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 suc￾cessful mucosal vaccines.31 Research has focused on the char￾acterization 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 activ￾ity. This is illustrated, in part, by peptides Gb-2 and Gb-3, which showed no binding to M-cell membranes when conju￾gated 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, particu￾larly 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 pep￾tides. 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 adju￾vant. Results show that Gb-1 peptide has immune stimula￾tory 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, phosphate￾buffered saline