Optimization of Intradermal Vaccination by DNA Tattooing in Human Skin
March 2009
Human gene therapy 20(3):181-9
DOI: 10.1089/hgt.2008.073
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Abstract and Figures
The intradermal administration of DNA vaccines by tattooing is a promising delivery technique for genetic immunization, with proven high immunogenicity in mice and in nonhuman primates. However, the parameters that result in optimal expression of DNA vaccines that are applied by this strategy to human skin are currently unknown. To address this issue we set up an ex vivo human skin model in which DNA vaccine-induced expression of reporter proteins could be monitored longitudinally. Using this model we demonstrate the following: First, the vast majority of cells that express DNA vaccine-encoded antigen in human skin are formed by epidermal keratinocytes, with only a small fraction (about 1%) of antigen-positive epidermal Langerhans cells. Second, using full randomization of DNA tattoo variables we show that an increase in DNA concentration,needle depth, and tattoo time all significantly increase antigen expression ( p < 0.001), with DNA concentration forming the most critical variable influencing the level of antigen expression. Finally, in spite of the marked immunogenicity of this vaccination method in animal models, transfection efficiency of the technique is shown to be extremely low, estimated at approximately 2 to 2000 out of 1 x 10(10) copies of plasmid applied. This finding, coupled with the observed dependency of antigen expression on DNA concentration, suggests that the development of strategies that can enhance in vivo transfection efficacy would be highly valuable. Collectively, this study shows that an ex vivo human skin model can be used to determine the factors that control vaccine-induced antigen expression and define the optimal parameters for the evaluation of DNA tattoo or other dermal delivery techniques in phase 1 clinical trials.
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Optimization of Intradermal Vaccination by DNA
Tattooing in Human Skin
Joost H. van den Berg,
1–3
Bastiaan Nuijen,
1
Jos H. Beijnen,
1,4
Andrew Vincent,
5
Harm van Tinteren,
5
Jo¨ rn Kluge,
6
Leonie A.E. Woerdeman,
7
Wim E. Hennink,
2
Gert Storm,
2
Ton N. Schumacher,
3
and John B.A.G. Haanen
3
Abstract
The intradermal administration of DNA vaccines by tattooing is a promising delivery technique for genetic
immunization, with proven high immunogenicity in mice and in nonhuman primates. However, the parameters
that result in optimal expression of DNA vaccines that are applied by this strategy to human skin are currently
unknown. To address this issue we set up an ex vivo human skin model in which DNA vaccine-induced
expression of reporter proteins could be monitored longitudinally. Using this model we demonstrate the fol-
lowing: First, the vast majority of cells that express DNA vaccine-encoded antigen in human skin are formed by
epidermal keratinocytes, with only a small fraction (about 1%) of antigen-positive epidermal Langerhans cells.
Second, using full randomization of DNA tattoo variables we show that an increase in DNA concentration,
needle depth, and tattoo time all significantly increase antigen expression ( p< 0.001), with DNA concentration
forming the most critical variable influencing the level of antigen expression. Finally, in spite of the marked im-
munogenicity of this vaccination method in animal models, transfection efficiency of the technique is shown to
be extremely low, estimated at approximately 2 to 2000 out of 110
10
copies of plasmid applied. This finding,
coupled with the observed dependency of antigen expression on DNA concentration, suggests that the devel-
opment of strategies that can enhance in vivo transfection efficacy would be highly valuable. Collectively, this
study shows that an ex vivo human skin model can be used to determine the factors that control vaccine-induced
antigen expression and define the optimal parameters for the evaluation of DNA tattoo or other dermal delivery
techniques in phase 1 clinical trials.
Introduction
Skin has become increasingly used as a successful de-
livery route for DNA vaccines (Mitragotri, 2005). The
excellent immunogenicity of dermal DNA vaccination is
probably related to the high prevalence of antigen-presenting
cells (APCs) in the skin, in the form of Langerhans cells (LCs)
in the epidermis and dendritic cells in the dermis (Kanitakis,
2002; Mathers and Larregina, 2006). Several techniques have
been developed for the intradermal administration and cel-
lular uptake of naked DNA, such as gene gun and particle
injection systems (Klinman et al., 1998; Mitragotri, 2005; Steitz
et al., 2006); jet injectors (Mitragotri, 2005; Bahloul et al., 2006);
electroporators (Maruyama et al., 2001; Heller et al., 2007;
Hooper et al., 2007; Hirao et al., 2008); and a technique, de-
veloped at our institute, that has been called ‘‘DNA tattooing’’
(Bins et al., 2005; Pokorna et al., 2008). DNA tattooing delivers
naked plasmid DNA into the skin through thousands of
punctures made with a multiple-needle tattoo device. We
have demonstrated that DNA tattooing results in local trans-
fection and expression of the encoded antigen by cells in
murine skin. More importantly, the efficacy of DNA tattooing
in inducing strong vaccine-specific immune responses has
been established in murine models (Bins et al., 2005) and the
1
Department of Pharmacy and Pharmacology, Slotervaart Hospital, 1066 EC Amsterdam, The Netherlands.
2
Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands.
3
Division of Immunology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
4
Department of Bioanalysis, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, 3584 CA Utrecht, The Netherlands.
5
Division of Biostatistics, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
6
MT.DERM, 14167 Berlin, Germany.
7
Department of Plastic and Reconstructive Surgery, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
HUMAN GENE THERAPY 20:181–189 (March 2009)
ªMary Ann Liebert, Inc.
DOI: 10.1089=hum.2008.073
181
superiority of DNA tattooing over intramuscular DNA vac-
cination has been demonstrated in nonhuman primates
(Verstrepen et al., 2008).
A major difficulty when translating novel dermal delivery
techniques, such as DNA tattooing, toward clinical applica-
tion is that they have been developed and optimized in non-
human skin. Because mouse and macaque skin have a higher
density of hair follicles than human skin and a different thick-
ness when compared with human skin (Godin and Touitou,
2007), direct translation of vaccination protocols to clinical
application is difficult. To prepare for the use of DNA tattoo
vaccination in a phase 1 clinical trial, we have therefore de-
veloped an ex vivo human skin model that allows the mea-
surement of vaccine-induced gene expression in real time.
Having established this ex vivo human skin model, we have
used the model to define optimized conditions for DNA tattoo
vaccination of human skin.
Materials and Methods
Plasmids
DNA vaccines were generated by the insertion of reporter
genes into the minimal pVAX1 plasmid backbone (Invitrogen,
Carlsbad, CA). pVAX:Luc was generated by insertion of the
gene encoding firefly luciferase into the EcoRI=NotI site of
pVAX1. pVAX:GFP was generated by inserting green fluo-
rescent protein (GFP)-encoding DNA into the BamHI=NotI
site of pVAX1. pVAX:LacZ, encoding b-galactosidase, was
purchased from Invitrogen. Histone-2B-GFP (H2B-GFP) fu-
sion protein-encoding DNA cloned into the pN1 vector
(Clontech, Palo Alto, CA) was described previously (Kanda
et al., 1998). pVAX:Luc was produced in one large batch for all
experiments according to a uniform process described previ-
ously (Quaak et al., 2008). All other plasmids were purified
with an EndoFree plasmid kit (Qiagen, Hilden, Germany). All
plasmids were dissolved in water for injections (Braun, Mel-
sungen, Germany). The purity and concentration of plasmid
DNA were assessed by agarose gel electrophoresis and ul-
traviolet spectroscopy, respectively.
Tattooing and injection of human skin
Healthy human abdominal skin from female patients (41–
63 years of age) was obtained from the plastic surgery de-
partment, according to institutional guidelines. Subcutaneous
fat was directly removed by blunt dissection. Skin was trans-
ported on ice and used within 2 hr of surgical removal.
Before DNA tattooing, skin was cleaned with sterile phos-
phate-buffered saline (PBS) and pinched onto a polypropylene
board with drawing pins. Next, a black marker was used to
apply a chessboard pattern on the skin to define the various
areas for tattooing. DNA tattooing was performed by appli-
cation of 10 ml of DNA solution onto the skin in a custom-
fabricated mold to keep the area of tattooing consistent
(diameter, 8 mm; surface, 50 mm
2
). The droplet of DNA
was subsequently administered onto skin with an Aella (now
called Permanent Make Up [PMU]) or Cheyenne tattoo ma-
chine (both machines and needles from MT.DERM, Berlin,
Germany). For all tattoos, nine-needle cartridges and an oscil-
lating frequency of 100Hz were used. During the experiments,
the tattoo depth, tattoo duration, DNA concentration, and the
two tattoo machines were varied on the basis of a randomi-
zation protocol (see Table 1). Needle amplitude was adjustable
with an accuracy of 0.1 mm, using a custom-built device that
contained a stroboscope and microscope (MT.DERM). For in-
tradermal injections, 50 ml of DNA solution at a concentration
of 1 mg=ml was injected intraepidermally with a 29-gauge,
12 mm needle in a side-by-side comparison with a tattoo of
10 mlof1mg=ml DNA solution at a 1.5 mm needle depth for
20 seconds with the Aella machine (n¼3, performed two times
in separate experiments).
After tattooing or injection, skin samples were kept at 5%
CO
2
,378C in complete keratinocyte serum-free medium
(SFM) containing 1% penicillin–streptomycin and amphoter-
icin B (0.25 mg=ml) (all from Invitrogen). During this incuba-
tion, skin was cultured at the air–medium interface with the
epidermis exposed to the air to mimic the natural situation.
For histology and flow cytometry experiments tattooed
areas of interest were removed from the intact skin with a
6-mm biopsy punch and transferred into 48-well plates.
Histology
Four-micrometer cryostat cross-sections of skin tattooed
with b-galactosidase encoding plasmid (pVAX:LacZ) were
prepared. Cryostat sections were fixed in acetone for 10 min
and washed for 10 min in PBS. Sections were stained for
10 min with X-Gal staining solution (Roche Applied Science,
Indianapolis, IN) to visualize b-galactosidase expression.
Subsequently, sections were stained with hematoxylin and
eosin according to standard procedures.
Flow cytometric analysis of DNA
vaccine-induced antigen expression
Directly after tattooing with pVAX:GFP, skin samples were
incubated for 1 hr in 10 mg=ml dispase II (Sigma-Aldrich, St.
Louis, MO) in keratinocyte medium at 378C, at which point
the epidermis was mechanically peeled from skin samples.
The obtained epidermal sheet and dermis were cultured
overnight in complete keratinocyte medium to allow accu-
mulation of vaccination-induced GFP expression. Eighteen
hours later, epidermal sheets were digested at 378C in com-
plete keratinocyte medium containing 0.05% trypsin and
DNase I (300 U=ml) (Roche Applied Science). After 15 min, the
epidermis was disrupted with a glass pipette and 10% fetal
calf serum (FCS) was added to the medium. Dermal samples
were digested with collagenase type IV (50 mg=ml; Sigma-
Aldrich) at 378C for 3 hr, after which the cells were filtered
through 70 mm (pore size) nylon gauze to remove debris. Fil-
tered epidermal cell suspensions and dermal cell suspensions
were washed with PBA (1PBS, 0.5% bovine serum albumin
[BSA], and 0.02% sodium azide) before antibody staining.
The antibodies used were phycoerythrin-conjugated mouse
Table 1. Fixed Effects (Tattoo Variations) Tested
in Linear Effect Model Skin Tattooing
Effect Parameters
DNA concentration (mg=ml) 0.2, 1, 5
Tattoo duration (sec) 5, 10, 20
Tattoo depth (mm) 0.5, 1.0, 1.5
Tattoo machine Aella, Cheyenne
182 VAN DEN BERG ET AL.
anti-human CD1a (Immunotech, Prague, Czech Republic),
allophycocyanin-conjugated mouse anti-human CD1a (Im-
munotech), and mouse anti-human cytokeratin (an equal
mixture of clones LP34 and MNF116, both from Dako,
Glostrup, Denmark), labeled with Alexa Fluor 647 (Invi-
trogen) according to the manufacturer’s protocol. Before
cytokeratin staining, epidermal cell suspensions were per-
meabilized with a BD Cytofix=Cytoperm kit (BD Biosciences,
San Jose, CA) according to the manufacturer’s protocol. Cell
suspensions were analyzed and sorted with a FACSCalibur or
a FACSAria (BD Biosciences) and Summit analysis software
(Dako). In the case of anti-CD1a staining, live cells were se-
lected on the basis of propidium iodide exclusion.
Confocal laser scanning microscopy
Skin samples were tattooed with an H2B-GFP-encoding
construct as described previously. The next day, epidermal
cell suspensions were prepared and GFP-positive epider-
mal keratinocytes and Langerhans cells were isolated by
fluorescence-activated cell sorting on the basis of CD1a ex-
pression and subsequently analyzed by confocal laser scan-
ning microscopy (Leica SP2) in PBA.
Calculation of transfection efficiency
The transfection efficiency with DNA tattooing was calcu-
lated by determining the number of GFP-expressing cells per
tattoo area, using flow cytometry. The amount of adminis-
tered molecules of plasmid DNA was calculated on the basis
of the molecular mass of the construct (2.2610
3
kDa, 3724 bp)
and the dose used per tattoo. Transfection efficiency was
calculated as follows:
(Cells transfected ·estimated number of plasmid molecules
per transfected cell ⁄ plasmid molecules administered
by tattooing) ·100%
Imaging of luciferase expression
Luciferase expression was measured in intact skin samples
3, 18, 24, 48, and 72 hr after tattooing. The substrate luciferin
(Xenogen=Caliper Life Sciences, Hopkinton, MA) was added
to the medium to a final concentration of 45 mg=ml. During
this procedure extra medium was added to the box in which
skin was incubated, to completely cover the epidermis of skin
samples with fluid to guarantee full accessibility of luciferin to
the tattooed areas. Thirty minutes after the addition of lucif-
erin, luminescence produced by active luciferase was ac-
quired during 30 sec with an IVIS system 100 charge-coupled
device (CCD) camera (Xenogen=Caliper Life Sciences).
Signal intensity was quantified as the sum of all detected
light within the tattoo area of interest. During each mea-
surement, background luminescence was measured to allow
correction during data analysis. After each measurement,
medium was refreshed to remove residual luciferin.
Linear mixed effects model
To study the effect of various tattoo parameters on the level
of antigen (i.e., luciferase) expression, the natural log trans-
form of the area under the curve (AUC) over the 72-hr period
was analyzed. To account for possible within-skin correlation
of the repeated measurements of antigen expression (at vari-
ous parameter levels), a linear mixed effects model was con-
structed. Fixed effects included the four variables of primary
interest: the DNA concentration, the duration of tattooing, the
depth of tattoo, and the type of tattoo machine (see Table 1).
Patient age, background luminescence (measured in non-
tattooed regions), the location of the tattoo in the piece of skin
(edge vs. center), and the time from surgery to tattooing were
also included as fixed effects to adjust for any possible con-
founding influences. Patient identifier was implemented as
the random grouping variable. Pair-wise interactions were
examined between all fixed effects. Backward stepwise se-
lection was performed, removing terms at the 0.05 signifi-
cance level. Conventional residual analysis was performed to
assess model fit. Data analysis was performed with S-PLUS
version 6.2 Pro (Insightful=TIBCO Software, Seattle, WA).
Results
Histology of DNA tattoo-treated ex vivo human skin
In view of the documented value of DNA tattoo in murine
and nonhuman primate models (Bins et al., 2005; Pokorna
et al., 2008; Verstrepen et al., 2008), we set out to further de-
velop this technique toward clinical application. As the mor-
phology of animal skin differs substantially from that of
human skin, an ex vivo human skin model was deemed es-
sential to allow translation of this new DNA vaccination
technique toward clinical testing. In a first set of experiments
we aimed to determine whether DNA tattooing results in
transfection of human skin cells and to assess the effect of
DNA tattoo application on general skin structure and trans-
fection. To this purpose, a b-galactosidase-encoding plasmid
was introduced into ex vivo human skin by DNA tattooing.
Application of DNA to human skin by tattooing with a needle
FIG. 1. Expression of LacZ in a cryosection (original mag-
nification,20) of human skin after application of LacZ DNA
by tattooing. Eighteen hours after tattooing, cross-sections of
skin were prepared and stained with X-Gal solution to
generate a blue precipitate in transfected cells (indicated
by arrows). The skin was tattooed with pVAX:LacZ at 5 mg=
ml for 20 sec with an Aella tattoo machine (needle depth,
1.0 mm).
DNA TATTOOING IN HUMAN SKIN 183
at a depth of 1.0 mm resulted in substantial disruption of the
epidermal layer of human skin, but only a slight disturbance
of the underlying dermal layer (Fig. 1). Furthermore, consis-
tent with the data obtained in mouse models (Bins et al., 2005),
DNA tattooing resulted in sporadic transfection of cells in the
epidermis (Fig. 1). Transfection of cells in the dermal layer was
not observed.
Visualization of vaccine-induced antigen
expression in human skin
To determine which specific cell type(s) expressed the
vaccine-encoded antigen after DNA tattoo vaccination, skin
samples were tattooed with a GFP-encoding plasmid and cell
suspensions of these samples were analyzed by flow cytom-
etry, using specific markers for epidermal keratinocytes and
LCs.
Of the viable cells (75.3 6.3% of total cells, based on pro-
pidium iodide [PI] exclusion, mean SD of three separate
experiments) recovered from epidermal preparations, ap-
proximately 2% expressed the vaccine-encoded GFP (1.80
1.35%, mean SD of three different patients, all measured in
triplicate). The vast majority (>98%) of these epidermal GFP
þ
cells appeared to be keratinocytes, as based on expression of
cytokeratin (Fig. 2). The fact that antigen expression is almost
exclusively restricted to keratinocytes was confirmed by the
observation that of all GFP
þ
cells, only 1% (1.29 0.53%,
mean SD, three different patients, all measured in triplicate)
expressed the Langerhans cell marker CD1a. In the dermal
layer of the skin only a small number of GFP
þ
cells could be
detected (approximately 2.5% of all GFP
þ
cells in the skin
sample were detected in the dermis, measured at a tattoo
depth of 1.5 mm; data not shown).
Collectively, these data indicate that transfection on DNA
tattooing is almost exclusively restricted to keratinocytes
within the epidermal layer. Transfection of CD1a
þ
LCs is also
observed but is relatively rare, and essentially proportional to
the relative frequency of LCs and keratinocytes in human skin
(a ratio of 2:98).
Quantification of the number of cells expressing the
vaccine-encoded antigen allowed us to determine the ‘‘bio-
logical availability’’ of DNA vaccines, administered by the
tattoo technique. For this calculation, we assumed that the
bulk of the GFP-expressing cells was directly transfected with
the plasmid, as there is no evidence in the literature that
keratinocytes (which constitute >98% of the GFP-positive
cells) are able to cross-present antigens. The total number of
GFP-expressing cells per tattoo area of 50 mm
2
was 2848 762
(mean SD, three different patients, all measured in tripli-
cate), using a DNA concentration of 5 mg=ml (20-sec appli-
cation at a needle amplitude of 1.5 mm, using the Aella tattoo
machine). Prior work has shown that on tattooing mouse skin,
using a mixture of two different fluorescent reporter plas-
mids, coexpression of two reporter genes in a single cell occurs
in some but not all cells (Bins et al., 2007a). On the basis of this
observation and on the fact that the log difference in GFP
expression is about 3-fold (see Fig. 2), we consider it reason-
able to assume that a single transfected cell can take up ap-
proximately 1–1000 DNA molecules, indicating that the
2.810
3
antigen-expressing cells were transfected with a total
of 2.810
3
2.810
6
DNA molecules. As the number of ad-
ministered molecules of plasmid DNA per tattoo was calcu-
lated to be 1.3310
13
, this observation indicates that the in vivo
transfection efficiency is between 210
–8
and 210
–5
% (i.e., 2
to 2000 out of 10,000,000,000 applied plasmid copies are taken
up and translated into protein). These data indicate that
transfection of epidermal cells by DNA tattooing represents a
highly inefficient process, when compared with the currently
available in vitro cell transfection methods. As an example, on
transfection of cells in in vitro systems, using cationic lipo-
somes, approximately 8% of administered DNA molecules
have been shown to become expressed, a 410
5
–410
8
-fold
FIG. 2. Flow cytometric analysis of
epidermal cell suspension of tattooed
skin. Control skin or skin tattooed
at 1.5 mm for 20 sec per 50 mm
2
with pVAX:GFP (5 mg=ml) or empty
pVAX backbone (5 mg=ml) was
stained with anti-CD1a antibody or
with anti-cytokeratin antibody.
184 VAN DEN BERG ET AL.
higher efficiency than that observed here on intradermal DNA
vaccination (Tseng et al., 1997).
Longitudinal measurements of gene expression
To explore the possibility of monitoring antigen expression
in a longitudinal fashion, human skin was tattooed with
firefly luciferase reporter plasmid (pVAX:Luc) and expression
was measured at several time points in intact skin by optical
imaging.
Luciferase expression could readily be detected with a
light-sensitive camera and expression was restricted to the
areas of tattooing (Fig. 3B). Expression was observed at sig-
nificant levels as early as 2 hr after tattooing, indicating that
DNA transfection, translation, and expression of the protein
take place rapidly after DNA tattooing. Luciferase expression
peaked between 2 and 18 hr after tattooing and remained
detectable for approximately 2–3 days (Fig. 4). On the basis of
the preceding data it seems plausible that the longitudinal
measurement of gene expression may be used as a preclinical
model to assess different methods of intradermal genetic
vaccination (Mitragotri, 2005; Giudice and Campbell, 2006).
As a first step toward such a comparison, we evaluated the
capacity of intradermal tattoo and intradermal DNA injection
to induce luciferase expression in intact skin. Remarkably, on
intradermal injection, luciferase expression levels were not
above background levels (whereas a tattoo with the same
solution gives an expression level that is at least 10- to 20-fold
higher as background), indicating that within ex vivo human
skin, the expression on DNA tattooing is at least 10-fold
higher than that obtained on classical intradermal injection.
Optimization of DNA tattooing
Having established the feasibility of performing longitu-
dinal measurements of DNA vaccine-induced antigen ex-
pression in human skin, we aimed to optimize variables that
we considered likely to influence the efficiency of DNA vac-
cination. To this purpose, a total of 428 skin areas with a
constant surface of 50 mm
2
were tattooed with a luciferase-
encoding plasmid, using 10 samples of healthy abdominal
skin. The tattoo variables tested for were as follows: DNA
concentration, duration of tattooing, needle depth, and tattoo
machine (see Table 1).
FIG. 3. Tattooing procedure of human skin (A) and typical expression of luciferase (B), visualized with a light-sensitive
camera, 18 hr after tattooing. Each area of 50 mm
2
was tattooed with a different tattoo setting. Note the marked variation in
luciferase signal obtained under different vaccination conditions.
FIG. 4. Typical longitudinal luciferase expression kinetics
in intact ex vivo human skin on DNA tattooing. Lines rep-
resent expression in three different tattooed areas of 50 mm
2
,
on three different skin explants. The skin was tattooed with
pVAX:Luc at 5 mg=ml for 20 sec, using the Aella tattoo ma-
chine at a depth of 1.5 mm. Note that the kinetics of luciferase
expression are comparable in skin samples derived from
different donors.
DNA TATTOOING IN HUMAN SKIN 185
The relationship between log AUC and the various fixed
effects tested with the Aella machine is shown in Fig. 5 and
data obtained with the Cheyenne machine showed a similar
profile (data not shown). All the tested fixed effects (Table 1),
except the two different tattoo machines, had a significant
effect ( p<0.001) on antigen expression. Specifically, within
the tested range of values an increase in DNA concentration,
needle depth, and tattoo time resulted in an increase in anti-
gen expression.
Patient age and tattoo location (edge or center) were found
not to be significantly related to gene expression AUC. In-
teractions that were significant were between tattoo machine
and pretattoo time ( p¼0.01), between tattoo machine and
tattoo depth ( p¼0.0007), between the log of DNA concen-
tration and pretattoo time ( p¼0.002), and between tattoo
duration and tattoo depth ( p¼0.03). However, none of these
interactions had an influence on the tattoo settings that
resulted in maximal antigen expression. For both tattoo ma-
chines the relationship between the AUC of antigen expres-
sion and the variations is expressed by the equation
log(AUC) ¼log (104)þ(0:35 þ0:13 ·A)·log (Conc)
þ(0:07 0:023 ·C)·BþX
where Xis different for the different tattoo machines (Aella,
X¼6.9 0.76Aþ0.84C; Cheyenne, X¼7.1 0.47Aþ
0.41C) and where Ais pretattoo time (hr), Bis tattoo dura-
tion (sec), and Cis tattoo depth (mm).
When varying each term separately (and fixing the others
at their median values), the AUC changes by a factor of 5.8
over the range of DNA concentrations tested (0.2 to 5 mg=ml),
by a factor of 2.0 over the range of tattoo durations (5 to
20 sec), by a factor of 1.8 and 1.2 over the range of tattoo
depths (0.5 to 1.5 mm) for tattoo machines Aella and Chey-
enne, respectively, and by a factor of 1.2 for Cheyenne versus
Aella. These data demonstrate that, of the variables tested, the
concentration of the DNA solution that is applied is by far the
major determinant of antigen expression in human skin (see
Fig. 6). The lines in Fig. 5 visualize the predicted AUC of
antigen expression obtained from the model. This figure
shows that the obtained equation fits the experimental data.
Discussion
Several dermal delivery techniques are currently moving
from preclinical to clinical evaluation, and consequently there
FIG. 5. Results of the randomization
study, demonstrating the relationship
between the log area under the curve
(AUC) and the pVAX:Luc DNA con-
centration, needle depth, and tattoo
duration for the Aella tattoo machine.
Log AUC is visualized as a box plot
(showing the lowest observation, the
lowest 25% of data, the median, the
highest 25% of data, and highest ob-
servation). Lines visualize the pre-
dicted AUCs obtained from the model
equation.
FIG. 6. Results from the linear mixed effect model, dem-
onstrating the influence of changing each tattoo variation
over its full range (with fixing of the others at median value)
on change in AUC of antigen expression for the Aella tattoo
machine.
186 VAN DEN BERG ET AL.
is a strong need to optimize these methods in a clinically
relevant model. To allow such evaluation, we developed an
ex vivo human skin model and used this model to characterize
and optimize a DNA tattooing strategy for clinical application.
As a first step toward developing this model we analyzed
vaccination-induced antigen expression by flow cytometry
and histology. Flow cytometric analysis of tattooed human
skin showed that only a small fraction of the epidermal cells,
approximately 2%, is transfected. This low frequency of trans-
fection was confirmed in an analysis of treated skin sections
by histology that also showed an infrequent transfection of
cells in this cell layer. This rather inefficient transfection of
cells in the epidermis is comparable to that observed with
other intradermal delivery techniques, such as the gene gun
(Mitragotri, 2005), and to a prior analysis of DNA tattoo-
induced transfection in murine skin (Bins et al., 2005). We
assume that the large amount of DNA that is not taken up by
skin cells is rapidly degraded in the skin by endonucleases,
similar to observations in murine skin (Barry et al., 1999).
We subsequently demonstrated that vaccination-induced
antigen expression can be quantified by longitudinal moni-
toring of luciferase activity in human skin samples. Tattooing
of firefly luciferase encoding DNA in intact skin showed fast
and reproducible expression of the antigen, which we could
measure longitudinally and was retained for 2–3 days. This
kinetic profile is comparable to the in vivo antigen kinetics
observed on DNA tattooing of mice (Bins et al., 2005). This
rather transient nature of vaccine-induced antigen expression
in skin may possibly be explained by the high turnover of the
epidermis (Gelfant, 1982) but could also be due to gene si-
lencing. Importantly, it has previously been shown that fresh
skin in culture does not lose its viability in the first 30 hr of
culturing, with a viability decrease of 50% after 60 hr in me-
dium at 378C (Castagnoli et al., 2003; Messager et al., 2003).
Therefore measurement of ex vivo antigen expression over a
maximal time span of 3 days in this study was deemed ap-
propriate.
As the level and duration of vaccination-induced antigen
expression are correlated with the magnitude of vaccine-
specific cytotoxic T lymphocyte (CTL) response in mice on
tattoo immunization (Bins et al., 2005), we have used the
ex vivo human skin model to determine the relationship be-
tween different vaccination parameters and antigen expres-
sion. From these experiments, analyzed in a linear mixed
effects model, we conclude that the effect of DNA concen-
tration forms the most important factor influencing the AUC
of antigen expression in human skin (Fig. 6). We did not reach
saturation for DNA concentration in these experiments. This
observation is in line with data obtained in mouse models, in
which intramuscular or intradermal injections of naked DNA
were used (Wolff et al., 1990). In contrast, it was reported that
transfection on intradermal injection of messenger RNA is
saturated at a concentration of 0.05 mg=ml. This suggests a
different uptake mechanism between intradermal mRNA in-
jection and the tattooing of double-stranded DNA (Probst
et al., 2007).
Using the ex vivo human skin model, we also showed that
the effects of tattoo time and tattoo depth on antigen expres-
sion are significant, but less dominant. The fact that trans-
fection of skin cells is almost exclusively observed in the
epidermal layer of the skin may explain why an increase in
needle depth has only a small effect on antigen expression.
The epidermis of the abdominal skin samples used in this
study was between 200 and 300 mm in depth. Thus, the min-
imal needle depth of 0.5 mm that was used here should be
enough to reach the complete epidermal layer when consid-
ered a fixed object. However, it is important to stress that
injection depth is probably lower than the needle depth, be-
cause of the flexibility and resistance characteristics of the
skin.
To what extent can data on vaccination-induced antigen
expression obtained in the ex vivo human skin model that we
have developed here be expected to translate to vaccination-
induced antigen expression and vaccine-induced immune
responses in human subjects? A first issue of relevance here is
whether the observed luciferase signal that is primarily de-
rived from keratinocytes forms a good measure of vaccine
efficacy. Specifically, can expression in keratinocytes be ex-
pected to correlate with immunogenicity?
Murine studies using a plasmid carrying a K14 promoter,
which is active only in keratinocytes, suggest that cross-
presentation is an important route in the induction of cyto-
toxic T cell responses by DNA tattooing (Bins et al., 2007b).
These findings were in line with a study showing that exclu-
sive transfection of dendritic cells (DCs) (using a CD11c pro-
moter) by gene gun immunization cannot trigger strong
CD4
þ
and CD8
þ
cell responses in mice (Lauterbach et al.,
2006), suggesting that antigen expression in non-APC cell
types may be required for the generation of strong cellular
immunity. Thus, antigen expression within keratinocytes is
likely to be in large part responsible for the immunogenicity of
intradermal DNA vaccines and the measurement of antigen
expression in human skin samples is therefore likely to have
predictive value. Interestingly, it has previously been shown
that phagocytosed GFP is quenched in the acidic environment
of the endosomes (Tsien, 1998). As the small population of
GFP-positive LCs that is observed in human skin samples
does show a lower GFP intensity than the GFP-positive ker-
atinocyte population (Fig. 2), it may be speculated that the
GFP in these LCs may possibly be derived from phagocytosed
material. To further unravel the source of the GFP signal ob-
served in LCs, we tattooed skin with a construct encoding a
histone–GFP fusion protein. Direct transfection of cells with
this construct results in expression of GFP exclusively in the
nucleus of the cells, where GFP taken up by cross-presentation
should be present in the endosomal pathway and possibly
also in the cytosol=nucleus. By sorting GFP
þ
CD1a
cells and
GFP
þ
CD1a
þ
cells and analyzing these separated populations
by confocal laser microscopy, we aimed to reveal the subcel-
lular localization of the GFP expressed in these cells. As
shown in Fig. 7, GFP expression in CD1a-negative keratino-
cytes is clearly restricted to the nucleus, demonstrating their
direct transfection. Unfortunately, because of their low num-
bers and fragility during the sorting procedure, we were not
able to successfully visualize GFP-positive Langerhans cells
by confocal laser scanning microscopy. Therefore, the source
of their GFP signal remains unknown at present.
On accepting that the antigen expression observed in
ex vivo skin samples has predictive value for in vivo immu-
nogenicity, it is also of importance to consider the effect of
the variables that have been tested here. With regard to the
influence of DNA concentration on antigen expression, it may
be expected that the influence of this parameter on antigen
expression will be similar in vitro and in vivo, as an increase in
DNA TATTOOING IN HUMAN SKIN 187
DNA concentration is not expected to alter the location of
antigen expression or cell type involved. In addition, high
antigen expression due to increased DNA concentration is
likely to translate directly into increased antigen presentation
and immunogenicity. Also, with respect to the influence of
DNA tattoo time on antigen expression, the in vitro skin model
is likely to translate well as the site of antigen expression and
cell types involved are unlikely to be influenced by this pa-
rameter. It does, however, seem possible that the effect of
DNA tattoo time on immunogenicity is greater than predicted
by the monitoring of ex vivo antigen expression, as the in-
creased tissue damage that occurs on prolonged vaccination
may have an adjuvant effect. Finally, it may be argued that the
effect of tattoo depth on antigen expression measured here
may perhaps have the smallest predictive value for in vivo
antigen expression and immunogenicity, as the flexibility of
skin in patients may well be greater than that of the fixed
ex vivo skin samples used here.
On the basis of the analyses performed in this study we will
initiate a phase 1 clinical trial, in which we will administer a
DNA vaccine at a concentration of 5 mg=ml for 20 sec per
50 mm
2
of skin surface, at a needle depth of 1.5 mm, the set-
tings that showed the maximal level of antigen expression
in this study. In this trial, a DNA vaccine encoding the HLA-
A2-restricted MART-1 (melanoma antigen recognized by T
cells-1) epitope will be administered to patients with meta-
static melanoma. This will be a dose escalation study, in which
an increased dose of the DNA vaccine will be achieved by a
simple increase in the skin area used for DNA tattoo appli-
cation, ranging from 4 cm
2
up to 32 cm
2
.
In conclusion, we here demonstrate that ex vivo human skin
is an adequate model for the characterization and optimiza-
tion of intradermal DNA vaccines. Furthermore, we have
shown that at fixed volumes, DNA concentration is the most
important parameter influencing vaccination-induced anti-
gen expression. In ongoing experiments, the skin model de-
veloped in this study is being used to determine the value of
nonviral DNA carriers and other dermal delivery techniques
for their ability to improve dermal antigen delivery. It seems
reasonable to assume that the preclinical testing of such DNA
vaccine formulations in this ex vivo human skin model will
form an efficient strategy to select promising vaccination
strategies for subsequent testing in clinical trials.
Acknowledgments
The authors thank Johan Westerga, Inge Heins, and
Chantal Lamers-de Ruiter for help with histology experi-
ments, and Silvia Ariotti for help with confocal laser scanning
microscopy.
Author Disclosure Statement
No competing financial interests exist.
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Address reprint requests to:
Dr. John B.A.G. Haanen
Division of Immunology
Netherlands Cancer Institute
Plesmanlaan 121
1066 CX, Amsterdam, The Netherlands
E-mail: j.haanen@nki.nl
Received for publication June 2, 2008;
accepted after revision November 25, 2008.
Published online: January 21, 2009.
DNA TATTOOING IN HUMAN SKIN 189
Citations (48)
References (30)
... Mechanisms by which DNA vaccines induce in vivo antigenspecific immunity are still the subject of intense research. Antigen encoding-DNA can be introduced into the body by intramuscular or intradermal inoculation through a variety of delivery tools, including intramuscular injection with or without in vivo electroporation [9], intradermal injection by gene gun [10], tattooing [11], microneedles [12], or low-frequency ultrasound [13]. At the inoculum locus, upon reaching the nucleus of the transfected cell, which may be a myocyte, primary keratinocyte, or even a resident antigen-presenting cell (APC), the antigen gene is processed by cell expression machinery ( Figure 1). ...
... However, DNA tattooing has low transfection efficiency and, therefore, it is still not an attractive alternative to methods already in clinical trials, such as gene gun and in vivo electroporation. Other parameters that may affect intradermal DNA tattooing transfection include DNA concentration, needle depth, and injection frequency during delivery [11]. Consequently, studies that optimize this technique in human patients are still necessary before any clinical trial conduction. ...
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... This strategy was shown to lead to a more rapid induction of cellular immunity as compared to conventional application methods of DNA vaccination in mice [20]. Furthermore, DNA tattooing outperforms classical intramuscular DNA vaccination by 10-to 100-fold when tested in non-human primates [21] and was optimized in an ex vivo human skin model [22]. ...
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... As the vaccine is distributed over a wide surface, more cells are likely to be transfected (Kis et al. 2012). Antigen expression levels are dependent on the needles depth, the tattoo duration type and the DNA concentration (van den Berg et al. 2009). Tattoo delivery systems promote higher specific cellular and humoral immune responses than subcutaneous administration. ...
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... In these skin damage models, sensitization occurs comparable to tattooing strategies that are used to improve vaccination efficiency during the induction of a protective immune response. 47 As repeatedly demonstrated in intraperitoneal oval- bumin sensitization models, skin sensitization also results in the production of allergen-specific IgE and IgG1 anti- bodies. 48 Furthermore, ex vivo cultured cells isolated from the axillary, subscapular, and inguinal draining lymph nodes of ovalbumin exposed mice produced increased amounts of IL4 in the presence of the allergen. ...
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