacuole. Lang et al. ( 1 9 9 4 ) have suggested that the para­

sitophorous v a c u o l e acquires its b i o c h e m i c a l characte­

ristics within 4 8 hours after infection. In a c c o r d a n c e with these results, it is demonstrated here that the immunoprecipitation profile o f the excreted-secreted a n t i g e n s w a s qualitatively c o m p l e x o n day 2 and remained more simple on days 3 and 4. In addition, Lang et al. ( 1 9 9 4 ) have s h o w n that a GTP-binding pro­

tein rab (marker o f the prelysosomal compartment) was highly expressed in/on the parasitophorous vacuole. In a similar manner, w h e n polyclonal antibodies directed against excreted-secreted antigens, w e r e used to screen a cDNA library o f L infantum amastigote stages, among t h e different cDNA c l o n e s o b t a i n e d , o n e o f t h e m

expressed an excreted-secreted GTP-binding protein rab (unpublished results).

With regard to the excretion timing o f the amastigote excreted-secreted antigens during the in vitro life cycle, a strong humoral response against a 67, 6 5 and 6 3 k D a triplet was observed from day 2 to day 4 o f culture. A polyclonal antibody directed against the major surface glycoprotein g p 6 3 is able to recognize these excreted- secreted antigens (data not s h o w n ) . Different authors [Medina-Acosta et al. ( 1 9 8 9 , 1 9 9 3 ) , F r o m m e l et al.

(1990)] have demonstrated that the g p 6 3 can b e found in the amastigote stage. T h e amastigote gp63, unlike the promastigote form o f the protein, lacks a phos­

phatidyl inositol m e m b r a n e a n c h o r and is an hydro- philic protein that can b e found in the flagellar p o c k e t o f t h e parasite. M o r e o v e r , F r o m m e l et al. ( 1 9 9 0 ) demonstrated that at least two higher-Mr gp63 bands can b e detected in an amastigote lysate. Medina-Acosta et al. ( 1 9 9 3 ) have shown that the gp63 genes expressed in the amastigote e n c o d e an extended (41 amino acids) carboxyl terminus. In addition, Frommel et al. ( 1 9 9 0 ) have also s u p p o s e d that these differences in the mole­

cular weigth possibly reflected differences in the post- translational processing o f the amastigote gp63. In this experiment, it can b e o b s e r v e d that, although the 67 kDa antigen was expressed from day 2 to day 4, the 65 and 6 3 kDa excreted-secreted antigens w e r e only e x p r e s s e d o n days 3 and 4 and could not b e immu­

noprecipitated o n day 2. That s e e m s to indicate that these different g p 6 3 proteins a n d / o r post-translational modifications probably possess a biological significance for the development o f the parasite inside the host-cell.

In conclusion, w e demonstrated the release o f extra­

cellular antigens from L. infantum amastigote and pro­

mastigote forms and the e x i s t e n c e o f a host humoral r e s p o n s e directed against these excreted-secreted anti­

gens. Moreover, the time course studies have s h o w n that the secretion o f leishmanial antigens exhibits both qualitative and quantitative variations according to the d a y o f t h e in vitro c y c l e . Currently, s t u d i e s are underway to demonstrate the efficiency o f e x c r e t e d - e x c r e t e d a n t i g e n s to i n d u c e an i m m u n o p r o t e c t i o n against L. infantum. Moreover, the b i o c h e m i c a l cha­

racterization o f e x c r e t e d - s e c r e t e d antigens will allow us to speculate about their possible functions in the survival o f amastigotes within the m a c r o p h a g e .

REFERENCES

ABRANCHES P., SANTOS GOMES G . , CAMPINO L., SCHNUR L.F. &

JAFFE C.L. An experimental model for canine visceral leish­

maniasis. Parasite Immunology, 1991, 13, 537-550.

ALEXANDER J . & RUSSEL D . G . The interaction of Leishmania species with macrophages. Advances in Parasitology, 1992, 31, 175-254.

CIBRELUS P., PRECIGOUT E.. SERENO D. ET ΑΙ..

BATES P.Α., HERMES I. & DWYER D . M . Leishmania donovani:

immunochemical localization and secretory mechanism of soluble acid phosphatase. Experimental Parasitology, 1989, 68, 335-346.

BATES P.Α., HERMES I. & DWYER D.M. Golgi-mediated post-

translational processing of secretory acid phosphatase by Leishmania donovani promastigotes. Molecular and Bio­

chemical Parasitology. 1990, 39. 247-255.

BATES P.Α., ROBERTSON C D . , TETLEY L. & COOMBS G . H . Axenic

cultivation and characterization of Leishmania mexicana amastigote-like forms. Parasitology, 1992, 105, 193-202.

BERMAN J. & DWYER D . M . Expression of Leishmania antigen on the surface membrane of infected human macrophages

in vitro. Clinical of Experimental Immunology, 1981. 44, 342-348.

BLUM J J . & OPPERDOES F . R . Secretion of sucrase by Leishmania donovani. Journal of Eukaryotic Microbiology, 1994, 41.

228-231.

CARVALHO Ε.M., TEIXEIRA R . S . & JOHNSON W . D . Cell-mediated

immunity in American visceral leishmaniasis: reversible immunostipression during acute infection. Infection and Immunity. 1981. 33, 498-500.

CHAKRABORTY P. & BASU Μ.К. L e i s h m a n i a phagolysosome:

drug trafficking and protein sorting across the compart­

ment. Critical Review of Microbiology, 1997, 23, 253-268.

CORREIA DA COSIA J . M . , NEOCY ASIT В., VOULDOUKIS I., SILVA SAMPAIO M.L., GENTIUM M . & MONJOUR L. Antigenic com­

ponents of partially purified antigens of Leishmania dono­

vani infantum recognized by sera from dogs with asymp­

tomatic or active visceral leishmaniasis. American Journal of Tropical Medicine and Hygiene, 1996, 55, 511-515.

DOYLE P.S. & DWYER D . M . Leishmania. immunochemical comparison of the secretory (extracellular) acid phospha­

tases from various species. Experimental Parasitology.

1993. 77, 435-444.

EL.-ON J., BRADLEY D J . & FREEMAN J.C. Leishmania donovani:

action of excreted factors on hydrolytic enzyme activity of macrophages from mice with genetically resistance to infection. Experimental Parasitology, 1980, 49, 167-174.

FROMMEL Т . О . . BITTON L.L., FUJIKURA Y . & MCMASTER W . R . The

major surface glycoprotein (GP63) is present in both life stages of Leishmania. Molecular and Biochemical Para­

sitology 1990. 38, 25-32.

GALVAO-CASTRO В . , SA FERREIRA J . A . , MARZOCHI К.F., MAR- ZOCHI M.C., COUTINHO S.G. & LAMBERT P.H. Polyclonal В cell

activation, circulating immune complexes and autoimmu­

nity in human american visceral leishmaniasis. Clinical and Experimental Immunology, 1984, 56, 58-66.

HANDMAN E., JARVIS H . M . & MITCHELL G.M. Leishmania major, identificai ion of stage-specific antigens and antigens shared by promastigotes and amastigotes. Parasite Immunology.

1984, 6. 223-233.

ILG T., STIERHOF Y . D . , ETGES R.. ADRIAN M., HARBECKE D . & ОVE­

NATH P. Secreted acid phosphatase of Leishmania mexi­

c a n a : a filamentous phosphoglycoprotein polymer. Pro­

ceedings of the National Academy of Science USA, 1991.

88, 8774-8778.

ILG T . , STIERHOF Y.D., MCCONVILLE M J . & OVERATH P. Purifi­

cation, partial characterization and immunolocalization of a proteophosphoglycan secreted by Leishmania mexi­

cana amastigote. European Journal of Cell Biology, 1995, 66. 205-215.

LANG T . , HELLIO R . . KAYE P.M. & ANTOINE J . C . Leishmania donovani-infected macrophages: characterization of the parasitophorous vacuole and potential role of this orga­

nelle in antigen presentation. Journal of Cell Science, 1994, 107. 2137-2150.

KING D.L., CHANG Y.D. & TURCO S . J . Cell surface lipophos- phoglycan of Leishmania donovani. Molecular and Bio­

chemical Parasitology, 1987, 24, 47-53.

K W E I D E R M . , LEMESRE J . L . , S A N T O R O F., KUSNIERZ J . P . , S A D I -

GURSKY M. & CAPRON A. Development of metacyclic Leish­

mania promastigotes is associated with the increasing expression of GP65, the major surface antigen. Parasite Immunology, 1989. 11. 197-209.

LAEMMLI U . K . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227, 680-685.

LEMESRE J . L . , SERENO D . , DAULOUEDE S., VEYRET В . , BRAYON N .

& V i N C E N D E A U P. Leishmania spp.: nitric oxide-mediated metabolic inhibition of promastigote and axenically grown amastigote forms. Experimental Parasitology. 1997, 86, 58- 68.

LONDNER M . V . , FRANKENBURG SLUTZKY G . M . &. GREENBLATT C.L.

Action of leishmanial excreted factors ( F F ) on human lymphocyte blast transformation. Parasite Immunology.

1983. 5. 249-256.

MAUËL J. Intracellular survival of protozoan parasites with spe­

cial reference to Leishmania spp.. Toxoplasma gondii and Trypanosoma cruzi. Advances in Parasitology, 1996, 38, 1-51.

MARY C . LAMOUROUX D., DUNAN S. & QUILICI M . Western blot

analysis of antibodies to Leishmania infantum antigens:

potential of the 14 KD and the 16 KD antigens for dia­

gnostis and epidemiologic purposes. American Journal of Tropical Medecine and Hygiene, 1992, 47. 764-771.

MEDINA-ACOSTA E., KARESS R.E.. SCHWARTZ H . & RUSSELL D . G .

The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage. Molecular and Biochemical Parasi­

tology, 1989, 37, 263-274.

MEDINA-ACOSTA E., KARESS R.E. & RUSSELL D . G . Structurally dis­

tinct genes for the surface protease of Leishmania mexi­

cana are developmentally regulated. Molecular and Bio­

chemical Parasitology, 1993, 57, 31-46.

MUKERJI K... DAS A.K.. BANERJEE N . & GHOSH D.K. Studies on

exoantigens of Leishmania donovani. Indian Journal of Biochemistry and Biophysics, 1986, 23. 148-151.

PRINA E., LANG T . , GLAICHENHAUS N . & ANTOINE J.C. Presenta­

tion of the protective parasite antigen LACK by Leish- mania-infected macrophages. Journal of Immunology, 1996. 156, 4318-4327.

PETERS C , STIERHOE Y.D. & ILG T. Proteophosphoglycan

secreted by Leishmania mexicana amastigote causes vacuole formation in macrophages. Infection and Immu­

nity, 1997, 65. 783-786.

LEISHMANIA INFANTUM SECRETED ANTIGENS

ROLLAND-BURGER L., ROLLAND X., GRIEVE G W . & MONJOUR L.

Immunoblot analysis of the humoral immune response to Leishmania donovani infantum polypeptides in human visceral leishmaniasis. Journal of Clinical Microbiology.

1991, 29. 1429-1435.

ROLLAND L., ZILBEKFARH V.. FURTADO A & GENTILINI M. Identi­

fication of a 94-kilodalton antigen on Leishmania pro­

mastigote forms and its specific recognition in human and canine visceral leishmaniasis. Parasite Immunology, 1994,

16, 599-608.

SCHLEIN Y . . JACOBSON R.L. & SHLOMAI J . Chitinase secreted by Leishmania functions in sandfly vector. Proceedings of the Royal Society of London. 1991. 245. 121-126.

TURCO S . J . & DESCOTEAUX A. The lipophosphoglycan of Leish- mania parasites. Annual Review of Microbiology, 1992, 46, 65-94.

WHITE A.C., CASTE M., GARCIA L., TRUJILLO D . & ZAMBRANO L.

Leishmania chagasi antigens recognized in cured visceral leishmaniasis and asymptomatic infection. American Journal of Tropical Médecine and Hygiene. 1992, 46, 123-

131.

WILLIAMS K . M . , SACCI J . B . & ANTHONY R.L. Identification and recovery of Leishmania antigen displayed on the surface membrane of mouse peritoneal macrophages infected in vitro. Journal of Immunology. 1986, 136, 1853-1858.

YAHIAOUI В . . LOYENS M;. TAIBI Α.. SCIIÒNECK К., DUHREMETZ J . F . .S: OUAISSI M.A.. Characterization of a Leishmania antigen associated with cytoplasmic vesicles ressembling endo- somal-like structure. Parasitology, 1993, 107, 497-507.

Reçu le 15 décembre 1998 Accepté le 26 mars 1999