Phage Display–Derived Ligand for Mucosal Transcytotic Receptor GP-2 Promotes Antigen Delivery to M Cells and Induces Antigen-Specific Immune Response

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.

Molecular insights into the mechanisms of M-cell differentiation and transcytosis in the mucosa-associated lymphoid tissues

“…In fact, orally administered biotinylated ovalbumin peptide (OVA) conjugated with anti-GP2-streptavidin efficiently induces OVA-specific fecal IgA secretion in a mouse model (Shima et al 2014). Additionally, GP2-binding peptide fused with green fluorescent protein (GFP) significantly increases the uptake of enhanced GFP (EGFP) by M cells and induces efficient mucosal and systemic immune responses measured at the level of antigen-specific serum and fecal antibodies, cytokine secretion, and lymphocyte proliferation (Khan et al 2017). Oral administration of tetanus toxoid-or botulinum toxoid-conjugated NKM 16-2-4, which is an M-cell-specific monoclonal antibody, induces high-level, antigen-specific serum IgG and mucosal IgA responses (Nochi et al 2007).…”

Immunoinformatic Identification of Multiple Epitopes of gp120 Protein of HIV-1 to Enhance the Immune Response against HIV-1 Infection

“…Mostly subunit vaccines typically possess an adjuvant to effectively activate innate immunity and induce an immune response. Given that antimicrobial peptides have shown potential in stimulating both innate and adaptive immune responses [22], our laboratory's recently discovered Gb-1 was selected as a suitable adjuvant for the vaccine. In the process of constructing the multiple-epitope vaccine in this study, a total of 5 B cell epitopes, 10 CTL epitopes, and 6 HTL epitopes were selected according to various immunogenic tests.…”

Current Progress and Challenges in the Study of Adjuvants for Oral Vaccines

“…Therefore, targeting GP-2 with specific ligands should increase antigen delivery to the immune initiation sites. Khan et al [ 82 ] selected a GP2-binding peptide ligand, Gb-1, through phage library screening, which showed high binding affinity to GP-2. When fused with EGFP, Gb-1 significantly enhanced the uptake of EGFP by M cells compared with EGFP alone.…”