Immunogenicity of Sm32 synthetic peptides derived from the Schistosoma mansoni adult worm

Immunology Letters 88 (2003) 211
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Immunogenicity of Sm32 synthetic peptides derived from the Schistosoma mansoni adult worm Oscar Noya a,*, Belkisyole´ Alarco´n de Noya a, Fanny Guzma´n b, Henry Bermu´dez a a

Escuela de Medicina ‘‘Luis Razetti’’ and Seccio´n de Biohelmintiasis, Instituto de Medicina Tropical, Universidad Central de Venezuela, Apartado 47624, Los Chaguaramos, Caracas 1010-A, Venezuela b Fundacio´n Instituto de Inmunologı´a, Bogota´, Colombia Received 30 March 2003; accepted 9 April 2003

Abstract The previously called ‘‘hemoglobinase’’ Sm32 molecule of the adult worm of Schistosoma mansoni was chemically synthesized in 22 polymeric peptides based on the t-boc strategy. Their immunogenicity was evaluated in rabbits to which a mixture of five to six peptides of 20 amino acids long were given in three doses with Freund’s adjuvant. Seventeen peptides were found to be immunogenic, and sera from immunized rabbits corresponding to the molecule from the first 335 amino acids, recognized the 32 kDa native protein from the adult worm antigen by western blot. Of those, the relevant peptides responsible of the recognition of the original molecule corresponded to amino acids 101
/120, 121
/140 and 244
/268, based on inhibition competitive assays. Because Sm32 is one of the excretory and secretory molecules released with the vomitus of the adult worm, it is one of the target antigens for detection in plasma of infected individuals. The production of these polyclonal monospecific antibodies against the synthetic peptides could be of value in the immunodiagnosis of this parasitosis. # 2003 Published by Elsevier Science B.V. Keywords: Schistosoma mansoni ; Sm32; Synthetic peptides; Asparagynil endoprotease; Hemoglobinase

1. Introduction The diagnosis of Schistosomiasis mansoni is one of the unsolved problems, particularly in areas of low transmission. Parasitological and immunodiagnostic tests based on the capture of circulating antigens have not reached the expected sensitivity in individuals with low parasite loads (B/100 eggs/g feces) [1]. Excretory
/ secretory molecules, especially those eliminated with the vomitus of the adult worm, in the circulation of infected hosts, would be of great value, not only for seroepidemiological surveys but also for the evaluation of potential candidate schistosomiasis vaccines. Previously, our laboratory identified a rich repertoire of adult worm parasite antigens [2] particularly a broad

* Corresponding author. Tel.: /58-212-605-3571/3563; fax: /58212-693-0454. E-mail address: [email protected] (O. Noya).

band of approximately 36 kDa, which corresponded to the duple 31
/32 kDa described by Ruppel et al. [3]. Sera from infected children strongly recognized these molecules, indicating a high degree of antigenicity [2]. The 31 kDa was identified as cathepsin B while the 32 kDa molecule as an asparagynil endopeptidase, both cloned and sequenced by Klinkert et al. [4]. This asparagynil endoprotease, initially cloned and sequenced by Davis et al. [5], was found to be very similar to the asparagynil endoprotease of the jack bean [6]. And it has been postulated that might play an important role in the hemoglobin digestion. However, the molecule expressed in cell insects by Gortz and Klinkert [7] did not degrade the hemoglobin. Sm32 does not appear to be directly involved in the hemoglobin digestion, but it probably modifies the post-translational protein, similar to other legumains [8]. Dalton and Brindley [9] proposed that Sm32 activate cathepsin L, B, D and C, playing an indirect but important role in the digestion of the hemoglobin.

0165-2478/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0165-2478(03)00086-5

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Sm32 is actively eliminated as exoantigen in the blood stream [10] and is recognized by infected S. mansoni human sera. We evaluated the antigenicity and immunogenicity of this molecule, using synthetic peptides based on the sequence previously reported [4]. The aim of the present work was to evaluate the immunogenicity of these peptides and the recognition of the native molecule, by antibodies of animals immunized with synthetic peptides of Sm32.

2. Materials and methods 2.1. Adult worm antigen (AWA) preparation Using the perfusion method of Smithers and Terry [11], adult worms were collected from hamsters, 7 weeks after infection. Worms were washed three times in saline solution (NaCl 0.85%), homogenized (Potter
/Elvehjem) in phosphate buffered saline (PBS) containing protease

Table 1 Sequences of the monomeric and polymeric synthetic peptides of the Sm-32 protein of the adult worm and their corresponding denomination IMT

MW

Sequencea

AA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 69 70 71 72 89 88 21 22 63 64 23 24 25 26 27 28 65 66 29 30 31 32 33 34 35 36

2327.50 2684.10 2263.40 2620.00 2136.60 2493.20 2396.30 2752.90 2364.10 2720.70 2349.90 2706.50 2025.00 2381.60 2173.80 2530.40 2739.60 3096.20 2433.90 2754.50 2227.60 2548.20 1926.40 2247.00 2368.70 2725.30 2473.50 2794.10 2285.60 2642.20 2493.00 2849.60 2576.20 2932.80 2415.30 2735.90 2218.60 2591.20 2334.70 2691.30 2156.60 2513.20 1949.70 2306.30

MMLFSLFLISILHILLVKCQ CGMMLFSLFLISILHILLVKCQGC LDTNYEVSDETVSDNNKWAV CGLDTNYEVSDETVSDNNKWAVGC LVAGSNGYPNYRHQADVCHA CGLVAGSNGYPNYRHQADVCHAGC YHVLRSKGIKPEHIITMMYD CGYHVLRSKGIKPEHIITMMYDGC DIAYNLMNPFLGKLFNDYNH CGDIAYNLMNPFLGKLFNDYNHGC KDWYEGVVIDYRGKKVNSKT CGKDWYEGVVIDYRGKKVNSKTGC FLKVLKGDKSAGGKVLKSGK CGFLKVLKGDKSAGGKVLKSGKGC NDDVFIYFTDHGAPGLIAFP CGNDDVFIYFTDHGAPGLIAFPGC DDELYAKEFMSTLKYLHSHKRY CGDDELYAKEFMSTLKYLHSHYRKGC YSKLVIYIEANESGSMFQQIL CGYSKLVIYIEANESGSMFQQILGC GSMFQQILPSNLSIYATTAAN CGGSMFQQILPSNLSIYATTAANGC PTECSYSTFCGDPTITTC CGPTECSYSTFCGDPTITTCGC LADLYSYNWIVDSQTHHLTQ CGLADLYSYNWIVDSQTHHLTQGC RTLDQQYKEVKRETDLSHVQ CGRTLDQQYKEVKRETDLSHVQGC VQRYGDTRMGKLYVSEFQGS CGVQRYGDTRMGKLYVSEFQGSGC RDKSSTENDESPMKPRHSIASR CGRDKSSTENDESPMKPRHSIASRGC DIPLHTLHRQIMMTNNAEDKSF CGDIPLHTLHRQIMMTNNAEDKSFGC LMQILGLKLKRRDLIEDTMK CGLMQILGLKLKRRDLIEDTMKGC LIVKVMNNEEIPNTKATIVQ CGLIVKVMNNEEIPNTKATIDQGC TLDCTESVYEQFKSKCFTLQ CGTLDCTESVYEQFKSKCFTLQGC QAPEVGGHFSTLYNYCADGY CGQAPEVGGHFSTLYNYCADGYGC CADGYTAETINEAIIKICG CGCADGYTAETINEAIIKICGGC

1
/20

IMT, Instituto de Medicina Tropical; MW, molecular weight; AA, aminoacids. a Based on Klinkert et al. [4].

21
/40 41
/60 61
/80 81
/100 100
/120 121
/140 141
/160 161
/182 182
/202 195
/215 216
/223 224
/243 244
/273 272
/291 292
/313 314
/335 336
/355 356
/375 376
/395 396
/415 416
/429

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inhibitors (1 mM PMSF and 1 mM 1 EDTA) in an ice bath, and centrifuged at 12 000/g for 2 h at 4 8C. The supernatant (AWA) was stored at /80 8C. Protein concentrations were determined as described by Lowry et al. [12].

213

Sm32 sequence of Klinkert et al. [4], a total of 44 monomeric and polymeric peptides were synthesized and identified as IMT (Instituto de Medicina Tropical) (Table 1). 2.3. Immunization of animals

2.2. Peptide synthesis Peptides were manually synthesized following the tBoc strategy as described by Merrifield [13] and Houghten et al. [14] at the Peptide Synthesis Laboratory of the Instituto de Medicina Tropical, UCV. Twentytwo polymeric peptides were synthesized adding glycine and cysteine amino acids at both carboxy and amino terminals to induce polymerization under oxidative conditions. Semipermeable polypropylene bags with a previously weighed BHA 4-methilbenzhidrilamine resin, were placed in a solution with the first amino acid and activated with dicyclohexylcarbodiimide (DCC, Amersham† ) for 45 min. Deprotection was then achieved with 55% trifluoroacetic acid (TFA) in dicloromethane (DCM). Afterwards, the amino acid was neutralized with 5% N ,N -diisopropyletilamine (DIEA, Amersham† ) in DCM. The bag was ready for immersion in the next amino acid solution and these steps were followed until the desired size of the peptide was reached. Finally, the peptide was cleaved from the resin as described by Tam et al. [15]. First, the ‘‘low’’ cleavage, in which only the protector groups of the lateral chains were cleaved, was achieved using 25% fluorhidric acid (HF). The second or ‘‘high’’ cleavage was performed with 90% HF and 10% anisol to separate the peptide from the resin. Once the peptide was isolated, it was extracted with 10% acetic acid, lyophilized and quality control assessed by analytical RP-HPLC and identified by MALDI-TOF mass spectrometry. Based on the Table 2 Groups of rabbits immunized with synthetic peptides of the Sm32 protein from the S. mansoni adult worm and control antigens Groups of rabbits

Rabbit identifica- Peptides or antigens tion

I

1, 3

II

4, 6,15

III

7, 8, 9

IV

10, 11,12

V VI VII

16 13, 14 E, G

IMT-2, IMT-6, IMT-10, IMT-4, IMT-8, IMT-12 IMT-14, IMT-18, IMT-72, IMT16, IMT-70 IMT-22, IMT-24, IMT-28, IMT64, IMT-26 IMT-66, IMT-32, IMT-36, IMT30, IMT-34 IMT-88 AWA CFA and IFA

IMT, Instituto de Medicina Tropical; AWA, adult worm antigen; CFA, complete Freund’s adjuvant; IFA, incomplete Freund’s adjuvant.

Seven groups of New Zealand rabbits were immunized as shown in Table 2. On days 0, 15 and 30, rabbits from groups I to V were immunized subcutaneously in the dorsal region, with a mixture of five to six polymeric peptides, with the exception of Group V that received only peptide IMT-88. Two hundred fifty microgram of each peptide, diluted in 0.5 ml distilled water and mixed with Complete Freund’s Adjuvant (CFA) 1:1, was inoculated to each rabbit in the first dose and Incomplete Freund’s Adjuvant (IFA) 1:1 was used for the second and third doses. Animals were bled on days 0 and 40; therefore, each animal had its own control serum. Group VI was immunized with AWA (850 mg per dose). Animals of Group VII received the CFA and IFA as control groups (0.5 ml per dose). For technical reasons and in order to decrease the number of rabbits, pools of peptides were used for the immunizations, instead of immunizing with individual peptides. Additionally, rabbits were bled under anesthesia (ketamine chlorhydrate: 10 mg/kg) and the manteinance and manipulations were carried out according to the legislation and guidelines established by the ‘‘Asociacio´n Venezolana de Bioterios’’. 2.4. Immunogenicity Immunogenicity was evaluated by the Multiple Antigen Blot Assay (MABA) [16]. Individual peptides, in their monomeric and polymeric forms, and AWA, at a concentration of 10 mg/ml, were poured in the channels of an acrylic template (Minibloter† 28 SL, Immunetics Inc., Cambridge, MA), containing a nitrocellulose membrane (Bio-Rad Laboratories, 0.45 mm) which adsorb synthetic peptides during 1 h. After three washes (PBS
/0.05% Tween) and blocking (PBS
/Tween
/5% dry milk for 2 h), the nitrocellulose membrane was cut into 2 mm wide strips perpendicular to the sensitized rows with antigens. Each strip, contained all monomeric or polymeric peptides plus AWA and it was incubated, with 1/100 control or immune rabbit sera. After 1 h and the respective washes (3 /), an anti-rabbit serum conjugated to horseradish peroxidase was used as a second antibody for 1 h. The reaction was developed by a chemiluminiscent substrate (Luminol† , Amersham) for 1 min and exposed to a photographic film (Hyperfilm† , ECL Detection System, Amersham). Immunogenicity was also evaluated by the predictive method of Hopp and Woods [17] using a window of 21 amino acids.

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2.5. PAGE and western blots AWA was separated by SDS-PAGE in a 9% mini-gel (6 /9 cm) under dissociating and reducing conditions as described by Laemmli [18]. Two hundred and fifty microliters of AWA at a protein concentration of 8.5 mg/ml diluted 1/10 in sampling buffer, were run at 25 mA in a preparative gel. Molecular weight standards (SDS-7, Sigma Chemicals Co.) were run simultaneously. AWA was electrotransferred onto nitrocellulose paper (0.45 mm) as described by Towbin et al. [19] at 125 mA for 90 min. Free reacting sites of the nitrocellulose membrane were blocked with PBS
/Tween
/5% dry milk solution for 2 h. Strips of 2 mm width were incubated individually with each control and immune rabbit serum. The next steps were followed as described above for the MABA technique in order to prove the recognition of the antigens contained in AWA by the sera from rabbits immunized with different synthetic peptides. 2.6. Western blot inhibition assay In order to identify the immunodominant peptide(s) responsible for the recognition of the Sm32 among the pool of peptides used to immunize each rabbit, a competitive western blot assay was designed. Fifty microliter of each peptide or pool of peptides at 1 mg/

ml concentration in distilled water, were incubated with the immune serum of each rabbit. After 30 min incubation, the nitrocellulose strip with electrotransferred AWA, was introduced into this mixture and incubated for 90 min. Next steps were similar to the immunoblot protocol described above. All data presented were the result of at least two different experiments.

3. Results 3.1. Immunogenicity of synthetic peptides of Sm32 None of the pre-immune sera recognized the peptides, and they were used as controls for each immunized rabbit. Sera from Group I of rabbits immunized with polymeric peptides IMT-2, 4, 6, 8, 10, 12 recognized peptides 4, 6, 10, and 12 in MABA as shown in Fig. 1. Sera from Group II recognized peptides IMT-14 and 18, giving the former a stronger signal. Rabbits from Group III strongly recognized peptides IMT-22, 24, 26, 28 and 64. Group IV sera recognized also in intense manner peptides IMT-30, 32, 34, and 66. One rabbit from Group VII recognized peptides IMT-88 and 89. Some cross reactivity appeared in all groups. In Group I, both animals recognized peptide IMT-30.

Fig. 1. Recognition of polymeric synthetic peptides by sera from rabbits (R) immunized with the different peptides, by MABA. Odd numbers belong to pre-immune sera and even numbers to immune sera. MABA of group V have different order of antigens. E and G; control rabbits immunized with FCA and IFA.

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Fig. 2. Recognition of monomeric synthetic peptides by sera from rabbits (R) immunized with the different peptides, by MABA. Odd numbers belong to pre-immune sera and even numbers to immune sera. E and G; control rabbits immunized with CFA and IFA.

Fig. 3. Predictive analysis of antigenicity based on hydrophilicity of the Sm32 molecule by Hopp and Woods, (b) aligment of synthetic peptides indicating the degree of immunogenicity in rabbits; j, high; b, low; I, negative.

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Two rabbits from Group II recognized peptide IMT-64. Two out of three rabbits from Group III recognized IMT-12 and 14 and two out three rabbits from Group IV recognized IMT-12 and 64. The two rabbits immunized with AWA and those immunized with CFA and IFA did not recognize any of the polymeric peptides of Sm32 (Fig. 1). Monomeric synthetic peptides were less well recognized than polymeric peptides (Fig. 2). Group I recognized monomerics IMT-3 and 9. Group II recognized IMT-11; Group III showed a weak recognition of IMT21, 23, 25 and 63. Group IV recognized IMT-27, 29 and 65. Also, animals immunized with AWA recognized AWA but did not recognize any monomeric peptides. Animals immunized with only CFA/IFA did not recognize AWA and any monomeric peptides.

3.3. Recognition of the original Sm32 by sera of rabbits immunized with synthetic peptides

3.2. Predictive studies of antigenicity

Identification was based on a competitive inhibition western blot assay with AWA as the source of antigen. A representative example of these experiments is shown in Fig. 5. In Group I, peptide IMT-12 was able to inhibit the recognition of the original Sm32, alone as well as when included in the pool. In Group II, peptide IMT-14 alone or in the pool was responsible for the inhibition. Peptide IMT-64 was the peptide responsible for the

Based on the analysis by Hopp and Wodds [16], five regions of the complete molecule showed hydrophilic properties (amino acids 30
/40; 100
/140; 170
/180; 250
/ 320 and 350
/370). These regions corresponded exactly with the immunogenic peptides demonstrated in the rabbit model (Fig. 3a and b).

With the exception of rabbits immunized with synthetic peptides of Group IV (IMT-66, 30, 32, 34 and 36) all immune sera corresponding to Groups I, II, III and V, recognized the 32 kDa molecule present in AWA by western blot (Fig. 4, Group V is not shown). The strongest recognition was observed with Group III. Animals immunized with the complete antigen (AWA) weakly recognized the molecule of 32 kDa. A human positive control serum (C/) intensely recognized Sm32.

3.4. Identification of the synthetic peptides of Sm32 responsible for recognition of the native protein

Fig. 4. Western blot of AWA exposed to sera from rabbits immunized with polimeric synthetic peptides of the Sm32 protein. Odd numbers correspond to pre-immune sera and even numbers to immune; /HS, positive human serum, /HS, negative human serum.

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Fig. 5. Western blot inhibtition assay of AWA. Immunized rabbit sera recognized native protein of Sm32 in western blot (immune sera of R-3,R-4 and R-8 rabbits). Each experiment corresponded to individual preincubation of the sera with their respective peptides and the pool of them (Ej; IMT 2, 4, 6, 8, 10, 12, pool). The predominant peptides, IMT-12, 14, 64, and the respective pools, inhibited the recognition of the native Sm32.

inhibition in Group III. Peptide IMT-88 of Group V was not able to inhibit the recognition of Sm32.

4. Discussion Synthetic peptides have become a very useful strategy in the development of diagnostic reagents and vaccine candidate molecules in several infectious diseases [20
/ 22]. When human sera are exposed to molecules of AWA, the most relevant recognized molecule is the broad band at 31 and 32 kDa. The high frequency of recognition of this broad band on immunoblots [2,3,23], its specificity [24] and the fact that it is the antigen of the AWA more strongly detected by all isotypes in children infected with S. mansoni [2], justify the study of the Sm31 and Sm32 molecules. In order to know the immunogenicity of synthetic peptides of one of the most important excretory and secretory components of S. mansoni known as Sm32, the complete molecule of the asparagynil endoprotease was chemically synthesized and its immunogenicity explored in rabbits. At this point, we must clarify that ELISA was the technique initially used for this research but MABA was found to be easier, quicker, cheaper, more sensitive and provided good results with some peptides not soluble in water. Peptides identified as

IMT-2, 8, 16, 70, 72 and 34 were not immunogenic or at least their recognition was not detected by these assays. Seventeen out of the 24 total peptides were immunogenic in their polymeric form. Peptides of group IV (IMT-66, 30, 34 and 36) resulted strongly immunogenic, however, rabbits from this group did not recognize the original molecule in the AWA. This could be due to the fact that our peptides did not imitate the conformation of this region or that in the original molecule correspond to discontinuous epitopes. Peptides IMT-2, 4, 6, 8 and 10, from Group I, corresponded to the pre-protein region and even though they were immunogenic, we did not expect that they would participate in the recognition of the Sm32 molecule. Dominant epitopes were found to be IMT-12, 14 and 64 peptides as demonstrated by the competitive inhibition western blot assay. These are responsible for the major recognition of the Sm32 protein. Sera from rabbits immunized with these three peptides are currently evaluated in an immunodiagnostic test, based on the capture of this circulating antigen. The regions recognized as B-cell epitopes by the rabbits were two regions comprised between amino acids 101 and 140 (IMT-12 and 14), and between 244 and 268 amino acids (IMT-64). Their immunogenic properties corresponded to the predictive pattern based on their hydrophilicity and accessibility of this region, pointing out the

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importance of this predictive tool in the selection of the most antigenic epitopes. The very low concentration of the excretory Sm32 molecule in the crude homogenate of the adult worm antigen might be the reason why animals immunized with AWA did not recognize Sm32 in the western blot. To our knowledge, this is the first attempt of immunizing animals with the synthetic peptides of Sm32. We also have produced antibodies raised from the immunization of synthetic peptides, which recognized the native molecule of the parasite. Sm32 has been located in the intestinal epithelium of the schistosomula and adult worm, in the ventral surface of the adult males and in the cephalic glands of the cercariae [25]. These observations and the indirect role in the activation of other proteolytic enzymes involved in the nutrition of the parasite such as cathepsin D and L could indicate that Sm32 is a relevant molecule for the parasite survival. Antibodies against the cephalic glands of the cercariae and the gastrodermis, as those produced in the present work, should also be explored for blocking cercarial penetration, schistosomula migration and adult worm nutrition. Note: aminoacid sequence data reported in this paper is available in the NCBI protein bank (http:// www.ncbi.nlm.nih.gov) under the accession number: P09841.

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13] [14]

Acknowledgements Financial support was partially provided by the Program of ‘‘Control de Enfermedades Ende´micas’’ PCEE-PNUD VEN/96. CDCH-UCV supported traveler expenses for the presentation of these results in international meetings. The authors thank Noraida Zerpa, Cecilia Colmenares and Sandra Losada for technical assistance.

[15]

[16]

[17]

[18]

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