A self-assembling peptide acting as an immune adjuvant

Abstract

The development of vaccines and other immunotherapies has been complicated by heterogeneous antigen display and the use of incompletely defined immune adjuvants with complex mechanisms of action. We have observed strong antibody responses in mice without the coadministration of any additional adjuvant by noncovalently assembling a T and B cell epitope peptide into nanofibers using a short C-terminal peptide extension. Self-assembling peptides have been explored recently as scaffolds for tissue engineering and regenerative medicine, but our results indicate that these materials may also be useful as chemically defined adjuvants. In physiological conditions, these peptides self-assembled into long, unbranched fibrils that displayed the epitope on their surfaces. IgG1, IgG2a, and IgG3 were raised against epitope-bearing fibrils in levels similar to the epitope peptide delivered in complete Freund's adjuvant (CFA), and IgM production was even greater for the self-assembled epitope. This response was dependent on self-assembly, and the self-assembling sequence was not immunogenic by itself, even when delivered in CFA. Undetectable levels of interferon-gamma, IL-2, and IL-4 in cultures of peptide-challenged splenocytes from immunized mice suggested that the antibody responses did not involve significant T cell help.

Fig. 1.

Schematic (A) and sequences (B) of epitope-bearing self-assembling peptides. The Q11 domain (blue) assembles into fibrillar aggregates, displaying the epitope sequence (red) at the end of a flexible spacer (green).

Fig. 2.

Q11-based peptides self-assembled into β-sheet fibrils that displayed functional epitopes on their surfaces. Q11 (A) self-assembled into long, unbranched fibrils that did not bind streptavidin-gold (B). O-Q11 (C) also formed long, unbranched fibrils, and biotin-O-Q11 bound streptavidin-gold (D), indicating availability of the N terminus on the fibril surface. O-Q11 possessed a predominant β-sheet structure by CD (E). OVA antisera reacted similarly to ELISA plates coated with OVA or O-Q11 (F). Each point represents one mouse’s serum; bars represent the mean. *p < 0.01 compared to OVA coat/OVA in PBS injection, by ANOVA with Tukey HSD post hoc testing. n.s., not statistically different.

Fig. 3.

Fibrillization by the Q11 domain strongly adjuvanted IgG responses to OVA. Similar titers of total IgG were raised against O-Q11 delivered in PBS and OVA delivered in CFA, whereas Q11 or OVA delivered in PBS did not elicit a response (A). Q11 was nonimmunogenic even in CFA, whereas CFA increased IgG titers for O-Q11 (B). O-Q11 antisera were strongly cross-reactive to OVA-coated ELISA plates and showed a small amount of reactivity to Q11-coated plates that was not statistically significant (C). The adjuvant activity of Q11 was entirely dependent on covalent conjugation between the fibrillizing domain and epitope domain, as mixtures of Q11 and OVA did not raise any OVA-specific IgG (D). Each point represents one mouse; bars represent the mean. *p < 0.01 by ANOVA with Tukey HSD post hoc testing, compared with OVA in PBS, or between groups as indicated.

Fig. 4.

Antibody isotypes in sera from mice immunized with peptides. Each point represents one mouse; bars represent the mean. *p < 0.01 compared with OVA delivered in PBS by ANOVA with Tukey HSD post hoc test. §p < 0.05 as indicated.

Fig. 5.

IFN-γ, IL-2, and IL-4 production in peptide-challenged (triangles) and unchallenged (circles) splenocyte cultures. Each point represents one mouse; bars represent the mean. ^∗p < 0.01 compared with corresponding unchallenged control, by ANOVA with Tukey HSD post hoc test.