Biomaterials 145 (2017) 256e265

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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Hepatitis B surface antigen incorporated in dissolvable microneedle

array patch is antigenic and thermostable
de
Danielle Poirier a, 1, Fre ric Renaud b, Vincent Dewar b, Laurent Strodiot b,
Florence Wauters b, 2, Jim Janimak b, *, Toshio Shimada c, Tatsuya Nomura c, Koki Kabata c,
Koji Kuruma c, Takayuki Kusano c, Masaki Sakai c, Hideo Nagasaki c, Takayoshi Oyamada c
a
GSK, Laval, Quebec, Canada
b
GSK, Rixensart, Belgium
c
FUJIFILM Corporation R&D, Kanagawa, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Alternatives to syringe-based administration are considered for vaccines. Intradermal vaccination with
Received 29 June 2017 dissolvable microneedle arrays (MNA) appears promising in this respect, as an easy-to-use and painless
Received in revised form method. In this work, we have developed an MNA patch (MNAP) made of hydroxyethyl starch (HES) and
16 August 2017
chondroitin sulphate (CS). In swines, hepatitis B surface antigen (HBsAg) formulated with the saponin
Accepted 25 August 2017
Available online 29 August 2017
QS-21 as adjuvant, both incorporated in HES-based MNAP, demonstrated the same level of immuno-
genicity as a commercially available aluminum-adjuvanted HBsAg vaccine, after two immunizations 28
days apart. MNAP application was associated with transient skin reactions (erythema, lump, scab),
Keywords:
Hydroxyethyl starch
particularly evident when the antigen was delivered with the adjuvant. The thermostability of the
Microneedle adjuvanted antigen when incorporated in the HES-based matrix was also assessed by storing MNAP at 37,
Hepatitis B antigen 45 or 50
C for up to 6 months. We could demonstrate that antigenicity was retained at 37 and 45
C and
QS-21 only a 10% loss was observed after 6 months at 50
C. Our results are supportive of MNAP as an attractive
Thermostability alternative to classical syringe-based vaccination.
Vaccine 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction
Vaccination has greatly helped to reduce disease, disability and
death worldwide. Nevertheless, access to vaccination may be
limited for some population groups, as most vaccines need to be
injected under the skin or deep into the muscle using needle-based
syringes, which require skilled health care professionals for the
administration, hence increasing the costs associated with the
immunization. In addition, needle fear is a reality for many people,
which may cause them to avoid vaccination [1,2]. New alternative
vaccination strategies that are less invasive might allow over-
coming the acceptance and access issues associated with injectable
vaccinations.
Another issue limiting vaccine access is the fact that most vac-
cines need to be kept and transported within a specic refrigeration
temperature range (usually 2e8
C). Beyond the logistic problems
* Corresponding author. GSK, Rue de lInstitut 89, 1330, Rixensart, Belgium.
E-mail address: JIM.J.JANIMAK@GSK.COM (J. Janimak).
1
Current address: PAIRimmune, Quebec, Canada.
2
Current address: MSD Belgium, Brussels, Belgium.
that can be encountered, the costs associated with vaccine storage
and transportation concur for a signicant part of the global im-
munization costs. So methods to produce thermostable vaccines
are needed, as they would contribute to render vaccination more
affordable for developing countries by decreasing these costs.
In this context, microneedle array patch (MNAP) has been
gaining attention as an alternative vaccine delivery method [3e6].
Visually MNAP is free from any needle and it is said to be painless as
it does not stimulate nerve ends that are associated with pain [7].
Both elements might be an asset for patients who are reluctant to
injection. In addition, MNAP is user-friendly, as it is ready-to-use
and does not require reconstitution, implying that it may be
administered by healthcare personnel with only minimal medical
training. Lastly, the use of microneedles has been shown to allow
antigen dose sparing while retaining equivalent degree of immune
responses as classical intramuscular injections in preclinical models
[8,9] and in humans [10].
First generation MNAs were made of silicon or metal. The an-
tigen in solution could be incorporated in hollow microneedles or
be coated on them [3,5,6]. However, this type of MNA may break off

http://dx.doi.org/10.1016/j.biomaterials.2017.08.038
0142-9612/ 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
 D. Poirier et al. / Biomaterials 145 (2017) 256e265
Fig. 1. Schematic view of the procedure to manufacture HBsAg-containing MNAP.
during administration, leaving tiny pieces of material in the skin,
which is a signicant safety concern. Here, we have developed a
dissolvable MNA. These microneedles are made of hydroxyethyl
starch, a polysaccharide already used in the pharmaceutical in-
dustry [11]. Due to the solubility of their components, such needles
are expected to resorb into the skin within minutes, releasing the
vaccine components and bringing them in contact with the cuta-
neous immune system. In addition, antigens in solid MNA are
known to be more thermostable as compared with antigens in
liquid vaccines [3], hence MNAP represent a possibility to store and
transport vaccines without the constraints and the costs of main-
taining the cold chain.
The present work describes the production of dissolvable MNA
containing hepatitis B surface antigen (HBsAg). Its capacity to
induce immune responses, with or without adjuvant was evaluated
in swines, whose skin is similar to that of humans in terms of
thickness, structure and immune function [12e14]. In addition,
antigen stability in our MNAP was also investigated.
2. Materials and methods
2.1. Antigen and adjuvant
HBsAg (GSK Vaccines) solution was concentrated using ultra-
ltration techniques involving Pellicon 3 with 88 cm3 Biomax 10KD
polyethersulfone (PES) kit (Merck Millipore) to reach a nal con-
centration of HBsAg 60 mg/ml, as determined by the Lowry
method [15].
QS-21 (Quillaja saponaria Molina, fraction 21; licensed by GSK
from Antigenics LLC, a wholly owned subsidiary of Agenus Inc., a
Delaware, USA corporation) is an immuno-enhancer formulated in
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes con-
taining cholesterol at 100 mg/ml. QS-21 was further concentrated
Table 1
Immunization groups in the study using MNA containing non-adjuvanted HBsAg.
Groups Prime
1 Ctrl
2 Ctrl
3 Ctrl
4 MNAP
5 MNAP
Ctrl, control vaccine, injected i.m; MNAP, microneedle array patch.
257
Table 2
Immunization groups in the study using MNA containing QS-21-adjuvanted HBsAg.
Groups Prime Boost
1 Ctrl Ctrl
2 Ctrl QS-21-MNAP
3 Ctrl Non-adjuvanted MNAP
4 QS-21-MNAP QS-21-MNAP
5 QS-21-MNAP None
Ctrl, control vaccine, injected i.m; MNAP, microneedle array patch.
approximately 60 times using Pellicon XL 50 cm3 Biomax 50KD PES
kit (Merck Millipore). The term QS-21 used throughout this
manuscript refers to QS-21 formulated in liposomes.
2.2. Production of MNAP
MNAPs were produced in an aseptic isolator of Grade A in a GMP
pilot facility. First, hydroxyethyl starch 70000 (HES, Fresenius Kabi)
was dissolved in distilled water and mixed with HBsAg (4:1 w/w).
Alternatively, QS-21 was incorporated into the mixture as an
adjuvant. In the latter case, HES and sucrose (Wako Chemicals)
were dissolved in distilled water and mixed with HBsAg and QS-21
(HES:sucrose:QS-21:HBsAg 18:11:6:1 w/w). In both cases, the
aqueous solution was sterilized through a 0.22-mm hydrophilic
polyethersulfone membrane (Millex SLGP033RS, Millipore) and
casted into thermosetting resin-based micromolds to shape the
microneedles. The same solution weight was distributed in each
microneedle. The procedure yielded MNAP comprising 100
microneedles, each approximately 600 mm long. After drying at
23
C for approximately 3.5 h, a polyethylene terephthalate (PET)
base lm coated with an aqueous solution containing chondroitin
sulphate (CS, Maruha Nichiro), was attached to the micromolds and
dried at 35
C for 19e24 h. After drying, the PET base lm bound to
the MNAP was easily separated from the micromolds. The shape
and length of the microneedles was conrmed under a digital
microscope (VHX-5000, Keyence). MNAP was placed in a plastic
case, packed with desiccant into a sealed aluminium foil bag and
Boost
stored at 4
C until use. Final water content of MNAP was <5% (w/
None w). The whole procedure is depicted in Fig. 1.
Ctrl
MNAP
MNAP 2.3. HBsAg distribution in MNAP
None
HBsAg localization in the microneedles was evaluated after
258 D. Poirier et al. / Biomaterials 145 (2017) 256e265

Table 3
Scoring criteria for evaluation of skin reactions.

Grade Erythema Lump Scab

0 None None None

1 Very slight (barely visible) Very slight (barely visible) Small (less than 3 mm)
2 Well-dened Slight (edges of area well-dened by denite raising) Medium (3e8 mm)
3 Moderate to severe Moderate (raising approximately 1 mm) Large (more than 8 mm)
4 Severe (beet redness) Severe (raising more than 1 mm and extending beyond the injection area) /

cutting at different levels by using a needle cutter (Fujilm pro-
prietary technology). The two different heights of cut, at 200 and
300 mm were dened after observation of the remaining micro-
needle length in pilot experiments. After cut, microneedle parts
were dissolved in phosphate-buffered saline (PBS) containing 0.1%
polysorbate 20 (PBS-T). HBsAg content was quantied by a
competitive enzyme-linked immunosorbent assay (ELISA) as
follows:
ELISA plates were rst coated with HBsAg overnight at 4
C and
then blocked by adding PBS containing 1% bovine serum albumin
(Sigma-Aldrich) for 30 min at 37
C. Concomitantly, dissolved
MNAP containing HBsAg was mixed with a known amount of anti-
hepatitis B human antibody (Nabi-HB, Biotest Pharma) and incu-
bated overnight at 25
C with shaking. Aggregated HBsAg/antibody
complex was sedimented by centrifugation and the supernatant
containing non-aggregated antibodies was transferred to the plates
and incubated for 2 h at 37
C. Thus, the more HBsAg in the dis-
solved MNAP, the less anti-hepatitis B human antibody left in the
supernatant. Horseradish peroxidase (HRP)-conjugated goat anti-
human IgG (Sigma-Aldrich) was added to the plate and incubated
for 1 h at 37
C. Between each process, plates were washed 6 times
with saline containing 0.1% polysorbate 20. Bound antibodies were
revealed by the addition of the peroxidase substrate tetrame-
thylbenzidine (TMB) for 20 min at room temperature in the dark.
The reaction was stopped by adding 1 N H2SO4, and the optical
density was measured at 450 nm and 620 nm. The intensity of the
reaction was inversely proportional to the original amount of
HBsAg in the MNAP.
2.4. In vivo study
2.4.1. Immunization
All animal studies were approved by the Institut National de la
Recherche Scientique e Institut Armand Frappier (INRS-IAF) An-
imal Care and Use Committee and were compliant with the Cana-
dian Council on Animal Care (CCAC) and the Association for
Assessment and Accreditation of Laboratory Animal Care (AAALAC)
recommendations and guidelines.
Twenty-eight-day-old female Yorkshire/Landrace F1(Y x L) x
Duroc swines were obtained from a porcine reproductive and res-
piratory syndrome virus (PRRSV)-seronegative farm. The animals
were pre-selected one week earlier, based on their health status
and appearance to have homogeneity of the swine population. For
all studies, animals were kept group-housed with 12 h light/dark
cycle and fed twice a day. They were four-month-old and weighed
approximately 35 kg at the start of the study.
Before immunization, the animals (n 6/group) were pre-
anaesthetized by intramuscular (i.m.) injection of ketamine/xyla-
zine (20 mg/kg: 2 mg/kg) in the neck region, and maintained under
isourane (1e3%). Atropine (0.5 mg/kg) was given in prevention for
tachycardia. The ank was rst carefully shaved using electric
clippers, followed by an electric shaver and a blade shaver in order
to get an injection site free of any hair residues. Thereafter, the
injection site was carefully wiped with warm water to clean the
skin before MNAP application. MNAP was administrated on the
shaved skin with a kinetic energy of 0.5 J using handheld applicator
(Fujilm proprietary technology) and held rmly in place for
10 min with a force of 5 N. A second immunization was made four
weeks later. As control, Engerix-B (GSK Vaccines), which contains
20 mg HBsAg/0.5 mL and Al(OH)3 adjuvant, was administered by
intramuscular injection in the gluteus supercialis muscle (this
vaccine when administered intramuscularly will be called control
vaccine [Ctrl] throughout the text). Tables 1 and 2 summarize the
different groups used in the studies.
An experiment was also performed to determine the adequate
dose of QS-21. In this experiment, 20 mg HBsAg in solution was
intradermally injected together with 1.5 mg, 5 mg, 15 mg or 50 mg QS-
21 in 0.1 mL using a ne hypodermic needle with the bevel facing
upwards to mimic a Mantoux injection.
2.4.2. Determination of humoral responses
Blood samples were collected 28 days after the boost. Serum

Fig. 2. A. Microphotograph of a whole microneedle array. B. View of porcine skin after insertion and removal of microneedles, showing delivery of a blue dye. C. Details of a
microneedle.
 D. Poirier et al. / Biomaterials 145 (2017) 256e265 259

Fig. 3. Distribution of HBsAg inside the microneedles. A. The needles were laser cut at different levels, dening different regions. The height of the cuts is shown with deviations. B.
The different regions were dissolved in phosphate-buffered saline and their HBsAg content was quantied by ELISA. ND not determined.

Fig. 4. Microphotographs of needles before and after administration in swine. In example 1, the microneedle was not entirely dissolved, as 200 mm remained, which means that 80%
of HBsAg content was administered. In example 2, the entire microneedle was dissolved, which means that 100% of the HBsAg content was administered.

antigen-specic IgG were quantied by ELISA. Briey, Maxisorp 96-
well at-bottom plates (Nunc cat: 439454) were coated with 1 mg/
ml HBsAg (GSK Vaccines) in carbonate-bicarbonate buffer 50 mM
pH 9.6 for 2 h at 37
C, For the reference curve, goat anti-swine IgG
capture antibody (1/100; Bethyl cat: A100-104A) was used for
coating. Plates were washed and aspecic sites were blocked with
Tris-buffered saline containing 0.05% polysorbate 20 and 1% bovine
serum albumin for 1 h at room temperature. Two-fold serial di-
lutions of reference serum (Bethyl cat: RS10- 107) diluted 1/8250 or
samples (pre-diluted if needed) were added for a 1 h incubation at
room temperature. After washing, HRP-conjugated anti-swine IgG
(Bethyl cat: A100-104P) diluted 1/75000 in blocking solution were
dispensed for a 30 min incubation at 37
C. Bound antibodies were
revealed by addition of TMB for 30 min at room temperature
protected from light. The colorimetric reaction was stopped with
H2SO4 1N. Results were read using a microplate reader at 450 nm
and concentrations were extrapolated from the standard curve
following a 4 parameters equation with the following criteria: % CV
below 20% between a minimum of 3 calculated concentrations.
2.4.3. Conrmation of MNA dissolution
Microneedle resorption was conrmed by measuring needle
length on microscopic images taken by a digital microscope (VHX-
5000, Keyence) after administration. A total of 48 MNAPs were
administered, 24 without adjuvant and 24 with adjuvant.
The percentage of antigen delivered to an animal was estimated
based on the remaining length of each microneedle and the known
HBsAg distribution in MNAP.
260 D. Poirier et al. / Biomaterials 145 (2017) 256e265

Table 4
Remaining needle length after administration (MNAP containing non-adjuvanted
HBsAg).

Total remaining Estimation of

needle length (mm) administered
antigen dose (mg)

Groups Swines Prime Boost Prime Boost

Group 3 Swine 3-1 / 66 / 17

Prime: Ctrl Swine 3-2 / 107 / 16
Boost: MNAP Swine 3-3 / 39 / 16
Swine 3-4 / 82 / 16
Swine 3-5 / 56 / 17
Swine 3-6 / 139 / 16
Group 4 Swine 4-1 46 276 16 11
Prime: MNAP Swine 4-2 48 29 17 16
Boost: MNAP Swine 4.3 9 40 17 16
Swine 4-4 13 107 18 17
Swine 4-5 39 17 17 16
Swine 4-6 55 45 15 16
Group 5 Swine 5-1 30 / 18 /
Prime: MNAP Swine 5-2 30 / 16 /
Boost: None Swine 5.3 35 / 17 /
Swine 5-4 16 / 17 /
Swine 5-5 35 / 18 / Fig. 5. IgG levels on Day 56 (Day 28 after boost immunization). Different combinations
Swine 5-6 15 / 18 / of prime and boost immunizations were used involving intramuscular administration
of an alum-adjuvanted control vaccine (Ctrl) and MNAP administration of non-
adjuvanted HBsAg, as indicated under the graph. One dot represents one animal,
and geomeans with 95% CI are shown. Statistical analyses evaluated the non-inferiority
2.4.4. Assessment of skin reaction of every group compared with Group 1 (benchmark).
Degree of erythema, lump and scab at the site of MNAP appli-
cation was measured for at least 7 days and at most 16 days after
administration, by using an adapted Draize scoring method, as per immunization route was at least non-inferior to the benchmark
the criteria listed in Table 3 [16,17]. consisting of two Ctrl injections. The non-inferiority [0.25, [ of a
group against the control group was assumed if the lower limit of
2.5. Stability of HBsAg in MNAP the 95% condence interval of the geometric mean ratio (group/
control) was greater than 0.25. Estimates of the geometric mean
MNAPs containing QS-21-adjuvanted HBsAg were stored at 37, ratios between groups and their 95% condence intervals (C I) were
45, or 50
C for up to 6 months in a sealed aluminum foil bag with derived from an Analysis of Variance (ANOVA) on the log10 values
dessicant. After storage, MNAs were dissolved in PBS-T and the with 1 factor (DQ escalation) using a heterogeneous variance
antigenicity of HBsAg was evaluated by ELISA as described in Sec- model. Adjustment for multiplicity was performed using the
tion 2.3. Dunnetts method.

2.6. Statistics 3. Results

The aim of each analysis was to evaluate whether the new 3.1. Shape of MNAP and HBsAg distribution

The picture of a whole MNA is shown in Fig. 2A. MNAP consisted

Table 5
of 100 microneedles/patch. When administered to porcine skin, it
Remaining needle length after administration (MNAP containing QS-21-adjuvanted
HBsAg). left a typical print consisting of 100 little pricks, as shown in Fig. 2B,
obtained with HES microneedles mixed with a blue dye.
Total remaining Estimation of
A microneedle was composed of 2 parts, the needle part (made
needle length (mm) administered
antigen dose (mg) of HES and containing HBsAg with or without QS-21) and the base
part (made of CS), as shown in Fig. 2C.
Groups Swines Prime Boost Prime Boost
The length of the needle part was between 582 and 647 mm for
Group 2 Swine 2-1 / 101 / 21 MNAP without QS-21 and between 594 and 650 mm for MNAP with
Prime: Ctrl Swine 2-2 / 151 / 19
Boost: MNAP Swine 2-3 / 47 / 22
QS-21. HBsAg content was between 16 and 19 mg for MNA without
Swine 2-4 / 107 / 20 QS-21, and between 22 and 24 mg for MNA with QS-21.
Swine 2-5 / 81 / 22 To measure HBsAg distribution in needle, the needles were cut
Swine 2-6 / 110 / 21 at the levels shown in Fig. 3A and HBsAg content was measured by
Group 4 Swine 4-1 46 47 21 23
ELISA after dissolution of the microneedles in buffer. No signicant
Prime: MNAP Swine 4-2 82 53 21 22
Boost: MNAP Swine 4.3 122 105 20 20 antigen aggregation was noticed, as evaluated by microscopic
Swine 4-4 90 69 20 22 observation. Cumulative HBsAg content is shown in Fig. 3B. When
Swine 4-5 36 116 22 21 formulated without QS-21, 75% of HBsAg was localized in the rst
Swine 4-6 33 83 22 22 400 mm of the needle tip, whereas it was 80% of HBsAg in the
Group 5 Swine 5-1 97 / 20 /
Prime: MNAP Swine 5-2 94 / 20 /
presence of QS-21.
Boost: None Swine 5.3 46 / 21 /
Swine 5-4 89 / 20 / 3.2. Conrmation of needle dissolution
Swine 5-5 33 / 21 /
Swine 5-6 220 / 17 /
After administration to the animals, the remaining length of
 D. Poirier et al. / Biomaterials 145 (2017) 256e265 261

Fig. 6. Skin reactions over time after administration of MNAP with non-adjuvanted HBsAg. A. Skin reactions after one MNAP immunization (priming). B. Skin reactions after the
second immunization done with MNAP. Pre indicates the reactions before immunization. Day 0 refers to the reactions measured 2 h after administration. Ctrl refers to the control
vaccine. The same color code as in Fig. 5 has been used for the groups. One dot represents one animal. (For interpretation of the references to colour in this gure legend, the reader
is referred to the web version of this article.)

every needle was measured, as shown in Fig. 4.
When non-adjuvanted, more than 15 mg of HBsAg was admin-
istered (except in 1 case out of 24 administrations) (Table 4). When
formulated with QS-21 more than 17 mg of HBsAg was administered
in all cases (Table 5).
3.3. Immunization with MNAP
3.3.1. MNAP containing non-adjuvanted HBsAg
3.3.1.1. MNAP-induced humoral responses. We evaluated whether
MNAP administration of the antigen could be efcient without
adjuvant, due to its unique way of administration and direct contact
with the skin immune cells. To this aim, serum IgG level was
measured 28 days after boost immunization (Fig. 5).
We observed, by comparing group 5 with group 1, that priming
with MNAP-administered non-adjuvanted HBsAg was not as
effective as priming with Ctrl. A boost with MNAP after priming
with MNAP (Group 4) did not induce humoral immunity either.
Further, administering a non-adjuvanted MNAP boost after priming
with Ctrl was not found non-inferior to the benchmark (twice Ctrl)
(Group 3 versus Group 2). It was not found inferior either, probably
due to a lack of power of the analysis. The ratio between these two
groups was 2.58 (95% CI: 0.42e15.85).
3.3.1.2. MNAP-induced reactogenicity in skin. After a single MNAP
application of non-adjuvanted HBsAg (priming; Fig. 6A), erythema
started to be visible in the majority of animals 2 h later. One day
after immunization with MNAP, all animals showed maximum
grade 2 erythema. No lump and no scab were detected. After 3 days,
no skin reactions remained visible.
Skin reactions were also evaluated after MNAP containing non-
adjuvanted HBsAg was used as second immunization (boost;
Fig. 6B). When priming was done with MNAP, the strongest ery-
thema reactions after MNAP boost were grade 1 and it took more
than 5 days for them to disappear, which was a bit longer than what
was observed after priming. When priming was done with Ctrl and
booster with non-adjuvanted MNAP erythema grade looked higher
and lasted longer than after two administrations with MNAP. Some
slight lump was observed in both groups (Fig. 6B). Scab was not
observed.
3.3.2. MNAP containing QS-21-adjuvanted HBsAg
The results described in the former section tended to indicate
that MNAP administration could not replace the use of an adjuvant
for priming. This prompted us to evaluate MNAP priming with
adjuvanted antigen.
3.3.2.1. Selection of QS-21 dose. QS-21 was selected as adjuvant and
the rst step was to determine the optimal dose of QS-21 able to
elicit similar anti-HBsAg antibody levels as those induced by Ctrl
which was needed to further compare MNA-induced with Ctrl-
induced antibody levels. For that, HBsAg in solution was
262 D. Poirier et al. / Biomaterials 145 (2017) 256e265

Fig. 7. Selection of QS-21 dose. A. IgG levels on Day 56 after intradermal immunization with HBsAg in solution, alone or adjuvanted with escalating doses of QS-21 (from 1.5 mg to
50 mg, as indicated under the graph). One dot represents one animal, and geomeans with 95% CI are shown. Statistical analyses evaluated the non-inferiority of the groups compared
with group 6 (benchmark: Control vaccine [Ctrl] prime and boost). An equal sign indicates a group that is non-inferior to the benchmark. B. Skin reactions over time after the second
intradermal immunization with HBsAg adjuvanted with escalating doses of QS-21. Pre indicates the reactions before immunization. Day 0 refers to the reactions measured 2 h
after immunization. In A and B the same color code has been used for the groups. (For interpretation of the references to colour in this gure legend, the reader is referred to the
web version of this article.)

intradermally injected in the presence of different doses of QS-21. equivalent humoral responses, which prompted us to choose 15 mg
Antibody levels and skin reactogenicity were evaluated to deter- QS-21 for further experiments.
mine the adequate dose of QS-21 to be used in MNA (Fig. 7).
It was observed that 1.5 and 5 mg QS-21 were not sufcient to
3.3.2.2. MNAP-induced humoral responses. Serum IgG level was
obtain anti-HBsAg antibody levels similar to those induced by Ctrl
measured 28 days after boost immunization (Fig. 8). We observed
(Fig. 7A). Moreover, the response was highly variable. With both
that single MNAP priming with QS-21-adjuvanted HBsAg was able
15 mg and 50 mg QS-21, anti-HBsAg antibody levels were not inferior
to elicit an anti-HBsAg antibody response (Group 5), but it was not
to those induced by Ctrl. When considering skin reactivity (Fig. 7B),
non-inferior to the benchmark (priming and boost with Ctrl; Group
it appeared that immunizing with 50 mg QS-21 was more reacto-
1). Boosting with QS-21-adjuvanted HBsAg led to antibody levels
genic than with 15 mg. Scab was particularly important when
not inferior to those obtained in the benchmark (Group 4 versus
immunizing with 50 mg QS-21, as compared with 15 mg, for almost
Group 1). Likewise, priming with Ctrl and MNA boosting with QS-
 D. Poirier et al. / Biomaterials 145 (2017) 256e265
Fig. 8. IgG levels on Day 56 (Day 28 after boost immunization). Different combinations
of prime and boost immunizations were used, involving intramuscular administration
of an alum-adjuvanted control vaccine (Ctrl) and MNAP administration of QS-21-
adjuvanted or non-adjuvanted HBsAg, as indicated under the graph. One dot repre-
sents one animal, and geomeans with 95% CI are shown. Statistical analyses evaluated
the non-inferiority of the groups compared with group 1 (benchmark: Control vaccine
[Ctrl] prime and boost). An equal sign indicates a group that is non-inferior to the
benchmark.
21-adjuvanted HBsAg was not inferior to the benchmark (Group 2
versus Group 1). We could also observe, after priming with Ctrl,
that MNA administration of non-adjuvanted HBsAg elicited lower
specic antibody levels than MNA administration of QS-21-
adjuvanted HBsAg. This group was not found non-inferior to the
benchmark (Group 3 versus Group 1), as observed earlier (see
Fig. 5). A meta-analysis was performed that included the two ex-
periments. In this meta-analysis, the ratio between the two groups
was 2.5 (95% CI: 1.2e5) and the Ctrl/non-adjuvanted MNAP group
was found inferior to the benchmark (p < 0.05).
3.3.2.3. MNAP-induced reactogenicity in skin. Skin reactions at the
MNAP application site are shown in Fig. 9. They were more
important than when applying MNAP containing non-adjuvanted
antigen. Particularly, after a single application (priming; Fig. 9A),
lumps and scabs were visible, which was not the case with non-
adjuvanted antigen. More grade 3 erythema was also recorded,
contrary to what was observed in the absence of QS-21. It took also
longer for erythema to dissipate, in approximately 10 days, as
compared with non-adjuvanted MNAP priming. Similarly, reactions
were more intense and lasted longer after booster with adjuvanted
MNAP, independently of the type of priming, than after booster
with non-adjuvanted MNAP (Fig. 9B). Up to grade 4 erythema and
lump were observed, but they did not last longer than one week.
Grade 3 scab was common and in one animal it lasted more than 16
days.
3.4. Stability of HBsAg in MNAP
The stability of HBsAg inside MNAP was evaluated by measuring
its antigenicity after storage for up to 6 months at different tem-
peratures (37
C, 45
C, 50
C). No reduction in antigenicity was
observed when stored at 37
C or 45
C. When stored at 50
C, a 10%
decrease in antigenicity was observed after 6 months (Fig. 10).
263
4. Discussion
We have produced a patch incorporating dissolvable MNA and
shown that it can be successfully used for HBsAg immunization.
Contrary to i.m. injections, MNAP immunization delivers the anti-
gen into the dermal layer of the skin where immunocompetent
antigen-presenting cells (including Langerhans cells) are densely
distributed [18]. Consequently, it was reasonable to think that
adjuvant could be avoided to a certain extent by using MNAP.
However, we observed that a boost with non-adjuvanted MNAP
was inferior to a boost with Ctrl (which is injected intramuscularly).
An adjuvant was also found necessary to prime with MNAP, and
when an adjuvant was used similar antibody levels were found
after two MNAP applications as after two Ctrl injections. In contrast,
two MNAP applications without adjuvant did not induce antibody
production in 5/6 animals.
Medium to high grade skin reactions were observed after MNAP
administration of QS-21-adjuvanted HBsAg. Skin reactions after
vaccination are not unusual as they reect the activation of the
innate immune system that takes place locally. Reactions can be
particularly strong when an adjuvanted vaccine is administered
through the cutaneous route in contrast to the intramuscular route
[19]. The reactions in swine skin were all transient, with erythema
and lump disappearing in approximately ten days. Scabs lasted
longer, up to two weeks. We found no correlation between IgG
levels and the degree of skin reactions (data not shown). It must be
emphasized that the skin reactions observed in this work do not
necessarily reect MNAP reactogenicity per se, as the reactions
depend more on the antigen and certainly of the adjuvant used. The
safety of MNAP administration will need to be investigated on a
case-by-case basis, so more work is needed, particularly in humans,
to determine whether the observed reactions ranged within an
acceptable reactogenicity prole. This is particularly true, as for
example the extent of pain could not be investigated in our study.
The development of MNAPs also aims to relieve the cold chain
constraints. Engerix-B, the commercially available alum-
adjuvanted HBsAg has a three-year shelf-life if stored at 4e8
C
[20] but is less stable at higher temperatures. Engerix-B lost
approximately 20% of antigenicity after 3 months at 37
C and 45%
after 3 months at 50
C [21]. Similarly, in a study with another
alum-adjuvanted HBsAg vaccine, a decrease in potency was
observed after 12 months at 25
C and after one month onwards at
37
C [22]. Here, we have seen that the antigenicity of HBsAg when
incorporated within the HES microneedles was maintained during
6 months at 37 or 45
C. Only a 10% loss was measured after
6 months at 50
C. The thermostability of the adjuvanted antigen
has been considerably increased when incorporated into our HES-
based MNAP. This nding was not investigated further but we may
hypothesize that the low water content in MNAP is a critical factor.
Indeed, a number of physical and chemical processes can lead to a
loss of vaccine potency, such as unfolding, aggregation, hydrolysis
or oxidation of the antigen. Most of these deleterious reactions
occur in aqueous solution and they are aggravated at higher tem-
peratures. Therefore, removing water appears among the most
efcacious ways of impeding these reactions, which is the reason
why lyophilisation is efcient at preserving protein integrity. Here,
we did not use lyophilisation, but the antigen was incorporated
inside a matrix that contained less than 5% of water after drying. It
was also protected from the oxidative activity of the air. These
reasons may explain why the thermostability of the antigen was
increased when incorporated inside MNAPs.
Overall, our results support the concept of dissolvable MNAP for
the administration of vaccines. Dissolvable MNAPs have already
been investigated as vaccine administration tool [23,24], but our
challenging combination of antigen with QS-21 adjuvant
264 D. Poirier et al. / Biomaterials 145 (2017) 256e265

Fig. 9. Skin reactions over time after administration of MNAP with QS-21-adjuvanted HBsAg. A. Skin reactions after one MNAP immunization (priming). B. Skin reactions after the
second immunization done with MNAP. Pre indicates the reactions before immunization. Day 0 refers to the reactions measured 2 h after immunization. Ctrl refers to the control
vaccine. The same color code as in Fig. 8 has been used for the groups. One symbol represents one animal. (For interpretation of the references to colour in this gure legend, the
reader is referred to the web version of this article.)

formulated in liposomes in a single device is unique. Further, the intramuscular injections with less dependence to cold chain re-
antigen was found very stable when incorporated in the HES-based quirements. These quality attributes will need to be further
matrix that constitutes the microneedles. Therefore, dissolvable conrmed in subsequent human studies.
MNAP could represent a suitable alternative to conventional
 D. Poirier et al. / Biomaterials 145 (2017) 256e265
Fig. 10. Evaluation of the antigenicity of HBsAg in MNAP after storage for up to
6 months at 37, 45 or 50
C. Antigenicity was measured by ELISA and compared with
antigenicity at Day 0. Means with SD are shown and the sample size (n) is indicated.
Author contributions
TO, JJ, FR, LS, FW and DP were involved in the conception and
design of the study. DP, VD, LS, JJ, HN, KKa, KKu, MS, TK, TN, TS
participated to the acquisition of data. DP, FR, VD, LS, JJ, HN, KKa,
KKu, MS, TK, TN, TO, TS and FW were involved in the analysis of
data. TS, TO, JJ, and DP were involved in drafting the manuscript and
revising it critically for important intellectual content. All authors
had full access to the data, reviewed the nal version of the
manuscript and approved it before it was submitted by the corre-
sponding author, and agreed to be accountable for all aspects of the
work.
Conicts of interest
All authors have declared the following interests: DP, VD, LS, FW,
FR and JJ are, or were at the time of the study, employees of the GSK
group of companies. JJ reports ownership of GSK shares and/or
restricted GSK shares. TS, TN, KKa, KKu, T K, MS, HN, and TO are
employees of FUJIFILM Corporation R&D. KKa, KKu, TS, MS, TO are
inventors on patents related to MNAP, owned by Fujilm.
Trademark statement
Engerix-B is a trade mark of the GSK group of companies.
Acknowledgments
This work was done under a collaboration agreement between
FUJIFILM Corporation R&D and GlaxoSmithKline Biologicals SA. The
authors wish to thank Kiyohito Takada, Kazunori Komatsu, Katsu-
hiro Nishimaki, Katsuko Sato, Yohei Takahashi from FUJIFILM Cor-
poration R&D, Kanagawa, Japan, for assistance with statistical
analysis, sample preparation, manufacture of MNAPs, and
Demostene Sifakakis, Bernard Chafwehe and Martin Plante from
GSK Vaccines, for their laboratory support and technical discus-
sions throughout the course of the collaboration and in the prep-
aration of this manuscript. We thank Pascal Cadot (Xpe-Pharma &
265
Science) for editorial input and Ulrike Krause (GSK Vaccines) for the
coordination of manuscript development.
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