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Research paper
Targeting the deep lungs, Poloxamer 407 and a CpG oligonucleotide optimize
immune responses to Mycobacterium tuberculosis antigen 85A following
pulmonary delivery
Julie Todoroff
a
, Bernard Ucakar
a
, Malory Inglese
a
, Sophie Vandermarliere
a
, Catherine Fillee
b
,
Jean-Christophe Renauld
c
, Kris Huygen
d
, Rita Vanbever
a,
⇑
a
Université catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery Research Group, Brussels, Belgium
b
Université catholique de Louvain, Cliniques Universitaires Saint-Luc, Department of Clinical Biology, Brussels, Belgium
c
Université catholique de Louvain, Ludwig Institute for Cancer Research, Experimental Medicine Unit, Brussels, Belgium
d
Scientific Institute of Public Health (WIV-ISP Site Ukkel), Service Immunology, Brussels, Belgium
article info
Article history:
Received 31 July 2012
Accepted in revised form 11 November 2012
Available online 11 December 2012
Keywords:
Tuberculosis
Pulmonary vaccination
Antigen 85A
Poloxamer 407
CpG oligonuleotide
abstract
The current Bacille Calmette–Guérin vaccine provides variable protection against tuberculosis and new
vaccination approaches are urgently needed. Pulmonary vaccination could be the best way to induce a
protective immunity against Mycobacterium tuberculosis as it targets its natural site of infection. The
aim of this study was to investigate the potential of Poloxamer 407 (P407) combined with a CpG oligo-
nucleotide (CpG) to enhance immune responses to M.tuberculosis antigen 85A (Ag85A) following pul-
monary delivery in BALB/c mice. An additional goal of this study was to localize the optimal delivery
site of Ag85A within the lungs for generating the most intense immunity. We also investigated the capac-
ity of P407 to prolong the residence time of the antigen within the lungs and we studied the safety of the
adjuvants following pulmonary delivery. Targeting the antigen to the deep lungs produced more intense
specific immune responses than targeting it to the upper airways. P407 and CpG further increased
humoral immune responses and splenocyte proliferation in vitro. CpG strongly increased the Th-1
immune response with high IgG2a/IgG1 ratio, high IFN-
c
and TNF-
a
productions by spleen mononuclear
cells in vitro. P407 tended to induce a Th-2 response, as indicated by the slight decrease in IgG2a/IgG1
ratio and the slight increase in IL-5 levels. The combination of P407 and CpG produced the highest Th-
1 and Th-17 responses by generating IFN-
c
, TNF-
a
, IL-2, and IL-17A cytokines. Targeting the antigen to
the deep lungs and the presence of P407 increased the residence time of the antigen within the lungs.
This might explain the enhancement of immune responses induced by these factors. CpG did not induce
inflammation in the lungs while P407 produced a reversible alteration of the alveolo-capillary barrier.
Adding CpG to P407 did not further increase this alteration of the alveolo-capillary barrier. In conclusion,
delivery of Ag85A formulated in a combination of P407 and CpG to the deep lungs induced strong
immune responses, with a polyfunctional T cells phenotype.
Ó2012 Elsevier B.V. All rights reserved.
1. Introduction
Tuberculosis (TB) is a global health problem, with one-third of
the world’s population currently infected with the TB bacillus
Mycobacterium tuberculosis. In 2010, 1.4 million people died of
tuberculosis (including 450.000 patients infected with human
immunodeficiency virus, HIV) and there were an estimated
8.8 million new TB cases [1]. Human immunodeficiency virus
infection dramatically increases the risk of TB and TB disease is
the leading killer in HIV-infected patients. Although the Bacille Cal-
mette–Guérin vaccine (BCG) is widely administered, its protective
efficacy against TB is highly variable. BCG protects against systemic
tuberculosis in children but fails to prevent pulmonary tuberculo-
sis in adults. In addition, BCG is a full contraindication in HIV-in-
fected infants due to the risk of BCG dissemination [2]. Therefore,
an adequate and truly effective vaccine is lacking and new vaccina-
tion approaches are being developed, some of which are currently
being tested in phase 1 and phase 2 clinical trials [3,4].
Because M. tuberculosis uses the respiratory tract as portal of en-
try into the body, pulmonary vaccination could be the best way to
induce a specific immunity in the lungs and to protect against the
disease [5]. Pulmonary vaccination has the advantage to be needle-
free and to generate a mucosal but also a systemic immunity [6].
0939-6411/$ - see front matter Ó2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejpb.2012.11.020
⇑
Corresponding author. Université catholique de Louvain, Louvain Drug Research
Institute, Pharmaceutics and Drug Delivery Group, 73 Avenue Emmanuel Mounier,
1200 Brussels, Belgium. Tel.: +32 2 764 73 25; fax: +32 2 764 73 98.
E-mail address: rita.vanbever@uclouvain.be (R. Vanbever).
European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb
Author's personal copy
However, there remains a crucial need to optimize the efficacy of
pulmonary vaccination. Live attenuated vaccines present the po-
tential disadvantage of being reactogenic. For instance, BCG pro-
duces an ulcerative lesion at the cutaneous vaccination site that
drains for several weeks [7]. Therefore, an alternative would be
to develop vaccines made of highly purified antigens formulated
in appropriate adjuvants. So far, administration of adjuvanted vac-
cines to the lungs has been little studied and particularly requires
demonstration of safety due to the vital character of the organ [8].
P407 is a non-ionic hydrophilic triblock copolymer of ethylene
oxide (EO) and propylene oxide (PO) conforming to the formula
polyEO–polyPO–polyEO, in which the polyPO portion has an aver-
age molecular weight of 4000 Da (first two digits in 407 multiplied
by 100) and the polyEO portion has a percentage by weight of 70%
(third digit in 407 multiplied by 10). This gives a molecular weight
of about 12,600 Da for P407 [9]. P407 is a pharmaceutical excipient
approved by the Food and Drug Administration and used in a vari-
ety of oral, ophthalmic, topical and injectable formulations for its
emulsifying, solubilizing, and stabilizing properties. Above the crit-
ical micellar concentration and temperature, copolymer molecules
self-aggregate into micelles. Additionally, P407 presents a reverse
thermogelation in which aqueous solutions are liquid at or below
ambient temperature and form gels at body temperature. There-
fore, P407 can be administered in liquid form and acts as sustained
release depot at body temperature [10].
The goal of this study was to investigate the potential of P407
combined with synthetic CpG C274 oligonucleotide to enhance im-
mune responses to antigen 85A (Rv3804c) of M. tuberculosis fol-
lowing pulmonary delivery as well as to orientate immunity
toward a polyfunctional T cells phenotype [17,18]. While the CpG
oligonucleotide was used as Th1-immunostimulant, P407 was
used as a vaccine vehicle that could prolong the duration of contact
of the antigen and immunostimulant with the respiratory mucosa
through its gelation properties. The mycolyl-transferase Ag85A is a
member of the well characterized antigen 85 complex of M. tuber-
culosis. Ag85A is a promising vaccine candidate that induces strong
protective responses in experimental animal models [11] and
which is actually used (as Modified Vaccinia Ankara 85A) in phase
2b clinical trials as a boosting vaccine of BCG vaccinees and PPD-
positive persons [12]. An additional goal of this study was to
localize the optimal delivery site of Ag85A within the lungs for
generating the most intense immunity. We also investigated the
capacity of P407 to prolong the residence time of the antigen
within the lungs and we studied the safety of the adjuvants follow-
ing pulmonary delivery.
2. Materials and methods
2.1. Mice
Specific-pathogen-free female BALB/c mice, aged between 8 and
10 weeks, were used for the experiments (Janvier elevage; Le Gen-
est-St-Isle, France). All experimental protocols in mice were ap-
proved by the Institutional Animal Care and Use Committee of
the Université catholique de Louvain.
2.2. Materials
Hexa-histidine tagged Ag85A protein from M. tuberculosis was
purified from recombinant Escherichia coli, as described before
[13]. Cloning in expression vector pQE-80L (QIAGEN) and purifica-
tion were performed, as described before [11]. The endotoxin level,
measured with the Limulus Amebocyte Lysate kinetic chromogenic
assay (Lonza Verviers Sprl, Belgium), was less than 25 EU/ml
(endotoxin units per milliliter) or 0.065 EU/
l
g of purified protein.
The I-E
d
restricted, immunodominant peptide 11, spanning
amino acids 101–120 sequence of the mature Ag85A sequence
[14], was synthesized by ProImmune Ltd., Oxford, UK. The peptide
was initially dissolved in dimethyl sulfoxide. Stock solutions were
subsequently prepared in RPMI 1640 culture medium at 1 mg/ml.
Aliquots were stored frozen at 20 °C.
P407 solutions of 10% to 20% w/v were prepared by dissolving
P407 in PBS buffer under stirring in ice. The solution was kept in
the fridge overnight to improve the dissolution. The viscosity of
the solutions was measured by using a Rheometer RM180 Rheom-
at (Lamy Rheology, Champagne Au Mont d’Or, France). For the 13%
w/v solution, particle size distribution was determined using dy-
namic light scattering (NanoSizer ZS; Malvern Instruments, Mal-
vern, UK). Several concentrations of P407 (Sigma, USA) were
administered by intratracheal instillation (IT) in BALB/c mice
namely, 10%, 13%, 15%, and 20% (w/v). The survival of mice was ob-
served immediately after delivery. Deposition in the deep lungs
was verified by adding the patent blue violet (Sigma, USA) to the
solutions and by visually assessing the blue coloration of resected
lungs.
2.3. Vaccination protocol
BALB/c mice were anesthetized by intraperitoneal injection of
ketamine/xylazine at 75 mg/kg and 8.4 mg/kg, respectively. Next,
mice received the vaccine by IT either in the upper airways (UA)
or in the deep lung (DL). A 10
l
l sterile solution was instilled in
the trachea of the mouse lying horizontally on its back in order
to reach upper respiratory airways. In order to reach the deep
lungs, a 20
l
l sterile solution was instilled in the trachea of the
mouse positioned with a 45°angle of tilt followed by insufflation
of a 500
l
l air bolus. Mice were instilled three times at 3 weeks
intervals (on days 1, 21, 42), with 5
l
g of purified recombinant
Ag85A alone or combined with 13% (w/v) P407 and/or 5
l
g CpG
C274 (sequence 5
0
-TCG-TCG-AAC-GTT-CGA-GAT-GAT-3
0
, Eurogen-
tec S.A, Seraing, Belgium). Control mice were injected subcutane-
ously (SC) in the back three times, at 3 week intervals (on days 1,
21, 42), with 5
l
g of purified recombinant Ag85A alone or emulsi-
fied in Gerbu adjuvant in a total volume of 100
l
l. Gerbu adjuvant
is a colloidal suspension composed of biodegradable cationic lipid
nanoparticles (octa-/hexa-decane). The suspension is completed
with a cell wall subunit of Lactobacillus bulgaricus (GMDP), which
is a powerful immunomodulator inducing T cell response and long
lasting cellular immunity. Additional immunomodulators, cimeti-
dine and saponin, are also included as general enhancers of the im-
mune response (GERBU Biochemicals, Germany). Additional
control mice were instilled in the deep lungs with saline (PBS),
P407, and/or CpG without Ag85A.
2.4. Samples collection and antibody ELISA assays
Sera were collected from the retro-orbital plexus before the first
vaccination (day 0) and 3 weeks after the third vaccination (day
63) and stored at 20 °C until assayed. On day 70, mice were killed
with an overdose of pentobarbital, spleens were removed asepti-
cally and broncho-alveolar lavages (BALs) were performed with
1 ml Hanks’ Balanced Salt solution (Sigma, USA) by positioning a
cannula in the trachea toward the lungs, as described before
[15]. Ag85A-specific IgG, IgG1, and IgG2a were measured by ELISA
in sera. Ag85A-specific IgA and Ag85A-specific IgG were measured
in BAL fluid. Specific anti-Ag85A antibodies were determined, on 2-
fold serial dilutions of sera and BALs, using recombinant Ag85A for
coating (8
l
g/ml), appropriate detection antibodies (rat antimouse
total IgG, IgG1, IgG2a, IgA labeled with peroxidase from IMEX, UCL,
Brussels, Belgium) and orthophenyldiamine for revelation (Sigma,
USA). Optical densities were read at 492 nm. Antibody titers were
J. Todoroff et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48 41
Author's personal copy
defined as the dilution corresponding to an optical density of 0.2 at
492 nm. Individual IgG2a/IgG1 ratios were calculated by dividing
individual IgG2a titers by individual IgG1 titers. Antigen-specific
antibody levels were undetectable in the PBS group and in the con-
trol group vaccinated in the deep lung with the combination of the
adjuvants alone (data not shown).
2.5. Proliferative responses and cytokines production
Splenocytes were adjusted to a concentration of 4 10
6
white
blood cells/ml and cultured 180
l
l per well in 96-well round-bot-
tom microtiter plates (Escolab, Kruibeke, Belgium) in RPMI 1640
medium supplemented with 10% (v/v) fetal calf serum, 0.05 mM
2-mercaptoethanol, 1% (v/v) sodium pyruvate, antibiotics, and 1%
(v/v) non-essential amino acid (MEM) (Gibco, Merelbeke, Belgium).
Cells were incubated at 37 °C in a humidified CO
2
incubator and
stimulated with purified recombinant Ag85A (5
l
g/ml), synthetic
Ag85A
101–120
peptide (10
l
g/ml) or Concanavalin A (5
l
g/ml). Neg-
ative control cultures were left unstimulated. All cultures were
incubated for 48 h and then pulsed overnight with 0.4
l
Ci [
3
H] thy-
midine per well. Cells were harvested onto microplate unifilters
and the activity was counted using a TopCount scintillation coun-
ter (PerkinElmer, Zaventem, Belgium). Results are expressed in
counts/min (c.p.m). Supernatants were collected either after 24 h
for interleukin-2 (IL-2) or after 72 h for interleukin-5 (IL-5), inter-
leukin-17A (IL-17A), tumor necrosis factor-alpha (TNF-
a
), and
interferon-gamma (IFN-
c
) assay. Cytokine concentrations were
measured in supernatants using mouse IL-2, IL-5, TNF-
a
, and
IFN-
c
Duoset ELISA development kit (R&D Systems Europe Ltd.,
Abingdon, UK), according to the manufacturer’s protocols. IL-17A
levels were quantified by sandwich ELISA using coating antibody
MM173G9, biotinylated antibody MM17F3 (Experimental Medi-
cine Unit, UCL, Belgium) and the standard murine recombinant
IL-17A (R&D Systems Europe Ltd., Abingdon, UK). The detection
limits were 4, 7.8, 15, 10.4, and 21 pg/ml for IL-17A, IL-2, IL-5,
TNF-
a
, and IFN-
c
, respectively.
2.6. Residence time
BALB/c mice were anesthetized by intraperitoneal injection of
ketamine–xylazine before IT of 5
l
g of ovalbumin (Grade V, Sig-
ma, USA) with or without 13% (w/v) P407 in the upper airways
or in the deep lungs. The endotoxin level, measured with the Lim-
ulus Amebocyte Lysate kinetic chromogenic assay (Lonza Verviers
Sprl, Belgium), was 1.78 UE/
l
g of protein. We verified that no pul-
monary inflammation resulted from the administration of 5
l
gof
OVA to the deep lungs (data not shown). Mice were instilled once.
BAL fluid was collected at 0, 24, and 48 h after instillation. The
lungs were lavaged four times with 1 ml Hanks’ Balanced Salt
solution. The supernatants of the lavages were stored frozen at
20 °C until assay for ovalbumin content. Ovalbumin concentra-
tion was measured by ELISA using an affinity purified rabbit
anti-ovalbumin antibody for coating and a peroxidase conjugated
rabbit anti-ovalbumin for detection (MyBioSource, San Diego,
USA). The detection limit was 0.6 ng/ml. Ovalbumin, instead of
Ag85A, was used because sensitivity of Ag85A-specific ELISA was
insufficient using available custom-made monoclonal antibodies
against Ag85A.
2.7. Toxicity study protocol
BALB/c mice were anesthetized by intraperitoneal injection of
ketamine–xylazine before receiving adjuvants alone by instilla-
tion in the deep lungs, as described above. Mice were instilled
once with 13% (w/v) P407 and/or 5
l
g CpG C274. Negative and
positive control mice were instilled with PBS and 5
l
g of LPS
(E.coli O111:B4, Sigma, USA), respectively. BAL fluid was collected
4 h, 24 h, 72 h, 7 days, and 14 days after intratracheal administra-
tion. The lungs were lavaged twice with 1 ml Hanks’ Balanced Salt
solution, as described above. The lavages were then centrifuged
(281gat 4 °C for 10 min). The supernatants of the first lavages
were stored frozen at 20 °C for TNF-
a
and serum albumin
assays.
2.8. BAL biochemical parameters and cellular components
BAL total protein content and lactate dehydrogenase (LDH)
activity were spectrophotometrically assayed in the supernatants
of the first lavages using the Synchron LX Systems (Beckman
Coulter Inc.; Brea, USA). Total protein content was assayed by mon-
itoring the formation of a purple color complex of pyrogallol red
and molybdate at 600 nm using human serum albumin as standard
while LDH activity was assayed by monitoring the reduction of
NAD
+
to NADH in the presence of lactate at 340 nm.
Serum albumin and TNF-
a
levels were measured by ELISA
according to the manufacturer’s protocols. Mouse albumin assay
was performed using a commercial kit (Imtec Diagnostics N.V.,
Antwerpen, Belgium) with a detection limit of 3 ng/ml and mouse
serum albumin as standard. TNF-
a
assay was performed using a BD
Biosciences kit (BD Biosciences, San Jose, USA) with a detection
limit of 33 pg/ml.
The cells of both BAL aliquots were pooled and the total number
of live cells in BAL fluid was determined by Türk’s solution method
(VWR International, Leuven, Belgium). Differential cell counts were
obtained by cytocentrifugation and coloration with Diff quick
(Dade NV/SA).
2.9. Statistical analysis
All results are expressed as mean ± standard error of the mean
(SEM). One-way ANOVA and Tukey’s post-test were performed
on normalized data to demonstrate statistical differences using
the software GraphPad Prism 5 for Windows. Groups of 6 to 8 mice
were used for the immunogenicity, toxicity, and residence time
studies. A value of p< 0.05 was considered statistically significant.
3. Results
3.1. The choice of P407 concentration
In a first step, we selected the maximal concentration of P407
that could be readily delivered deeply in the lungs of mice. From
a concentration of 20% w/v and higher, the mice did not survive
the intratracheal instillation of P407 because the extremely viscous
solution obstructed respiratory airways and prevented mice to
properly breathe (Table 1). For a concentration of 15% w/v and
higher, the viscosity of the solution prevented it to go deep into
the lungs. A P407 concentration of 13% w/v allowed delivery of
the solution to the lungs periphery (Table 1). Therefore, a P407
concentration of 13% was chosen for further experiments. The
average viscosity of the 13% solution was 17 mPasat37°C, which
means a 17-times increased viscosity as compared to pure water
(Table 1). The average size of P407 micelles at this concentration
was 27 nm.
3.2. The choice of the delivery site of Ag85A within the lungs
Both the delivery site of Ag85A within the lungs and P407 had
an impact on serum anti-Ag85A IgG titers (Fig. 1). In order to reach
the deep lungs, a 20
l
l solution of Ag85A was instilled in the
trachea of the mouse tilted at an angle of 45°and an air bolus
42 J. Todoroff et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48
Author's personal copy
was immediately insufflated in the trachea following administra-
tion. In order to deposit the Ag85A solution in upper airways, a
10
l
l solution was instilled in the trachea of the mouse lying hor-
izontally on its back and no air bolus was insufflated. The targeting
of the different lung regions obtained using these techniques was
previously demonstrated using ovalbumin as a tracer compound
[15]. The administration of Ag85A to the deep lungs generated
2log
10
higher anti-Ag85A IgG titers in sera than its delivery to
upper airways. Both in the deep lungs and upper airways, P407
strongly enhanced antigen-specific serum IgG titers (Fig. 1A). The
administration of Ag85A to the deep lungs tended to induce
increased splenocyte proliferation and IFN-
c
production as
compared to its administration to upper airways (Fig. 1B and C).
These results confirm the importance of the antigen delivery site
within the lungs for an optimal immune response [15]. They also
highlight the potential of P407 as a carrier for pulmonary vaccina-
tion. Because targeting the antigen to the deep lungs enhanced im-
mune responses, this lung region was chosen as antigen delivery
site for the subsequent experiments.
3.3. Humoral immune responses to Ag85A following its pulmonary
delivery in combination with P407 and CpG
P407 and CpG C274 both strongly increased humoral immune
responses to Ag85A following delivery to the deep lungs
(Fig. 2A). Serum anti-Ag85A IgG titers increased by 2log
10
Table 1
Dynamic viscosity of P407 at increasing concentrations and two temperatures.
Temperature 10 °C37°C Mouse survival Deep lungs deposition
Concentration% (w/v) Dynamic viscosity (mPa s)
10 9 ± 1 8 ± 1 Yes Yes
13 13 ± 1 17 ± 0.7 Yes Yes
15 15 ± 0.6 37 ± 0.7 Yes No
20 22 ± 0.7 4310 ± 650
a
No No
a
At these concentration and temperature, P407 did not follow a Newtonian behavior and viscosity is given at 6.5 cm shear rate.
Ag85A SC
Ag85A-Gerbu SC
Ag85A UA
Ag85A DL
Ag85A-P407 UA
Ag85A-P407 DL
0
1
2
3
4
5
6
###
###
***
***
###
###
###
+++
Serum Anti-Ag85A IgG (log10)
Ag85A SC
Ag85A-Gerbu SC
PBS DL
Ag85A UA
Ag85A DL
Ag85A-P407 UA
Ag85A-P407 DL
0
500
1000
1500
2000
2500 Ag85A
Peptide 11
No stimulant
LD
§§§ §§
##
##
#
##
#
+
++
IFN-γ (pg/ml)
Ag85A SC
Ag85A-Gerbu SC
PBS DL
Ag85A UA
Ag85A DL
Ag85A-P407 UA
Ag85A-P407 DL
5
10
15
20
25
30
35 Ag85A
Peptide 11
No stimulant
§§§
§§§
###
### ###
#
§
+++
+++
+
Cpm (*103)
ABC
Fig. 1. Impact of Ag85A delivery site on humoral and cellular immune responses. (A) Serum anti-Ag85A IgG titers. (B) Proliferation of spleen mononuclear cells following
in vitro Ag85A or peptide 11 stimulation or without stimulation. (C) The supernatants from the culture of spleen mononuclear cells were assayed for IFN-
c
, limit of
detection = 21 pg/ml.
indicates a significant difference between the upper airways (UA) and deep lung (DL) groups. # indicates significant difference with Ag85A-Gerbu SC
group. + indicates significant difference with the Ag85A delivered alone in the deep lung. § indicates difference with PBS delivered in the deep lung. One symbol indicates
p< 0.05. Two symbols indicate p< 0.01. Three symbols indicate p< 0.001.
Ag85A SC
Ag85A-Gerbu SC
Ag85A DL
Ag85A-P407 DL
Ag85A-CpG DL
Ag85A-P407-CpG DL
2
3
4
5
6
##
###
##
###
+++
+++
+++
+++
Serum Anti-Ag85A IgG (log10 )
Ag85A SC
Ag85A-Gerbu SC
Ag85A DL
Ag85A-P407 DL
Ag85A-CpG DL
Ag85A-P407-CpG DL
2
3
4
5
6
IgG2a
IgG1
0.69
0.86
0.77
1.20 0.97
0.92
ratio IgG2a/IgG1
++
+++
+++
Serum Anti-Ag85A IgG subclasses (log10)
Ag85A SC
Ag85A-Gerbu SC
Ag85A DL
Ag85A-P407 DL
Ag85A-CpG DL
Ag85A-P407-CpG DL
0
1
2
3
### ### ###
+++ ++
+++
BAL Anti-Ag85A IgG (log10)
CBA
Fig. 2. Impact of P407 and CpG on antigen-specific humoral immune responses. (A) Serum anti-Ag85A IgG titers. (B) BAL anti-Ag85A IgG titers. (C) Serum anti-Ag85A IgG
subclass titers. # indicates significant difference with Ag85A-Gerbu SC group. + indicates significant difference with the Ag85A delivered alone in the deep lung. Two symbols
indicate p< 0.01, and three symbols indicate p< 0.001.
J. Todoroff et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48 43
Author's personal copy
when P407 or CpG was added to the antigen. The pulmonary
administration of Ag85A together with either P407 or CpG induced
similar serum anti-Ag85A IgG titers as the subcutaneous adminis-
tration of a same dose of Ag85A without adjuvant. Yet, when com-
bining P407 and CpG with the antigen, serum anti-Ag85A IgG
levels were comparable to those obtained following subcutaneous
administration of Ag85A formulated in Gerbu adjuvant (Fig. 2A).
The BAL anti-Ag85A IgG levels paralleled serum IgG levels, in sup-
port of IgG passive diffusion from serum into the lungs (Fig. 2B).
Antigen-specific IgA levels in broncho-alveolar lavages could not
be detected in any of the groups, probably due to their high dilu-
tion in BAL samples.
Serum IgG2a and IgG1 subclasses were analyzed as an indirect
assessment of the polarization of T-helper cell populations. As
compared to subcutaneous administration, pulmonary vaccination
of Ag85A alone tended to generate a higher IgG2a to IgG1 ratio
(Fig. 2C), indicating a slight shift toward a T-helper type-1 pheno-
type. Adding the CpG oligonucleotide to the antigen increased
IgG2a to IgG1 ratio following pulmonary vaccination as compared
to the pulmonary vaccination with Ag85A alone, with a ratio reach-
ing over 1. However, P407 presented the reverse tendency to CpG
and decreased the IgG2a to IgG1 ratio as compared to the pulmon-
ary vaccination with Ag85A alone. The formulation of Ag85A in a
combination of P407 and CpG induced a IgG2a to IgG1 ratio
Ag85A SC
Ag85A-Gerbu SC
P407-CpG DL
PBS DL
Ag85A DL
Ag85A-P407 DL
Ag85A-CpG DL
Ag85A-CpG-P407D
L
5
10
15
20
25
30
35
40
45 Ag85A
Peptide 11
No stimulant
§§§
§§§
§§§
§§§
+++
+++
+++
+++
###
+
###
###
##
§
Cpm (*103)
Ag85ASC
Ag85A-Gerbu SC
P407-CpG DL
PBS DL
Ag85A DL
Ag85A-P407DL
Ag85A-CpG DL
Ag85A-P407-CpG DL
0
500
1000
1500
2000
2500
3000
3500
4000 Ag85A
Peptide 11
No stimulant
LD
§§
§§§
§§§
§§§
+
++
+++
++
###
###
IFN- γ (pg/ml)
Ag85A SC
Ag85A-Gerbu SC
P407-CpG DL
PBS DL
Ag85A DL
Ag85A-P407DL
Ag85A-CpG DL
Ag85A-P407-CpG DL
0
50
100
150
200
LD
§
§§§
++
Peptide 11
Ag85A
No stimulant
#
IL-2 (pg/ml)
Ag85A SC
Ag85A-Gerbu SC
P407-CpG DL
PBS DL
Ag85A DL
Ag85A-P407 DL
Ag85A-CpG DL
Ag85A-P407-CpG DL
0
200
400
600
800
1000
LD
**
Ag85A
Peptide 11
No stimulant
IL-5 (pg/ml)
Ag85A SC
Ag85A-Gerbu SC
P407-CpG DL
PBS DL
Ag85ADL
Ag85A-P407 DL
Ag85A-CpG DL
Ag85A-P407-CpG DL
0
2000
4000
6000
8000
10000
12000 Ag85A
Peptide 11
No stimulant
LD
###
§§§ §§§
+++ +++
###
###
###
### ###
IL-17A (pg/ml)
BA
CD
Ag85AS
C
Ag85A-Gerbu SC
P407-CpG DL
PBS DL
Ag85ADL
Ag85A-P407 DL
Ag85A-CpG DL
Ag85A-CpG-P407 DL
0
50
100
150
200 Ag85A
Peptide 11
No stimulant
*
§§§ §§§
+++ +++ +++
§§§
###
### ###
##
LD
TNF-α (pg/ml)
EF
Fig. 3. Impact of P407 and CpG on the cellular immune responses. (A) Proliferation of spleen mononuclear cells following in vitro Ag85A or peptide 11 stimulation or without
stimulation. The supernatants from the culture of spleen mononuclear cells were assayed for (B) IFN-
c
, limit of detection (LD) = 21 pg/ml (C) TNF-
a
, LD = 10.4 pg/ml. (D) IL-2,
LD = 7.8 pg/ml. (E) IL-5, LD = 15 pg/ml and (F) IL-17A, LD = 4 pg/ml. # indicates significant difference with Ag85A-Gerbu SC group. + indicates significant difference with the
Ag85A delivered alone in the deep lung.
indicates difference with all the other groups. § indicates difference with PBS delivered in the deep lung. One symbol indicates
p< 0.05. Two symbols indicate p< 0.01. Three symbols indicate p< 0.001.
44 J. Todoroff et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48
Author's personal copy
intermediate between the Ag85A–CpG and Ag85A–P407 groups
(Fig. 2C).
3.4. Cellular immune responses to Ag85A following its pulmonary
delivery in combination with P407 and CpG
Both P407 and CpG enhanced cellular immune responses to
Ag85A following pulmonary administration (Fig. 3). Positive
splenocyte proliferation was seen in the groups vaccinated in the
deep lungs against M. tuberculosis with P407, CpG, or the combina-
tion of both molecules (Fig. 3A). Positive splenocyte proliferation
was also seen in the groups vaccinated subcutaneously without
or with Gerbu. Yet, the antigen delivered alone to the deep lungs
did not induce proliferative responses. The combination of P407
and CpG delivered to the lungs induced the highest splenocyte pro-
liferation and a proliferation even higher than that generated by
the subcutaneous injection of Ag85A with Gerbu (Fig. 3A). When
stimulated with Ag85A
101–120
peptide, splenocytes proliferated in
the same proportions as following stimulation with the whole
antigen.
Similarly to the IgG2a to IgG1 ratios, the in vitro splenocyte
cytokine production indicated that formulation in CpG oligonu-
cleotide induced a shift of the Ag85A-specific immune response
toward a Th-1 phenotype. The IFN-
c
production by Ag85A-stimu-
lated splenocytes in vitro was 7-fold and 4-fold increased in the
groups of mice vaccinated with Ag85A in CpG and in the combina-
tion of P407 and CpG, respectively, in comparison with the group
vaccinated with Ag85A alone (Fig. 3B). CpG as well as the combina-
tion of P407 and CpG significantly increased TNF-
a
production as
compared to the group vaccinated with Ag85A alone (Fig. 3C).
P407 alone did not enhance splenocyte production of IFN-
c
, TNF-
a
, and IL-2 levels. These cytokines were only increased by the com-
bined use of P407 and CpG (Fig. 3D). Significant IL-5 levels were
only detected in the group vaccinated subcutaneously with
0 24 48
0
20
40
60
80
100
120
OVA UA
OVA-P407 UA
OVA DL
OVA-P407 DL
***
Hours after instillation
OVA content
(% of the dose recovered at time 0)
0 24 48
0.0
0.5
1.0
1.5
3.0
4.5
6.0
7.5
OVA UA
OVA-P407 UA
OVA DL
OVA-P407 DL
*
Hours after instillation
OVA amount/BAL (µg)
B
A
Fig. 4. Impact of the delivery site and the carrier P407 on the residence time of OVA
antigen in the lungs. (A) Amount of OVA/BAL (
l
g). (B) OVA content in % of the
average dose recovered per group at time 0.
indicates difference with all the other
groups. One symbol indicates p< 0.05, and three symbols indicate p< 0.001.
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
100
200
300
400
500
600
700
PBS
CpG
P407
LPS
§§§
§§§
§§§
P407-CpG
§§§ §§§
§§§
§§§
§§§
###
###
###
###
++
+++
Days after instillation
Total proteins (µg/ml)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
200
400
600
800
1000
1200
1400
1600
1800
PBS
CpG
P407
LPS
##
§§§ **
§§§
P407-CpG
§
###
###
§§§
#
#
§
§§§
++
Days after instillation
Albumin (µg/ml)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
100
200
300
400 PBS
CpG
P407
LPS
***
P407-CpG
§§§
§
§§§
§§§
#
###
Days after instillation
LDH (UI/L)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
500
1000
1500 PBS
CpG
P407
LPS
**
P407-CpG
Da
y
s after instillation
TNFa (pg/ml)
A
B
C
D
Fig. 5. Impact of P407 and CpG on inflammatory biochemical parameters. (A) Total
proteins; (B) albumin; (C) LDH and (D) TNF-
a
. § indicates significant difference
from the PBS group.
indicates significant difference from all other groups. +
indicates significant difference between P407 and P407–CpG groups. # indicates
significant difference from the LPS group. One symbol indicates p< 0.05. Two
symbols indicate p< 0.01. Three symbols indicate p< 0.001.
J. Todoroff et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48 45
Author's personal copy
Ag85A alone or vaccinated in the deep lungs with Ag85A formu-
lated in P407 (Fig. 3E). IL-17A levels were increased both by the
combined use of P407 and CpG and by the Gerbu adjuvant
(Fig. 3F). BAL samples were concentrated to assay local IFN-
c
. Nev-
ertheless, IFN-
c
levels were undetectable in the BALs of all the
groups.
3.5. Residence time
In order to assess whether antigen delivery to the deep lungs
and P407 led to increased residence time of the antigen within
the lungs, we measured ovalbumin content in broncho-alveolar
fluid in terms of time after instillation of ovalbumin in the upper
airways or deep lungs, without and with P407 (Fig. 4A). Twenty-
four hours post-instillation, the lungs retained 20% and 11% of
the time zero-recovered ovalbumin dose following delivery to
the deep lungs and upper airways, respectively. When P407 was
added, 24 h lung retention increased to, respectively, 36% and
27% (Fig. 4B). Forty-eight hours post-instillation, 20% of the ovalbu-
min dose was still present in the lungs following deep lung deliv-
ery in P407 while only 3% to 8% of ovalbumin remained for the
other groups (Fig. 4B).
3.6. BAL biochemical parameters
We analyzed several biochemical markers in broncho-alveolar
lavages in order to check whether P407 and CpG induced inflam-
mation in the lungs. Total proteins and albumin were measured
to detect permeability alteration to the alveolo-capillary barrier.
The extracellular release of lactate dehydrogenase (LDH), an intra-
cellular enzyme, allows detecting injury to pulmonary cells. TNF-
a
was measured in BAL as an early inflammation marker. Total pro-
teins were significantly increased in the P407 group from day 1 un-
til day 14 after pulmonary administration but tended to return to
baseline values at day 14. The combination of P407 and CpG signif-
icantly increased total proteins at day 3 and day 14 as compared
with the P407 group (Fig. 5A). Albumin followed the same rise as
total proteins following P407 delivery and tended to return to
baseline values at day 14 as well. Albumin was similarly increased
in the P407–CpG group but decreased faster than in the P407 alone
group. Therefore, albumin was significantly lower in the combina-
tion group versus the P407 alone group at day 7 (Fig. 5B). Total pro-
teins distributed on a lower scale than albumin and this might be
explained by the use of a non-specific standard for the quantifica-
tion of total proteins (Fig. 5B). Intratracheal instillation of P407 in-
duced a slight and apparently reversible increase in LDH content.
P407–CpG tended to induce a higher increase in LDH than P407
alone. However, there were no significant differences between
both groups (Fig. 5C). P407 and P407–CpG did not induce modifi-
cation to TNF-
a
levels (Fig. 5D). Pulmonary delivery of CpG as pul-
monary delivery of saline, the negative control, did not modify the
levels of any of these biochemical markers (Fig. 5A–D). Intratra-
cheal instillation of LPS, the positive control, induced a sharp and
reversible increase in total proteins, LDH and TNF-
a
but no alter-
ation of albumin (Fig. 5A–D).
3.7. BAL cellular components
Counting of total cells and analysis of cell distribution in BAL
fluid can give an indication of pulmonary inflammation as well. To-
tal cells were not increased following pulmonary administration of
P407 and/or CpG (Fig. 6A). However, the number of macrophages
increased in the P407 and P407–CpG groups and was significantly
different from the saline group at day 14 (Fig. 6B). The number of
lymphocytes tended to increase in the group P407–CpG at day 7
(Fig. 6D). In contrast, intratracheal instillation of LPS (the positive
control) induced a large increase in the number of total cells recov-
ered, and this increase was due to an influx of neutrophils from day
1 to day 3, an influx of lymphocytes from day 3 to day 7 and an in-
flux of macrophages at day 7 (Fig. 6A–D).
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
1000
2000
3000
4000
5000
6000 PBS
LPS
CpG
P407
***
***
*
P407-CpG
Days after instillation
Total cells (x103/BAL)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
500
1000
1500
2000
PBS
CpG
P407
LPS
*
§§
P407-CpG
§§§
+++
Days after instillation
Macrophages (x103/BAL)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
1000
2000
3000
4000
5000
PBS
CpG
P407
LPS
***
***
P407-CpG
++
Days after instillation
Neutrophils (x103/BAL)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0
100
200
300
400
500
PBS
CpG
P407
LPS
***
***
***
**
P407-CpG
Da
y
s after instillation
Lymphocytes (x103/BAL)
A
B
C
D
Fig. 6. Impact of P407 and CpG on inflammatory cellular parameters. (A) Total cells;
(B) macrophages; (C) neutrophils; and (D) lymphocytes. § indicates significant
difference from the PBS group.
indicates significant difference from all other
groups. + indicates significant difference between P407 and P407–CpG groups. One
symbol indicates p< 0.05. Two symbols indicate p< 0.01. Three symbols indicate
p< 0.001.
46 J. Todoroff et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48
Author's personal copy
4. Discussion
The goal of this study was to assess the potential of a formula-
tion composed of P407 and a CpG to optimize immune responses
to Ag85A following pulmonary delivery. Our objective was to gen-
erate a TB-specific immunity that could protect the host against
infection by airborne M. tuberculosis through non-invasive pul-
monary vaccination. In a first step, we showed that targeting
Ag85A to the deep lungs, as compared to the upper airways, in-
duced higher antigen-specific immune responses (Fig. 1). In a sec-
ond step, we demonstrated that formulation of Ag85A in P407 and
CpG further increased humoral and cellular responses and gener-
ated a polyfunctional T cells phenotype (Figs. 2 and 3). In a third
step, we showed that deep lung targeting and P407 enhanced the
residence time of the antigen within the airway lumen, in support
of our starting hypothesis (Fig. 4). Finally, we showed that intratra-
cheal instillation of CpG did not cause inflammation in the lungs
but that intratracheal instillation of P407 induced an alteration of
the alveolo-capillary barrier (Figs. 5 and 6).
Pulmonary delivery of Ag85A combined with P407 and CpG
C274 generated a strong immune response needed for protection
against infection with M. tuberculosis [7]. IgG diffusing from the
bloodstream can block initial infection at the mucosal surface by
preventing the binding of the mycobacterium to host receptors
(Fig. 2B). T lymphocytes migrate back and forth across the mucosal
epithelium and production of interferon
c
, tumor necrosis factor
a
,
and interleukin 2 by CD4 + Th-1 cells can activate alveolar macro-
phages initially infected with aerosolized M. tuberculosis to pro-
duce intracellular microbicidal activities (Fig. 3). CD4 + T cells
expressing IL-17 may also contribute to the adaptive immune re-
sponses to mycobacteria by triggering expression of chemokines
in the lung, which in turn may mediate recruitment of Th1 cells
to the airways (Fig. 3F) [16]. The generation of multifunctional T
cells has been shown to correlate with protection against tubercu-
losis (Figs. 3B–D and F) [17,18].
Poloxamer 407 alone or combined with CpG increased immune
responses to Ag85A following pulmonary delivery. Our starting
hypothesis was that P407 would enhance the residence time of
the antigen and adjuvant within the lungs due to the increased vis-
cosity of the solution made of P407. This enhancement was in fact
verified by measuring ovalbumin content in BAL fluid in terms of
time after delivery of an ovalbumin solution formulated or not in
P407 (Fig. 4). P407 did not induce inflammation in the lungs (Figs. 5
and 6), but P407 modulated the immune responses and acted as an
adjuvant probably by forming an antigen depot and extending the
delivery of the antigen (Fig. 4). Although P407 altered the perme-
ability of the alveolo-capillary barrier (Fig. 5), this did not translate
in increased antigen transport from the alveolar space into the lung
interstitium since the antigen amount recovered from the alveolar
space was higher when P407 was present in the instillate (Fig. 4).
No synergy between P407 and CpG was observed as to the toxicity
to the alveolo-capillary barrier since adding CpG to P407 did not
further increase albumin content in BAL (Fig. 5B). Sustaining the
release of the antigen and adjuvant within the lungs in order to in-
crease immune responses is a strategy that has not been tested yet
by the pulmonary route, in contrast to particulate carriers well ex-
plored to target vaccines to antigen presenting cells in the lungs
[19,20]. Pulmonary administration of poly(propylene sulfide)
nanoparticles, stabilized with P407 as emulsifier surfactant, tar-
geted lymphoid tissue and led to enhanced Ag85B-specific mucosal
and systemic Th-1 and Th-17 immune responses [19]. P407 has
also been used as adjuvant for vaccines delivered by the intranasal
route or by injection and has already been shown to increase hu-
moral immune responses. After intranasal delivery, P407 combined
with chitosan increased IgA antibodies to Bordetella bronchiseptica
antigens [21] and IgG and IgA antibodies specific to tetanus toxoid
[22]. P407 has previously been shown to be synergistic with CpG
following injection. Their combination formulated with tetanus
toxoid, diphtheria toxoid, and anthrax recombinant protective
antigen resulted in enhancement of IgG antibody responses as
compared to the antigens mixed with CpG alone [23]. Yet, the
mechanism behind the adjuvant action and the sustained antigen
release P407 provides has not been verified in these studies. Cellu-
lar immune responses have also been little analyzed [24,25].
Targeting Ag85A to the deep lungs induced more intense anti-
gen-specific immunity than targeting it to upper airways (Fig. 1).
This confirms our previous results on a split influenza virus vac-
cine, which generated stronger specific immunity following deliv-
ery to the lung periphery than following delivery to the nasal
cavity or to the upper or central airways [15]. At that time, we
hypothesized that the increased efficacy of deep lung vaccination
might originate from the ensuing longer residence time of the anti-
gen within the lungs. Here, we confirm this hypothesis by directly
measuring ovalbumin content in BAL fluid in terms of time after
delivery to the deep lungs or upper airways (Fig. 4). Ronan et al.
have compared intranasal instillations targeting a recombinant
adenovirus expressing Ag85A to the nasal cavities or to the whole
respiratory tract in mice. They showed that only deep lung immu-
nization induced strong immune responses in the lungs and that
these correlated with protection against an aerosol M. tuberculosis
challenge [26].
The analysis of IgG subclasses and cytokines secreted by spleno-
cytes in culture indicated that the CpG induced a Th-1 cellular re-
sponse (Figs. 2 and 3). This effect can be related to the binding of
CpG to Toll-like receptor-9 in the intracellular compartment of im-
mune cells [27]. The particular CpG used in this study (CpG C274)
belongs to class C CpG oligonucleotides, which provide both the ef-
fect of class A (activation of plamacytoid dendritic cells) and class B
(activation of B cells). In addition, CpG C274 is a particularly potent
inducer of IL-12, IFN-
a
, IFN-
c
and IL-6, as compared to other class C
oligonucleotides [28]. Formulation of Ag85A in P407 induced a Th-
2 polarization of the immune response, but the mechanism behind
this orientation remains to be determined (Figs. 2 and 3). Newman
et al. have shown that poloxamers with 10% polyOE increased Th-2
responses to ovalbumin, whereas more lipophilic copolymers aug-
mented both Th-1 and Th-2 responses [29]. Lipophilic copolymers
rapidly adhere to cell membranes, incorporate into them, and in-
crease membrane fluidization, thereby facilitating the non-specific
transfer of protein antigens into the cytoplasm. On the contrary,
hydrophilic copolymers as P407 adhere to cell membranes but do
not incorporate into them [30]. P407 could increase the efficiency
of antigen delivery to dendritic cells through endocytosis of mi-
celles and this could augment the T cell response. Formulation in
CpG adjuvant and P407 carrier produced the highest splenocyte
proliferation (Fig. 3A) and induced a mixed Th-1 and Th-17-ori-
ented response with high levels of IFN-
c
, TNF-
a
, IL-2, and IL-17A
(Fig. 3B–F).
The CpG and P407 did not induce any inflammation in the lungs
but P407 increased the permeability of the alveolo-capillary barrier
(Fig. 5). Adding CpG to P407 did not further increase the alteration
of the alveolo-capillary barrier. By adhering to the surface of cell
plasma membranes, P407 limits the lateral mobility of membrane
lipids and thereby causes membrane solidification [30]. There
might be a link between this adherence to cell surfaces and the in-
crease in epithelial permeability. However, the alteration of the
alveolo-capillary barrier by P407 is reversible over time and its
reversible nature could come from the clearance of the polymer
from the lungs. P407 has a molecular weight of 12,600 Da, and
macromolecules of this size are known to be mostly cleared from
the lungs within 1 day. Yet, P407 micelles might present a slower
clearance than P407 unimers since nanoparticles can remain in
the lungs for weeks [31]. After absorption in the systemic
J. Todoroff et al. / European Journal of Pharmaceutics and Biopharmaceutics 84 (2013) 40–48 47
Author's personal copy
circulation, P407 unimers are rapidly excreted in the urine [32].
The alteration of the alveolo-capillary barrier induced by P407
could possibly be reduced by decreasing P407 concentration in
the solution delivered. Because sodium chloride and phosphates
have been shown to increase the strength of P407 gels, these ions
could be added to the P407 solution in order to keep its viscosity
constant using lower polymer concentrations [33].
5. Conclusion
This study showed that targeting the deep lungs, P407 and CpG
all increased immune responses to Ag85A. CpG led to a T-helper
type-I phenotype and the combination with the carrier P407 gen-
erated a more polyfunctional T cells phenotype. This work also
showed that these effects could be explained, at least in part, by
the enhanced residence time of the antigen within the airway lu-
men. However, in order to consider further development of this
formulation, the key experiment will involve the assessment of
the protection it confers against a M. tuberculosis challenge as com-
pared to BCG.
Acknowledgements
The authors thank Pr. François Huaux for its helpful discussion
and Pr. Jacques Van Snick for providing antibodies for IL-17A assay.
This work was supported by the Fonds Spécial de Recherche de
l’Université catholique de Louvain, by the Fonds de la Recherche
Scientifique Médicale, and by the Fonds National de la Recherche
Scientifique (FNRS, Belgium). Rita Vanbever is Senior research
associate of the FNRS.
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