Modular design of triple a-helical fibrillar peptides as an epitope delivery platform

Abstract

Event Abstract Back to Event Modular design of triple α-helical fibrillar peptides as an epitope delivery platform Yaoying Wu1 and Joel H. Collier1 1 University of Chicago, Department of Surgery, United States Introduction: Self-assembly of peptides has been widely explored for biomaterials applications, including cell delivery, drug delivery, and vaccine platform. It was previously demonstrated that epitope-bearing β-sheet fibrillizing peptides could elicit strong and specific antibody response without supplemental immune adjuvants[1]. However, β-sheet fibrillizing peptides lack structural precision, and the kinetics of their assembly and disassembly are difficult to control. To address these shortcomings, we now report the design of a new self-assembly platform based on peptides that fold and assemble into α-helical nanofibers. Herein, we report our effort to develop a unique triple α-helical fibrillizing peptide system as vaccine delivery platform. Based upon Woolfson and coworkers’ previous works[2],[3], we engineered a self-assembling system composed of two peptides containing heterodimer and trimer domain (A+C and CB-), and one peptide as trimer (TriC). (Fig. 1) OVA epitope bearing peptide was synthesized via N-terminal extension on TriC peptide. This abstract reports the peptide design and self-assembly characteristics of this system. Materials and Methods: The peptides were synthesized using standard Fmoc solid-phase peptide synthesis, and purified via high performance liquid chromatography (HPLC), and marix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was utilized to confirm the molecular weight. Peptide fibers were formed by mixing equal molar of three peptides in 10 mM MOPS buffer at final concentration of 2 mM. The morphology of the self-assembled fiber was studied using both circular dichroism (CD) and transmission electron microscopy (TEM). Results and Discussion: A+C and CB- peptides are composed of both heterodimer and homotrimer sequences, while TriC peptide contains only the homotrimer sequence. Acidic and basic heterodimer peptides complement each other and function as overhanging “sticky-end” leading to fiber formation in our design, and the homotrimer sequence not only facilitates the fibrilization, but also provides a “docking position” for the epitope peptide on nanofibers. To incorporate epitopes in the fiber, OVA-TriC is synthesized by N-terminal extension with TriC as the anchor. The CB- and TriC did not self-assemble in the buffer, but the A+C peptide fibrilized in buffer possibly due to its β-sheet characteristics. (Fig. 2 A, B, C) Upon mixing the three peptides in buffer (ABC peptide assembly), nanofibers were formed after overnight incubation at room temperature. (Fig. 2D) The morphology of the obtained nanofibers was noticeably different from A+C peptide fiber, which was thicker and shorter (about 500 nm) according to TEM. Replacing TriC peptide by OVA-TriC peptide, the triple peptide system still self-assembled into nanofiber in buffer, despite of the presence of long epitope sequence. (Fig. 2 E, F) Moreover, the morphology of the obtained peptide fibers was not altered by the amount of OVA-TriC peptide in the mixture, indicating that the integration of OVA epitope did not significantly impact the fibrilization process. Fig. 1. Schematics for the design of triple α-helical fibrillar system. Left to right: helical-wheel diagram representing the heptad repeats of heterodimer sequence (acidic sequence, red; basic sequence blue) and homotrimer (green); schematic of the epitope-bearing fiber composed of three different peptides. Fig. 2. TEM images and CD plots of various peptides and mixtures: A) A+C peptide, B) CB- peptide, C) TriC peptide, D) ABC peptide assembly, E) ABC-1/2 OVA peptide assembly, F) ABC-OVA peptide assembly. Conclusion: Three α-helical peptides were designed to form nanofibers by overhanging “sticky-ends”. Neither CB- nor TriC self-assembled by itself in the MOPS buffer, only A+C peptide. However, mixing the three peptides in MOPS buffer led to formation of short nanofibers. It was also established that this triple peptide fibrilization system can tolerate OVA epitopes in a range of feeding ratios. This α-helical peptide system showed promising potential to modulate the peptide fiber length, and the dose of epitopes in nanofibers, both important in vaccine design. NIH/NIAID 5R01AI118182