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

Khan et al A Figure I. Immunohistochemical analysis of Interaction of selected peptides with GP-2 nd internalization into PPs through M cells Anti-GP-2+EGFP-GbI Anti GP-2 EGFP-GbI was evaluated by immunohistochemical after administration of enhanced green fluorescent protein(EGFP) into ligated ops as described in the Materia ethods. (A)Whole-mounted PPs Anti-GP-2 +EGFP incubated with Gbl-EGFP and stained with DAPI op ligation and staining with GP-2 and FAE 4, 6-diamidino-2-Phenylindole(DAPI)as indicated Lower right panel shows merged image of anti-GP-2(red), EGFP-Gbl (green), and DAPl(blue).(C)Internalization EGFP-Gb1 EGFP- Gb2 f EGFPs assisted by selected ligands or without ligands as indicated, 60 min after gut loop assay: ()EGFP-Gbl,(EGFP-Gb2 (i)EGFP-Gb3, and (iM)EGFP alone. In each FAE case, the left and right panels show results rom light and fluorescence microscopy. ely, taken at lox magnification EGFP- Gb3 FAE, follicle-associated epithelium. Scale EGFPbars: 50 um stained whole PPs and frozen sections with anti-GP-2 after with the ligand-fused EGFPs increased the induction of incubation with either ligand-conjugated EGFPs or EGFP EGFP-specific serum IgG compared to EGFP alone. In par- alone. Immunohistochemical staining of the whole-mount ticular, Gb-1-conjugated EGFP enhanced the induction of and frozen sections of PPs confirmed the binding of only EGFP-specific IgG more efficiently and increased the level Gb-1-conjugated EGFP to M cells that specifically express of EGFP-specific serum gG by >2-fold compared to other iP-2(Fig. 1A, B). However, Gb-2 and Gb-3 conjugated to ligand-fused EGFPs(Fig 2A). The enhancement in immune EGFP were not preferentially bound to M cells. To confirm response was further analyzed at the IgG subclass level.As the ligand-assisted internalization of EGFPs into PPs, we shown in the Figure 2C, D, a-2-fold increase in the induc analyzed and compared the fluorescence intensity of EGFP tion of EGFP-specific IgGl was detected for Gb-l, but no fluorescence under the dome area of PPs in frozen sections significant increase in the level of Ag-specific IgG2a could I h after oral feeding. We found that ligand-fused EGFPs be detected even with the Gb-1-fused EGFP. were internalized into the PPs successfully; in particular, At the mucosal level. mice immunized with Gbl-fused peptide Gb-1 most efficiently transferred its fluorescence EGFP induced EGFP-specific fecal IgA level >1. 5-fold into PPs(Fig. IC). However, unconjugated EGFP did not stronger than those immunized with other ligand-fused show such internalization. These results show that Gb-1 EGFPs or with eGFP alone( Fig 2B).IL-4, IL-5, and IL-6 binds to GP-2 on M cells and can promote Ag transcytosis known to be associated with isotype switching to secretory IgA, oral immunization with Gb-1[AQ: 6] not only enhanced the induction of Gb-I-specific fecal IgA but also Gb-1 Enhances Ag-Specific Immune Response increased the number of Gb-1-specific IgA-secreting cells Induction in PPs compared with EGFP alone(Fig. 3B). In addition, oral administration of gbl-fused egfp also enhanced the To test the ability of the selected peptides to induce immune priming of EGFP-specific lymphocytes(Fig 3C response, we administered ligand-conjugated or only EGFF Interferon-Y(IFN-Y) is the representative cytokine for to mice orally and evaluated antibody response in systemic Thl-type response, and IL-4 and IL-6 represent the Th2 and mucosal compartments using ELISA. Immunization type response. We analyzed the pattern of cytokine

Khan et al. 5 stained whole PPs and frozen sections with anti–GP-2 after incubation with either ligand-conjugated EGFPs or EGFP alone. Immunohistochemical staining of the whole-mount and frozen sections of PPs confirmed the binding of only Gb-1–conjugated EGFP to M cells that specifically express GP-2 (Fig. 1A,B). However, Gb-2 and Gb-3 conjugated to EGFP were not preferentially bound to M cells. To confirm the ligand-assisted internalization of EGFPs into PPs, we analyzed and compared the fluorescence intensity of EGFP fluorescence under the dome area of PPs in frozen sections 1 h after oral feeding. We found that ligand-fused EGFPs were internalized into the PPs successfully; in particular, peptide Gb-1 most efficiently transferred its fluorescence into PPs (Fig. 1C). However, unconjugated EGFP did not show such internalization. These results show that Gb-1 binds to GP-2 on M cells and can promote Ag transcytosis to PPs. Gb-1 Enhances Ag-Specific Immune Response Induction To test the ability of the selected peptides to induce immune response, we administered ligand-conjugated or only EGFP to mice orally and evaluated antibody response in systemic and mucosal compartments using ELISA. Immunization with the ligand-fused EGFPs increased the induction of EGFP-specific serum IgG compared to EGFP alone. In particular, Gb-1–conjugated EGFP enhanced the induction of EGFP-specific IgG more efficiently and increased the level of EGFP-specific serum IgG by >2-fold compared to other ligand-fused EGFPs (Fig. 2A). The enhancement in immune response was further analyzed at the IgG subclass level. As shown in the Figure 2C,D, a ~2-fold increase in the induction of EGFP-specific IgG1 was detected for Gb-1, but no significant increase in the level of Ag-specific IgG2a could be detected even with the Gb-1–fused EGFP. At the mucosal level, mice immunized with Gb1-fused EGFP induced EGFP-specific fecal IgA level >1.5-fold stronger than those immunized with other ligand-fused EGFPs or with EGFP alone (Fig. 2B). IL-4, IL-5, and IL-6 known to be associated with isotype switching to secretory IgA, oral immunization with Gb-1[AQ: 6] not only enhanced the induction of Gb-1–specific fecal IgA but also increased the number of Gb-1–specific IgA-secreting cells in PPs compared with EGFP alone (Fig. 3B). In addition, oral administration of Gb1-fused EGFP also enhanced the priming of EGFP-specific lymphocytes (Fig. 3C). Interferon-γ (IFN-γ) is the representative cytokine for Th1-type response, and IL-4 and IL-6 represent the Th2- type response.19,20 We analyzed the pattern of cytokine Figure 1. Immunohistochemical analysis of peptide–glycoprotein-2 (GP-2) interaction and internalization to Peyer’s patches (PPs). Interaction of selected peptides with GP-2 and internalization into PPs through M cells was evaluated by immunohistochemical staining of whole-mount specimens and cryosections of mouse PPs 20 min after administration of enhanced green fluorescent protein (EGFP) into ligated loops as described in the Materials and Methods. (A) Whole-mounted PPs incubated with Gb1-EGFP and stained with anti-GP2 antibody. (B) Frozen sections of mouse PPs prepared 20 min after gut loop ligation and staining with GP-2 and 4′,6-diamidino-2-phenylindole (DAPI) as indicated. Lower right panel shows merged image of anti–GP-2 (red), EGFP-Gb1 (green), and DAPI (blue). (C) Internalization of EGFPs assisted by selected ligands or without ligands as indicated, 60 min after gut loop assay: (i) EGFP-Gb1, (ii) EGFP-Gb2, (iii) EGFP-Gb3, and (iv) EGFP alone. In each case, the left and right panels show results from light and fluorescence microscopy, respectively, taken at 10× magnification. FAE, follicle-associated epithelium. Scale bars: 50 µm

SLAS Discovery Serum IgG fecalIgA 客宗 1 EGFP Gb-1 Gb-2 Gb-3 EGFP Gb-1 Gb-2 Gb-3 Figure 2. Enhanced green fluorescent protein(EGFP)-specific Serum IgG1 Serum IgG2a mucosal and systemic immune responses in orally immunized mice. Level of EGFP-specific immune esponse in mice orally immunized evaluated 5 days after the last munosorbent assay(ELISA).(A) and(D)IgG2a. Results are expresse as the reciprocal of the geometric n log2 titer and are the mean t EGFP Gb-1 Gb-2 Gb-3 EGFP Gb-1 Gb-2 Gb-3 and pp <0.00I indicate significant differences between the value compared secretion in splenocytes restimulated with EGFPs. IL-4 membrane of intestinal epithelial cells is a major limitation level was significantly raised in the Gb-l-immunized group to be overcome for the successful development of an oral compared to the eGFP group(Fig 3A). Since IgGl produc- vaccine. In the case of subunit vaccines, denaturation tion and IL-4 secretion are indicators of Th2-type response, caused by the acidic environment of the stomach is also a this implies that oral immunization with Gb-1 ligand barrier to oral vaccines. Encapsulation of vaccines in induces predominantly Th2-type immunity against the sub- nano- and microparticulate forms such as polylactide Ject antigen (PLA), polylactide-coglycolide(PLga), or liposomes, and the use of enteric-coated capsules may offer some protec Discussion tion against acidic and enzymatic degradatic ever, these strategies have affected the delivery of vaccines Oral vaccination induces not only antibody response in the across intestinal epithelial cells ystemic compartment, but also the serum IgG response is One approach to enhance drug and particulate delivery sys- strengthened by IgA production at the mucosal surface. tems across the intestinal epithelial barrier is to target Ags to This makes oral vaccination superior compared to systemic specific transcytotic ic receptor of the intestine. Cells are spe vaccination where only the IgG response is elicited. Low cialized cells that internalize luminal Ags across the epithelial cost and needle-free delivery are other attractive features of layer to the underlying immune induction sites without degra- mucosal/oral vaccination, so it is a feasible and economic dation and play an important role in initiating Ag-specific vaccination strategy, especially in developing countries. mucosal immune responses by inducing the production However, low permeability of vaccines across the plasma of secretory IgA. Therefore, the M-cell-mediated Ag

6 SLAS Discovery secretion in splenocytes restimulated with EGFPs. IL-4 level was significantly raised in the Gb-1–immunized group compared to the EGFP group (Fig. 3A). Since IgG1 production and IL-4 secretion are indicators of Th2-type response, this implies that oral immunization with Gb-1 ligand induces predominantly Th2-type immunity against the subject antigen. Discussion Oral vaccination induces not only antibody response in the systemic compartment, but also the serum IgG response is strengthened by IgA production at the mucosal surface.19,21 This makes oral vaccination superior compared to systemic vaccination where only the IgG response is elicited.22 Low cost and needle-free delivery are other attractive features of mucosal/oral vaccination,4 so it is a feasible and economic vaccination strategy, especially in developing countries. However, low permeability of vaccines across the plasma membrane of intestinal epithelial cells is a major limitation to be overcome for the successful development of an oral vaccine. In the case of subunit vaccines, denaturation caused by the acidic environment of the stomach is also a barrier to oral vaccines.23 Encapsulation of vaccines in nano- and microparticulate forms such as polylactide (PLA), polylactide-coglycolide (PLGA), or liposomes, and the use of enteric-coated capsules may offer some protection against acidic and enzymatic degradation24–29; however, these strategies have affected the delivery of vaccines across intestinal epithelial cells. One approach to enhance drug and particulate delivery systems across the intestinal epithelial barrier is to target Ags to specific transcytotic receptor of the intestine.23 M cells are specialized cells that internalize luminal Ags across the epithelial layer to the underlying immune induction sites without degradation and play an important role in initiating Ag-specific mucosal immune responses by inducing the production of secretory IgA.2,30 Therefore, the M-cell–mediated Ag Figure 2. Enhanced green fluorescent protein (EGFP)–specific mucosal and systemic immune responses in orally immunized mice. Level of EGFP-specific immune response in mice orally immunized with the indicated antigens was evaluated 5 days after the last immunization by enzyme-linked immunosorbent assay (ELISA). (A) Serum IgG, (B) fecal IgA, (C) IgG1, and (D) IgG2a. Results are expressed as the reciprocal of the geometric mean log2 titer and are the mean ± SE of five mice per group. **p < 0.01 and ***p < 0.001 indicate significant differences between the values compared

Khan et al A IFN丫 IL-4 IL-5 IL- 60 40 30 20 EGFP Gb-1 EGFP Gb-1 EGFP Gb-1 EGFP Gb-1 PBS EGFP Gb-1 Gb-2 Gb-3 Figure 3. Levels of cytokines and cytokine-secreting cells and splenocyte proliferation in Gb-I-immunized mice. Levels of cytokines and cytokine-secreting cells were determined using enzyme- linked immunosorbent assay(ELISA)and enzyme- linked immunospot (ELISPOT), respectively, as described in the Materials and Methods, after in vitro stimulation of cells from spleen and Peyers patches (PPs).(A) Cytokine level from splenocytes. (B)Number of cytokine-secreting cells in PPs. Results are expressed as ng/mL and cell Imber per cells stimulated, respectively, and are the mean t se of five mice per group. ( C) Enhanced green fluorescent protein (EGFP)-specific splenocyte proliferation was determined CCK-8 reagent [AQ: 8] and results are expressed as stimulation indices. " p 0.05 indicates significant differences compared with the EGFP-immunized group IL, interleukin; IFN-y, interferon-G PBS, phosphate- buffered saline sampling is believed to be an essential and critical step for extend its half-life. The conformation and/or dimerization eliciting a successful mucosal immune response. of the peptide when presented as a conjugate to a protein may In this regard, identifying mucosal vaccine adjuvants that differ from the conformation or presentation of a free peptide exploit M cells would be an effective strategy to develop suc- in solution. Thus, conformation constraint or polymerization cessful mucosal vaccines. Research has focused on the char- may be important for full retention of receptor binding activ acterization of M-cell-specific markers and their exploitation ity. This is illustrated, in part, by peptides Gb-2 and Gb-3 to enhance M-cell targeting efficiency of the antigen in oral which showed no binding to M-cell membranes when conju vaccination. The identification of M-cell-specific markers gated to EGFP peptide but demonstrated binding to GP-2 made M cells possible to be targeted with delivery vehicles. when presented as free peptides in solution. GP-2 was shown to express specifically on M cells and acts as Next, we evaluated the ability of selected lig mucosal transcytosis receptor. stimulate the immune system. We immunized mice orally We used a phage display library to screen peptide ligands with a ligand-fused Ag and monitored immune induction against GP-2 and validated their binding to GP-2 in vitro and in systemic and mucosal compartments. Gb-I significantly in vivo by ELISA and immunohistochemistry (IHC). Selected (p <0.001)enhanced antigen-specific serum IgG, particu- peptides showed high binding affinity to recombinant GP-2 in larly IgGl in the systemic compartment as well as mucosal vitro, especially peptide Gb-1. Competition ELISA results IgA, and the concentration of IgA-switching cytokines showed that the selected peptides do not bind to a common or (IL-5 and IL-6)compared to EGFP alone or the other pep- overlapping epitope on GP-2. Immunohistochemical staining tides. Similarly, elevated levels of IFN-y and IL-4 were of whole-mount and frozen sections of PPs confirmed the detected in supernatant from lymphocytes collected from binding of Gb-l to M cells(Fig. lA, B) and hence promoted the Gb-l-immunized group compared to control groups the internalization of EGFP into PPs significantly compared In summary, we used phage library biopanning to screen to EGFPalone. However, we could not demonstrate this for peptide ligands against mucosal transcytosis receptor GP-2 the other two peptides profiled, suggesting that other factors and validated an identified peptide's role as a mucosal adju also function. The selected peptides most likely interact vant. Results show that Gb-l peptide has immune stimula- with the target receptor in a multivalent fashion. Multivalent tory ability and that it induces Th2 immune response. These presentation of a ligand is a well-accepted approach to findings suggest that peptide Gb-I can be used as adjuvant increase the biological potency of a ligand through enhanced for the development of mucosal vaccines ligand-receptor interaction and, in some cases, cellular internalization by surface receptor oligomerization. Peptide multimerization can also improve peptide stability and [AQ: 7]

Khan et al. 7 sampling is believed to be an essential and critical step for eliciting a successful mucosal immune response. In this regard, identifying mucosal vaccine adjuvants that exploit M cells would be an effective strategy to develop successful mucosal vaccines.31 Research has focused on the characterization of M-cell–specific markers and their exploitation to enhance M-cell targeting efficiency of the antigen in oral vaccination. The identification of M-cell–specific markers made M cells possible to be targeted with delivery vehicles. GP-2 was shown to express specifically on M cells and acts as mucosal transcytosis receptor.10 We used a phage display library to screen peptide ligands against GP-2 and validated their binding to GP-2 in vitro and in vivo by ELISA and immunohistochemistry (IHC). Selected peptides showed high binding affinity to recombinant GP-2 in vitro, especially peptide Gb-1. Competition ELISA results showed that the selected peptides do not bind to a common or overlapping epitope on GP-2. Immunohistochemical staining of whole-mount and frozen sections of PPs confirmed the binding of Gb-1 to M cells (Fig. 1A,B) and hence promoted the internalization of EGFP into PPs significantly compared to EGFP alone. However, we could not demonstrate this for the other two peptides profiled, suggesting that other factors also function. The selected peptides most likely interact with the target receptor in a multivalent fashion. Multivalent presentation of a ligand is a well-accepted approach to increase the biological potency of a ligand through enhanced ligand-receptor interaction and, in some cases, cellular internalization by surface receptor oligomerization. Peptide multimerization can also improve peptide stability and extend its half-life.32 The conformation and/or dimerization of the peptide when presented as a conjugate to a protein may differ from the conformation or presentation of a free peptide in solution. Thus, conformation constraint or polymerization may be important for full retention of receptor binding activity. This is illustrated, in part, by peptides Gb-2 and Gb-3, which showed no binding to M-cell membranes when conjugated to EGFP peptide but demonstrated binding to GP-2 when presented as free peptides in solution. Next, we evaluated the ability of selected ligands to stimulate the immune system. We immunized mice orally with a ligand-fused Ag and monitored immune induction in systemic and mucosal compartments. Gb-1 significantly (p < 0.001) enhanced antigen-specific serum IgG, particularly IgG1 in the systemic compartment as well as mucosal IgA, and the concentration of IgA-switching cytokines (IL-5 and IL-6) compared to EGFP alone or the other peptides. Similarly, elevated levels of IFN-γ and IL-4 were detected in supernatant from lymphocytes collected from the Gb-1–immunized group compared to control groups. In summary, we used phage library biopanning to screen peptide ligands against mucosal transcytosis receptor GP-2 and validated an identified peptide’s role as a mucosal adjuvant. Results show that Gb-1 peptide has immune stimulatory ability and that it induces Th2 immune response. These findings suggest that peptide Gb-1 can be used as adjuvant for the development of mucosal vaccines. Acknowledgments [AQ: 7] Figure 3. Levels of cytokines and cytokine-secreting cells and splenocyte proliferation in Gb-1–immunized mice. Levels of cytokines and cytokine-secreting cells were determined using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT), respectively, as described in the Materials and Methods, after in vitro stimulation of cells from spleen and Peyer’s patches (PPs). (A) Cytokine level from splenocytes. (B) Number of cytokine-secreting cells in PPs. Results are expressed as ng/mL and cell number per cells stimulated, respectively, and are the mean ± SE of five mice per group. (C) Enhanced green fluorescent protein (EGFP)–specific splenocyte proliferation was determined CCK-8 reagent,[AQ: 8] and results are expressed as stimulation indices. *p < 0.05 indicates significant differences compared with the EGFP-immunized group. IL, interleukin; IFN-γ, interferon-γ; PBS, phosphatebuffered saline

SLAS Discovery Declaration of Conflicting Interests Mucosal Immune Responses in Oral Immunization Microbes The authors declared no potential conflicts of interest with respec Infect.2013,l5,895-902 to the research, authorship, and/or publication of this articl 5. Kim, S H. Seo, K. W; Kim, J. et al. The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces Antigen- Specific Immune Responses in Mucosal Vaccination. J. Immunol.2010,185,5787-5795 The authors disclosed receipt of the following financial support 16. Hackett, D J; Zhang, C. Stefanescu, C; et al. Enzyme-Linked for the research, authorship, and/or publication of this article: This work was supported by the Major National Projects for Infectious Glycoprotein B Antibody in Serum. Clin. Vaccine Immunol. Diseases research(2008zX10002002,2012ZX10002-006002- 2010,17,836-839 003)and for New Drug Research Development(2009ZX0913- 17. Maciel, M; Fusaro, A. E; Oliveira, C.R.; et al. IgA Response 710, 2011AA02A114), the National Natural Science Foundation in Serum and gut secretion in sensitized mice Fed with the of China(30571650, 31370927), and project of the Science and Dust Mite Dermatophagoides pteronyssinus Extract. Braz J. Technology Commission of Shanghai(13431900602) Med.Biol.Res.2004,37,817-826 Tominaga, H. Ishiyama, M. Ohseto, F, et al. A Water- Soluble tetrazolium salt Useful for Colorimetric Cell References Viability Assay. Anal. Commun. 1999, 36, 47-50 1. Strugnell,R. A, Wijburg, O. L. The Role of Secretory 19. Fagarasan, S; Kawamoto, S: Kanagawa, O; et al. Adaptive Antibodies in Infection Immunity. Nat Rev Microbiol. 2010, Immune Regulation in the Gut: T Cell-Dependent and T Cell 8,656667 Independent IgA Synthesis. Annu. Rev. Immunol. 2010, 28, 2. Neutra M.R.: Mantis. N.J. Kraehenbuhl J. P Collaboration 243-273. of Epithelial Cells with Organized Mucosal Lymphoid 20. Cerutti, A. The Regulation of IgA Class Switching. Nat Rev Tissues. Nat Immunol. 2001. 2. 1004-1009 Immunol.2008.8.421-434 3. Chen, K, Cerutti, A. Vaccination Strategies to Promote 21. Iwasaki, A. Mucosal Dendritic Cells. Annu. Rev. Immunol Mucosal Antibody Responses. Immunity 2010, 33, 479-491 2007,25,381-418 4. Neutra, M.R., Kozlowski, P A Mucosal Vaccines: The Promise 22. Lamm. M. E. Interaction of Antigens and Antibodies at ind the Challenge. Nat Rev Immunol. 2006.6.148-158. Mucosal Surfaces. Annul. Rev. Microbiol. 1997.51.311-340 5. Kopecky-Bromberg, S.A. Fraser, K.A., Pica, N, et al. Alpha- 23. Higgins, L. M. Lambkin, I Donnelly, G. et al. In Viv C-Galactosylceramide as an Adjuvant for a Live Attenuated Phage Display to Identify M Cell-Targeting Ligands. Pharn Influenza Virus Vaccine Vaccine 2009.27. 3766-3774 Res.2004,21,695-70: 6. Brayden, D. J; Jepson, M. A; Baird, A. W et al. Keynote 24. Clark, M. A; Blair, H. Liang, L; et al. Targeting Polymerised Review: Intestinal Peyers Patch M Cells and Oral Vaccine Liposome Vaccine Carriers to Intestinal M Cells. Vaccine Targeting Drug Discov Today 2005, 10, 1145-1157 2001,20,208-217 7. Sun, J B; Czerkinsky, C; Holmgren, J Mucosally Induced Mantis, N.J. Frey, A: Neutra, M.R.; et al. Accessibility of Immunological Tolerance, Regulatory T Cells and the Glycolipid and Oligosaccharide Epitopes on Rabbit Villus and Adjuvant Effect by Cholera Toxin B Subunit. Scand. J. Follicle-Associated Epithelium. Am J Physiol. Gastrointest Nakato, G Hase, K. Suzuki, M.; et al. Cutting Edge: Brucella 26. Conacher, M. Alexander, J; Brewer, J M. Oral Immunisation abortus Exploits a Cellular Prion Protein on Intestinal M Cells with Peptide and Protein Antigens by Formulation in Lipid as an Invasive Receptor. J. Immunol. 2012, 189, 1540-1544 Vesicles Incorporating Bile Salts(Bilosomes) Vaccine 2001 9. Pavot, V: Rochereau, N; Genin, C. et al. New Insights in 19.2965-2974 Mucosal Vaccine Development. Vaccine 2012, 30, 142-154. 27. Cusi, M. G. Zurbriggen, R, Correale, P; et al. Influenza 10. Hase, K, Kawano, K, Nochi, T, et al. Uptake through Virosomes Are an Efficient Delivery System for Respiratory Glycoprotein 2 of FimH(+) Bacteria by M Cells Initiates Syncytial Virus-F Antigen Inducing Humoral and Cell Mucosal Immune Response. Nature 2009, 462, 226-230. Mediated Immunity Vaccine 2002, 20, 3436-3442 11. Vodnik, M; Zager, U, Strukelj, B, et al. Phage Display: 28. Gluck, R. Adjuvant Activity of Immunopotentiating Recon- electing Straws Instead of a Needle from a Haystack. stituted Influenza Virosomes (IRIVs). Vaccine 1999, 17, Molecules 2011.16. 790-817. 1782-1787 12. Padmanaban. G: Park. H. ChoiJ. S: et al. Identific 29. Senior, K. Bilosomes: The Answer to Oral Vaccine Delivery? Peptides That Selectively Bind to Myoglobin by Bi Drug Discov. Today 2001, 6, 1031-1032. f Phage Displayed-Peptide Library. J. biotechno 30. Kyd, J. M: Cripps, A. W. Functional Differences between l87.43-50 M Cells and Enterocytes in Sampling Luminal Antigens 13. Huang. J: Khan, I; Liu, R; et al. Single Primer-Mediated accIne2008,26,6221-6224 Circular Polymerase Chain Reaction for Hairpin DNA 31. Kuolee, R; Chen, w. M Cell-Targeted Delivery of Vaccines Cloning and Plasmid Editing. Anal. Biochem. 2016, 500, 18- and Therapeutics. Exp. Opin. Drug Deliv. 2008, 5, 693-702 20 32. Singh, A. N. McGuire, M. J: Li, S; et al. Dimerization of a 14. Kim, S.H. ; Yang, I. Y; Jang, S.H. et al C5a Receptor- Phage-Display Selected Peptide for Imaging of Alphavbeta6- Targeting Ligand-Mediated Delivery of Dengue Virus Integrin: Two Approaches to the Multivalent Effect. Theranostics Antigen to M Cells Evokes Antigen-Specific Systemic and 2014,4,745-760

8 SLAS Discovery Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Major National Projects for Infectious Diseases Research (2008ZX10002002, 2012ZX10002-006-002- 003) and for New Drug Research & Development (2009ZX0913- 710, 2011AA02A114), the National Natural Science Foundation of China (30571650, 31370927), and project of the Science and Technology Commission of Shanghai (13431900602). References 1. Strugnell, R. A.; Wijburg, O. L. The Role of Secretory Antibodies in Infection Immunity. Nat. Rev. Microbiol. 2010, 8, 656–667. 2. Neutra, M. R.; Mantis, N. J.; Kraehenbuhl, J. P. Collaboration of Epithelial Cells with Organized Mucosal Lymphoid Tissues. Nat. Immunol. 2001, 2, 1004–1009. 3. Chen, K.; Cerutti, A. Vaccination Strategies to Promote Mucosal Antibody Responses. Immunity 2010, 33, 479–491. 4. Neutra, M. R.; Kozlowski, P. A. Mucosal Vaccines: The Promise and the Challenge. Nat. Rev. Immunol. 2006, 6, 148–158. 5. Kopecky-Bromberg, S. A.; Fraser, K. A.; Pica, N.; et al. AlphaC-Galactosylceramide as an Adjuvant for a Live Attenuated Influenza Virus Vaccine. Vaccine 2009, 27, 3766–3774. 6. Brayden, D. J.; Jepson, M. A.; Baird, A. W.; et al. Keynote Review: Intestinal Peyer’s Patch M Cells and Oral Vaccine Targeting. Drug Discov. Today 2005, 10, 1145–1157. 7. Sun, J. B.; Czerkinsky, C.; Holmgren, J. Mucosally Induced Immunological Tolerance, Regulatory T Cells and the Adjuvant Effect by Cholera Toxin B Subunit. Scand. J. Immunol. 2010, 71, 1–11. 8. Nakato, G.; Hase, K.; Suzuki, M.; et al. Cutting Edge: Brucella abortus Exploits a Cellular Prion Protein on Intestinal M Cells as an Invasive Receptor. J. Immunol. 2012, 189, 1540–1544. 9. Pavot, V.; Rochereau, N.; Genin, C.; et al. New Insights in Mucosal Vaccine Development. Vaccine 2012, 30, 142–154. 10. Hase, K.; Kawano, K.; Nochi, T.; et al. Uptake through Glycoprotein 2 of FimH(+) Bacteria by M Cells Initiates Mucosal Immune Response. Nature 2009, 462, 226–230. 11. Vodnik, M.; Zager, U.; Strukelj, B.; et al. Phage Display: Selecting Straws Instead of a Needle from a Haystack. Molecules 2011, 16, 790–817. 12. Padmanaban, G.; Park, H.; Choi, J. S.; et al. Identification of Peptides That Selectively Bind to Myoglobin by Biopanning of Phage Displayed-Peptide Library. J. Biotechnol. 2014, 187, 43–50. 13. Huang, J.; Khan, I.; Liu, R.; et al. Single Primer-Mediated Circular Polymerase Chain Reaction for Hairpin DNA Cloning and Plasmid Editing. Anal. Biochem. 2016, 500, 18– 20. 14. Kim, S. H.; Yang, I. Y.; Jang, S. H.; et al. C5a ReceptorTargeting Ligand-Mediated Delivery of Dengue Virus Antigen to M Cells Evokes Antigen-Specific Systemic and Mucosal Immune Responses in Oral Immunization. Microbes Infect. 2013, 15, 895–902. 15. Kim, S. H.; Seo, K. W.; Kim, J.; et al. The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces AntigenSpecific Immune Responses in Mucosal Vaccination. J. Immunol. 2010, 185, 5787–5795. 16. Hackett, D. J.; Zhang, C.; Stefanescu, C.; et al. Enzyme-Linked Immunosorbent Assay for Measurement of Cytomegalovirus Glycoprotein B Antibody in Serum. Clin. Vaccine Immunol. 2010, 17, 836–839. 17. Maciel, M.; Fusaro, A. E.; Oliveira, C. R.; et al. IgA Response in Serum and Gut Secretion in Sensitized Mice Fed with the Dust Mite Dermatophagoides pteronyssinus Extract. Braz. J. Med. Biol. Res. 2004, 37, 817–826. 18. Tominaga, H.; Ishiyama, M.; Ohseto, F.; et al. A WaterSoluble Tetrazolium Salt Useful for Colorimetric Cell Viability Assay. Anal. Commun. 1999, 36, 47–50. 19. Fagarasan, S.; Kawamoto, S.; Kanagawa, O.; et al. Adaptive Immune Regulation in the Gut: T Cell-Dependent and T CellIndependent IgA Synthesis. Annu. Rev. Immunol. 2010, 28, 243–273. 20. Cerutti, A. The Regulation of IgA Class Switching. Nat. Rev. Immunol. 2008, 8, 421–434. 21. Iwasaki, A. Mucosal Dendritic Cells. Annu. Rev. Immunol. 2007, 25, 381–418. 22. Lamm, M. E. Interaction of Antigens and Antibodies at Mucosal Surfaces. Annu. Rev. Microbiol. 1997, 51, 311–340. 23. Higgins, L. M.; Lambkin, I.; Donnelly, G.; et al. In Vivo Phage Display to Identify M Cell–Targeting Ligands. Pharm. Res. 2004, 21, 695–705. 24. Clark, M. A.; Blair, H.; Liang, L.; et al. Targeting Polymerised Liposome Vaccine Carriers to Intestinal M Cells. Vaccine 2001, 20, 208–217. 25. Mantis, N. J.; Frey, A.; Neutra, M. R.; et al. Accessibility of Glycolipid and Oligosaccharide Epitopes on Rabbit Villus and Follicle-Associated Epithelium. Am. J. Physiol. Gastrointest. Liver Physiol. 2000, 278, G915–G923. 26. Conacher, M.; Alexander, J.; Brewer, J. M. Oral Immunisation with Peptide and Protein Antigens by Formulation in Lipid Vesicles Incorporating Bile Salts (Bilosomes). Vaccine 2001, 19, 2965–2974. 27. Cusi, M. G.; Zurbriggen, R.; Correale, P.; et al. Influenza Virosomes Are an Efficient Delivery System for Respiratory Syncytial Virus-F Antigen Inducing Humoral and CellMediated Immunity. Vaccine 2002, 20, 3436–3442. 28. Gluck, R. Adjuvant Activity of Immunopotentiating Reconstituted Influenza Virosomes (IRIVs). Vaccine 1999, 17, 1782–1787. 29. Senior, K. Bilosomes: The Answer to Oral Vaccine Delivery? Drug Discov. Today 2001, 6, 1031–1032. 30. Kyd, J. M.; Cripps, A. W. Functional Differences between M Cells and Enterocytes in Sampling Luminal Antigens. Vaccine 2008, 26, 6221–6224. 31. Kuolee, R.; Chen, W. M Cell–Targeted Delivery of Vaccines and Therapeutics. Exp. Opin. Drug Deliv. 2008, 5, 693–702. 32. Singh, A. N.; McGuire, M. J.; Li, S.; et al. Dimerization of a Phage-Display Selected Peptide for Imaging of Alphavbeta6- Integrin: Two Approaches to the Multivalent Effect. Theranostics 2014, 4, 745–760

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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