FEMS MicrobiologyLetters 99 (1992) 7-14 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier
FEMSLE 05150
Immunological characterization of a 17-kDa antigen from Cryptosporidium parvum recognized early by mucosal IgA antibodies J e a n - M i c h e l R e p e r a n t a, M u r i e l N a c i r i
b Thierry
C h a r d e s a a n d D a n i e l T. B o u t a
a Institut National de la Recherche Agronomique (LN.R.A.) and UFR des Sciences Pharmaceutiques de Tours Unit~ de Recherche Universitd-INRA d'Immunologie Parasitaire, Laboratoire de Pathologie lnfectieuse & lmmunologie, Nouzilly, France and b Unitd des Maladies h Protozoaires, Station de Pathologie Aviaire & Parasitologie, Nouzilly, France
Received 29 July 1992 Revision received 26 August 1992 Accepted 26 August 1992
Key words: Cryptosporidium p a r v u m ; IgA; Mucosal antigen
1. S U M M A R Y C r y p t o s p o r i d i u m p a r v u m antigens were characterized by immunoblot analysis of sera and intestinal secretions of B A L B / c mice orally infected with 10 5 oocysts. A major band at 17 kDa under non-reduced conditions and at 18 kDa under reduced conditions was recognized by antiC. p a r v u m IgA and IgG in serum and intestinal secretions from day 15 post-infection. This recognition persisted throughout the experiment (day 30). Mouse-serum antibodies raised against the 17-kDa purified antigen (P17) showed no crossreactivity with other C. p a r v u m antigens. Ira-
Correspondence to." J.-M. Reperant, Unit6 de Recherche Universit6-1NRA, Laboratoire de Pathologie Infectieuse et Immunologie INRA, Centre de Tours, 37380 Nouzi|ly, France.
munofluorescence study revealed that this antigen is located on the sporozoite. It is suggested that this antigen could be a good candidate for studies of mucosal immune response to C. p a r v u m and for vaccination.
2. I N T R O D U C T I O N Cryptosporidium p a r v u m is an opportunistic pathogen belonging to the phylum Apicomplexa. It was found for the first time in the small intestine of laboratory mice [1]. The first case of human cryptosporidiosis was described in 1976 [2,3]. Cryptosporidium was considered as a rare parasite and rare pathogen until 1981. Since this date, however, it has been shown to be responsible for severe pathology in A I D S patients and the number of fatal cases has increased significantly.
Most of the time, the parasite is rapidly eliminated in immunocompetent humans although persistent cryptosporidiosis has been reported in some patients [4]. In immunocompromised individuals such as people under immunosuppressive therapy [5] or more frequently AIDS patients [6], it is responsible for cholera-like diarrheas with severe dehydration often leading to death. Cryptosporidium is also of a great veterinary importance. In newborn animals, especially ruminants, cryptosporidiosis causes weight loss, dehydration and death [7]. Presently, there is no readily available therapy. Many drugs have been tested without success [8,9]. Only chemical treatment with lasalocid [10] or halofuginone lactate [11,12] are efficient in animals but these are not yet commercially available. Immune therapy using hyperimmune bovine colostrum has sometimes given good results [13,14] but the efficiency of this kind of treatment must be confirmed. Cryptosporidium part,urn is usually found in intestinal or bronchial mucosa where secretory IgA is abundant. Other parasites located in the intestinal compartment or passing through the intestine induce the production of specific secretory IgA (Eimeria, Toxoplasma) [15-17]. Local immunity and, particularly, secretory IgA could play a role in coccidian infection [16] by inhibiting the penetration of the cell by the parasite and its subsequent intracellular development. Polymeric or monoclonal IgA are also active in various infections such as, for example, those caused by influenza and Sendal viruses [18,19]. Important insights into the mechanisms involved during infection to confer resistance to cryptosporidiosis may be gained by studying the C~ptosporidium part, urn antigens which induce a mucosal IgA response. It would be of value to investigate the target antigens in an experimental infection model prior to studying their protective ability. Thus, we monitored the evolution of antigen recognition by serum and intestinal antibodies in B A L B / c mice orally inoculated with the parasite. We particularly focused on one major 17-kDa antigen recognized by mucosal IgA antibodies following infection.
3. M A T E R I A L S AND M E T H O D S
3. I. Parasites Oocysts of Cryptosporidium part'urn were obtained from calves inoculated orally with 105 parasites isolated from an infected child [20]. Calf feces were kept at +4°C in a 2.5% potassium dichromate solution until use. Feces were passed through filters from 1 mm to 100 p~m in diameter. They were then centrifuged and the pellets were suspended in water plus a 2(1% diethyl ether solution in water and shaken vigorously. They were centrifuged at 2000 × g for 10 min at +4°C. The supernatant was treated again with ether, shaken and centrifuged. This process was repeated on the resultant supernatant to collect the remaining oocysts. The oocysts were filtered through 20-#m meshes, then treated in 10% sodium hypochlorite for 10 rain and washed three times in distilled water. They were then purified using a discontinuous sucrose gradient centrifugation technique [21]. Sporozoites were obtained by excysting oocysts in a 0.2% sodium taurocholate solution in PBS at 37°C for 1 h under gentle shaking. Sporozoites and remaining oocysts were washed in PBS, numerated and then used immediately for immunofluorescent studies. 3. 2. Infection of mice Twenty male B A L B / c mice aged three or six weeks were inoculated on day 0 with an oral dose of 105 purified oocysts in distilled water. They had free access to food and water. Three mice were not inoculated, and three mice were inoculated with 105 frozen oocysts. 3.3. Sample collection On days 8, 15, 24 and 30 post-infection, sera and intestinal secretions were collected. Intestinal secretions were recovered and processed by the lavage technique described by Elson et al. [22] except that the dose of pilocarpine was 0.2 mg per mouse. They were stored at - 2 0 ° C until use. 3.4, Monoclonal antibodies Two mAbs provided by A. Bonnin [23,24] were used to evaluate the molecular mass of the bands
separated in the blots. These antibodies consisted of one IgM (IGO) binding to a 250-kDa targetantigen and one IgG (BIA) binding to a 20-kDa antigen.
3.5. lmmunoblot procedure 5 x/~ [ 0 7 ooeysts were submitted to three cycles of freezing-thawing at - 7 0 ° C in sample buffer (50 mM Tris. HCI pH 6.8; 10% glycerol; 2% sodium dodecyl sulfate (SDS); 0.1% Bromophenol blue) in non-reducing conditions, then heated at 100°C for 5 min. After centrifugation at 7800 × g for 10 rain, the supernatant was subjected to electrophoresis in 15% polyacrylamide gel with 5% SDS on a Mini Protean I1 gel apparatus (Bio-Rad) according to the method of Laemmli [25]. A standard molecular mass kit (Sigma) ranging from 14.4 to 66 kDa migrated on the same gel. The run was done with a constant current of 20 mA per gel at room temperature. A small part of the gel was stained with Coomassie blue or silver stained. The remaining part was transferred onto nitrocellulose (0.20 # m porosity; Schleicher & Schluell). The molecular mass markers were separately stained with Ponceau red. Nitrocellulose sheets were incubated for 1 h with T N T (15 mM Tris, 140 mM NaCI, 0.05% Tween 20) with 5% non-fat powdered milk (PM). Sera (diluted 1/100 in TNT) or intestinal secretions (diluted 1 / 3 in TNT) were added for 1 h. After three washes with TNT-PM 5%, alkaline phosphatase conjugate anti-lgG antibodies (goat-anti-mouseIgG, tt + L) or M (goat-anti-mouse-IgM,/x chain specific) or A (goat-anti-mouse-IgA, a chain specific) isotypes (Sigma) were incubated for 1 h. After three washes with T N T and one with R buffer (100 mM Tris, 100 mM NaC1, 5 mM MgC12, pH 9.5), the substrate preparation (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) was added in R buffer. The reaction was stopped with 20 mM Tris, 5 mM EDTA, pH 8 in distilled water. An electrophoresis was performed using, for one part of the gel, a sample buffer (50 mM T r i s - H C I pH 6.8; 10% glycerol; 2% SDS; 0.1% Bromophenol blue) with dithiothreitol (DTT, 200 mM) and for the other part of the gel the sample buffer without DTT. After transfer onto nitrocel-
lulose, the apparent molecular masses of the bound antigens were compared by immunoblotting.
3.6. Purification of the 17-kDa antigen A blot was performed on a 0.5-cm wide nitrocellulose strip transferred from the SDS-PAGE, using anti-C, parcum mouse sera, to localize the 17-kDa antigen on the gel. The corresponding band was cut on the polyacrylamide gel previously stained with Coomassie blue (R250, Sigma). The Coomassie blue colored bands above and behind the main band were also taken separately. They were stored at - 20°C before electroelution. The bands were minced and then put into dialysis tubing with exclusion limits of 3500 with 50 mM Tris, 380 mM glycin and 0.01% SDS. Etution was accomplished in 50 mM Tris, 380 mM glycin and 0.1% SDS with a constant voltage of 70 V, in a horizontal electrophoresis apparatus (Mini Sub DNA Cell, Bio-Rad). The tubings were dialysed for 24 h in ultrapure water, changing the water three times. Then, the proteins were concentrated using 20000 M r PEG and stored at + 4°C. 3. 7. Inoculation of the 17-kDa antigen to anbnats The three eluted extracts were examined in Western blots with infected mouse sera to ensure that the 17-kDa antigen was present. The positive extracts were emulsified in an equal volume of Freund's complete adjuvant for the first injection. A total volume of 200 ~xl was injected subcutaneously at multiple loci at the back of the neck of six B A L B / c mice (two mice per extract). Mice were inoculated with Freund's complete adjuvant and uninoculated mice served as negative controls. The same protocol was followed 16 days later, except that Freund's incomplete adjuvant was given instead of the complete adjuvant. Two mice orally infected with 105 oocysts in distilled water and boosted 16 days later served as positive controls. Blood was collected four weeks post-inoculation by intracardiac puncture after etherchloroform anesthesia. 3.8. Location of the antigen on the parasite Sera raised against the 17-kDa antigen, previously tested by immunoblotting with a soluble
l0
extract of C. parvum to ensure reactivity, were used for immunofluorescence studies with sporozoites and oocysts. Ten /xl containing 5 × 10 4 excysted purified oocysts were put on microscope multitest slides. The slides were air-dried at room temperature, then fixed in acetone at - 2 0 ° C for 15 min. Sera, diluted 1 / 1 0 and 1/100 in PBS, were put on the slide, 20 gl per circle for 30 rain at 37°C in a humidified room. After three washes in PBS and standing for 10 min in PBS, a 1/20 dilution in PBS of fluorescein conjugated antibodies (sheep-anti-mouse IgG whole molecule FITC conjugate; Sigma) and a 1/10000 dilution of Evans blue counterstain (Sigma) were added followed by incubation for 30 min at 37°C. Three washes of PBS and then 10 min in PBS in the dark were followed by the deposit of antifading (0.1 g p-phenylene diamine, 50 ml glycerol, adjusted p H 8-8.5 with a 0.5 M, pH 9 carbonate-bicarbonate buffer in PBS). Observations were made on an Olympus BH-2 microscope equipped for fluorescein fluorescence under 1000 × magnification. A negative control was performed with the serum of uninfected mice and positive controls were made by using the mAb IGO which binds to oocyst surface antigens.
~
~1_i
ii
~
ii!i!!~
iili¸¸ i!i
14.4--
8 15 24 30
8 15 24 30
8 15 24 30
Days post-infection Fig. 1. Immunoblot performed with the intestinal secretions of mice infected with Cryptosporidium parvum. Ig A, G and M were tested. Bands appear only with IgA and |gG. The major antigen is located between 16 and 18 kDa. This antigen is first recognized between day 8 and day 15 post-infection.
Several antigens were characterized on the immunoblots, most of them recognized by anti-C. parvum serum IgG. The main ones showed apparent molecular masses of 13 kDa, 16-18 kDa,
4. R E S U L T S IgA, G and M in both serum and intestinal secretions were studied. The intestinal antibody response to the parasite was characterized by a strong recognition of antigens by IgA and a similar but less important pattern of binding with local IgG (Fig. 1). No specific IgM was detected in intestinal secretions. Specific IgA, IgG and IgM were found in the sera of infected mice (Fig. 2). The antibody response appeared in serum and intestinal secretions between day 8 and day 15 post-infection. Control mice and mice orally inoculated with frozen oocysts had no detectable antibodies against Cryptosporidium parvum (data not shown). Furthermore, foetal calf serum showed no band on the nitrocellulose. However, no oocyst was found in the feces or in the intestine of the infected mice from day 4 to day 8 post-inoculation.
ii
ii
IgM
IgG
lgA
lgA
lgG
lgM
8 15 24 30
........ 8 15 24 30
8 15 24 30
4,5-36-29--
20.1--
Days post-infection Fig. 2. | m m u n o b l o t performed with the sera of mice infected with Cryptosporidium parvum.Ig A, G and M were tested. A major band recognized by IgA and G is located between 16 and 18 kDa.
11
24 kDa, 35-40 kDa and 65 kDa. The 24-kDa antigen was also recognized by serum IgA. Antigens of 40 and 42 kDa were recognized by serum IgM on day 15 only. The most strongly recognized antigen, detected by mucosal and systemic IgA and IgG, in nearly all animals, was one localized between 16 and 18 kDa in non-reduced conditions, which migrated to between 17 and 19 kDa in reduced conditions (Fig. 3). Most of this study was performed under non-reduced conditions (except for the definition of the pattern in reduced conditions) and the major antigen always appearing between 16 and 18 kDa on the immunoblots was named P17. Serum IgM bound this antigen faintly on day 15 and on day 30 post-infection. Purified P17 antigen injected subcutaneously in mice produced specific antibodies, as revealed in immunoblots performed on the C. pare, urn soluble extracts with the sera of these mice (Fig. 4). There was no cross-reactivity with other antigens of the parasite. Immunofluorescence on oocysts and excysted sporozoites performed with polyclonal anti-P17
~iii!i~ iii~ii~
~!~i!i~i!~i~
,i!,%i'i
1 2 3 TF TN Inf Fig. 4. Immunoblot performed with the sera of mice inoculated subcutaneously with purified antigens of Cryptosporidium parvum. Lanes 1, 2 and 3 are revealed with the sera of mice inoculated with the band above P17, P17 and the band below P17, respectively. There is no cross-reactivity with other antigens. TF: mouse inoculated with Freund's adjuvant. TN: negative control. Inf: mouse orally infected with 105 oocysts.
serum revealed that this antigen was located on the sporozoite (Fig. 5A). No fluorescence was observed with this serum on the surface of the oocyst, as compared to that seen with the IGO mAb (Fig. 5B). The fluorescence pattern was characterized by small dots inside the sporozoite body, sometimes distributed evenly throughout the organism and sometimes in a restricted location, usually in the middle of the sporozoite body (Fig. 5A).
5. DISCUSSION ~J~i ~~
12 Fig. 3. Immunoblot performed with the intestinal secretions of mice 30 days after infection. Lane 1: oocysts for electrophoresis were treated with DTT (reducing). The major antigen migrates to 18 kDa. Lane 2: sample buffer did not contain DTT (non-reducing). The major antigen migrates to 17 kDa.
After oral infection with Cryptosporidium paruurn, intestinal secretions contained parasitespecific IgA and IgG first detected on day 15 post-infection. A study performed on experimentally infected Iambs revealed only the presence of IgA antibodies in fecal extracts appearing on day 10 post-infection [26]. In calves, specific IgA, M and G were present in feces, appearing by days 4-6 post-infection [27]. These discrepancies can
12
It Fig. 5. Immunofluorescence on ooeysts and sporozoites under l{1011 × magnification performed with: (A) sera of mice immunized with the purified P17; (B) the mAb IGO which binds to oocyst surface antigens. be explained by the animal species and the sensitivity of the techniques used to reveal the production of antibodies. In our mouse model, immunoblots showed the presence of a major antigen from day 15 post-infection until the end of the experiment. It appeared as a large and intense band located between 16 and 18 kDa under non-reducing conditions and between 17 and 19 kDa under reducing conditions. This antigen, which we called P17, bound IgA and IgG present in serum and intestinal secretions from mice orally infected with C. part'urn. Serum lgM only faintly recognized this band by day 15 post-infection. Further investigations are needed to determine if the 17-kDa antigen represents only one protein because several antigens could be located among these molecular masses. The production of a monoclonal antibody directed to this band should lead to improved characterization. Antigens of a similar molecular mass have already been characterized and described in other studies [28,29]. These antigens are recognized by human
[29,30], calf [30,31] and horse serum [30] and by lamb secretory antibodies [26]. In mice, however, such antigens had not been reported [32]. In hyperimmune bovine colostrum, anti-C, p a r r u m IgA and lgG1 recognize many antigens and along with IgG2 and IgM, they recognize a major band at 14.5-16.5 kDa, which could be a surface glycoprotein [33]. Antigens from different host species detected by the same monoclonal antibody may differ in size [34]. Thus, the 17-kDa antigen could correspond.with this 14.5-16.5-kDa antigen or with a strongly recognized l l-kDa antigen [35] which migrates at 13 kDa in non-reduced conditions, though a 13-kDa antigen is faintly recognized in our experiment by serum IgG on day 15 post-inoculation. A 23-kDa antigen characterized many times [26,29,36,37] with immune bovine serum [38] could correspond with the 24-kDa antigen recognized by serum lgG and A. The polyclonal serum produced in mice immunized with the purified band was used to localize the P17. lmmunofluorescence indicated that this antigen is present on the sporozoite. The P17 can
13 t h e r e f o r e be c o n s i d e r e d as an a n t i g e n o f i n t e r e s t since it is p r e s e n t o n the infective f o r m a n d c o u l d b e t h e p o t e n t i a l t a r g e t o f a local i m m u n e res p o n s e in mice. A 15.5-kDa a n t i g e n was shown by t25I surface l a b e l l i n g to b e p r e s e n t on the surface of t h e oocyst [28] a n d so w o u l d be d i f f e r e n t to the o n e we d e s c r i b e here. T h e 17-kDa a n t i g e n , w h e n p u r i f i e d a n d inj e c t e d into mice, is a b l e to i n d u c e a specific a n t i b o d y r e s p o n s e , with no cross-reactivity with o t h e r antigens. T h u s this a n t i g e n m a y not corres p o n d to a 15-kDa a n t i g e n d e s c r i b e d by Tilley et al. [39] b e c a u s e t h e s e a u t h o r s p r o d u c e d a m A b 5C3 d i r e c t e d a g a i n s t this a n t i g e n which also recognizes a d o u b l e t of 5 5 - 6 0 k D a ( p e r s o n a l c o m m u n i c a t i o n ) . T h e reactivity with t h e p o l y c l o n a l s e r u m against the P17 was s t r o n g e r t h a n with a s e r u m r a i s e d a g a i n s t the w h o l e p a r a s i t e at the s a m e dilution. T h e i m m u n o g e n i c i t y o f this b a n d is thus very great. T h i s r e s u l t offers the possibility of p r o d u c t i o n o f a large a m o u n t o f a n t i b o d i e s (in i m m u n i z e d rabbits, for e x a m p l e ) to purify the a n t i g e n on affinity c h r o m a t o g r a p h y , or, m o r e interestingly, to s c r e e n a g e n o m i c library o f t h e p a r a s i t e to find a c l o n e p r o d u c i n g t h e antigen. T h e ability o f the 17-kDa a n t i g e n to induce b o t h a local a n d systemic h u m o r a l i m m u n e res p o n s e has b e e n shown in this work. T h e c e l l u l a r i m m u n e r e s p o n s e a g a i n s t this a n t i g e n will be t e s t e d . F u r t h e r i n v e s t i g a t i o n s will b e c a r r i e d o u t with t h e i m m u n o a f f i n i t y p u r i f i e d antigen, a n d the g e n e e n c o d i n g for t h e 17-kDa a n t i g e n will be d e t e r m i n e d . T h e s e studies a r e a p r e r e q u i s i t e to envisaging t h e study o f its p r o t e c t i v e role in cryptosporidiosis.
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by t h e C o n s e i l R~gional du C e n t r e , the A N R S ( A g e n c e N a t i o n a l e d e R e c h e r c h e sur le S I D A ) , a n d t h e C o m i t 6 D 6 p a r t e m e n t a l d ' I n d r e - e t - I _ ~ i r e d e la Ligue Nat i o n a l e c o n t r e le C a n c e r . W e g r a t e f u l l y acknowle d g e P i e r r e Yvor6 w h o a l l o w e d a p a r t o f this w o r k to b e p e r f o r m e d in his l a b o r a t o r y , A l a i n B o n n i n for p r o v i d i n g t h e m o n o c l o n a l a n t i b o d i e s
u s e d on the i m m u n o b l o t s a n d g o o d advice, and, especially, I s a b e l l e B o u r g u i n for assistance.
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14 [24] Bonnin, A., Dubremetz, J.F. and Camerlynck, P. (1991) Infect. Immun. 59, 1703-1708. [25] Laemmli, U.K. (1970) Nature 227, 680-685 [26] Hill, B.D., Blewett, D.A., Dawson, A.M. and Wright, S. (1990) Res. Vet. Sci, 48, 76-81. [27] Williams, R.O. and Burden, D.J. (1987) Res. Vet. Sci. 43, 264-265. [28] Lumb, R., Lanser, J.A. and O'Donoghue, P.J. (1988) Immunol. Cell Biol. 66, 369-376. [29] Ungar, B.L.P. and Nash, T.E. (1986) Infect. Immun. 53, 124-128. [30] Mead, J.R., Arrowood, M.J. and Sterling, C.R. (1988) J. Parasitol. 74, 135-143. [31] Peeters, J.E., Villacorta, I., Vanopdenbosch, E., Vandergheynst, D., Naciri, M., Ares-Mazas, E. and Yvor6, P. (1992) Infect. Immun. 60, 2309-2316. [32] Luft, B.J., Payne, D., Woodmansee, D.B. and Kim, C.K. (1987) Infect. Immun. 55, 2436 2441.
[33] Tilley, M., Fayer, R., Guidry, A., Upton, S.J. and Blagburn, B.L. (1990) Infect. Immun. 58, 2966-2971. [34] Nina, J.M.S., McDonald, V., Deer, R.M.A., Wright, S.E., Dyson, D.A., Chiodini, P.L. and McAdam, K.P.W.J. (1992) Parasitol. Immunol. 14, 227-232 [35] Whitmire, W.M. and Harp, J.A. (1991) Infect. Immun. 59, 990-995. [36] Lumb, R., Smith, P.S., Davies, R., O'Donoghue, P.J., Atkinson, H.M. and Lanser, J.A. (1989) Immunol. Cell Biol. 67, 267-270. [37] Whitmire, W.M., and Harp, J.A. (1990) J. Parasitol. 76, 450-452. [38] Riggs, M.W., McGuire, T.C., Mason, P.H. and Perryman, L.E. (1989) J. Immunol. 143, 1340-1345. [39] Tilley, M., Upton, S.J., Fayer, R., Barta, J.R., Chrisp, C.E., Freed, P.S., Blagburn, B.L., Anderson, B.C. and Barnard, S.M. (1991) Infect. Immun. 59, 1002-1007.
FEMSLE 05150
Immunological characterization of a 17-kDa antigen from Cryptosporidium parvum recognized early by mucosal IgA antibodies J e a n - M i c h e l R e p e r a n t a, M u r i e l N a c i r i
b Thierry
C h a r d e s a a n d D a n i e l T. B o u t a
a Institut National de la Recherche Agronomique (LN.R.A.) and UFR des Sciences Pharmaceutiques de Tours Unit~ de Recherche Universitd-INRA d'Immunologie Parasitaire, Laboratoire de Pathologie lnfectieuse & lmmunologie, Nouzilly, France and b Unitd des Maladies h Protozoaires, Station de Pathologie Aviaire & Parasitologie, Nouzilly, France
Received 29 July 1992 Revision received 26 August 1992 Accepted 26 August 1992
Key words: Cryptosporidium p a r v u m ; IgA; Mucosal antigen
1. S U M M A R Y C r y p t o s p o r i d i u m p a r v u m antigens were characterized by immunoblot analysis of sera and intestinal secretions of B A L B / c mice orally infected with 10 5 oocysts. A major band at 17 kDa under non-reduced conditions and at 18 kDa under reduced conditions was recognized by antiC. p a r v u m IgA and IgG in serum and intestinal secretions from day 15 post-infection. This recognition persisted throughout the experiment (day 30). Mouse-serum antibodies raised against the 17-kDa purified antigen (P17) showed no crossreactivity with other C. p a r v u m antigens. Ira-
Correspondence to." J.-M. Reperant, Unit6 de Recherche Universit6-1NRA, Laboratoire de Pathologie Infectieuse et Immunologie INRA, Centre de Tours, 37380 Nouzi|ly, France.
munofluorescence study revealed that this antigen is located on the sporozoite. It is suggested that this antigen could be a good candidate for studies of mucosal immune response to C. p a r v u m and for vaccination.
2. I N T R O D U C T I O N Cryptosporidium p a r v u m is an opportunistic pathogen belonging to the phylum Apicomplexa. It was found for the first time in the small intestine of laboratory mice [1]. The first case of human cryptosporidiosis was described in 1976 [2,3]. Cryptosporidium was considered as a rare parasite and rare pathogen until 1981. Since this date, however, it has been shown to be responsible for severe pathology in A I D S patients and the number of fatal cases has increased significantly.
Most of the time, the parasite is rapidly eliminated in immunocompetent humans although persistent cryptosporidiosis has been reported in some patients [4]. In immunocompromised individuals such as people under immunosuppressive therapy [5] or more frequently AIDS patients [6], it is responsible for cholera-like diarrheas with severe dehydration often leading to death. Cryptosporidium is also of a great veterinary importance. In newborn animals, especially ruminants, cryptosporidiosis causes weight loss, dehydration and death [7]. Presently, there is no readily available therapy. Many drugs have been tested without success [8,9]. Only chemical treatment with lasalocid [10] or halofuginone lactate [11,12] are efficient in animals but these are not yet commercially available. Immune therapy using hyperimmune bovine colostrum has sometimes given good results [13,14] but the efficiency of this kind of treatment must be confirmed. Cryptosporidium part,urn is usually found in intestinal or bronchial mucosa where secretory IgA is abundant. Other parasites located in the intestinal compartment or passing through the intestine induce the production of specific secretory IgA (Eimeria, Toxoplasma) [15-17]. Local immunity and, particularly, secretory IgA could play a role in coccidian infection [16] by inhibiting the penetration of the cell by the parasite and its subsequent intracellular development. Polymeric or monoclonal IgA are also active in various infections such as, for example, those caused by influenza and Sendal viruses [18,19]. Important insights into the mechanisms involved during infection to confer resistance to cryptosporidiosis may be gained by studying the C~ptosporidium part, urn antigens which induce a mucosal IgA response. It would be of value to investigate the target antigens in an experimental infection model prior to studying their protective ability. Thus, we monitored the evolution of antigen recognition by serum and intestinal antibodies in B A L B / c mice orally inoculated with the parasite. We particularly focused on one major 17-kDa antigen recognized by mucosal IgA antibodies following infection.
3. M A T E R I A L S AND M E T H O D S
3. I. Parasites Oocysts of Cryptosporidium part'urn were obtained from calves inoculated orally with 105 parasites isolated from an infected child [20]. Calf feces were kept at +4°C in a 2.5% potassium dichromate solution until use. Feces were passed through filters from 1 mm to 100 p~m in diameter. They were then centrifuged and the pellets were suspended in water plus a 2(1% diethyl ether solution in water and shaken vigorously. They were centrifuged at 2000 × g for 10 min at +4°C. The supernatant was treated again with ether, shaken and centrifuged. This process was repeated on the resultant supernatant to collect the remaining oocysts. The oocysts were filtered through 20-#m meshes, then treated in 10% sodium hypochlorite for 10 rain and washed three times in distilled water. They were then purified using a discontinuous sucrose gradient centrifugation technique [21]. Sporozoites were obtained by excysting oocysts in a 0.2% sodium taurocholate solution in PBS at 37°C for 1 h under gentle shaking. Sporozoites and remaining oocysts were washed in PBS, numerated and then used immediately for immunofluorescent studies. 3. 2. Infection of mice Twenty male B A L B / c mice aged three or six weeks were inoculated on day 0 with an oral dose of 105 purified oocysts in distilled water. They had free access to food and water. Three mice were not inoculated, and three mice were inoculated with 105 frozen oocysts. 3.3. Sample collection On days 8, 15, 24 and 30 post-infection, sera and intestinal secretions were collected. Intestinal secretions were recovered and processed by the lavage technique described by Elson et al. [22] except that the dose of pilocarpine was 0.2 mg per mouse. They were stored at - 2 0 ° C until use. 3.4, Monoclonal antibodies Two mAbs provided by A. Bonnin [23,24] were used to evaluate the molecular mass of the bands
separated in the blots. These antibodies consisted of one IgM (IGO) binding to a 250-kDa targetantigen and one IgG (BIA) binding to a 20-kDa antigen.
3.5. lmmunoblot procedure 5 x/~ [ 0 7 ooeysts were submitted to three cycles of freezing-thawing at - 7 0 ° C in sample buffer (50 mM Tris. HCI pH 6.8; 10% glycerol; 2% sodium dodecyl sulfate (SDS); 0.1% Bromophenol blue) in non-reducing conditions, then heated at 100°C for 5 min. After centrifugation at 7800 × g for 10 rain, the supernatant was subjected to electrophoresis in 15% polyacrylamide gel with 5% SDS on a Mini Protean I1 gel apparatus (Bio-Rad) according to the method of Laemmli [25]. A standard molecular mass kit (Sigma) ranging from 14.4 to 66 kDa migrated on the same gel. The run was done with a constant current of 20 mA per gel at room temperature. A small part of the gel was stained with Coomassie blue or silver stained. The remaining part was transferred onto nitrocellulose (0.20 # m porosity; Schleicher & Schluell). The molecular mass markers were separately stained with Ponceau red. Nitrocellulose sheets were incubated for 1 h with T N T (15 mM Tris, 140 mM NaCI, 0.05% Tween 20) with 5% non-fat powdered milk (PM). Sera (diluted 1/100 in TNT) or intestinal secretions (diluted 1 / 3 in TNT) were added for 1 h. After three washes with TNT-PM 5%, alkaline phosphatase conjugate anti-lgG antibodies (goat-anti-mouseIgG, tt + L) or M (goat-anti-mouse-IgM,/x chain specific) or A (goat-anti-mouse-IgA, a chain specific) isotypes (Sigma) were incubated for 1 h. After three washes with T N T and one with R buffer (100 mM Tris, 100 mM NaC1, 5 mM MgC12, pH 9.5), the substrate preparation (nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) was added in R buffer. The reaction was stopped with 20 mM Tris, 5 mM EDTA, pH 8 in distilled water. An electrophoresis was performed using, for one part of the gel, a sample buffer (50 mM T r i s - H C I pH 6.8; 10% glycerol; 2% SDS; 0.1% Bromophenol blue) with dithiothreitol (DTT, 200 mM) and for the other part of the gel the sample buffer without DTT. After transfer onto nitrocel-
lulose, the apparent molecular masses of the bound antigens were compared by immunoblotting.
3.6. Purification of the 17-kDa antigen A blot was performed on a 0.5-cm wide nitrocellulose strip transferred from the SDS-PAGE, using anti-C, parcum mouse sera, to localize the 17-kDa antigen on the gel. The corresponding band was cut on the polyacrylamide gel previously stained with Coomassie blue (R250, Sigma). The Coomassie blue colored bands above and behind the main band were also taken separately. They were stored at - 20°C before electroelution. The bands were minced and then put into dialysis tubing with exclusion limits of 3500 with 50 mM Tris, 380 mM glycin and 0.01% SDS. Etution was accomplished in 50 mM Tris, 380 mM glycin and 0.1% SDS with a constant voltage of 70 V, in a horizontal electrophoresis apparatus (Mini Sub DNA Cell, Bio-Rad). The tubings were dialysed for 24 h in ultrapure water, changing the water three times. Then, the proteins were concentrated using 20000 M r PEG and stored at + 4°C. 3. 7. Inoculation of the 17-kDa antigen to anbnats The three eluted extracts were examined in Western blots with infected mouse sera to ensure that the 17-kDa antigen was present. The positive extracts were emulsified in an equal volume of Freund's complete adjuvant for the first injection. A total volume of 200 ~xl was injected subcutaneously at multiple loci at the back of the neck of six B A L B / c mice (two mice per extract). Mice were inoculated with Freund's complete adjuvant and uninoculated mice served as negative controls. The same protocol was followed 16 days later, except that Freund's incomplete adjuvant was given instead of the complete adjuvant. Two mice orally infected with 105 oocysts in distilled water and boosted 16 days later served as positive controls. Blood was collected four weeks post-inoculation by intracardiac puncture after etherchloroform anesthesia. 3.8. Location of the antigen on the parasite Sera raised against the 17-kDa antigen, previously tested by immunoblotting with a soluble
l0
extract of C. parvum to ensure reactivity, were used for immunofluorescence studies with sporozoites and oocysts. Ten /xl containing 5 × 10 4 excysted purified oocysts were put on microscope multitest slides. The slides were air-dried at room temperature, then fixed in acetone at - 2 0 ° C for 15 min. Sera, diluted 1 / 1 0 and 1/100 in PBS, were put on the slide, 20 gl per circle for 30 rain at 37°C in a humidified room. After three washes in PBS and standing for 10 min in PBS, a 1/20 dilution in PBS of fluorescein conjugated antibodies (sheep-anti-mouse IgG whole molecule FITC conjugate; Sigma) and a 1/10000 dilution of Evans blue counterstain (Sigma) were added followed by incubation for 30 min at 37°C. Three washes of PBS and then 10 min in PBS in the dark were followed by the deposit of antifading (0.1 g p-phenylene diamine, 50 ml glycerol, adjusted p H 8-8.5 with a 0.5 M, pH 9 carbonate-bicarbonate buffer in PBS). Observations were made on an Olympus BH-2 microscope equipped for fluorescein fluorescence under 1000 × magnification. A negative control was performed with the serum of uninfected mice and positive controls were made by using the mAb IGO which binds to oocyst surface antigens.
~
~1_i
ii
~
ii!i!!~
iili¸¸ i!i
14.4--
8 15 24 30
8 15 24 30
8 15 24 30
Days post-infection Fig. 1. Immunoblot performed with the intestinal secretions of mice infected with Cryptosporidium parvum. Ig A, G and M were tested. Bands appear only with IgA and |gG. The major antigen is located between 16 and 18 kDa. This antigen is first recognized between day 8 and day 15 post-infection.
Several antigens were characterized on the immunoblots, most of them recognized by anti-C. parvum serum IgG. The main ones showed apparent molecular masses of 13 kDa, 16-18 kDa,
4. R E S U L T S IgA, G and M in both serum and intestinal secretions were studied. The intestinal antibody response to the parasite was characterized by a strong recognition of antigens by IgA and a similar but less important pattern of binding with local IgG (Fig. 1). No specific IgM was detected in intestinal secretions. Specific IgA, IgG and IgM were found in the sera of infected mice (Fig. 2). The antibody response appeared in serum and intestinal secretions between day 8 and day 15 post-infection. Control mice and mice orally inoculated with frozen oocysts had no detectable antibodies against Cryptosporidium parvum (data not shown). Furthermore, foetal calf serum showed no band on the nitrocellulose. However, no oocyst was found in the feces or in the intestine of the infected mice from day 4 to day 8 post-inoculation.
ii
ii
IgM
IgG
lgA
lgA
lgG
lgM
8 15 24 30
........ 8 15 24 30
8 15 24 30
4,5-36-29--
20.1--
Days post-infection Fig. 2. | m m u n o b l o t performed with the sera of mice infected with Cryptosporidium parvum.Ig A, G and M were tested. A major band recognized by IgA and G is located between 16 and 18 kDa.
11
24 kDa, 35-40 kDa and 65 kDa. The 24-kDa antigen was also recognized by serum IgA. Antigens of 40 and 42 kDa were recognized by serum IgM on day 15 only. The most strongly recognized antigen, detected by mucosal and systemic IgA and IgG, in nearly all animals, was one localized between 16 and 18 kDa in non-reduced conditions, which migrated to between 17 and 19 kDa in reduced conditions (Fig. 3). Most of this study was performed under non-reduced conditions (except for the definition of the pattern in reduced conditions) and the major antigen always appearing between 16 and 18 kDa on the immunoblots was named P17. Serum IgM bound this antigen faintly on day 15 and on day 30 post-infection. Purified P17 antigen injected subcutaneously in mice produced specific antibodies, as revealed in immunoblots performed on the C. pare, urn soluble extracts with the sera of these mice (Fig. 4). There was no cross-reactivity with other antigens of the parasite. Immunofluorescence on oocysts and excysted sporozoites performed with polyclonal anti-P17
~iii!i~ iii~ii~
~!~i!i~i!~i~
,i!,%i'i
1 2 3 TF TN Inf Fig. 4. Immunoblot performed with the sera of mice inoculated subcutaneously with purified antigens of Cryptosporidium parvum. Lanes 1, 2 and 3 are revealed with the sera of mice inoculated with the band above P17, P17 and the band below P17, respectively. There is no cross-reactivity with other antigens. TF: mouse inoculated with Freund's adjuvant. TN: negative control. Inf: mouse orally infected with 105 oocysts.
serum revealed that this antigen was located on the sporozoite (Fig. 5A). No fluorescence was observed with this serum on the surface of the oocyst, as compared to that seen with the IGO mAb (Fig. 5B). The fluorescence pattern was characterized by small dots inside the sporozoite body, sometimes distributed evenly throughout the organism and sometimes in a restricted location, usually in the middle of the sporozoite body (Fig. 5A).
5. DISCUSSION ~J~i ~~
12 Fig. 3. Immunoblot performed with the intestinal secretions of mice 30 days after infection. Lane 1: oocysts for electrophoresis were treated with DTT (reducing). The major antigen migrates to 18 kDa. Lane 2: sample buffer did not contain DTT (non-reducing). The major antigen migrates to 17 kDa.
After oral infection with Cryptosporidium paruurn, intestinal secretions contained parasitespecific IgA and IgG first detected on day 15 post-infection. A study performed on experimentally infected Iambs revealed only the presence of IgA antibodies in fecal extracts appearing on day 10 post-infection [26]. In calves, specific IgA, M and G were present in feces, appearing by days 4-6 post-infection [27]. These discrepancies can
12
It Fig. 5. Immunofluorescence on ooeysts and sporozoites under l{1011 × magnification performed with: (A) sera of mice immunized with the purified P17; (B) the mAb IGO which binds to oocyst surface antigens. be explained by the animal species and the sensitivity of the techniques used to reveal the production of antibodies. In our mouse model, immunoblots showed the presence of a major antigen from day 15 post-infection until the end of the experiment. It appeared as a large and intense band located between 16 and 18 kDa under non-reducing conditions and between 17 and 19 kDa under reducing conditions. This antigen, which we called P17, bound IgA and IgG present in serum and intestinal secretions from mice orally infected with C. part'urn. Serum lgM only faintly recognized this band by day 15 post-infection. Further investigations are needed to determine if the 17-kDa antigen represents only one protein because several antigens could be located among these molecular masses. The production of a monoclonal antibody directed to this band should lead to improved characterization. Antigens of a similar molecular mass have already been characterized and described in other studies [28,29]. These antigens are recognized by human
[29,30], calf [30,31] and horse serum [30] and by lamb secretory antibodies [26]. In mice, however, such antigens had not been reported [32]. In hyperimmune bovine colostrum, anti-C, p a r r u m IgA and lgG1 recognize many antigens and along with IgG2 and IgM, they recognize a major band at 14.5-16.5 kDa, which could be a surface glycoprotein [33]. Antigens from different host species detected by the same monoclonal antibody may differ in size [34]. Thus, the 17-kDa antigen could correspond.with this 14.5-16.5-kDa antigen or with a strongly recognized l l-kDa antigen [35] which migrates at 13 kDa in non-reduced conditions, though a 13-kDa antigen is faintly recognized in our experiment by serum IgG on day 15 post-inoculation. A 23-kDa antigen characterized many times [26,29,36,37] with immune bovine serum [38] could correspond with the 24-kDa antigen recognized by serum lgG and A. The polyclonal serum produced in mice immunized with the purified band was used to localize the P17. lmmunofluorescence indicated that this antigen is present on the sporozoite. The P17 can
13 t h e r e f o r e be c o n s i d e r e d as an a n t i g e n o f i n t e r e s t since it is p r e s e n t o n the infective f o r m a n d c o u l d b e t h e p o t e n t i a l t a r g e t o f a local i m m u n e res p o n s e in mice. A 15.5-kDa a n t i g e n was shown by t25I surface l a b e l l i n g to b e p r e s e n t on the surface of t h e oocyst [28] a n d so w o u l d be d i f f e r e n t to the o n e we d e s c r i b e here. T h e 17-kDa a n t i g e n , w h e n p u r i f i e d a n d inj e c t e d into mice, is a b l e to i n d u c e a specific a n t i b o d y r e s p o n s e , with no cross-reactivity with o t h e r antigens. T h u s this a n t i g e n m a y not corres p o n d to a 15-kDa a n t i g e n d e s c r i b e d by Tilley et al. [39] b e c a u s e t h e s e a u t h o r s p r o d u c e d a m A b 5C3 d i r e c t e d a g a i n s t this a n t i g e n which also recognizes a d o u b l e t of 5 5 - 6 0 k D a ( p e r s o n a l c o m m u n i c a t i o n ) . T h e reactivity with t h e p o l y c l o n a l s e r u m against the P17 was s t r o n g e r t h a n with a s e r u m r a i s e d a g a i n s t the w h o l e p a r a s i t e at the s a m e dilution. T h e i m m u n o g e n i c i t y o f this b a n d is thus very great. T h i s r e s u l t offers the possibility of p r o d u c t i o n o f a large a m o u n t o f a n t i b o d i e s (in i m m u n i z e d rabbits, for e x a m p l e ) to purify the a n t i g e n on affinity c h r o m a t o g r a p h y , or, m o r e interestingly, to s c r e e n a g e n o m i c library o f t h e p a r a s i t e to find a c l o n e p r o d u c i n g t h e antigen. T h e ability o f the 17-kDa a n t i g e n to induce b o t h a local a n d systemic h u m o r a l i m m u n e res p o n s e has b e e n shown in this work. T h e c e l l u l a r i m m u n e r e s p o n s e a g a i n s t this a n t i g e n will be t e s t e d . F u r t h e r i n v e s t i g a t i o n s will b e c a r r i e d o u t with t h e i m m u n o a f f i n i t y p u r i f i e d antigen, a n d the g e n e e n c o d i n g for t h e 17-kDa a n t i g e n will be d e t e r m i n e d . T h e s e studies a r e a p r e r e q u i s i t e to envisaging t h e study o f its p r o t e c t i v e role in cryptosporidiosis.
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by t h e C o n s e i l R~gional du C e n t r e , the A N R S ( A g e n c e N a t i o n a l e d e R e c h e r c h e sur le S I D A ) , a n d t h e C o m i t 6 D 6 p a r t e m e n t a l d ' I n d r e - e t - I _ ~ i r e d e la Ligue Nat i o n a l e c o n t r e le C a n c e r . W e g r a t e f u l l y acknowle d g e P i e r r e Yvor6 w h o a l l o w e d a p a r t o f this w o r k to b e p e r f o r m e d in his l a b o r a t o r y , A l a i n B o n n i n for p r o v i d i n g t h e m o n o c l o n a l a n t i b o d i e s
u s e d on the i m m u n o b l o t s a n d g o o d advice, and, especially, I s a b e l l e B o u r g u i n for assistance.
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