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 were2 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 spe￾cifically expressed on M cells and serves as transcytotic recep￾tor 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 effi￾cient 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 sys￾temic 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 mecha￾nisms 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 signifi￾cantly high levels of serum IgG and mucosal IgA, as well as cytokines secreting cells in various lymphoid tissues, espe￾cially interleukin (IL)–4, IL-5, and IL-6, which are associ￾ated 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 pro￾vided 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 incu￾bated for 2 h at room temperature (RT). The loosely/nonspe￾cifically 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, quanti￾tated on agarose gel by comparing with 0.5 µg of purified sin￾gle-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 enzyme￾linked 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 reac￾tion 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-back￾ground 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