Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis.

Pull-down assay, immunofluorescence antibody test (IFAT) and mass spectrometry. These tests were carried out to identify and characterize the S. venezuelesis antigen that was targeted and bound by the scFv. The 15% SDS-PAGE silver stained, after pull-down assay, showed the profiles of the purified scFv (~29 kDa) and the S. venezuelesis antigenic fraction of ~65 kDa that bound to the scFv ( Fig. 2A). Furthermore, the scFv bound to the body periphery and digestive system (arrow), specifically intestine (Fig. 2B) and esophagus (Fig. 2C), from S. venezuelensis infective larvae (L3), as evidenced by IFAT. Taenia solium metacestodes showed no staining by anti-HA-FITC, but only the red color Bioinformatic in silico analysis between scFv and HSP60. Docking between scFv and HSP60 was performed to determine possible binding regions by in silico prediction tools. The most stable 3D structure of the scFv-HSP60 was submitted and analyzed by the PyMOL online tool, and identified the putative CDR regions that are responsible for the antibody binding to the HSP60 epitope with a strong association between them (Fig. 3A, B and C). The binding site analysis also showed that only the CDR2 of the light chain had no binding region to the HSP60 (Fig. 3D,E).
Sandwich ELISA for immune complexes detection in human sera. The scFv was immobilized in high affinity microtiter plates for the serological detection of immune complexes. The TG-ROC curve obtained the best cut-off (0.7175) (Fig. 4A). The data of reactivity index demonstrated a significant discrimination between sera from S. stercoralis positive individuals (G1) in relation to serum samples from individuals that were positive for other parasitic diseases (G2) and healthy controls (G3) (P < 0.001) (Fig. 4B). Diagnostic parameters of sensitivity and specificity were 97.5% (86.84 -99.94) and 98.81% (93.54 -99.97), respectively. The high efficiency of the test was further demonstrated by the significant positive likelihood ratio (LR+ = 81.90) and area under the curve (AUC = 0.9993) (Fig. 4C).

Discussion
The engineering of novel functional combinatorial antibodies expressed on the surface of bacteriophages, such as scFvs, with high specificity for antigens coupled with a phage display strategy is the most promising technology for improved diagnostics and therapeutics of many pathologies 10,11 . This is due to their small size, great reactivity and specificity, and easiness to maintain and express in bacteria, which surpass the hybridoma technology in all aspects. The scFv selected in this study was structurally characterized and applied in the immune complexes detection using human serum samples positive for strongyloidiasis. This diagnostic strategy is an important tool because it is indicative of active infection [12][13][14][15] .
In this study, we have selected an scFv clone through Phage Display against total proteins of S. venezuelensis as a model for S. stercoralis infection in humans, with a successful application in the serodiagnosis of human strongyloidiasis. Our novel scFv showed strong reactivity to a parasite protein fraction of ~65 kDa from S. venezuelensis, which further identified and characterized to be the heat shock protein 60 (HSP60) from Strongyloides sp., a 64 kDa protein shown in experimental models to have relevance to the parasite-host relationship, with potential significance for the host immune response 16 . This newly scFv was localized by immunofluorescence staining in the entire parasite body periphery and to regions of the digestive system from the infective larvae. Moreover, the scFv did not bind to T. solium metacestode, indicating specificity.
The scFv functionality was confirmed by a sandwich ELISA using this antibody fragment to detect immune complexes in serum samples from individuals with strongyloidiasis. The TG-ROC curve provided the optimum cut-off value, and ROC curve confirmed its high sensitivity and specificity, both values higher than 97%. The test performance indicated by the high LR+ (81.9) and the AUC value close to the maximum (1.00) conferred a high diagnostic value. The assay was able to discriminate all positive serum samples from patients with strongyloidiasis in relation to sera from healthy individuals and with other parasitic diseases, except one false-positive result for an individual with Ascaris lumbricoides, suggesting a possible cross-reaction. However, the very low reactivity indices for the other seven positive sera for A. lumbricoides that was below the threshold suggest a possible co-infection of A. lumbricoides and S. stercoralis, which was not detected by the conventional parasitological method. To date there have been few studies in the literature employing scFv to diagnose parasitic diseases. The most recent articles have demonstrated its important applicability in the detection of antigenic surface components of Schistosoma mansoni 17 and the selection of antigens for diagnosis of neurocysticercosis 18 . Moreover, there is only one serological assay based on ELISA for the detection of immune complexes in serum samples of individuals with strongyloidiasis 19 , but with a sensitivity of 93.3% and specificity of 86.1%. Unlike this later study, our present investigation used a phage display technology that enabled the in vitro production of antibody fragments with high affinity for the target molecule and, consequently, did not require animal models for their synthesis, dramatically reducing the time required to obtain the reagent.
Furthermore, this is the first description of the HSP60 as a diagnostic target for the human strongyloidiasis, with additional data on the epitope and paratope binding sites of the scFv-HSP60 by in silico prediction tools. This result confirms previous studies that described HSPs as major immune dominant antigens in several infections, in addition to their original role as molecular chaperones. It has been demonstrated that HSP60 represents a major component of Strongyloides ratti ESPs (excretory/secretory proteins) and presents functions as a target to the humoral and cellular immune response. Interestingly, the vaccination with alum-precipitated HSP60 or a passive immunization with a monoclonal anti-HSP60 IgM antibody, both specifics for Strongyloides ratti, conferred protection to mice against a challenge infection 20,21 .
This is the first study that has successfully selected and used an scFv biomarker for the diagnosis of human strongyloidiasis, which was applied in the detection of immune complexes in serum samples of patients with great accuracy, aiming the newly found HSP60 as the target antigen. The serological detection of circulating immune complexes of this helminthiasis is indicative of infection, regardless the serum antibody titers and the release of larvae in feces. The development of a better, powerful and innovative diagnostic tool is of public health importance, since strongyloidiasis may be fatal, particularly in immunocompromised individuals.

Preparation of total proteins from Strongyloides venezuelensis. Strongyloides venezuelensis
total proteins were isolated using L3 larvae 25 . Larvae were resuspended in 0.01 M PBS (pH 7.2) containing protease inhibitors and disrupted with five cycles of freezing (1 min, -196 °C) and thawing/sonication (5 min, 40 kHz, 4 °C) (Thornton, Impec Eletrônica, São Paulo, Brazil). After overnight incubation at 4 °C with constant agitation, the suspension was centrifuged at 12,400 × g for 30 min at 4 °C. Protein quantification of the supernatant was performed 26 and the extract was stored at -20 °C until use.

Selection of scFvs clones against total proteins of S. venezuelensis. The selection of scFv
clones was performed using a human scFv phage library, with approximately 2 × 10 6 combinatorial sequences with variability for multiple diseases 27 , against total proteins of S. venezuelensis as target. Two cycles of selection were performed 18 , being preceded by scFv library reamplification in the competent Escherichia coli XL1-Blue strain and infection by VCSM13 helper phage for assembly and replication of viral proteins 28 . A microtiter plate well (Nunc MaxiSorp TM ) was coated with 50 μ L of S. venezuelensis total proteins (1 μ g/well) diluted in 0.1 M sodium bicarbonate buffer (pH 8.6) and incubated for 18 h at 4 °C. The well was then blocked with 250 μ L PBS/0.05% Tween 20 (PBST) (v/v) and 5% BSA (w/v) for 1 h at 37 °C, and washed three times with PBS. Then, 100 μ L of the library was added to the well and the plate was incubated for 1 h at 37 °C. Subsequently, the plate was washed 10 times with PBS/0.1% Tween 20, and bound phages to total proteins were eluted with 100 μ L of 100 mM glycine-HCl (pH 2.2), followed by neutralization with 16.5 μ L of 2 M Tris (pH 9.1). The resulting phages from the first selection were reamplified in E. coli XL1-Blue, and a second round of selection was then performed as described above.
Expression of scFv in E. coli TOP 10. Plasmids were extracted from Escherichia coli XL1-Blue bacterial cells infected in the 2nd cycle of selection for further transformation in E. coli TOP 10, a non-suppressor strain 28 . Aliquots of the transformed cells were plated on LB (Luria-Bertani) agar containing carbenicillin (100 μ g/mL). Plates were incubated at 37 °C for 16 h to allow growth of recombinant colonies. Each colony was transferred to a well in a deep well plate containing 1 mL of Super broth (SB) medium containing carbenicillin (100 μ g/mL) and 2% 2 M glucose (v/v). After centrifugation, the expression of soluble scFv was induced by incubation with isopropyl β -D-thiogalactopiranoside (IPTG) (Sigma, USA) to a final concentration of 2.5 mM and carbenicillin (100 μ g/mL) overnight at 250 × g and 30 °C. The plates were centrifuged at 4000 × g for 15 min at 4 °C, and the supernatant containing soluble scFv was transferred to another 96-well plate and stored at 4 °C 28 .
Analysis of expression and specificity of scFv clones. For analysis of scFv expression, a microtiter plate was coated with suspensions of 96 scFv clones. For analysis of scFv specificity, total proteins of S. venezuelensis (10 μ g/mL) were immobilized in a micotiter plate and the suspension of 96 scFv clones was added. In both cases anti-HA (tag) peroxidase conjugated (1:1000) (Roche, Switzerland) was added. Reactions were revealed by addition of substrate (30% H 2 O 2 ) and the OPD chromogen (orthophenylenodiamine) diluted in 0.1 M citrate-phosphate (pH 5.0). The optical densities were determined at 492 nm in an ELISA reader (Titertek Plus, Flow Laboratories, USA). An irrelevant scFv clone, from the same library but selected against another target, was used as a negative control. following primers: MMB4 (5'-GCT TCC GGC TCG TAT GTT GTG T-3') for the light chain and MMB5 (5'-CGT TTG CCA TCT TTT CAT AAT C-3') for the heavy chain. The samples were sequenced using an automatic capillary sequencer (MegaBACE 1000 Genetic Analyzer, Amersham Biosciences, USA), and deduced amino acid sequences were then analyzed by IgBlast at NCBI (http://www.ncbi.nlm.nih. gov/igblast). Furthermore, sequences were submitted to the Raptor-x (http://raptorx.uchicago.edu/) and PyMOL (http://www.pymol.org/) bioinformatics online tools to obtain and analyze the 3D structure of the scFv molecule.
Medium-scale production of soluble scFv and purification of selected scFv by HPLC. The expression of the selected clone was produced in a 1000 mL Erlenmeyer cell culture flask, as described above (expression of scFv section). For medium-scale purification, His 6 -tagged scFv fragments were purified by immobilized-metal (Nickel) affinity chromatography (HisTrap TM HP, GE Healthcare Life Sciences, USA) according to the manufacturer's instructions in a HPLC system (AKTA TM purifier). The purified scFv was concentrated using a 10 kDa Amicon apparatus (Millipore, USA), the protein concentration was determined 26 and a dot-blot assay of the purified scFv was performed 18 to confirm the efficiency of the purification process.
Pull-down assay. To characterize the specific protein target that bound to the scFv from the extract of total proteins of S. venezuelensis, 100 μ L of mouse anti-His mAb Mag Beads (GenScript Transforming Biology Research, USA) were incubated with 100 μ L of purified scFv according to the manufacturer´s instructions. After washing, 100 μ L of S. venezuelensis total proteins (1000 μ g/mL) were added and, after incubation and additional washings, 20 μ L of the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) sample buffer were added, and the tube was incubated at 100 °C for 5 min. Proteins were eluted from beads using a magnetic apparatus, and the supernatant was submitted to a 15% SDS-PAGE. Proteins were visualized by silver staining 29 , and the electrophoretic profile was determined by Image J 1.44 (National Institute of Health, Bethesda, EUA).

Immunofluorescence antibody test (IFAT). Strongyloides venezuelensis larvae were embedded in
Tissue-tek (Tissue Freezing Medium, Durham, NC, USA) and frozen at -70 °C. Sections of 2 μ m in thickness were cut using a cryomicrotome and placed on microscope slides. The sections were left at room temperature for 30 min to dry. Twenty microliters of scFv (750 μ g/mL) was added to each section and the slides were incubated in a humidified chamber at 37 °C for 1 h. The sections were incubated with fluorescein isothiocyanate conjugated (FITC) mouse anti-HA (1:1000) and counterstained with 2% Evans blue for 40 min at 37 °C. Between each step three washes were performed in PBS. The slides were mounted using glycerol/PBS (pH 9.0) and coverslips. The staining profile of the scFv on the larvae was verified using a LSM 510 confocal microscope (Meta, Carl Zeiss, Germany). A section of Taenia solium metacestode was used as a negative control for staining.

Mass spectrometry (CID-MS/MS) and bioinformatic analyses.
For mass spectrometry, the pull-down assay was repeated; however, the gel was staining with Comassie blue G-250, and the band related to the protein of S. venezuelensis that bound to the scFv was trypsinized and submitted to CID-MS/MS 30 (Quattro II, Micromass, Manchester, UK). Peptide fragments obtained were submitted and analyzed by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) in relation to their similarity to proteins of Strongyloides sp. (taxid: 6247). Furthermore, the highest similar protein sequence was submitted to bioinformatics online tools. The Raptor-x (http://raptorx.uchicago.edu/) was used to obtain the 3D structure of the protein. For identification of potential binding regions of the protein and scFv it was performed a docking (http://bioinfo3d.cs.tau.ac.il/PatchDock/). The regions of scFv-protein structure were selected and analyzed by PyMOL (http://www.pymol.org/). Sandwich ELISA for immune complexes detection in human sera. High affinity microtiter plates (Nunc MaxiSorp TM ) were incubated with 50 μ L of scFv (10 μ g/mL) in carbonate bicarbonate buffer (0.06 M, pH 9.6), overnight at 4 °C. The plates were blocked with PBS/5% BSA for 45 min at 37 °C. Serum samples were diluted 1:50 in PBST, added to the wells and the plates were incubated for 45 min at 37 °C. Subsequently, mouse anti-human I gG peroxidase conjugated diluted in PBST (1:10,000) was added to the wells and the plates were incubated for 45 min at 37 °C. Between each step three washes were performed in PBST. The reaction was revealed by adding orthophenylenediamine (OPD) diluted in 0.1 M citrate-phosphate (pH 5.0) and 30% H 2 O 2. Plates were incubated for 15 min at room temperature, and reactions were stopped by adding 2N H 2 SO 4 . The optical densities (OD) were determined at 492 nm in an ELISA reader (Titertek Plus, Flow Laboratories, USA). The cut-off value was determined by the TG-ROC curve 31 , based on the ELISA results. The ELISA reactivity index (RI) was obtained from the ratio: OD of each sample/cut-off value; RI values > 1 were considered positive. under the curve (AUC) was obtained from the ROC curve in order to determine the diagnostic accuracy of the method 33 . Values were considered statistically significant when p < 0.05 and 95% confidence intervals (CI) were provided for Se, Sp and AUC statistical calculations. The Analysis of Variance (ANOVA) followed by the Tukey post hoc was used to determine differences among groups.