Influenza virus-like particles coated onto microneedles can elicit stimulatory effects on Langerhans cells in human skin

Abstract

Virus-like particles (VLPs) have a number of features that make them attractive influenza vaccine candidates. Microneedle (MN) devices are being developed for the convenient and pain-free delivery of vaccines across the skin barrier layer. Whilst MN-based vaccines have demonstrated proof-of-concept in mice, it is vital to understand how MN targeting of VLPs to the skin epidermis affects activation and migration of Langerhans cells (LCs) in the real human skin environment. MNs coated with vaccine reproducibly penetrated freshly excised human skin, depositing 80% of the coating within 60 s of insertion. Human skin experiments showed that H1 (A/PR/8/34) and H5 (A/Viet Nam/1203/04) VLPs, delivered via MN, stimulated LCs resulting in changes in cell morphology and a reduction in cell number in epidermal sheets. LC response was significantly more pronounced in skin treated with H1 VLPs, compared with H5 VLPs. Our data provides strong evidence that MN-facilitated delivery of influenza VLP vaccines initiates a stimulatory response in LCs in human skin. The results support and validate animal data, suggesting that dendritic cells (DCs) targeted through deposition of the vaccine in skin generate immune response. The study also demonstrates the value of using human skin alongside animal studies for preclinical testing of intra-dermal (ID) vaccines.

Figure 1. Microneedle targeted delivery of vaccines to Langerhans cells in the epidermis

(A) An en face image of a single LC in epidermal sheet prepared from human skin showing distinctive dendritic cell morphology (bar = 10μm). (B) At lower magnification the extensive network typical of LCs is apparent (bar = 10μm). (C) Schematic showing the principle steps involved in delivering vaccines via coated MNs.

Figure 2. Microneedles effectively penetrate human skin

(A) SEM study of a MN penetrating human SC in situ (bar = 300μm). Human skin treated with MNs resulted in the formation of discreet channels through the SC, as revealed by methylene blue staining (B) and histological sections (5μm) that reveal MN penetration through the SC and epidermis and into the upper reaches of the dermis; red arrow indicates direction of MN application (C; bar = 150μm). MN penetration was associated with an increase in trans-epidermal water loss (D); data presented as mean ± SD (n = 10), two replicates. Significance was determined relevant to blank skin (*p≤0.05).

Figure 3. Coating microneedles and dissolution of coating in excised human skin

(A) SEM images of a single MN coated with fluorescent nanospheres (bar = 200μm). (B) Bright field plus fluorescence microscopy of a single MN coated with fluorescent nanospheres (bar = 200μm). (C) Amount of fluorescently coated material remaining on MN surface following insertion into human skin; data presented as mean percentage fluorescence remaining ± SD (n=4), two replicates. Representative images of entire single needles from corresponding time points are shown as inserts (bar= 50 μm). (D) MNs coated with methylene blue demonstrate dissolution and deposition of the coated material within the skin (bar = 1mm).

Figure 4. Reduction in LC numbers in epidermal sheets

(A) blank skin (◆); blank coated MNs containing no VLP (●); H1 VLPs (▲) and H5 VLPs (■) delivered from coated MNs; data presented as mean ± SD (n=4),one replicate. (B) Representative images of LC from each time point and treatment (bar = 100μm).

Figure 5. Area and distribution of LC in histological sections

(A) The area of histological skin sections staining positive for CD207; Blank skin (black bars), H5 VLP MN treated (white bars), H1 VLP MN treated (grey bars). Data presented as mean ± SD (n=4), one replicate. Significance was determined relative to blank skin at corresponding time point (*p≤0.05 **p≤0.01, ***p≤0.001). (B-D) The distribution of LC in histological skin sections: blank skin (B); skin treated with H5 and H1 VLPs delivered from coated MNs (C & D respectively). All images taken from sections of cultured skin samples 24 hours post treatment (bar = 50μm in all cases).

Figure 6. Comparison of immunogenicity of H1 and H5 MN vaccines delivered to mice

Groups of mice (n=6) were vaccinated in the skin using MNs coated with 0.4 μg of H1 VLPs (A/PR8) or H5 VLPs (A/Viet Nam/1203/04). Placebo is the group that received MNs without vaccine. At week 4 after vaccination, serum samples were collected from individual mice and used to determine virus specific antibody responses. Inactivated A/PR8 (H1N1) and reassortant A/Viet Nam/1203/04 (H5N1) viruses were used as a coating antigen for ELISA of H1 VLP and H5 VLP groups respectively. Values of optical density at 450 nm were shown with 400 x diluted samples. Results represent data from at least two independent experiments and three independent serum antibody analyses. **p<0.01 compared with H5 VLP group.