Download Immune responses to vaccines involving a combined antigen

Biomaterials 35 (2014) 6086e6097
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Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Immune responses to vaccines involving a combined
antigenenanoparticle mixture and nanoparticle-encapsulated
antigen formulation
Weifeng Zhang a, b,1, Lianyan Wang a,1, Yuan Liu a, b, Xiaoming Chen a, b, Qi Liu a, b, Jilei Jia a, b,
Tingyuan Yang a, Shaohui Qiu c, Guanghui Ma a, *
a
National Key Laboratory of Biochemical Engineering, PLA Key Laboratory of Biopharmaceutical Production & Formulation Engineering,
Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China
University of Chinese Academy of Sciences, Beijing 100049, PR China
c
National Institutes for Food and Drug Control, Beijing 100050, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2014
Accepted 6 April 2014
Available online 26 April 2014
Many physicochemical characteristics significantly influence the adjuvant effect of micro/nanoparticles;
one critical factor is the kinetics of antigen exposure to the immune system by particle-adjuvanted
vaccines. Here, we investigated how various antigenenanoparticle formulations impacted antigen
exposure to the immune system and the resultant antigen-specific immune responses. We formulated
antigen with poly(lactic-co-glycolic acid) (PLGA) nanoparticles by encapsulating antigen within nanoparticles or by simply mixing soluble antigen with the nanoparticles. Our results indicated that the
combined formulation (composed of antigen encapsulated in nanoparticles and antigen mixed with
nanoparticles) induced more powerful antigen-specific immune responses than each single-component
formulation. Mice immunized with the combined vaccine formulation displayed enhanced induction of
antigen-specific IgG antibodies with high avidity, increased cytokine secretion by splenocytes, and
improved generation of memory T cell. Enhanced immune responses elicited by the combined vaccine
formulation might be attributed to the antigen-depot effect at the injection site, effective provision of
both adequate initial antigen exposure and long-term antigen persistence, and efficient induction of
dendritic cell (DC) activation and follicular helper T cell differentiation in draining lymph nodes. Understanding the effect of antigenenanoparticle formulations on the resultant immune responses might
have significant implications for rational vaccine design.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Adjuvant
Nanoparticles
Vaccine formulations
Immune response
Mechanism of action
1. Introduction
Vaccination, considered as one of the most significant achievements in medicine, plays an important role in preventing infectious
diseases, and saves more than 3 million people every year [1e3].
However, inadequate immunogenicity and/or safety concerns are
still significant obstacles to developing ideal vaccines. While
endogenous adjuvants endow traditional vaccines based on attenuated or inactivated pathogens with sufficient immunogenicity, but
* Corresponding author. Chinese Academy of Sciences, Institute of Process Engineering, National Key Laboratory of Biochemical Engineering, Bei-Er-Jie No. 1,
Zhong-Guan-Cun, Haidian District, Beijing 100190, PR China. Tel./fax: þ86 10
82627072.
E-mail address: [email protected] (G. Ma).
1
Both authors contributed equally.
http://dx.doi.org/10.1016/j.biomaterials.2014.04.022
0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
the side effects and safety concerns limit the extent to which they
can be used against various pathogens, such as HIV and Hepatitis C.
Although subunit vaccines based on protein antigens are usually
better tolerated and regarded as safer alternatives to traditional
vaccines, they are usually poorly immunogenic when used alone
and therefore require exogenous adjuvants to augment resultant
immune responses [3e5]. Alum is a conventional adjuvant and the
only one widely licensed for human use. Despite being used for over
80 years in vaccines [6], alum has some disadvantages, including
side effects and safety concerns [7], contributing little to or even
suppressing cell-mediated immunity and subsequent CTL responses
[8,9], and providing poor adjuvanticity for recombinant protein
vaccines [9]. These disadvantages necessitate the development of
new adjuvants for subunit vaccines.
As one potential alternative, particulate-based adjuvants can
act as antigen delivery systems that facilitate the access of antigen
W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
to antigen presenting cells (APCs) and regulate the antigen presentation pathway, or as immune potentiators that enhance the
subsequent antigen-specific immune responses [3,10,11]. Ever
since Kreuter et al. evaluated the adjuvanticity of polymethylmethacrylate nanoparticles for the influenza virus in 1976
[12], biodegradable polymer-based micro/nanoparticles have been
extensively investigated as adjuvants for subunit-based vaccines.
The efficacy of particle vaccines are significantly influenced by
various physicochemical characteristics of micro/nanoparticles
(such as particle size [11,13e15], surface charge [14e16], hydrophobicity [17,18]), administration route [19,20], and antigen
release kinetics [13,21e23]. Antigen release kinetics affects the
efficiency of particulate-based vaccines by regulating antigen
exposure to the immune system. For example, Kanchan et al. reported that slow and continuous antigen release from polymerbased particles played a critical role in eliciting memory antibody responses after a single immunization [21]. Demento et al.
reported that sustained antigen release from poly(lactic-coglycolic acid) (PLGA) nanoparticles favored long-term effectormemory cellular responses [22], and Johansen et al. demonstrated
that antigenic stimulation increasing exponentially over days
induced more potent CD8þ T cell responses and antiviral immunity than a single dose or multiple doses (equivalent doses,
administered daily) [23].
In order to achieve these different antigen-exposure kinetics,
most studies fabricated particulate-based vaccines with different
antigen-release profiles by regulating particle structure [21],
employing different types of particles [22,24], preparing particles
with stimuli-responsive polymers [3], and so on. However, relatively little attention was focused on ways to optimally formulate
antigens with the particles. Antigen can be formulated with particles through attachment (e.g., conjugation, encapsulation, adsorption) or simple mixing [25]. Although various antigeneparticle
formulations significantly affect the kinetics of antigen exposure to
the immune system, how various antigeneparticle formulations
impact the resultant antigen-specific immune responses remains
unknown.
We hypothesized that encapsulating antigen in PLGA nanoparticles would increase its antigen persistence in vivo, and
combining soluble antigen with the nanoparticle vaccine would
improve initial antigen exposure to immune system after vaccination, both together allowing for the generation of stronger and
more prolonged adjuvant-induced, antigen-specific immune responses. In the present study, our objectives were therefore to
determine the type and strength of the immune responses elicited
by different PLGA nanoparticle-based vaccine formulations using
ovalbumin (OVA) as the model antigen, and to elucidate the underlying mechanisms of action.
2. Materials and methods
2.1. Mice, reagents, and materials
Female Balb/c mice used in this study were purchased from Vital River Laboratories (Beijing, China). All animal experiments were performed in accordance with
the Guide for the Care and Use of Laboratory Animals, and were approved by the
Experimental Animal Ethics Committee in Beijing. PLGA (75/25, Mw z 13 kDa) was
purchased from Lakeshore Biomaterials (Birmingham, AL, USA). Poly(vinyl alcohol)
(PVA-217, degree of polymerization 1700, degree of hydrolysis 88.5%) was ordered
from Kuraray (Tokyo, Japan). Ovalbumin (OVA) was supplied by SigmaeAldrich (St.
Louis, MO, USA). Premix membrane emulsification equipment (FMEM-500M) was
provided by the National Engineering Research Center for Biotechnology (Beijing,
China). Shirasu porous glass (SPG) membrane was provided by SPG Technology Co.
Ltd. (Sadowara, Japan). The medium for splenocytes culture was RPMI 1640 (Gibco,
Carlsbad, CA, USA) with 10% (v/v) fetal bovine serum (Gibco, Carlsbad, CA, USA). All
mouse cytokines ELISA and fluorochrome-conjugated anti-mouse antibodies for
flow cytometric use, were obtained from eBioscience (San Diego, CA, USA), unless
otherwise indicated. ELISpotPLUS kits were obtained from Mabtech AB (Nacka Strand,
Sweden). All other reagents were of analytical grade.
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2.2. Preparation and characterization of PLGA nanoparticles
PLGA nanoparticles were prepared using a two-step procedure by combining
the solvent extraction method and the premix membrane emulsification technique, as described before with some modifications [26] (Fig. S1). Briefly, 1 mL
deionized water containing 100 mg OVA (internal water phase) was added into
12 mL ethyl acetate containing 600 mg PLGA; the primary water in oil (W/O)
emulsion was formed by sonication (120 W; Digital Sonifier 450, Branson Ultrasonics Corp., Danbury, CT, USA) in a tube over an ice bath for 1 min (4 s on and 2 s
off). To prepare the double emulsion, the resulting primary emulsion was added
into 65 mL external water phase containing 1.5% w/v PVA and 0.9% w/v NaCl, and
magnetically stirred at 450 rpm for 90 s. Then, the resulting coarse double
emulsion was extruded through the SPG membrane under a certain nitrogen
pressure for 8 times and nanodroplets with narrow size distribution were obtained. The obtained double emulsion was poured into 800 mL deionized water
containing 0.9% w/v NaCl (solidification solution) under magnetic stirring for 4 h to
solidify the nanoparticles. The obtained PLGA nanoparticles encapsulating OVA
were collected by centrifugation at 15,000 g for 5 min, washed 3 times with
deionized water to remove residual PVA, and then lyophilized and stored at 4 C
for later use. To prepare blank PLGA nanoparticles, 1 mL of deionized water
without OVA was used as the internal water phase.
The hydrodynamic size and zeta potential of PLGA nanoparticles were measured
by a Nano-ZS Zeta Sizer (Malvern Instruments Ltd., Malvern, UK). Morphology of the
PLGA nanoparticles was characterized by scanning electron microscopy (JEM-6700F,
JEOL Ltd., Tokyo, Japan). Nano Measurer 1.2 software was employed to measure the
size of PLGA nanoparticles according to the scanning electron micrographs.
The OVA content of PLGA nanoparticles was determined by incubating 3 mg of
lyophilized PLGA nanoparticles in 1 mL of 0.1 M NaOH solution under gentle shaking
overnight. Protein concentration in the solution was determined using Micro-BCA
protein assay kit (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. OVA dissolved in 0.1 M NaOH was used to establish a standard
curve, and blank PLGA nanoparticles were used as the control.
2.3. Immunization studies
As shown in Table S1, 30 mice were randomly divided into 5 groups (n ¼ 6) and
intramuscularly immunized with 100 mL (50 mL/hind leg) of different vaccine formulations containing 25 mg of antigen (OVA) (Fig. 1E). Mice were immunized 3 times
at 2-week intervals (Fig. S2). Blood samples were collected from the caudal vein
before each immunization and 10 days after the third immunization. Sera was
separated and stored at 70 C for later analysis. At 10 days after the third immunization, splenocytes were collected for in vitro proliferation, cytokine response, and
flow cytometric assays.
2.4. Determination of OVA-specific IgG and IgG subclasses
OVA-specific IgG, IgG1, and IgG2a in the serum were quantitatively determined by enzyme-linked immunosorbent assay (ELISA) in accordance with a
protocol described previously [27]. Briefly, 96-well ELISA plates (Costar, Corning,
New York, USA) were coated overnight at 4 C with 2 mg of OVA per well in
coating buffer (0.05 M CBS, pH 9.6). Plates were washed with PBST (0.01 M PBS
containing 0.05% [m/v] Tween 20, pH 7.4) and blocked by incubating with 2% (m/
v) BSA (Roche, Basel, Switzerland) in PBST for 60 min at 37 C. After washes with
PBST, 100 mL per well of appropriate sera dilutions were added to the plates,
serially diluted two-fold in dilution buffer (PBST containing 0.1% [m/v] BSA), and
incubated for 30 min at 37 C. Plates were then washed and incubated with
100 mL horseradish peroxidase-conjugated goat antibodies against either mouse
IgG (SigmaeAldrich, St. Louis, MO, USA), IgG1, or IgG2a (Santa Cruz, CA, USA) (IgG
diluted 1:20,000; IgG1 and IgG2a diluted 1:2000) for 30 min at 37 C. Thereafter,
the plates were washed again with PBST, and 100 mL of 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate was added to each well and incubated for 20 min
at room temperature. After stopping the reaction by adding 50 mL of 2 M H2SO4 to
each well, the optical density (OD, 450 nm) was measured by an Infinite M200
Microplate Spectrophotometer (Tecan, Männedorf, Switzerland). Titers were
given as the reciprocal sample dilution corresponding to twice higher OD than
that of the negative sera.
OVA-specific IgG avidity measurement of was carried out by ELISA with a ureaelution step [28]. The antigeneantibody complexes with low-affinity were dissociated by incubating the plates with urea (SigmaeAldrich, St. Louis, MO, USA) at 20 C
for 10 min. Avidity index (AI) was calculated in accordance with the following formula: AI ¼ (IgG titer incubated with urea/IgG titer incubated without urea) 100.
2.5. Determination of cytokine levels by ELISA
Splenocytes were harvested from vaccinated mice 10 days after the third immunization and restimulated with OVA (50 mg/mL) for 60 h at 37 C in a humid
atmosphere with 5% CO2, and the supernatant was collected. IL-4, IL-10, IL-12, and
IFN-g levels in the supernatant were measured by Ready-to-use Sandwich ELISA kits
(eBioscience, San Diego, CA) according to the manufacturer’s instructions.
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W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
Fig. 1. Scanning electron micrographs (A, B) and size distribution (C, D) of PLGA nanoparticles, and schematic illustration of different vaccine formulations (E). The scale bar in (A, B)
represents 1 mm.
2.6. ELISpot assays
The frequency of IFN-g- and IL-4-secreting cells in splenocytes harvested from
vaccinated mice 10 days after the third immunization were measured by ELISpotPLUS
kits (Mabtech AB, Nacka Strand, Sweden) according to the manufacturer’s instructions. In brief, 2.5 105 splenocytes were seeded into each well in triplicate and
restimulated with 50 mg/mL OVA. The plates were incubated for 36 h in a 37 C
humidified incubator with 5% CO2. After emptying and washing the plates, detection
antibodies were added, the plates were incubated for 2 h at room temperature,
Streptavidin-ALP was added, and the plates were incubated for 1 h at room temperature. Finally, the ready-to-use substrate solution (BCIP/NBT-plus) was added to
the plates, and color development was stopped by washing extensively in tap water
after distinct spots emerged. Spots were inspected and counted in an ELISpot
analysis system (SageCreation, Beijing, China). Results were expressed as the
number of antigen-specific spot-forming cells (SFCs) per 106 splenocytes.
2.7. Splenocyte proliferation assay
Based on previously described methods [27], the splenocyte proliferation assay
was performed to evaluate antigen-specific splenocyte activation. Splenocytes
(2.5 106 cells/mL), stimulated with OVA (50 mg/mL) or not, were seeded in triplicate (100 mL/well) in a 96-well plate and incubated at 37 C in a humid atmosphere
with 5% CO2. After 72 h, 10 mL of CCK-8 solution (Dojindo, Japan) was added to each
well, and the plates were incubated for an additional 4 h. The absorbance at 450 nm
(with 620 nm as reference) was measured by an Infinite M200 Microplate Spectrophotometer (Tecan, Männedorf, Switzerland). The results were expressed as the
proliferation index (PI), calculated based on the following formula: PI ¼ OD (450 nm)
for stimulated cultures/OD (450 nm) for non-stimulated cultures.
2.8. Determination of memory T cell responses by flow cytometry
Flow cytometry was performed to measure the percentage of memory T cells in
splenocytes collected from immunized mice 10 days after the third immunization.
Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and
restimulated with OVA (50 mg/mL) for 60 h. Cells were then stained with the
following set of fluorochrome-conjugated anti-mouse antibodies: eFluor 450eantiCD4, PerCP-Cy5.5eanti-CD8a, PEeanti-CD44, and APCeanti-CD62L (eBioscience).
After washing, cell samples were examined by Beckman Coulter CyAnÔ ADP flow
cytometer, and data were analyzed by Summit software (version 4.3).
2.9. Antigen persistence at injection sites
To monitor antigen persistence at injection sites in vivo, antigen was labeled
with the near-infrared dye Cy7 mono-reactive NHS ester (Fanbo Biochemicals,
Beijing, China). Balb/c mice (n ¼ 6) were intramuscularly injected in the hind legs
with different vaccine formulations containing antigen labeled with the nearinfrared dye Cy7 (25 mg OVA in 100 mL buffer per mouse, half dose at each site).
Antigen persistence at the injection sites was documented by Carestream FX PRO
in vivo imaging system at the indicated time points (ex: 730 nm; em: 790 nm).
Carestream Molecular Imaging Software was used to quantify sum fluorescence
intensity at the injection sites.
2.10. Determination of available antigen in draining lymph nodes by
immunohistochemical assay
The hind legs (n ¼ 4) of Balb/c mice were intramuscularly injected with 50 mL of
different vaccine formulations containing 1.25 mg of antigen. At various time points
(2 or 7 days after injection), sciatic and popliteal lymph nodes were isolated from
the euthanized mice, fixed in 4% paraformaldehyde, embedded into paraffin, and
cut to yield 4-mm thick sections. After deparaffinization, sections were incubated
with 3% H2O2 for 20 min to inhibit endogenous peroxidase activity. Then, sections
were blocked by goat serum and immunostained with antibody against OVA
(LifeSpan BioSciences, Inc., Seattle, USA). A secondary antibody from PicTureÔPV6000 kit (Zymed Laboratories Inc., South San Francisco, CA, USA) and diaminobenzidine (ZSGB-Bio, Beijing, China) were used for color development.
Specimens were then stained with hematoxylin for cell nucleus identification.
Immunohistochemical micrographs were documented by an Olympus BX51
microscope.
W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
Table 1
Characteristics of PLGA nanoparticles. Data are shown as mean SEM.
Particles
Diameter
(DLS, nm)
PDIa
Diameter
(SEM, nm)
Zeta potential OVA content
(mV)b
(mg/mg)
PLGA NPs 481.2 5.12 0.04 0.01 331.6 3.16 11.93 0.55 e
(Blank)
PLGA NPs 590.8 4.51 0.17 0.04 341.6 2.90 11.43 0.45 59.65 3.62
(OVA)
a
b
PDI, polydispersity index from dynamic light scattering (DLS).
Zeta potential of different nanoparticles was detected in deionized water.
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2.13. Statistical analysis
All values in the present study were expressed as the mean standard error of
the mean (SEM). Statistical analysis was performed using GraphPad Prism 5.0
software (San Diego, CA, USA). Differences between two groups were tested using an
unpaired, two-tailed Student’s t-test. Differences among multiple groups were
tested by one-way ANOVA followed by Tukey’s multiple comparison. Significant
differences between the groups were expressed as follows: *p < 0.05, **p < 0.01, and
***p < 0.001.
3. Results
3.1. Preparation and characterization of PLGA nanoparticles
2.11. Expression of MHC and co-stimulatory molecules on dendritic cells in draining
lymph nodes
Balb/c mice (n ¼ 3) were intramuscularly vaccinated with different vaccine
formulations (25 mg antigen in 100 mL buffer per mouse, half dose at each site), and
mice were euthanized at 1, 2, and 7 days post-immunization. Popliteal lymph nodes
were harvested and processed into single cell suspension. Cells were stained with a
mixture of anti-mouse antibodies (eFluor 450eanti-CD11c, APCeanti-MHC I, FITCe
anti-MHC II, and PEeanti-CD86; all from eBioscience). Expression of MHC I, MHC II,
and CD86 on CD11cþ DCs was determined by CyAnÔ ADP flow cytometer (Beckman
Coulter, California, USA) and analyzed using Summit software (version 4.3.02).
2.12. Determination of follicular helper CD4þ T cells in draining lymph nodes
Balb/c mice (n ¼ 3) were intramuscularly vaccinated with different vaccine
formulations (25 mg antigen in 100 mL buffer per mouse, half dose at each site), and
mice were euthanized 9 days after immunization. Popliteal lymph nodes were
harvested and processed into single cell suspension. Cells were stained with a
mixture of anti-mouse antibodies (eFluor 450eanti-CD4, APCeanti-CXCR5, and PEe
anti-PD-1; all from eBioscience). The percentage of follicular helper CD4þ T cells
(CD4þCXCR5hiPD-1hi) was determined by CyAnÔ ADP flow cytometer (Beckman
Coulter, California, USA) and analyzed using Summit software (version 4.3.02).
To generate different PLGA nanoparticle-adjuvanted vaccine
formulations, PLGA nanoparticles were prepared by emulsificationesolvent extraction method combined with the premix membrane emulsification technique [26]. The double emulsion was
prepared by simply magnetic stirring which has little harm on
antigen structure (Fig. S8), compared to ultrasonication or homogenization. Scanning electron micrographs revealed that both
blank PLGA nanoparticles and PLGA nanoparticles encapsulating
OVA were spherical particles with narrow size distributions (Fig. 1A
and B), and their respective diameters were 481.2 5.12 nm and
590.8 4.51 nm (Table 1, Fig. 1C and D). Thus, similar sizes were
acquired for blank and antigen-encapsulated PLGA nanoparticles.
The zeta potential of the blank and antigen-encapsulated particles
were also similar at 11.93 0.55 mV and 11.43 0.45 mV,
respectively. The antigen content in the antigen-encapsulated
particles was 59.65 3.62 mg/mg, as determined by Micro-BCA
kit (Table 1). As shown in Fig. S3, the in vitro release profile revealed
Fig. 2. Antigen-specific IgG antibody responses in Balb/c mice immunized with different vaccine formulations. Mice (n ¼ 6) were intramuscularly vaccinated on day 0, 14, and 28 as
described in the Methods section. (A) IgG titers in the serum at the indicated time points after first immunization. (B) Antigen-specific IgG1 and IgG2a levels and (C) IgG2a/IgG1
ratios in the serum of immunized mice 10 days after the third immunization (serum diluted 1:200). (D) Avidity index of anti-OVA IgG in the serum of immunized mice 10 days after
the third immunization. Data are expressed as the mean SEM (n ¼ 6). *p < 0.05; **p < 0.01; ***p < 0.001.
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Fig. 3. Cytokine secretion by splenocytes. Mice (n ¼ 6) were immunized three times as described in the Methods section. Splenocytes were harvested 10 days after the third
immunization. For ELISA assay, splenocytes were restimulated with OVA (50 mg/mL) in culture for 60 h ex vivo. IFN-g (A), IL-12 (B), IL-4 (C), and IL-10 (D) levels in the supernatant
were measured by ELISA. For evaluating the frequency of cytokine-secreting splenocytes, the frequency of (E) IFN-g- and (F) IL-4-secreting cells were analyzed by ELISpot.
SFC ¼ spot-forming cells. Data are expressed as the mean SEM (n ¼ 6). *p < 0.05; **p < 0.01; ***p < 0.001.
that the antigen encapsulated within PLGA nanoparticles was
released in a sustained manner for at least the 15-day evaluation
period.
3.2. Systemic antibody responses in vaccinated mice
To investigate the effect of various antigenenanoparticle formulations on antibody response, serum was collected over time
from mice intramuscularly immunized with soluble antigen alone
(Ag), antigen encapsulated within particles (Encapsulation), soluble
antigen mixed with blank particles (Mixture), and a combined
vaccine
formulation
(antigen
encapsulated
within
particles þ soluble antigen mixed with blank particles, Combination). Serum IgG, IgG1, and IgG2a titers were then evaluated by
ELISA. As shown in Fig. 2A, the combined vaccine formulation
induced significantly higher antigen-specific IgG titers than soluble
antigen alone and antigen mixed with PLGA nanoparticles on day
14 after primary immunization, and significantly higher titers than
all other formulations on day 28 and 38. On day 28 after primary
immunization, serum IgG titers elicited by antigen encapsulated
within PLGA nanoparticles were significantly higher than that elicited by soluble antigen alone or antigen mixed with particles. On
W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
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levels were produced by splenocytes from mice immunized with
antigen encapsulated in particles and the combined vaccine
formulation, which were significantly higher than those for soluble
antigen mixed with blank particles or soluble antigen alone. Results
of ELISpot assay revealed that the frequencies of IL-4- and IFN-gsecreting splenocytes in mice immunized with Encapsulation and
Combination formulations were significantly higher than those for
Mixture formulation and soluble antigen alone (Fig. 3E and F, and
Fig. S4). On the whole, the Encapsulation formulation and the
Combination formulation induced higher levels of both Th1 and
Th2 cytokines secretion by splenocytes, indicating stronger immune responses.
3.4. Splenocyte proliferation assay
Fig. 4. Proliferative responses of splenocytes responding to antigen ex vivo. Mice
(n ¼ 6) were immunized three times as described in the Methods section. Splenocytes
were harvested 10 days after the third immunization and restimulated ex vivo with
antigen. Splenocyte proliferation was measured using CCK-8 kit, and the proliferation
index was calculated. Data are expressed as the mean SEM (n ¼ 6). *p < 0.05.
day 38, IgG response induced by antigen entrapped within particles
was more potent than that by soluble antigen alone.
IgG2a antibody production reveals a Th1-polarized immune
response, and the ratio of IgG2a/IgG1 is indicative of Th1-biased
immune response [35]. Both Encapsulation and Combination vaccine formulations induced stronger IgG1 and IgG2a responses than
soluble antigen (p < 0.05, Fig. 2B). However, soluble antigen mixed
with blank particles induced stronger IgG2a response than soluble
antigen, but comparable IgG1 response (Fig. 2B). All PLGA nanoparticles-adjuvanted vaccine formulations generated similar IgG2a/
IgG1 ratios, which were significantly larger than that for antigen
alone (Fig. 2C).
To evaluate the quality of the antibody response, the avidity
between antigen and IgG antibody in the serum was determined by
ELISA combined with a urea-elution step. As shown in Fig. 2D, the
avidity of the IgG antibody induced by these PLGA nanoparticlesbased vaccines was higher than that for antigen alone. Immunization with the Mixture formulation elicited IgG responses with
significantly higher avidity than the encapsulation formulation. The
combined vaccine formulation elicited IgG antibody with significantly higher avidity than that for antigen alone.
Taken together, all nanoparticles-adjuvanted vaccine formulations (Encapsulation, Mixture, and Combination) induced more
potent antibody responses than the soluble antigen. Furthermore,
compared to Encapsulation and Mixture formulations, mice
immunized with the combined vaccine formulation developed
more potent antigen-specific IgG antibody responses, with higher
titers and relatively higher avidity.
3.3. Cytokine levels secreted by ex vivo restimulated splenocytes
Since we observed that PLGA nanoparticles-adjuvanted vaccine
formulations induced a Th1-biased antibody response in mice
compared to soluble antigen alone, we next asked whether these
formulations could also alter the cytokine profiles in the mice toward a Th1 bias. Splenocytes harvested from vaccinated mice were
restimulated ex vivo with OVA, and Th1 (IFN-g, IL-12) and Th2 (IL-4,
IL-10) cytokines in the supernatant were determined by ELISA. As
shown in Fig. 3AeD, similar IFN-g, IL-12, IL-4, and IL-10 cytokine
An ex vivo splenocyte proliferation assay was performed to
assess the influence of various antigenenanoparticle formulations
on splenocyte proliferative responses. As shown in Fig. 4, under the
stimulation of OVA, splenocytes collected from mice immunized
with the combined vaccine formulation proliferated more efficiently than those collected from mice immunized with soluble
antigen mixed with nanoparticles or soluble antigen alone
(p < 0.05). Hence, we believed that the combined vaccine formulation developed more potent antigen-specific immune responses
than other formulations.
3.5. Memory T cell responses
The ultimate goal of vaccination is to generate immune memory
that can rapidly respond to pathogens upon reinfection, and
memory T cells are important components of these memory immune responses. Since CD44hiCD62Llow and CD44hiCD62Lhi were
regarded as markers for effector-memory and central-memory T
cells, respectively [29], we evaluated their frequency by flow
cytometry. The frequency of CD44hiCD62Lhi central-memory CD4þ
(Fig. 5A) and CD8þ (Fig. 5C) T cells was similar between splenocytes
harvested from mice immunized with the Encapsulation formulation and the Combination formulation, both of which were significantly higher than that for soluble antigen mixed with blank
particles or soluble antigen alone. With respect to CD44hiCD62Llow
effector-memory T cells, the frequency of memory CD4þ (Fig. 5B)
and CD8þ (Fig. 5D) T cells was significantly higher for mice
immunized with the combined vaccine formulation, as compared
to mice immunized with soluble antigen mixed with blank particles or soluble antigen alone (p < 0.05). Representative FACS plots
of the mean percentages are shown in Fig. 5E. In summary, the
combined vaccine formulation induced the strongest memory T cell
responses among the formulations tested, indicating that this
formulation might provide better protection against reinfection.
3.6. Antigen persistence at injection sites and antigen transport into
draining lymph nodes
To uncover the mechanisms underlying the enhanced immune
responses elicited by the combined vaccine formulation, we first
determined antigen persistence at the injection sites as well as the
amount of available antigen in draining lymph nodes by in vivo
imaging and immunohistochemistry, respectively. To track and
visualize the various formulations by in vivo imaging, we injected
formulations containing fluorescently labeled antigen. As shown in
Fig. 6A and B, none of the initial fluorescence remained at the injection sites 6 h after injection in animals immunized with the
formulations containing soluble antigen (soluble antigen alone or
soluble antigen mixed with blank particles), and there was no
significant difference between their decreasing rates of
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Fig. 5. Frequency of central (CD44hiCD62Lhi)/effector (CD44hiCD62Llow) memory CD4þ and CD8þ T cells. Mice (n ¼ 6) were immunized three times as described in the Methods
section. Splenocytes were harvested 10 days after the third immunization and restimulated ex vivo with antigen for 60 h. The frequency of CD44hiCD62Lhi CD4þ T cells (A),
CD44hiCD62Llow CD4þ T cells (B), CD44hiCD62Lhi CD8þ T cells (C), and CD44hiCD62Llow CD8þ T cells (D) were measured by flow cytometry. FACS plots in (E) are representative of the
mean percentages of 6 mice in each group. Data in (A), (B), (C), and (D) are expressed as the mean SEM (n ¼ 6). *p < 0.05; **p < 0.01.
fluorescence intensity. In contrast, antigen encapsulated within
nanoparticles and antigen in the combined formulation persisted at
the injection sites for as long as 168 h (7 days) after injection.
Moreover, compared to the combined vaccine formulation, more
antigen was detained at sites injected with the Encapsulation
vaccine formulation.
Considering that significantly different antigen persistence
profiles at injection sites existed between various formulations, we
wondered whether these differences affected antigen transport
into draining lymph nodes. We therefore determined the available
antigen contained in the draining lymph nodes over time by
immunohistochemistry. At the early stage (2 days postimmunization), the most abundant antigen was detected in
lymph nodes of mice immunized with the Mixture formulation
(soluble antigen mixed with blank nanoparticles), followed by mice
immunized with the combined vaccine formulation (Fig. 6C, upper
panel). At the later stage (7 days post-immunization), immunization with either the Encapsulation formulation or the Combination
W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
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Fig. 6. Antigen depot at injection sites and antigen transport into draining lymph nodes. (AeB) Antigen persistence at the sites of injection. Balb/c mice (n ¼ 6) were intramuscularly
injected in the hind legs with different vaccine formulations containing antigen labeled with the near-infrared dye Cy7. Antigen persistence at injection sites was evaluated and
documented by an in vivo imaging system at the indicated time points. Carestream Molecular Imaging Software was employed to quantify the sum fluorescence intensity at the
injection sites. (A) Representative fluorescence images and (B) quantitative fluorescence intensity of antigen persisting at injection sites. (C) Antigen level in draining lymph nodes
determined by immunohistochemical assay. Balb/c mice were intramuscularly injected with 50 mL of different vaccine formulations containing 12.5 mg of antigen. At 2 days (upper
panels) or 7 days (lower panels) after injection, draining lymph nodes were isolated from euthanized mice, embedded in paraffin, and cut into sections. Sections were stained with
antibody against antigen (OVA) and hematoxylin for coloration of antigen and the cell nucleus, respectively. Data in (A) and (C) are representative of the 4 mice in each group. Yellow
areas indicated by arrows in (C) represent antigen. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
formulation resulted in more abundant antigen in the draining
lymph nodes than immunization with soluble antigen alone or the
Mixture formulation (Fig. 6C, lower panel). Taking these results into
consideration, our data suggested that the combined vaccine
formulation effectively provided not only adequate initial antigen
exposure but also long-term antigen persistence.
3.7. Expression of MHC and the co-stimulatory molecule CD86 on
DCs in draining lymph nodes
Since we observed that the various nanoparticleeantigen formulations differentially affected antigen transport to the draining
lymph nodes over time, we next wondered how they each affected
vaccine potency in terms of activating DCs in the draining lymph
nodes. To address this question, MHC molecules (MHC I and MHC II)
and co-stimulatory molecule (CD86) expression on DCs in draining
lymph nodes were measured by flow cytometry. As shown in
Fig. 7AeF and Fig. S57, compared to soluble antigen alone, all
nanoparticles-adjuvanted vaccine formulations (Encapsulation,
Mixture, and Combination) induced significantly higher MHC I,
MHC II, and CD86 expression 24 h after vaccination; no significant
differences were observed among any of the three nanoparticleadjuvanted vaccine formulations. After 48 h, the following observations were made: the nanoparticle-adjuvanted vaccine formulations (Encapsulation, Mixture, and Combination) induced
comparable MHC II expression, all significantly higher than soluble
antigen alone (Fig. 7D and Fig. S6). The Mixture vaccine formulation
and the Combination formulation elicited significantly higher
expression levels of MHC I than the Encapsulation vaccine formulation and soluble antigen alone (Mixture vs Ag, Combination vs Ag:
p < 0.001; Mixture vs Encapsulation, Combination vs Encapsulation: p < 0.05; Fig. 7B and Fig. S5). With respect to CD86 expression,
the Mixture vaccine formulation and the Combination formulation
elicited significantly higher levels (Mixture vs Ag: p < 0.01; Combination vs Ag: p < 0.05; Fig. 7F and Fig. S7). At 7 days after immunization, MHC I, MHC II, and CD86 expression was significantly
higher on DCs from mice immunized with the combined vaccine
formulation than all of the other vaccine formulations (Fig. 7AeF
and Fig. S5e7). Taken together, the combined vaccine formulation
could induce effective expression of MHC and co-stimulatory
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W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
Fig. 7. Expression of MHC I, MHC II, and co-stimulatory molecule CD86 on DCs in draining lymph nodes of immunized mice. Balb/c mice (n ¼ 3) were intramuscularly vaccinated
with different vaccine formulations. At the indicated time points (1, 2, or 7 days post-immunization), mice were euthanized, and popliteal lymph nodes were isolated. The percentage and MFI of MHC I (A, B), MHC II (C, D), and CD86 (E, F) expression on CD11cþ DCs was determined by flow cytometry. Data are expressed as the mean SEM (n ¼ 3).
*p < 0.05; **p < 0.01; ***p < 0.001.
molecules on DCs in the draining lymph node, both at the initial
stage and in the later stages after immunization.
3.8. Frequency of follicular helper CD4þ T cells in draining lymph
nodes
Follicular helper CD4þ T cells play an important role in helping B
cells produce high-affinity antibody responses [30]. It has been
reported that follicular helper T cell differentiation requires
continuous antigen presentation [31]. In order to elucidate whether
the combined vaccine formulation might have favored the generation of antibody responses by improving follicular helper T cell
activation, we determined the frequency of follicular helper CD4þ T
cells in the draining lymph nodes of immunized mice. Indeed, the
frequency of CD4þCXCR5hiPD-1hi follicular helper T cells was
significantly higher in the draining lymph nodes of mice immunized with the combined vaccine formulation, as compared to
those for other vaccine formulations (p < 0.05, Fig. 8A and B).
4. Discussion
Vaccination plays an important role in protecting individuals
against pathogens, as well as in preventing and controlling infectious diseases. With the development of modern vaccines, the
development of more efficacious adjuvants is imperative for the
successful induction of sufficient immune responses. Among
various potential adjuvants, polymeric micro/nanoparticles represent a kind of promising vaccine adjuvant, as they can simultaneously augment both humoral and cellular immune responses.
Many physicochemical characteristics significantly affect the
adjuvant effect of micro-/nano-particles, and one critical factor is
the kinetics of antigen exposure to the immune system. In this
study, we investigated how various antigenenanoparticle formulations impacted antigen exposure to the immune system and
evaluated the resultant antigen-specific immune responses. Our
findings indicated that the combined vaccine formulation
composed of antigen encapsulated in nanoparticles plus soluble
antigen mixed with blank nanoparticles induced the most powerful
antigen-specific immune responses among any of the other formulations tested. Indeed, mice immunized with the combined
vaccine formulation displayed enhanced induction of antigenspecific IgG antibodies with high avidity, increased cytokine
secretion by splenocytes, and increased generation of memory T
cells.
Overall, the results presented here suggest that combining antigen encapsulated within nanoparticles with soluble antigen
mixed with blank nanoparticles improved the efficacy of
nanoparticle-based vaccine. By monitoring antigen persistence at
the injection site and detecting antigen levels in draining lymph
nodes over time, we found that encapsulating antigen into
W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
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Fig. 8. The frequency of follicular helper CD4þ T cells in the draining lymph nodes of immunized mice. Balb/c mice (n ¼ 3) were intramuscularly vaccinated with different vaccine
formulations. Mice were euthanized 9 days later, and popliteal lymph nodes were isolated. The frequency of follicular helper CD4þ T cells (CD4þCXCR5hiPD-1hi) was determined by
flow cytometry. (A) Representative flow cytometry plots and (B) percentage of follicular helper CD4þ T cells (CD4þCXCR5hiPD-1hi) in CD4þ T cells. Data are expressed as the
mean SEM (n ¼ 3). *p < 0.05.
nanoparticles significantly prolonged antigen retention at the injection site (Fig. 6A and B) and achieved sustained antigen transport
into the draining lymph node (Fig. 6C). Prolonged antigen presentation is propitious to inducing powerful immune responses
[32,33]. Indeed, antigen persistence is required throughout the
expansion phase of CD4þ T cell responses [34], and is required for
DC licensing and CD8þ T cell cross-priming [35,36]. Demento et al.
demonstrated that sustained antigen availability mediated by
particulate platforms facilitated a long-term memory T cell
response [22]. Moreover, although the duration of antigen availability plays a critical role, initial antigen presentation is also pivotal
to successful induction of antigen-specific immune responses.
Indeed, Blair et al. showed that the initial engagement of TCR
through interaction with cognate peptideeMHC was required for T
cell activation and antigen specificity [33]. Here, the combined
vaccine formulation effectively provided not only long-term antigen persistence but also adequate initial antigen exposure (Fig. 6C).
Following antigen exposure, maturation and activation of DCs is a
prerequisite of effective antigen presentation and subsequent T cell
activation. And follicular helper T cells play an important role in
helping B cells activate and produce antibodies with high avidity.
Here, the combined vaccine formulation induced higher DC activation (Fig. 7) and a higher frequency of follicular helper T cells
(Fig. 8) in the draining lymph nodes. Thus, the aforementioned
mechanisms could help to explain why and how the combined
vaccine formulation elicited more powerful antigen-specific immune responses than other nanoparticle-adjuvanted vaccine formulations with single component (Encapsulation and Mixture),
although further investigations are required to understand the
precise underlying mechanisms.
Based on the results in the present study, we therefore proposed
the following model to explain the underlying mode of action by
which the combined vaccine formulation acted as a vaccine adjuvant to promote antigen-specific immune responses toward a given
antigen (Fig. 9). First, the soluble antigen component in the combined vaccine formulation provided adequate initial antigen to
prime the immune system (Fig. 6C, upper panel). At the same time,
the nanoparticle-encapsulated antigen component in the combined formulation created an antigen depot at the injection site
(Fig. 6A and B), providing a supply of persistent antigen to the
immune system (Fig. 6C, lower panel). Effective provision of both
adequate initial antigen exposure and long-term antigen persistence likely resulted in higher expression of both MHC molecules
(MHC I and MHC II) and the co-stimulatory molecule CD86 on DCs
(Fig. 7) as well as a higher frequency of follicular helper T cells in the
draining lymph nodes (Fig. 8). Then, more potent antigen-specific
immune responses were elicited (Figs. 2e5). Therefore, by optimizing the antigenenanoparticle formulation, enhanced antigenspecific immune responses could be elicited to achieve better
prevention of infectious diseases.
5. Conclusions
This study investigated the effect of various antigenenanoparticle formulations on the immune responses elicited by
nanoparticle-adjuvanted vaccines. Our findings demonstrated that
the combined vaccine formulation (antigen encapsulated within
nanoparticles, and soluble antigen mixed with blank nanoparticles)
elicited more potent antigen-specific immune responses than
single-component nanoparticle-adjuvanted formulations (antigen
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W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
Fig. 9. Schematic illustration of the proposed mode of action of the combined vaccine formulation composed of PLGA nanoparticles-encapsulated antigen and soluble antigen
mixed with blank nanoparticles.
encapsulated within nanoparticles, or soluble antigen mixed with
blank nanoparticles). The enhanced immune responses elicited by
the combined vaccine formulation might be attributed to the
antigen-depot effect at the injection site, effective provision of both
adequate initial antigen exposure and long-term antigen persistence, and efficient induction of DC activation and follicular helper
T cell differentiation in draining lymph nodes. Understanding the
effect of the antigenenanoparticle formulations on the resultant
immune responses might have significant implications for rational
vaccine design.
Acknowledgments
This work was financially supported by the 973 Program (Grant
No. 2013CB531500), Special Fund for Agro-scientific Research in the
Public Interest (Grant No. 201303046), and the 863 Program (Grant
No. 2012AA02A406).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2014.04.022.
References
[1] Ulmer JB, Valley U, Rappuoli R. Vaccine manufacturing: challenges and solutions. Nat Biotech 2006;24:1377e83.
[2] Pulendran B, Ahmed R. Immunological mechanisms of vaccination. Nat
Immunol 2011;131:509e17.
[3] Broaders KE, Cohen JA, Beaudette TT, Bachelder EM, Fréchet JMJ. Acetalated
dextran is a chemically and biologically tunable material for particulate
immunotherapy. Proc Natl Acad Sci USA 2009;106:5497e502.
[4] Baumgartner CK, Malherbe LP. Regulation of CD4 T-cell receptor diversity by
vaccine adjuvants. Immunology 2010;130:16e22.
[5] Sokolova V, Knuschke T, Kovtun A, Buer J, Epple M, Westendorf AM. The use of
calcium phosphate nanoparticles encapsulating toll-like receptor ligands and
the antigen hemagglutinin to induce dendritic cell maturation and T cell
activation. Biomaterials 2010;31:5627e33.
[6] Marrack P, McKee AS, Munks MW. Towards an understanding of the adjuvant
action of aluminium. Nat Rev Immunol 2009;9:287e93.
[7] Zaharoff DA, Rogers CJ, Hance KW, Schlom J, Greiner JW. Chitosan solution
enhances both humoral and cell-mediated immune responses to subcutaneous vaccination. Vaccine 2007;25:2085e94.
[8] Chen X, Kim P, Farinelli B, Doukas A, Yun S-H, Gelfand JA, et al. A novel laser
vaccine adjuvant increases the motility of antigen presenting cells. PLoS One
2010;5:e13776.
[9] Singh M, O’Hagan DT. Recent advances in vaccine adjuvants. Pharm Res
2002;19:715e28.
[10] De Temmerman ML, Rejman J, Demeester J, Irvine DJ, Gander B, De Smedt SC.
Particulate vaccines: on the quest for optimal delivery and immune response.
Drug Discov Today 2011;16:569e82.
[11] Oyewumi MO, Kumar A, Cui ZR. Nano-microparticles as immune adjuvants:
correlating particle sizes and the resultant immune responses. Expert Rev
Vaccines 2010;9:1095e107.
[12] Kreuter J, Speiser PP. New adjuvants on a polymethylmethacrylate base. Infect
Immun 1976;13:204e10.
[13] Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol 2010;10:787e96.
[14] Foged C, Brodin B, Frokjaer S, Sundblad A. Particle size and surface charge
affect particle uptake by human dendritic cells in an in vitro model. Int J
Pharm 2005;298:315e22.
[15] Kohli AK, Alpar HO. Potential use of nanoparticles for transcutaneous vaccine
delivery: effect of particle size and charge. Int J Pharm 2004;275:13e7.
W. Zhang et al. / Biomaterials 35 (2014) 6086e6097
[16] Gomez JMM, Csaba N, Fischer S, Sichelstiel A, Kundig TM, Gander B, et al.
Surface coating of PLGA microparticles with protamine enhances their
immunological performance through facilitated phagocytosis. J Control
Release 2008;130:161e7.
[17] Seong SY, Matzinger P. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol
2004;4:469e78.
[18] Moyano DF, Goldsmith M, Solfiell DJ, Landesman-Milo D, Miranda OR, Peer D,
et al. Nanoparticle hydrophobicity dictates immune response. J Am Chem Soc
2012;134:3965e7.
[19] Mohanan D, Slutter B, Henriksen-Lacey M, Jiskoot W, Bouwstra JA, Perrie Y,
et al. Administration routes affect the quality of immune responses: a crosssectional evaluation of particulate antigen-delivery systems. J Control
Release 2010;147:342e9.
[20] Lesterhuis WJ, de Vries IJM, Schreibelt G, Lambeck AJA, Aarntzen EHJG,
Jacobs JFM, et al. Route of administration modulates the induction of dendritic
cell vaccine-induced antigen-specific t cells in advanced melanoma patients.
Clin Cancer Res 2011;17:5725e35.
[21] Kanchan V, Katare YK, Panda AK. Memory antibody response from antigen
loaded polymer particles and the effect of antigen release kinetics. Biomaterials 2009;30:4763e76.
[22] Demento SL, Cui WG, Criscione JM, Stern E, Tulipan J, Kaech SM, et al. Role of
sustained antigen release from nanoparticle vaccines in shaping the T cell
memory phenotype. Biomaterials 2012;33:4957e64.
[23] Johansen P, Storni T, Rettig L, Qiu ZY, Der-Sarkissian A, Smith KA, et al. Antigen
kinetics determines immune reactivity. Proc Natl Acad Sci USA 2008;105:
5189e94.
[24] Rizwan SB, McBurney WT, Young K, Hanley T, Boyd BJ, Rades T, et al. Cubosomes containing the adjuvants imiquimod and monophosphoryl lipid A
stimulate robust cellular and humoral immune responses. J Control Release
2013;165:16e21.
[25] Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu CZ, et al. Nanoparticle
vaccines. Vaccine 2014;32:327e37.
6097
[26] Zhang W, Wang L, Liu Y, Chen X, Li J, Yang T, et al. Comparison of PLA microparticles and alum as adjuvants for H5N1 influenza split vaccine: adjuvanticity evaluation and preliminary action mode analysis. Pharm Res
2014;31:1015e31.
[27] Yuan L, Wu LH, Chen JA, Wu QA, Hu SH. Paclitaxel acts as an adjuvant to
promote both Th1 and Th2 immune responses induced by ovalbumin in mice.
Vaccine 2010;28:4402e10.
[28] Moon JJ, Suh H, Li AV, Ockenhouse CF, Yadava A, Irvine DJ. Enhancing humoral
responses to a malaria antigen with nanoparticle vaccines that expand Tfh
cells and promote germinal center induction. Proc Natl Acad Sci USA
2012;109:1080e5.
[29] Kaech SM, Wherry EJ, Ahmed R. Effector and memory T-cell differentiation:
implications for vaccine development. Nat Rev Immunol 2002;2:251e62.
[30] Yu D, Vinuesa CG. The elusive identity of T follicular helper cells. Trends
Immunol 2010;31:377e83.
[31] Deenick EK, Chan AN, Ma CS, Gatto D, Schwartzberg PL, Brink R, et al. Follicular
helper T cell differentiation requires continuous antigen presentation that is
independent of unique B cell signaling. Immunity 2010;33:241e53.
[32] Obst R, van Santen HM, Melamed R, Kamphorst A, Benoist C, Mathis D. Sustained antigen presentation can promote an immunogenic T cell response,
like dendritic cell activation. Proc Natl Acad Sci USA 2007;104:15460e5.
[33] Blair DA, Turner DL, Bose TO, Pham QM, Bouchard KR, Williams KJ, et al.
Duration of antigen availability influences the expansion and memory differentiation of T cells. J Immunol 2011;187:2310e21.
[34] Obst R, van Santen HM, Mathis D, Benoist I. Antigen persistence is required
throughout the expansion phase of a CD4þ T cell response. J Exp Med
2005;201:1555e65.
[35] Jusforgues-Saklani H, Uhl M, Blachere N, Lemaitre F, Lantz O, Bousso P, et al.
Antigen persistence is required for dendritic cell licensing and CD8þ T cell
cross-priming. J Immunol 2008;181:3067e76.
[36] Cockburn IA, Chen YC, Overstreet MG, Lees JR, van Rooijen N, Farber DL, et al.
Prolonged antigen presentation is required for optimal CD8þ T cell responses
against malaria liver stage parasites. PLoS Pathog 2010;6:e1000877.