Self-assembled peptide nanofibers raising durable antibody responses against a malaria epitope

Abstract

Biomaterials that modulate innate and adaptive immune responses are receiving increasing interest as adjuvants for eliciting protective immunity against a variety of diseases. Previous results have indicated that self-assembling β-sheet peptides, when fused with short peptide epitopes, can act as effective adjuvants and elicit robust and long-lived antibody responses. Here we investigated the mechanism of immunogenicity and the quality of antibody responses raised by a peptide epitope from Plasmodium falciparum circumsporozoite (CS) protein, (NANP)(3),conjugated to the self-assembling peptide domain Q11. The mechanism of adjuvant action was investigated in knockout mice with impaired MyD88, NALP3, TLR-2, or TLR-5 function, and the quality of antibodies raised against (NANP)(3)-Q11 was assessed using a transgenic sporozoite neutralizing (TSN) assay for malaria infection. (NANP)(3)-Q11 self-assembled into nanofibers, and antibody responses lasted up to 40 weeks in C57BL/6 mice. The antibody responses were T cell- and MyD88-dependent. Sera from mice primed with either irradiated sporozoites or a synthetic peptide, (T1BT*)(4)-P3C, and boosted with (NANP)(3)-Q11 showed significant increases in antibody titers and significant inhibition of sporozoite infection in TSN assays. In addition, two different epitopes could be self-assembled together without compromising the strength or duration of the antibody responses raised against either of them, making these materials promising platforms for self-adjuvanting multi-antigenic immunotherapies.

Figure 1

(NANP)₃-Q11 fibril formation and structural analysis. TEM images of (NANP)₃-Q11 nanofibers after incubation in PBS for 30 min (a) and overnight (b). Scale bar = 100 nm. Secondary structure of (NANP)₃-Q11 (open circles) and Q11 (solid circles) showing beta sheet structure.

Figure 2

Antibody responses to (NANP)₃-Q11 were durable in B6 mice (a). In contrast, Q11 did not elicit any antibody responses (b). Antibody responses were not observed in BALB/c mice (c).

Figure 3

Antibody responses to (NANP)₃-Q11 were T cell-dependent. T cell receptor knockout mice (open circles) did not produce antibodies against (NANP)₃-Q11, but did respond to a T-independent antigen, NP-Ficoll. B6 mice (solid circles) responded to both antigens.

Figure 4

Anti-(NANP)₃-Q11 responses in knockout mouse models, measured by ELISA. Antibody production was abolished in MyD88-knockout mice (a), but not in knockout mice lacking functional TLR-2 (b), TLR-5 (c), or NALP3 (d).

Figure 5

Recovery of antibody titers in mice boosted with (NANP)₃-Q11, after priming with irradiated sporozoites (a) or (T1BT*)₄-P3C peptide (b). Closed circles represent boosted mice, open circles indicate mice that did not receive a boost, and the error bars indicate SEM. TSN assay showing sporozoite neutralizing activity in HepG2 cells after incubation with antisera from immune mice (c). Immune sera from mice immunized with PBS and the monoclonal antibodies 2A10 or 3D11 were used as controls. Mice primed with irradiated sporozoites or (T1BT*)₄-P3C are labeled Irr. Spz. and (T1BT*) respectively. Mice that received (NANP)₃-Q11 only are labeled NANP. Pre, +, and - denote antibody titers pre-boost, post-boost with (NANP)₃-Q11, or no-boost within each group. The levels of parasites in HepG2 cells were measured using qPCR. * p<0.05 by ANOVA.

Figure 6

Fibril formation was not disrupted when peptides bearing two different epitopes were mixed together. TEM images of OVA-Q11 fibrils (a), (NANP)₃-Q11 fibrils (b), and co-assembled fibrils of OVA-Q11 and (NANP)₃-Q11 (c). Antibody levels in mice raised against (NANP)₃-Q11 (d, e) and OVA-Q11 (f, g) after immunization with individual epitopes (d, f) or co-assembled epitopes (e, g) respectively. * p<0.05 by t-test between groups at corresponding time points.