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复旦大学：《免疫学 Immunology》教学论文_Phage Display–Derived Ligand for Mucosal Transcytotic Receptor GP-2 Promotes Antigen Delivery to M Cells and Induces Antigen-Specific Immune Response

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Original Research SLAS Discovery Phage Display-Derived Ligand for Mucosal Transcytotic Receptor GP-2 Promotes Dok:0.77/2472555217690483 journals. sagepub. com/home/jt Antigen Delivery to M Cells and Induces ⑤SAGE Antigen-Specific Immune Response Inam Ullah Khan, Jiansheng Huang, Rui Liu, Jingbo Wang Jun Xie, and Naishuo Zh Abstract Successful oral immunization depends on efficient delivery of antigens(Ags) to the mucosal immune induction site Glycoprotein-2(GP-2) is an integral membrane protein that is expressed specifically on M cells within follicle-associated epithelium(FAE) and serves as transcytotic receptor for luminal Ags. In this study, we selected peptide ligands against recombinant human GP-2 by screening a phage display library and evaluated their interaction with GP-2 in vitro and ex ivo Selected peptides were conjugated to the C-terminal of enhanced green fluorescence protein(EGFP)and evaluated for their ability to induce an immune response in mice. One of our selected peptides, Gb-I, showed high binding affinity o GP-2 and, when fused to EGFP, significantly increased the uptake of EGFP by M cells compared to EGFP alone. After oral administration, the Gbl-EGFP fusion induced efficient mucosal and systemic immune responses in mice measured at the level of antigen-specific serum and fecal antibodies, cytokine secretion, and lymphocyte proliferation. Furthermore, the IgG subclasses and cytokine secretion showed that ligand Gb-I induced a Th2-type immune response. Collectively, our findings suggest that the ligand we selected through phage library screening is capable of targeting Ags to GP-2 on M cells and can be used as an oral vaccine adjuvant. Keywords glycoprotein-2, M cells, transcytosis, adjuvant, Peyers patches Introduction the efficiency of antigen delivery to mucosal lymphoid tis- sue and to avoid possible oral tolerance induction is of great The mucosal surface of the gastrointestinal and respiratory importance for the development of successful mucosal tract is continuously exposed to microorganisms, and a vaccine large number of pathogens invade the body through the The luminal side of the GALT lymphoid follicles is covered mucosal surface. Gut-associated lymphoid tissue(GALT) by follicle-associated epithelium(FAE). M cells are special- serves as sentinel for the recognition of intestinal microbes ized cells in the fae that actively transport luminal antigens immunization is superior to systemic immunization since it and macromolecules across the epithelial membrane, a process referred to as transcytosis. The basal plasma membrane of M elicits immune responses at systemic and mucosal levels. cells forms a pouch-like structure called the"M-cell pocket, Consequently, it seems logical to develop oral vaccines Oral vaccination also offers additional advantages over sys- are challenges to the oral application of vaccines, and only PR Chin g School of Life Sciences, Fudan University, Shanghai 200433 tion, low cost, and needle-free application. However, there Engin a few oral vaccines are currently available. Most of the cur- Received Oct 7, 2016, and in revised form Dec 29, 2016, Accepted for rently available vaccines are administered through the sys- blication Dec 31, 2016 temic route and induce an immune response predominantly the systemic compartment . Difficulty in efficient deliv- Corresponding Authors ery of antigens to the mucosal immune induction site is one Naishuo Zhu and Jun Xie, Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan of the main obstacles in the development of oral mucosal University, Shanghai 200433, PR China vaccines.Consequently,devisingnewstrategiestoenhanceEmails:nzhu@fudan.edu.cnandxiejun@fudan.edu.cn

https://doi.org/10.1177/2472555217690483 SLAS Discovery 1–8 © 2017 Society for Laboratory Automation and Screening DOI: 10.1177/2472555217690483 journals.sagepub.com/home/jbx Original Research Introduction The mucosal surface of the gastrointestinal and respiratory tract is continuously exposed to microorganisms, and a large number of pathogens invade the body through the mucosal surface.1 Gut-associated lymphoid tissue (GALT) serves as sentinel for the recognition of intestinal microbes and initiation of immune responses.2 Theoretically, mucosal immunization is superior to systemic immunization since it elicits immune responses at systemic and mucosal levels.3 Consequently, it seems logical to develop oral vaccines. Oral vaccination also offers additional advantages over systemic administration such as convenience of administration, low cost, and needle-free application.4 However, there are challenges to the oral application of vaccines, and only a few oral vaccines are currently available. Most of the currently available vaccines are administered through the systemic route and induce an immune response predominantly in the systemic compartment.5,6 Difficulty in efficient delivery of antigens to the mucosal immune induction site is one of the main obstacles in the development of oral mucosal vaccines.7 Consequently, devising new strategies to enhance the efficiency of antigen delivery to mucosal lymphoid tissue and to avoid possible oral tolerance induction is of great importance for the development of successful mucosal vaccines. The luminal side of the GALT lymphoid follicles is covered by follicle-associated epithelium (FAE). M cells are specialized cells in the FAE that actively transport luminal antigens and macromolecules across the epithelial membrane, a process referred to as transcytosis.8 The basal plasma membrane of M cells forms a pouch-like structure called the “M-cell pocket,” XXX10.1177/2472555217690483SLAS DiscoveryKhan et al. research-article2017 1 Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, PR China Received Oct 7, 2016, and in revised form Dec 29, 2016, Accepted for publication Dec 31, 2016. Corresponding Authors: Naishuo Zhu and Jun Xie, Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, PR China. Emails: nzhu@fudan.edu.cn and xiejun@fudan.edu.cn Phage Display–Derived Ligand for Mucosal Transcytotic Receptor GP-2 Promotes Antigen Delivery to M Cells and Induces Antigen-Specific Immune Response Inam Ullah Khan1 , Jiansheng Huang1 , Rui Liu1 , Jingbo Wang1 , Jun Xie1 , and Naishuo Zhu1 Abstract Successful oral immunization depends on efficient delivery of antigens (Ags) to the mucosal immune induction site. Glycoprotein-2 (GP-2) is an integral membrane protein that is expressed specifically on M cells within follicle-associated epithelium (FAE) and serves as transcytotic receptor for luminal Ags. In this study, we selected peptide ligands against recombinant human GP-2 by screening a phage display library and evaluated their interaction with GP-2 in vitro and ex vivo. Selected peptides were conjugated to the C-terminal of enhanced green fluorescence protein (EGFP) and evaluated for their ability to induce an immune response in mice. One of our selected peptides, Gb-1, showed high binding affinity to GP-2 and, when fused to EGFP, significantly increased the uptake of EGFP by M cells compared to EGFP alone. After oral administration, the Gb1-EGFP fusion induced efficient mucosal and systemic immune responses in mice measured at the level of antigen-specific serum and fecal antibodies, cytokine secretion, and lymphocyte proliferation. Furthermore, the IgG subclasses and cytokine secretion showed that ligand Gb-1 induced a Th2-type immune response. Collectively, our findings suggest that the ligand we selected through phage library screening is capable of targeting Ags to GP-2 on M cells and can be used as an oral vaccine adjuvant. Keywords glycoprotein-2, M cells, transcytosis, adjuvant, Peyer’s patches

SLAS Discovery where dendritic cells (DCs) and lymphocytes migrate. phages were eluted with 100 AL of 0. 2 M glycine-HCI (pH Although M cells appear to be ideal targets for eliciting 2.2)and l mg/mL BSA. The rescued phages were amplified Ag-specific immune responses through oral vaccination, there in Escherichia coli ER2738, titred on isopropyl B-D-l are challenges to using these cells for Ag delivery thiogalactopyranoside (IPTG)Xgal plates, and a known titer Efficient uptake of antigens by M cells requires specific of the amplified phages was used for next round of screening surface receptor molecules. Identification of M-cell-specific After four rounds of biopanning, DNA was extracted, quanti- markers made it possible to target these cells for antigen(Ag) tated on agarose gel by comparing with 0.5 ug of purified sin- delivery to improve vaccine efficacy. Glycoprotein-2(GP-2)is gle-stranded M13mp18 DNA (NEB 4040), and sequenced a glycosylphosphatidyl inositol anchored protein that is spe- Amino acid sequences were deduced according to the vendor cifically expressed on M cells and serves as transcytotic recep- instructions using Chromas Lite software [AQ: 1] tor for intestinal Ags. Also, GP-2 was shown to be associated The resulting amino acid sequences were scanned usin with specific uptake of Fimht bacteria from the gut. These SarOtup(htTp://immunet.cn/sarotup/index.html)tocheck findings led us to consider GP-2 as a target molecule for effi- whether they match any known target-unrelated peptide(TUP) cient mucosal vaccine delivery. Targeting GP-2 with specific motif or if any submitted peptide has also been selected by ligands should increase Ags delivery to the immune initiation other groups with other targets or to confirm that the phage sites and hence induce enhanced immune responses in sys- clones achieved in the biopanning results are without any temic and mucosal compartments. Therefore, exploiting GP-2 propagation advantage and are true binders to the target for vaccine delivery would be a realistic approach to develop Insight Il program[AQ: 2] was used to align the amino acid mucosal vaccines sequences and find regions of structural conservation among Phage display library is a powerful tool for screening the selected peptides peptide ligands against proteins and other macromolecules both in vitro and in vivo and has been used in basic and applied research for studying molecular biology mecha- Phage-Binding Enzyme-Linked nisms involving protein-protein interactions. In this study, Immunosorbent Assay we used a phage display library to screen short peptide To identify high-affinity binding clones, 96-well enzyme- ands against the transcytosis receptor GP-2. The affinity linked immunosorbent assay(ELISA) plates were coate of the selected ligands to bind to GP-2 and their ability in 150 pL GP-2(5 ug/mL) in 0. 1 M NaHCO, (pH 9.6)at 4C immune induction were assessed in mice. We selected three overmight. Plates were blocked with 5% BSA in 0.1 M peptide ligands, hereafter called GP2-binding peptides NaHCO3(pH 9.6)at 4C for 2 h. Then, 100 HL phage clones ( Gbs),of which one peptide, Gb-1, showed significant (1 x 10 pfu/well) was added and incubated at RT for I h results in immune induction. Gb-l fusion increased the After washing, horseradish peroxidase(HRPh-conjugated uptake of Ag by M cells through GP-2 and elicited signifi- anti-M13 antibody(1: 5000 dilution)was added and incubated cantly high levels of serum IgG and mucosal IgA, as well at RT, followed by TMB substrate incubation. Finally, the reac- cytokines secreting cells in various lymphoid tissues, espe- tion was stopped using 2 M H SO, and plates were read at 450 cially interleukin(ILA4, IL-5, and IL-6, which are associ- nm using a SpectraMax M5( Molecular Devices, Sunnyvale ated with isotype switching to secretory IgA. Our results CA)microplate reader. Phage clones giving target-to-back suggest that peptide Gb-l selected through biopanning has ground absorbance >4 were selected for further assays mmune-stimulating ability and can be used as an adjuvant for mucosal vaccines Peptides Synth aterials and methods The deduced 12-mer peptides were synthesized by standard Fmoc method(China Peptides Co., Shanghai). The Phage Library Biopanning N-terminal of peptides was kept free while the C-terminal The 12-mer peptides phage display library of filamentous was amidated and conjugated to fluorescein isothiocyanate phage M13KE was biopanned according to the guideline pro.(FITC)using the GGGS linker. vided by the vendor(New England BioLabs, Ipswich, MA) lstaetly, 5 ug recombinant GP-2(ProsPec Bio, Ness-Ziona, Measurement of Peptide-GP2 Binding Israel) was adsorbed to a 96-well plate(Corning, Corming, NY)at 4C. After overnight incubation, wells were blocked Affinity(Kd Values) with 5% bovine serum albumin(BSA)in H,O for 2 h at 4C. To measure the binding strength of the selected peptide ligands Approximately l x 10 phages were added to the GP-2-coated to GP-2, saturation binding assays were performed as wells after washing with TBST(.05% Tween 20) and incu- described. 2 Then, 150 HL (5 ug/mL)GP-2 in assay diluent bated for 2 h at room temperature(RT). The loosely/nonspe-(BioLegend, San Diego, CA)was adsorbed to 96-well plates at cifically bound phages were washed and the strongly bound 4C ovemight. The solution was discarded and wells were

2 SLAS Discovery where dendritic cells (DCs) and lymphocytes migrate. Although M cells appear to be ideal targets for eliciting Ag-specific immune responses through oral vaccination, there are challenges to using these cells for Ag delivery.9 Efficient uptake of antigens by M cells requires specific surface receptor molecules. Identification of M-cell–specific markers made it possible to target these cells for antigen (Ag) delivery to improve vaccine efficacy. Glycoprotein-2 (GP-2) is a glycosylphosphatidyl inositol anchored protein that is specifically expressed on M cells and serves as transcytotic receptor for intestinal Ags. Also, GP-2 was shown to be associated with specific uptake of FimH+ bacteria from the gut.10 These findings led us to consider GP-2 as a target molecule for efficient mucosal vaccine delivery. Targeting GP-2 with specific ligands should increase Ags delivery to the immune initiation sites and hence induce enhanced immune responses in systemic and mucosal compartments. Therefore, exploiting GP-2 for vaccine delivery would be a realistic approach to develop mucosal vaccines. Phage display library is a powerful tool for screening peptide ligands against proteins and other macromolecules both in vitro and in vivo and has been used in basic and applied research for studying molecular biology mechanisms involving protein-protein interactions.11 In this study, we used a phage display library to screen short peptide ligands against the transcytosis receptor GP-2. The affinity of the selected ligands to bind to GP-2 and their ability in immune induction were assessed in mice. We selected three peptide ligands, hereafter called GP2-binding peptides (Gbs), of which one peptide, Gb-1, showed significant results in immune induction. Gb-1 fusion increased the uptake of Ag by M cells through GP-2 and elicited significantly high levels of serum IgG and mucosal IgA, as well as cytokines secreting cells in various lymphoid tissues, especially interleukin (IL)–4, IL-5, and IL-6, which are associated with isotype switching to secretory IgA. Our results suggest that peptide Gb-1 selected through biopanning has immune-stimulating ability and can be used as an adjuvant for mucosal vaccines. Materials and Methods Phage Library Biopanning The 12-mer peptides phage display library of filamentous phage M13KE was biopanned according to the guideline provided by the vendor (New England BioLabs, Ipswich, MA). Briefly, 5 µg recombinant GP-2 (ProsPec Bio, Ness-Ziona, Israel) was adsorbed to a 96-well plate (Corning, Corning, NY) at 4 °C. After overnight incubation, wells were blocked with 5% bovine serum albumin (BSA) in H2 O for 2 h at 4 °C. Approximately 1 × 1011 phages were added to the GP-2–coated wells after washing with TBST (.05% Tween 20) and incubated for 2 h at room temperature (RT). The loosely/nonspecifically bound phages were washed and the strongly bound phages were eluted with 100 µL of 0.2 M glycine-HCl (pH 2.2) and 1 mg/mL BSA. The rescued phages were amplified in Escherichia coli ER2738, titred on isopropyl β-D-1- thiogalactopyranoside (IPTG)/Xgal plates, and a known titer of the amplified phages was used for next round of screening. After four rounds of biopanning, DNA was extracted, quantitated on agarose gel by comparing with 0.5 µg of purified single-stranded M13mp18 DNA (NEB 4040), and sequenced. Amino acid sequences were deduced according to the vendor instructions using Chromas Lite software.[AQ: 1] The resulting amino acid sequences were scanned using SAROTUP (http://immunet.cn/sarotup/index.html) to check whether they match any known target-unrelated peptide (TUP) motif or if any submitted peptide has also been selected by other groups with other targets or to confirm that the phage clones achieved in the biopanning results are without any propagation advantage and are true binders to the target. Insight II program[AQ: 2] was used to align the amino acid sequences and find regions of structural conservation among the selected peptides. Phage-Binding Enzyme-Linked Immunosorbent Assay To identify high-affinity binding clones, 96-well enzymelinked immunosorbent assay (ELISA) plates were coated with 150 µL GP-2 (5 µg/mL) in 0.1 M NaHCO3 (pH 9.6) at 4 °C overnight. Plates were blocked with 5% BSA in 0.1 M NaHCO3 (pH 9.6) at 4 °C for 2 h. Then, 100 µL phage clones (1 × 1010 pfu/well) was added and incubated at RT for 1 h. After washing, horseradish peroxidase (HRP)–conjugated anti-M13 antibody (1:5000 dilution) was added and incubated at RT, followed by TMB substrate incubation. Finally, the reaction was stopped using 2 M H2 SO4 , and plates were read at 450 nm using a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) microplate reader. Phage clones giving target-to-background absorbance >4 were selected for further assays. Peptides Synthesis The deduced 12-mer peptides were synthesized by standard Fmoc method (China Peptides Co., Shanghai). The N-terminal of peptides was kept free while the C-terminal was amidated and conjugated to fluorescein isothiocyanate (FITC) using the GGGS linker. Measurement of Peptide-GP2 Binding Affinity (Kd Values) To measure the binding strength of the selected peptide ligands to GP-2, saturation binding assays were performed as described.12 Then, 150 µL (5 µg/mL) GP-2 in assay diluent (BioLegend, San Diego, CA) was adsorbed to 96-well plates at 4 °C overnight. The solution was discarded and wells were

Khan et al Table I. Primers Used for Fusion of Peptides to Enhanced Green Fluorescent Protein and Plasmid Circularization. Gb- GbI-F:5-GATCGCATGCATACCCAGTAACAAAGCCCGAAAGGAAGCTG-3 GbI-R: 5-ATGATCATAGTTCGGATGGCTGCCGCCGCCCTTGTACA-3 Gb.5-AGCCATCCGAACTATGATCATGATCGCATGCATACCCAG-3 Gb2-F: 5-GATATGCCGGGCGCGCATTAACAAAGCCCGAAAGGAAGCTG-3 Gb2-R: 5-ATGCGCGCCCGGCATATCGCTGCCGCCGCCCTTGTACA-3 Gb2-cir: 5-AGCTTTCGCGTGATTTATACCGATATGCCGGGCGCGCAT-3 Gb-3 Gb3-F: 5-CATCCGGTGAACACCGGCTAACAAAGCCCGAAAGGAAGCTG-3 Gb3-R: 5-TCCGCATACTGCATGCTGCTGCCGCCGCCCTTGTACA-3 Gb3-cir: 5-AGCAGCATGCAGTATGCGGATCATCCGGTGAACACCGGC-3 pRimers used for circularization of linear constructs blocked with 5% BSA in water for 2 h at 4C. After from overnight fasted mice and 0. 2 mg of recombinant wells were subjected to incubation with different co Ag-fused EGFPs or EGFP alone was injected into the ligated tions of FITC-conjugated peptides for I h, followed by segment. After l-h incubation at 37C, the ligated segment tion with HRP-conjugated anti-FITC antibody was excised, washed with phosphate-buffered saline(PBs)to dilution). The reaction was visualized with 100 HL HRP sub- remove the internal contents, and fixed with 4% PFA. [AQ: 4] strate and ODs was measured. Specific binding of each pep- For whole-mount staining, PPs were stained with anti-GP2 ide to GP-2 was calculated by subtracting the OD value of (Abcam, xXX, UK) followed by allophycocyanin-conjugated each peptide binding to BSA-coated wells from the OD value anti-rabbit IgG, whole mounted with antifade medium. Frozen that peptide binding to GP-2. Assays were performed three sections(10 um) were prepared after freezing PPs in OC times in duplicate. Binding affinity of each peptide was calcu-(Takara, XXX, Japan), blocked with 2.5% BSA and stained lated by nonlinear regression and transformed to Scatchard with anti-GP2, counterstained with 4, 6-diamidino-2-phenyl plot using the GraphPad Prism 6 program( GraphPad Software, indole ( DAPD), and analyzed by confocal laser scanning La Jolla, CA). microscopy(CLSM)(LSM 710: Carl Zeiss, Thornwood Next, we performed competition ELISA to investiga NY).[AQ: 5] the epitope binding sites on GP-2 using the above method. Production of Peptide-Conjugated Mice Immunization and Measurement of Ag- Specific Immune Responses We used enhanced green fluorescence protein(EGFP) as a Groups of five female BALB/c mice between 4 and 6 weeks model antigen and fused selected peptides to the C-terminal of of age were immunized with 100 ug of experimental anti EGFP. The EGFP gene was amplified by PCR from pEGFP- gen/PBS by oral gavage once every week for 6 weeks CI(Clontech, XXX) using primers F: 5'-GTGAGCAAGG Five days after the last immunization, serum and fecal GCGAGGAGCTG-3 and R: 5'-CTTGTACAGCTCGTCCA extracts were collected to monitor EGFP-specific systemic TGCCG-3. Fusion of DNA sequences of the selected peptides IgG and mucosal Ig A as described by Hackett et al. Since and insertion of the resulting construct into pET-28a was IgA in fecal extracts is rapidly degraded by the proteases achieved by our newly introduced single primer-mediated cir- from enteric bacteria, I mM phenylmethyl sulfonyl fluoride cular PCR using primers as shown in Table 1 Protein(PMFS) protease inhibitor was included in the extraction was expressed in BL21 (DE3)and purified on HisTrap FF col- cocktail. Ab titers were expressed as the reciprocal log2 of umns(GE Healthcare Bio-Sciences, xXX) according to the the highest sample dilutions that gave an ODsn of 0.08 manufacturer's instructions Purified protein was confirmed by which was the value of the PBS blank. sodium dodecyl sulfate polyacrylamide gel electrophoresis Lymphocytes from spleen(SPLs)and PPs(PPLs)were iso (SDS-PAGE)and Western blot using anti-His Tag and anti- lated, minced, and digested with 300 U/mL Collagenase D EGFP antibodies. [AQ: 3] (Roche, Mannheim, Germany) for 30 min at 37C. The digested mixture was passed through a nylon mesh to remove Ex Vivo Ag Uptake Assay undigested tissue and subjected to Percoll(Sigma-Aldrich, Louis, MO)density gradient centrifugation. Cells at the inter- Ex vivo Ag uptake of the selected ligands by M cells was face between 40% and 75% Percoll were collected as mono- assessed by gut loop assay as described previously. A gut nuclear cells and subjected to characterization for lymphocyte loop containing one or two Peyers patches(PPs)was prepared proliferation and cytokine secretion

Khan et al. 3 blocked with 5% BSA in water for 2 h at 4 °C. After washing, wells were subjected to incubation with different concentrations of FITC-conjugated peptides for 1 h, followed by incubation with HRP-conjugated anti-FITC antibody (1:5000 dilution). The reaction was visualized with 100 µL HRP substrate and OD450 was measured. Specific binding of each peptide to GP-2 was calculated by subtracting the OD value of each peptide binding to BSA-coated wells from the OD value of that peptide binding to GP-2. Assays were performed three times in duplicate. Binding affinity of each peptide was calculated by nonlinear regression and transformed to Scatchard plot using the GraphPad Prism 6 program (GraphPad Software, La Jolla, CA). Next, we performed competition ELISA to investigate the epitope binding sites on GP-2 using the above method. Production of Peptide-Conjugated Recombinant Ag We used enhanced green fluorescence protein (EGFP) as a model antigen and fused selected peptides to the C-terminal of EGFP. The EGFP gene was amplified by PCR from pEGFPC1 (Clontech, XXX) using primers F: 5′-GTGAGCAAGG GCGAGGAGCTG-3′ and R: 5′-CTTGTACAGCTCGTCCA TGCCG-3′. Fusion of DNA sequences of the selected peptides and insertion of the resulting construct into pET-28a was achieved by our newly introduced single primer–mediated circular PCR method13 using primers as shown in Table 1. Protein was expressed in BL21 (DE3) and purified on HisTrap FF columns (GE Healthcare Bio-Sciences, XXX) according to the manufacturer’s instructions. Purified protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using anti-HisTag and antiEGFP antibodies.[AQ: 3] Ex Vivo Ag Uptake Assay Ex vivo Ag uptake of the selected ligands by M cells was assessed by gut loop assay as described previously.14 A gut loop containing one or two Peyer’s patches (PPs) was prepared from overnight fasted mice and 0.2 mg of recombinant Ag-fused EGFPs or EGFP alone was injected into the ligated segment. After 1-h incubation at 37 °C, the ligated segment was excised, washed with phosphate-buffered saline (PBS) to remove the internal contents, and fixed with 4% PFA.[AQ: 4] For whole-mount staining, PPs were stained with anti-GP2 (Abcam, XXX, UK) followed by allophycocyanin-conjugated anti-rabbit IgG, whole mounted with antifade medium. Frozen sections (10 µm) were prepared after freezing PPs in OCT (Takara, XXX, Japan), blocked with 2.5% BSA and stained with anti-GP2, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and analyzed by confocal laser scanning microscopy (CLSM) (LSM 710; Carl Zeiss, Thornwood, NY).[AQ: 5] Mice Immunization and Measurement of AgSpecific Immune Responses Groups of five female BALB/c mice between 4 and 6 weeks of age were immunized with 100 µg of experimental antigen/PBS by oral gavage once every week for 6 weeks.15 Five days after the last immunization, serum and fecal extracts were collected to monitor EGFP-specific systemic IgG and mucosal IgA as described by Hackett et al.16 Since IgA in fecal extracts is rapidly degraded by the proteases from enteric bacteria, 1 mM phenylmethyl sulfonyl fluoride (PMFS) protease inhibitor was included in the extraction cocktail.17 Ab titers were expressed as the reciprocal log2 of the highest sample dilutions that gave an OD450 of 0.08, which was the value of the PBS blank. Lymphocytes from spleen (SPLs) and PPs (PPLs) were isolated, minced, and digested with 300 U/mL Collagenase D (Roche, Mannheim, Germany) for 30 min at 37 °C. The digested mixture was passed through a nylon mesh to remove undigested tissue and subjected to Percoll (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation. Cells at the interface between 40% and 75% Percoll were collected as mononuclear cells and subjected to characterization for lymphocyte proliferation and cytokine secretion. Table 1. Primers Used for Fusion of Peptides to Enhanced Green Fluorescent Protein and Plasmid Circularization. Peptide Primer Gb-1 Gb1-F: 5-GATCGCATGCATACCCAGTAACAAAGCCCGAAAGGAAGCTG-3 Gb1-R: 5-ATGATCATAGTTCGGATGGCTGCCGCCGCCCTTGTACA-3 Gb1-cira : 5-AGCCATCCGAACTATGATCATGATCGCATGCATACCCAG-3 Gb-2 Gb2-F: 5-GATATGCCGGGCGCGCATTAACAAAGCCCGAAAGGAAGCTG-3 Gb2-R: 5-ATGCGCGCCCGGCATATCGCTGCCGCCGCCCTTGTACA-3 Gb2-cir: 5-AGCTTTCGCGTGATTTATACCGATATGCCGGGCGCGCAT-3 Gb-3 Gb3-F: 5-CATCCGGTGAACACCGGCTAACAAAGCCCGAAAGGAAGCTG-3 Gb3-R: 5-TCCGCATACTGCATGCTGCTGCCGCCGCCCTTGTACA-3 Gb3-cir: 5-AGCAGCATGCAGTATGCGGATCATCCGGTGAACACCGGC-3 a Primers used for circularization of linear constructs

SLAS Discovery The level of cytokine secretion was determined by cyto- Table 2. Summary of Phage Library Biopanning against kine-specific ELISAs with culture supernatants from lym- Glycoprotein-2 phocytes restimulated with the Ags for 24 h as described Input Phages previously. Cytokine concentrations were calculated from Round Phages(pfu) Enrichment" the plotted standard curves of serial dilutions of the recom- binant cytokine and expressed as mean t standard error First round ×10 2.3x10 2.3×10 (SE)(ng/mL)of each group. To measure the number of Second round 5x10% antigen-specific cytokine(interferon [INFIY, interleukin Third roun 2.0x [IL1, IL-5 and IL-6)secreting cells, enzyme-linked Fourth round 4x10 3.7×105 9.3×10 lospot(ELISPOT) assays were performed with lym- Enrichment is expressed as the relative percentage of the total number phocytes isolated from spleens and PPs as described of phages recovered compared to the input phages Table 3. Binding Affinity(K, values) of Selected Peptides Ag-Specific Lymphocyte Proliferation Ag-specific lymphocyte stimulation was determined using Sequence K, (HM) Dojindo CCK-8 reagent(Dojindo Laboratories, Kumamoto, HPNYDHDRMHTQGb-I16/50(32)0.068 Japan). Purified lymphocytes were plated in flat-bottom FRVIYTDMPGAH Gb-2 9/50(18) 0250 96-well plates at a density of 5 x 10- cells per well and SMQYADHPVNTG Gb-3 13/50(26) simulated with recombinant EGFP(1 mg/mL) or PBS (negative control) at 37C in a 5%Co, incubator. Plates were incubated for 72 h and pulsed with 10 uL CCK-8 obtained seven different amino acid sequences and named reagent(Dojindo Laboratories) per well for another 4 h. them GP-2 binding peptides(Gb-l to Gb-7), of which three Absorbance was measured at 450 nm, and the stimulation sequences appeared more frequently than the others and index(SI)was calculated as the ratio of the average ODsn were selected for further characterization. SAROTUP scan- value of wells containing antigen-stimulated cells to the ning results showed that none of these sequences have been average OD4so value of wells containing cells stimulated reported before for other targets or contain any known TUP with PBs. All assays were performed in triplicate motif or confer propagation advantages. Alignment analysis using Insight II showed that no conserved structural fea Statistical Analysis tures exist among the selected peptides The results are expressed as mean+ SE(SEM) using Microsoft Excel 2016(Microsoft Corp, Redmond, WA), Selected Peptides Bind to GP-2 with High Affinity and at least three independent experiments were performed The ability of the selected peptides ligands to bind to GP-2 unless otherwise stated. An unpaired Student t test(two- was measured in terms of dissociation constant(K)values nificant nonlinear regression analysis and Scatchard transformation using GraphPad Prism 6. The binding of peptides to GP-2 Results was specific and hyperbolic. All three peptides showed K values in the nanomolar range: Gb-1(K-68 nM), Gb-2 Biopanning of Phage Library (K,=250 nM), and Gb-3(K,=272 nM)as shown in Table To select peptide ligands against mucosal transcytotic 3. The binding pattern of these peptides to GP-2 measured receptor GP-2, we screened a 12-mer peptide phage display by saturation binding assay was consistent with the binding library against recombinant GP-2 as described in Materials pattern measured by phage-binding ELISA. Competition Ind Methods. Four rounds of biopanning were performed, ELISA results showed that the selected peptides do not bind and comparatively stringent conditions were applied in to any common or overlapping epitope on GP-2 each round to ensure enrichment in favor of GP-2. Phage titer increased more than 4000-fold from the first round to Binding of Ligand-Fused EGFPs to GP-2 and the fourth round (Table 2). The overall enrichment of the Transcytosis to M Cells library suggests that enrichment for selectively binding phage was achieved. After four rounds of biopanning, 3.7x To test the GP-2 targeting ability of selected peptides, we 10 phages were recovered and 50 plaques were randomly expressed peptide-fused EGFPs and analyzed their binding picked for sequence determination. From 50 plaques, we to M cells on PPs. We performed ligated gut loop assay and

4 SLAS Discovery The level of cytokine secretion was determined by cytokine-specific ELISAs with culture supernatants from lymphocytes restimulated with the Ags for 24 h as described previously. Cytokine concentrations were calculated from the plotted standard curves of serial dilutions of the recombinant cytokine and expressed as mean ± standard error (SE) (ng/mL) of each group. To measure the number of antigen-specific cytokine (interferon [INF]–γ, interleukin [IL]–4, IL-5 and IL-6) secreting cells, enzyme-linked immunospot (ELISPOT) assays were performed with lymphocytes isolated from spleens and PPs as described previously.15 Ag-Specific Lymphocyte Proliferation Ag-specific lymphocyte stimulation was determined using Dojindo CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan).18 Purified lymphocytes were plated in flat-bottom 96-well plates at a density of 5 × 105 cells per well and stimulated with recombinant EGFP (1 mg/mL) or PBS (negative control) at 37 °C in a 5% CO2 incubator. Plates were incubated for 72 h and pulsed with 10 µL CCK-8 reagent (Dojindo Laboratories) per well for another 4 h. Absorbance was measured at 450 nm, and the stimulation index (SI) was calculated as the ratio of the average OD450 value of wells containing antigen-stimulated cells to the average OD450 value of wells containing cells stimulated with PBS. All assays were performed in triplicate. Statistical Analysis The results are expressed as mean ± SE (SEM) using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA), and at least three independent experiments were performed unless otherwise stated. An unpaired Student t test (twotailed) was used to compare groups, and p values <0.05 were considered statistically significant. Results Biopanning of Phage Library To select peptide ligands against mucosal transcytotic receptor GP-2, we screened a 12-mer peptide phage display library against recombinant GP-2 as described in Materials and Methods. Four rounds of biopanning were performed, and comparatively stringent conditions were applied in each round to ensure enrichment in favor of GP-2. Phage titer increased more than 4000-fold from the first round to the fourth round (Table 2). The overall enrichment of the library suggests that enrichment for selectively binding phage was achieved. After four rounds of biopanning, 3.7 × 105 phages were recovered and 50 plaques were randomly picked for sequence determination. From 50 plaques, we obtained seven different amino acid sequences and named them GP-2 binding peptides (Gb-1 to Gb-7), of which three sequences appeared more frequently than the others and were selected for further characterization. SAROTUP scanning results showed that none of these sequences have been reported before for other targets or contain any known TUP motif or confer propagation advantages. Alignment analysis using Insight II showed that no conserved structural features exist among the selected peptides. Selected Peptides Bind to GP-2 with High Affinity The ability of the selected peptides ligands to bind to GP-2 was measured in terms of dissociation constant (Kd ) values of the synthetic peptides. Kd values were determined by nonlinear regression analysis and Scatchard transformation using GraphPad Prism 6. The binding of peptides to GP-2 was specific and hyperbolic. All three peptides showed Kd values in the nanomolar range: Gb-1 (Kd = 68 nM), Gb-2 (Kd = 250 nM), and Gb-3 (Kd = 272 nM) as shown in Table 3. The binding pattern of these peptides to GP-2 measured by saturation binding assay was consistent with the binding pattern measured by phage-binding ELISA. Competition ELISA results showed that the selected peptides do not bind to any common or overlapping epitope on GP-2. Binding of Ligand-Fused EGFPs to GP-2 and Transcytosis to M Cells To test the GP-2 targeting ability of selected peptides, we expressed peptide-fused EGFPs and analyzed their binding to M cells on PPs. We performed ligated gut loop assay and Table 2. Summary of Phage Library Biopanning against Glycoprotein-2. Round Input Phages (pfu) Recovered Phages (pfu) Enrichmenta First round 1 × 1011 2.3 × 105 2.3 × 10–6 Second round 5 × 109 5.3 × 105 1.1 × 10–4 Third round 1 × 107 2.0 × 104 2.0 × 10–3 Fourth round 4 × 107 3.7 × 105 9.3 × 10–3 a Enrichment is expressed as the relative percentage of the total number of phages recovered compared to the input phages. Table 3. Binding Affinity (Kd Values) of Selected Peptides. Sequence Name Frequency, No. (%) Kd (µM) HPNYDHDRMHTQ Gb-1 16/50 (32) 0.068 FRVIYTDMPGAH Gb-2 9/50 (18) 0.250 SMQYADHPVNTG Gb-3 13/50 (26) 0.272

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复旦大学：《免疫学 Immunology》教学论文_Phage Display–Derived Ligand for Mucosal Transcytotic Receptor GP-2 Promotes Antigen Delivery to M Cells and Induces Antigen-Specific Immune Response