Chrysanthemum sporopollenin: A novel vaccine delivery system for nasal mucosal immunity

Abstract

Objective: Mucosal immunization was an effective defender against pathogens. Nasal vaccines could activate both systemic and mucosal immunity to trigger protective immune responses. However, due to the weak immunogenicity of nasal vaccines and the lack of appropriate antigen carriers, very few nasal vaccines have been clinically approved for human use, which was a major barrier to the development of nasal vaccines. Plant-derived adjuvants are promising candidates for vaccine delivery systems due to their relatively safe immunogenic properties. In particular, the distinctive structure of pollen was beneficial to the stability and retention of antigen in the nasal mucosa.

Methods: Herein, a novel wild-type chrysanthemum sporopollenin vaccine delivery system loaded with a w/o/w emulsion containing squalane and protein antigen was fabricated. The unique internal cavities and the rigid external walls within the sporopollenin skeleton construction could preserve and stabilize the inner proteins. The external morphological characteristics were suitable for nasal mucosal administration with high adhesion and retention.

Results: Secretory IgA antibodies in the nasal mucosa can be induced by the w/o/w emulsion with the chrysanthemum sporopollenin vaccine delivery system. Moreover, the nasal adjuvants produce a stronger humoral response (IgA and IgG) compared to squalene emulsion adjuvant. Mucosal adjuvant benefited primarily from prolongation of antigens in the nasal cavity, improvement of antigen penetration in the submucosa and promotion of CD8+ T cells in spleen.

Disccusion: Based on effective delivering both the adjuvant and the antigen, the increase of protein antigen stability and the realization of mucosal retention, the chrysanthemum sporopollenin vaccine delivery system has the potential to be a promising adjuvant platform. This work provide a novel idea for the fabrication of protein-mucosal delivery vaccine.

Keywords: adhesion; chrysanthemum; nasal mucosal immunity; sporopollenin; vaccine delivery system.

Figure 1

Preparation process of Spo-Squ/OVA and the shape and morphology of sporopoll en in carrie. (A) Schematic diagram of Spo-Squ/OVA preparation process flow chart. (B) SEM images of pollen, DE-pollen, Spo and Spo-Squ/OVA samples at different magnifications. (C) Morphology of Spo under SEM. (D) Morphologies under Spo-Squ/OVAfluorescence microscope. Scale bars: (B) 10 μm, 5 µm, (C) 20 μm, (D) 20 µm.

Figure 2

The characterization of sporopollenin carrier. (A) grain size patterns and (B) Infrared analysis of Pollen, DE-pollen, Spo and Spo-Squ/OVA. (C) Contact Angle diagram of Spo and Spo­ Squ/OVA. (D) Chemical composition analysis of pollen, DE-pollen and Spo. (E) The nitrogen content of pollen, DE- pollen and Spo.

Figure 3

Spo-Squ /OVA enhanced systemic and mucosal immun e responses after intran asal vaccination. BALB/c mice were immunized intranasally wi th 25 µg of OVA or mixed with 25 µg of adjuvant and boosted 2 weeks later with the same formulations. Control groups re present samples coll ected from mice immuni zed with PBS witho ut antigens. (A) Timeline representation of experimental porceduures. (B) Mucosal slgA titers in nasal wash. (C) IgG and (D) IgA titers in serum. (E) The percentage of CD8+ T and CD4+ T cells in the spleens after ex vivo stimulation with the OVA (5 µg/ mL) for 24 hours. Significant differences were determined by double-tail t-test. Values were repotied as mean ± SEM (n=3). The asterisks indicate significant differences ("*" P<0.05, "**" P<0.01, "ns" P>0.05).

Figure 4

Sporopollenin increase the retention and release of antigen. (A) Retention of Squ/OVA and Spo-Squ/OVA in nasal mucosa. (B) Spo-Squ /OVA enters the nasal mucosa 4 hours after administration. (C) The appearance of the Squ /OVA and Spo-Squ/OVA w/o/w emulsion. (D) The fluorescence microscopy images of Spo-Squ/OVA. Scale bar: 50 µm. (E) The microscopy images of Spo-Squ /OVA. Scale bar: 1 µm. (F) In vitro release curve of OVA from Spo-Squ/OVA in phosphate­ buffered saline. (G) The particle size of Squ/OVA and Spo-Squ/OVA.

Figure 5

Biosafety of Spo-Squ/OVA in v ivo. (A) H&E staining and hi stopathological exam i nati on of different organs on day 28 after primary immunization. Scale bar: 200 µm (B) Body weight of mice during vaccination. (C) H&E staining and histopathological examination of nasal mucosa in rabbits after 7 days of continuous administration of Spo-Squ/OVA. Scale bar: 200 µm, 50 µm.