Development of the H3N2 influenza microneedle vaccine for cross-protection against antigenic variants

Abstract

Due to the continuously mutating nature of the H3N2 virus, two aspects were considered when preparing the H3N2 microneedle vaccines: (1) rapid preparation and (2) cross-protection against multiple antigenic variants. Previous methods of measuring hemagglutinin (HA) content required the standard antibody, thus rapid preparation of H3N2 microneedle vaccines targeting the mutant H3N2 was delayed as a result of lacking a standard antibody. In this study, H3N2 microneedle vaccines were prepared by high performance liquid chromatography (HPLC) without the use of an antibody, and the cross-protection of the vaccines against several antigenic variants was observed. The HA content measured by HPLC was compared with that measured by ELISA to observe the accuracy of the HPLC analysis of HA content. The cross-protection afforded by the H3N2 microneedle vaccines was evaluated against several antigenic variants in mice. Microneedle vaccines for the 2019-20 seasonal H3N2 influenza virus (19-20 A/KS/17) were prepared using a dip-coating process. The cross-protection of 19-20 A/KS/17 H3N2 microneedle vaccines against the 2015-16 seasonal H3N2 influenza virus in mice was investigated by monitoring body weight changes and survival rate. The neutralizing antibody against several H3N2 antigenic variants was evaluated using the plaque reduction neutralization test (PRNT). HA content in the solid microneedle vaccine formulation with trehalose post-exposure at 40℃ for 24 h was 48% and 43% from the initial HA content by HPLC and ELISA, respectively. The vaccine was administered to two groups of mice, one by microneedles and the other by intramuscular injection (IM). In vivo efficacies in the two groups were found to be similar, and cross-protection efficacy was also similar in both groups. HPLC exhibited good diagnostic performance with H3N2 microneedle vaccines and good agreement with ELISA. The H3N2 microneedle vaccines elicited a cross-protective immune response against the H3N2 antigenic variants. Here, we propose the use of HPLC for a more rapid approach in preparing H3N2 microneedle vaccines targeting H3N2 virus variants.

Figure 1

Schematic diagram of H3N2 microneedle vaccine development based on HPLC analysis of HA for cross-protection of influenza H3N2 virus. (a) Preparation of 19–20 A/KS/17 H3N2 microneedle vaccine, (b) pretreatment of HA, (c) establishment of HPLC-based analysis of HA content of H3N2 vaccine stability and comparison with ELISA, and (d) observation of cross-reactivity immune response against 10–12 A/PE/09, 14–15 A/TX/12, and 15–16 A/SW/13 H3N2 viruses in animal experiments.

Figure 2

Images of coated microneedles. (a) Optical microscopic image (scale bar = 500 μm) and (b) fluorescence image of FITC-dextran coating layer on microneedles (scale bar = 500 μm). (c) Enlarged SEM image of H3N2 microneedle vaccine. (d) SEM image of H3N2 microneedle vaccine array (scale bar = 500 μm).

Figure 3

(a) Optical image of the porcine skin surface after administration and removal of trypan blue microneedles (Scale bar = 1 mm). (b) Optical image of a cross-section of the microneedles passing through the blue dots (Scale bar = 500 μm). ‘SC’, ‘EP’, and ‘DE’ refer to stratum corneum, epidermis, and dermis, which are components of the skin layer, respectively. Trypan blue containing the vaccine was transferred to the epidermis and dermis of the skin.

Figure 4

(a) Fluorescence image on the microneedle surface observed with a confocal microscope at 15, 30, 60, 90, 150, 210, 270, and 330 min (scale bar = 200 μm). (b) Change in fluorescence intensity on the surface of microneedle tips from 15 to 330 min.

Figure 5

Brief schedule of in vivo experiment. BALB/c mice were immunized intradermally with 19–20 A/KS/17 H3N2 microneedle vaccines (3 μg/mouse) or intramuscularly with vaccine formulations (3 μg/mouse). Mice were infected with the mouse-adapted 15–16 A/SW/13 H3N2 virus at 3 weeks after vaccination (50MLD₅₀, 30 μl).

Figure 6

(a) Body weight change and (b) survival rate monitored for 14 days after challenge with mouse-adapted 15–16 A/SW/13 H3N2 virus in mice. MN-Tre20: Group administered transdermally with 20 times the amount of trehalose compared to the HA content of the vaccine, MN-Tre100: Group administered transdermally with 100 times the amount of trehalose compared to the HA content of the vaccine. IM: Group administered vaccine through intramuscular injection. (c) Neutralizing antibody titers of 19–20 A/KS/17 H3N2 vaccine microneedle and IM groups mouse sera at 14 days after challenge against A/PE/09 H3N2 virus, A/TX/12 H3N2, and A/SW/13 H3N2 virus. (d) Hemagglutination inhibition (HI) titers of 19–20 A/KS/17 H3N2 vaccine microneedle and IM groups mouse sera at 14 days after challenge against A/PE/09 H3N2 virus, A/TX/12 H3N2, and A/SW/13 H3N2 virus.