INTRODUCTION
The skin acts as a mechanical barrier against the environment and provides the first line of defense against pathogens. The skin-associated lymphoid tissue (SALT), which was first described by Streilein (
34), represents an ideal target for skin-based vaccinations because it contains keratinocytes, Langerhans cells (LC), dermal dendritic cells (DDC), and T cells. Skin-associated antigen-presenting cells (APC) and keratinocytes have been shown to express several pattern recognition receptors (PRRs), including TLR9, TLR2, and TLR3 (
19,
20), which are important enhancers of the immune response. The LC and DDC present in the epidermis and dermis, respectively, have been shown to take up antigen, migrate to draining lymph nodes, and induce an antigen-specific adaptive immune response (
36). Therefore, targeting vaccine to the skin has been shown to enhance immunogenicity (
2,
3,
15,
21).
The 2009 swine-origin influenza pandemic illustrates the need for rapid and effective vaccination. Skin-based influenza vaccines have utilized approaches including tape stripping (
32), epidermal powder immunization (
4), and microneedles (MNs) (
9). These strategies have used diverse antigens including virus-like particles (VLP) (
25), inactivated influenza virus (
24), and hemagglutinin (HA) DNA vaccines (
1). However, a recombinant HA subunit vaccine, which has the advantage of rapid, high-yield production in an expression system with a high level of purity, has not been evaluated.
Microneedle arrays are designed to penetrate the stratum corneum, the outer layer of the skin, and deposit vaccine or drug into the epidermis and dermis. Using this approach, vaccine is applied as a coating to the surface of metal microneedles or encapsulated in a polymer making up the microneedle (
22). Delivery of soluble protein via coated microneedles suggests that antigen can be delivered quickly into the skin. Furthermore, this immunization method generated an antigen-specific antibody response that was superior to those induced by subcutaneous (s.c.) and intramuscular (i.m.) routes (
17,
18,
35,
41).
We previously demonstrated that a modified form of soluble HA (sHA) derived from the H3N2 influenza virus A/Aichi/2/68 containing the GCN4pII trimerization repeat stabilized the trimeric structure of the HA protein (
20). In the current study, we tested the hypothesis that MN delivery of the recombinant vaccine would induce levels of protective immune responses superior or at least equivalent to those induced by subcutaneous immunization. Specifically, we investigated the efficacy of skin delivery of stabilized trimeric influenza virus HA from the H3 virus A/Aichi/2/68 via coated microneedles. In addition we determined whether the stabilized trimeric sHA microneedle vaccination induces improved humoral and cellular responses compared with those induced by s.c. immunization. To compare the effects of immunization on postchallenge virus clearance, we determined virus lung titers after challenge infection. The work presented here illustrates the first analysis of transdermal delivery of a recombinant influenza virus subunit HA vaccine using microneedle technology.
MATERIALS AND METHODS
Recombinant trimeric soluble influenza virus hemagglutinin (sHA).
The HA gene derived from the H3N2 influenza virus A/Aichi/2/68 was truncated, and the trimeric GCN4pII sequence from
Saccharomyces cerevisiae, encoding the trimerization motif, was fused to the C terminus and cloned into the recombinant baculovirus (rBV) pFastBac1 expression vector as previously described (
37). rBVs carrying genes for the sHA and sHA.GCN4pII proteins were generated, and recombinant proteins were expressed and purified as previously described (
37). For purification, a His tag was added to the C terminus of each protein construct.
Microneedle fabrication and coating.
Microneedles were fabricated from stainless steel sheets (Trinity Brand Industries, Georgia; SS 304; 50 μm thick) by wet etching. Individual microneedles had a length of 750 μm and a width of 200 μm.
The coating solution was composed of 1% (wt/vol) carboxymethyl cellulose sodium salt (low viscosity, USP grade; Carbo-Mer, San Diego, CA), 0.5% (wt/vol) Lutrol F-68 NF (BASF, Mt. Olive, NJ), and soluble HA protein at 5 mg/ml. In order to reach a high vaccine concentration in coating solution, we used evaporation for 5 to 10 min at room temperature (+23°C) at the final step of preparation (Vacufuge; Eppendorf, New York). The coating step was performed by a dip coating process (
12). The apparatus had a chamber with coating solution and a microneedle holder which was attached to a linear stage that allowed the microneedle array to move in two dimensions with 0.4-μm accuracy. The coating was performed automatically and was monitored by a video camera (Prosilica, Massachusetts) attached to a computer.
To measure the amount of vaccine applied as a coating per row of microneedles, three rows out of each batch of coated microneedles were each submerged into 200 μl of phosphate-buffered saline (PBS) buffer for 5 min. The concentration of protein in the solution was measured by bicinchoninic acid (BCA) protein assay and was consistent within each batch (Pierce, Rockford, IL).
BS3 cross-linking.
The oligomeric status of purified recombinant proteins was determined using the water-soluble BS3 (bis[sulfosuccinimidyl] suberate) cross-linker (Pierce, Rockford, IL). Cross-linking was performed as described by De Fillette et al. (
8), with the following modifications. Briefly, 1 μg of recombinant protein was incubated at room temperature in the presence of BS3 (final concentration, 3 mM) for 30 min. Cross-linking was stopped by the addition of 1 M Tris-HCl, pH 8.0, to a final concentration of 50 mM. After cross-linking, proteins were separated on a 5 to 15% SDS-PAGE gel under reducing conditions (1% mercaptoethanol) and then blotted and analyzed by Western blotting using anti-six-His antibody and developed using ECL-Plus.
Vaccinations.
Female BALB/c mice (6 to 8 weeks old) were anesthetized with a xylazine-ketamine cocktail intraperitoneally (i.p.), and hair on the lower back was removed with a hair-removal cream (Nair; Church & Dwight Co.) 2 days prior to microneedle vaccination. Mice were anesthetized again, and microneedle arrays were inserted into the skin on days 0 (prime) and 28 (boost) and left in place for 5 min to allow the vaccine coating to dissolve.
Tissue and mucosal secretion collection.
Vaginal washes were collected as previously described (
7). For lung homogenates, mice were anesthetized with a xylazine-ketamine cocktail and perfused with sterile PBS to remove circulating blood. Lung tissue was removed, stored in PBS with a protease cocktail (Thermo Scientific, Rockford, IL), and homogenized.
Virus and challenge.
To determine vaccine efficacy, vaccinated mice were lightly anesthetized with isoflurane and challenged by slow intranasal inoculation of 50 μl containing 5 50% lethal doses (LD50) of live-mouse-adapted A/Aichi/2/68 (H3N2). Body weight loss and survival rates were monitored daily for 14 days postchallenge. Weight loss of ≥25% was used as the endpoint at which mice were euthanized according to IACUC guidelines.
Viral lung titers.
Mouse lungs were collected on day 4 postchallenge as previously described (
17). MDCK cells were maintained at a low passage number in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (HyClone, ThermoFisher, Rockford, IL). Plaque assays were performed on lung homogenates from challenged mice as previously described (
31).
ELISA.
IgG enzyme-linked immunosorbent assay (ELISA) was performed on serum and lung homogenates as previously described (
17). All horseradish peroxidase (HRP)-conjugated secondary antibodies to mouse IgG, IgG1, IgG2a, IgG2b, and IgG3 were purchased from Southern Biotechnology Associates (Birmingham, AL). Briefly, 96-well immunoplates (Nunc Co., Rochester, NY) were coated overnight at 4°C with 4 μg of inactivated A/Aichi/2/68 virus per well. Plates were washed with PBS-Tween (0.05%) and blocked with PBS-Tween supplemented with 3% bovine serum albumin (BSA). Sera were diluted 1:100 and incubated for 1.5 h at 37°C. Plates were washed 3 times with PBS-Tween (0.05%) and incubated for 1.5 h at 37°C in a 1:1,000 dilution of goat, anti-mouse HRP-conjugated secondary antibody (Southern Biotechnology, Birmingham, AL). Plates were washed 3 times with PBS-Tween (0.05%),
o-phenylenediamine (OPD) substrate (Invitrogen, Carlsbad, CA) was added to each well, and color was allowed to develop. Color development was stopped using 1 M phosphoric acid. Absorbances were read at 450 nm on a Bio-Rad Model 680 microplate reader. Immunoglobulin concentrations were determined by linear regression of a standard curve of known concentrations.
The IgA ELISA procedure was modified from the work of Rodriguez et al. (
28). Briefly, 96-well immunoplates (Nunc Co., Rochester, NY) were coated as described above and blocked with 1% BSA. Vaginal washes were diluted 1:5, and sera were diluted 1:100 and then incubated overnight at 4°C. A 1:500 dilution of biotin-conjugated rat anti-mouse IgA was used, and streptavidin-HRP followed by OPD substrate was used as a detection method (obtained from BD Pharmingen, San Jose, CA).
HAI and microneutralization.
Hemagglutination inhibition (HAI) tests were performed on vaccinated animal sera based on the WHO protocol (
38). Briefly, sera were treated with receptor-destroying enzyme (Denka Seiken Co. Ltd., Tokyo, Japan) for 16 h at 37°C and then heat inactivated for 30 min at 56°C. Treated sera were diluted to a final concentration of 1:10 in PBS and incubated with packed chicken erythrocytes (RBC) for 1 h at 4°C to remove cryoglobulins. Treated sera were serially diluted and incubated with 4 HA units of A/Aichi/2/68 virus for 30 min at room temperature. An equal volume of 0.5% chicken RBC was added to each well and incubated for 30 min at room temperature. The HAI titer was read as the reciprocal of the highest dilution of serum that inhibited hemagglutination. Values were expressed as the geometric mean with a 95% confidence interval.
The microneutralization assay was performed as described by Rowe et al. (
29) with modifications. Briefly, mouse sera were heat inactivated for 30 min at 56°C and serially diluted in virus diluent (DMEM plus 1% BSA) in a 96-well tissue culture plate. Virus (200 50% tissue culture infective doses [TCID
50]) was added to diluted serum in virus diluent supplemented with 2 μg/ml of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin and incubated for 2 h at 37°C. Freshly trypsinized MDCK cells were added to all wells and incubated overnight at 37°C. Cells were then fixed in 80% acetone-PBS and washed 3 times with PBS-Tween (0.05% Tween 20). A 1:2,000 dilution of biotin-conjugated anti-influenza A virus nucleoprotein (clone A3) (Milipore, Billerica, MA) and streptavidin-HRP was added for the detection of infected cells. For each plate, the 50% specific signal value was calculated as follows: [(average of virus-only wells) − (average of cell-only well)]/2 + average of cell-only well. The 50% endpoint neutralization titer is reported as the last dilution to score below the 50% specific signal value.
Cellular immune responses.
Single-cell suspensions were prepared from spleens of mice 14 days postvaccination or from those of naïve mice, by mincing the tissue through a 70-μm cell strainer (BD Falcon), followed by incubation in red blood cell lysing buffer (Sigma) to remove red blood cells. CD4+ T cells were purified by negative selection using BD iMag magnetic cell separation (BD Biosciences, San Jose, CA). Naïve splenocytes were treated with mitomycin C for 30 min and used as accessory cells after incubation with 20, 5, or 0 μg of vaccine in complete RPMI medium overnight at 37°C in a 5% CO2 incubator. Purified CD4+ T cells were added to accessory cells at a ratio of 2:1 (responder/accessory cells) in complete RPMI medium supplemented with 30 U of recombinant human interleukin-2 (IL-2; BD Biosciences, San Jose, CA). In vitro antigen-specific stimulation was measured after incubation for 5 days by intracellular cytokine staining.
Antibodies and flow cytometry.
Cells were washed with PBS-1% BSA buffer and surface stained with fluorochrome-conjugated antibodies to CD4 and CD3, followed by intracellular staining of gamma interferon (IFN-γ) and IL-4. Antibodies were purchased from eBiosciences and BD Biosciences. For intracellular cytokine staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm manufacturer's protocol and reagents (BD Biosciences, San Jose, CA). The data were acquired on a BD LSR-II flow cytometer and analyzed with FlowJo Software (Tree Star, Inc.; v7.6.1).
Statistics.
Statistical analysis was done using the one-way analysis of variance (ANOVA) of grouped data with GraphPad Prism5 software. Survival curves were analyzed using the log rank test. All data followed normal Gaussian distributions unless otherwise noted. Viral lung titers were analyzed using one-way ANOVA. A P value of less than 0.05 was defined as a significant difference.
DISCUSSION
The recent H1N1 influenza pandemic has illustrated the need for convenient alternatives to traditional influenza vaccine. Previous studies from our lab and others have demonstrated the successful use of both coated and dissolving microneedles for transdermal delivery of influenza vaccines using virus-like particles or inactivated virus (
17,
23,
35). However, little information is available on using this approach to deliver a protein subunit vaccine. Microneedles have the advantage of delivering vaccine to an area rich in APC such as the epidermis and dermis without causing pain (
11). These vaccinations induced robust antibody and cellular responses, resulting in protection against lethal challenge with several subtypes of influenza virus.
We have previously demonstrated that the influenza virus HA protein modified at the C terminus with the trimerization repeat, GCN4pII, generates stable trimeric soluble HA. Following subcutaneous vaccination, the modified trimeric sHA was able to induce higher virus-specific serum IgG and HAI titers (
37). These enhanced humoral responses translated to a greater vaccine-induced protection following challenge with homologous virus. In the present study, we have tested the hypothesis that MN-mediated delivery via the transdermal route would be effective in enhancing the immune response to an influenza virus subunit vaccine. We demonstrated that the structure of the recombinant trimeric sHA was preserved when 15% (wt/vol) trehalose was included in the coating formulation. However, the unmodified sHA remained as a mixture of trimers, dimers, and monomers in the presence or absence of 15% trehalose. A stabilizing effect of trehalose on the HA activity of influenza virus was previously observed when coating microneedles with the whole inactivated virus (
24).
Microneedle vaccination with the modified trimeric sHA induced an improved humoral systemic response compared with that induced by unmodified sHA as determined by ELISA. This improved response to trimeric sHA over that to unmodified sHA was particularly evident in HAI and microneutralization titers. These results strongly support the view that the trimeric sHA induces higher levels of functional antibodies than does the unmodified sHA, while both recombinant proteins induce binding antibodies as measured by ELISA. This difference in induction of functional antibodies could be attributed to the better presentation of native epitopes present in the stabilized trimeric structure of the modified sHA corresponding to those in the live virus. The unmodified sHA, in contrast, probably presents additional epitopes found at the interfaces between monomers, which are not exposed in the native virus. Importantly, sHA.GCN4pII immunization also induced 100% protection while sHA vaccination provided only partial protection.
The postchallenge viral lung titers observed in mice vaccinated with recombinant soluble HA-coated microneedles demonstrate that the soluble trimeric HA induced immune responses that were efficient at clearing replicating virus. In addition, comparing the postchallenge viral lung titers of mice vaccinated subcutaneously and of mice vaccinated with coated microneedles indicated that the skin-based immunization resulted in greater clearance of replicating virus independently of the recombinant protein used. These data support the conclusion that skin-based delivery of a recombinant protein antigen using microneedles increases the efficacy of such vaccines.
Further investigation of the vaccine-induced responses indicated that sHA.GCN4pII induced a balanced IgG subtype profile, contrasting with sHA, which induces a predominantly IgG1 profile. The cytokines expressed by CD4
+ helper T cells influence the B cell isotype switch; therefore, we assessed the CD4 T cell phenotype in terms of a Th1 cytokine (IFN-γ) and a Th2 cytokine (IL-4) by intracellular cytokine staining. We observed that both recombinant sHA vaccines induced both IFN-γ-producing T cells and IL-4-producing T cells. Notably, sHA.GCN4pII induced a higher frequency of IFN-γ
+ CD4
+ T cells. Helper T cells which express IFN-γ have been shown to play an important role in inducing isotype switching to IgG2 in BALB/c mice (
5). In addition, IFN-γ induces an antiviral state, upregulation of major histocompatibility complex (MHC) classes I and II, and costimulatory molecules on APC (
30). Here, we showed that both Th1 and Th2 effector cells are generated after antigen delivery to the skin and that Th1 effector CD4 T cells are predominant in the sHA.GCN4pII group, correlating with the highest vaccine efficacy. The microneedle route for vaccination has the potential to enhance the CD4
+ T cell response over that with traditional routes of immunization (
10). Thus, the role of CD4
+ T cells should be further examined to determine their significance in determining the efficacy of transdermal vaccination.
For influenza virus infection, secretory IgA (sIgA) has been shown to bind and neutralize virus in the upper respiratory tract and nasal cavity (
27). In addition, transcytosis of influenza virus-specific IgG across the lung epithelial cell layer via FcRn plays an important role in preventing lung pathology (
27,
33). We observed that mice receiving sHA.GCN4pII induced higher levels of tissue-localized virus-specific IgG and mucosal sIgA, suggesting that microneedle vaccination induced the critical antibody responses at the site of infection. The differences observed at the mucosal site could be the result of lower immunogenicity of sHA, or perhaps sHA results in a homing pattern different from that of sHA.GCN4pII.
These results demonstrate the efficacy of transdermal vaccinations using recombinant sHA derived from the A/Aichi/2/68 (H3N2) virus. Microneedle vaccination with the stabilized HA trimers induced protective immune responses in mice. It is noteworthy that in comparison to our previous work with subcutaneous vaccination, the MN immunization with the trimeric HA induced similar serum IgG and HAI titers and induced the same level of protection against lethal challenge as that in the s.c. vaccinated mice. Comparisons between intramuscular, intranasal, subcutaneous, and microneedle vaccinations using virus-like particles (VLP) and inactivated virus antigens have all demonstrated the enhanced immunogenicity of the transdermal route (
16,
17,
23). Taken together, the results emphasize the conclusion that the delivery route as well as the nature of the vaccine antigen is important in determining the efficacy of an influenza vaccine.
ACKNOWLEDGMENTS
We thank Erin-Joi Collins for her valuable assistance in the preparation of the manuscript.
This project was supported in part by NIH grants EB006369 (M.R.P.) and AI074579 (R.W.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
M.R.P. serves as a consultant and is an inventor on patents licensed to companies developing microneedle-based products. This possible conflict of interest has been disclosed and is being managed by Georgia Tech and Emory University.