Increased immunogenicity of avian influenza DNA vaccine delivered to the skin using a microneedle patch

Abstract

Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700-μm long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip-coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic.

Figure 1

H5 influenza HA DNA vaccine. (A) Schematic diagram of H5 HA in the pCAGGS protein expression vector. The synthesized HA gene from influenza A/Vietnam/1203/04 (H5N1) virus was cloned into the pCAGGS vector between chicken beta actin promoter and rabbit beta globin polyA site. Multi-basic amino acids (RRRK) in the HA1/HA2 cleavage site were deleted and codon usage was optimized for the sf9 insect cell. (B) Western blotting analysis of H5 HA expression. HA expression was confirmed by Western blotting of culture supernatants from CV1 cells transfected with pCAGGS/H5 HA vaccine plasmid at 30 h (Lane 2) or 60 h (Lane 3) after transient transfection. Culture supernatant from non-transfected cells (Lane 1) and recombinant HA proteins (Lane 4) were used as negative and positive control, respectively.

Figure 2

Microneedles coated with Cy3-stained HA DNA vaccine. (A) Representative bright-field (i) and fluorescence (ii) micrographs of microneedles coated with HA DNA vaccine (scale bar= 1 mm). (B) Representative bright-field (i) and fluorescence (ii–vi) micrographs of a microneedle coated with HA DNA vaccine (i, ii) before insertion and (iii) 30 s (iv) 60 s (v) 120 s and (vi)180 s after insertion into mouse skin in vitro (scale bar = 200 µm).

Figure 3

HA DNA vaccine coated on microneedles as a function of coating parameters. Amount of DNA vaccine coated on an array of five stainless-steel microneedles according to (A) the number of dip-coating cycles (5 mg/ml DNA solution) and (B) the initial concentration of DNA in the coating solution (with 10 dipings). Data represent the average of n = 9 replicates. Error bars represent the standard error.

Figure 4

Antibody responses after vaccination with HA DNA. (A) Total virus-specific antibody responses and (B) antibody isotype ratio (IgG2a/IgG1) and (C) hemagglutination inhibition (HAI) titers in mouse sera after vaccination with microneedles or IM injection at 3 weeks after a three-dose vaccination regimen. Mock = negative control immunization using microneedles coated only with coating solution only; IM = intramuscular immunization with HA DNA coated on microneedles, dissolved off and injected; MN = microneedle immunization. *p<0.05 compared to Mock and IM.

Figure 5

Protection of mice after immunization with HA DNA vaccine. (A) Survival rate, (B) body weight change in infected mice. Mice were challenged by intranasal inoculation with H5N1 influenza virus (20 LD₅₀) 21 days after a three-dose vaccination regimen. Survival rate and body weight change were monitored in n=6 mice.

Figure 6

Effect of coating solution formulation on DNA stability. Lipofectamine-mediated transfection of DU-145 cells was determined using GFP-encoding DNA after formulation and coating under different conditions. (A) Representative multiphoton microscope images of cell transfection (green cells have been transfected). (i) no DNA added (negative control). (ii) DNA in DI water added (positive control), (iii) DNA dried in coating solution (CS), (iv) DNA in coating solution, (v) DNA dried in DI water. Scale bar = 100 µm. (B) Quantitative cell transfection rates determined by flow cytometry. Data represent the average of n=6 replicates. Error bars represent the standard error. (C) DNA molecule size determined by dynamic light scattering. Data represent the average of n=4 replicates. Error bars represent the standard error. DIW : deionized water, CS : coating solution.