Modulating adaptive immune responses to peptide self-assemblies

Abstract

Self-assembling peptides and peptide derivatives have received significant interest for several biomedical applications, including tissue engineering, wound healing, cell delivery, drug delivery, and vaccines. This class of materials has exhibited significant variability in immunogenicity, with many peptides eliciting no detectable antibody responses but others eliciting very strong responses without any supplemental adjuvants. Presently, strategies for either avoiding strong antibody responses or specifically inducing them are not well-developed, even though they are critical for the use of these materials both within tissue engineering and within immunotherapies. Here, we investigated the molecular determinants and immunological mechanisms leading to the significant immunogenicity of the self-assembling peptide OVA-Q11, which has been shown previously to elicit strong antibody responses in mice. We show that these responses can last for at least a year. Using adoptive transfer experiments and T cell knockout models, we found that these strong antibody responses were T cell-dependent, suggesting a route for avoiding or ensuring immunogenicity. Indeed, by deleting amino acid regions in the peptide recognized by T cells, immunogenicity could be significantly diminished. Immunogenicity could also be attenuated by mutating key residues in the self-assembling domain, thus preventing fibrillization. A second self-assembling peptide, KFE8, was also nonimmunogenic, but nanofibers of OVA-KFE8 elicited strong antibody responses similar to OVA-Q11, indicating that the adjuvant action was not dependent on the specific self-assembling peptide sequence. These findings will facilitate the design of self-assembled peptide biomaterials, both for applications where immunogenicity is undesirable and where it is advantageous.

Figure 1

Antibody responses to self-assembled peptide antigens were epitope-dependent, long-lived, and primarily directed against the N-terminal epitope domain. List of peptides investigated in this study (a, fibrillization domain shown in blue, spacer in green, and epitope in red). Durable antibody responses lasting at least one year were raised against OVA-Q11 and OVA peptide in CFA, but not RGD-Q11 or Q11 (b, a single boost containing ½ the primary immunization dose was given after 4 weeks for all groups). Using ELISA, antisera raised against OVA-Q11 were probed against various peptide fragments (c–d, sera collected 5 weeks (c) or 24 weeks (d) after primary immunizations). No significant differences in titers were observed between antibodies reactive to the free OVA antigen and the fibrillized OVA-Q11 at either time point. Some antibodies were raised against the SGSG-Q11 peptide at week 5 (c), but these diminished by week 24 (d). * p<0.05 by ANOVA using Tukey post hoc test.

Figure 2

OT-II CD4+ T cells proliferated in response to fibrillized OVA-Q11. The gating process is shown for distinguishing adoptively transferred CFSE-labeled OT-II cells from endogenous CD4+ T cells (a). Proliferation of adoptively transferred OT-II cells in the spleens (b) and lymph nodes (c) of mice immunized with Q11, OVA-CFA, or OVA-Q11. Percentage of proliferating cells in the spleen and lymph nodes (d). p<0.01 by ANOVA using Tukey’s post hoc test.

Figure 3

Antibody responses to OVA-Q11 were T cell-dependent. T cell receptor knockout mice (TCR KO, Tcrb^−/−Tcrd^−/−), did not produce antibodies detectable by ELISA against OVA-Q11, but they did respond to a known T-independent antigen, nitrophenyl-Ficoll (NP-Ficoll). WT mice responded to both OVA-Q11 and NP-Ficoll.

Figure 4

Nanofibers were formed by OVA(T)-Q11, which lacked the putative B cell epitope of OVA_323–339, (a); by OVA(B)-Q11, which lacked the putative T cell epitope (b), and by co-fibrillized 1:1 mixtures of the two peptides (c). Scale bar is 100 nm. Mice immunized with OVA(B)-Q11, OVA(T)-Q11, or co-fibrillized mixtures (OVA(B+T)-Q11) did not raise strong antibody responses comparable to OVA-Q11 (d).

Figure 5

The peptides P3-Q11 (a) and P3-OVAQ11 (b) did not form fibrillar structures by TEM. OVA-Q11 is shown for comparison (c), scale bar is 100 nm for (a–c). P3-Q11 and P3-OVAQ11 adopted a random coil secondary structure, whereas Q11 adopted a β-rich secondary structure by CD (d, 100 μM peptide in 10 mM phosphate buffer/140 mM KF/pH 7.4). Immunizing mice with P3-Q11 or P3-OVAQ11 led to significantly diminished antibody responses compared to OVA-Q11 (e, *p<0.01 by ANOVA with Tukey post-hoc. All mice received a booster immunization after four weeks containing ½ the dose of the primary immunization).

Figure 6

The peptides KFE8 (a), RGD-KFE8 (b), and OVA-KFE8 (c) all formed fibrillar assemblies by TEM (scale bar 100 nm for all images). In mice, only OVA-KFE8 was immunogenic, consistent with the behavior of Q11 previously (d). KFE8 was not immunogenic by itself, in CFA, as RGD-KFE8, or when mixed with OVA_323–339.