Long-term protective immunity from an influenza virus-like particle vaccine administered with a microneedle patch

Abstract

Skin vaccination with influenza virus-like particles (VLPs) using microneedles has been shown to induce protection similar to or better than that induced by intramuscular immunization. In this study, we examined the long-term protective efficacy of influenza (H1N1 A/PR/8/34) VLPs after skin vaccination using microneedle patches coated with the vaccine. Microneedle vaccination of mice in the skin induced 100% protection against lethal challenge infection with influenza A/PR/8/34 virus 14 months after a single vaccine dose. Influenza virus-specific total IgG response and hemagglutination inhibition (HAI) titers were maintained at high levels for over 1 year after microneedle vaccination. Microneedle vaccination also induced substantial levels of lung IgG and IgA antibody responses, and antibody-secreting plasma cells from spleen and bone marrow, as well as conferring effective control of lung viral loads, resulting in complete protection 14 months after vaccination. These strong and long-lasting immune responses were enabled in part by stabilization of the vaccine by formulation with trehalose during microneedle patch fabrication. Administration of the stabilized vaccine using microneedles was especially effective at enabling strong recall responses measured 4 days after lethal virus challenge, including increased HAI and antibody-secreting cells in the spleen and reduced viral titer and inflammatory response in the lung. The results in this study indicate that skin vaccination with VLP vaccine using a microneedle patch provides long-term protection against influenza in mice.

Fig 1

Influenza A/PR8 virus-specific IgG responses. Groups of mice (n = 12) immunized with a single dose of VLPs. Mice (n = 12 per group) were immunized with microneedles coated with 4 μg of influenza VLPs. At weeks 1 and 2 (W1 and W2) and months 1, 2, 3, 7, 10, and 13 (M1, M2, M3, M7, M10, and M13) after a single-dose vaccination, blood samples were collected. At month 14, mice were challenged with a high lethal dose of A/PR8 virus (10× LD₅₀) and IgG levels were measured 4 days after challenge (AC). The groups of immunized mice were designated V (microneedle vaccine without trehalose), VT (microneedle vaccine with trehalose as a stabilizer), and Mock (placebo microneedles with coating buffer only). Significant differences were detected between W1 and W2 (*, P < 0.05), W2 and M1 (**, P < 0.01), and M13 and AC (**, P < 0.01). Three independent experiments have been performed, and the data shown in the figures consist of the averages of several independent experiments. Data show averages ± standard errors of the means from 6 mice.

Fig 2

Hemagglutination inhibition titers. HAI titers were determined at weeks 0, 1, and 2 (W0, W1, and W2) and months 1, 2, 3, 7, 10, and 13 (M1, M2, M3, M7, M10, and M13) postimmunization and at day 4 postchallenge (AC). Significantly higher HAI titers were found from week 2, and higher titers were found from month 1 postvaccination and maintained until month 13 compared to week 1 or week 2 (*, P < 0.05; **, P < 0.01). V, microneedle vaccine without trehalose; VT, microneedle vaccine with trehalose. Data show averages ± standard errors of the means from 6 mice.

Fig 3

Body weight changes and survival. At month 14 after microneedle vaccination, mice were challenged with a lethal dose (A/PR8 virus, 10 LD₅₀) and were monitored daily to record body weight changes (A) and survival (B). V, microneedle vaccine without trehalose; VT, microneedle vaccine with trehalose; Mock, microneedles with coating buffer only. Data show averages ± standard errors of the means from 6 mice out of 12 mice per group.

Fig 4

Lung virus titers and lung IFN-γ and IL-6 responses. Lungs from individual mice in each group were collected on day 4 postchallenge, and lung virus titers (PFU) and lung IFN-γ and IL-6 responses were determined in the lung extracts at day 4 postchallenge. V, microneedle vaccine without trehalose; VT, microneedle vaccine with trehalose; Mock, microneedles with coating buffer only; Naïve, normal mice. Either no virus or lower virus titers were detected in VT and V groups (*, P < 0.01). No inflammatory cytokines IFN-γ and IL-6 were determined in the VT group compared to the V or mock control group (*, P < 0.01). Data show averages ± standard errors of the means from 6 mice.

Fig 5

Lung IgG (A) and IgA (B) responses after lethal challenge. Lung IgG and IgA responses were determined from the lung extracts collected at day 4 postchallenge. Significantly higher IgG and IgA antibody responses were found in the VT group than in the V (*, P < 0.01) or mock challenge control (*, P < 0.01) group. V, microneedle vaccine without trehalose; VT, microneedle vaccine with trehalose; Mock, microneedles with coating buffer only. Data show averages ± standard errors of the means from 6 mice.

Fig 6

Antibody-secreting cells (ASC) induced by microneedle VLP vaccination. Spleen (Sp) (A) and bone marrow (BM) (B) samples were collected from individual mice in each group at day 4 postchallenge. Significantly higher numbers of ASC were found in VT groups in both spleen and bone marrow than in V and mock control groups (P < 0.05). V, microneedle vaccine without trehalose; VT, microneedle vaccine with trehalose; Mock, microneedles with coating buffer only. Data show averages ± standard errors of the means from 6 mice.