Formulation of microneedles coated with influenza virus-like particle vaccine

Abstract

Mortality due to seasonal and pandemic influenza could be reduced by increasing the speed of influenza vaccine production and distribution. We propose that vaccination can be expedited by (1) immunizing with influenza virus-like particle (VLP) vaccines, which are simpler and faster to manufacture than conventional egg-based inactivated virus vaccines, and (2) administering vaccines using microneedle patches, which should simplify vaccine distribution due to their small package size and possible self-administration. In this study, we coated microneedle patches with influenza VLP vaccine, which was released into skin by dissolution within minutes. Optimizing the coating formulation required balancing factors affecting the coating dose and vaccine antigen stability. Vaccine stability, as measured by an in vitro hemagglutination assay, was increased by formulation with increased concentration of trehalose or other stabilizing carbohydrate compounds and decreased concentration of carboxymethylcellulose (CMC) or other viscosity-enhancing compounds. Coating dose was increased by formulation with increased VLP concentration, increased CMC concentration, and decreased trehalose concentration, as well as increased number of dip coating cycles. Finally, vaccination of mice using microneedles stabilized by trehalose generated strong antibody responses and provided full protection against high-dose lethal challenge infection. In summary, this study provides detailed analysis to guide formulation of microneedle patches coated with influenza VLP vaccine and demonstrates effective vaccination in vivo using this system.

Fig. 1

Microneedles coated for vaccine delivery. a Scanning electron micrograph of a microneedle (scale bar 100 μm), b microneedle array containing five microneedles coated with influenza virus-like particle (VLP) vaccine in standard coating solution containing trehalose (scale bar 400 μm)

Fig. 2

Influenza VLP vaccine delivery from coated microneedles into skin. a Representative fluorescence micrograph of microneedles coated with red-fluorescent, R18-stained VLPs (left) and after insertion into human cadaver skin for 30, 60, and 120 s. As a positive control to confirm complete release of VLPs from the microneedles, microneedles were incubated in PBS for 1 h (right; scale bar 500 μm). b Multiphoton fluorescence micrograph of cryosectioned human cadaver skin after insertion of R18-stained VLP-coated microneedle (white arrow microneedle insertion site, scale bar 300 μm)

Fig. 3

Determination of VLP dose coated onto microneedles. Amount of VLP coated per microneedle as a function of a the number of dips into coating solution containing 2 mg/mL VLPs and b the VLP concentration in the coating solution after 6 dips. Standard coating solution was used. Replicate data from n = 4 samples is shown as the mean value with error bars indicating the standard error of the mean (SEM)

Fig. 4

The effect of microneedle formulation and material on HA activity. HA activity of VLPs is shown after coating and dissolution in PBS. Coatings were made using a standard coating solution containing 15% trehalose and substituting CMC for various stabilizers (*p < 0.05 for comparison between xanthan gum and other viscous enhancers); b standard coating solution containing various carbohydrate stabilizers at a concentration of 15% (w/v) (*p < 0.05 for comparison between trehalose and other stabilizers); and c microneedles made from various materials as indicated in the figure legend using standard coating solution containing 15% trehalose (*p < 0.05 for comparison between stainless steel and other metals; n = 4, mean ± SEM)

Fig. 5

The effect of trehalose stabilizer concentration on HA activity and coating dose of VLPs. a HA activity of VLPs is shown after coating and dissolution in PBS. Coatings were made using standard coating solution with trehalose added over a range of concentrations at 25°C. b Trade-off between HA activity and coated dose of VLPs is shown at different trehalose concentrations (*trehalose concentration, n = 4, mean ± SEM)

Fig. 6

The effect of CMC concentration on HA activity and coating dose of VLPs. a HA activity and b coating dose of VLPs is shown after coating and dissolution in PBS. Coatings were made by use of standard coating solution containing 15% trehalose and modified to contain various concentrtations of CMC. c Trade-off between HA activity and coated dose of VLPs is shown at different CMC concentrations (F-68 Lutrol F-68 NF surfactant, Treh trehalose, *CMC concentration, n = 4, mean ± SEM)

Fig. 7

Humoral antibody response and protection efficacy after microneedle immunization with influenza VLP vaccine. a Total influenza virus-specific serum antibody response (IgG) measured 2 and 4 weeks after immunization with VLP vaccine-coated microneedles. (⁺ p < 0.01 for comparison between microneedle and naïve, *p < 0.05 for comparison between formulation with trehalose and without trehalose). b Body weight change and c survival of mice immunized using antigen-coated microneedles after lethal challenge infection (MN microneedle, Treh trehalose)