The mouse single-chain variable fragment (SCFV) antibody specifically recognizes the epitope on cathepsin f protein of the human liver fluke opisthorchis viverrini ( ovcatf) (amino acid residues 11 to 30) selected by biopanning from the murine naÏve single-chain variable fragments (SCFV) library with the production procedures

Description

[0001] THE MOUSE SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) ANTIBODY SPECIFICALLY RECOGNIZES THE EPITOPE ON CATHEPSIN F PROTEIN OF THE HUMAN LIVER FLUKE OPISTHORCHIS VIVERRINI (OFCATF) (AMINO ACID RESIDUES 11 TO 30) SELECTED BY BIOPANNING FROM THE MURINE NAIVE SINGLE-CHAIN VARIABLE FRAGMENTS (SCFV) LIBRARY WITH THE PRODUCTION PROCEDURES

[0002] TECHNICAL FIELD

[0003] Molecular Biology, Immunology, Parasitology, Microbiology

[0004] SUMMARY OF THE INVENTION

[0005] Cysteine protease is a group of protease enzymes that have been characterized in numerous infectious pathogens. In parasitic helminths, their functions include metacercaria excystation, tissue invasion, catabolism of host proteins for nutrition, and immune evasion. Previous reports indicated that several cysteine proteases were released during O. viverrini infection, and one of the highest amounts is cathepsin F. It is an interesting candidate because cathepsin F is an enzyme with a half-life that is highly released during the infection; detecting this protein could reflect the current infection rather than the past infection. Hence, the primary objective of this study is to produce single-chain variable fragment (scFv) antibodies against cathepsin F of O. viverrini (OvCatF) by using phage display technologies. This novel approach holds great potential in the field of parasitology and infectious diseases, and the characterization of their immunological properties could pave the way for the development of an effective diagnosis in the future.

[0006] BACKGROUND ART

[0007] The infection of human fiver fluke Opisthorchis viverrini (O. viverrini) is a significant health problem in Southeast Asia, especially in the Great Mekong Subregion, which includes Laos PDR, Cambodia, Vietnam, and Thailand, which houses more than 328 million population. The recent prevalence in some areas of this region demonstrated the remaining high infection rate. The infection causes a wide range of symptoms, from mild to severe, that could lead to death in the chronic infection. In the acute phase, the infected patient may be asymptomatic or have mild symptoms, such as abdominal discomfort and non-specific mild diarrhea. On the other hand, longterm infection can lead to cholangiocarcinoma (CCA) or liver biliary cancer, which is one of the most aggressive cancers with short survival. The infection of O. viverrini, coupled with the consumption of carcinogens such as nitrosamine that exist in fermented fish, also the source of the parasite’s infective stage, is recognized as an essential risk factor for people in this region. Therefore, an urgent need for an early diagnostic procedure for O. viverrini infection, as proposed in this research, is crucial to stopping carcinogenesis and saving fives.

[0008] Nowadays, stool examination techniques by light microscopes, such as simple wet smear and formalin-ethyl acetate concentration technique (FECT), are used as a standard procedure for diagnosing O. viverrini infection. However, the standard methods have several limitations, including being unable to samples with a low number of parasites, being time-consuming, and requiring a professional investigator for morphological differentiation. Apart from the microscopic-based methods, molecular techniques have been reported targeting several molecules such as internal transcribed spacer (ITS-1 and ITS-2), cytochrome c oxidase 1 (coxl), and NADH dehydrogenase (NAD) subunits by using various methods, including conventional PCR, qPCR, and LAMPs. However, the variation in the sensitivity and specificity of the target genes is still under investigation. Moreover, the instrument requirement and the price of reagents are significant limitations.

[0009] In the past, there has been a vast effort to develop the immunodiagnosis of O. viverrini infection. Still, it remains unusable due to several factors, such as specificity, cross-reactivities, and the discrimination of past and present infections. Among the established targets, excretory / secretory (ES) products are famously in demand. The previous studies demonstrated that some proteins in the parasite ES products could be detected in serum and urine specimens, which have a high potential to be diagnostic targets. However, ES products contain many enzymes that are concerned with stability and half-life. Proteins in the parasite’s eggs, such as eggshell protein, are another interesting candidate. For example, the glycine-tyrosine-rich eggshell protein (OvESP) has been molecularly characterized, but unfortunately, the detectable sensitivity is dramatically low. Another attractive target is rhophilin-associated tail protein 1 -like (OvROPNIL), the sperm- associated protein that was previously demonstrated diagnostic potential. However, although the detection efficiency is good, the scFv against OvROPNIL is still under development.

[0010] BRIEF DESCRIPTION OF DRAWINGS

[0011] FIG. 1 Amino acid residues 11 to 30 (labeled as Pl) on OvCatF compared with other cathepsins from related flatworm parasites and humans. FIG. 2 SDS-PAGE of recombinant scFv antibodies against OvCatF epitope (amino acid residues 11 to 30) after induction by 0.1 M Isopropyl 0-D-1 -thiogalactopyranoside (IPTG) for 3 h. (M; tricolor broad range protein ladder [Vivantis, Malaysia], 1; non-induced, 2; 0.1 M IPTG induced) FIG. 3 SDS-PAGE of purified scFv antibodies against OvCatF epitope (amino acid residues 11 to 30) using Ni-NTA affinity chromatography (M = Whole blue broad range protein ladder [Vivantis, Malaysia]; BP = before purify; FT = flow through, W1 = wash fraction 1, W2 = wash fraction 2, E1-E10 = elution fraction 1-10)

[0012] FIG. 4 Indirect ELISA result shows the binding of scFv antibodies against OvCatF epitope (amino acid residues 11 to 30) and OvCatF recombinant protein (rOvCatF). (Antigen; rOvCatF, Control; phosphate buffer saline, scFv; scFv antibodies against OvCatF [amino acid residues 11 to 30], poAb; polyclonal antibodies against OvCatF recombinant protein [rOvCatF]).

[0013] FIG. 5 Western blot analysis illustrates the specific binding of scFv antibodies against OvCatF epitope (amino acid residues 11 to 30) and OvCatF recombinant protein (rOvCatF). (Antigen; rOvCatF, M; tricolor broad range protein ladder [Vivantis, Malaysia], Negative control [NC]; phosphate buffer saline; scFv; scFv antibodies against OvCatF [amino acid residues 11 to 30], poAb; polyclonal antibodies against OvCatF recombinant protein [rOvCatF]).

[0014] FIG. 6 The molecular docking of the scFv antibodies against OvCatF [amino acid residues 11 to 30] (green) to OvCatF (brown) shows the antibodies' specific interaction with the specific protein target.

[0015] DISCLOSURE OF INVENTION

[0016] A. B-cell epitope prediction: The B-cell-specific epitopes of OvCatF (GenBank accession no. FJ346536.1) are predicted using BepiPred-2.0 (available at https: / / services.healthtech.dtu.dk / services / BepiPred-2.0Z). The obtained B-cell epitopes are located in the deduced amino acid sequence of OvCatF and multiply aligned with other cysteine proteases from the closely related parasites (flukes) as well as humans, including Clonorchis sinensis (GenBank accession no. ABK91811.1), Paragonimus westermani (GenBank accession no. KAA3674842.1), Schistosoma mansoni (GenBank accession no. XP_018649823.1), Fasciola hepatica (GenBank accession no. AAB30089.1), and Homo sapiens (GenBank accession no. AAD4 1790.1). The candidature sequences that showed low conservation to the other parasites are identified and subjected to synthesizing specific peptides conjugated with biotin at the N-terminus. The epitope containing amino acid residues 11 to 30 containing the following sequence “TTIWSVFARTTPFEPDDARA” is selected. B. Immobilization of antigen: The O vC at F- specific scFv antibodies are selected from the murine naive single-chain variable fragments (scFv) library by biopanning. The biotinylated OvCatF synthetic peptide (amino acid residues 11 to 30) is dissolved with PBS pH 7.4 to a final concentration of 2 pg / mL. The synthetic peptide is immobilized in the streptavidin-coated 96- well microtiter plate. 100 pL of the biotinylated capture antibody is added to each well. The plate is incubated for 2 hours at room temperature with gentle shaking. When the incubation time is reached, the wells are washed thrice with 200 pL of wash buffer. One pg of biotinylated synthetic peptide is prepared for 100 pL in the carbonate coating buffer pH 9.6 and added to each well. The plate is then incubated at 37°C overnight until dried to allow complete binding. After incubation, the wells are washed three times with 200 pL of wash buffer and blocked with 3% BSA in PBS, pH 7.4 at 37 °C, for 1 h in a moisture chamber. The excess antigens are removed by washing with a wash buffer. The wells are filled with PBS and kept at 4 °C before use in the biopanning step.

[0017] C. Biopanning, phage rescuing, and titration: OvCatF-specific scFv is selected by subtractive and positive biopanning with non-infected human fecal extract, biotin, and biotinylated OvCatF synthetic peptide (residues 11 to 30), respectively. Nonspecific binders are removed from the library by subtractive biopanning. The subtraction is performed by incubating 107CFU of the scFv-phage library with non-infected human fecal extract coated in an ELISA well at 37 °C for 1 h. Then, the unbound scFv-phages are transferred and incubated with biotin coated on the well. Consecutively, positive biopanning is carried out by transferring and incubating the unbound scFv- phages with the immobilized target peptide at 25 °C for 1 h on an oscillating agitator. The unbound phages are washed out with PBS-T and discarded; then, the wells are incubated with 500 pL trypsin at 37 °C for 15 min to elute the peptide-bound scFv-phages. The eluted scFv-phages are harvested by magnetic separation twice using streptavidin-coated magnetic beads; then, these two eluted fractions are pooled. Five hundred microliters of the eluted scFv-phages are mixed with an equal volume of log-phase XL-1 Blue Escherichia coli and incubated at 37 °C for 15 min to allow phagemid transduction into bacterial cells. The transformed E. coli harboring recombinant phagemids are selected by growing on the Luria-Bertani (LB) agar plate containing 100 pg / mL ampicillin antibiotics. The ampicillin-resistant colonies are screened for the scFv insert sequence by direct colony PCR using pelB forward primer (5'-ATACCTATTGCCTAC GGCAGC-3') and gill reverse primer (5'-TAGCATTCCACAG ACAGCCC-3'). The PCR conditions are initial denaturing at 95 °C for 5 mins, 30 cycles of denaturation at 95 °C for 30 seconds, annealing at 58 °C for 45 seconds, and extension at 72 °C for 1 min, followed by final extension at 72 °C for 3 mins. The E. coli clones, which have PCR amplicons with the expected size of approximately or slightly above 900 bps, are counted as scFv positive. The individual scFv-positive E. coli clones are sub-cultured and co-infected with M13KO7 helper phages to rescue the scFv-phage progenies. The titers of the rescued scFv-phages are evaluated and normalized by colony-forming unit count.

[0018] D. Screening of the binding of scFv-phages with rOvCatF: The rescued scFv- phages are determined for the binding efficiency to the recombinant protein of OvCatF (rOvCatF) using indirect ELISA. One microgram ofrOvCatF is mixed with 100 pl of carbonate coating buffer pH 9.6 and loaded into each well of the 96-well ELISA plate. The plate is then incubated at 37 °C overnight to allow drying. After overnight incubation, the wells are washed three times with PBS- T and non-specific bindings are blocked with 3% BSA in PBS at 37°C for 1 hour. Each clone of rescued scFv-phage is diluted 10-fold in PBS-T, added into the antigen (rOvCatF) coated well, and incubated at 37°C for 1 hour. After incubation, the wells are washed with PBS-T five times to remove the excess contents. All the wells with scFv-phages are subsequently incubated with mouse anti-M13 antibodies (dilution 1:6,000) at 37°C for 1 hour. The 1:10,000 Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP-conjugated, is added and further incubated at room temperature for 1 hour. The wells are washed thrice with PBS-T, and the TMB-ELISA Substrate Solution is added. The scFv-phages that yielded more than two times over the background are selected for isolation and engineered.

[0019] E. Isolation of pSEX81 phagemid, subcloning, and verifying of scFv properties: The selected clones of scFv-phages are cultured, and the phagemid, pSEX81, is isolated. The scFv coding sequence of the selected clones from the pSEX81 phagemid is digested with restriction endonucleases (NotUNcoT) and subcloned into the pOPElOl expression plasmid. The ligation of pOPElOl and scFv fragment is transformed into XL 1 -Blue E. coli using chemical transformation. The positive transformants are selected by direct colony PCR. The PCR-positive clones are subjected to plasmid DNA extraction and verified by DNA sequencing. The pOPElOl containing scFv fragment is predicted for the complementarity determining regions (CDRs) and immunoglobulin framework regions (FRs) by the IMGT / V-QUEST tool (the International ImMunoGeneTics Information System (IMGT®).

[0020] F. Production of soluble scFv anti-OvCatF antibodies: The clones of XL-1 Blue E. coli containing pOPElOl with correct scFv sequence (both VH and VL fragments) are grown in LB broth by inducing with 1 mM isopropyl -d-l -thiogalactopyranoside (IPTG, Sigma- Aldrich, Saint Louis, MO, USA) at 37 °C for 3 hours in the incubator shaker. The cells are harvested by centrifugation at 6,000 xg at 4 °C for 30 min and lysed using denaturing lysis buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, adjusted pH to 8.0). The scFv is purified under denaturing conditions using Ni-NTA sepharose affinity chromatography. The purified scFv is dialyzed against PBS, pH 7.4, using a dropwise dialysis procedure for refolding the produced scFv. The purified soluble scFv is verified by Western blot analysis using mouse monoclonal anti-c-Myc, followed by the Goat anti-Mouse IgG (H+L) Secondary Antibody, AP-conjugated, followed by NBT / BCIP Substrate Solution.

[0021] G. Molecular modeling and docking: The deduced amino acid sequences of scFv and OvCatF are individually submitted to AlphaFold2 to generate the proteins' 3D model structure. After generation, the scFv and OvCatF are docked by submitting them to the HADDOCK server version 2.4 to verify the specific bindings between these proteins. The possible models are selected using the HADDOCK and PRODICGY web server, and the interactions are analyzed using the Discovery Studio Visualizer 2021 version 21.

[0022] BEST MODE FOR CARRYING OUT THE INVENTION

[0023] Same as mentioned in the production procedures

Claims

CLAIMS1. The single-chain variable fragment (scFv) antibody produced from murine naive singlechain variable fragments (scFv) library. This scFv antibody recognizes the epitope containing amino acid residues 11 to 30 (amino acid sequence: TTIWSVFARTTPFEPDDARA) on cathepsin F of Opisthorchis viverrini, the human Ever fluke (OvCatF). The clone no. is scFv#Pl-020, which contains the following amino acid sequence (from 5' to 3')“ PWRRCSWRSLEEACVQPGDSLRLSCATSGFAFTDYYMNWVRQPPGKALEWLG FIRNKANGYTTEYSASVKGRFTISRDHSQSILYLQMNTLRAEDSATYYCARDIPSMDYW GQGTSVTVSSAKTTPPKLEEGEFSEARVDIVMTQAAPSVPVTPGESVSTSCRSSKSLLHS NGNTYLYWSLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVY YCMQHLEYPYTFGGGTKLEIKRADAAPTVSAAA”2. The production procedure consists of the following steps:A.B-cell epitope prediction: The B-cell-specific epitopes of OvCatF (GenBank accession no. FJ346536.1) are predicted using BepiPred-2.0 (available at https: / / services.healthtech.dtu.dk / services / BepiPred-2.0Z). The obtained B-cell epitopes are located in the deduced amino acid sequence of OvCatF and multiply aligned with other cysteine proteases from the closely related parasites (flukes) as well as humans, including Clonorchis sinensis (GenBank accession no. ABK91811.1), Paragonimus westermani (GenBank accession no. KAA3674842.1), Schistosoma mansoni (GenBank accession no. XP_018649823.1), Fasciola hepatica (GenBank accession no. AAB30089.1), and Homo sapiens (GenBank accession no. AAD4 1790.1). The candidature sequences that showed low conservation to the other parasites are identified and subjected to synthesizing specific peptides conjugated with biotin at the N-terminus. The epitope containing amino acid residues 11 to 30 containing the following sequence “TTIWSVFARTTPFEPDDARA” is selected.B. Immobilization of antigen: The O vC at F- specific scFv antibodies are selected from the murine naive single-chain variable fragments (scFv) library by biopanning. The biotinylated OvCatF synthetic peptide (amino acid residues 11 to 30) is dissolved with PBS pH 7.4 to a final concentration of 2 pg / mL. The synthetic peptide is immobilized in the streptavidin-coated 96- well microtiter plate. 100 pL of the biotinylated capture antibody is added to each well. The plate is incubated for 2 hours at room temperature with gentle shaking. When the incubation time is reached, the wells are washed thrice with 200 pL of wash buffer. One pg of biotinylated synthetic peptide is prepared for 100 pL in the carbonate coating buffer pH 9.6 and added to each well. The plate is then incubated at 37°C overnight until dried to allow complete binding. After incubation,the wells are washed three times with 200 pL of wash buffer and blocked with 3% BSA in PBS, pH 7.4 at 37 °C, for 1 h in a moisture chamber. The excess antigens are removed by washing with a wash buffer. The wells are filled with PBS and kept at 4 °C before use in the biopanning step.C. Biopanning, phage rescuing, and titration: OvCatF-specific scFv is selected by subtractive and positive biopanning with non-infected human fecal extract, biotin, and biotinylated OvCatF synthetic peptide (residues 11 to 30), respectively. Nonspecific binders are removed from the library by subtractive biopanning. The subtraction is performed by incubating 107CFU of the scFv-phage library with non-infected human fecal extract coated in an ELISA well at 37 °C for 1 h. Then, the unbound scFv-phages are transferred and incubated with biotin coated on the well. Consecutively, positive biopanning is carried out by transferring and incubating the unbound scFv- phages with the immobilized target peptide at 25 °C for 1 h on an oscillating agitator. The unbound phages are washed out with PBS-T and discarded; then, the wells are incubated with 500 pL trypsin at 37 °C for 15 min to elute the peptide-bound scFv-phages. The eluted scFv-phages are harvested by magnetic separation twice using streptavidin-coated magnetic beads; then, these two eluted fractions are pooled. Five hundred microliters of the eluted scFv-phages are mixed with an equal volume of log-phase XL-1 Blue Escherichia coli and incubated at 37 °C for 15 min to allow phagemid transduction into bacterial cells. The transformed E. coli harboring recombinant phagemids are selected by growing on the Luria-Bertani (LB) agar plate containing 100 pg / rnL ampicillin antibiotics. The ampicillin-resistant colonies are screened for the scFv insert sequence by direct colony PCR using pelB forward primer (5'-ATACCTATTGCCTAC GGCAGC-3') and gill reverse primer (5'-TAGCATTCCACAG ACAGCCC-3'). The PCR conditions are initial denaturing at 95 °C for 5 mins, 30 cycles of denaturation at 95 °C for 30 seconds, annealing at 58 °C for 45 seconds, and extension at 72 °C for 1 min, followed by final extension at 72 °C for 3 mins. The E. coli clones, which have PCR amplicons with the expected size of approximately or slightly above 900 bps, are counted as scFv positive. The individual scFv-positive E. coli clones are sub-cultured and co-infected with M13KO7 helper phages to rescue the scFv-phage progenies. The titers of the rescued scFv-phages are evaluated and normalized by colony-forming unit count.D. Screening of the binding of scFv-phages with rOvCatF: The rescued scFv- phages are determined for the binding efficiency to the recombinant protein of OvCatF (rOvCatF) using indirect ELISA. One microgram ofrOvCatF is mixed with 100 pl of carbonate coating buffer pH 9.6 and loaded into each well of the 96-well ELISA plate. The plate is then incubated at 37 °C overnight to allow drying. After overnight incubation, the wells are washed three times with PBS- T and non-specific bindings are blocked with 3% BSA in PBS at 37°C for 1 hour. Each clone ofrescued scFv-phage is diluted 10-fold in PBS-T, added into the antigen (rOvCatF) coated well, and incubated at 37°C for 1 hour. After incubation, the wells are washed with PBS-T five times to remove the excess contents. All the wells with scFv-phages are subsequently incubated with mouse anti-M13 antibodies (dilution 1:6,000) at 37°C for 1 hour. The 1:10,000 Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP-conjugated, is added and further incubated at room temperature for 1 hour. The wells are washed thrice with PBS-T, and the TMB-ELISA Substrate Solution is added. The scFv-phages that yielded more than two times over the background are selected for isolation and engineered.E. Isolation of pSEX81 phagemid, subcloning, and verifying of scFv properties: The selected clones of scFv-phages are cultured, and the phagemid, pSEX81, is isolated. The scFv coding sequence of the selected clones from the pSEX81 phagemid is digested with restriction endonucleases (Notl / Ncol) and subcloned into the pOPElOl expression plasmid. The ligation of pOPElOl and scFv fragment is transformed into XL 1 -Blue E. coli using chemical transformation. The positive transformants are selected by direct colony PCR. The PCR-positive clones are subjected to plasmid DNA extraction and verified by DNA sequencing. The pOPElOl containing scFv fragment is predicted for the complementarity determining regions (CDRs) and immunoglobulin framework regions (FRs) by the IMGT / V-QUEST tool (the International ImMunoGeneTics Information System (IMGT®).F. Production of soluble scFv anti-OvCatF antibodies: The clones of XL-1 Blue E. coli containing pOPElOl with correct scFv sequence (both VH and VL fragments) are grown in LB broth by inducing with 1 mM isopropyl 0-d-l -thiogalactopyranoside (IPTG, Sigma- Aldrich, Saint Louis, MO, USA) at 37 °C for 3 hours in the incubator shaker. The cells are harvested by centrifugation at 6,000 xg at 4 °C for 30 min and lysed using denaturing lysis buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, adjusted pH to 8.0). The scFv is purified under denaturing conditions using Ni-NTA sepharose affinity chromatography. The purified scFv is dialyzed against PBS, pH 7.4, using a dropwise dialysis procedure for refolding the produced scFv. The purified soluble scFv is verified by Western blot analysis using mouse monoclonal anti-c-Myc, followed by the Goat anti-Mouse IgG (H+L) Secondary Antibody, AP-conjugated, followed by NBT / BCIP Substrate Solution.G. Molecular modeling and docking: The deduced amino acid sequences of scFv and OvCatF are individually submitted to AlphaFold2 to generate the proteins' 3D model structure. After generation, the scFv and OvCatF are docked by submitting them to the HADDOCK server version 2.4 to verify the specific bindings between these proteins. The possible models are selectedusing the HADDOCK and PRODICGY web server, and the interactions are analyzed using the Discovery Studio Visualizer 2021 version 21.

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