Experimental antivenom against serine proteases from the Bothrops jararaca venom obtained in mice, and its comparison with the antibothropic serum from the Butantan Institute

Toxicon 169 (2019) 59–67

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Experimental antivenom against serine proteases from the Bothrops jararaca venom obtained in mice, and its comparison with the antibothropic serum from the Butantan Institute

T

Alexandre Kazuo Kuniyoshia, Roberto Tadashi Kodamaa, Daniela Cajado-Carvalhoa, Leo Kei Iwaib, Eduardo Kitanob, Cristiane Castilho Fernandes da Silvaa, Bruno Duzzia, Wilmar Dias da Silvaa, Fernanda Calheta Portaroa,∗ a b

Immunochemistry Laboratory, Butantan Institute, São Paulo, Brazil Special Laboratory of Applied Toxinology/ Center of Toxins, Immune-Response and Cell Signaling (CeTICS), Butantan Institute, São Paulo, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Bothrops jararaca Serine protease Venom Antivenom

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (< 1%) at the dose of 500:1 (weight of antivenom:weight of venom). These results show that a different immunization method using isolated serine proteases improves the toxins neutralizing efficacy and could lead to a better end product to be used as a supplemental medicine to the currently used immunotherapy.

1. Introduction In Brazil about 27,000 snake accidents are registered each year (Ministério da Saúde, 2017), and the Bothrops jararaca is the species responsible for most of these cases (Cardoso et al., 1993). The recommended and clinical treatment in case of snake envenomation is the specific antivenom serum therapy (World Health Organization, 2017). In this scenario, improvement in production, storage and distribution, as well as further studies on the quality and safety of antivenoms, are of great importance. There are four Brazilian producers of antivenoms for snakebite envenomation: Butantan Institute, Vital Brazil Institute, Ezequiel Dias Foundation (FUNED) and Immunobiological Production

∗

and Research Center (CPPI). These producers offer, through the Ministry of Health, the bothropic antivenom (BAV) free of charge to the general public. The BAV produced by the Butantan Institute is obtained by immunizing horses with five different bothropic venoms: Bothrops alternatus (12.5%), B. jararaca (50%), B. jararacussu (12.5%), B. moojeni (12.5%) and B. neuwiedi (12.5%), being termed as a pentavalent antiserum. The BAV produced by the Butantan Institute presents a high specific activity in neutralizing the lethality of bothropic venoms, even against those of species not included in the antigenic mixture used in the immunization of horses (Dias da Silva and Tambourgi, 2011; Segura et al., 2010). Studies analyzing the neutralization efficacy of the commercial

Corresponding author. Immunochemistry Laboratory, Butantan Institute, Av. Prof. Vital Brazil, 1500, CEP, 05503-900, São Paulo, SP, Brazil. E-mail address: [email protected] (F.C. Portaro).

https://doi.org/10.1016/j.toxicon.2019.09.001 Received 23 May 2019; Received in revised form 15 August 2019; Accepted 1 September 2019 Available online 05 September 2019 0041-0101/ © 2019 Elsevier Ltd. All rights reserved.

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2. Methodology

antivenom have been published, and despite its proven efficacy, some show that the currently antivenom used could have some therapeutic limitations. For example, Battellino et al. (2003) demonstrated that, even after the pre-incubation of the Bothrops jararaca venom with BAV, the hemorrhage was not totally neutralized. da Silva et al. (2007) evaluated the efficacy of BAV along with two other Brazilian commercial antibothropic sera, and even with the pre-incubation of the B. jararaca venom (BjV) using 10 times the recommended dose of antivenoms, both hemorrhagic and miotoxic effects were not totally neutralized. Aiming to verify the effectiveness of BAV produced by the Butantan Institute in neutralizing the hyperalgesia and oedema effects induced by B. jararaca and B. asper venoms, it was demonstrated that hyperalgesia was efficiently blocked in pre-incubation assays of venoms with the antivenom. However, the oedema had a significant reduction (80%) in the venom pre-incubation assays with BAV, but there was no complete inhibition of this symptom (Picolo et al., 2002). Also, in vivo studies showed that the treatment of animals with the BAV before and after the B. moojeni venom inoculation was able to reduce oedema formation. In all cases of post-envenoming treatments, a residual oedematogenic effect could be observed, which was only totally abolished 60 min after the application of the BAV (Galvão Nascimento et al., 2010). Proteomics and transcriptomics studies have shown that the venom of B. jararaca consists mainly of proteolytic enzymes of metalloproteases (SVMP, Snake Venom MetalloProtease) and serine proteases (SVSP, Snake Venom Serine Proteases) classes (Fox and Serrano, 2008; Goncalves-Machado et al., 2016; Nicolau et al., 2017). These enzymes are responsible for several symptoms observed in snakes accidents with humans, such as: local pain, oedema, blisters, hemorrhage, coagulation cascade disorders, hypotension and renal failure (Ministério da Saúde, 2017). In general, SVMP activities are the main ones responsible for hemorrhagic effects, degrading proteins such as laminin, fibronectin and collagen (Markland and Swenson, 2013), whereas SVSPs affect hemostasis, acting on several components of the coagulation cascade and in the kallikrein-kinin system (Serrano and Maroun, 2005). Nevertheless, it was demonstrated that the inhibition of BjV with the serine protease inhibitor PMSF before injection reduces the oedema in the assayed mice paws. It is important to mention that oedema reduction observed by the use of PMSF was lower when compared to the experiments of metalloproteases inhibition, but, even so, demonstrated the contribution of SVSPs to this local symptom (Zychar et al., 2010). In addition, two serine proteases, BpirSP27 and BpirSP41, from Bothrops pirajai venom have also been described as oedema promoters (Menaldo et al., 2013). The mechanisms of action of the SVSPs of snake venoms of the Bothrops genus in the local injuries are not yet fully clarified, but it has been proposed that some of these enzymes are capable of activating matrix metalloproteases, such as MMP-2 (Saravia-Otten et al., 2007), thus, inducing the degradation of the extracellular matrix and causing tissue damage (Bode and Maskos, 2003). The lack of a complete neutralization of envenomation symptoms, in addition to the fact that in vitro enzymatic assays have shown that serine proteases from BjV are not well neutralized by the BAV from the Butantan Institute, while fully neutralizing metalloproteases (Kuniyoshi et al., 2017, 2012), was the reason for the development of the present study. Facing these facts, it is possible that the inability of the immunotherapeutically currently used BAV of neutralizing Bothrops jararaca venom serine proteases is due to a lack of intrinsic immunogenicity of its toxic domains or to its unappropriated exposition to the immune cells. In order to clarify these facts, a modification of the immunization schedule, including the use of adjuvant, was used in this study to immunize mice with purified serinoproteases from Bothrops jararaca venom. Therefore, the aim of this work was to obtain and characterize experimental antivenoms against non-BAV-inhibited serine proteases and to evaluate their efficacy in neutralizing the enzymatic activity of these proteases in vitro, in comparison to the commercial product.

2.1. Venom, adjuvant and antivenoms The lyophilized venom of Bothrops jararaca was provided by the Venom Section of the Butantan Institute, SP, Brazil. Stock solution was prepared in ammonium acetate 100 mM at pH 4.0 (23.3 mg/mL final concentration). The bothropic antivenom (BAV) batches 0506110 (BAV 1) and 0901004 (BAV 2) were obtained from the Hyperimmune Plasmas Processing Section, Butantan Institute, São Paulo, Brazil. The protein concentration of antivenoms batches used was 40 mg/mL and 10.5 mg/mL, respectively. The adjuvant ordered mesoporous SBA-15 silica was kindly donated by Dr. Osvaldo Sant'Anna, from the Immunochemistry Laboratory, Butantan Institute. 2.2. Determination of protein concentration Protein concentration measurements were carried out using a Quick Start Bradford assay kit (Bio Rad Protein Assay; Bio Rad Laboratories) according to the manufacturer's instructions. An albumin curve was set as control and the absorbance was measured in a spectrophotometer (Labsystems, Finland) at λ 595 nm. 2.3. Fluorimetric assays: serine proteases screening and sera neutralization The proteolytic activity assays were conducted in a phosphate-buffered saline (PBS, 8.1 mM sodium phosphate, 1.5 mM potassium phosphate, 137 mM sodium chloride and 2.7 mM potassium chloride solution) at pH 7.4 (final volume: 100 μL), using Corning 96 well plates, and the FRETs substrates (Abz-RPPGFSPFRQ-EDDnp = Abz-Serine and AbzFASSAQ-EDDnp = Abz-Metal) in a final concentration of 5 μM. These substrates were previously described as selective for serine proteases and metalloproteases from the B. jararaca venom, respectively (Kuniyoshi et al., 2012). Active site-directed inhibitors, PMSF (3 mM) and EDTA (50 mM) (serine and metalloprotease inhibitors, respectively) were used in the screening of the purification process of serine proteases. The reactions occurred at 37 °C and were initiated by the addition of 0.25 μg of protein samples. The reactions were monitored (fluorescence at λEM 420 nm and λEX320 nm) for 15 min (one read per minute) in a fluorescence spectrophotometer (Victor 3 Perkin–Elmer, Boston, MA, USA). Specific proteolytic activity was expressed as units of free fluorescence of cleaved substrate per minute per μg of venom. In addition to PMSF, to select non-BAV-inhibited serine proteases, the ability of the commercial antivenom to neutralize the proteolytic activity of chromatographic fractions was estimated as previously described (Kuniyoshi et al., 2017). This method was also used to verify the neutralizing potential of antivenoms (commercial serum and experimental sera) on the proteolytic activity of the pool of serine proteases (termed as SVSPpool) used in the immunization processes. Briefly, the antivenoms in doses of 50:1, 200:1 and 500:1 (weight of antivenom: weight of venom) were pre incubated for 30 min with 0.25 μg of SVSPpool at room temperature followed by the addition of the AbzSerine. The assays were conducted in triplicate and the results were calculated as the mean value percentage inhibition (+standard deviation, SD) when compared to the positive control. 2.4. Purification of serine proteases from Bothrops jararaca venom that are not blocked by the commercial antivenom The BjV (23.3 mg) was suspended in 1.0 mL of 50 μM ammonium acetate pH 4.0 solution and submitted to anion exchange chromatography in an HPLC system (Shimadzu Co., Kyoto, Japan) using a ShimPack PA-DEAE column (20 mm × 100 mm) at 5 mL/min flow. The gradient used was 0%–80% B for 80 min (buffer A containing 20 mM Tris, 20 mM NaCl, pH 8.2 and buffer B composed of buffer A with addition of 500 mM NaCl, pH 8.2). Five fractions were shown to contain 60

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serine proteases not inhibited by BAV, however, as these fractions also showed metalloprotease activity, a second chromatographic step was used. Thus, the selected fractions were submitted to a Shim Pack-C8RP-HPLC column using an isocratic elution (20% of B for 5 min) followed by a gradient of 20–50% of B for 35 min [buffer A (H2O/ 0.1%TFA) and buffer B (acetonitrile/buffer A, 9:1)], with UV detection at 280 nm. All fractions were screened by fluorescent enzymatic assays as described above, and only those which were both free of metalloprotease activities and were not inhibited by BAV were selected for further studies.

Fig. 1. Immunization schedule of BALB/c and C57BL/6 mice with the SVSPpool. The animals were injected with SVSPpool in the presence (*) or in absence (#) of SBA-15. Retro-orbital blood samples were collected at days 0, 42 and 85.

2.5. SDS-PAGE and SVSPs identification

2.8. Processing the mice plasma and IgG purification

The selected fractions were analyzed by 13% SDS-PAGE (Laemmli, 1970). Samples were solubilized in non-reducing sample buffer, and protein profiles were visualized by silver staining. The protein bands corresponding to the non-neutralized serine proteases, obtained after the second purification step, were subjected to an in-gel digestion with trypsin (Sigma-Aldrich, St. Louis, MO, USA) (Shevchenko et al., 2006). The mixture was then desalted, concentrated, and resuspended in 0.1% formic acid. Mass spectrometric analysis was performed by online liquid chromatography in an Easy-nLC Proxeon nanoHPLC system coupled with an LTQ-Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Raw data files were analyzed on MASCOT Search Engine against the Serpentes database containing 49,427 protein sequences. A more refined analysis were made on PEAKS Studio X (Bioinformatics Solutuin, Waterloo, Canada), using the sequences of KN-BJ (accession number: O13069), HS112 (Q5W960), BjSP (Q5W959) and BPA (Q9PTU8) as data base. The search parameters were: trypsin cleavage specificity (maximum 2 missed cleavages); ion fragment mass tolerance set to 0.5 Da; and a peptide mass tolerance of 10 ppm. Regarding Post Translational Modifications (PTM), carbamidomethylation was set as fixed modification and oxidized methionine as a variable modification. After confirmation of the nature of serine proteases in the samples, they were pooled for use in immunization procedures.

Approximately 200 μL of blood from each animal was collected for pre-immune control, and the same volume was extracted at the final bleed, eight days after the last injection. Since plasma proteins could interfere with the subsequent proteolytic assays, the IgGs purification was performed. For that, sera from the final bleed of all seven mice from both lineages were submitted to affinity chromatography using 1 mL of HiTrap Protein A HP column (GE Healthcare Life Sciences, IL, USA), according to the manufacturer's instructions.

2.9. Immunological analyses: ELISA and relative avidity assays A pool made up with approximately 200 μL of serum from each mouse, collected before immunization with the SVSPpool, was used as pre-immune control. The immunogenic responses of the SVSPpool in both animal strains were evaluated by ELISA, as described below, using the pre-immune sera samples in comparison with the immunized ones. The results showed that the SVSPpool is immunogenic (data not shown) and, after final bleed, IgGs produced by BALB/c and C57BL/6 mice were purified for further studies. Microtitration plates (Costar, Corning, MA, USA) were coated with 1 μg of SVSPpool per each well and incubated at 4 °C overnight in carbonate buffer pH 9.6. After blocking (3% powdered milk in PBS) and washing (0.05% Tween-saline), the plates were washed and incubated (in duplicate) with purified IgGs (from both lineages) and two batches of the commercial antivenom from the Butantan Institute. Dilutions of 1:1000 to 1:512,000 (1 ng and 1.9 pg, respectively) were used and the samples were incubated for 1 h at 37 °C. The plate was washed 3 times with PBS-BSA 0.1%/Tween 0.05% and then incubated for 1 h at 37 °C with peroxidase-conjugated rabbit anti-horse IgG (1:20000; Sigma-Aldrich, St. Louis, USA) when using the commercial antivenom or peroxidase-conjugated goat antimouse IgG (1:2000; Sigma-Aldrich, St. Louis, USA) when analyzing mice sera. After washing (PBS), the peroxidase activity was measured by adding 100 μL of o-phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, St. Louis, USA) and the absorbance values at a wavelength of 490 nm were obtained using a FLUOstar Omega (BMG Labtech) spectrophotometer. The highest dilution that produced an O.D. of at least twice the mean of the pre-immune blood (used as control) was considered the sample titer (U-ELISA/mg/mL). The assays were conducted in duplicate and the results were calculated as the mean value (+SD). The relative avidity of samples was measured as described above, with exception to experimental sera, which were used in a single concentration (1:3000). The samples were incubated for 1 h at 37 °C, washed with PBS-BSA 0.1%/Tween20 0.05% and, after, potassium thiocyanate (KSCN, 4M) was added to the wells for 15 min. Plates were washed again, and all subsequent steps were the same as the ELISA titration, described above. The experiments were performed in triplicate and the relative avidity was determined as the percentage reduction (+SD) in O.D. (measured at 490 nm) when compared to the negative control (without KSCN addition).

2.6. Animals A total of fourteen male adult mice was used, with seven BALB/c and seven C57BL/6, weighing 18–22 g in both groups. The animals were obtained from the Central Animal Facility of the Butantan Institute. The animals were kept in standard conditions, with ad libitum access to food and water. Experimental procedures were in accordance with the ethical principles in animal research adopted by the Brazilian Society of Animal Science and the National Brazilian Legislation no.11.794/08 and the National Institutes of Health guide for the care and use of Laboratory animals. Animal care and experimental procedures were approved by the Institutional Committee for the Care and Use of Laboratory Animals from Butantan Institute (CEUIB protocol number 1161/13).

2.7. Antigen preparation and immunization schedule Immunogenicity analysis of the SVSPpool was performed using two isogenic mouse strains, BALB/c and C57BL/6. Each mouse received six injections of 10 μg of SVSPpool by the subcutaneous route, with intervals of 14 days. The first three immunizations occurred in the presence of the adjuvant SBA-15, where the SVSPpool (80 μg) was added to SBA15 (1.0 mg) diluted in PBS in a final volume of 1600 μL. The final three immunizations were performed using only the SVSPpool (80 μg) (Carvalho et al. 2010). After 24 h at 4 °C, 200 μL of this solution was injected into each mouse. Retro-orbital samples collection and the immunization schedule can be seen in Fig. 1. 61

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5 and 19-9 was produced and was named as SVSPpool (2 mg of total protein). Then, mice from two lineages, BALB/c and C57BL/6, were injected with the serine protease pool six times along 85 days, following the immunization schedule described in Fig. 1. The IgGs present in the serum of both mice lineages were purified by affinity chromatography, protein A Sepharose, to eliminate plasma protein contaminants, which could interfere with the subsequent assays of proteolytic activity. In addition to that, the commercial serum produced by the Butantan Institute is also made up of purified immunoglobulins, which is posteriorly reduced into F(ab')2 fragments to diminish adverse effects. Thus, a comparative analysis between the BAV and the experimental sera results could be of higher quality, as the samples would show a higher similarity (comparing IgGs with IgGs F(ab')2 fragments) than without purification. The SDS-PAGE analyses of resulting purified IgGs from both experimental sera are shown in Fig. 6. SDS-PAGE analyses of purified IgGs under reducing conditions, from both mice lineage show major protein bands with around 50 kDa, which correspond to the heavy chains. The proteins with approximately 30 kDa indicate the light chains of IgGs. Under non-reducing conditions, the purified IgG samples display a major protein band between 140 and 260 kDa. BALB/ c mice yielded approximately 1.4 mg of purified IgGs, while C57BL/6 mice yielded about 3 mg.

Fig. 2. Fractionation of the Bothrops jararaca venom (23.3 mg/mL) by anion-exchange chromatography on a PA-DEAE column in HPLC system. The open boxes show the collected fractions and the blue line indicates the acetonitrile gradient used. The serine protease activity which is not blocked by BAV was detected in fractions F10–F13 and F19 by using the FRET substrate Abz-Serine, which was previously established as specific for this class of protease from the Bothrops jararaca venom (Kuniyoshi et al., 2012).

3. Results

3.3. 3- ELISA, relative avidity and serine protease activity inhibition: comparison of BAV and experimental sera

3.1. 1- purification and identification of serine proteases from Bothrops jararaca venom that are not blocked by BAV

Experimental sera and commercial antivenom were tested for crossreactivity by ELISA using the SVSPpool as antigen. The titration was analyzed taking into account the protein concentration of the samples, and all sera were able to interact with serine proteases present in the SVSPpool (Fig. 7). However, both experimental antivenoms presented higher specific antibody levels when compared with the results obtained with two batches of BAV (Fig. 7). Furthermore, the serum obtained from BALB/c mice was more efficient in interacting with the SVSPpool in comparison to the serum produced by C57BL/6. The chaotropic agent KSCN is able to dissociate interactions between proteins and, in this case, it was used to measure the antigenantibody binding (relative avidity). Fig. 8 shows that treatment with 4 M of KSCN for 15 min was able to reduce by almost 78% and 79% the binding of the experimental sera obtained from BALB/c and C57BL/6 mice, respectively, to the SVSPs present in the pool of purified serinoproteases. In comparison, KSCN reduced the binding of BAV 1 and BAV 2 batches with SVSPpool by only 26% and 35%, respectively. Three doses of each of the antivenoms (experimental and commercial) were tested for their ability to inhibit the catalytic activity of the serine protease pool (SVSPpool), as shown in Fig. 9. Both experimental antivenoms were able to neutralize the proteolytic activity of the SVSPpool upon the FRET substrate Abz-Serine in different extensions. BALB/c serum presented a better neutralization potential at the 1:500 dose (~80%) when compared to serum obtained from C57BL/6 (~25%). When smaller doses of the two experimental antivenoms (1: 200 and 1:50) were used, no significant differences were observed in their neutralizing potential on the catalytic activity of the SVSPpool (Fig. 9). In accordance to previous results (Kuniyoshi et al., 2017, 2012), both batches of commercial antivenoms - at all doses tested were not able to neutralize SVSPpool's catalytic activity. Similar results were obtained when the venom of B. jararaca was used instead of SVSPpool (data not shown).

The BjV was submitted to a DEAE-HPLC and 20 fractions were collected (Fig. 2). All of them were screened through proteolytic fluorimetric assay using FRETs substrates, Abz-RPPGFSPFFQ-EDDnp (Abz-Serine) and Abz-FASSAQ-EDDnp (Abz-Metal), which were previously described to be selective to serine proteases and metalloproteases, respectively (Kuniyoshi et al., 2012). Active fractions were examined through PMSF, EDTA and BAV inhibition. Fractions F10 to F13 and F19 were selected for the next purification step, since they exhibited serine protease activities that are not blocked by BAV. However, these samples also presented metalloprotease activity (data not shown). Thus, these samples were separately submitted to a reverse-phase C8-HPLC chromatography (Fig. 3) and the same proteolytic screening assay described above was performed. Subfractions 105, 11-5, 12-5, 13-5 and 19-9 were selected for the continuity of the study since they presented activity of serine proteases not inhibited by the commercial serum and for not being able to hydrolyze the substrate Abz–Metal. Thus, these five sub-fractions were analyzed by 13% SDSPAGE, as shown in Fig. 4. All proteins present in the samples 10-5, 11-5, 12-5 and 13-5 showed similar retention times on column C8, around 22.5 min (Fig. 3), and also the same SDS-PAGE profiles (Fig. 4). As shown in Fig. 4, SDSPAGE analysis of 10-5, 11-5, 12-5 and 13-5 samples identified three proteins with molecular weights of approximately 36 kDa (B1), 28 kDa (B2) and 25 kDa (B3). Analysis of fraction 19-9 revealed the presence of a protein band of about 70 kDa (B4). Protein bands indicated as B1, B2, B3 and B4 were separately submitted to mass spectrometric analysis. Then, as expected, mass spectrometric analysis of the proteins indicated as B1, B2 and B3 in the different samples resulted in the identification of the same protein in each one of the bands. B1 was identified as KNBJ2, B2 as HS112, B3 as BjSP and F19-9 as BPA (Fig. 5). Based on the molecule sequences in their mature forms, the results of mass spectrometric analyzes covered 35% of KN-BJ2, 35% of HS112, 12% of BjSP and 15% of BPA. All of them are serine proteases from Bothrops jararaca venom (Table 1).

4. Discussion Ophidism is considered a serious public health problem, affecting individuals virtually everywhere in the world. In case of human envenomation by snakes, the immunotherapy is the clinical treatment recommended by the World Health Organization. The Butantan Institute was the first producer of antivenom sera in Latin America and

3.2. 2- Obtainment and purification of the experimental sera produced in BALB/c and C57BL/6 animal models A pool of serine proteases made up of fractions 10-5, 11-5, 12-5, 1362

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Fig. 3. - Rechromatography on reverse phase C8 HPLC of selected fractions from anion exchange chromatography. A) fraction 10, B) fraction 11, C) fraction 12, D) fraction 13 and E) fraction 19. The arrows indicate the peaks containing serine protease activity over the FRET substrate Abz-Serine which is not blocked by BAV. The blue lines show the used gradient.

stimulated the advance of research in the field of ophidism (De Franco and Kalil, 2014). Like other antivenoms, the bothropic antivenom has saved thousands of lives in Brazil since it was created almost 100 years ago. The initial product was modified over the years, and is currently composed of F(ab')2 fragments produced in horses immunized with a mixture of five bothropic venoms (listed in the Introduction section). Despite the good efficacy of BAV in blocking the main effects of Bothrops snake envenoming, it may be that the product could still be

improved, as the neutralization of some symptoms of accidents is still not achieved. The present work intends to contribute in this aspect, since it was previously verified the BAV's inability to neutralize toxins of the B. jararaca venom belonging to the serine protease class. This limitation has been demonstrated by our group with the use of FRETs substrates designed to be selective for proteases of the classes of serine proteases and metalloproteases, the Abz-Serine and Abz-Metal (Kuniyoshi et al., 2012). In addition, hydrolyses of several bioactive 63

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Table 1 Identification of serine proteases that are not blocked by the commercial antivenom produced by the Butantan Institute. Identification of Proteins by Mass Spectrometry Band gel extracted

Protease

Mass

Score

Matches

Sequences

B1 B2 B3 B4

O13069|KN-BJ2 Q5W960|HS112 Q5W959|BjSP Q9PTU8|BPA

25212 25290 25160 25408

938 686 6585 459

101 (54) 88 (46) 301 (225) 43(26)

13 (13) 13 (11) 14 (14) 6 (5)

peptides, which are substrates for serine proteases present in the B. jararaca venom, are not inhibited by the BAV (Kuniyoshi et al., 2017). In this work, four BjV serine proteases were isolated and identified in order to better understand why the present BAV does not neutralize at least two of these toxins.

Fig. 4. SDS-PAGE 13% of fractions A) 10-5, 11-5, 12-5, 13-5 and B) F19–9. All fractions (2 μg) were analyzed under non-reduced conditions. Protein bands indicated as B1, B2, B3 and B4 were excised, separately submitted to an in-gel digestion by trypsin and analyzed by LC-MS/MS.

Fig. 5. Identification and coverage of BjV serine proteases which made up the SVSPpool . A) KN-BJ, accession number: O13069 (66% of coverage); B) HS112, Q5W960 (40% of coverage); C) BjSP, Q5W959 (74% of coverage); D) BPA, Q9PTU8 (39% of coverage). Highlighted and underlined amino acids in blue represent the peptides sequenced by PEAKS Studio X, the amino acids underlined in gray represent the sequences found in the de novo sequencing. 64

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form. BjSP shows hydrolytic action on fibrinogen, but without fibrin formation (Carone et al., 2018). The third one, HS112, is a putative serine protease sequence obtained through a KN-BJ2 probe in a cDNA library from the Bothrops jararaca venom gland (Saguchi et al., 2005), and, to the best of our knowledge, this is the first time that it has been identified as a toxin present in the venom. The last one, BPA, is a 67 kDa fibrinogenolytic protease - highly glycosylated - and therefore stable against high temperatures (86 °C for 10 min) and pH variation (pH 3–9) (Henriques et al., 1958; Paes Leme et al., 2008). These four serine proteases have a high degree of identity between their primary sequences, with KN-BJ2 and HS112 having the lowest identity with each other (64.07%) and HS112 and BPA with the highest identity among them (75.32%). Since three SVSPs have been obtained together, KNBJ2, BjSP and HS112, it cannot be guaranteed that all enzymes are cleaving the FRET substrate used and that this hydrolysis is not inhibited by antibothropic serum. However, as these SVSPs show a high level of primary structure identity, it is possible that these enzymes feature similar activities. Due to the low quantity obtained of these serine proteases, a pool composed of these four molecules was made up for the immunogenicity studies. This work demonstrated that the SVSPpool is able to induce the production of specific and neutralizing IgGs, therefore activating the immune system when inoculated in two mouse strains. Although the SVSPpool resulted in the production of specific antibodies in both animal models used, BALB/c mice serum presented better titers and, more importantly, a better proteolytic in vitro neutralizing potential then the C57BL/6 serum. This difference was expected, since the same phenomenon has been previously demonstrated for different antigens, and this was the reason why these two lineages were selected for the studies with the SVSPpool (Bryan et al., 2010; Vlkova et al., 2012). Since BALB/c mice predominantly present TH1 response while C57BL/6 have TH2 (Quimby and Luong, 2007), our results point out that the choice of animals can contribute to a different final product. Despite this difference, even the C57BL/6 IgGs, which presented the lower inhibitory potential when compared to BALB/c IgGs, was more efficient than BAV to block serine protease activity of SVSPpool and BjV. Commercial antivenom shows higher avidity antibodies against the serine proteases present in the SVSPpool when compared to the IgGs present in the two experimental sera. However, in spite of the high avidity, the antibodies present in the BAV probably recognize regions of the SVSPs molecules that do not cause allosteric changes or interaction with the active sites, since they are not able to block the enzymatic activity of these proteases. It is important to mention that the commercial serum, although presenting good titers against serine proteases present in the BjV, is not capable to effectively neutralize their activity. Perhaps this is due to a smaller IgG repertoire against epitopes of serine proteases in the BAV when compared to the experimental sera, which were obtained by immunization using isolated molecules. Also, it is known that immunizing BALB/c mice with low doses of immunogen helps to narrow the spectrum specificity of antibodies when compared to higher-doses (Li et al., 2017), once the immunizing protocol in horses is in the mg/kg scale while ours is on μg/kg. The use of the adjuvant SBA-15 could be another decisive factor for the production of efficient neutralizing antibodies, since it has been shown that this silica is efficient in producing immune response for antivenom manufacture (Ferreira Junior et al., 2010). The SVSPs that make up the pool used to obtain the experimental sera have been reported to be coagulant (BPA, BjSP and KN-BJ2) or kinin-releasing (KN-BJ2) enzymes, in in vitro studies. As an exception, macromolecular substrates for HS112 have not been described. Thus, a more efficient inhibition of these enzymes is likely to control the coagulation and hypotension disorders observed in victims of B. jararaca accidents. This work demonstrated that immunization of animals with isolated serine proteases may be a way to improve product quality even more. Another important point to note was the immunization schedule used,

Fig. 6. Electrophoretic profile of IgGs purified by protein A affinity chromatography. The IgGs (5 μg) from BALB/c and C57BL/6 mice were subjected to electrophoresis under reducing and non-reducing conditions. MW = molecular weight.

Fig. 7. ELISA assays. ELISA titration of BALB/c and C57BL/6 mice purified IgGs compared to BAV over SVSPpool (1 μg).

Fig. 8. Relative avidity assays. The plates were sensitized with the SVSPpool (1 μg) and the avidity of the experimental sera from BALB/c and C57BL/6 mice and two batches of BAV (1 and 2) were evaluated in the presence of the chaotropic agent KSCN 4 M.

The first one, KN-BJ2, is a serine protease with calculated molecular weight of 25 kDa, and its glycosylated form featured about 38 kDa. The activity of KN-BJ2 is reported as both coagulant and kinin-releasing (Serrano et al., 1998). The second, BjSP, was recently purified for the first time and presents molecular weight of 28 kDa in its glycosylated 65

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Fig. 9. In vitro proteolytic sera neutralization assay. The ability of experimental antivenoms and two batches of BAV to neutralize SVSPpool (0.25 μg) was evaluated. The SVSPpool was pre-incubated for 30 min with three concentrations of each antivenom and, after this period, the activities were determined through fluorimetric assays using 5 μM of the AbzSerine FRET substrate.

including the adjuvant SBA-15. Thus, although the objective of the present work was to demonstrate that it is possible to obtain IgGs capable of inhibiting the activity of SVSPs present in the B. jararaca venom, future studies can use the information based on the results presented here in order to improve antivenom efficacy.

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