Surface potentials determine the potency of supramolecular nanofibrous vaccines

摘要

Introduction: Self-assembling peptides have been developed recently for immunotherapies and vaccines. Because of their exceptional modularity, functional groups or immune epitopes can be displayed with precise ratios, allowing the tuning of the immune phenotype. When T cell and B cell epitopes are conjugated to the self-assembling peptide QQKFQFQFEQQ (Q11), supramolecular nanofibers displaying the antigenic epitopes are formed, and these elicit strong immune responses. However, there are not yet known design rules for strengthening, weakening, or ablating these immune responses. Such knowledge is important not only for designing nanomaterials as vaccines, but for other applications in which immune responses must be avoided. In the current study, antigen displaying nanofibers with different surface properties were engineered, and their potency on stimulating immune responses were investigated. We found that surface charge, and in particular negative surface charge, had a surprising ability to diminish immune responses against self-assembling peptide materials. Methods: The N-terminus of Q11 was modified with lysine (K), glutamic acid (E), polyethylene glycol (PEG), and propyl groups to introduce positive charge (KQ11 and KKKQ11), negative charge (EQ11 and EEEQ11), hydrophilicity (PEGQ11), and hydrophobicity (PropylQ11), respectively. The peptide antigen OVA_(323-339) was conjugated to Q11 and used as the model antigen (0VAQ11). All peptides were synthesized using standard Fmoc based solid phase peptide synthesis as previously reported. Each modified peptide was mixed with OVAQ11 at a molar ratio of 9:1 and allowed to co-assemble into nanofibers. Thioflavin T (ThT) binding assay, Transmission Electron Microscopy (TEM), and zeta potential measurements were employed to confirm fibrillization and physiochemical properties of the formed nanofibers. To study the effect of surface properties on nanofiber immunogenicity in vivo, co-assembled nanofibers were injected intraperitoneally, and dendritic cell (DC) uptake was measured by flow cytometry (with TAMRA conjugated OVAQ11). Presentation of the peptides by DCs was measured by IL-2 production from DOBW cell co-cultures. Antibody titers were measured by ELISA after subcutaneous immunizations. Results and Discussion: Similar to our previous reported formulation (0VAQ11/Q11), all modified peptides were able to fibrilize and co-assemble with OVAQ11 in beta-strand conformations. Zeta potential measurements revealed that co-assembled nanofibers had altered surface properties. Q11/OVAQ11 had a slightly negative zeta potential (-2.1 mV). KQ11/OVAQ11 and KKKQ11/OVAQ11 had strong positive potential of +20.2 mV and +29.1 mV, respectively, while EQ11/OVAQ11 and EEEQ11/OVAQ11 had strong negative potential of-17.8 mV and -26.5 mV, respectively. PEGQ11/OVAQ11 (-1.9 mV) and PropylQ11/OVAQ11 (-2.6 mV) exhibited near neutral potential like their Q11/OVAQ11 counterparts. Q11/OVAQ11 nanofibers were internalized by DCs efficiently, with 19% MHCII high CD11c+ cells showing positive fluorescence signals. Noticeably, nanofibers with negative surface potentials exhibited negligible uptake by DCs, while KQ11/OVAQ11 and PrapylQ11/OVAQ11 had higher uptake efficiencies. Consequently, Q11/OVAQ11 and positively charged nanofibers led to efficient presentation of OVA epitopes in MHC-Ⅱ, evidenced by strong IL-2 production from DOBW cells when co-cultured with DCs from immunized mice. In contrast, negatively charged nanofibers did not lead to any detectable presentation. Consistent with the DC uptake and presentation results, antibody responses in mice immunized with negatively charged nanofibers were significantly compromised (EQ11/OVAQ11) or completely abolished (EEEQ11/OVAQ11). Conclusions: These results suggest that the immunogenicity of self-assembling peptide nanofibers can be strongly tuned by altering the nanofibers' surface properties. In particular, negative surface charges have the ability to strongly curtail or even abolish immunogenicity. This information provides an efficient way to manipulate the immunogenicity of self-assembled peptide biomaterials and facilitates the design of effective vaccines.