Intradermal vaccination with influenza virus-like particles by using microneedles induces protection superior to that with intramuscular immunization

Abstract

Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated. Microneedle vaccination of mice in the skin with a single dose of stabilized influenza VLPs induced 100% protection against challenge infection with a high lethal dose. In contrast, unstabilized influenza VLPs, as well as intramuscularly injected vaccines, provided inferior immunity and only partial protection (</=40%). The stabilized microneedle vaccination group showed IgG2a levels that were 1 order of magnitude higher than those of other groups and had the lowest lung viral titers after challenge. Also, levels of recall immune responses, including hemagglutination inhibition titers, neutralizing antibodies, and antibody-secreting plasma cells, were significantly higher after skin vaccination with stabilized formulations. Therefore, our results indicate that HA stabilization, combined with vaccination via the skin using a vaccine formulated as a solid microneedle patch, confers protection superior to that with intramuscular injection and enables potential dose-sparing effects which are reflected by pronounced increases in rapid recall immune responses against influenza virus.

FIG. 1.

Characterization of influenza VLPs and coating onto microneedles. (A) Coomassie blue staining of influenza VLPs. Influenza VLP proteins (total protein of VLPs in lane 1, 10 μg; lane 2, 5 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was stained with Coomassie blue. The second major band between the molecular masses of 37 kDa and 50 kDa indicates unidentified insect cell or baculovirus-derived protein. (B) Influenza HA and M1 proteins on Western blot. The equivalent amount (5 μg of protein) of inactivated A/PR8/1934 virus (lane 1) and VLPs (lane 2) were run on an SDS-PAGE gel, and influenza proteins (HA and M1) were visualized by blotting with mouse anti-A/PR8/1934 virus serum. (C) Determination of total protein in influenza VLPs dissolved from coated microneedles. Equivalent amounts of influenza VLPs were coated onto microneedles in the presence of trehalose (H1 VLPs +T) or absence of trehalose (H1 VLPs). (D) Hemagglutination activities in influenza VLP vaccines dissolved from coated microneedles without trehalose (H1 VLPs) or with trehalose (H1 VLPs+T).

FIG. 2.

Influenza A/PR8 virus-specific IgG responses. (A) Groups of mice (n = 12) immunized with a single dose of VLPs using different formulations and different routes of administration. Mice (n = 12 per group) were immunized with microneedles coated with 0.35 μg of influenza VLPs or by intramuscular injection with 0.35 μg of influenza VLPs dissolved from coated microneedles or by intramuscular injection with 0.35 μg of unprocessed intact inactivated influenza vaccine. At 7 weeks after vaccination, mice were challenged with a high lethal dose of A/PR8 virus (100 LD₅₀). Groups were divided as follows: MN, microneedle vaccine without trehalose; MN+T, microneedle vaccine with trehalose; IM, intramuscular injection of reconstituted vaccine without trehalose; IM+T, intramuscular injection of reconstituted vaccine from trehalose-formulated microneedles; Mock, microneedles with trehalose coating buffer only (without vaccine). (B) Virus-specific total IgG antibody responses. Blood samples were collected at weeks 1, 2, and 4 after vaccination. Virus-specific antibody responses measured by ELISA are expressed as the reciprocals of serum dilutions that give antibody titers 2-fold higher than the standard deviations of naïve serum samples. Statistical significance among groups compared are as follows: at week 1, for IM+T and IM-intact versus MN+T, MN, and IM, P < 0.05; at week 2, for MN+T versus MN, IM, and IM+T, P < 0.01; for IM-intact versus IM, P < 0.05; at week 4, for MN+T versus MN, IM+T, and IM-intact, P < 0.05; for MN+T versus IM, P < 0.01. (C) Lack of trehalose effect on inducing antibody responses. Virus-specific total IgG antibodies were determined by ELISA from blood samples collected at weeks 1, 2, and 4 after a single intramuscular VLP immunization (n = 6 BALB/c mice per group) with or without 15% trehalose. IM(T), 0.35 μg of VLPs plus trehalose; IM, 0.35 μg of VLPs.

FIG. 3.

Body weight changes, survival, lung virus titers, and lung IgG response after lethal challenge. (A) Body weight changes. (B) Survival rates. At week 7 after a single vaccination, mice (n = 12) were challenged with a lethal dose (A/PR8 virus, 100 LD₅₀) and were monitored daily to record body weight changes and survival rates. (C) Lung virus titers. Lungs from individual mice in each group (n = 6 out of 12 mice per group) were collected on day 4 postchallenge, and lung virus titers (number of PFU) were determined. (D) Lung IgG antibody responses. Lung IgG responses were determined from the lung extracts collected at day 4 postchallenge. OD, optical density.

FIG. 4.

Virus-specific isotype antibody responses. (A) Antibody isotypes before challenge. (B) Antibody isotypes after challenge. A/PR8 virus-specific isotype antibody responses (IgG1, IgG2a, and IgG2b) were determined before and after challenge. Results are expressed as averages of optical density readings at 450 nm (OD₄₅₀) with 100-fold diluted serum samples in each group of mice (n = 12). Groups of mice are described in the legend of Fig. 2. Antibody titers are provided in Table 2.

FIG. 5.

Hemagglutination inhibition titers. (A) HAI titers before challenge. (B) HAI titers at day 4 postchallenge. HAI titers were determined at weeks 1, 2, and 4 (W1, W2, and W4, respectively) postimmunization and at day 4 postchallenge. Significant differences were found among groups of mice: at week 4, for MN+T versus IM-intact, P < 0.05; for MN+T versus MN, and IM+T, P < 0.01; At postchallenge, for MN+T versus MN, IM, IM+T, and IM-intact, P < 0.001.

FIG. 6.

Neutralizing activities determined at day 4 postchallenge. Serial dilutions of serum samples were incubated with infectious influenza viruses and percentages of PFU were determined. Neutralizing activities were expressed as the percentage of plaque reduction compared to the medium control. Significant differences were found among groups of mice: for neutralizing titers at both 270 and 810 dilutions for plaque reduction, P was <0.001 between MN+T and other groups.

FIG. 7.

Antibody-secreting cells (ASC) induced by microneedle VLP vaccine. Spleen and bone marrow samples were collected from individual mice in each group (n = 6) at day 4 postchallenge. Spots representing antibody-secreting cells were determined by ELISPOT assay. Significant differences were found among the groups: in spleen (A) for MN+T versus MN, and IM+T, IM-intact, P < 0.05; for MN versus IM, P < 0.05; in bone marrow (B), for MN+T versus MN, IM+T, and IM-intact, P < 0.01; for MN versus IM, P < 0.05.

FIG. 8.

IFN-γ production in spleen cells. Groups of mice (n = 6) were immunized with stabilized microneedle VLP vaccines (MN+T) or with unprocessed influenza VLPs (IM-intact) and challenged with A/PR8 virus at week 7 after vaccination. To collect spleen cells, mice were sacrificed at day 4 postchallenge. Levels of IFN-γ secreted into splenocyte culture supernatants were determined using a cytokine ELISA. Cells (1 × 10⁶/well) from groups of mice (see legend of Fig. 2 for descriptions) were stimulated with A/PR8 HA-specific MHC-I and MHC-II peptides. Mock+Cha, group administered microneedles with trehalose coating buffer only (without vaccine) after challenge.