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. 2012 Apr 17;20(7):1472–1480. doi: 10.1038/mt.2012.69

DNA Vaccination in the Skin Using Microneedles Improves Protection Against Influenza

Jae-Min Song 1,2, Yeu-Chun Kim 3,4, Eunju O 1, Richard W Compans 5,*, Mark R Prausnitz 3,*, Sang-Moo Kang 1,*
PMCID: PMC3392990  PMID: 22508490

Abstract

In this study, we tested the hypothesis that DNA vaccination in the skin using microneedles improves protective immunity compared to conventional intramuscular (IM) injection of a plasmid DNA vaccine encoding the influenza hemagglutinin (HA). In vivo fluorescence imaging demonstrated the expression of a reporter gene delivered to the skin using a solid microneedle patch coated with plasmid DNA. Vaccination at a low dose (3 µg HA DNA) using microneedles generated significantly stronger humoral immune responses and better protective responses post-challenge compared to IM vaccination at either low or high (10 µg HA DNA) dose. Vaccination using microneedles at a high (10 µg) dose further generated improved post-challenge protection, as measured by survival, recall antibody-secreting cell responses in spleen and bone marrow, and interferon (IFN)-γ cytokine T-cell responses. This study demonstrates that DNA vaccination in the skin using microneedles induces higher humoral and cellular immune responses as well as improves protective immunity compared to conventional IM injection of HA DNA vaccine.

Introduction

There are often limitations in manufacturing capacity and production time of conventional vaccines, in particular, during the outbreaks of pandemics. Plasmid DNA vaccines are relatively easy to produce and may have the potential to prevent diseases for which there are no currently available vaccines, thus representing an attractive vaccine strategy. The safety and immunogenicity of DNA vaccines have been demonstrated in clinical studies involving monovalent influenza DNA vaccines.1 Immunization with DNA vaccines using needle and syringe injections has been the most common method of administration. Recent animal and clinical studies demonstrated that DNA vaccine priming significantly enhanced the efficacy and breadth of subsequent influenza vaccination.2,3,4,5 Despite the potential attractive features of DNA vaccines, their low immunogenicity has been an obstacle for approving their application.6

Vaccination in the skin is receiving increased attention as an alternate route of immunization. The skin layers are known to be highly populated with professional antigen-presenting cells, which play an important role in effectively inducing immune responses.7,8 Therefore, it is possible that delivering DNA vaccines to the highly immunoresponsive layers of the skin may improve their immunogenicity. However, the outer stratum corneum layer of skin represents a significant barrier to the delivery of genes and other high molecular weight agents, so improved delivery strategies are required to overcome this skin exclusion property.

The conventional method of skin vaccination involves intradermal injection with a hypodermic needle. This method, however, requires special training, is painful and is unreliable at targeting the skin.9 Bifurcated needles and multipuncture devices such as dermaroller have also been used, but suffer from multiple doses, low and poorly reproducible delivery efficiency.10 Alternate approaches for delivering vaccines to the skin have been reported, including physical disruption methods such as tape-stripping, microdermabrasion, jet injection, or electroporation to breach or permeate the skin's stratum corneum barrier.11,12,13,14 Gene gun, microdermabrasion, and electroporation require complex vaccination equipment and high cost, which limit their widespread application to humans. Therefore, it is a high priority to develop a convenient and low-cost method for delivering DNA vaccines through the skin.

Microneedles measure hundreds of microns in length and can be precoated with vaccines that rapidly dissolve in the skin's interstitial fluid.15 Coated microneedles are especially attractive as a method for rapid administration of vaccines and can be prepared as adhesive patch-like devices for self-application with little or no training.15 Recently, microneedles prepared with influenza vaccines in a dry state were demonstrated to induce protective immune responses.16,17,18 DNA vaccines have also been administered using microneedles, such as model DNA vaccines against hepatitis C.19

We hypothesized that microneedles coated with influenza hemagglutinin (HA) DNA vaccine for delivery to the skin would improve protective immunity compared to conventional intramuscular (IM) DNA immunization. In the present study, we tested this hypothesis by investigating the immunogenicity and protective efficacy of DNA microneedle vaccination. To our knowledge, this study provides the first evidence that delivery of DNA vaccines to the skin via dry-coated microneedles is superior to conventional IM immunization in inducing binding antibodies, antibody-secreting recall responses, and interferon (IFN)-γ secreting T cells, as well as improved protection.

Results

Delivery of plasmid DNA to mouse skin using coated microneedles

Concentrated DNA was effectively coated on the surfaces of metal microneedles (Figure 1). Fluorescently labeled DNA was observed on the surfaces of microneedle shafts after coating and was imaged by white light (Figure 1a) and fluorescence (Figure 1b) microscopy. To determine the kinetics of plasmid DNA delivery into the skin, microneedles were imaged after various periods of insertion time (Figure 1c–f). As shown in Figure 1, DNA was rapidly dissolved off microneedles into the skin within 5 minutes of insertion.

Figure 1.

Figure 1

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Kinetics of influenza HA DNA vaccine delivery from coated microneedles into skin. (a) White light and (b) fluorescence images of a microneedle coated with fluorescently labeled HA DNA before insertion and fluorescence images of a microneedle after insertion into human cadaver skin for (c) 0.5 minute, (d) 1 minute, (e) 3 minutes, and (f) 5 minutes. Bar = 250 µm. These images are representative of data from two replicate experiments. HA, hemagglutinin.

For effective DNA vaccination, delivered DNA should transfect skin cells. Therefore, we first tested delivery of plasmid DNA expressing a reporter luciferase. We inserted two types of microneedles; one as a negative control without DNA coating and the other coated with the plasmid DNA encoding luciferase. One day after microneedle delivery, mice were visualized by an in vivo imaging system. As shown in Figure 2, mice that received microneedles coated with plasmid DNA displayed a strong bioluminescence signal compared to control mouse. These results suggest that plasmid DNA coated onto microneedles is rapidly dissolved into the skin and enter cells to express its encoded gene.

Figure 2.

Figure 2

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Analysis of gene expression after microneedle delivery of reporter DNA. The mice were inserted with (a) microneedle arrays only and (b) microneedle arrays coated with 3 µg phMGFP/CBL plasmid DNA. Microneedle arrays were inserted for 20 minutes and mice were imaged 24 hours after DNA delivery by microneedles using the in vivo imaging system (IVIS). Color presentation indicates the intensity of relative bioluminescence, as shown in the bar. These images are representative of data from two replicate experiments. CBL, click beetle luciferase.

Immunization with influenza DNA vaccine in the skin using microneedles shows enhanced immunogenicity compared to IM vaccination

To prepare a HA DNA vaccine-coated microneedle patch, microneedle arrays were dip-coated in a solution of DNA vaccine expressing HA of A/PR8 virus20 to achieve doses of 3 or 10 µg HA DNA vaccine per mouse. To assess vaccine immunogenicity, groups of BALB/c mice (13 per group) were immunized with 3 or 10 µg of DNA vaccine either using coated microneedles applied to the skin (MN-3, MN-10) or using IM injection(IM-3 and IM-10) at 0 and 4 weeks.

At 3 weeks after each vaccination, we determined levels of A/PR8 virus-specific antibodies by enzyme-linked immunosorbent assay (ELISA) (Figure 3a). At the 3 µg dose, the IM injection (IM-3) failed to induce virus-specific antibody response even after two immunizations. In contrast, the group that was immunized with 3 µg DNA in the skin via coated microneedles (MN-3) raised a detectable anti-PR8–specific IgG response even after a single vaccination, and the IgG level was increased more than fourfold after two immunizations (Figure 3a). This group (MN-3) induced similar or higher levels of A/PR8-specific antibody responses compared to those induced by 10 µg of DNA vaccine injected intramuscularly (IM-10) (P = 0.5418). The 10 µg-dose microneedle group (MN-10) induced three to fourfold higher IgG antibody levels than the 10 µg IM group (IM-10) after boost (Figure 3a). Therefore, these results indicate that a smaller dose of microneedle DNA vaccines delivered to the skin would be effectively immunogenic compared to IM immunization, because the MN-3 group had stronger antibody responses than the IM-10 group.

Figure 3.

Figure 3

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Influenza-specific total IgG, isotype (IgG1, IgG2a) antibody, and HAI activity responses in sera. Female BALB/c mice were immunized in a regimen of prime and boost at a 4-week interval with 3 µg or 10 µg of pCAG-HA WPRE DNA immunization via intramuscular injection (IM) or microneedles (MN). (a) Total IgG. Sera were collected on 3 weeks after prime and boost vaccination and IgG antibodies were measured by ELISA. Cut-off values (0.140) were set as mean OD ± 2 SD of naive control sera. Error bars indicates mean ± SD. N = 10 for all immunized groups and 5 for control. Asterisks indicate titers that are significantly different between groups (**P < 0.01). (b) IgG isotype titers. Cut-off values (0.128) were set as mean OD ± 2 SD of naive control sera. (c) IgG2a/IgG1 ratios. Isotype antibody ratios (IgG2a/IgG1) were calculated based on ELISA results. (d) HAI titers were detected by hemagglutination inhibition assay. Detection limit was 4. Error bars indicate mean ± SD. N = 10 for all immunized groups and 5 for control. IM_3: intramuscular immunization with 3 µg DNA vaccine, MN_3: microneedle immunization with 3 µg of DNA vaccine, IM_10: intramuscular immunization with 10 µg of DNA vaccine, MN_10: microneedle immunization with 10 µg DNA vaccine. Control: PBS buffer-coated microneedle vaccination. ELISA, enzyme-linked immunosorbent assay; HAI, hemagglutination inhibition; OD, optical density; PBS, phosphate-buffered saline.

Microneedle vaccination in the skin with DNA vaccine induces higher levels of IgG2a isotype antibody and HAI activity

To better understand types of immune responses induced, the IgG isotypes raised by DNA vaccination via the MN and IM routes were also determined by ELISA. As shown in Figure 3b, MN vaccination induced IgG2a antibodies as the predominant isotype, which represents a T helper type 1(Th1) response. This Th1-biased response induced by MN vaccination is similar to that observed with typical IM immunization using DNA vaccines. In contrast, IgG1 isotype antibodies were found at only marginal levels in all groups (Figure 3b). The groups of mice that received a 10 µg DNA dose showed higher ratios of IgG2a to IgG1 isotype antibodies than the 3 µg groups for both MN and IM vaccination (Figure 3c).

Next, we determined the hemagglutination inhibition (HAI) titers as a HA-specific functional antibody response. No significant HAI titers were detected in IM groups with 3 or 10 µg DNA dose (Figure 3d). Importantly, low but significant levels of HAI antibodies were detected in immune sera from the 10 µg dose MN vaccinated group (MN-10, Figure 3d). Altogether, these results suggest that microneedle delivery of influenza HA DNA vaccine induces predominantly IgG2a isotype antibody and is more effective in inducing humoral immune responses than IM injection.

Microneedle vaccinated mice exhibit improved protective immunity

The main purpose of vaccination is to provide protective immunity. To determine the protective efficacy of microneedle DNA vaccination, immunized and mock control (microneedles coated with phosphate-buffered saline (PBS) buffer only) mice were intranasally challenged with a lethal dose (5 × LD50) of A/PR8 virus at 4 weeks after boost immunization. We monitored survival rates and body weight changes of mice post-challenge daily for 2 weeks to evaluate the protective capability of the HA DNA vaccine. As shown in Figure 4a, all mice in the mock control and IM immunized group with 3 µg of DNA (IM-3) rapidly lost body weight and died by day 7 post-challenge. The IM-immunized group with 10 µg of HA DNA vaccine (IM-10) and MN-immunized group with 3 µg of HA DNA vaccine (MN-3) showed partial protection resulting in 20% survival rates. The surviving mice in these groups displayed body weight loss up to 19% (Figure 4a,b). Importantly, the group of mice that received microneedle vaccination with 10 µg of HA DNA (MN-10) was 100% protected with only a transient body weight loss of 6% at day 7. In support of superior protection by microneedle vaccination, the MN group (MN-10) also showed partial protection against a high dose (25 LD50) of challenge virus but the IM group with same DNA vaccine dose (IM-10) was not protected with 0% survival (data not shown).

Figure 4.

Figure 4

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DNA vaccine-coated microneedle vaccination induces improved protective immunity. Groups of mice that were immunized via intramuscular injection (IM) or microneedle (MN) delivery to the skin were intranasally challenged with a lethal dose (5 × LD50) of homologous influenza A/PR8 (H1N1) virus 4 weeks after boost (N = 13). (a) Average body weight changes and (b) survival rates were monitored for 14 days. Error bars indicates SD. P value means a significant difference between MN_10 and all other groups in marked duration. LD, lethal dose.

Lung viral titers and inflammatory cytokine levels at an early time point post-challenge would be informative for assessing the vaccine efficacy. Four days after challenge, three mice from each group were killed and lung tissues were harvested for titration of viral titers and the inflammatory cytokine IFN-γ. After microneedle vaccination with 10 µg DNA (MN-10), mice had 12-fold lower lung viral titers compared to that in the control group (Figure 5a, P < 0.01). In contrast, the 10 µg IM group (IM-10) had only two to threefold lower lung viral titers compared to the control group (Figure 5a, P < 0.05). Also, the 3 µg MN group (MN-3) showed lower lung viral titers than those in the 3 µg IM group (IM-3) (Figure 5a, P < 0.05).

Figure 5.

Figure 5

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Microneedle delivery of DNA vaccine to the skin is effective in reducing lung viral titers and inflammatory cytokine IFN-γ (a) Lung viral titers. (b) IFN-γ in lung extracts. Lung viral titers were determined by a plaque assay at day 4 after challenge (N = 4) and IFN-γ was determined by cytokine ELISA (N = 4). IM_3 and IM_10, intramuscular 3 and 10 µg DNA vaccine; MN_3 and MN_10, microneedle 3 and 10 µg DNA vaccine; control, no vaccine. Data represent mean ± SD. Asterisk indicates significant difference between immunized and control groups (*P < 0.05, **P < 0.01). IFN, interferon; IM, intramuscular; MN, microneedle.

As an indicator of lung inflammation due to influenza viral replication, proinflammatory IFN-γ cytokine levels in lung extracts were detected at lowest levels in the microneedle vaccination group with 10 µg of HA DNA (MN-10) (Figure 5b, P < 0.01). Even the low-dose microneedle group (MN-3) showed a lower level of IFN-γ compared to that observed after IM immunization with 10 µg DNA vaccine dose (IM-10) (Figure 5b, P < 0.01). Taken together, these results show that microneedle vaccination with influenza HA DNA provided improved protective immunity against lethal influenza challenge and is superior to the conventional IM injection of a DNA vaccine in controlling lung viral replication and in inducing protective immunity.

Enhancement of recall humoral immune responses by microneedle DNA vaccination

The dose range of DNA vaccines used in this study was not high enough to induce significant levels of cellular immune responses after IM vaccination. Thus, to understand the induction of host cellular immunity by microneedle vaccination, we compared the recall antibody-secreting cell responses in bone marrow and spleen. At day 4 post-challenge, bone marrow cells were isolated from challenged mice and incubated in culture plate with or without antigenic stimulation with inactivated A/PR8 virus for 2 days. Culture supernatants were harvested at day 1 and 2, and the A/PR8-specific IgG levels were measured by ELISA. Significant levels of IgG antibodies secreted by bone marrow cells was detected in the microneedle group vaccinated with 10 µg HA DNA (MN-10) in in vitro culture supernatants at day 2 (P < 0.05) in the absence (Figure 6a) or presence (Figure 6b) of antigenic stimulation. The in vitro production of antibodies was observed regardless of antigenic stimulation, indicating the induction of antibody-secreting plasma cells in bone marrow from the microneedle vaccination group but not in the IM groups.

Figure 6.

Figure 6

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Microneedle DNA vaccination induces enhanced recall humoral immune responses. (a,b) Bone marrow and (c,d) spleen cells were isolated from mice at day 4 post-challenge (N = 4) and were incubated in the absence (as shown in a,c) or in the presence (as shown in b,d) of inactivated A/PR8 virus antigen coated on the culture plates for in vitro stimulation. Culture supernatants were harvested at different time points as shown in figures. Influenza-specific IgG levels were determined by ELISA. Data represent mean ± SD. Asterisk indicates significant difference (*P < 0.05, **P < 0.01). BM, bone marrow; ELISA, enzyme-linked immunosorbent assay; IM, intramuscular; MN, microneedle; OD, optical density.

In contrast, spleen cells did not show detectable levels of antibodies in the absence of antigenic stimulation (Figure 6c). Under conditions of antigenic stimulation, in vitro antibody production by splenocytes was observed at significant levels after an extended time of culture for 6 days from the MN-10 group of mice but was not detected at day 1 (Figure 6d). These results indicate the induction of memory B cells in spleens from mice that received microneedle DNA vaccination, which can differentiate into antibody-secreting plasma cells upon antigenic stimulation. Therefore, microneedle skin immunization is more effective in inducing recall humoral immune responses than IM DNA injection.

Enhancement of recall cellular immune responses by microneedle DNA vaccination

To study recall cellular immune responses, we determined the cytokine-producing cell populations in spleen by the ELISPOT assay. Splenocytes at day 4 post-challenge were stimulated with HA major histocompatibility complex (MHC) class I or II restricted peptides, then IFN-γ or interleukin (IL)-4 secreting cell spots were counted. The IM injection of DNA vaccines raised only background levels of IFN-γ producing T cells even in the 10 µg-dose vaccinated group upon MHC-I peptide stimulation. Most importantly, the microneedle delivery of 10 µg HA DNA (MN-10) elicited significantly higher frequencies of IFN-γ producing T cells upon either MHC class I or class II peptide stimulation (Figure 7a, P < 0.01). As shown in Figure 7b, microneedle immunization with 10 µg DNA vaccine (MN-10) induced IL-4 secreting spleen cell spots that were equally well-induced by stimulation with MHC class I or class II peptides. Whereas, IM delivery of 10 µg HA DNA (IM-10) induced similar levels of IL-4 secreting cell spots upon MHC class II stimulation but showed approximately fivefold lower levels of IL-4 secreting cell spots with MHC class I peptide stimulation compared to that induced by microneedle vaccination. Overall, these results suggest that microneedle skin DNA immunization can generate more effective cellular immune responses than IM DNA immunization.

Figure 7.

Figure 7

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Microneedle DNA vaccination is effective in inducing recall cellular immune responses. Splenocytes were isolated from mice at day 4 post-challenge (N = 4) and incubated on the (a) IFN-γ and (b) IL-4 capture antibody-coated plates in the presence of an influenza A/PR8 HA MHC class I and class II peptide pool as a stimulator. The plates were developed with substrate and IL4 and IFN-γ cytokine-producing cells were counted by ELISPOT reader. Data represent mean ± SD. Asterisk indicates significant difference (*P < 0.05, **P < 0.01). IFN, interferon; IL, interleukin; IM, intramuscular; MHC, major histocompatibility complex; MN, microneedle; NS, not significant.

Protective roles of immune sera by microneedle DNA vaccination

To have better insight into the potential immune mechanisms by which microneedle DNA vaccination confers improved protection, we investigated roles of immune sera in providing protection in naive mice when infected with mixtures of a lethal virus dose and sera in different conditions. Naive mice that were infected with a mixture of virus (A/PR8) and naive sera showed severe body weight loss reaching to below 75% of original weights by day 7 postinfection and all had to be euthanized (Figure 8). However, microneedle immune sera (MN-10) provided complete protection to naive mice without a sign of illness as indicated by no body weight loss (Figure 8). Meanwhile, transfer of immune sera from the IM immunization (IM-10) group yield suboptimal responses with significant weight loss (up to 15%) and only 75% survival (Figure 8).

Figure 8.

Figure 8

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Protective efficacy of immune sera and effects of clodronate-liposome treatments. Some groups of naive mice (n = 4 BALB/c) were intranasally treated with clodronate-liposomes to deplete dendritic and macrophage cells. Twofold diluted immune sera of microneedle (MN_10) or intramuscular (IM_10) DNA vaccination were incubated with a lethal dose (10 LD50) of influenza A/PR8 (H1N1) virus at room temperature for 30 minutes. Mixtures of virus and immune sera or naive serum were used to infect clodronate-liposome–treated or naive mice. (a) Body weight and (b) survival rate were monitored for 14 days. MN: microneedle DNA immune sera + (MN_10) + virus, MN/ΔDC: microneedle DNA immune sera (MN_10) + virus in clodroante-treated mice, IM: IM immune sera (IM_10) + virus, IM/ΔDC: IM immune sera (IM_10) + virus in clodroante-treated mice, naive: naive sera + virus in naive mice. DC, dendritic cells; IM. intramuscular; LD, lethal dose; MN, microneedle.

The dendritic and macrophage cells may play a role in clearing pathogens by taking up immune complexes. To determine the roles of these cells in conferring protection by microneedle immune sera, naive mice were pretreated with clodronate-liposomes as previously described.21,22 We demonstrated that clodronate-liposome treatment depleted 55–70% of both CD11c+ dendritic and CD11b+ macrophage cells.23,24 Microneedle immune sera (MN-10) were found not to be affected by clodronate-liposome treatment in conferring protection to naive mice (Figure 8). In contrast, the protective efficacy in the group received IM immune sera (IM-10) was significantly reduced as shown by a lower survival rate (25%) and severe loss in body weight (Figure 8). Therefore, these results suggest that dendritic and macrophage cells do not have a major contribution to the improved protection conferred by microneedle DNA vaccination, which might be different from the partial protection observed in the intramuscularly immunized mice.

Discussion

The skin contains a high population of Langerhans cells and dermal dendritic cells, which are potent immature dendritic cells well-equipped for immune surveillance. However, the physical barrier posed by the stratum corneum layer of the epidermis prevents effective passive delivery of antigens through the skin, and there has been limited success in use of simple, reliable, and low-cost methods of transdermal vaccine delivery. In this study, we investigated the feasibility of delivering influenza HA DNA vaccine to the skin using dry-coated microneedles and compared the immunogenicity and protective efficacy with those induced by conventional IM immunization. A DNA vaccine is made using bacterial plasmid DNA encoding a gene of interest under a promoter to be expressed in the host. Delivery of a reporter gene luciferase into the mouse skin via dry-coated micorneedles resulted in the expression of the gene at the delivered site and its vicinities. It was reported that Langerhans and dermal dendritic cells are most likely responsible for expressing an antigen and trafficking it to the local lymph nodes.8,25 In our study, vaccination by influenza HA DNA vaccine in the skin using dry-coated microneedles was found to be superior to IM injection for inducing protective immune responses, potentially enabling DNA skin vaccination to be a feasible reality.

In addition to convenience for mass vaccination, means of increasing the available supply of vaccines are of great interest. Intradermal vaccination has been demonstrated to have potential dose-sparing effects,26,27 which can reduce the cost of vaccines and make it possible to vaccinate more of the population during a time of vaccine shortage. We observed that a low dose of 3 µg HA DNA vaccine (MN-3) delivered by microneedles induced substantial levels of antibody responses, which is equivalent to or higher than those by IM immunization with 10 µg of HA DNA vaccine (IM-10). In a previous study comparing intradermal and IM immunization with a liquid form of influenza HA DNA vaccines, an approximately fivefold dose-sparing effect was observed after intradermal immunization, where protective efficacy was not assessed.28 IM immunization with 10 µg HA DNA vaccine (IM-10) was found not to be sufficient to induce complete protective immunity, which is consistent with results using the same dose and DNA vaccine in a previous study.20 Previous studies demonstrated that 100 µg of influenza DNA vaccines delivered by IM immunization induced 100% protection.20,29,30 In this present study, microneedle vaccination with 10 µg HA DNA vaccine (MN-10) provided complete protection against lethal challenge. Therefore, microneedle vaccination with DNA vaccine in the skin can be an effective method of gene delivery for inducing stronger immune responses and improved protection compared to conventional IM immunization as evidenced in this study.

Interestingly, we found that microneedle delivery of HA DNA vaccine to the skin in a solid state induced predominantly IgG2a isotype antibody responses indicating Th1-type immune responses. In contrast, previous studies reported that gene gun immunization of skin induced IgG1 as a predominant isotype antibody, while standard needles used for intradermal or IM immunization with high doses of DNA vaccines in solution resulted in the induction of skewed IgG2a antibody responses.13,31 It is not clear how the pattern of antibody isotypes induced by gene gun immunization was different from that by microneedle vaccination, despite the fact that both were apparently intended to deliver DNA vaccines to the skin. One possible explanation might be due to a difference in delivery methods that affect the target antigen-presenting cells. In a recent gene gun immunization study using Langerhans cell-deficient skin, the Langerhans cells were suggested to be responsible for inducing IgG1 isotype antibody responses.8 Also, Nagao et al. demonstrated that langerin+ dermal dendritic cells were required for the induction of IgG2a antibody response from a study of differential repopulation kinetics of skin dendritic cells.8 Microneedle delivery of DNA vaccine may be more likely to target the dermal dendritic cells, whereas gene gun immunization may target epidermal Langerhans cells. Previous studies demonstrated that the induction of IgG2a isotype antibodies contributed to enhanced clearance of influenza viruses.32,33,34,35 The detailed mechanisms still remain unknown although complement activation or Fc receptor interactions were suggested to be involved in IgG2a antibody-mediated effector functions.36,37 As implicated in previous studies above, it is possible that a significantly higher level of IgG2a induced by the microneedle immunization with HA DNA vaccine might have contributed to more effective control of the challenge virus compared to IM immunization.

A major goal of vaccination is to induce memory immune responses that can rapidly respond upon antigen exposure or infection. In the present study, microneedle vaccination induced significant levels of antibody-secreting plasma cell responses in bone marrow, which can produce antibodies independent of antigen stimulation. Also, functional memory B cells in spleens that can differentiate into antibody-secreting cells upon antigen stimulation were observed in mice immunized via microneedle delivery of DNA vaccine to the skin. In addition, IFN-γ secreting cell spots were highly induced by microneedle delivery to the skin but not by IM immunization, and were observed in a dose-dependent manner. It is also worth noting that microneedle vaccination did induce IFN-γ and IL-4 secreting splenocytes equally well in response to MHC I (stimulating CD8 T cells) and II (stimulating CD4 T cells) peptides, respectively, whereas IM immunization seemed to induce more MHC II peptide-stimulated IL-4 cytokine responses. In a previous study, gene gun delivery of DNA vaccines was also shown to induce both IFN-γ and IL-4 secreting splenocyte responses.31 In a recent relevant study, Gill et al. reported that cutaneous vaccination using microneedles coated with hepatitis C DNA vaccine effectively primed cytotoxic CD8 T lymphocyte responses.19 Nanopatches dry-coated with antigen, adjuvant, and/or DNA vaccines were demonstrated to effectively target antigen-presenting cells.38 Taken together, microneedle vaccination with DNA vaccine in the skin can be effective in inducing protective immunity and long-lived memory immune responses.

It is important to better understand the protective immune mechanisms and to explain improved protection by microneedle vaccination. The protection conferred by immune sera of microneedle DNA vaccination was not dependent on the intact presence of dendritic and macrophage cells because both naive mouse groups showed similar protection with or without clodronate-liposome treatment (Figure 8). Therefore, virus-neutralizing activity, although it was weak as evidenced by low HAI titers observed in microneedle immune sera, might have played an important role in providing enhanced protection. However, the protection efficacy of immune sera from IM immunization was significantly lower in mice treated with clodronate-liposomes, implicating that dendritic and macrophage cells might have contributed to the protection in low protective immune sera where no HAI titers were observed. Therefore, nonneutralizing antibody-mediated protection is likely to be weak and may require the help from dendritic and macrophage cells in clearing viral antigen–antibody complexes. This is consistent with the results demonstrated in previous studies that dendritic and macrophage cells are important for cross-protection in the absence of virus-neutralizing activities.23,24

The immunogenicity of the DNA vaccine was relatively weak even in microneedle vaccination, not being able to provide a sterilizing immunity. Recent clinical and preclinical studies demonstrated that priming with DNA vaccines and boosting with protein vaccines offered superior immune responses,3,4,5 suggesting that heterologous prime and boost strategies would be beneficial compared to either vaccine alone. Therefore, it will be important to investigate heterologous prime and boost microneedle vaccination studies as a future direction.

In conclusion, microneedle vaccination with HA DNA vaccine induced an enhanced antibody response particularly IgG2a isotype, improved protective efficacy, and increased recall humoral and cellular immune responses compared to IM immunization. The present study provides evidence that microneedles can be used for targeting gene-based therapeutics and vaccine antigens to the skin immune system and that microneedles could be developed as a promising approach for a simple and reliable delivery.

Materials and Methods

Plasmid DNA and virus. The pCAG-HA-WPRE plasmid DNA vaccines used in this study contains influenza A/PR/8/34 virus HA DNA encoding the entire HA protein under the hybrid cytomegalovirus enhancer/chicken β-actin (CAG) promoter and the messenger RNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus (WPRE) as previously described.20 This plasmid DNA vaccine was propagated in Escherichia coli DH-5α strain (Invitrogen, Carlsbad, CA) and purified using a QIAGEN plasmid GIGA-purification kit (QIAGEN, Valencia, CA). Influenza virus A/PR/8/34 (H1N1, abbreviated as A/PR8) was grown in 10-day-old embryonated hen's eggs and purified from allantoic fluid by using discontinuous sucrose gradient (15, 30, and 60%) layers. The purified virus was inactivated as described39 and used as a coating antigen for ELISA assay. Madin Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium and used for a plaque assay of influenza virus.

For labeling the DNA vaccine, a Label IT Tracker Cy 3 kit (Mirus Bio, Madison, WI) was used. Briefly, 37.5 µl sterile water (DNase and RNase free), 5 µl 10× labeling buffer A, 5 µl HA DNA (1 mg/ml), and Label IT Tracker reagent (Mirus Bio) were mixed and incubated at 37 °C for 1 hour. Unreacted reagents were removed by ethanol precipitation. Labeled DNA pellet was obtained by centrifugation for 10 minutes at 10,000g and washed with 500 µl of 70% ethanol. Finally, labeled DNA vaccine was resuspended by adding sterile water.

The phMGFP/CBL (clone 138) plasmid was kindly provided by Stanford University (Stanford, CA) and TransDerm (Santa Cruz, CA). This plasmid contains a fusion of humanized Montastrea cavernosa (“monster”) green fluorescence protein (hMGFP) and click beetle luciferase (CBL), which is expressed under the control of the cytomegalovirus promoter.40

Fabrication and coating of microneedles with influenza HA DNA. Metal microneedle arrays each consisting of a row of five microneedles were fabricated by infrared laser (Resonetics Maestro, Nashua, NH) cutting of stainless steel sheets (SS304, 75 µm thickness: McMaster-Carr, Atlanta, GA). Each microneedle measured 750 µm in length and 200 µm in width at its base. An array of microneedles was dip-coated by horizontally dipping the microneedle three times into an 8 mg/ml DNA solution held in a dip-coating device, as described previously.41 After air-drying for 5 minutes, microneedles were again dip-coated nine times into a 6 mg/ml DNA solution. To determine the amount of DNA vaccine coated on microneedles, coated microneedles were incubated in distilled water for 12 hours at 4 °C, and the amount of released DNA was determined by ultraviolet spectrophotometric absorption at 260/280 nm wavelengths.

In vivo bioluminescence imaging. Six- to eight-week-old female CD1 mice (Charles River, Wilmington, MA) were used according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Stanford University. Mice were treated with microneedles arrays coated with or without plasmid DNA (phMGFP/CBL). Three arrays of five microneedles each were inserted into the back skin of mouse for 20 minutes. Mice were imaged 24 hours after DNA delivery by microneedles, facilitated by D-Luciferin (Biosynth International, Naperville, IL) injection (100 µl of a 30 mg/ml solution) into the peritoneal cavity of the mice under isoflurane anesthesia. The mice were imaged 10 minutes later using an In Vivo Imaging System (IVIS Spectrum; Caliper LifeSciences, Alameda, CA).42

Immunization and infection. Six- to eight-week-old female BALB/c mice (n = 13, Harlan, Indianapolis, IN) were immunized with 3.3 ± 0.2 µg (denoted 3 µg) or 9.9 ± 0.6 µg (denoted 10 µg) of DNA via coated microneedle insertion into the back skin or IM injection in the quadriceps muscles of mice, as previously described.43,44 A booster vaccination of the same dose was given 4 weeks after the priming dose. Vaccine doses of 3 and 10 µg were achieved by administering one and three microneedle arrays to each mouse, respectively. Vaccinated and naive mice were intranasally challenged with homologous influenza A/PR8 virus at a dose of five times the 50% mouse lethal dose (LD50, equivalent to 160 pfu) in 50 µl PBS at 4 weeks after boost immunization. After virus challenge, body weight changes and survival rates were recorded daily for 14 days. Mice with weight loss exceeding 25% of their initial weight were euthanized. All animal experiments were performed in the animal facility following the approved IACUC protocols of Emory University and Georgia State University.

Serum antibody responses. Serum samples were collected at 3 weeks after primary and secondary immunization of mice and used to measure influenza virus-specific antibody responses by ELISA assays, as previously described.45 Briefly, 96-well microtiter plates (Nunc, Rochester, NY) were coated with 100 µl of 4 µg/ml inactivated A/PR8 virus in coating buffer (0.1 mol/l NaHCO3, pH 8.5), blocked with 3% bovine serum albumin in PBS (pH 7.0), and incubated with serum samples diluted to 1:100 in 0.05% Tween 20 in PBS for 1 hour at 37 °C. After washing, wells were incubated with horse radish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL) for 1 hour at 37 °C. After washing, plates were incubated with an o-phenylenediamine dihydrochloride substrate solution. H2SO4 (0.5 mol/l) was added to stop the reaction and the signal was quantified using a plate reader at 450 nm. HAI titers were determined as described previously.45

Virus-specific recall immune responses. On day 4 after virus challenge, bone marrow and spleen cells were isolated from challenged mice and single cells were cultured in 96-well plates coated with inactivated A/PR8 virus.45 The culture supernatants were harvested at 1, 2, and 6 days and diluted with the equal volume of phosphate-buffered saline with Tween 20 (PBST) to determine the levels of virus-specific IgG responses by ELISA. Absorbance was read at 450 nm and expressed mean value ± SD.

ELISPOT assay for determination of T-cell responses. Splenocytes (1.0 × 106 cells/well) were cultured in Multiscreen 96-well filtration plates (Millipore, Billerica, MA) coated with capture antibodies against mouse IFN-γ and IL-4 cytokines (3 µg/ml in coating buffer; BD Biosciences, San Diego, CA) with or without A/PR8 HA-specific MHC class I and II peptide stimulator (10 µg/ml), as described previously.39 Peptide stimulators were either a mixture of two MHC class I peptides (IYSTVASSL, LYEKVKSQL) or a pool of five MHC class II peptides (SFERFEIFPKE, HNTNGVTAACSH, CPKYVRSAKLRM, KLKNSYVNKKGK, and NAYVSVVTSNYNRRF).46,47 After washing with PBS, the plates were incubated with biotinylated rat anti-mouse IFN-γ or IL-4 (1 µg/ml in PBST with 5% fetal bovine serum) antibodies over night at 4 °C. After three additional washes, horse radish peroxidase-conjugated streptavidin solution was added to each well for 3 hours at room temperature. The spots were developed with stable DAB (3,3-diaminobenzidine) and counted using an ImmunoSpot ELISpot reader (Cellular Technology, Shaker Heights, OH).

Lung viral titer. Four days after viral challenge, lung homogenates were prepared from each infected mouse and virus titration was performed by a plaque assay in MDCK cells as previously described.45

Clodronate-liposome treatment and in vivo protective efficacy of immune sera. Liposome-encapsulated clodronate was prepared as previously described.48 Twelve hours before infection with a virus-serum mixture, clodronate-liposomes were intranasally administered to some groups of naive mice (n = 4 BALB/c) to deplete dendritic and macrophage cells as described.21,22,23,24,49 Clodronate was a kind gift of Roche Diagnostics GmbH, Mannheim, Germany. To test protective efficacy of immune sera in vivo, serum samples (twofold diluted) from immunized (MN-10, IM-10 boost vaccination) and naive control mice were preincubated with a lethal dose of influenza virus at room temperature for 30 minutes as described.24 A mixture of a lethal infectious dose (10 × LD50) of A/PR8 (H1N1) influenza virus and sera was administered to naive mice (n = 4 BALB/c), and body weight changes and survival rates were monitored daily.

Statistical analysis. To determine the statistical significance, a two-tailed Student's t-test and analysis of variance were used when comparing two or more different groups, respectively. A P value less than 0.05 was considered to be significant.

Acknowledgments

This work was supported by NIH/NIBIB grant EB006369 (M.R.P.), NIH/NIAID grant AI0680003 (R.W.C.), and NIH/NIAID grants AI093772 (S.-M.K.) and AI087782 (S.-M.K.). We thank Emilio González-González and, Ryan Spitler for carrying out animal studies involving IVIS imaging at Stanford University and Christopher Contag, Roger Kaspar, and Robyn Hickerson and Joshy Jacob for providing phMGFP/CBL and A/PR8 HA plasmid DNA vaccine respectively. We also thank Fu-shi Quan for discussion of immunization studies, Dae-goon Yoo for technical help, and Mark Allen for use of his laser microfabrication facilities. M.R.P. is an inventor of patents that have been licensed to companies developing microneedle-based products, is a paid advisor to companies developing microneedle-based products, and is a founder/shareholder of companies developing microneedle-based products. For this reason, this study could affect the personal financial status of M.R.P. This potential conflict of interest has been disclosed and is overseen by Georgia Tech and Emory University. The other authors declared no conflict of interest.

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