Figure 2. Microneedle-based immunization induces antigen-specific CTLs.

Groups of 4-8 week old female C57BL/6 or Balb/c mice (Charles River, Uppsala, Sweden) were immunized cutaneously with microneedles or gene gun, or intramusculary using hypodermic needles.For microneedle-based immunization two or five rows per mouse were used to control the dose. Each microneedle row was coated with 1.6 μg DNA. DNA-coated microneedle rows were manually inserted into trimmed abdominal or back skin and held for 1 min to allow dissolution of coated DNA into skin. For gene gun-based immunization mice were immunized by gene gun (Bio-Rad Laboratories, Hercules, CA, USA) at a dose of 4 μg/mouse as previously described ¹¹. Plasmid DNA was linked to 1-μm diameter gold particles for gene gun-based immunizations according to protocols supplied by the manufacturer. For intramuscular immunization mice were injected with 100 μg DNA in the tibialis anterior using a hypodermic needle. Un-treated (naive) mice were used as a negative control. All experimental protocols were approved by the ethical committee for animal research at Karolinska Institutet. (A) C57B1/6 mice immunized once with codon-optimized NS3/4A DNA using either microneedles (1.6 μg per row × 5 microneedle rows per mouse = 8 μg dose; n=4 mice; gray bars), gene gun (4 μg dose; n=4 mice; black bars) or no immunization (n=2 mice; white bars) and euthanized after two weeks. As described previously ¹¹,¹³, cells harvested from the spleen (effector cells, 2.5×10⁷ cells per mouse) were restimulated with a NS3 H-2D^b-specific peptide and 2.5×10⁷ irradiated naïve C57B1/6 spleenocytes cells. After five days, 5×10³ RMA-S cells pulsed with NS3 H-2D^b-specific peptide and labeled with ⁵¹Cr were used as target cells. The specific cell lysis of target cells was then measured at different effector-to-target cell ratios by measuring ⁵¹Cr released from lysed target cells. (B) Balb/C mice were either immunized intramuscularly (100 μg; n=5 mice),cutaneously with microneedles (1.6 μg per row × 2 microneedle rows per mouse = 3.2 μg dose; n=5 mice) or were not immunized (n=2 mice). Presence of in vivo functional CTLs was determined using a tumor challenge model ¹³. Two weeks after immunization, mice were subcutaneously injected with 1×10⁶ SP2/0 myeloma cells stably transfected with NS3/4A. Tumor growth was then monitored daily through skin by recording mean tumor size (thickness of skin flap at tumor injection site) for 14 days and compared to growth of the same tumor cell line in non-vaccinated mice. Mean tumor sizes were compared by analysis of variance (ANOVA, α=0.05). Error bars represent SD; Symbol ‡ represents p<0.05; NS means not significant.