DNA vaccination in the skin using microneedles improves protection against influenza

Abstract

In this study, we tested the hypothesis that DNA vaccination in the skin using microneedles improves protective immunity compared to conventional intramuscular (i.m.) 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 i.m. 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 i.m. injection of HA DNA vaccine.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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.

Figure 7

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.

Figure 8

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.