Microneedle vaccination with stabilized recombinant influenza virus hemagglutinin induces improved protective immunity

Abstract

The emergence of the swine-origin 2009 influenza pandemic illustrates the need for improved vaccine production and delivery strategies. Skin-based immunization represents an attractive alternative to traditional hypodermic needle vaccination routes. Microneedles (MNs) can deliver vaccine to the epidermis and dermis, which are rich in antigen-presenting cells (APC) such as Langerhans cells and dermal dendritic cells. Previous studies using coated or dissolvable microneedles emphasized the use of inactivated influenza virus or virus-like particles as skin-based vaccines. However, most currently available influenza vaccines consist of solubilized viral protein antigens. Here we test the hypothesis that a recombinant subunit influenza vaccine can be delivered to the skin by coated microneedles and can induce protective immunity. We found that mice vaccinated via MN delivery with a stabilized recombinant trimeric soluble hemagglutinin (sHA) derived from A/Aichi/2/68 (H3) virus had significantly higher immune responses than did mice vaccinated with unmodified sHA. These mice were fully protected against a lethal challenge with influenza virus. Analysis of postchallenge lung titers showed that MN-immunized mice had completely cleared the virus from their lungs, in contrast to mice given the same vaccine by a standard subcutaneous route. In addition, we observed a higher ratio of antigen-specific Th1 cells in trimeric sHA-vaccinated mice and a greater mucosal antibody response. Our data therefore demonstrate the improved efficacy of a skin-based recombinant subunit influenza vaccine and emphasize the advantage of this route of vaccination for a protein subunit vaccine.

Fig. 1.

Trehalose-supplemented microneedle coating solution preserves the sHA.GCN4pII trimeric structure. BS3 cross-linking of recombinant baculovirus produced sHA and sHA.GCN4pII after dissolution of the coating from the microneedles coated in the presence or absence of 15% trehalose. Lane 1, sHA without 15% trehalose; lane 2, sHA with 15% trehalose; lane 3, sHA.GCN4pII without 15% trehalose; lane 4, sHA.GCN4pII with 15% trehalose. Cross-linked proteins were separated on 5 to 15% SDS-PAGE gels, and Western blotting was performed using mouse anti-His primary antibody.

Fig. 2.

Microneedle vaccination with sHA.GCN4pII induces higher serum levels of A/Aichi/2/68-specific IgG. Mice were primed and boosted 3 weeks later with 3 μg of sHA or sHA.GCN4pII or blank microneedles (naïve). Blood was collected 28 days after priming (prime) and 28 days after boosting (boost). ELISA plates were coated with 4 μg/ml of A/Aichi/2/68 virus, and antigen-specific serum IgG for prime and boost was measured by ELISA. HRP-conjugated goat anti-mouse IgG was used for detection (n = 12).

Fig. 3.

IgG subtype as determined by ELISA. Sera collected 21 days after boosting with antigen-coated microneedles were tested for antigen-specific IgG subclasses by ELISA as described in Materials and Methods. ELISA plates were coated with 4 μg/ml of A/Aichi/2/68, and antigen-specific IgG subclasses were detected using HRP-conjugated goat anti-mouse IgG1 (A), IgG2a (B), or IgG2b (C) (n = 6 per group).

Fig. 4.

Transdermal vaccination with sHA.GCN4pII induces higher HAI and microneutralization titers. (A) Hemagglutination inhibition testing of prime and boost sera was performed as described in Materials and Methods using MDCK cell-grown A/Aichi/2/68 virus. HAI titers were reported as the reciprocal of the last dilution of serum to inhibit agglutination of RBC. (B) A microneutralization assay using boost serum was performed as described in Materials and Methods with 200 TCID₅₀ of MDCK cell-grown A/Aichi/2/68. Virus infection was detected using biotin-conjugated antinucleoprotein monoclonal antibody and streptavidin-HRP. Fifty percent endpoint titers were reported as the dilution of serum able to inhibit 50% of signal observed in virus-infected MDCK cells.

Fig. 5.

sHA.GCN4pII induces higher serum IgA levels and mucosal antibody responses. IgA ELISA was performed as described in Materials and Methods. ELISA plates were coated with 4 μg/ml of A/Aichi/2/68, and IgA was detected using biotin-conjugated rat anti-mouse and streptavidin-HRP. IgG ELISA was performed as described previously. IgA was assayed in vaginal washes (A) and serum (B). IgG was assayed in lung homogenate 21 days after boosting (C).

Fig. 6.

MN immunization with trimeric sHA induces improved protective immune responses against lethal challenge with mouse-adapted A/Aichi/2/68. (A and B) Mice primed and boosted were challenged with 5 LD₅₀ of mouse-adapted H3N2 A/Aichi/2/68. Body weights and survival were followed for 14 days postchallenge. Symbols: circles, sHA; squares, sHA.GCN4; inverted triangles, PBS. Open symbols with a dashed line indicate percent mean initial body weight of surviving mice for each group. Closed symbols with a solid line indicate percent mean initial body weight of mice showing signs of morbidity (n = 6). (C) Sublethal infection of microneedle-vaccinated mice. Body weights were followed for 14 days postinfection (n = 6 per group). Symbols: circles, sHA; squares, sHA.GCN4; triangles, PBS.

Fig. 7.

MN vaccination with trimeric sHA induces improved clearance of virus from the lung. Mice primed and boosted with 3 μg of recombinant soluble HA by the microneedle or subcutaneous routes were challenged with 5 LD₅₀ of mouse-adapted A/Aichi/2/68. Lungs were removed on day 4 postchallenge and processed for virus plaque assays. Lung viral titers are reported as the arithmetic mean ± standard deviation (n = 3). ND, not detectable.

Fig. 8.

Microneedle vaccination with trimeric recombinant sHA induces greater Th1 responses. Purified CD4⁺ T cells were restimulated with 20, 5, or 0 μg/ml of vaccine for 5 days supplemented with 30 U/ml of recombinant human IL-2. (A and B) On day 5, restimulated cells were fixed and stained for surface markers and the indicated cytokines. (C) Ratios of frequencies of IFN-γ⁺ to IL-4⁺ helper T cells in microneedle-vaccinated mice.