A Polymer/Oil Based Nanovaccine as a Single-Dose
Sara Vicente1, Belen Diaz-Freitas2, Mercedes Peleteiro2, Alejandro Sanchez3,4, David W. Pascual5,6,
Africa Gonzalez-Fernandez2, Maria J. Alonso1*
1Pharmacy and Pharmaceutical Technology Department, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela,
Santiago de Compostela, Spain, 2Immunology, Institute of Biomedical Research (IBIV), Biomedical Research Center (CINBIO), University of Vigo, Vigo, Spain, 3Pharmacy
and Pharmaceutical Technology Department, School of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain, 4Health Research Institute of
Santiago de Compostela (IDIS), Santiago de Compostela, Spain, 5Department of Immunology and Infectious Diseases, Montana State University, Bozeman, Montana,
United States of America, 6Department of Infectious Diseases & Pathology, University of Florida, Gainesville, Florida, United States of America
The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing
immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily
nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can
associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this
first characterization of the system, we intended to study the influence of different antigen organizations on the
nanocarrier’s surface (using the recombinant hepatitis B surface antigen –rHBsAg– as a model antigen) on their long-term
immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC
(CSNC+ and CSNC2), exhibiting similar particle size (200 nm) and high antigen association efficiency (.80%), were
developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively).
The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-
rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+
led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by
CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its
physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting
features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAg.
Citation: Vicente S, Diaz-Freitas B, Peleteiro M, Sanchez A, Pascual DW, et al. (2013) A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization
Approach. PLoS ONE 8(4): e62500. doi:10.1371/journal.pone.0062500
Editor: Prosper N. Boyaka, The Ohio State University, United States of America
Received December 11, 2012; Accepted March 21, 2013; Published April 22, 2013
Copyright: ? 2013 Vicente et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the Bill and Melinda Gates Foundation (www.gatesfoundation.org), Consolider Ingenio 2010 CSD2006-00012
(Ministry of Science and Innovation, Spain) and Competitive Reference Groups SUDOE-FEDER (SOE1/P1/E014). SV and MP acknowledge a fellowship from the
Spanish Ministry of Education (FPU predoctoral grants). DWP was in part supported by Montana Agricultural Experiment Station and US Department of
Agriculture Formula Funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Aluminum salts (alum), mainly in the form of aluminum
hydroxide (Al(OH)3) or aluminum phosphate (AlPO4), have been
traditionally used worldwide (since 1930s) as adjuvants in many
vaccines . Unfortunately, despite their wide use, these adjuvants
exhibit important drawbacks, such as inflammatory reactions at
the injection site, irreversible loss of potency upon freezing and
induction of strong biased Th2-type immune responses .
Consequently, many efforts have been oriented to the search of
new adjuvants and delivery carriers, which could help induce,
strengthen, and simultaneously prolong the immune response.
Ideally, the delivery carrier could also contribute to the
development of single-dose vaccines capable of generating
protective immunity upon only one antigenic exposure. This is
especially relevant for subunit antigens, characterized by their
improved safety profile, but low immunogenicity, which usually
require multiple doses to assure immunological protection .
A promising strategy towards this goal relies in the design of
polymeric nanocarriers, which are known to protect the associated
antigen from degradation, facilitate antigen uptake by antigen-
presenting cells (APCs), and control antigen release . Within this
frame, the increasing understanding of the influence of the
nanocarrier characteristics (composition, size, charge) on their
effectiveness is gradually paving the way to the rational design of
nanovaccines . For instance, both size and surface properties of
uptake and activation of APCs  and, ultimately, on the type of
immune response generated (preferentially cellular vs. humoral)
Chitosan (CS) is one of the most commonly used biomaterials in
vaccine delivery. So far, its use has been found particularly
promising for mucosal immunization because of its mucoadhesive
and penetration enhancing properties . In fact, chitosan itself,
as a dry powder mixed with monophosphoryl lipid A (MPLA) and
the antigen, is currently ongoing phase I of clinical evaluation for
intranasal vaccination against norovirus [10,11]. On the other
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hand, our group, among others, has developed CS nanoparticles
specifically adapted for the protection and delivery of antigens
. In particular, CS nanoparticles have demonstrated great
potential for nasal vaccination [13–17]. Recently, their application
as vaccine adjuvants has also been investigated against a variety of
protein and DNA-encoded antigens [14,18–21], suggesting
additional immunological properties of these CS-based nanocar-
Taking this background information into account, the aim of
our work has been to design a new CS-based nanocarrier that
might offer advanced properties in terms of antigen localization
and the possibility to incorporate additional immunomodulating
agents. This new nanocarrier, named CS nanocapsules (CSNC),
has a core-corona architecture, which enables the surface
presentation of different types of antigens while co-delivering
immunoactive molecules included in the oily core. However, in the
present work, we intended to study the effect of the organization of
the antigen molecules on the nanocarrier’s surface, without
including any additional immunostimulant, on their ability to
promote antigen specific immune responses. For this purpose, we
have selected the recombinant hepatitis B surface antigen
(rHBsAg, since now HB in the text) as a model antigen, which
could benefit from this technology. Therefore, in our aim to
explore the potential of CSNC as adjuvant and single-dose vaccine
formulation, we have developed and evaluated different HB-
surface assembled CSNC exhibiting different surface characteris-
tics (both surface charge and composition) Overall, we have
designed a formulation able to elicit long-lasting and protective
immune responses against subunit antigens, such as the HB, in
order to offer an alternative to alum as adjuvant agent, as well as
possibly reduce the number of doses to elicit long-lasting immune
Materials and Methods
Immunization studies involving fresh formulations of CSNC
prototypes were conducted in the University of Vigo (Spain), and
all protocols were adapted to the guidelines of the Spanish
regulations (Royal Decree 1201/2005) regarding the use of
animals in scientific research and under approval of the ethical
committee of the University of Vigo. In the case of freeze-dried
CSNC+ (trehalose 5%), the dried formulation was properly
arranged and shipped to Montana State University (Bozeman,
MT) where the biological evaluation was performed. All animal
care and procedures were in accordance with institutional policies
for animal health and well-being and approved by Montana State
University Institutional Animal Care and Use Committee
(IACUC) under protocol 58.
Female BALB/c mice (4–5 weeks old) were housed in filter-top
cages in a 12 h light/12 h dark cycle with constant temperature
environment of 22uC and provided with food and water ad libitum.
Ultrapure chitosan (CS) hydrochloride salt (Protasan UP CL
113, MW 125 kDa, acetylation degree of 14%) was purchased
from Novamatrix (Sandvika, Norway). MiglyolH 812 (M812) is a
neutral oil composed of triglycerides of medium chain fatty acids
(6–8 C) and was donated by Sasol Germany GmbH (Witten,
Germany). The emulsifier soybean L-a-lecithin Epikuron 145V
was a gift from Cargill (Barcelona, Spain). The recombinant
hepatitis B surface antigen (rHBsAg or HB) (MW 24 kDa) was
kindly donated by Shantha Biotechnics Ltd. (Hyderabad, India) as
an aqueous suspension in PBS containing a protein concentration
of 0.16 mg/ml.
Preparation of HB-surface-assembled chitosan
nanocapsules and physicochemical characterization.
Blank chitosan nanocapsules (CSNC) were prepared by the
solvent displacement technique, as previously reported (,
Protocol S1). The formation of HB-surface-assembled CSNC
was achieved by the strong electrostatic interaction between the
cationic polysaccharidic surface of CSNC and the negatively
charged particle antigen (220 mV in water). For this purpose, HB
stock solution was desalted and concentrated to 0.5 mg/mL by
ultrafiltration (Amicon Ultra4, Millipore; Cork, Ireland). The
resulting HB aqueous solution was immediately mixed with blank
CSNC (CS concentration 1 mg/mL) and incubated for 1 hour at
room temperature. Several nanosystems were prepared following
two different strategies (Figure S1): 1) increasing the antigen
amount (25, 50, 100, 250 mg) in the formulation or 2) maintaining
constant the antigen amount (25 mg), but changing the CSNC
concentration. Following these two methods, it was possible to
prepare a series of nanosystems defined by their CSNC:HB ratio,
ranging from 1:0.025 to 1:12.8.
The particle size and polydispersity index were measured by
photon correlation spectroscopy (PCS) and f potential by laser
doppler anemometry (LDA).
Quantification of HB association to CSNC
The amount of HB associated to CSNC surface was indirectly
quantified by measuring the concentration of free antigen
remaining in supernatant after ultracentrifugation (420006 g,
1 h, 15uC) of the nanostructures. An ELISA commercial kit
(Murex HBsAg Version 3, Murex Biotech Ltd; Dartford, UK), was
used to quantify HB concentration in the samples. The analysis
protocol was conducted as specified by the manufacturer. The
association efficiency for HB (A.E. %) was then calculated by the
difference between the concentration of free antigen detected in
the supernatant and the total concentration in the initial
Stability of cationic HB-surface-assembled CSNC
Storage at 46C of CSNC+ + aqueous suspension.
stability of the CSNC+ suspension was assessed during storage at
4uC. Samples were collected each week during one month.
Particle size and HB association to CSNC were analyzed
according to the techniques already described.
Freeze-drying of CSNC+ +.
lyophilization, was used to enhance stability of CSNC+ by
converting the aqueous suspension into a dry powder. For this
purpose, prototype CSNC+ was freeze-dried in the presence of
different sugar cryoprotectants (sucrose and trehalose) at concen-
trations 0, 1, 2.5, and 5%. CSNC+ suspension and cryoprotectant
solution were mixed 1:1 (v:v) in 5 ml freeze-drying glass vials. Vials
were slowly frozen at 220uC and then placed on the stainless-steel
shelf plates of the Labconco Freeze Dry System (Kansas City, MI),
operating at 235uC. The primary drying (sublimation) was carried
out at this temperature under high vacuum (1023mBar) for
approximately 40 hours. The second drying step (moisture
desorption) lasted 8 hours, during which the temperature gradu-
ally rose until +20uC under vacuum. The final freeze-dried
product was reconstituted with ultrapure water by gentle pipette
mixing and analyzed for its physicochemical properties.
The freeze-drying technique, or
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ide (HB-alum) was used as positive control. HB and aluminum
hydroxide (AlhydrogelTM; Sigma-Aldrich, St. Louis, MO) solu-
tions were incubated in a volumetric ratio 3:1 (HB:alum) for
30 minutes at 4uC under moderate agitation. Then, the suspen-
sion was centrifuged (100006g, 10 minutes, 4uC), and the pellet
was resuspended in adequate volume of isotonic saline solution.
Immunizations and sample collection.
capacity of CSNC was evaluated either in their fresh or freeze-
dried forms. Groups of 10 female BALB/c mice were immunized
with 10 mg of HB incorporated in the selected fresh CSNC:HB
prototypes (CSNC2 and CSNC+) or adsorbed to alum, at weeks 0
and 4 (two doses) or in a single dose. Formulations of CSNC and
HB-alum were injected intramuscularly on the posterior leg of
mice while animals were conscious. Subsequent sampling was
performed monthly, post-primary immunization, during a total
period of 27 weeks. Blood samples were collected from the
maxillary vein without anesthesia.
For the biological evaluation of freeze-dried formulation, mice
were randomly distributed in two groups of 10 animals and then
immunized intramuscularly with reconstituted freeze-dried for-
mulation or HB-alum as control following a boost-dose schedule (0
and 4 weeks). For reconstitution of freeze-dried formulations,
0.5 mL of ultrapure water was added, and gentle pipette mixing
was applied in order to properly disperse the nanocapsules. Blood
samples were collected at selected time points post-immunization
(days 28, 56, 84, 112) and specific serum anti-HB IgG titers were
analyzed by ELISA.
Serum anti-HB IgG endpoint titers were measured
by ELISA. Maxisorp microtiter wells were coated with 5 mg/mL
of HB in carbonate buffer (pH 9.6) overnight at 4uC. Plates were
then blocked with BSA 1% in PBS for 1 hour at 37uC in order to
minimize non-specific interactions. Serum samples and a mouse
IgG monoclonal antibody directed against HB (Biokit; Barcelona,
Spain) (used as control in the calibration curve) were serially
diluted and incubated for 2 hours at 37uC. All serum samples were
tested at least twice and in duplicate. Control rabbit antiserum of
known concentration (mIU/mL) (Acris Antibodies GmbH;
Hiddenhausen, Germany) was used in order to transform serum
titers into international units (IU). Goat anti-mouse and anti-rabbit
IgG conjugated with horseradish peroxidase (Southern Biotech;
Birmingham, AL) were added to each well and incubated for
1 hour at 37uC. Bound antibodies were revealed with ABTS and
the titers were expressed in mg/ml or in mIU/mL.
Antigen specific IgG subclasses (G1 and G2a) were also
quantified in mouse serum in order to know the IgG1/IgG2a
ratio. For this study, pooled sera from all mice from each group
were prepared and analyzed following a similar ELISA protocol
described for total anti-HB specific IgG. In this case, polyclonal
goat anti-mouse IgG1 and IgG2a antibodies, both conjugated with
horseradish peroxidase (Southern Biotech; Birmingham, AL), were
used as secondary antibodies. Then, a ratio between IgG1/IgG2a
was calculated in relation to the optical density levels.
The analysis of variance (ANOVA) was
performed using Statgraphics Plus 5.1. Tukey post-hoc analysis
was employed to establish significant differences between groups.
Differences were considered significant at a level of p,0.05.
HB adsorbed to aluminum hydrox-
Development and characterization of HB-surface-
assembled chitosan nanocapsules
For the purpose of loading HB onto CS nanocapsules (CSNC),
we first prepared blank nanocapsules, and then the antigen was
associated to their polymeric surface. Blank CSNC composed of
an oily core of MiglyolH812 (M812) and lecithin and a CS shell
were obtained by the solvent displacement technique. The
resulting CSNC exhibited a particle size in the nanometer range
(around 200 nm) with spherical shape and high positive j potential
provided by the CS coating (Table 1 and Figure 1).
Because of the cationic nature of the polymeric coating, it was
possible to associate negatively charged particulated viral proteins,
such as the HB (22 nm size and f potential of 220 mV), to the
surface of the CSNC. In order to identify the most adequate
association conditions, we prepared different prototypes displaying
different surface properties by associating the antigen at different
Incubating increasing amounts of HB (25, 50, 100, 250 mg) in a
1 mL suspension of blank CSNC (CS concentration of 1 mg/mL)
resulted in a series of nanosystems with CSNC:HB ratios between
1:0.025 and 1:0.25. The particle size of all formulations was
around 250 nm, regardless the amount of antigen initially
included, although slightly larger if compared to blank CSNC
(Table 1). Additionally, it was observed that the association
efficiency increased with the amount of HB added, reaching a
value of around 80% of HB for the ratio CSNC:HB 1:0.25.
Despite this high association rate, the j potential of the
nanocapsules remained highly positive (+40 mV), thus, indicating
the prevalence of the cationic polysaccharide on the surface of the
nanostructure. On the other hand, increments in the antigen
concentration beyond 0.25 mg/mL led to unstable nanosystems.
we chose an alternative incubation protocol. A suspension of blank
CSNC was sequentially diluted and then incubated with constant
antigen amount (25 mg). Using this method, we observed an
inversion of the surface charge of the nanosystem for the ratio
CSNC:HB, 1:3.2. Although, as shown in Figure 2, this inversion
was initially accompanied by a significant size increment (larger than
1mm), it was possible to preserve the nanometric size by reducing the
concentration of CSNC. Indeed, for the CSNC:HB ratio of 1:12.8,
we obtained nanostructures of a size around 310 nm, a negative
surface charge (220 mV) and high association efficiency (83%).
Therefore, two different prototypes were obtained with similar
particle size, but opposite surface characteristics in terms of
composition and f potential: CSNC:HB with ratios of 1:0.25
(CSNC+) and 1:12.8 (CSNC2).
Influence of surface charge in the adjuvant capacity of
HB-surface-assembled CSNC prototypes
In order to assess the adjuvant capacity of CSNC+ and
CSNC2, we administered the anionic and cationic prototypes by
intramuscular (i.m.) injection to female BALB/c mice. Briefly,
prime and boost doses of 10 mg of HB associated to CSNC, as well
as to aluminium hydroxide (conventional vaccine: HB-alum) at the
same dose, were given in a 4 week interval.
As observed in Figure 3A, CSNC2 was not able to induce a
significant response against HB as compared to the vaccine
containing alum, although anti-HB IgG levels remained over
seroprotective levels (.10 mUI/mL for humans ) during the
27-week study. In contrast, the cationic formulation (CSNC+) was
able to generate a more potent antibody response than that elicited
by the HB-alum at the same dose (p,0.05), thus, proving the
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immune potentiating effect of CSNC+. These results represent the
proof-of-concept of the adjuvant capacity of HB-surface assembled
Induction of immune protection after a single injection
of cationic HB-surface- assembled CSNC prototype
Taking into account the adjuvant effect observed for the
CSNC+, we decided to evaluate the immune response elicited by
this prototype after a single administration (10 mg) and compare it
to two doses of HB-alum (10 mg at 0 and 4 weeks). As noticed in
Figure 3B, although slightly lower, the antibody levels induced by
only one shot of CSNC+ were similar to those elicited upon
vaccination with two doses of 10 mg of HB-alum. In fact, no
significant differences were observed between the vaccination
regimens, except for two time-points (at 5 and 19 weeks post-
immunization) (p,0.05). Additionally, a single dose of CSNC+
induced protective anti-HB IgG levels (.10 mU/ml in humans),
which remained around 200 mIU/mL until the end of the study
(week 27). IgG levels higher than 100 mIU/mL are considered to
Figure 1. Morphology of CSNC. (A) Structure of HB-surface assembled CSNC showing the different components of the system. (B) TEM
micrographs of blank CSNC (left) and HB-surface assembled CSNC (right).
Figure 2. Physicochemical characterization of anionic HB-
surface assembled CSNC prototypes. Particle size, polydispersion
(PdI) and j potential evolution of CSNC:HB when CSNC suspension was
sequentially diluted. The ratio CSNC:HB which exhibited the most
adequate physicochemical properties (black column) was: particle size
310 nm 634; polydispersity index 0.3360.04; j potential 223 mV 63.
Mean 6 SD.
Figure 3. Evaluation of the efficacy of HB-surface assembled
CSNC prototypes. Immune response induced by the nanovaccine
prototypes (CSNC2 and CSNC+) and the positive control (HB-alum)
administered intramuscularly following different vaccination protocols.
(A) IgG anti-HB levels generated after prime-boost immunization
(arrows, weeks 0–4) with both prototypes: CSNC2 (blue –m–); CSNC+
(green –N–) and HB-alum (black –?X?–) at the same dose (10 mg).
Results are presented as mean 6 SEM.*Alum or CSNC+ vs. CSNC2
(p,0.05). ** CSNC+ vs. Alum and CSNC- (p,0.05). (B) IgG anti-HB (mIU/
ml) levels elicited after a single dose (arrow) of CSNC+ (10 mg; green –
N–) compared to alum-HB administered twice (10 mg, weeks 0 and 4)
(black –?X?–). Results are presented as mean 6 SEM.*(p,0.05).
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provide proper immune protection against HB, suggesting that no
additional boosters are further required .
Effect of cationic HB-surface assembled CSNC on the
modulation of the elicited immune response
The effect of CSNC+ on the type of T helper (Th1 or Th2)
responses preferentially involved was evaluated by measuring the
serum anti-HB IgG subtypes (IgG1 and IgG2a) and calculating the
ratio between both. While higher levels of antigen specific IgG1 in
mice are associated to a predominant humoral-Th2 type response,
the presence of IgG2a is mostly related to a cellular-Th1 type.
IgG1/IgG2a ratios represented in Figure 4 indicate that HB-
alum vaccine induced a predominant humoral Th2-type response
(higher levels of IgG1 with IgG1/IgG2a ratios ranging from 2 to
5), as expected . Higher production of IgG1 than IgG2a was
also found after a single immunization with CSNC+. However, a
second administration of this nanovaccine lessened the IgG1/
IgG2a ratio, suggesting a cellular immune response (Th1 type) was
also induced. These results show the impact of the nanovaccine
booster upon the modulation of the immune response by CSNC+.
Stability of the aqueous suspension of cationic HB-
surface-assembled CSNC prototype
The stability of the prototype CSNC+ as aqueous suspension
was assessed during its storage at 4uC. The particle size,
polydispersity index and HB association were analyzed each week
during one month. The nanosystem maintained its original
particle size and homogeneous distribution during at least one
month of storage under refrigerated conditions (Figure S2).
Similarly, the antigen remained associated to CSNC during this
Freeze-drying of cationic HB-surface-assembled CSNC
The aqueous suspension of CSNC+ was transformed into a
dried powder using standard freeze-drying techniques. Two
different cryoprotectants, sucrose and trehalose, at increasing
concentrations were added to the CSNC+ suspension to achieve a
final concentration of 1, 2.5 or 5%. After freeze-drying, the
resulting dried cakes had an overall good appearance without signs
of collapse. Samples were rehydrated and redispersed without
appreciable macroscopic aggregates. Particle size of the reconsti-
tuted suspension was then analyzed. On those samples without
cryoprotectant or with low sugar concentration (1 and 2.5%), the
particle size was found to be much larger compared to their sizes
before freeze-drying (Figure 5). At 5% sucrose or trehalose, the
original particle size of the fresh suspension was properly
Trehalose is usually preferred for cryoprotection of biomacro-
molecules because of its lower hygroscopicity, capacity of
formation of more flexible hydrogen bonds and very low reactivity
. Because the HB was associated to the CSNC surface and
therefore more exposed to freezing stress, this disaccharide was
selected as the most adequate cryoprotectant to produce a freeze-
dried product from this nanosystem suspension when included in a
concentration of 5%.
Immunization with freeze-dried cationic HB-surface-
To assess the preservation of the adjuvant properties of CSNC
and immunogenicity of associated antigen after freeze-drying,
dried CSNC+ with trehalose 5% was properly reconstituted and
administered to BALB/c mice. Animals were intramuscularly
vaccinated at 0 and 4 weeks with either the reconstituted CSNC+
prototype or freshly prepared HB-alum (as positive control), both
containing 10 mg of HB. Dried CSNC+ was able to induce IgG
titers similar to those achieved by standard alum-based vaccine
(Figure S3). This result indicates that the freeze-dried product
Table 1. Physicochemical characterization of positively
charged CSNC:HB prototypes.
ratioHB (mg)Size (nm) PdI
(mV) A.E. (%)
1:0.05 50 254617
+ +41±7 78±3
Results are presented as mean 6 SD. CSNC: chitosan nanocapsules; HB:
recombinant hepatitis B surface antigen; PdI: polydispersity index; A.R.: association
efficiency; n.d.: not determined.
Figure 4. Modulation of the immune response by CSNC+ +. Ratio of IgG1/IgG2a anti-HB for prototype CSNC+ (10 mg; green –N–) after single (A)
or double immunization separated 4 weeks (B) (indicated in arrows) compared to HB-alum (10 mg) administered in two doses (0, 4 weeks) (black –
?X?–). Results are presented as mean 6 SD.
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preserved a marked adjuvant capacity after the freeze-drying
process and during transportation and storage without cold chain
Nanocapsules are colloidal structures showing a characteristic
core-corona architecture, consisting of an oily liquid core
surrounded by a polymeric shell. Until now, this type of structure
has been used for the encapsulation of drugs with the idea of
improving their solubility, as well as their transporting through
mucosal barriers . Herein, the technology of chitosan
nanocapsules (CSNC) was adapted to explore its potential for
antigen delivery. Particularly, we studied the possibility to entrap
and present the antigen molecules onto the surface of the
nanocarrier and the influence of the physicochemical properties
of the resulting nanovaccine on the generation of specific immune
responses. Given the multifunctional character of these nanocar-
riers, the results of this work are expected to lead the way for their
rational optimization based on the association of different
immunostimulants and antigens.
Two different HB-surface-assembled CSNC prototypes were
developed by modifying the proportions between CSNC and the
antigen. CSNC:HB with ratios of 1:0.25 (CSNC+) and 1:12.8
(CSNC2) were obtained with similar particle size, but opposite
surface characteristics in terms of composition and f potential
(Table 1 and Figure 2). The cationic surface charge of CSNC+
indicated the prevalence of CS on the surface of the nanostructure
despite the high antigen association rate. In contrast, the negative
charge of CSNC2 indicated the exposure of the antigen molecules
entrapped in the CS shell towards the external medium. This
surface antigen display approach has been proposed as a way to
mimic the natural structure of pathogens . In fact, the
exposure of repetitive copies of the antigenic epitope on the surface
of nanoparticles has been demonstrated to enhance the immune
response against weakly immunogenic antigens . In addition,
the surface charge of the nanocarriers has been shown to affect
their adjuvant properties, and in particular, the cationic surface
charge has been determined to enhance the immune responses
. Therefore, the influence of the antigen exposure and the
surface properties (charge and composition) of the nanocarriers on
the immune response was then investigated.
The immune response observed for CSNC+, CSNC2 and the
control HB-alum administered following a boost-dose immuniza-
tion schedule evidenced clear differences between both CSNC
prototypes. Namely, CSNC2, which contained a high proportion
of antigen molecules on the surface of the nanostructure, was not
efficient at improving the immune response elicited by HB-alum,
suggesting that the presentation of repetitive copies of HB
molecules onto a particulate carrier is not enough to trigger a
potent immune response and other characteristics of the nanos-
tructure are more crucial. In contrast, the prototype exhibiting an
excess of CS on their surface (CSNC+), was able to stimulate the
generation of a pronounced IgG response against HB, which was
even higher than for the HB-alum (Figure 3A). This positive
effect of the CS coating being exposed on the surface is in
agreement with the promising results recently reported by our
group for nanogelled CS encapsulating HB . The intense
humoral immune response achieved with both CS-based nano-
carriers provides additional evidence of the immunostimulatory
properties of nanostructured CS [14,18–21].
The adjuvant properties of CS itself have been previously
studied in vivo with other model antigens, such as b-galactosidase
. The adjuvant effect of CS was mainly attributed to its
capacity to form a depot at the injection site in vivo  and also to
attract immunocompetent cells to the area . On the other
hand, some studies have raised the issue of a possible direct
immunostimulation mechanism of CS through the Toll-like
receptor 4 (TLR4) [32,33]. Unfortunately the results published
are inconclusive due to the different nature and purity of the CS
used in these studies. Irrespective of the potential inherent
immunostimulatory behavior of CS, it is known that the
association of antigens to particulate carriers facilitates their
uptake by APCs and subsequent activation [4,5]. In particular,
nanostructures composed of different cationic biomaterials, such
as cationized gelatin , poly-L-lysine or protamine , have
been shown to positively promote recognition and uptake by
APCs. Likewise, CS-based nanoparticles increased antigen inter-
nalization due to their strong association to the outer membrane of
the dendritic cells . Therefore, the association of HB to
CSNC+, characterized by a cationic surface charge and predom-
inant presence of CS on their surface, could have importantly
contributed to its adequate presentation to the immune system and
Figure 5. Physicochemical characterization of reconstituted CSNC+ + freeze dried under different conditions. Particle size recovery after
freeze-drying and reconstitution of CSNC+ using different cryoprotectants. (A) Particle size after reconstitution of freeze-dried formulation using
sucrose (blue columns) and trehalose (yellow columns) as cryoprotectants at different concentrations. (B) Detailed physicochemical characterization
of dried formulations in the presence of sucrose or trehalose (both at 5%) compared to the original characteristics of the suspension (before). Size is
shown in yellow columns and polydispersity index (PdI) in black line. Results are presented as mean 6 SD.
Nanovaccine for Single-Dose Vaccination
PLOS ONE | www.plosone.org6 April 2013 | Volume 8 | Issue 4 | e62500
moreover, to an enhanced activation of APCs and further
development of a strong adaptive immune response.
One of the main goals in vaccination has been to reduce the
number of injections but achieving an efficient immune protection.
Indeed, a vaccine able to generate long-term immune responses
after a single immunization offers many advantages, such as
reducing the risk of patients’ under-protection due to a deficient
compliance to the immunization schedule, minimizing waste
disposal, and decreasing the costs related to vaccination . The
sustained effect observed for CSNC+ after a single administration
(Figure 3B) could be attributed to a prolonged antigen delivery to
peripheral APCs possibly due to an accumulation of the
nanostructures at the injection site (depot effect) and subsequent
drainage to the lymph nodes. Additionally, the strong interaction
between HB and the polysaccharidic shell of CSNC may have also
contributed to this effect, enhancing the retention of the associated
antigen and enabling its cellular uptake for long periods of time
. These results provide the first evidence of the ability of
CSNC to elicit high specific, long-lasting and protective IgG levels
against HB after a single-dose vaccination regimen.
To study the effect of CSNC+ prototype on modulating the
elicited immune response, IgG1 and IgG2a levels were analyzed in
sera collected from vaccinated mice. The ratio IgG1/IgG2a is
used as indicator of the predominant immune response and cells
involved (predominant Th1 or Th2 immune response). The type
of immune response elicited by CSNC+ was strongly dependent
on the immunization regimen. A single dose with CSNC+ resulted
in a predominant Th2-mediated response (humoral), whereas both
Th1- and Th2-type responses were induced after booster
immunization (Figure 4). This type of response was also reported
for CS aqueous solutions mixed with model antigen (b-galactosi-
dase)  and for ovalbumin-loaded CS nanoparticles adminis-
tered subcutaneously . Overall, these findings suggest the
potential of CS and CS nanocarriers to promote a mixed Th1/
Th2 immune response.
A critical feature of an antigen delivery carrier is its ability to
preserve the stability of the associated antigen during storage.
Initially in this study, we observed CSNC+ maintained its
physicochemical properties for one month during storage under
refrigerated conditions (Figure S2). In a second instance, we
explored the freeze-drying method as a way to improve long-term
stability of colloidal formulations and avoid cold chain restrictions.
In a dry environment, nanostructures and the associated bioactive
molecule can be protected from degradation, while their original
physicochemical properties can be recovered upon rehydration
The results evidenced the feasibility to transform the aqueous
suspension of CSNC+ into a dry powder that can be rehydrated,
while maintainingthe original
(Figure 5). Furthermore, upon reconstitution, the dried formu-
lation maintained the adjuvant properties following in vivo
administration (Figure S3). As the dry form may be a suitable
presentation for long-term storage, the preservation of the immune
behavior of the nanovaccine upon freeze-drying is critical .
This is indeed an advantage over common aluminum salts that
cannot be frozen and consequently freeze-dried. The particulate
structure of alum can be destroyed after freezing, leading to a loss
of potency of the vaccine , which is an important drawback
leading to the necessity of strictly maintaining the cold chain
during transportation and storage, a particularly difficult require-
ment for developing countries with deficient logistic infrastructures
The results of this work represent the first proof-of-principle of
the potential of CSNC as antigen delivery adjuvants, in particular
for a vaccine using the HB antigen as model antigen. Furthermore,
these results provide preliminary evidence of the value of this
technology as a single-dose immunization strategy against the
disease. With this first study, we were able to establish the optimal
organization of the antigen molecules on the nanocarrier’s surface
to enhance the specific immune response using the CSNC
platform. However, from the multifunctional structure of the
CSNC it could also be deduced that further improvements could
be achieved by incorporating additional immunostimulants in the
CSNC’s core. In addition, this new technology may also represent
a way to preserve the stability of the antigen in a freeze-dried form.
Finally, from the conceptual point of view, the results of this work
further assess the value of positively charged nanocarriers as a way
to facilitate antigen presentation to the immune cells.
protocols to obtain both HB-surface-assembled CSNC
prototypes. (A) CSNC+ (ratio CSNC:HB 1:0.25) and (B)
CSNC2 (ratio CSNC:HB 1:12.8).
Stability of CSNC+ +. Both particle size (blue
columns) and percentage of associated HB to CSNC (dark blue
line) are shown at different time points (0–4 weeks) during storage
at 4uC. Results are presented as mean 6 SD.
Efficacy of freeze-dried CSNC+ + upon storage
and reconstitution. Humoral immune response (IgG titers)
after two i.m. administrations of reconstituted freeze-dried
CSNC+ (green –N–) and compared to HB-alum (black –?X?–) at
the same dose (10 mg, weeks 0 and 4). Results are presented as
mean 6 SEM.
Illustration of the different preparation
for the preparation of blank CSNC.
Solvent displacement technique protocol
We would like to thank Shantha Biotechnics Limited (Hyderabad, India)
for providing us the antigen (rHBsAg) and the advice provided by Martin
Friede from the World Health Organization. The technical assistance on
animal experimentation of Rafael Romero, Andrea Herna ´ndez and
Christian Sa ´nchez Espinel is highly appreciated.
Revised the manuscript: BDF MP AS DWP AGF MJA. Conceived and
designed the experiments: SV BDF AS DWP AGF MJA. Performed the
experiments: SV BDF MP DWP. Analyzed the data: SV BDF MP DWP
AGF MJA. Wrote the paper: SV.
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