TLR4 ligand formulation causes distinct effects on antigen

Vaccine 31 (2013) 5848–5855
Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
TLR4 ligand formulation causes distinct effects on antigen-speciﬁc
cell-mediated and humoral immune responses
Christopher B. Fox ∗ , Magdalini Moutaftsi, Julie Vergara, Anthony L. Desbien,
Ghislain I. Nana, Thomas S. Vedvick, Rhea N. Coler, Steven G. Reed
Infectious Disease Research Institute, 1616 Eastlake Avenue, Suite 400, Seattle, WA 98102, USA
a b s t r a c t
i n f o
Article history:
Received 16 March 2013
Received in revised form
14 September 2013
Accepted 30 September 2013
Available online 10 October 2013
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The formulation of TLR ligands and other immunomodulators has a critical effect on their vaccine adjuvant
activity. In this work, the synthetic TLR4 ligand GLA was formulated with three distinct vaccine delivery
system platforms (aqueous suspension, liposome, or oil-in-water emulsion). The effect of the different
formulations on the adaptive immune response to protein subunit vaccines was evaluated in the context of a recombinant malaria antigen, Plasmodium berghei circumsporozoite protein (PbCSP). Antibody
responses in vaccinated mice were similar for the different formulations of GLA. However, cell-mediated
responses differed signiﬁcantly depending on the adjuvant system; in particular, the emulsion formulation of the TLR4 ligand induced signiﬁcantly enhanced cellular IFN-␥ and TNF-␣ responses compared
to the other formulations. The effects of differences in adjuvant formulation composition and physical
characteristics on biological activity are discussed. These results illustrate the importance of formulation
of immunostimulatory adjuvants (e.g. TLR ligands) on the resulting immune responses to adjuvanted
vaccines and may play a critical role for combating diseases where T cell immunity is advantageous.
© 2013 Elsevier Ltd. All rights reserved.
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Keywords:
Glucopyranosyl lipid adjuvant
Vaccine adjuvant formulation
Th1-type immunity
Oil-in-water emulsion
Liposome
Aqueous nanosuspension
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1. Introduction
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A signiﬁcant challenge of modern vaccine development based
on subunit protein vaccines is the induction of effective immune
responses. Subunit protein vaccines generally show excellent
safety proﬁles, but are weakly immunogenic without the addition of adjuvants. Adjuvants may be particulate delivery systems
(e.g. emulsions, liposomes) or immunostimulatory molecules (e.g.
TLR agonist such as MPL® ; saponin such as QS21) [1]. However,
some delivery systems have also shown immunostimulatory functions, such as emulsions (e.g. MF59® , AS03) or aluminum salts, even
without the addition of TLR agonists or other immunostimulants
[2,3].
Combination of delivery systems and immunopotentiators can
dramatically alter the resulting immunomodulatory properties as
shown by the development history of a recombinant malaria antigen based on the circumsporozoite protein (CSP) and known as
RTS,S. Differences in formulations evaluated in C57Bl/6 immunized
with RTS,S showed that strong CD8+ and some CD4+ T cell responses
were generated when adjuvants (MPL® and QS21) were formulated
in liposomes (AS01B), but not when formulated in an oil-in-water
emulsion (AS02A) [4]. These results were supported by clinical
∗ Corresponding author. Tel.: +1 206 858 6027; fax: +1 206 381 3678.
E-mail address: [email protected] (C.B. Fox).
RTS,S studies where AS01 induced higher antigen-speciﬁc antibodies and IFN-␥-producing CD4+ T cells and potentially higher
efﬁcacy than AS02 [5–8], emphasizing the critical role of appropriate adjuvant formulation for the generation of T cell and antibody
responses, although the mouse studies did not prove predictive
regarding CD8+ responses in non-human primates and humans
(which were generally undetectable) [4,6,9].
Glucopyranosyl lipid adjuvant (GLA, or PHADTM ) is a synthetic TLR4 agonist with potent adjuvant activity [10,11]. The
glycolipid structure of GLA is amenable to formulation in various
lipid-based delivery platforms, including oil-in-water emulsions,
liposomes, and aqueous nanosuspensions. We previously showed
that emulsion-based formulations of GLA with a recombinant
leishmaniasis vaccine elicited more of a Th1-biased response compared to an aqueous nanosuspension of GLA, with higher ratios of
IgG2a:IgG1 antigen-speciﬁc antibodies and more IFN-␥-producing
cells [12]. In the present work, we expand on GLA delivery system comparisons to include a liposomal formulation along with
emulsion and aqueous nanosuspension formulations in the context of a model antigen, a recombinant malaria protein (Plasmodium
berghei circumsporozoite protein, PbCSP). Besides antigen-speciﬁc
antibodies, we evaluate the adjuvant formulation effects on stimulation of CD4+ T cells, which could be important for protection
against many complex diseases lacking vaccines, including malaria.
Therefore, we discuss the differences in formulation structure and
composition between the different delivery platforms and their
0264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.09.069
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C.B. Fox et al. / Vaccine 31 (2013) 5848–5855
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with new lots of each formulation prepared in a similar manner
as the initial lots, with the exception that the GLA-Liposomes
contained 0.025% w/v GLA stock concentration (instead of 0.1%
w/v) and the recirculating water bath during homogenization for
the liposomes was 20–25 ◦ C and for the emulsion was 10–15 ◦ C.
potential effects on antigen-speciﬁc humoral and cellular adjuvant
activity.
2. Materials and methods
2.1. Ethics
2.3. Mice
Animal protocols were approved by the Infectious Disease
Research Institute (IDRI) Institutional Animal Care and Use Committee (IACUC) under the protocol/approval number #2008/14.
Six-twelve week old, female C57BL/6 mice were purchased from
Charles River (Wilmington, MA) or Jackson Laboratories (Bar Harbor, ME). Mice were maintained and housed under pathogen-free
conditions at the IDRI.
2.2. Adjuvant formulations
2.4. Antibody ELISA
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Serum antibodies were determined by ELISA. Brieﬂy, MaxiSorp plates (Nunc, NY) were coated with 2 ␮g/ml PbCSP in 0.1 M
bicarbonate buffer and incubated at 4 ◦ C overnight. Plates were
washed using 0.1% Tween 20 in PBS and blocked for at least 2 h
at room temperature with 1% bovine serum albumin (BSA) in 0.25%
Tween 20/PBS. Serial dilution of mouse serum were incubated on
coated/blocked plates for 2 h at room temperature, washed and
incubated for at least 1 h with HRP-conjugated goat anti-mouse
IgG, IgG1 or IgG2c (Southern Biotech). Plates were washed using
0.1% Tween 20/PBS and developed using SureBlue teramethylbenzidine (TMB) substrate (KPL, MD). The reaction was stopped after
1–2 min in 1 M NH2 SO4 and data were collected using an ELISA
reader at 450 nm wavelength. Reciprocal dilutions corresponding
to endpoint titers were determined with GraphPad Prism (GraphPad Software Inc.) with a cut-off of 3x the standard deviation of the
average of the naïve sample on the plate.
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Shark liver squalene (≥98% purity) was purchased from
Sigma–Aldrich (St. Louis, MO). Poloxamer 188 (Pluronic® F68)
and glycerol were purchased from Spectrum Chemical (Gardena, CA). Egg phosphatidylcholine (egg PC), 1,2-dipalmitorylsn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-snglycero-3-phospho-(1 -rac-glycerol) (DPPG) were obtained from
Avanti Polar Lipids, Inc. (Alabaster, AL). Cholesterol and ammonium phosphate buffer components were obtained from J. T. Baker
(Phillipsburg, NJ). PBS was obtained from Invitrogen (Carlsbad, CA).
Saline was obtained from Teknova (Hollister, CA).
The emulsion formulations were prepared by making separate aqueous and oil phases. Poloxamer 188, glycerol, and buffer
components were dissolved in the aqueous phase with stirring,
whereas egg phosphatidylcholine was dissolved in a squalene
oil phase with heating and sonication. Aqueous and oil phases
were then mixed with a Silverson Heavy Duty Laboratory Mixer
Emulsiﬁer (3/4 in. tubular square hole high shear screen attachment; East Longmeadow, MA) at ∼7000 rpm for 10 min to yield
a crude emulsion. The crude emulsion was processed through a
Microﬂuidics M110P (Newton, MA) high-pressure homogenizer for
12 passes at ∼207 MPa (∼30,000 psi). The recirculating product
was cooled by a water bath at 25–35 ◦ C. The liposome formulation was prepared by combining DPPC, DPPG, and cholesterol in
chloroform:methanol:water, and evaporating the solvent under
vacuum, followed by hydration with PBS. Water bath sonication
was used to disperse the lipid components from the sides of the
glass into the buffer. This crude dispersion was then processed on
the Microﬂuidics 110P high-pressure homogenizer for 12 passes
at ∼207 MPa, with the recirculating water bath set above 40 ◦ C. A
small-scale GLA-Liposome batch for the antigen-adjuvant association study (below) was prepared by water bath sonication instead
of microﬂuidization. The aqueous nanosuspension was prepared
by combining DPPC with GLA in chloroform:methanol, followed by
evaporation under vacuum. The lipid ﬁlm was hydrated with ultrapure water and sonicated at ∼60 ◦ C until a translucent appearance
was obtained, up to ∼4 h.
Formulations were monitored for stability for 12 months at
5 ◦ C and room temperature. Particle size, zeta potential, and
hemolysis assay measurements were performed according to
previous descriptions [13]; particle size of emulsions was measured on the Malvern Zetasizer APS while other formulations were
measured using the Malvern Zetasizer Nano-S or Nano-ZS. HPLC
with charged aerosol detection (Corona, ESA Biosciences) was used
to quantify GLA concentration using a method slightly modiﬁed
from an earlier publication [12]. Brieﬂy, a mobile phase gradient
of methanol:chloroform:aqueous buffer (A: 75/15/10, v/v/v; B:
50/50/0, v/v/v; both mobile phases contained 20 mM ammonium
acetate buffer and 1% v/v acetic acid) was employed over 30 min.
Samples were diluted 1:20 in mobile phase before injection on to
a C18 column (Atlantis, Waters). GLA peak area was compared to
a standard curve. Cryo-transmission electron microscopy (cryoTEM) was performed by NanoImaging Services (San Diego, CA)
2.5. Immunization and cell preparation
PbCSP was produced in-house using the codon-harmonized
construct kindly provided by Dr. Evelina Angov from the Walter Reed Army Institute of Research. Endotoxin levels in PbCSP
batches were ≤13 EU/mg. Mice were immunized intramuscularly
(i.m.) three times at the base of the tail with 10 ␮g of recombinant
PbCSP in the presence or absence of 5 ␮g GLA in each formulation platform in a total volume of 100 ␮l, with three-week intervals
between immunizations. Negative control mice received the antigen in saline without adjuvant. Spleens were harvested seven days
following the last immunization. Single cell solutions were prepared by homogenization through a 70 ␮m cell strainer, followed
by red blood cell lysis (eBioscience, CA). The total cell number was
determined using a Guava Cell Counter (Millipore) according to
manufacturer’s instructions.
2.6. Flow cytometry
Splenocytes (1–2 × 106 ) were antigen-pulsed for no more than
12 h in the presence of brefeldin A (10 ␮g/ml) prior to surface staining. Cells were stained in staining buffer (PBS containing 1% FBS),
containing the ﬂuorophore-conjugated monoclonal surface antibodies (CD3, CD44, CD62L, CD4, CD8) for 30 min at 4 ◦ C. Cells were
ﬁxed and permeablized with Cytoﬁx/Cytoperm kit (BD Biosciences)
according to manufacturer’s instructions, followed by intracellular
staining with ﬂuorochrome conjugated antibodies (IFN-␥ and TNF␣). Cells were collected using the BD Fortessa cytometer and data
were analyzed using FlowJo software (Tree Star Inc.).
2.7. Antigen-adjuvant association
Mixtures of PbCSP and representative adjuvant formulations
were prepared according to the same protocol used for the mouse
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IgG2c Endpoint Titers
(Log10)
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IgG2c/lgG1 Ratios
(Log10)
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IgG Endpoint Titers
(Log10)
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IgG1 Endpoint Titers
(Log10)
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Fig. 1. Evaluation of PbCSP-speciﬁc antibody responses following immunization with different adjuvants compared to antigen alone (Saline).
Total IgG and IgG isotype antibody titers measured 17–20 days after the ﬁrst boost immunization. (a) IgG1 endpoint titers. (b) IgG2c endpoint titers. (c) Total IgG endpoint
titers. (d) IgG2c/IgG1 ratios. The compiled results of two-three compiled independent experiments (six to nine individual mice) are shown. P-values < 0.05 were considered
signiﬁcant (*). Dots represent individual mice and lines represent the means. For simplicity, the following statistical differences (P < 0.05) were not shown in the ﬁgure: (a)
SE vs. GLA-AF, (b) GLA-SE vs. Liposomes, GLA-AF vs. Liposomes, (c) SE vs. Liposomes, GLA-SE vs. Liposomes, GLA-AF vs. Liposomes, (d) GLA-SE vs. Liposomes, GLA-AF vs.
Liposomes, GLA-AF vs. SE.
immunization studies, with saline as diluent and ﬁnal concentrations of PbCSP and GLA as 0.1 mg/ml and 0.05 mg/ml, respectively.
The mixtures were ultracentrifuged for 2 h at 180,000 × g in a
Beckman Optima XP (Brea, CA). 20-␮l supernatant samples were
prepared for SDS-PAGE by mixing with 20 ␮l of 4x reducing sample buffer and 40 ␮l 20% SDS. Gold-stained PVDF membrane blots
from the SDS–PAGE gels were then performed.
2.8. Statistical analysis
Data handling, analysis and graphic representation were performed using GraphPad Prism. For antibody titers and cellular ﬂow
cytometry results, P-values were calculated by one-way ANOVA
with Tukey’s correction for multiple group comparison.
3. Results
3.1. TLR4-containing vaccines induce similar humoral responses
regardless of adjuvant formulation/delivery system
Antibody endpoint titers from serum obtained from PbCSPimmunized C57Bl/6 mice measured 17–20 days after the second
immunization indicated that the overall magnitude of total IgG
titers varied between the delivery systems (Fig. 1). The oil-in-water
emulsion (SE) showed increased antibody titers overall, while liposomes induced similar levels compared to antigen alone (Saline).
Addition of the immunopotentiator adjuvant GLA (TLR4 ligand) to
any delivery system increased total IgG titers compared to antigen
alone and was similar to the SE.
Analysis of IgG isotypes (IgG1, IgG2c) is critical for the characterization of adaptive immune responses as distinct isotypes
have been associated with different ability to recruit innate cells,
ﬁx complement and engage Fc␥-receptors [14]. The liposome formulation (without GLA) did not increase IgG1 or IgG2c antibody
titers compared to the antigen alone, whereas the vaccine with SE
induces higher IgG1 antibodies than the antigen alone or the vaccine with GLA-SE or GLA-AF (Fig. 1). However, IgG2c antibodies
were highest in the vaccines containing GLA (GLA-SE, GLALiposomes, GLA-AF), each of which showed signiﬁcantly higher
responses than the antigen alone. Moreover, IgG2c/IgG1 ratios
conﬁrm the tendency of GLA-containing formulations to induce
a Th1-type immune response compared to vaccines without GLA
that favor Th2-biased immune responses as previously observed
[11,15].
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and different adjuvants were tested for IFN-␥ and TNF-␣ production
by ﬂow cytometry seven days following last boost immunization. Statistically signiﬁcant IFN-␥− TNF-␣+ , IFN-␥+ TNF-␣− and
IFN-␥+ TNF-␣+ production was observed from CD4+ T cells obtained
from mice immunized with PbCSP admixed with GLA-SE (Fig. 2).
The majority of the Th1 cytokine producing CD4+ T cells shown in
Fig. 2 produced both IFN-␥ and TNF-␣, which is in good agreement
with a related study by Orr et al. [17]. In contrast, immunization
3.2. Formulation of TLR4 agonist is critical for induction of
cellular immunity
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The induction of cellular immunity has been shown to play a
critical role in protection from many infectious diseases, including malaria [16]. We evaluated the effect of different formulations
of the TLR4 ligand adjuvant GLA on CD4+ T cell responses (Fig. 2).
Splenocytes obtained from C57Bl/6 mice immunized with PbCSP
Fig. 2. Evaluation of PbCSP-speciﬁc T cell responses following immunization with different adjuvants.
C57Bl/6 mice were immunized i.m. three times in three weeks interval with PbCSP (10 ␮g) admixed with different adjuvants: saline, oil-in-water emulsion (SE), GLA-SE,
Liposomes, GLA-Liposomes, GLA-AF. Splenocytes were removed one week (effector) after the last immunization and tested by ICS staining for CD4+ T cell responses following
in vitro stimulation with PbCSP (5 ␮g/ml). Stimulation with media results are shown in the Supplementary Information for comparison. (a) Gating strategy. (b) Percent
Th1-type cytokine producing CD4+ T cells. (c) Number of Th1-type cytokine producing CD4+ T cells per spleen. (d) Number of Th1-type cytokine producing CD4+ T cells per
million splenocytes. The average of two-three compiled independent experiments (six to nine individual mice) are represented by the bar. P-values <0.05 were considered
signiﬁcant (*).
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Fig. 2. (continued)
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of mice with PbCSP in the presence of any other adjuvant formulations did not result in any signiﬁcant IFN-␥-producing T cells,
despite the presence of the Th1-inducing TLR4 agonist GLA. Mice
immunized in the presence of GLA-Liposomes or GLA-AF did not
induce signiﬁcant levels of IFN-␥ or TNF-␣ producing CD4+ T cells,
indicating that formulation of TLR4 agonist is critical in generating cellular immune responses. Control mice immunized with
PbCSP alone (Saline) did not produce any signiﬁcant IFN-␥ or
TNF-␣ measurable by ﬂow cytometry. Single-cell solutions from
any group stimulated with media (control) did not produce any
signiﬁcant IFN-␥ or TNF-␣ measurable by ﬂow cytometry (see
Supplementary Information). Finally, detection of any appreciable IFN-␥+ or TNF-␣+ CD8+ T cells from immunized mice was
inconclusive.
3.3. Formulations differ in morphological characterization
Key features of vaccine delivery systems are their size, shape
and surface molecule organization, with each parameter affecting
antigen uptake and presentation by APC and the resulting adaptive immune response [18]. Although all the formulations tested
in the current study are lipid-based, they differ signiﬁcantly from
each other in morphological characteristics as evidenced by the
cryo-transmission electron microscopy (cryo-TEM) images (Fig. 3).
The aqueous suspension, GLA-AF, is a heterogeneous mix of small
and large lipid particles, including lipid vesicles, disks, and micelles
(Fig. 3a). Besides GLA itself, the GLA-AF formulation contains a small
amount of synthetic phosphatidylcholine (Table 1) as a suspensionforming agent; thus, GLA-AF contains far fewer particles compared
to the other formulations. The GLA-liposome formulation consists
mostly of homogeneous, unilamellar ∼30–40 nm lipid bilayer vesicles composed of synthetic phospholipid (DPPC and DPPG) and
cholesterol (Fig. 3b). Some larger multilamellar vesicles and disks
are also present. GLA-SE is an oil-in-water emulsion consisting of
droplets of the metabolizable oil squalene, emulsiﬁed with natural
phosphatidylcholine and poloxamer (Fig. 3c). Cryo-TEM analysis
indicates oil droplets of uniform density ranging in size from ∼20
to 120 nm. In addition, the emulsion formulations contain a few
lipid vesicles (white arrow in Fig. 3c).
The average light scattering intensity-based particle size (Z-ave)
conﬁrmed the particle sizes seen in the cryo-TEM images for the
emulsion and liposome formulations. SE and GLA-SE particle sizes
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Fig. 3. Evaluation of formulation size and morphology by Cryo-transmission electron microscopy.
Cryo-transmission electron micrographs of (a) GLA-AF, (b) GLA-Liposomes, and (c) GLA-SE reveal particle size and morphology characteristics. Scale bar in each image
represents 200 nm. The white arrow in (c) indicates a lipid vesicle.
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Overall, the formulations show good size stability for multiple months at 5 ◦ C (Fig. 4a) and room temperature (RT; Fig. 4b).
An arbitrary stability standard utilized in our laboratory speciﬁes
that particle size should not grow more than 50% of initial size
over time to be considered stable [19]. All of the formulations
at 5 ◦ C met this standard even at ≥9 months post-manufacture
(Fig. 4). At this temperature, the SE, GLA-SE, and GLA-AF formulations show little change in size or PdI over time, whereas the
liposome formulations grow gradually in size and PdI. At RT, the
adjuvant formulations showed good size stability for 6 months or
more, with GLA-AF and the liposome formulation showing little
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were ∼90–100 nm with very low polydispersity indices (PdI), indicating high homogeneity (Fig. 4a and b). PdI for liposomes was low,
and average initial particle size was ∼40 nm. In contrast to the cryoTEM results, the Z-ave particle size of GLA-AF was similar to the
emulsion formulations. This is most likely due to the heterogeneous
composition of GLA-AF, which is apparent in the high PdI value.
Moreover, the Z-ave particle size is heavily weighted toward larger
particles even when they are low in number due to the much greater
laser scattering intensity of larger particles (Rayleigh’s approximation of the proportionality factor of scattering intensity to particle
diameter is 106 ).
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GLA-SE
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Parcle Size (Z-ave, nm)
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Parcle Size (Z-ave, nm)
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Polydispersity Index
Polydispersity Index
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0.2
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0
2
4
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8
Time at 5 ⁰C (months)
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0
2
4
6
8
Time at RT (months)
10
12
Fig. 4. Evaluation of formulation size and polydispersity over time by dynamic light scattering.
Particle size and polydispersity index (PdI) of adjuvant formulations are shown over time and at different temperatures. Size is measured by dynamic light scattering and
reported as the scattering intensity-based average (Z-ave). (a) Size and PdI of formulations stored at 5 ◦ C after manufacture. (b) Size and PdI of formulations stored at room
temperature (RT) after manufacture. Error bars represent the standard deviation from nine total size measurements from three separate sample aliquots.
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Table 1
Adjuvant formulation composition and physicochemical characterization.
Formulation
Componentsa (% w/v)
Hemolysisb (%)
SE
Squalene (8.6%), Egg PC (1.9%),
Poloxamer 188 (0.1%), Glycerol
(1.8%), Ammonium phosphate
buffer
Same as above with GLA
(0.025%)
DPPC (1.8%), DPPG (0.2%),
Cholesterol (0.5%), PBS
Same as above with GLA (0.1%)
DPPC (0.02%), GLA (0.025%)
0.1
(−)5.2 ± 1.3
0.1
GLA-SE
Liposomes
GLA-Liposomes
GLA-AF
Zeta potentialc (mV)
Dynamic viscosityd (cP)
pHe
HPLC–CADf (Pass/Fail)
NM
5.5 ± 0.0
NM
(−)11.4 ± 2.5
1.8 ± 0.2
5.3 ± 0.2
Pass
0.2
(−)48.1 ± 4.6
1.2 ± 0.1
7.2 ± 0.0
NM
0.2
0.3
(−)42.4 ± 15.8
(−)47.8 ± 12.2
1.2 ± 0.1
NM
7.2 ± 0.1
NM
Pass
Pass
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Fc␥-receptors [14], resulting in a qualitatively different outcome of
the adaptive immune response. A recent study showed that IgG2c,
but not IgG1 isotype antibodies of identical speciﬁcity, were able
to protect mice from lethal inﬂuenza challenge [23], underscoring the importance to induce the appropriate antibody isotype for
protection. In our study, the IgG2c/IgG1 ratio of the vaccine is not
altered by the presence of SE or liposome, resulting in Th2-biased
immune responses, and indicating that some adjuvant formulations may increase the magnitude of the immune response but do
not shape the quality, unless a TLR ligand such as GLA is included.
A similar ﬁnding has been reported for MF59 with the TLR9 agonist
CpG or the TLR4 agonist E6020 [24]. In contrast, all TLR4-containing
4. Discussion
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change in size for at least 12 months (GLA-Liposomes were not
monitored at RT).
Other initial physicochemical characterization included
hemolytic activity, zeta potential, viscosity, pH, and GLA quantiﬁcation (Table 1). All of the formulations had negative zeta potentials
indicating a negative surface charge. SE had the zeta potential of
lowest magnitude, which was expected since this formulation
does not include any charged emulsiﬁers. The presence of GLA
causes a more negative zeta potential in SE, a phenomenon that
we have reported earlier and that is indicative of localization of
GLA at the oil-water interface [12]. Further evidence for emulsion
droplet-associated localization of GLA in GLA-SE, or lipid bilayerassociated GLA in GLA-Liposomes, comes from recent in vitro
bioactivity analyses, where reduced in vitro bioactivity appears to
be due to the association of GLA with the lipid particles, reducing
the immediate availability of the GLA [20]. All formulations also
show good hemocompatibility, which is desirable from a safety
perspective since hemolytic adjuvants (such as unformulated
saponins) may cause local reactogenicity [21]. The oil-in-water
emulsions are more viscous than the liposomal formulations
and are buffered at lower pH values. HPLC with charged aerosol
detection (CAD) indicates the expected GLA target concentration in
all GLA-containing formulations. A description of the composition
of each formulation is shown in Table 1.
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a
Component concentrations listed are stock formulations, which are then diluted 5-fold for immunization. Viscosity and pH measured using stock formulation concentration; hemolysis measured at 5-fold dilution; zeta potential and HPLC measured at 20-fold dilution.
b
Reported hemolysis represent an average of data collected from two separate donors within 1 month of formulation manufacture.
c
Reported zeta potential values are averages of measurements collected at 0, 3, and 6 months after manufacture, except for GLA-AF which only had 3 and 6 month
measurements.
d
Reported viscosity values represent the average of measurements collected at 0 and 3 months after manufacture.
e
Reported pH values represent averages from data collected at 0, 3, and 6 months after manufacture.
f
HPLC–CAD “Pass” indicates peak area representing GLA corresponded to within 20% of the target concentration. Abbreviations: SE, stable emulsion; PC, phosphatidylcholine; NM, not measured.
The present results indicate that classiﬁcation of adjuvants as delivery systems or immunomodulatory molecules is
an over-simpliﬁcation, since liposomes (without GLA) appear
immunologically inert, whereas SE (without GLA) shows immunostimulatory activity (enhanced IgG1 antibodies). This may be due
to the presence of emulsiﬁed squalene, which has demonstrated
enhanced adjuvant activity compared to other emulsiﬁed oils
[13,22]. Thus, SE is not simply a delivery system but an adjuvant
in its own right. The resulting enhancement in cellular immune
responses may thus be due to synergistic activity between the
squalene-based emulsion and the TLR4 agonist GLA. Therefore the
prime difference in formulations of GLA affecting the induction of
cellular immunity appears to be in the composition, although other
physical differences existed between the formulations (size, surface charge) and further study is merited to determine their explicit
effects.
Distinct IgG isotypes (IgG1, IgG2c) have been associated with
different ability to recruit innate cells, ﬁx complement and engage
Fig. 5. Assessment of adjuvant interactions with PbCSP antigen.
Reduced SDS-PAGE proﬁles of PbCSP obtained from supernatants of ultracentrifuged mixtures of PbCSP and adjuvant formulations do not appear to
indicate differences in the amount of association of PbCSP with formulation
particles. Lane 1: PbCSP + saline; Lane 2: PbCSP + SE; Lane 3: PbCSP + GLASE; Lane 4: PbCSP + Liposomes; Lane 5: PbCSP + GLA-Liposomes; Lane 6:
PbCSP + GLA-AF; Lane 7: PbCSP + ammonium phosphate buffer (pH 5.8); Lane 8:
PbCSP + sodium/potassium phosphate-buffered saline (pH 7.2). The control samples
in Lanes 7 and 8 were not ultracentrifuged and indicate that different buffers and pH
values representative of the various formulation systems did not themselves affect
the SDS–PAGE proﬁle of PbCSP. PbCSP molecular weight is 21 kDa but its SDS–PAGE
proﬁle is anomalous due to its complex structure, as reported previously [31].
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C.B. Fox et al. / Vaccine 31 (2013) 5848–5855
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[16]
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or
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ad
adjuvants biased toward Th1 immune responses (increased IgG2c
levels), conﬁrming previous observations [11,15] that the inclusion of a TLR4 agonist shapes the quality of the resulting immune
response [25,26].
When comparing vaccines containing PbCSP and GLA with different formulation platforms (liposome, AF and SE), we observed
that the induction of cellular immune responses was highly
dependent on the delivery system, despite similar induction
of humoral immunity. We focused on IFN-␥ and TNF-␣ as
indicative of Th1-type responses which are believed to be
important for protection induced by CSP-based vaccines [27].
However, related work with GLA-SE and the CelTOS malaria
antigen involving a panel of induced cytokines indicated that
vaccines containing GLA-SE enhanced IL-17 production (in addition to IFN-␥), while SE induced higher IL-5, IL-13, and IL-10
responses indicative of a Th2-type response [28]. Likewise, GLASE with a tuberculosis antigen induced enhanced multifunctional
CD4+ Th1-type cytokine responses (IFN-␥, TNF-␣, IL-2) compared to GLA-Liposomes and GLA-AF, although GLA-Liposomes
also elicited appreciable responses [17]. The present work is
generally consistent with these previous results, with the oilin-water (O/W) emulsion of GLA enabling the highest activation
of CD4+ and T cell responses; however, since GLA-Liposomes
and GLA-AF induce appreciable IgG2c antibodies, it is possible
that IFN-␥-independent mechanisms may be partly responsible
[29,30].
Another critical determinant of formulation effects is association with the antigen after admixing and in vivo distribution. For the
present study, we attempted ultracentrifugal separation of PbCSPadjuvant mixtures followed by SDS-PAGE of supernatants, but did
not see evidence of extensive PbCSP association with representative SE, GLA-SE or GLA-Liposome formulations (Fig. 5), indicating
that antigen-adjuvant association may not be the critical determinant of bioactivity in this case. In summary, these studies indicate
that the formulation of particulate adjuvants is crucial for the generation of T cell responses, even as antibody responses appeared
similar between the different formulations as long as they contained the TLR4 ligand.
5855
Acknowledgments
Co
pi
We thank Dr. Evelina Angov from Walter Reed Army Institute
of Research for providing the codon-harmonized PbCSP construct
and for helpful input regarding the manuscript. In addition, we
appreciate insightful discussions with Dr. Mark Orr and Dr. Malcolm Duthie. We are also grateful to NanoImaging Services for the
cryo-TEM images. Finally, we gratefully acknowledge the excellent
technical assistance of Sandra Sivananthan, Traci Mikasa, Kristen
Forseth, Tim Dutill, and Susan Lin.
Appendix A. Supplementary data
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.vaccine.2013.09.069.
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08/07/2014

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