Figure 4. Supramolecular PEG-peptide nanofibers enabled sublingual immunization against peptide epitopes.

(a) Adjuvant plus soluble OVA peptide (pOVA) alone fails to elicit sublingual antibody responses, even when the peptide is PEGylated. Mice were primed and boosted at weeks 1 and 3 with 7 μL of 5.6 mM peptide with or without 2 μg of cholera toxin (CT) adjuvant, and serum pOVA-specific IgG was measured by ELISA at week 6, n=5/group. (b) PEGylated nanofibers were immunogenic sublingually with CT, regardless of whether they were sheared. Nanofibers lacking PEG were non-immunogenic sublingually. Mice were primed and boosted at weeks 1, 3, and 20 with 7 μL of 5.6 mM peptide with 2 μg of cholera toxin per mouse, and pOVA-specific IgG was measured by ELISA. *p < 0.05, one-way ANOVA, Tukey’s multiple comparisons test, n=4/group. Arrows indicate timepoints of immunizations. (c) Antibody subclasses at week 6 were balanced between IgG1, IgG2b, and IgG2c. *p < 0.05, two-way ANOVA, n=4/group. (d) Sublingually delivered OVAQ11-PEG nanofibers also raised IgG antibodies in the reproductive tract at week 10. *p<0.05, unpaired, two-tailed t-test, n=5/group. (e) PEGylated nanofibers elicited T cell responses, measured by splenocyte ELISPOT at week 21 (same mouse groups as shown in (b) and Fig. S7). SFC (spot-forming cells) after pOVA stimulation are shown. *p < 0.05, one-way ANOVA, Tukey’s multiple comparisons test, n = 4/group (OVAQ11 + CT, OVAQ11-PEG + CT), n = 7 (OVAQ11-PEG (Sheared) + CT), or n = 5 (OVAQ11 (Sheared) + CT). (f) OVA-Q11-PEG nanofibers remained strongly and durably immunogenic using the more clinically translatable adjuvant CTB. n=5 mice, immunized sublingually at weeks 0, 1, 3, and 6 with 7 μL of 5.6 mM OVAQ11-PEG with 10 μg of cholera toxin subunit B, pOVA-specific serum IgG measured by ELISA. (g) At week 52 for the mice immunized with CTB-adjuvanted nanofibers shown in (e), Ig responses were balanced between IgG1, IgG2b, and IgG2c.