Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity

Abstract

Microneedle patches coated with solid-state influenza vaccine have been developed to improve vaccine efficacy and patient coverage. However, dip coating microneedles with influenza vaccine can reduce antigen activity. In this study, we sought to determine the experimental factors and mechanistic pathways by which inactivated influenza vaccine can lose activity, as well as develop and assess improved microneedle coating formulations that protect the antigen from activity loss. After coating microneedles using a standard vaccine formulation, the stability of influenza vaccine was reduced to just 2%, as measured by hemagglutination activity. The presence of carboxymethylcellulose, which was added to increase viscosity of the coating formulation, was shown to contribute to vaccine activity loss. After screening a panel of candidate stabilizers, the addition of trehalose to the coating formulation was found to protect the antigen and retain 48-82% antigen activity for all three major strains of seasonal influenza: H1N1, H3N2 and B. Influenza vaccine coated in this way also exhibited thermal stability, such that activity loss was independent of temperature over the range of 4-37 degrees C for 24h. Dynamic light scattering measurements showed that antigen activity loss was associated with virus particle aggregation, and that stabilization using trehalose largely blocked this aggregation. Finally, microneedles using an optimized vaccine coating formulation were applied to the skin to vaccinate mice. Microneedle vaccination induced robust systemic and functional antibodies and provided complete protection against lethal challenge infection similar to conventional intramuscular injection. Overall, these results show that antigen activity loss during microneedle coating can be largely prevented through optimized formulation and that stabilized microneedle patches can be used for effective vaccination.

Figure 1

Microneedle vaccine delivery system. (A) Scanning electron micrograph of a microneedle (700 µm length, 160 µm width, 50 µm thickness; scale bar = 100 µm) (B) Comparison between an array of five microneedles and a human hair (scale bar = 500 µm) (C) Bright-field (i,iii) and fluorescence (ii,iv) micrographs of a microneedle coated with red-fluorescent inactivated influenza virus before (i,ii) and 10 min after (iii,iv) insertion into human cadaver skin (scale bar = 200µm). (D) Histologic section of human cadaver skin fixed after insertion of a vaccine-coated microneedle imaged by (i) bright-field microscopy with H&E staining showing skin deformation and needle track across the epidermis and into superficial dermis and (ii) fluorescence microscopy showing deposition of red-fluorescent vaccine coating in skin (scale bar = 200µm).

Figure 2

Stabilization of influenza vaccine coatings. (A) Effects of various carbohydrates on retaining the HA activity of inactivated influenza virus during drying. Influenza vaccines in coating solution containing various carbohydrates were dried on pieces of stainless steel mimicking microneedles and then reconstituted to determine the HA activity. All carbohydrates were added at a concentration of 15% (w/v). Data are presented as the percent HA activity compared to the same amount of unprocessed inactivated whole virus in PBS solution without drying (n=4, *p <0.05 for pair wise comparisons between the “coating solution + trehalose” formulation and all other formulations). (B) Particle-size distribution determined by dynamic light scattering in coating solution (control) or reconstituted microneedle coatings prepared using coating solution (CS) with or without 15% trehalose.

Figure 3

Vaccine stability as a function of vaccine strain and storage temperature. HA activities of three different inactivated influenza virus strains dried in PBS or coating solution with or without 15% trehalose. H1 = inactivated influenza A/PR/8/34 (H1N1) virus; H3 = inactivated influenza A/Aichi/68 (H3N2). B = influenza B virus. CS= coating solution. Tre=trehalose.

Figure 4

Vaccine stability as a function of storage temperature. HA titers of coated inactivated virus after drying and storage at different temperatures for 24 h. CS = coating solution.

Figure 5

Effect of trehalose concentration on vaccine coating. (A) Effect of trehalose concentration in coating solution on () mass (per 5 microneedles) and () HA activity of coated inactivated influenza virus after reconstitution (n=4). (B) Effect of trehalose concentration in coating solution on the size of coated inactivated influenza virus particles after reconstitution (n=4).

Figure 6

Effect of CMC concentration in coating solution () with or () without 15% trehalose on (A) HA activity and (B) size of coated inactivated influenza virus particles after reconstitution

Figure 7

Antibody responses in mice after vaccination. (A) Virus-specific total IgG and isotype antibodies after a single vaccination. Influenza virus-specific serum antibody responses were determined at week 4 post-immunization. Antibody levels in 100× diluted serum samples were determined by ELISA using inactivated virus as a coating antigen and presented as optical densities (OD at 450 nm). (B) At week 4 after a single vaccination, hemagglutination inhibition (HAI) titers in sera were determined. Mock = microneedle immunization with coating solution only; MN = microneedle immunization with influenza vaccine formulated in the absence of trehalose; MN+Tre = microneedle immunization with influenza vaccine formulated in the presence of trehalose (15%); IM = intramuscular immunization with liquid influenza vaccine. (n=6 per group)

Figure 8

Protection of mice after a single vaccination. Immunized mice were challenged with a lethal dose (20×LD₅₀) of a highly pathogenic A/PR8 influenza virus 5 weeks after a single vaccination (n=6). Animal survival was monitored daily for 14 days (n=6). Groups of mice are as described in the legend of Figure 7.