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.cnhttps://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 sys￾temic administration such as convenience of administra￾tion, 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 cur￾rently available vaccines are administered through the sys￾temic route and induce an immune response predominantly in the systemic compartment.5,6 Difficulty in efficient deliv￾ery 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 tis￾sue 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 special￾ized 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