Vaccine 32 (2014) 2086–2092
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Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms C. Dias-Lopes a , L. Felicori a , L. Rubrecht b , S. Cobo b , L. Molina b , C. Nguyen b , P. Galéa b , C. Granier b , F. Molina b , C. Chávez-Olortegui a,∗ a b
Departamento de Bioquímica e Imunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil SysDiag, UMR3145,CNRS/Biorad, Montpellier, France
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 21 January 2014 Accepted 6 February 2014 Available online 22 February 2014 Keywords: Loxosceles spider venoms Sphingomyelinases D Monoclonal antibody Epitopes
a b s t r a c t We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29–36 kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The spiders Loxosceles intermedia, L. laeta and L. gaucho are a group of arachnids known as “brown spider” with medical importance in the South and South-east of Brazil [2,3]. The number of human accidents caused by spiders of this genus in Brazil has reached almost 7000 annually [4]. Loxoscelism, the term used for envenomations with Loxosceles spiders, can be observed as two well-defined clinical variants: cutaneous loxoscelism and systemic loxoscelism. Pain, edema, and livedoid plaque, which develop later into a necrotic scar, are the predominant local manifestations in cutaneous loxoscelism, occurring in around 83% of the cases. In systemic loxoscelism, hematuria and hemoglobinuria are always observed, whereas intravascular hemolysis and
∗ Corresponding author at: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, CEP: 31.270.901, 6627 Belo Horizonte, Brazil. Tel.: +55 31 3409 2625; fax: +55 31 3409 2613. E-mail address: [email protected] (C. Chávez-Olortegui). http://dx.doi.org/10.1016/j.vaccine.2014.02.012 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
coagulation, sometimes accompanied by thrombocytopenia and renal failure, occur in approximately 16% of the victims [5–7]. Antivenom therapy is used to neutralize the circulating venom and reduces the risk of fatal complications following human accidents [8]. The anti-loxoscelic antivenom is the polyspecific serum that containing antibodies against whole venoms of the three Loxosceles species (L. gaucho, L. laeta and L. intermedia) and is produced at Centro de Producão e Pesquisa de Imunobiológicos (CPPI) of the State of Paraná, Brazil [9]. Proteins of the phospholipase D family also named sphingomyelinase D (SMase D), or dermonecrotic proteins, are responsible for the necrotic skin lesions and also for systemic toxic effects following envenomation by Loxosceles spiders [10–13]. Loxosceles venoms express SMases D isoforms and these proteins constitute a family of homologs with 190 non-redundant sequences described in 21 species of the Sicariidae family [14]. SMase D proteins are also the most antigenic/immunogenic components of the venom [15]. Monoclonal and polyclonal antibodies against Loxosceles whole venoms generally recognize dermonecrotic proteins [16,17]. Neutralizing monoclonal antibodies (mAbs) open new perspectives to employment of these antibodies in loxoscelism
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treatment or diagnosis. However, all neutralizing mAbs produced until now recognize effectively only the venom species-specific [15,17]. In the present work, we produced a mAb able to recognize the venoms of the three major medically important species of Loxosceles spiders in Brazil. These mAb was obtained by immunization of mice with recombinant L. intermedia Smase D isoform (rLiD1) [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho whole venoms as antigens coated onto micro titer plates, so as to drive selection toward cross-reactive antibodies. The mAb namely LiD1mAb16 was able to recognize a large range of proteins antigens (at least 25) in each of the three Loxosceles venom. Epitope-mapping experiments revealed that it recognizes a highly conserved linear epitope located in the catalytic region of SMases D toxins. LiD1mAb16 displayed a protective activity in rabbits challenged with rLiD1, suggesting that this mAb may be a promising candidate for therapeutic serum development or diagnosis in the future. 2. Materials and methods 2.1. Animals Animals were maintained at the Centro de Bioterismo of the Instituto de Ciências Biológicas of the Federal University of Minas Gerais, Brazil and received water and food under controlled environmental conditions. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) (A5452-01) and was approved by local authorities (protocol #89/11 – Comitê de Ética em Experimentac¸ão Animal – Federal University of Minas Gerais). 2.2. mAbs production The rLiD1 from L. intermedia venom [1] was used as immunogen to BALB/c mice. The animals were immunized four times subcutaneously, at approximately 2 weeks intervals, with 10 g of protein in complete Freund’s adjuvant (Sigma) at the first injection, and incomplete Freund’s adjuvant (Sigma) at subsequent inoculations. A booster injection of rLiD1 was made 4 weeks after the fourth immunization. Throughout the immunization schedule, mice were bled and the reactivity of immune sera was tested against rLiD1, L. intermedia and L. laeta venoms. Three days after the last injection, spleen cells from immunized mice were fused with Sp2/0 myeloma cells (ATCC). Supernatants from resulting hybridomas were screening by ELISA using L. intermedia and L. laeta venoms. mAbs were purified on a protein A-sepharose column (GE Healthcare). 2.3. Indirect ELISA for the screening of hybridomes Maxisorp plates (Nunc) were coated overnight at 4 ◦ C with a solution of 1 g/mL of the protein or L. intermedia, L. gaucho or L. laeta venom in 50 mM Na2 CO3 , pH 9.0, and blocked with PBS containing 10 g/L BSA. Antibody binding was detected by horseradish peroxidase conjugated anti-mouse (Sigma) followed by addition of TMB solution (Bio-Rad). Washes between steps were done in a Tecan microplate washer. Absorbance values were determined at 450 nm with a Tecan infinite microplate reader. 2.4. Two-dimensional separation of crude venoms High-resolution 2D electrophoresis was performed according to [18] with some modifications. Precast, non-linear immobilized pH 3–10 gradient (IPG) strips 7 cm strips (Ready strip, Biorad) were
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rehydrated with 10 g or 20 g of L. intermedia, L. gaucho and L. laeta venom proteins for 4 h (no electric field) and then for 12 h at 30 V. Isoelectric focusing was carried out on the EttanTM IPGphorTM isoelectric at 20 ◦ C using a gradient mode to a total amount of 12 kVh. In the second dimension, proteins and molecular standards (6.5–200 kDa, Sigma) were separated on 12% SDS-PAGE gel. One of the gels was stained with silver nitrate and the other was blotted as follows: proteins were electro-transferred to a nitrocellulose membrane of 0.22 M that was blocked for 1 h with 3% non-fat milk in 0.05% Tween 20 in PBS and incubated with the antibody (hybridoma supernatant in 1:10 dilution) at room temperature for 1 h. Antibody binding was detected with a horseradish peroxidase (HRPO)-conjugated rabbit anti-mouse secondary antibody and visualized with a chemiluminescent substrate (Hybond ECL, Amershan biotech). 2.5. Peptide synthesis on cellulose membranes and immunoassays Ninety overlapping pentadecapeptides frameshifted by 3 residues covering the amino acid sequence of LiD1 protein were prepared by Spot synthesis as previously described [19]. The cellulose membranes were obtained from Intavis (Koln, Germany); fluorenylmethyloxycarbonyl amino acids and Nhydroxybenzotriazole were from Novabiochem. A Multipep robot (Intavis) was used for automated peptide synthesis. After the peptide sequences were assembled, the side-chain protecting groups were removed by treatment with trifluoroacetic acid. The peptide FDDNANPEYTYHGIP and fifteen of its alanine analogs, the epitope peptide (TYHGIP), peptides changing amino acids next to the epitope and homolog’s epitope region peptides were prepared as described above. After an overnight saturation step with 3% BSA, the set of membrane bound peptides was probed by incubation with LiD1mAb16 (5 g/mL). Antibody binding was detected as described in [1]. 2.6. Alignment of Loxosceles homolog sequences Multiple alignment of sequences already classified in SicTox groups [14] from L. intermedia, L. gaucho and L. laeta venoms was carried out using clustal W2 (http://www.ebi.ac.uk/Tools/msa/ clustalw2/). Alignment analysis and editing were done using Jalview 2.5 [20]. Weblogo was obtained according to [21]. 2.7. Molecular modeling and epitope localization The LiD1 model was achieved using LiRecDT1 (PDB accession code: 3RLH) as the template and employing the molecular modeling package SWISS MODEL [22]. Epitopes were localized in the three-dimensional model of LiD1 and visualized using PyMol 1.3 (Schrodinger, LLC). 2.8. In vivo neutralizing assay Neutralization of dermonecrotic activity of rLiD1 was estimated in rabbits by incubation of 2 MND of rLiD1 with 0.2 mL of LiD1mAb16 (2.5 mg/mL) for 1 h at 37 ◦ C (the MND-minimum necrotizing dose of the rLiD1 used throughout this study was 10 g). After incubation, the mixture was injected intradermally (i.d.) into rabbit dorsum. The local areas of lesions were inspected 48 h after injection. As the control, rLiD1 alone (2 MND) and rLiD1 after preincubation with the same amount of non-immune mouse IgG were injected into rabbits, under the same conditions. The diameters of hemorrhagic and edematogenic lesions were measured with a scale meter and tachymeters, respectively.
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Fig. 1. Antigenic reactivity of selected monoclonal antibodies (LiD1mAbs). (A) Reactivity of selected LiD1mAbs against L. intermedia venom, L. laeta venom and BSA (negative control) using culture cell supernatant (diluted 1:10) was measured by ELISA. (B) Reactivity of LiD1mAb16 against L. intermedia, L. gaucho and L. laeta venoms using different dilutions of culture cell supernatant was accessed by ELISA. Results are expressed as mean ± SEM of the absorbance value of triplicates.
3. Results 3.1. mAb production A panel of seventeen anti-rLiD1 secreting hybridomas were selected by screening using L. intermedia and L. laeta venoms as antigens coated to ELISA plates. All of the culture cell supernatants (1:10 dilution) of mAbs were reactive with L. intermedia venom antigens. Only one clone namely LiD1mAb16 was also capable of cross-reacting with the L. laeta venom. Culture cell supernatants did not bind to BSA, excluding the possibility of non-specific binding (Fig. 1A). Other ELISA experiments demonstrated the crossreactivity of LiD1mAb16 supernatant against L. intermedia, L. laeta and L. gaucho venoms (Fig. 1B) even using 1:1000 supernatant dilution.
To characterize the critical amino acid residues in the LiD1 epitope recognized by LiD1mAb16, a series of alanine analogs of peptide 13 (37 FDDNANPEYTYHGIP51 ) was prepared by SPOT synthesis. The results (Fig. 3D) revealed that some residues in the peptide sequence could not be modified by alanine without a decrease of at least 70% in the reactivity with the antibody: this is the case of Thr46 , His48 and Gly49 . Some other residues located close to the crucial residues, such as Asn42 , Pro43 , Tyr47 , Ile50 , seem also to be important for antibody binding. In order to investigate the role of neighboring amino acids we exchanged by alanine all the amino acids close to the common reactive region (46 TYHGIP51 ) disclosed by the epitope mapping. The results (Fig. 4A) show that the neighboring amino acids
3.2. Molecular characterization of LiD1mAb16 For the molecular characterization we have examined initially the cross-reactivity of LiD1mAb16 by western blot analysis in 2D SDS-PAGE electrophoresis against L. intermedia, L. gaucho and L. laeta venoms (Fig. 2). LiD1mAb16 was reactive with 34 protein spots in L. intermedia venom with molecular masses of 29–36 kDa corresponding to SMases D homologs (Fig. 2A). In L. gaucho venom LiD1mAb16 reacted with 33 proteins spots in same molecular weight range (Fig. 2B). Similar results were observed for L. laeta venom, for which LiD1mAb16 interacted with 27 spots in the same region (Fig. 2C). With the aim of mapping continuous epitopes on LiD1 recognized by LiD1mAb16 we used SPOT synthesis technique to prepare a set of 90 overlapping peptides (15 residues, frameshifted by 3 residues) corresponding to the primary sequence of the LiD1 protein. Fig. 3A and B shows the binding pattern of LiD1mAb16 with the overlapping peptides. Four peptides in the N-terminal part (spots 13–16) were strongly recognized. Fig. 3B shows the amino acid sequence of reactive peptides. They correspond to 37 FDDNANPEYTYHGIP51 , 40 NANPEYTYHGIPCDC54 , 43 PEYTYHGIPCDCGRN57 and 46 TYHGIPCDCGRNCKK60 . The reactive peptides exhibit a common 6-residue motif 46 TYHGIP51 . To visualize the position of experimentally determined epitopes, LiD1 protein structure was modeled by homology using the X-ray structure of LiRecDT1 from L. intermedia [23]. The localization of the 46 TYHGIP51 epitope in the context of the three-dimensional structure of the dermonecrotic protein is shown in Fig. 3 C. The epitopic region found is located in the catalytic loop region [23].
Fig. 2. LiD1mAb16 cross reactivity among Loxosceles venoms. L. intermedia (A), L. gaucho (B) and L. laeta (C) venoms were separated by 2DE electrophoresis (first dimension: IEF pH range 3–10 NL, second dimension: 12% SDS-PAGE) and directly visualized by silver nitrate (A–C – pink) or electroblotted onto a nitrocellulose membrane for immunoblot analysis (green). Membranes were blotted with 20 g of the venom and were incubated with LiD1mAb16. Merged areas are shown in black, images alignment was made using Progenesis Samespot® software. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. LiD1mAb16 key epitope residues. (A) Reactivity of 15-mer overlapping peptides derived from the amino acid sequence of LiD1 (top left). Peptides were prepared by the Spot method on cellulose membranes and LiD1mAb16 binding (5 g/mL) to cellulose-bound peptides was detected by a peroxidase-coupled anti-mouse antibody (diluted 1:2000). (B) The reactive peptides are numbered 13–16. The amino acid sequence shared in all peptides sequences is shown in red and it is localized in LiD1 3D structure (C). His48 from active site involved in the epitope is highlighted (red). (D) A series of alanine analogs of peptide 13 were prepared by Spot synthesis and further probed by LiD1mAb16. The reference peptide is at position 1 and each further spot corresponds to an alanine analog. Each bar represents the intensity of the binding of LiD1mAb16 with the reference peptide (first bar) or with an analog peptide in which the indicated residue has been replaced by alanine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
cannot be replaced by alanine without a loss of 60% or greater of reactivity with the antibody. The common region was also synthesized as such and surprisingly showed a better reactivity than the other peptides (70% of reactivity compared with the control
peptide). As the peptide containing only the six common amino acids was reactive, our next step was to examine the minimum epitope of LiD1mAb16. From the six residues of the epitope region (46 TYHGIP51 ), three were seen to be key in antibody recognition
Fig. 4. Importance of neighboring amino acids in LiD1mAb16 epitope and minimum epitope. (A) Analogs of the peptide 13 of LiD1 spot membrane were prepared by Spot synthesis and further probed by LiD1mAb16. The reference peptide 13 is at position 1. Each further spot corresponds to an alanine analog with only the epitope region having preserved sequence. The last peptide corresponds to epitope sequence alone. (B) Two peptides containing only the epitope sequence and excluding amino acid residues, which did not seem to be as key for binding were prepared by Spot synthesis and further probed by LiD1mAb16. The reference epitope peptide (TYHGIP) is at position 1. Each further spot corresponds to an analog excluding at least one C-terminal residue. Peptides were prepared by the Spot method and tested as described in the text.
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Fig. 5. LiD1mAb16 epitope in Loxosceles SMases D homologs. (A) Alignment of available L. intermedia, L. Laeta e L. gaucho SMases D homolog sequences. Key epitope residues are colored in orange. Secondary structure of SMase I (AAM21154; PDB accession code 1XX1), consensus logo and consensus sequence are shown underneath the alignment. In the right top of the panel, SicTox group of each sequence [14] and LiD1mAb16 epitope consensus logo of each SicTox group. (B) Two peptide sequences shifting key amino acids residues of the original epitope to residues occupying the same position (residue 46) in SMases D homologs were prepared by Spot synthesis and further probed by LiD1mAb16. The reference peptide is at position 1. Each further spot corresponds to peptides with shifting residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
by alanine scanning (Thr46 , His48 and Gly49 ). In addition, with this experiment we observed a loss of antibody binding in 40 and 75%, after deletion of the C-terminal residues: Ile50 and Pro51 (Fig. 4B). Fig. 5 shows the multiple sequence alignment of 33 mature sequences of SMase D homologs from the venom of L. intermedia, L. gaucho and L. laeta. The three key residues of the epitope identified by alanine scanning (shown in orange) are residues strictly (His48 and Gly49 ) or functionally (Thr46 ) conserved in all sequences analyzed. The His48 is conserved among sequences, since it is involved in the catalytic activity of SMases D. The sequences were organized according to their SicTox group and each group was submitted to a WebLogo at the epitope region (Fig. 5, top right), group SicTox ␣II and ID were not submitted, since there is only one sequence for each. WebLogo of the groups confirms that residue Thr46 can be replaced by serine in some sequences of the group SicTox ␣III and by methionine in the group SicTox IA (the same was observed in sequence ABD91847, which belongs to the group ID). Next, with the aim of determining whether shifting in position 46 would affect antibody recognition, the epitope region was synthesized replacing threonine by serine or methionine (Fig. 5, right bottom). The LiD1mAb16 was able to recognize all the peptides; however the peptide with a serine in the place of the threonine had a loss of 50% in the reactivity.
3.3. In vivo neutralization assay The percentage of inhibition of dermonecrotic, hemorrhagic and oedematogenic activities caused by rLiD1 was assessed by pre-incubation of LiD1mAb16 for 1 h at 37 ◦ C with two minimum necrotizing doses (2 MND) of rLiD1 corresponding to 20 g of protein in a total volume of 200 L and then injected intradermal into naïve rabbits (Fig. 6C). As control, the protein was injected alone (Fig. 6A) or after incubation with non-immune mouse IgG under same conditions (Fig. 6B). An inhibition of 80% of the dermonecrotic activity and of 82% of hemorrhagic activity by LiD1mAb16 was observed, while non-immune mouse IgG was not at all protective. Concerning the oedematogenic activity induced by rLiD1, it was inhibited to 48% after the pre-incubation with the mAb while non-immune mouse IgG was not capable of reducing this activity. 4. Discussion We have described the development of a neutralizing mAb with a broad cross-reactivity toward many antigens from three different venoms, which protects rabbits against dermonecrotic toxin activity. Other mAbs against L. laeta and L. intermedia spiders whole venoms have already been produced, however these mAbs failed
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Fig. 6. Neutralization capacity of LiD1mAb16 against rLiD1. Rabbits were injected i.d. with 2 MND of rLiD1 which was preincubated for 1 h, at 37 ◦ C with PBS (A) or non-immune mouse IgG (B) or LiD1mAb16. The dermonecrotic lesions were observed 48 h after injection.
to recognize and neutralize the toxic components of venoms from heterologous species [24,25]. Due to the similarity between sphingomyelinase D toxins, the major toxic components from the Loxosceles spider venoms, we used a recombinant dermonecrotic protein from L. intermedia [1,26] as immunogen to obtain mice hybridomas. Previous works support the feasibility of using recombinant SMases for the production of neutralizing polyclonal and polyvalent anti-Loxosceles antivenoms [27–29]. Seventeen anti-rLiD1 hybridomas were obtained from the initial screening, all displaying affinity for L. intermedia venom antigens. However, of 17 anti-rLiD1 reactive with L. intermedia mAbs, only one displayed cross-reactivity with proteins of L. laeta and L. gaucho venom antigens. This result confirms the previous observation that mAbs produced against Loxosceles venoms are mostly species-specific, differing from cross-reactive polyclonal antibodies [25,30]. The polyvalent mAb named LiD1mAb16 recognizes at least 25 proteins in each Loxosceles venom tested. The diversity of spots revealed by the antibodies could be explained by the site of recognition of the mAb (catalytic site, which is very conserved among SMase D homologs, as showed by Fig. 5) and also by the high number of homologs described until now (more than 168 sequences as reported by [14]). We believe that all spots recognized by the antibody are phospholipases D molecules since they are in the molecular range of these proteins. The other two main components of L. intermedia venom besides phospholipases D have lower molecular mass (insecticidal peptides) than the spots recognized by LiD1mAb16 or lower molecular mass and higher isoelectric points (astacin-like metalloproteases) [31,32]. Besides, since only one (out of seventeen) clones was cross-reactive between Loxosceles venoms, it would be hard to imagine that the only cross-reactive mAb found until know could recognize more than one class of enzymes from the venom. L. gaucho proteome analysis showed that venom from the same species could contain a large number of proteins (at least 8) [33]. The transcriptome of L. intermedia showed a total 160 ESTs encoding phospholipases D like toxins with 23 clusters formed by these ESTs, these data provide some evidence of the presence of a high number of homologs in the same venom [31]. We mapped the epitope recognized by LiD1mAb16 via its binding to a set of overlapping peptides covering the amino acid sequence of LiD1. The mapping of epitopes recognized by LiD1mAb16 indicated that the region 37 FDDNANPEYTYHGIPCDCGRNCKK60 , encompassed in the catalytic loop of SMases D and which includes histidine 48, an amino acid involved in the catalytic activity of these enzymes [23], is the main epitope recognized. All the peptides bound by LiD1mAb16 shared the sequence 46 TYHGIP51 that contains amino acids that were shown by alanine scanning experiments to be important for LiD1mAb16 binding, the catalytic charged residue (His48 ), a polar residue (Thr46 ) and a non-charged residue (Gly49 ). Two of three LiD1mAb16 key epitope residues (histidine 48 and glycine 49) are strictly conserved among the sequences of SMase D homologs
of L. intermedia, L. gaucho and L. laeta venoms, explaining the cross-reactivity of these antibody. The other residue (Thr46 ) is conserved in 67% of the sequences analyzed. In SicTox ␣III group it is replaced by serine and in SicTox IA and ID groups it is replaced by methionine. The hexapeptide 46 TYHGIP51 alone was recognized by LiD1mAb16, although with a lower efficacy than the reference peptide. When this epitope was surrounded by alanine residues, antibody reactivity with LiDmAb16 was lost, probably because the number of possible conformations of the peptide was increased or some residues important for binding were not replaceable by alanine. The original residues might play a role in the structure of the peptide, reducing the number of degrees of freedom and/or stabilizing the interaction with the antibody. We also checked out if 46 TYHGIP51 was the minimum epitope. Once this region comprises residues 46–51 and the key residues are 46, 48 and 49, we tried to eliminate residues 50 and 51. The removal of residues 50 and 51 provoked a loss of 75% of the reactivity compared to reference peptide, meaning that the six-residue sequence cannot be C-terminally further shortened. The interaction of LiD1mAb16 with an epitope inside the catalytic loop of LiD1, including the catalytic hystidine 48 and the surrounding glycine 49 and threonine 46, provides a plausible explanation for the neutralizing capacity of this antibody. About 80% of protection against necrotic and hemorrhagic activities was obtained by pre-incubation of the mAb with rLiD1. Concerning the protection against oedematogenic activity, the level of neutralization was smaller (48%), although significant. All these results are very similar with those obtained with polyclonal antibodies against synthetic epitopes [18,34,35] with, however, the advantage associated with monoclonal antibodies, i.e., a controlled and reproducible production. In conclusion, our results show the generation and characterization of a neutralizing mAb produced against a dermonecrotic protein of L. intermedia venom able to recognize more than 25 proteins (at SMases D molecular mass range) in each of the medically important Loxosceles whole venoms. These results confirm the perspective of utilization of mAbs for therapeutic approaches and also in diagnosis assays against loxoscelism.
Acknowledgments This research was supported by Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior, Brazil – CAPES (Toxinologia No. 23038000825/2011-63), Fundac¸ão de Amparo a Pesquisa do Estado de Minas Gerais, Brazil (FAPEMIG) and by funds of the INCTTOX Program of Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq). A PICS grant from the Center National de La Recherche Scientifique was also appreciated. We would like to express our gratitude to Dr. Michael Richardson for revising this manuscript.
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[19] Laune D, Molina F, Ferrieres G, Villard S, Bes C, Rieunier F, et al. Application of the Spot method to the identification of peptides and amino acids from the antibody paratope that contribute to antigen binding. J Immunol Methods 2002;267:53–70. [20] Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2 – a multiple sequence alignment editor and analysis workbench. Bioinformatics 2009;25:1189–91. [21] Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004;14:1188–90. [22] Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 2003;31:3381–5. [23] Giuseppe PO, Ullah A, Trevisan Silva D, Gremski LH, Wille ACM, Chaves Moreira D, et al. Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge. Biochem Biophys Res Commun 2011;409:622–7. [24] Guilherme P, Fernandes I, Barbaro KC. Neutralization of dermonecrotic and lethal activities and differences among 32–35 kDa toxins of medically important Loxosceles spider venoms in Brazil revealed by monoclonal antibodies. Toxicon 2001;39:1333–42. [25] Alvarenga LM, Martins MS, Moura JF, Kalapothakis E, Oliveira JC, Mangili OC, et al. Production of monoclonal antibodies capable of neutralizing dermonecrotic activity of Loxosceles intermedia spider venom and their use in a specific immunometric assay. Toxicon 2003;42:725–31. [26] Kalapothakis E, Araujo SC, de Castro CS, Mendes TM, Gomez MV, Magili OC, et al. Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom. Toxicon 2002;40:1691–9. [27] Araujo SC, Castanheira P, Alvarenga LM, Mangili OC, Kalapothakis E, ChavezOlortegui C. Protection against dermonecrotic and lethal activities of Loxosceles intermedia spider venom by immunization with a fused recombinant protein. Toxicon 2003;41:261–7. [28] de Almeida DM, Fernandes-Pedrosa Mde F, de Andrade RM, Marcelino JR, Gondo-Higashi H, de Azevedo IL, et al. A new anti-loxoscelic serum produced against recombinant sphingomyelinase D: results of preclinical trials. Am J Trop Med Hyg 2008;79:463–70. [29] Olvera A, Ramos-Cerrillo B, Estevez J, Clement H, de Roodt A, Paniagua-Solis J, et al. North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens. Toxicon 2006;48:64–74. [30] Barbaro KC, Eickstedt VR, Mota I. Antigenic cross-reactivity of venoms from medically important Loxosceles (Araneae) species in Brazil. Toxicon 1994;32:113–20. [31] Gremski LH, da Silveira RB, Chaim OM, Probst CM, Ferrer VP, Nowatzki J, et al. A novel expression profile of the Loxosceles intermedia spider venomous gland revealed by transcriptome analysis. Mol Biosyst 2010;12:2403–16. [32] Trevisan-Silva D, Bednaski AV, Gremski LH, Chaim OM, Veiga SS, Senff-Ribeiro A. Differential metalloprotease content and activity of three Loxosceles spider venoms revealed using two-dimensional electrophoresis approaches. Toxicon 2013;76:11–22. [33] Machado LF, Laugesen S, Botelho ED, Ricart CA, Fontes W, Barbaro KC, et al. Proteome analysis of brown spider venom: identification of loxnecrogin isoforms in Loxosceles gaucho venom. Proteomics 2005;5:2167–76. [34] Felicori L, Fernandes PB, Giusta MS, Duarte CG, Kalapothakis E, Nguyen C, et al. An in vivo protective response against toxic effects of the dermonecrotic protein from Loxosceles intermedia spider venom elicited by synthetic epitopes. Vaccine 2009;27:4201–8. [35] Dias-Lopes C, Guimaraes G, Felicori L, Fernandes P, Emery L, Kalapothakis E, et al. A protective immune response against lethal, dermonecrotic and hemorrhagic effects of Loxosceles intermedia venom elicited by a 27-residue peptide. Toxicon 2010;55:481–7.
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Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms C. Dias-Lopes a , L. Felicori a , L. Rubrecht b , S. Cobo b , L. Molina b , C. Nguyen b , P. Galéa b , C. Granier b , F. Molina b , C. Chávez-Olortegui a,∗ a b
Departamento de Bioquímica e Imunologia, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil SysDiag, UMR3145,CNRS/Biorad, Montpellier, France
a r t i c l e
i n f o
Article history: Received 15 May 2013 Received in revised form 21 January 2014 Accepted 6 February 2014 Available online 22 February 2014 Keywords: Loxosceles spider venoms Sphingomyelinases D Monoclonal antibody Epitopes
a b s t r a c t We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29–36 kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction The spiders Loxosceles intermedia, L. laeta and L. gaucho are a group of arachnids known as “brown spider” with medical importance in the South and South-east of Brazil [2,3]. The number of human accidents caused by spiders of this genus in Brazil has reached almost 7000 annually [4]. Loxoscelism, the term used for envenomations with Loxosceles spiders, can be observed as two well-defined clinical variants: cutaneous loxoscelism and systemic loxoscelism. Pain, edema, and livedoid plaque, which develop later into a necrotic scar, are the predominant local manifestations in cutaneous loxoscelism, occurring in around 83% of the cases. In systemic loxoscelism, hematuria and hemoglobinuria are always observed, whereas intravascular hemolysis and
∗ Corresponding author at: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, CEP: 31.270.901, 6627 Belo Horizonte, Brazil. Tel.: +55 31 3409 2625; fax: +55 31 3409 2613. E-mail address: [email protected] (C. Chávez-Olortegui). http://dx.doi.org/10.1016/j.vaccine.2014.02.012 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
coagulation, sometimes accompanied by thrombocytopenia and renal failure, occur in approximately 16% of the victims [5–7]. Antivenom therapy is used to neutralize the circulating venom and reduces the risk of fatal complications following human accidents [8]. The anti-loxoscelic antivenom is the polyspecific serum that containing antibodies against whole venoms of the three Loxosceles species (L. gaucho, L. laeta and L. intermedia) and is produced at Centro de Producão e Pesquisa de Imunobiológicos (CPPI) of the State of Paraná, Brazil [9]. Proteins of the phospholipase D family also named sphingomyelinase D (SMase D), or dermonecrotic proteins, are responsible for the necrotic skin lesions and also for systemic toxic effects following envenomation by Loxosceles spiders [10–13]. Loxosceles venoms express SMases D isoforms and these proteins constitute a family of homologs with 190 non-redundant sequences described in 21 species of the Sicariidae family [14]. SMase D proteins are also the most antigenic/immunogenic components of the venom [15]. Monoclonal and polyclonal antibodies against Loxosceles whole venoms generally recognize dermonecrotic proteins [16,17]. Neutralizing monoclonal antibodies (mAbs) open new perspectives to employment of these antibodies in loxoscelism
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treatment or diagnosis. However, all neutralizing mAbs produced until now recognize effectively only the venom species-specific [15,17]. In the present work, we produced a mAb able to recognize the venoms of the three major medically important species of Loxosceles spiders in Brazil. These mAb was obtained by immunization of mice with recombinant L. intermedia Smase D isoform (rLiD1) [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho whole venoms as antigens coated onto micro titer plates, so as to drive selection toward cross-reactive antibodies. The mAb namely LiD1mAb16 was able to recognize a large range of proteins antigens (at least 25) in each of the three Loxosceles venom. Epitope-mapping experiments revealed that it recognizes a highly conserved linear epitope located in the catalytic region of SMases D toxins. LiD1mAb16 displayed a protective activity in rabbits challenged with rLiD1, suggesting that this mAb may be a promising candidate for therapeutic serum development or diagnosis in the future. 2. Materials and methods 2.1. Animals Animals were maintained at the Centro de Bioterismo of the Instituto de Ciências Biológicas of the Federal University of Minas Gerais, Brazil and received water and food under controlled environmental conditions. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) (A5452-01) and was approved by local authorities (protocol #89/11 – Comitê de Ética em Experimentac¸ão Animal – Federal University of Minas Gerais). 2.2. mAbs production The rLiD1 from L. intermedia venom [1] was used as immunogen to BALB/c mice. The animals were immunized four times subcutaneously, at approximately 2 weeks intervals, with 10 g of protein in complete Freund’s adjuvant (Sigma) at the first injection, and incomplete Freund’s adjuvant (Sigma) at subsequent inoculations. A booster injection of rLiD1 was made 4 weeks after the fourth immunization. Throughout the immunization schedule, mice were bled and the reactivity of immune sera was tested against rLiD1, L. intermedia and L. laeta venoms. Three days after the last injection, spleen cells from immunized mice were fused with Sp2/0 myeloma cells (ATCC). Supernatants from resulting hybridomas were screening by ELISA using L. intermedia and L. laeta venoms. mAbs were purified on a protein A-sepharose column (GE Healthcare). 2.3. Indirect ELISA for the screening of hybridomes Maxisorp plates (Nunc) were coated overnight at 4 ◦ C with a solution of 1 g/mL of the protein or L. intermedia, L. gaucho or L. laeta venom in 50 mM Na2 CO3 , pH 9.0, and blocked with PBS containing 10 g/L BSA. Antibody binding was detected by horseradish peroxidase conjugated anti-mouse (Sigma) followed by addition of TMB solution (Bio-Rad). Washes between steps were done in a Tecan microplate washer. Absorbance values were determined at 450 nm with a Tecan infinite microplate reader. 2.4. Two-dimensional separation of crude venoms High-resolution 2D electrophoresis was performed according to [18] with some modifications. Precast, non-linear immobilized pH 3–10 gradient (IPG) strips 7 cm strips (Ready strip, Biorad) were
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rehydrated with 10 g or 20 g of L. intermedia, L. gaucho and L. laeta venom proteins for 4 h (no electric field) and then for 12 h at 30 V. Isoelectric focusing was carried out on the EttanTM IPGphorTM isoelectric at 20 ◦ C using a gradient mode to a total amount of 12 kVh. In the second dimension, proteins and molecular standards (6.5–200 kDa, Sigma) were separated on 12% SDS-PAGE gel. One of the gels was stained with silver nitrate and the other was blotted as follows: proteins were electro-transferred to a nitrocellulose membrane of 0.22 M that was blocked for 1 h with 3% non-fat milk in 0.05% Tween 20 in PBS and incubated with the antibody (hybridoma supernatant in 1:10 dilution) at room temperature for 1 h. Antibody binding was detected with a horseradish peroxidase (HRPO)-conjugated rabbit anti-mouse secondary antibody and visualized with a chemiluminescent substrate (Hybond ECL, Amershan biotech). 2.5. Peptide synthesis on cellulose membranes and immunoassays Ninety overlapping pentadecapeptides frameshifted by 3 residues covering the amino acid sequence of LiD1 protein were prepared by Spot synthesis as previously described [19]. The cellulose membranes were obtained from Intavis (Koln, Germany); fluorenylmethyloxycarbonyl amino acids and Nhydroxybenzotriazole were from Novabiochem. A Multipep robot (Intavis) was used for automated peptide synthesis. After the peptide sequences were assembled, the side-chain protecting groups were removed by treatment with trifluoroacetic acid. The peptide FDDNANPEYTYHGIP and fifteen of its alanine analogs, the epitope peptide (TYHGIP), peptides changing amino acids next to the epitope and homolog’s epitope region peptides were prepared as described above. After an overnight saturation step with 3% BSA, the set of membrane bound peptides was probed by incubation with LiD1mAb16 (5 g/mL). Antibody binding was detected as described in [1]. 2.6. Alignment of Loxosceles homolog sequences Multiple alignment of sequences already classified in SicTox groups [14] from L. intermedia, L. gaucho and L. laeta venoms was carried out using clustal W2 (http://www.ebi.ac.uk/Tools/msa/ clustalw2/). Alignment analysis and editing were done using Jalview 2.5 [20]. Weblogo was obtained according to [21]. 2.7. Molecular modeling and epitope localization The LiD1 model was achieved using LiRecDT1 (PDB accession code: 3RLH) as the template and employing the molecular modeling package SWISS MODEL [22]. Epitopes were localized in the three-dimensional model of LiD1 and visualized using PyMol 1.3 (Schrodinger, LLC). 2.8. In vivo neutralizing assay Neutralization of dermonecrotic activity of rLiD1 was estimated in rabbits by incubation of 2 MND of rLiD1 with 0.2 mL of LiD1mAb16 (2.5 mg/mL) for 1 h at 37 ◦ C (the MND-minimum necrotizing dose of the rLiD1 used throughout this study was 10 g). After incubation, the mixture was injected intradermally (i.d.) into rabbit dorsum. The local areas of lesions were inspected 48 h after injection. As the control, rLiD1 alone (2 MND) and rLiD1 after preincubation with the same amount of non-immune mouse IgG were injected into rabbits, under the same conditions. The diameters of hemorrhagic and edematogenic lesions were measured with a scale meter and tachymeters, respectively.
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Fig. 1. Antigenic reactivity of selected monoclonal antibodies (LiD1mAbs). (A) Reactivity of selected LiD1mAbs against L. intermedia venom, L. laeta venom and BSA (negative control) using culture cell supernatant (diluted 1:10) was measured by ELISA. (B) Reactivity of LiD1mAb16 against L. intermedia, L. gaucho and L. laeta venoms using different dilutions of culture cell supernatant was accessed by ELISA. Results are expressed as mean ± SEM of the absorbance value of triplicates.
3. Results 3.1. mAb production A panel of seventeen anti-rLiD1 secreting hybridomas were selected by screening using L. intermedia and L. laeta venoms as antigens coated to ELISA plates. All of the culture cell supernatants (1:10 dilution) of mAbs were reactive with L. intermedia venom antigens. Only one clone namely LiD1mAb16 was also capable of cross-reacting with the L. laeta venom. Culture cell supernatants did not bind to BSA, excluding the possibility of non-specific binding (Fig. 1A). Other ELISA experiments demonstrated the crossreactivity of LiD1mAb16 supernatant against L. intermedia, L. laeta and L. gaucho venoms (Fig. 1B) even using 1:1000 supernatant dilution.
To characterize the critical amino acid residues in the LiD1 epitope recognized by LiD1mAb16, a series of alanine analogs of peptide 13 (37 FDDNANPEYTYHGIP51 ) was prepared by SPOT synthesis. The results (Fig. 3D) revealed that some residues in the peptide sequence could not be modified by alanine without a decrease of at least 70% in the reactivity with the antibody: this is the case of Thr46 , His48 and Gly49 . Some other residues located close to the crucial residues, such as Asn42 , Pro43 , Tyr47 , Ile50 , seem also to be important for antibody binding. In order to investigate the role of neighboring amino acids we exchanged by alanine all the amino acids close to the common reactive region (46 TYHGIP51 ) disclosed by the epitope mapping. The results (Fig. 4A) show that the neighboring amino acids
3.2. Molecular characterization of LiD1mAb16 For the molecular characterization we have examined initially the cross-reactivity of LiD1mAb16 by western blot analysis in 2D SDS-PAGE electrophoresis against L. intermedia, L. gaucho and L. laeta venoms (Fig. 2). LiD1mAb16 was reactive with 34 protein spots in L. intermedia venom with molecular masses of 29–36 kDa corresponding to SMases D homologs (Fig. 2A). In L. gaucho venom LiD1mAb16 reacted with 33 proteins spots in same molecular weight range (Fig. 2B). Similar results were observed for L. laeta venom, for which LiD1mAb16 interacted with 27 spots in the same region (Fig. 2C). With the aim of mapping continuous epitopes on LiD1 recognized by LiD1mAb16 we used SPOT synthesis technique to prepare a set of 90 overlapping peptides (15 residues, frameshifted by 3 residues) corresponding to the primary sequence of the LiD1 protein. Fig. 3A and B shows the binding pattern of LiD1mAb16 with the overlapping peptides. Four peptides in the N-terminal part (spots 13–16) were strongly recognized. Fig. 3B shows the amino acid sequence of reactive peptides. They correspond to 37 FDDNANPEYTYHGIP51 , 40 NANPEYTYHGIPCDC54 , 43 PEYTYHGIPCDCGRN57 and 46 TYHGIPCDCGRNCKK60 . The reactive peptides exhibit a common 6-residue motif 46 TYHGIP51 . To visualize the position of experimentally determined epitopes, LiD1 protein structure was modeled by homology using the X-ray structure of LiRecDT1 from L. intermedia [23]. The localization of the 46 TYHGIP51 epitope in the context of the three-dimensional structure of the dermonecrotic protein is shown in Fig. 3 C. The epitopic region found is located in the catalytic loop region [23].
Fig. 2. LiD1mAb16 cross reactivity among Loxosceles venoms. L. intermedia (A), L. gaucho (B) and L. laeta (C) venoms were separated by 2DE electrophoresis (first dimension: IEF pH range 3–10 NL, second dimension: 12% SDS-PAGE) and directly visualized by silver nitrate (A–C – pink) or electroblotted onto a nitrocellulose membrane for immunoblot analysis (green). Membranes were blotted with 20 g of the venom and were incubated with LiD1mAb16. Merged areas are shown in black, images alignment was made using Progenesis Samespot® software. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. LiD1mAb16 key epitope residues. (A) Reactivity of 15-mer overlapping peptides derived from the amino acid sequence of LiD1 (top left). Peptides were prepared by the Spot method on cellulose membranes and LiD1mAb16 binding (5 g/mL) to cellulose-bound peptides was detected by a peroxidase-coupled anti-mouse antibody (diluted 1:2000). (B) The reactive peptides are numbered 13–16. The amino acid sequence shared in all peptides sequences is shown in red and it is localized in LiD1 3D structure (C). His48 from active site involved in the epitope is highlighted (red). (D) A series of alanine analogs of peptide 13 were prepared by Spot synthesis and further probed by LiD1mAb16. The reference peptide is at position 1 and each further spot corresponds to an alanine analog. Each bar represents the intensity of the binding of LiD1mAb16 with the reference peptide (first bar) or with an analog peptide in which the indicated residue has been replaced by alanine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
cannot be replaced by alanine without a loss of 60% or greater of reactivity with the antibody. The common region was also synthesized as such and surprisingly showed a better reactivity than the other peptides (70% of reactivity compared with the control
peptide). As the peptide containing only the six common amino acids was reactive, our next step was to examine the minimum epitope of LiD1mAb16. From the six residues of the epitope region (46 TYHGIP51 ), three were seen to be key in antibody recognition
Fig. 4. Importance of neighboring amino acids in LiD1mAb16 epitope and minimum epitope. (A) Analogs of the peptide 13 of LiD1 spot membrane were prepared by Spot synthesis and further probed by LiD1mAb16. The reference peptide 13 is at position 1. Each further spot corresponds to an alanine analog with only the epitope region having preserved sequence. The last peptide corresponds to epitope sequence alone. (B) Two peptides containing only the epitope sequence and excluding amino acid residues, which did not seem to be as key for binding were prepared by Spot synthesis and further probed by LiD1mAb16. The reference epitope peptide (TYHGIP) is at position 1. Each further spot corresponds to an analog excluding at least one C-terminal residue. Peptides were prepared by the Spot method and tested as described in the text.
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Fig. 5. LiD1mAb16 epitope in Loxosceles SMases D homologs. (A) Alignment of available L. intermedia, L. Laeta e L. gaucho SMases D homolog sequences. Key epitope residues are colored in orange. Secondary structure of SMase I (AAM21154; PDB accession code 1XX1), consensus logo and consensus sequence are shown underneath the alignment. In the right top of the panel, SicTox group of each sequence [14] and LiD1mAb16 epitope consensus logo of each SicTox group. (B) Two peptide sequences shifting key amino acids residues of the original epitope to residues occupying the same position (residue 46) in SMases D homologs were prepared by Spot synthesis and further probed by LiD1mAb16. The reference peptide is at position 1. Each further spot corresponds to peptides with shifting residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
by alanine scanning (Thr46 , His48 and Gly49 ). In addition, with this experiment we observed a loss of antibody binding in 40 and 75%, after deletion of the C-terminal residues: Ile50 and Pro51 (Fig. 4B). Fig. 5 shows the multiple sequence alignment of 33 mature sequences of SMase D homologs from the venom of L. intermedia, L. gaucho and L. laeta. The three key residues of the epitope identified by alanine scanning (shown in orange) are residues strictly (His48 and Gly49 ) or functionally (Thr46 ) conserved in all sequences analyzed. The His48 is conserved among sequences, since it is involved in the catalytic activity of SMases D. The sequences were organized according to their SicTox group and each group was submitted to a WebLogo at the epitope region (Fig. 5, top right), group SicTox ␣II and ID were not submitted, since there is only one sequence for each. WebLogo of the groups confirms that residue Thr46 can be replaced by serine in some sequences of the group SicTox ␣III and by methionine in the group SicTox IA (the same was observed in sequence ABD91847, which belongs to the group ID). Next, with the aim of determining whether shifting in position 46 would affect antibody recognition, the epitope region was synthesized replacing threonine by serine or methionine (Fig. 5, right bottom). The LiD1mAb16 was able to recognize all the peptides; however the peptide with a serine in the place of the threonine had a loss of 50% in the reactivity.
3.3. In vivo neutralization assay The percentage of inhibition of dermonecrotic, hemorrhagic and oedematogenic activities caused by rLiD1 was assessed by pre-incubation of LiD1mAb16 for 1 h at 37 ◦ C with two minimum necrotizing doses (2 MND) of rLiD1 corresponding to 20 g of protein in a total volume of 200 L and then injected intradermal into naïve rabbits (Fig. 6C). As control, the protein was injected alone (Fig. 6A) or after incubation with non-immune mouse IgG under same conditions (Fig. 6B). An inhibition of 80% of the dermonecrotic activity and of 82% of hemorrhagic activity by LiD1mAb16 was observed, while non-immune mouse IgG was not at all protective. Concerning the oedematogenic activity induced by rLiD1, it was inhibited to 48% after the pre-incubation with the mAb while non-immune mouse IgG was not capable of reducing this activity. 4. Discussion We have described the development of a neutralizing mAb with a broad cross-reactivity toward many antigens from three different venoms, which protects rabbits against dermonecrotic toxin activity. Other mAbs against L. laeta and L. intermedia spiders whole venoms have already been produced, however these mAbs failed
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Fig. 6. Neutralization capacity of LiD1mAb16 against rLiD1. Rabbits were injected i.d. with 2 MND of rLiD1 which was preincubated for 1 h, at 37 ◦ C with PBS (A) or non-immune mouse IgG (B) or LiD1mAb16. The dermonecrotic lesions were observed 48 h after injection.
to recognize and neutralize the toxic components of venoms from heterologous species [24,25]. Due to the similarity between sphingomyelinase D toxins, the major toxic components from the Loxosceles spider venoms, we used a recombinant dermonecrotic protein from L. intermedia [1,26] as immunogen to obtain mice hybridomas. Previous works support the feasibility of using recombinant SMases for the production of neutralizing polyclonal and polyvalent anti-Loxosceles antivenoms [27–29]. Seventeen anti-rLiD1 hybridomas were obtained from the initial screening, all displaying affinity for L. intermedia venom antigens. However, of 17 anti-rLiD1 reactive with L. intermedia mAbs, only one displayed cross-reactivity with proteins of L. laeta and L. gaucho venom antigens. This result confirms the previous observation that mAbs produced against Loxosceles venoms are mostly species-specific, differing from cross-reactive polyclonal antibodies [25,30]. The polyvalent mAb named LiD1mAb16 recognizes at least 25 proteins in each Loxosceles venom tested. The diversity of spots revealed by the antibodies could be explained by the site of recognition of the mAb (catalytic site, which is very conserved among SMase D homologs, as showed by Fig. 5) and also by the high number of homologs described until now (more than 168 sequences as reported by [14]). We believe that all spots recognized by the antibody are phospholipases D molecules since they are in the molecular range of these proteins. The other two main components of L. intermedia venom besides phospholipases D have lower molecular mass (insecticidal peptides) than the spots recognized by LiD1mAb16 or lower molecular mass and higher isoelectric points (astacin-like metalloproteases) [31,32]. Besides, since only one (out of seventeen) clones was cross-reactive between Loxosceles venoms, it would be hard to imagine that the only cross-reactive mAb found until know could recognize more than one class of enzymes from the venom. L. gaucho proteome analysis showed that venom from the same species could contain a large number of proteins (at least 8) [33]. The transcriptome of L. intermedia showed a total 160 ESTs encoding phospholipases D like toxins with 23 clusters formed by these ESTs, these data provide some evidence of the presence of a high number of homologs in the same venom [31]. We mapped the epitope recognized by LiD1mAb16 via its binding to a set of overlapping peptides covering the amino acid sequence of LiD1. The mapping of epitopes recognized by LiD1mAb16 indicated that the region 37 FDDNANPEYTYHGIPCDCGRNCKK60 , encompassed in the catalytic loop of SMases D and which includes histidine 48, an amino acid involved in the catalytic activity of these enzymes [23], is the main epitope recognized. All the peptides bound by LiD1mAb16 shared the sequence 46 TYHGIP51 that contains amino acids that were shown by alanine scanning experiments to be important for LiD1mAb16 binding, the catalytic charged residue (His48 ), a polar residue (Thr46 ) and a non-charged residue (Gly49 ). Two of three LiD1mAb16 key epitope residues (histidine 48 and glycine 49) are strictly conserved among the sequences of SMase D homologs
of L. intermedia, L. gaucho and L. laeta venoms, explaining the cross-reactivity of these antibody. The other residue (Thr46 ) is conserved in 67% of the sequences analyzed. In SicTox ␣III group it is replaced by serine and in SicTox IA and ID groups it is replaced by methionine. The hexapeptide 46 TYHGIP51 alone was recognized by LiD1mAb16, although with a lower efficacy than the reference peptide. When this epitope was surrounded by alanine residues, antibody reactivity with LiDmAb16 was lost, probably because the number of possible conformations of the peptide was increased or some residues important for binding were not replaceable by alanine. The original residues might play a role in the structure of the peptide, reducing the number of degrees of freedom and/or stabilizing the interaction with the antibody. We also checked out if 46 TYHGIP51 was the minimum epitope. Once this region comprises residues 46–51 and the key residues are 46, 48 and 49, we tried to eliminate residues 50 and 51. The removal of residues 50 and 51 provoked a loss of 75% of the reactivity compared to reference peptide, meaning that the six-residue sequence cannot be C-terminally further shortened. The interaction of LiD1mAb16 with an epitope inside the catalytic loop of LiD1, including the catalytic hystidine 48 and the surrounding glycine 49 and threonine 46, provides a plausible explanation for the neutralizing capacity of this antibody. About 80% of protection against necrotic and hemorrhagic activities was obtained by pre-incubation of the mAb with rLiD1. Concerning the protection against oedematogenic activity, the level of neutralization was smaller (48%), although significant. All these results are very similar with those obtained with polyclonal antibodies against synthetic epitopes [18,34,35] with, however, the advantage associated with monoclonal antibodies, i.e., a controlled and reproducible production. In conclusion, our results show the generation and characterization of a neutralizing mAb produced against a dermonecrotic protein of L. intermedia venom able to recognize more than 25 proteins (at SMases D molecular mass range) in each of the medically important Loxosceles whole venoms. These results confirm the perspective of utilization of mAbs for therapeutic approaches and also in diagnosis assays against loxoscelism.
Acknowledgments This research was supported by Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior, Brazil – CAPES (Toxinologia No. 23038000825/2011-63), Fundac¸ão de Amparo a Pesquisa do Estado de Minas Gerais, Brazil (FAPEMIG) and by funds of the INCTTOX Program of Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq). A PICS grant from the Center National de La Recherche Scientifique was also appreciated. We would like to express our gratitude to Dr. Michael Richardson for revising this manuscript.
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