(C-93) Gastrointestinal Delivery of α-helical Supramolecular Peptides

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row C - Poster # 93

Presenting Author(s)

Mia E. Woodruff

Student
Duke University
Winston-Salem, North Carolina, United States

Co-Author(s)

EC

Elizabeth J. Curvino

PhD Candidate
Duke University, United States
ER

Emily F. Roe

PhD Candidate
Duke University, United States

Primary Investigator(s)

JC

Joel Collier

Principal investigator
Duke University, United States

Introduction::

Vaccine hesitancy is classified by the World Health Organization as one of the top ten threats to global health and poses a significant problem to developed and developing nations alike.¹ Recently, efforts to reduce hesitancy have resulted in a major push towards needle-free vaccines that enable self-administration while minimizing the need for hiring and training healthcare personnel.²In pursuit of this, several alternative routes for delivery have been explored, such as sublingual, nasal, and oral, each with their own challenges. The common thread among these routes is their direct access to mucous membranes, which often act as the body’s first line of defense via the trapping and clearing of pathogens. In this project, we investigated how the mucoadhesive properties of vaccines affect efficacy in such routes and specifically focused on oral delivery as it provides access to the mucous membranes of the intestines. The self-assembling α-helical peptide Coil29 (QARILEADAEILRAYARILEAHAEILRAQ) is a promising vaccine platform as this peptide forms a supramolecular nanofiber structure which enables the presentation of epitopes and antigens in a highly multivalent fashion that enhances immune responses.³It has previously been shown that Coil29 nanofibers can modulate B- and T-cell responses, making it a candidate for vaccine delivery.⁴ However, pilot in vivo experiments have shown that the oral route’s challenges limit the immunogenicity of Coil29. Here, we investigate PASylation and trimethyl chitosan (TMC) coating as methods for improving muco-adhesivity, as steps towards ameliorating challenges of oral delivery while enhancing the immunogenicity of Coil29.

Materials and Methods::

Peptides were synthesized with Fmoc solid phase synthesis. HPLC was used for purification and MALDI was used to confirm molecular weights. 2 mM nanofibers were made by dissolving lyophilized peptides in 10 mM acetate buffer and heating to 95° C for 15 minutes. This solution was then left at room temperature overnight followed by the addition of 10X PBS at 10% the final volume to induce self-assembly. Transmission Electron Microscopy (TEM) was used to visualize nanofiber formation. For the in vivo experiment, groups of five 8-week-old C57BL/6 female mice were immunized with Coil29 nanofibers loaded with peptide epitope OVA_323-339 with and without the adjuvant cholera toxin subunit B (CTB). Mice were fasted before being orally gavaged with the vaccine for two days in a row and were boosted at weeks 2 and 4. Blood and fecal samples were collected biweekly. Anti-OVA IgG antibody responses were measured using ELISA. PASylation of Coil29 was achieved through the addition of proline, alanine, and serine onto the Coil29 backbone during peptide synthesis. Trimethyl chitosan was electrostatically coated onto pre-assembled Coil29 nanofibers and Coil29 nanofiber variants (10% EEECoil29, 25% EEECoil29). Zeta potential measurements confirmed coating and TEM images confirmed nanofiber morphology. The mucus adhesion assay used zeta potential measurements to quantify the binding rate of various nanofiber formations to soluble mucin, dissolved at 0.3 mg/mL. Zeta potentials were measured for different combinations of mucin volume to nanofibers after they had been allowed to mix for 4 hours while spinning at 60 RPM at 37° C.

Results, Conclusions, and Discussions:: We found that the addition of CTB adjuvant was necessary to render unmodified Coil29 immunogenic via the oral route, with serum anti-OVA IgG responses measured in 3 out of 5 mice receiving CTB (Fig. 1). This data suggests that while Coil29 has potential as an oral vaccine platform, there is a need for further design improvements to increase immunogenicity. We therefore investigated two forms of modification: PASylation and TMC coating. PASylation has been reported to prolong drug bioavailability and pharmacokinetics in vivo due to its hydrophilicity⁵_, and we have previously shown its ability to increase nanofiber muco-penetration in the sublingual route.⁶ TMC has been used to improve oral vaccine uptake due to its muco-adhesivity and ability to penetrate the mucous membrane.⁷TMC coating was achieved via electrostatically coating Coil29 loaded with glutamic acid (10% EEECoil29, 25% EEECoil29), which is negatively charged at a neutral pH, with positively charged TMC. Coating was confirmed using zeta potential, with a switch from negative to positive potential suggesting that TMC coating was successful (Fig. 2A). Neither PASylation nor TMC coating interfered with Coil29 nanofiber morphology as visualized with TEM (Fig. 2B-D). Furthermore, to determine the impacts of these modifications on nanofiber uptake, a mucus adhesion assay was performed using zeta potential to gauge binding. By quantifying the change in nanofiber zeta potential with varying volumes of mucin, the affinity for mucus was approximated, with decreasing zeta potential and increasing mucin concentration corresponding to increased muco-adhesivity. It was found that TMC coating made Coil29 significantly more muco-adhesive relative to plain Coil29, while PASylation decreased adhesivity (Fig. 3A-B). In conclusion, we have illustrated two strategies for modulating the mucoadhesivity of Coil29 nanofibers. PASylation improves muco-penetration, and TMC coating increases muco-adhesion, both of which impact the capacity of the nanofibers to function as mucosal vaccines. Future work includes investigating nanofiber transport in an in vitro model of the intestinal epithelium, evaluating nanofiber survival and their ability to preserve antigen presentation after exposure to simulated gastrointestinal environments, and finally determining the ability of modified Coi29 nanofibers to elicit immune responses through various routes in vivo, including oral immunization.

Acknowledgements (Optional): :

This work is supported by the National Institutes of Health (R01EB009701) and by the National Science Foundation Graduate Research Fellowship Program (DGE-1644868).

References (Optional): :

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