Eight-week-old female C57BL/6 (H2b) mice were purchased from Charles River (Sulzfeld, Germany) and kept under specific pathogen-free conditions at the animal facilities of the German Cancer Research Center in compliance with the regulations of the Germany Animal Protection Law.
Plasmid pUF3L1h  carrying the humanized HPV16 L1 gene under the control of the human cytomegalovirus immediate-early promoter (pCMV) was used for the induction of antigen-specific immune responses in the DNA immunization experiments. The L1 protein expression of pUF3L1h has been shown to be substantially increased due to the codon optimization.
The plasmid pBSC/GM-CSF (kindly provided by M. Smahel, Institute of Hematology and Blood Transfusion, Prague, the Czech Republic) was used as an adjuvant in the DNA immunization experiment. This plasmid contains the sequence coding for the mouse GM-CSF that was excised from the plasmid pBK-GM  by XhoI and SalI restriction enzymes and ligated into the XhoI-site of the plasmid pBSC . The production of GM-CSF was confirmed by transfecting 293T cells with the pBSC/GM-CSF plasmid and analyzing lysates using the mouse GM-CSF ELISA kit (OptEIA™, BD Biosciences Pharmingen, San Diego, CA, USA). The adjuvant effect of pBSC/GM-CSF plasmid has been evaluated in our previous immunization experiments .
Plasmid DNA was purified from E. coli DH5α using CsCl equilibrium density centrifugation and dissolved in TE buffer to a final concentration of 5 mg/ml. Anesthetized mice were immunized with DNA four times, on days 0, 14, 28 and 98. Each mouse received 50 μg of plasmid pUF3L1h (6 groups) or pBSC/GM-CSF (control group) in one immunization dose. Two groups of mice received a mixture of 50 μg pUF3L1h DNA and 50 μg pBSC/GM-CSF DNA per animal in a single dose. For intramuscular delivery, the DNA was injected into the tibia anterior muscle of the right leg in a final volume of 50 μl PBS. Tattooed DNA was delivered in 10 μl TE buffer for single plasmid administration or 20 μl TE buffer for the mixture of plasmids in one or two drops to the shaved skin at the dorsum followed by tattoo with a 7-linear tattoo needle using a commercial tattoo machine (Rotary 12000 PL, Bortech Tattoogrosshandel, Wuppertal, Germany). The tattoo device was adjusted to allow exposure of only 1–2 mm of the needle tip beyond the barrel guide. The depth of 1–2 mm for tattooing of the mouse skin was shown to result in the immediate location of tattooed inks mainly in the dermis and to a lower extent in the epidermis . A skin surface area of approximately 2 cm × 1 cm was tattooed by 30-times repeated two-second-lasting treatments with the tattoo needle oscillating at the voltage 17.4 V corresponding with the frequency 145 Hz (145 punctures per second) set on the power supply (DC POWER SUPPLY, DF 1730 SB3A, Bortech Tattoogrosshandel, Wuppertal, Germany). Thus, every tattooed mouse received during one immunization the total number of 60 900 (7 × 30 × 2 × 145 = 60 900) solid-needle punctures to deliver 50 μg DNA in 10 μl TE buffer or 121 800 (2 × 60 900 = 121 800) solid-needle punctures to deliver 100 μg DNA in 20 μl TE buffer. The tattoo procedure was well tolerated, however local trauma involving minor swelling and reddening of the skin was observed.
In addition, some mice were pretreated with 50 μl of cardiotoxin (10 μM, Latoxan, Valence, France) five days before the first DNA immunization in the loci of vaccination. Thus, cardiotoxin was applied either into the tibia anterior muscle by needle injection or to the dorsal skin by tattoo.
Blood of immunized mice was collected 10 days after the third and 9 days after the fourth DNA immunization. For detection and endpoint-titration assays of HPV 16 L1-specific antibodies an antigen capture ELISA was used. For this, microtiter plates were coated overnight at 4°C with 50 μl PBS containing purified rabbit polyclonal IgG anti-HPV16 L1 antibodies at a 1:200 dilution. Plates were blocked with 100 μl 3% milk/PBS-0.3% Tween 20 for 1 h at 37°C followed by the addition of 50 μl of the HPV16 L1 VLPs (5 mg/ml) diluted 1:1500 in 1.5% milk/PBS-0.3% Tween 20 for 1 h at 37°C. Plates were washed with PBS-0.3% Tween 20 and 50 μl of mouse serum were added in 2-fold dilutions starting at 1:50 and ending at 1:13107200 and incubated for 1 h at 37°C. Non-specific binding was determined using the dilution 1:50 of the mouse sera on plates coated with PBS only. Plates were washed and incubated with 50 μl/well of a sheep anti-mouse IgG polyclonal antibody conjugated to peroxidase (Sigma) diluted 1:3000 in 1.5% milk/PBS-0.3% Tween 20 for 1 h at 37°C. After the final washing, 100 μl/well of ABTS [2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)] staining solution (1 mg/ml in a 100 mM sodium acetate-phosphate buffer, pH 4.2, 0.015% H2O2) was used for enzyme reaction. Absorptions were measured at 405 nm in a Titertek automated plate reader after 40–60 minutes.
IFN-γ-enzyme-linked immunosorbent (ELISPOT) assay
The ELISPOT assay was performed 9 days after the fourth DNA immunization as described in our previous work . MultiScreen IP sterile plates (96 well; Millipore, Eschborn, Germany) were pre-soaked with 70% ethanol for 1 min, and the ethanol was removed by extensive rinsing with PBS. The plates were coated with 600 ng per well of anti-mouse interferon gamma (IFN-γ) capture antibody (BD Pharmingen, Heidelberg, Germany) in 100 μl of PBS overnight at 4°C. Unbound antibody was removed by washing twice with PBS and twice with medium (RPMI-1640, Sigma; 10% fetal calf serum, 2 mM L-glutamine, 1% penicillin-streptomycin). Plates were blocked for 7 h with 100 μl of medium at 37°C, and splenocytes from individual mice were seeded in four serial dilutions: 2, 1, 0.5 and 0.25 × 106 cells per well in 100 μl of medium. Splenocytes from each mouse were left either untreated (background control), or stimulated with 900 ng of pokeweed mitogen (Sigma) in 100 μl of medium (positive control), or with 0.2 μM L1 aa165-173 peptide  in 100 μl of medium. Plates were incubated for 20 h at 37°C. Cells were removed by six washes with PBS-0.01% Tween 20 and one wash with sterile water. Then, 200 ng of sterile-filtered biotinylated rat anti-mouse IFN-γ detection antibody (BD Pharmingen) in 100 μl of PBS were added per well, and the plates were kept at 4°C overnight. The plates were washed six times with PBS-0.01% Tween 20 and once with PBS, and this was followed by the addition of 100 μl of a 1:1000 dilution of streptavidin-alkaline phosphatase (BD Pharmingen) in PBS. Plates were incubated for 30 min at room temperature and then washed three times with PBS-0.01% Tween 20, followed by three washing steps with PBS alone. Plates were developed with 5-bromo-4-chloro-3-indolylphosphate (BCIP/Nitro Blue Tetrazolium Liquid Substrate System; Sigma), 100 μl per well. The reaction was stopped after 15 minutes by rinsing the plates with water. Spots were quantified using an ELISPOT reader (AID EliSpot Reader ELR04; AID GmbH, Strassberg, Germany).
Data of end-point titration of ELISA assay were analyzed by Wilcoxon Rank sum test. For ELISPOT assay analysis, we performed two tailed unpaired t-test using Prism 4 software (GraphPad Software, Inc., San Diego, CA, USA). A difference between groups was considered significant for p < 0.05.