Mucosal Adjuvants Delivered by a Mucoadhesive Patch for Sublingual Administration of Subunit Vaccines

Abstract

Among mucosal administration routes for vaccines, the sublingual route has been proven capable of inducing a potent systemic and mucosal immune response. However, the absence of a simple and compliant delivery system and the lack of robust mucosal adjuvants impede the development of sublingual vaccines. Here, we describe a mucoadhesive patch made of a layer-by-layer assembly of polysaccharides, chitosan, and hyaluronic acid. The mucoadhesive patch was covered by adjuvanted nanoparticles carrying viral proteins. We showed that the nanoparticles effectively cross the outer layers of the sublingual mucosa to reach the epithelium. Furthermore, the encapsulated adjuvants, 3M-052 and mifamurtide, targeting toll-like receptor (TLR) 7/8 and nucleotide-binding oligomerization domain-2 (NOD2), respectively, remain fully active after encapsulation into nanoparticles and exhibit a cytokine/chemokine signature similar to the mucosal gold-standard adjuvant, the cholera toxin. However, the particulate adjuvants induced more moderate levels of proinflammatory interleukin (IL)-6 and keratinocyte chemoattractant (KC), suggesting a controlled activation of the innate immune response.

Keywords: adjuvant; cytokine profiling; layer by layer; mucoadhesive; mucosal vaccine; nanoparticle; sublingual.

Figure 1

Mucosal NP delivery by a mucoadhesive LbL patch. (A) (CHI/HyA)₁₀₀ patch and PLA-NP adsorption on the surface. (B) Optical section of the PLA-NP (bodipyTR, red) adsorbed on the mucoadhesive LbL patch (CHI^FITC, green).

Figure 2

Cytotoxicity of NPs and the LbL patch on (A) human epithelial cells (HeLa), (B) human buccal epithelial cells (Ho-1u-1), and (C) murine dendritic cells (DC2.4). The percentage of viability was normalized to untreated cells. Data are presented as mean ± SD; statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test (not significant (ns): p > 0.05; **: p < 0.001, and ****: p < 0.00001). (D) PLA-NP uptake by DC2.4 cells after release by the LbL patch. The LbL patches were degraded for 24h in artificial saliva before addition to cells. Actin (phalloidin-TRITC) and nucleus (DAPI) labelling were performed 20 min after incubation with an LbL patch, PLA-NP, or the combination thereof. NP internalization was observed using fluorescein–labelled PLA-NP. 3D reconstructions of optical sections were obtained from pictures taken by confocal microscopy. Scale bar: 10 µm.

Figure 3

PLA-NP residence and transport through the SL mucosa. (A) Fluorescence molecular tomography of PLA-NP^ICG (indocyanine green) in solution or incorporated in the LbL patch. Fluorescent signal was detected 1, 10, or 30 min after administration. (B) PLA-NP visualized by confocal microscopy 10 min after administration as a liquid dosage or by a mucoadhesive LbL patch made of CHI or VIS combined with HyA. The pictures were taken with a 40× objective lens with a zoom factor of 4×. White arrows indicate fluorescent PLA-NPs, and dashed lines represent the outer surface of the SL mucosa. The panels on the right side of the figure are 3D reconstructions (maximum-intensity projection) of optical sections from 40 µm thick cuts of SL mucosa (ventral part of the tongue).

Figure 4

Bioactivity of mucosal adjuvants. (A) Quantification of intracellular cAMP produced after 24 h incubation of DC 2.4 cells with CT either formulated as a liquid solution (H₂O or NaCl) or as an LbL patch in 0.15 mol/L NaCl. The percentage was normalized to the average amount of cAMP produced after contact with CT in H₂O. (B) Schematics of 3M-052 and mifamurtide incorporated in PLA-NP and their respective immune receptors. (C) Staining of Nod2 receptors in the SL mucosa of mice. Nod2 receptors (red) and nuclei (DAPI, blue) were stained in naïve mice. Scale bar, 100 µm. (D,E) Quantification of the SEAP produced after the activation of (D) HEK-Blue hTLR7 cells by 3M-052 formulations and (E) HEK-Blue hNOD2 cells by mifamurtide formulations, either in their free form, encapsulated in NP, or released from the LbL patch. Data are presented as mean ± SD and were statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (not significant (ns): p > 0.05).

Figure 5

Cytokine/chemokine profiling in serum after SL administration of adjuvanted p24 vaccine formulations. (Mifa: mifamurtide; CT: cholera toxin; NP: nanoparticles; DNFB: 1-fluoro-2,4-dinitrobenzène). (A) A total of 25 multiplexed cytokines from the ‘mouse immune panel’ sera were collected either 6 or 24 h after SL administration. Values were calculated as a log2 fold change of the ratio between the mean measured cytokine quantity for 3 mice in each condition over the mean cytokine quantity of control mice (dPBS administration). (B–G) Quantification of selected cytokines 6 h after SL administration of formulations. Data are presented as mean ± SD and were statistically analyzed using one-way ANOVA followed by Tukey’s multiple comparison test (****: p < 0.00001).