Carbohydrate-containing nanoparticles as vaccine adjuvants

Abstract

Introduction
: Adjuvants are essential to vaccines for immunopotentiation in the elicitation of protective immunity. However, classical and widely used aluminum-based adjuvants have limited capacity to induce cellular response. There are increasing needs for appropriate adjuvants with improved profiles for vaccine development towards emerging pathogens. Carbohydrate-containing nanoparticles (NPs) with immunomodulatory activity and particulate nanocarriers for effective antigen presentation are capable of eliciting a more balanced humoral and cellular immune response.

Areas covered
We reviewed several carbohydrates with immunomodulatory properties. They include chitosan, β-glucan, mannan, and saponins, which have been used in vaccine formulations. The mode of action, the preparation methods, characterization of these carbohydrate-containing NPs and the corresponding vaccines are presented.

Expert opinion
Several carbohydrate-containing NPs have entered the clinical stage or have been used in licensed vaccines for human use. Saponin-containing NPs are being evaluated in a vaccine against SARS-CoV-2, the pathogen causing the on-going worldwide pandemic. Vaccines with carbohydrate-containing NPs are in different stages of development, from preclinical studies to late-stage clinical trials. A better understanding of the mode of action for carbohydrate-containing NPs as vaccine carriers and as immunostimulators will likely contribute to the design and development of new generation vaccines against cancer and infectious diseases.

Full text

Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ierv20
Expert Review of Vaccines
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ierv20
Carbohydrate-containing nanoparticles as vaccine
adjuvants
Xinyuan Zhang, Zhigang Zhang, Ningshao Xia & Qinjian Zhao
To cite this article: Xinyuan Zhang, Zhigang Zhang, Ningshao Xia & Qinjian Zhao (2021):
Carbohydrate-containing nanoparticles as vaccine adjuvants, Expert Review of Vaccines, DOI:
10.1080/14760584.2021.1939688
To link to this article: https://doi.org/10.1080/14760584.2021.1939688
Published online: 15 Jun 2021.
Submit your article to this journal
Article views: 9
View related articles
View Crossmark data

REVIEW
Carbohydrate-containing nanoparticles as vaccine adjuvants
Xinyuan Zhang
a
, Zhigang Zhang
a
, Ningshao Xia
a,b,c
and Qinjian Zhao
a
a
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in
Infectious Diseases, School of Public Health, Xiamen University, Xiamen, Fujian, PR China;
b
School of Life Sciences, Xiamen University, Xiamen,
Fujian, PR China;
c
The Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen University,
Xiamen, Fujian, PR China
ABSTRACT
Introduction: Adjuvants are essential to vaccines for immunopotentiation in the elicitation of protec-
tive immunity. However, classical and widely used aluminum-based adjuvants have limited capacity to
induce cellular response. There are increasing needs for appropriate adjuvants with improved profiles
for vaccine development toward emerging pathogens. Carbohydrate-containing nanoparticles (NPs)
with immunomodulatory activity and particulate nanocarriers for effective antigen presentation are
capable of eliciting a more balanced humoral and cellular immune response.
Areas covered: We reviewed several carbohydrates with immunomodulatory properties. They include
chitosan, β-glucan, mannan, and saponins, which have been used in vaccine formulations. The mode of
action, the preparation methods, characterization of these carbohydrate-containing NPs and the
corresponding vaccines are presented.
Expert opinion: Several carbohydrate-containing NPs have entered the clinical stage or have been
used in licensed vaccines for human use. Saponin-containing NPs are being evaluated in a vaccine
against SARS-CoV-2, the pathogen causing the on-going worldwide pandemic. Vaccines with carbohy-
drate-containing NPs are in different stages of development, from preclinical studies to late-stage
clinical trials. A better understanding of the mode of action for carbohydrate-containing NPs as vaccine
carriers and as immunostimulators will likely contribute to the design and development of new
generation vaccines against cancer and infectious diseases.
ARTICLE HISTORY
Received 13 March 2021
Accepted 3 June 2021
KEYWORDS
Antigen presentation;
carbohydrate; immune
potentiation; mechanism;
nanoparticle; vaccine
adjuvant
1. Introduction
The presence of an adjuvant in most vaccines is critical for
stabilizing immunogens and for enhancing the elicitation of
a protective immune response [1]. Classical adjuvants based
on aluminum salts have been used for decades in human
vaccines. However, while aluminum-based adjuvants (referred
to as ‘Alum’ from this point on) are highly effective in eliciting
high levels of protective antibodies, their ability to elicit a cell-
mediated immune response is quite limited. Antigen-specific
cytotoxic lymphocytes could be essential for eliminating intra-
cellular pathogens [2–4]. In addition, the recent development
of advanced biotechnologies facilitates the emergence of
a new generation of vaccine antigens, such as recombinant
proteins, peptides and nucleic acids. In comparison to tradi-
tional attenuated vaccines, the safety profile and wider range
of disease coverage of these new immunogens make them
better candidates for human vaccines [5]. However, some of
these new immunogens are poorly immunogenic (such as
some recombinant proteins and peptides), and Alum showed
only a limited effect on improving the potency of vaccines
containing non-virus-like particle antigens [4–6]. Thus, there
are increasing demands for more appropriate and effective
adjuvants for these immunogens to be used in prophylactic
or therapeutic vaccines to stimulate protective immune
responses [3,4].
With the recent development of nanotechnology, nanopar-
ticles (NPs) varying in size, shape and surface properties could
be prepared according to specific designs and meeting differ-
ent needs [7,8]. Some of these NPs, including certain poly-
meric NPs, inorganic NPs and liposomes, could be used in
vaccines as both vehicles and adjuvants [9]. NPs can be further
modified to introduce additional functionality [10,11].
Carbohydrates are essential components of an organism.
Most carbohydrates are biocompatible and biodegradable.
Some carbohydrates possess attractive bioactivities, including
immunomodulatory activities [6,12–16]. Different carbohy-
drate patterns in pathogens can be recognized as pathogen-
associated molecular patterns by pattern recognition recep-
tors on the immune cell surface. Recognition could facilitate
the endocytosis of pathogens and trigger downstream path-
way activation, leading to chemokine and cytokine secretion
[6,16,17]. These molecular or cellular events result in an
increased presentation of antigens to the relevant antigen-
presenting cells (APCs) and the induction of an adaptive
immune response [6,16,18,19]. Soluble assembly of carbohy-
drates into NPs could further enhance cellular uptake and
potentiate their function as vaccine adjuvants [5].
CONTACT Qinjian Zhao qinjian_zhao@xmu.edu.cn State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health,
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian, PR China
EXPERT REVIEW OF VACCINES
https://doi.org/10.1080/14760584.2021.1939688
© 2021 Informa UK Limited, trading as Taylor & Francis Group

Polysaccharides, such as chitosan and β-glucan, could be
synthetically prepared as NPs [20,21]. In addition, mannan
could be coated onto the surface of NPs or encapsulated
inside NPs to enhance the adjuvanticity of the particles [22].
In addition, naturally occurring immunostimulant saponins
could form NPs together with cholesterol and lipids under
appropriate conditions [6,23]. These carbohydrate-containing
NPs not only have been extensively used in biomedical pro-
ducts, cosmetics and foods but also are studied for their
potential application as vaccine adjuvants [5,24,25].
Carbohydrate-containing NPs mimic the particulate nature
of pathogens in some aspects [26]. The size of NPs is similar to
that of viruses and bacteria, and carbohydrate patterns of NPs
can bind to pattern recognition receptors on immune cells
[10]. When used in vaccines as adjuvants, carbohydrate-
containing NPs together with the associated immunogens
could be effectively taken up by APCs, leading to an enhanced
level of immune response [5,6,10]. This review summarizes the
characterization and applications of nanoparticulate adju-
vants, including chitosan-, β-glucan-, mannan- and saponin-
containing adjuvants. Specific focus is given on on-going
efforts in the development of carbohydrate-containing NPs
as adjuvants in human vaccines. The proposed mechanism of
action, different preparation methods and characterization of
these carbohydrate-containing NPs will be discussed
(Figure 1). Additionally, the potency of vaccines using carbo-
hydrate-containing NPs as adjuvants, as well as the character-
istics of the immune response stimulated by these vaccines,
will be discussed in detail (Tables 1 & 2).
2. Nanoparticulate adjuvants containing chitosan
As a naturally occurring and readily available polysaccharide,
chitosan could be made into particles of different sizes under
different processing conditions. Chitosan is obtained through
deacetylation of chitin. Chitosan is present in the exoskeletons
of marine arthropods, such as shrimp and crabs, and in fungal
cell walls [27]. Chitosan has been extensively studied for bio-
medical applications due to its biocompatibility and attractive
bioactivity [28]. Chitosan is able to mediate the maturation of
dendritic cells (DCs) and drive T helper type 1 (Th1)-biased
immune response dependent on cGAS and STING activation
and type I interferon (IFN) production (Figure 2) [29]. In addi-
tion, chitosan is an activator of the NOD-like receptor protein 3
(NLRP3) inflammasome and consequently enhances the
interleukin (IL)-1β signaling pathway [30]. This activity is essen-
tial for Th17 and Th1 response and host defense against
fungal pathogens [16,28]. Both cGAS-STING and NLRP3 path-
ways are dependent on phagocytosis and lysosomal destabi-
lization [16]. Chitosan could be made into nanometer-scaled
particles or used to decorate other NPs. These carbohydrate-
containing NPs exhibit potent immunostimulatory properties
as vaccine adjuvants.
2.1. Chitosan-based NPs as vaccine adjuvants
The immunostimulatory properties of chitosan-based NPs
have been extensively studied since the 1990s [31]. Most
chitosan-based NPs are prepared based on an ionic gelation
method [6,20,27,32]. Chitosan with molecular weights from
200 to 400 kDa could be used for this purpose. After dissolving
chitosan powder in acetic acid (1%-2%) and then adding
anions, such as tripolyphosphate, into the chitosan solution,
NPs are formed through ionic gelation. The formation of NPs is
closely related to the concentration and volumetric mixing
ratio of chitosan and tripolyphosphate, as well as pH condi-
tions. Tripolyphosphate crosslinked chitosan NPs only formed
under specific conditions (chitosan concentration less than
2.5 mg/mL, mixing ratio of chitosan/tripolyphosphate greater
than 2 and pH between 3.5 to 5.5), otherwise it will lead to
coacervation [33]. The prepared chitosan NPs are nearly sphe-
rical, with smooth surfaces and diameters between 50 nm and
300 nm (Figure 1) [20,27]. To further assess their adjuvanticity,
chitosan-based NPs have been combined with different anti-
gens. Tetanus toxin-loaded chitosan NPs elicited higher anti-
body titers in mice than Alum-adjuvanted formulations [34].
Hepatitis B surface antigen (HBsAg) could be encapsulated
into chitosan NPs [20]. HBsAg-encapsulated NPs not only
increased the endocytosis of DCs but also elicited a stronger
and more durable immune response than commercial vac-
cines. More recently, to further enhance the adjuvanticity of
chitosan-based NPs, different cross-linkers have been used to
replace tripolyphosphate in the synthesis of chitosan NPs. As
reported by Lebre et al., chitosan NPs were prepared by using
aluminum sulfate as a cross-linkers [27]. Chitosan-aluminum
NPs induced DC maturation, while Alum did not. After sub-
cutaneous immunization with HBsAg, chitosan-aluminum NPs
stimulated higher antibody titers than Alum in both serum
and mucosal secretions. Besides, dextran, a carbohydrate with
immunostimulating properties, could also be used as cross-
linkers in the preparation of chitosan-based NPs. Dextran perse
is an effective adjuvant, which has been used in bacille
Calmette-Guerin (BCG) vaccine (Phase 3 clinical trials in pro-
gress) and influenza vaccine [35]. The combination of dextran
and chitosan could further enhance the level of immune
response. Sharma and coworkers developed chitosan-based
NPs by using dextran sulfate as ionic cross-linkers. The chito-
san-dextran NPs formulation of pertussis toxoid induced
a strong Th2 biased immune response, characterized by sig-
nificantly higher IgG and IgG1 titers as compared to Alum
formulation [36].The immunostimulatory properties of chito-
san-based NPs are closely related to their particle size. As
reported by Wang et al., chitosan particles of different sizes
were combined with hemagglutinin of H5N1. Higher levels of
Article highlights
●Novel adjuvants with improved properties could boost the immune
response of non-virus-like particle antigens.
●Carbohydrate-containing nanoparticles help facilitate antigen deliv-
ery and elicit a desirable immune response.
●Cationic polysaccharide chitosan-containing NPs are capable of elicit-
ing a balanced cellular and humoral immune response.
●β-glucan and mannan could act as motif-targeting molecules to
achieve targeted delivery.
●Saponin-containing NPs can elicit robust cellular response and have
been used in licensed human vaccines. The evaluation of vaccines
against COVID-19 is on-going in late-stage clinical trials.
2X. ZHANG ET AL.

Figure 1. Different processing methods of carbohydrate-containing NPs and the interaction modes between carbohydrate-containing NPs and cargos. The
preparation of carbohydrate-containing NPs can be demonstrated as in (a). Polysaccharide chitosan can form NPs under the action of ionic linker agents or can
be coated onto poly(lactic-co-glycolic acid) (PLGA) or liposomes. Formed NPs are spherical [20]. β-glucan is in triple helix structure. The preparation of β-glucan-
based NPs requires the dissociation of β-glucan into single stands using DMSO or NaOH. β-glucan strands could form NPs under the action of ionic linkers or paired
nucleic acids. β-glucan could also be encapsulated into PLGA NPs. Formed NPs are irregular and nearly spherical [21]. Mannan could be coated onto PLGA or
liposomes [22]. Saponins, cholesterol and lipids can form nanocomplexes with or without antigens. These NPs are in cage-like structures with a diameter of 40 nm
[23]. The interaction modes between carbohydrate-containing NPs and antigens can be elucidated as in (b). The interaction between carbohydrate-containing NPs
and antigens includes three modes: 1) antigens interact with NPs and are encapsulated in NPs; 2) antigens form nanocomplexes together with other materials;
and 3) antigens simply mix with carbohydrate-containing NPs and are adsorbed onto NPs.
EXPERT REVIEW OF VACCINES 3

antibodies and cytokines were detected in mice immunized
with relatively smaller chitosan particles [37].
Another important characteristic of chitosan-based NPs is
the cationic nature of chitosan. Chitosan-based NPs with posi-
tive charges on the particle surface can effectively adsorb
antigens that are negatively charged. However, the solubility
of chitosan in neutral and alkaline solutions is poor, which
could have an impact on the stability of chitosan-based NPs
and antigen absorption [38]. Therefore, it is especially mean-
ingful to introduce chemical modifications to increase the
solubility of chitosan at physiological pH. Trimethylation of
chitosan achieved this goal [39]. The synthesis of trimethyl
chitosan is dependent on quaternization (methylation) of
amino groups in chitosan. The quaternization degree can be
altered by controlling reaction steps and conditions, such as
increasing the number of reaction steps or the reaction time
[39]. The quaternization degree of chitosan influences its adju-
vanticity. As an adjuvant for the H5N1 vaccine, chitosan-based
NPs with moderate quaternization degrees (41% and 60%)
elicited higher levels of cytokines and antibodies [37].
Trimethyl chitosan-based NPs are an ideal adjuvant for muco-
sal vaccination. Mucosal vaccination has the potential to
induce a mucosal immune response, which is particularly
meaningful for protection against pathogens entering via the
mucosa [40]. Trimethyl chitosan-based NPs with positive
charges are able to adhere to the mucosa and are taken up
by local APCs. Together with ovalbumin (OVA), trimethyl chit-
osan-based NPs induced a robust mucosal immune response
characterized by a high level of IgA in mucosal secretions.
Moreover, mucosal vaccination with trimethyl chitosan-based
NPs and OVA also elicited a balanced systematic immune
response. Higher levels of Th1 cytokines and antibodies
(both IgG1 and IgG2) were detected in serum with trimethyl
chitosan-based NPs than in Alum [41]. Apart from trimethyla-
tion, the conjunction of ethylene glycol branches can also
increase the solubility of chitosan at neutral pH [42]. Glycol
chitosan-based NPs were also reported to enhance both
mucosal and systematic immune responses. Upon nasal
administration, HBsAg-loaded glycol chitosan-based NPs eli-
cited higher levels of antibodies in both serum and mucosal
Figure 2. Chitosan stimulates cytoplasmic DNA and ROS release, which engages the cGAS-STING pathway to mediate DC activation and Th1 response induction
(adapted from Carroll, 2016) [29]. Polysaccharide chitosan could induce mitochondrial damage in DCs, which causes the release of reactive oxygen species (ROS) and
endogenous DNA. Cytoplasmic DNA activates the cGAS-STING pathway and causes the production of type I IFN. Cytokines mediate the maturation of DCs and drive
Th1-biased immune responses.
4X. ZHANG ET AL.

secretions than native chitosan-based NPs. Moreover, glycol
chitosan-based NPs increased the production of both Th1 and
Th2 cytokines, while Alum increased the production of only
Th2 cytokines [32]. Overall, chitosan-based NPs are able to
elicit a more balanced immune response than Alum. With
appropriate chemical modifications, chitosan derivatives
show increased solubility under physiological conditions.
These chitosan derivative-based NPs are able to induce both
mucosal and balanced systematic immune responses upon
mucosal vaccination. It is especially meaningful for the design
of a needle-free vaccine defending against pathogens enter-
ing through the mucosa.
2.2. Chitosan-modified NPs as vaccine adjuvants
As an attractive polysaccharide with immunostimulatory prop-
erties, chitosan could act as a functional motif and be com-
bined with other biocompatible polymeric nanocarriers
[43,44]. These polymeric NPs are not powerful adjuvants them-
selves, and chitosan coating on polymeric NPs could signifi-
cantly enhance their capacity to induce an immune response
(Figure 1). Chitosan of low molecular weight (low viscosity) is
appropriate for this purpose (the viscosity of chitosan is
approximately 8 cP). This preparation could be achieved via
an emulsion method. Chitosan-coated polymeric NPs formed
by injecting the organic phase containing polymers into the
water phase contain chitosan. Chitosan-coated polymeric NPs
are spherical, with diameters of approximately 200 nm [43]. As
reported by Jesus et. al, chitosan-modified poly-ε-caprolactone
NPs can effectively absorb HBsAg and elicit higher levels of
specific antibodies than the commercial HBsAg vaccine [43].
Moreover, these NPs enhanced the production of IFN-γ and IL-
17, indicating the induction of Th1 and Th17 immune
responses [43]. In addition, glycol chitosan-coated polymeric
NPs are a powerful mucosal adjuvant. Upon the intranasal
coadministration of a recombinant antigen derived from
Chlamydia trachomatis, glycol chitosan-coated NPs signifi-
cantly increased the levels of IgA and IgG in both vaginal
washes and serum [44]. Overall, the emulsion method com-
bines the immunostimulatory polysaccharide chitosan and
other biocompatible polymeric NPs. These polymeric NPs
could be favorable carriers for antigen delivery but not power-
ful adjuvants themselves, and the combination with chitosan
potentiates their function to enhance the immune response.
Table 1. Recent studies on nanoparticulate adjuvants containing carbohydrates in the early stage of development.
Carbohydrates
Nanoparticulate
adjuvants Preparation
Size
(nm) Morphology Immunogens Effects References
Chitosan Chitosan NPs Ionic gelation 100–
250
Uniform and
spherical
TT, HBsAg ✔ Induced balanced Th1 and Th2 response
✔ Stimulated Th17 immune response
✔ Elicited mucosal immune response
✔ Achieved dose-sparing effect
[20,34]
Chitosan-Aluminum
NPs
281 Spherical and
smooth
HBsAg [27]
Chitosan-dextran NPs 300–
350
Uniform and
spherical
Pertussis
toxoid
[36]
Trimethyl chitosan NPs 178 Uniform and
spherical
OVA [37,41]
Glycol chitosan NPs Complexation 200 Almost
spherical
HBsAg [32]
Chitosan-coated PCL
NPs
Emulsification 208 Spherical HBsAg [43]
Glycol chitosan-coated
LPNs
220 Spherical CHT522 [44]
β-glucan β-glucan-based NPs Nucleic acid
hybridization
30–60 Rod-like OVA ✔ APCs targeting effect
✔ Elicited humoral and cellular response
✔ Induce mucosal immune response
[48,51]
Aminated β-glucan
NPs
Ionic
complexation
200 Irregular
spherical
OVA [21]
Aminated β-glucan-
based NPs
Electrostatic
interaction
200–
300
Nearly
spherical
H1N1 PR8 [52]
β-glucan encapsulated
in PLGA NPs
Emulsification 781 Spherical HPV E7 [53]
Mannan Mannan-coated PLGA
NPs
Emulsification 806 Spherical HBsAg ✔ APCs targeting effect
✔ High potential in anti-HIV infection
[61]
Mannan-coated
liposomes
Thin film
method
150–
200
Spherical HIV-1 DNA
plasmid
[65]
Saponin Saponin-based
nanocomplexes
Ethanol
injection
47 Spherical and
cage-like
OVA ✔ Elicited robust cellular response
✔ Induced Th2 immune response
✔ Conferred 100% protective efficacy
against virus and bacteria challenge
✔ Achieved dose-sparing effect
[89,90]
Saponin-based
nanocomplexes
Dialysis 60 Spherical and
cage-like
H7N1
glycoprotein
[91]
Ginsenoside-based
NPs 70–
107
Spherical OVA [92]
Ophiopogonin-based
nanocomplexes
Emulsification 76 Spherical MRSA protein [93]
Platycodin-loaded
liposomes
Film dispersion 136 Uniform
and
spherical
OVA [94]
*(footnote to the table) NPs: nanoparticles, PCL: poly-ε-caprolactone, LPNs: lipid-polymer hybrid NPs, PLGA: poly(lactic-co-glycolic acid), TT: tetanus toxoid, HBsAg:
Hepatitis B surface antigen, OVA: ovalbumin, CHT522: recombinant Chlamydia trachomatis fusion protein, H1N1 PR8: inactivated antigen derived from Influenza A/
PuertoRico/8/34, HPV E7: human papillomavirus E7 protein, HIV-1: human immunodeficiency virus-1, H7N1 glycoprotein: Influenza virus strain A/FPV/Rostock/34
(H7N1) isolated glycoprotein antigens, MRSA protein: Methicillin-resistant Staphylococcus aureus (MRSA) recombinant protein, Hla
H35L
IsdB
348-465
.
EXPERT REVIEW OF VACCINES 5

3. Nanoparticulate adjuvants containing β-glucan
β-glucan is another natural polysaccharide with immunosti-
mulatory properties [45]. NPs containing β-glucan could be
prepared through different methods. The prepared NPs have
shown great potential in antigen delivery and enhancement of
the immune response [46,47]. β-glucan can be found in plants,
fungi, and bacteria. β-glucans in different species vary in chain
length as well as distribution [5]. β-1,3-
D
-glucan (referred to as
‘β-glucan’ from this point on), which mainly exists in fungi, is
in a triple helix structure and is reported to be able to mod-
ulate the immune response [46,48]. β-glucan can interact with
pattern recognition receptors on the immune cell surface.
Dectin-1 of the C-type lectin receptor family is the main
receptor that recognizes β-glucan (especially particulate β-
glucan) [16,49]. The ligand-binding signal results in Th1 and
Th17 cytokine and chemokine production, which conse-
quently enhances the immune response [19,50]. β-glucan-
containing NPs can target APCs due to the interaction
between β-glucan and pattern recognition receptors. Thus, β-
glucan-containing NPs have great advantages in antigen deliv-
ery. Moreover, cytokine production induced by β-glucan-
containing NPs could enhance the level of the immune
response.
3.1. β-glucan-based NPs as vaccine adjuvants
In consideration of the favorable characteristics of β-glucan,
different methods have been used to prepare β-glucan-based
NPs. β-glucan-based NPs were assessed in different vaccines
as adjuvants. For the synthesis of β-glucan-based NPs, disso-
ciating the triple helix structure of β-glucan into single strands
is necessary [46,51]. Single β-glucan strands could form NPs
under cross-linking. CpG oligonucleotides (a nucleic acid-
based adjuvant) can be used as linkers in the preparation of β-
glucan NPs (Figure 1). β-glucan strands could form rod-like
NPs under hybridization between CpG and complementary
CpG [51]. These β-glucan-based NPs exhibited great potential
as adjuvants for antitumor vaccines [51]. Coadministration of
β-glucan-based NPs and OVA significantly increased the num-
ber of antigen-specific CD8
+
T cells and enhanced the produc-
tion of IFN-γ. These results indicated the induction of a robust
Th1 immune response. In addition, mice immunized with a β-
glucan-based NP formulation exhibited delayed tumor growth
and an improved survival rate [48,51]. Apart from forming NPs
through pairing of nucleic acids, β-glucan can be ionically
crosslinked to form NPs. Amine modification is needed for
this purpose, which endows β-glucan with a cationic nature.
Aminated β-glucan can form NPs under the action of different
anionic linkers [21,52]. The prepared NPs have exhibited
potent adjuvanticity in different vaccines. CpG can act as an
ionic linker in the preparation of aminated β-glucan-based NPs
as well. Aminated β-glucan NPs containing CpG are irregular
spheres, with a particle size of approximately 200 nm
(Figure 1). Cell culture experiments revealed that these ami-
nated β-glucan NPs significantly enhanced the uptake of
macrophages. Upon subcutaneous administration, aminated
β-glucan NPs with the antigen OVA resulted in greater secre-
tion of the Th1 cytokine IFN-γ and elicited higher levels of
antibodies (both IgG1 and IgG2a) than Alum (Table 1) [21]. In
addition, the anionic antigen itself could function as a linker in
the preparation of aminated β-glucan-based NPs. As reported
by Lee et al, aminated β-glucan-based NPs formed through
the electrostatic interaction between aminated β-glucan and
the antigen derived from influenza A. These aminated β-
glucan-based NPs were assessed for oral vaccination [52].
Aminated β-glucan-based NPs remarkably enhanced the anti-
body titers in serum and the intestine. Cell culture experi-
ments verified that aminated β-glucan-based NPs could
target microfold-cells (microfold-cells are responsible for trans-
porting antigens selectively from the intestinal cavity to
underlying lymphoid cells) [52]. Overall, β-glucan-based NPs
could be prepared with different processing conditions. β-
glucan-based NPs have the advantage of targeting immune
cells and thus could be an appropriate choice for antigen
delivery. The combination of other immunostimulants or the
introduction of chemical modifications could further improve
the function of β-glucan-based NPs as a vaccine adjuvant.
3.2. β-glucan modified NPs as vaccine adjuvants
β-glucan could be encapsulated into biocompatible polymeric
NPs, such as poly(lactic-co-glycolic acid) (PLGA) (Figure 1).
Introduction of β-glucan endows PLGA NPs with the ability
to boost the immune response [53]. Chemical treatment to
dissociate the β-glucan triple helix structure is not necessary
for this purpose. Encapsulation could be achieved through the
emulsion method. As reported by Piyachat et al, β-glucan
together with human papillomavirus recombinant protein
was encapsulated into PLGA NPs [53]. The size of prepared
NPs was approximately 700 nm. After subcutaneous adminis-
tration, the level of IgG antibodies was significantly increased
by β-glucan-encapsulated NPs. Moreover, the cellular immune
response was significantly enhanced compared to that with
PLGA NPs, characterized by an increased number of CD4
+
and
CD8
+
T cells. Overall, the introduction of β-glucan could
enhance the adjuvanticity of PLGA NPs. The emulsion method
for the preparation of β-glucan-modified NPs is relatively sim-
ple. β-glucan-modified NPs are able to induce both humoral
and cellular immune responses.
4. Nanoparticulate adjuvants containing mannan
The natural polysaccharide mannan possesses immunomodu-
latory properties [54,55]. NPs containing mannan are able to
enhance the immune response, as vaccine adjuvants, espe-
cially in vaccines against human immunodeficiency virus (HIV).
Mannan is extensively distributed in plants and the microbial
cell wall, consisting of β-1,4 glycosidic bonds linked to
D
-
mannose [5]. Mannan could promote the maturation of DCs
dependent on TLR4 and activate the inflammasome to mod-
ulate immune response [16]. Mannan could also interact with
mannan-binding lectin and C-type lectin receptors that are
widely expressed on immune cells (including all myeloid
cells and lymphocytes) [16,56,57]. Mannan-binding signals
activate pathways to induce the production of cytokines and
chemokines, promoting Th cell differentiation [58]. In consid-
eration of the capacity of mannan to interact with pattern
6X. ZHANG ET AL.

recognition receptors and mediate the immune response,
mannan-containing NPs have been assessed in vaccines as
adjuvants to achieve antigen target delivery and boost the
immune response.
4.1. Mannan-modified NPs as vaccine adjuvants
Mannan perse has not been reported to be made into NPs but
can act as a functional molecule to modify other biocompa-
tible NPs. Mannan-modified NPs have exhibited powerful
capacity to target APCs, and their adjuvanticity was further
assessed in different vaccines [6,22,59,60]. As reported by Li
et al, mannan could be coated onto PLGA through the emul-
sion method. Mannan-coated PLGA NPs are spherical, with
a diameter of approximately 700 nm [61]. Cell culture experi-
ments verified that mannan coating on PLGA particles signifi-
cantly enhanced uptake by macrophages. Mannan-coated NPs
loaded with HBsAg increased the level of antibodies and the
production of Th1 cytokines. In addition, mannan-modified
NPs have been assessed in vaccines against HIV infection.
HIV envelope glycoproteins comprise high-mannose-type oli-
gosaccharides [62]. These glycoproteins are immunodominant
and essential for virus entry into target cells [63]. The recogni-
tion of these glycoproteins is important for the immune
response against HIV [62,64]. Mannan-coated NPs faithfully
mimic the mannosylated nature of native virion surfaces and
thus are appropriate adjuvants to be used in vaccines against
HIV. As reported by Toda et al, mannan-coated liposomes
were assessed in an anti-HIV vaccine [65]. The coating process
was achieved by mixing mannan and liposomes together. The
size of prepared NPs is approximately 200 nm. Loaded with
DNA plasmids of HIV, mannan-coated liposomes significantly
enhanced HIV-specific cytotoxic T lymphocyte production and
IgG2a levels. In addition, a therapeutic plasmid DNA vaccine
for HIV treatment containing mannose-modified NPs has pro-
gressed to clinical studies (NCT00711230) (Table 2). Mannose-
modified NPs could target APCs and induce an HIV-specific
T cell response [66]. In conclusion, the interaction between
mannan and receptors on immune cells defines the capacity
of mannan-modified NPs to target APCs. Thus, mannan-
modified NPs could be appropriate vehicles for antigen deliv-
ery. Enhanced uptake of mannan-modified NPs together with
antigens leads to further boosting of the immune response.
Mannan-modified NPs exhibited potent adjuvanticity, espe-
cially in experimental vaccines against HIV.
5. Nanoparticulate adjuvants containing saponins
The potent adjuvanticity of saponins has been known for
decades [67]. Saponin-containing NPs have been evaluated
in different vaccines and exhibit powerful adjuvanticity [68].
Several saponin-containing vaccines have been approved for
Table 2. Vaccine formulations with nanoparticulate adjuvants containing different carbohydrates in the clinical development stage.
Carbohydrates Vaccines Particulate adjuvants Immunogens Phase Identifier
Chitosan Haemophilus influenzae
type b vaccine
Viscogel antigen derived from
Heamophilus influenza type b
1/2a NCT01578070
Mannan HIV therapeutic vaccine Mannose-modified NPs HIV plasmid DNA 2 NCT00711230
Saponin Herpes Zoster vaccine
(Shingrix)
AS01B (QS-21 and MPL) recombinant VZV gE Licensed by FDA for marketing (2017)
Malaria vaccine
(Mosquirix)
AS01E (QS-21 and MPL) RTS and S Licensed by EMA for marketing (2015)
HIV vaccine AS01B (QS-21 and MPL) HIV gp120-NefTat 1 NCT03368053
Tuberculosis vaccine AS01E (QS-21 and MPL) M72 2 NCT01755598
NY-ESO-1 ISCOMATRIX
vaccine
ISCOMATRIX (QH-A and
QH-C)
NY-ESO-1 protein 2 NCT00518206
H5N1 influenza vaccine Matrix M (Fraction
A and Fraction C)
Hemagglutinin 1 NCT00868218
H7N9 influenza vaccine Matrix M (Fraction
A and Fraction C)
H7N9 VLP 1/2 NCT02078674
Malaria vaccine Matrix M (Fraction
A and Fraction C)
ChAd63 and MVA 1 NCT01669512
COVID-19 vaccine (NYX-
CoV2373)
Matrix M (Fraction
A and Fraction C)
S glycoprotein 3 NCT04583995
*(footnote to the table) MPL: Monophosphoryl Lipid A, VZV gE: varicella-zoster virus glycoprotein E, HIV: human immunodeficiency virus, gp 120: glycoprotein 120,
M72: tuberculosis antigens Mtb39A and Mtb32, NY-ESO-1: melanoma-associated antigen, VLP: virus like particle, ChAd63: Chimpanzee I adenovirus serotype 63,
MVA: modified vaccinia Ankara, S: spike protein of SARS-CoV-2. Data from: https://clinicaltrials.gov/ (Accessed on 4 May 2021)
Table 3. Characteristics, applications and mechanisms of the promising adju-
vant Matrix M.
Matrix M Reference
Saponin
components
Fraction A (QS-7) and Fraction C (QS-21) [80]
Particles Two individual particles with cage-like
structure, both with diameter of 40 nm
[80]
Vaccines in clinical
trials
H5N1 influenza vaccine [82]
H7N9 influenza vaccine [83]
Malaria vaccine [84]
COVID-19 vaccine [85]
Adjuvanting effect
in COVID-19
vaccine
High neutralization titer and T cell
response (Phase 1/2)
[86]
60% protective efficacy against South
Africa variant (Phase 2b)
[87]
89% protection against symptomatic
cases (Phase 3)
[87]
100% protection against severe cases
(Phase 3)
[88]
Mechanism of
action
Pro-inflammatory cytokines and
chemokines production, endosomal
escape and forming intracellular lipid
bodies, MyD88 and NLRP3
[16]
*(footnote to the table) Matrix M is a Matrix adjuvant (similar to ISCOMATRIX)
developed from ISCOM, formulated with Fraction A and Fraction C in separate
particles. DCs: dendric cells, MyD88: myeloid differentiation factor 88, NLRP3:
NOD-like receptor protein 3.
EXPERT REVIEW OF VACCINES 7

marketing (Table 2) [69,70]. Saponins extensively exist in ter-
restrial higher plants comprising lipophilic steroidal or triter-
pene aglycones linked to hydrophilic carbohydrate chains
[67,71,72]. Triterpene saponins derived from the bark of the
tree Quillaja saponaria are known for their potent adjuvant
effects. Quil A, the most widely studied extraction from this
source, comprises heterogeneous mixtures of structurally
related saponins [67]. QS-21 is one of the saponin fractions
purified from Quil A, which has potent adjuvanticity with low
toxicity among all fractions [16,73]. Efforts on formulation
optimization were aimed at reducing the hemolytic activity
of the saponins from Quil A [68]. Nanoparticulate adjuvants
with QS-21 or other Quil A fractions exhibited good safety
profiles and are used in different licensed or test vaccines.
5.1. Vaccines under clinical development with
saponin-containing NPs
It has been a long journey for saponin-containing NPs from
the bench to patients. Improving the toxicity of Quil
A fractions is the first consideration. The hemolytic activity of
triterpene saponins is closely related to their structure [68,74].
The presence of lipids and cholesterol in the formulation could
improve the hemolytic activity of triterpene saponins [75].
Adjuvant system 01 (AS01) of GlaxoSmithKline (GSK) is
a liposome-based adjuvant that contains QS-21 and mono-
phosphoryl lipid A (QS-21 and monophosphoryl lipid A have
synergetic effect), which can induce robust cellular and
humoral immune response [6,76]. AS01 was first licensed in
2017 for use in a vaccine against varicella zoster virus and then
in 2019 for use in another vaccine against malaria (Table 2)
[70,77]. Another saponin-containing nanoparticulate adjuvant
system, termed the immune-stimulating complex (ISCOM), is
composed of Quil A fractions, cholesterol, lipids and antigens.
ISCOM particles are approximately 40 nm in size, with cage-
like structures (Figure 1) [78]. ISCOM could be prepared
through various methods, including dialysis, centrifugation,
lipid-film hydration, ethanol injection and ether injection
[75]. ISCOM could induce long-lasting antibodies and cytotoxic
T cells [78,79]. However, the physical incorporation of antigens
is a challenge in the manufacturing of ISCOM-based vaccines.
Reproducible and efficient incorporation with antigens, such
as recombinant DNA protein, could be difficult [80]. This issue
led to the development of matrix formulations. Matrix adju-
vants and antigens do not need to be physically incorporated
to achieve potent immune stimulation [80]. The matrix adju-
vant ISCOMATRIX is a stable NP that is identical to ISCOM but
does not contain antigens [78]. ISCOMATRIX could be asso-
ciated with antigens through chelating or electrostatic inter-
actions [78]. ISCOMATRIX performs well with most types of
antigens. The adjuvanticity of the ISCOMATRIX formulation
could be comparable to that of ISCOM. ISCOMATRIX vaccines
have progressed to clinical trials for the treatment of advanced
malignant melanoma (Table 2) [81]. Another matrix adjuvant,
the Matrix M adjuvant of Novavax, has attracted much atten-
tion, its characteristics, corresponding vaccines and mechan-
isms of action are summarized in Table 3 [16]. Matrix
M consists of two individual 40 nm-sized particles [80]. Two
types of particles are formed each with a different saponin
fraction purified from Quil A (QS-7 and QS-21, also known as
Fraction A and Fraction C). Fraction A and Fraction C have
complementary properties. Fraction C (QS-21) is highly active
in adjuvant effects. Fraction A (QS-7) is weaker but well toler-
ated [80]. Matrix M could induce potent and balanced humoral
and cellular immune response, and could be a safe and effi-
cient adjuvant. Matrix M has been evaluated in a human
vaccine against influenza and a vaccine against malaria
(Table 2) [82–84]. Recently, the world-wide COVID-19 emer-
gency has been caused by SARS-CoV-2 infection. There is an
urgent need for the development of an efficient vaccine
against SARS-CoV-2 [85]. Matrix M is being evaluated in
a COVID-19 vaccine (NVX-CoV2373) as the adjuvant to boost
the immune response against SARS-CoV-2. This Phase 1/2
clinical trial has been completed and has progressed to
Phase 3 now (Table 2). The neutralization titer elicited by NVX-
CoV2373 was 4-fold higher than that of convalescent sera
from patients recovered from COVID-19. Matrix M also
induced multifunctional CD4
+
T cells, of which most had
a Th1-favored phenotype (Table 3) [86]. NVX-CoV2373 has
demonstrated 89.3% efficacy in the ongoing UK Phase 3 trial
with over 50% cases attributed to UK variant, and 60% efficacy
in Phase 2b South Africa trial with over 90% cases attributed
to prevalent South Africa variant [87]. The latest results con-
firmed 96% protection against original COVID-19 strain and
100% protection against severe cases (Table 3) [88]. Matrix
M is a potent adjuvant and the corresponding vaccines are
particularly promising for marketing. However, saponin com-
ponents of Matrix M rely on natural extraction, and the output
of Matrix M is closely related to the abundance of saponins in
Quillaja Saponaria [16]. Overall, Quil A saponin-containing NPs
have been studied for decades and ultimately used in different
human vaccines. Quil A saponin-containing NPs are powerful
immunostimulants that induce cellular immune responses and
thus could be an appropriate choice to be applied in different
vaccines against viruses and tumors.
5.2. Saponin-containing NPs as vaccine adjuvants in the
preclinical stage
The capacity of saponins to induce a robust immune response
is very attractive. Some other fractions purified from Quil A (in
addition to those mentioned above) also possess immunosti-
mulatory properties. Apart from Quillaja saponaria, adjuvant-
active saponins could also be purified from many other plants.
Most studies on NPs containing these saponins are in the
preclinical stage. Being assessed in different vaccines, NPs
containing these saponins also exhibited potent adjuvanticity.
These saponins could form NPs similar to ISCOM and
ISCOMATRIX as well. As reported by Cibulsk et al., a saponin
fraction also purified from Quil A was made into an immune-
stimulating nanocomplex together with cholesterol, phospho-
lipids and OVA, named IQB-90 [89,90]. The preparation of IQB-
90 is dependent on a modified ethanol injection method. The
IQB-90 particle is spherical, with a cage-like structure. The
diameter of the IQB-90 particle is between 40 nm and
50 nm. Cell culture experiments revealed that IQB-90 could
be effectively taken up by DCs with no toxicity. Following
8X. ZHANG ET AL.

subcutaneous administration, IQB-90 elicited a robust anti-
body response (both IgG1 and IgG2a) and increased Th1
cytokine secretion. In another study [91], three immunostimu-
lating saponins with low toxicity were isolated from plants in
Kazakhstan. A nanocomplex consisting of these saponins,
lipids and H7N1 influenza virus antigens were prepared via
an extensive dialysis method. The prepared nanocomplex was
approximately 60 nm in diameter. After subcutaneous immu-
nization, the nanocomplex containing these three saponins
stimulated robust Th1 and Th2 response. This effect was char-
acterized by high antibody titers (both IgG1 and IgG2a) and
enhanced Th1 and Th2 cytokine production. Moreover, mice
preimmunized with these nanocomplexes exhibited 100%
protective efficacy against H7N1 influenza virus challenge. In
addition, saponins with adjuvanticity have been reported to
be isolated from herbs [92–94]. These herbal saponins could
also be prepared as nanocomplexes and were assessed in
vaccines. As reported by Song et al, ginsenoside-containing
NPs were prepared via a dialysis method similar to
ISCOMATRIX technology. Ginsenoside-containing NPs are
spherical, with diameters ranging from 70 nm to 107 nm
[92]. Coadministration of ginsenoside-containing NPs and
OVA promoted T cell proliferation and enhanced both Th1
and Th2 response. Additionally, ophiopogonin-D derived
from Ophiopogonis could be a powerful immunopotentiator
[93]. Different from methods used for the preparation of
ISCOM®, ophiopogonin-D-containing NPs were synthesized
through emulsion methods. The spherical ophiopogonin-
D-containing NPs are approximately 76 nm in diameter.
Administration of ophiopogonin-D-containing NPs and recom-
binant MRSA protein upregulated the level of the Th2 immune
response and secretion of IL-17. Following a lethal challenge
of MRSA, ophiopogonin-D-containing NP formulations
achieved a 100% survival rate, while formulations containing
Alum only achieved 60% survival. Overall, the potent adjuvan-
ticity of saponins has been proven by numerous studies. The
main aim of current studies on saponin-containing NPs is to
find saponins with both potent adjuvanticity and an improved
safety profile. NPs containing saponins from other sources
exhibit potent adjuvanticity with less toxicity. Thus, vaccines
using these novel saponin-containing NPs are also promising
for progression to clinical studies for human use.
5.3. Proposed mechanism of action of Quillaja
saponins
A better understanding of the mechanism of action of sapo-
nins could help in the design of adjuvants containing sapo-
nins. Quillaja saponins in the adjuvants AS01, ISCOM,
ISCOMATRIX and Matrix M contribute much to adjuvant activ-
ity. The adjuvanticity of these Quillaja saponins is closely
related to their structure (Figure 3) [71,95]. Reaction between
the triterpene aldehydes and amino groups of receptors on
the T cell surface delivers alternative costimulatory signals,
Figure 3. Proposed mechanism of action for Quillaja saponin, an active component of the adjuvants AS01, ISCOM, ISCOMATRIX and Matrix M (adapted from
Marciani, 2018) [95]. Quillaja saponin can act on both DCs and T cells. DCs: The hydrophobic triterpene parts and lipophilic acyl chain are able to interact with cell
membranes, thus facilitating the delivery of exogenous antigens into DCs. (a) Exogenous antigens and saponin adjuvants enter DCs by endocytosis, (b) saponin
adjuvants disrupt the endosomal membrane, and c) early escape of antigens occurs for further processing inside the cell into peptides. Properly degraded antigens
and additionally processed antigens are loaded onto MHCs. After binding to MHCs, peptides are presented on the DC surface to T cells. Additionally, oligosaccharide
chains of saponins can activate DCs by binding to C-type lectin receptors (CLRs) expressed on the surface of DCs. T cells: The aldehyde group on triterpene saponin
forms an imine with an amino group from a T cell surface receptor, most likely CD2, delivering a costimulatory signal to the T cell. This signal converges with T cell
receptor-mediated signaling at the level of tyrosyl phosphorylation of a mitogen-activated protein (MAP) kinase (ERK2), which together with changes in cell K
+
and
Na
+
transport stimulates T cell activation biased to the Th1 response and the resultant secretion of Th1 cytokines [95].
EXPERT REVIEW OF VACCINES 9

which are essential for T cell activation and Th1 cytokine
production (Figure 3) [71,95,96]. In contrast, converting the
aldehyde to a secondary amine abrogates its adjuvanticity
[95,97]. In addition, hydrophobic triterpene part of Quillaja
saponins has high affinity for cholesterol and thus are able
to intercalate into the cholesterol-rich regions of cell mem-
branes [72,95]. This activity facilitates the endocytosis and
destabilization of endosomes–lysosomes, leading to presenta-
tion of exogenous antigens by APCs (mainly DCs and macro-
phages) [95]. The lipophilic acyl chains of Quillaja saponins
also facilitate the delivery of exogenous antigens (Figure 3)
[71,95]. Moreover, oligosaccharide components in triterpene
saponins are also capable of mediating the immune response
by interacting with C-type lectin receptors on immune cells
(Figure 3) [71,95]. Overall, these proposed mechanisms of
action for adjuvant-active triterpene saponins improve our
understanding of the great immunostimulatory properties of
saponin compounds. These could also serve as a basis for the
design of novel saponin-containing adjuvants with improved
adjuvant function and reduced toxicity.
6. Nanoparticulate adjuvants containing other
carbohydrates
One of the most common carbohydrates, sucrose, is used as
an immunostimulant in vaccines [35]. Evaluation of immuno-
genicity of a sucrose-containing nanoparticulate adjuvant was
conducted [98,99]. Wang and coworkers synthesized sucrose-
containing NPs by taking silica as template [99]. The prepared
NPs are uniform and spherical with diameter of 470 nm, which
have large mesopores and macropores. Following oral admin-
istration of formulations of bovine serum albumin (BSA), sig-
nificantly higher level of IgA titers was induced by sucrose-
containing NPs as compared to Freund’s adjuvant. Besides,
high level of IgG2a antibodies was elicited (almost equal to
IgG1 titers). These results indicated that these sucrose-
containing NPs could induce both mucosal and systematic
immune response, which is balanced in Th1 and Th2 [99].
Herbal polysaccharides are also reported to have immuno-
modulatory activities. In this case, efforts have been made to
assess the function of herbal polysaccharide-containing NPs as
vaccine adjuvants. As reported by Wusiman et al, herbal poly-
saccharides termed Alhagi-honey with immunostimulatory
properties were encapsulated into PLGA NPs via an emulsion
method [100]. After coadministration of functionalized NPs
and OVA, the specific T cell response was significantly upre-
gulated. Moreover, Th1 cytokines were induced. Apart from
herbal polysaccharides, phytoglycogen (a dendrimer similar to
alpha-
D
-glucan) isolated from sweet corn was used to prepare
NPs. The prepared NPs are nearly spherical, with a diameter of
500 nm. Together with influenza virus antigen, phytoglyco-
gen-containing NPs were administered intranasally and intra-
muscularly. The phytoglycogen-containing NP formulations
were capable of stimulating Th1 and Th2 response via both
administration routes. This effect was characterized by the
expression of transcription factors (GATA-3 and T-bet) and
the enhanced production of cytokines and antibodies (both
IgG1 and IgG2a). Antigen-specific mucosal IgA was detected in
nasal secretions [101].
7. Conclusion
As vaccine adjuvants, carbohydrate-containing NPs are able to
elicit robust and more balanced immune response than tradi-
tional Alum. On the one hand, carbohydrate-containing NPs
mimic the particulate nature of pathogens and thus can be
easily recognized. On the other hand, immunostimulating
carbohydrates improve the capacity to boost the immune
response. Carbohydrates with different characteristics can
endow NPs with different functions. For instance, chitosan-
based NPs with a cationic nature could effectively absorb
antigens, and NPs containing β-glucan and mannan could
target immune cells. The function of carbohydrate-containing
NPs as vaccine adjuvants could be further improved through
additional chemical modifications. Several vaccines using car-
bohydrate-containing NPs as adjuvants have successfully pro-
gressed to the clinical stage or have been approved for
marketing. Anti-influenza vaccines using chitosan-based NPs
and anti-HIV vaccines using mannose-modified NPs are under
evaluation in clinical trials. One of the most promising adju-
vants could be saponin-containing NPs due to their ability to
induce potent cellular responses. Two vaccines using saponin-
containing NPs as adjuvants have been approved for market-
ing, and several other vaccines are under evaluation in clinical
trials. A vaccine for COVID-19 using saponin-containing NPs as
adjuvants has exhibited potent effects. Although most carbo-
hydrate-containing nanoparticulate adjuvants are still in the
preclinical development stage, they have great potential for
use in human vaccines in the future. NPs containing chitosan
derivatives that are able to induce mucosal responses should
be applied in vaccines for mucosal vaccination. β-glucan-
containing NPs induced powerful T cell response, making
them an appropriate choice for antitumor vaccines.
Carbohydrate-containing NPs may have some advantages
when compared to Alum. Carbohydrate-containing nanoparti-
culate adjuvants with different functionalities may satisfy the
different needs of vaccine development against emerging
infectious diseases and against cancer.
8. Expert opinion
For decades, efforts have been made to develop vaccine
adjuvants. It has been a long journey for these vaccine adju-
vants from the bench to patients. Factors such as the compat-
ibility, stability, effectiveness and complexity of the
manufacturing process are considered [2,4]. Carbohydrates
widely exist in nature and are important components in
organisms. Carbohydrates expressed on cellular glycoproteins,
glycolipids and secretions of cells are essential in cell signaling
[102]. Carbohydrates regulate cell adhesion, recognition,
migration, morphogenesis and differentiation [103–105].
Therefore, the presence of carbohydrates in NPs increases
their biocompatibility. Carbohydrate-containing NPs integrate
better into the physiological environment than NPs without
carbohydrate modifications. Thus, carbohydrate-containing
NPs could easily access immune cells, which may enhance
cellular uptake. In addition, compared to inorganic NPs, car-
bohydrate chains and branches increase the fluidity and plia-
bility of carbohydrate-containing NPs, facilitating the cellular
10 X. ZHANG ET AL.

uptake of vaccine/antigen particles. Dynamic remodeling of
the NP surface and lateral diffusion increase the contact area
with APCs [106]. The increased interaction with APCs leads to
enhanced uptake of carbohydrate-containing NPs along with
antigens, which enables the presentation of antigens and the
induction of the immune response. Moreover, carbohydrate
chains and branches also increase the looseness and surface
area of NPs, which endow both the adsorption of antigens and
adhesion to the immune cell surface.
In addition, manufacturability and scalability are important
for vaccine adjuvants in their journey from the bench to
patients. The manufacturing process of carbohydrate-
containing NPs could be relatively simple and highly repeata-
ble [107]. Carbohydrate-containing NPs have been extensively
applied in pharmaceutical products [108,109]. For example,
chitosan-based NPs are easy to obtain at low cost. The particle
size and electrostatic properties of chitosan-based NPs could
be controlled by modulating the acetylation degree and intro-
ducing chemical modifications. Large amounts of uniform
chitosan-based NPs could be obtained for preclinical and
clinical evaluation [107].
Furthermore, a number of infectious diseases are trans-
mitted via the mucosa [110]. The ability of NPs containing
chitosan and aminated β-glucan to elicit a mucosal immune
response could effectively defend against pathogens that
enter via the mucosa. Assessment of carbohydrate-
containing NPs in vaccines for mucosal vaccination is espe-
cially meaningful for protection against pathogens entering
via the mucosa.
However, effort and investment in optimizing the character
of carbohydrate-containing NPs have not been adequate.
Additional studies are needed to control the electrostatic
properties and particle size of NPs [111,112]. The electrostatic
properties of carbohydrate-containing NPs are essential for
antigen adsorption, mucosal adhesion as well as immune
receptor interaction [44,112]. Cationic NPs are able to adsorb
antigens that are negatively charged under physiological con-
ditions. The electrostatic properties of carbohydrate-
containing NPs can be controlled through modifying carbohy-
drates by chemical groups, such as carboxylation and amina-
tion. Particle size is closely related to the recognition and
uptake of carbohydrate-containing NPs. Little is known about
controlling the particle size of carbohydrate-containing NPs. In
the next five years, methods to control the electrostatic prop-
erties and particle size of carbohydrate-containing NPs need
to be further investigated.
In addition, as emerging molecular modalities in the vac-
cine arena, nucleic acid-based vaccines (plasmid DNA, viral
vectors or RNA) are being developed as effective means to
elicit robust humoral and cellular immune response [4,113].
The data from the test vaccines against COVID-19 have shown
great promise. Therefore, the application of carbohydrate-
containing NPs in nucleic acid-based vaccines could be a key
advancement. A derivative of dextran is the first used cationic
polymer-based nanodelivery system for mRNA [114]. Nucleic
acids are negatively charged under physiological conditions,
and the potency of vaccines based on cationic carbohydrate-
containing NPs and nucleic acids needs to be investigated in
the next five years.
Last but not least, the effect of vaccines that consist of
antigenic glycoprotein and carbohydrate-containing NPs is
worth studying. Protein glycosylation is very abundant and
structurally diverse [115]. Glycoproteins present on tumor
cells and virions could be useful vaccine candidates. SARS-
CoV-2, for instance, has caused the world-wide emergency of
COVID-19. SARS-CoV-2 spike glycoproteins mediate the entry
of coronavirus into cells and are the main target for the anti-
body response [116]. In addition, hemagglutinin is the anti-
genic structure of influenza virus [117]. Hemagglutinin
mediates receptor binding and membrane fusion activity and
is a major target for neutralizing antibodies [117,118].
Carbohydrate-containing NPs are highly compatible with
these glycosylated proteins, and the hydrophilic groups of
carbohydrates could stabilize protein conformations.
Therefore, the effect of carbohydrate-containing NPs in vac-
cines against pathogens carrying antigenic glycoproteins, such
as SARS-CoV-2 and influenza virus, is worth studying in the
next few years.
Author contributions
Qinjian Zhao contributed to the conceptualization and Funding acquisi-
tion of this manuscript. Xinyuan Zhang wrote and edited this manuscript.
Zhigang Zhang edited this manuscript. Ningshao Xia contributed to the
Funding acquisition of this manuscript.
Declarations of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other
relationships to disclose.
Declaration of interest
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by National Natural Science Foundation of China
[32070925].
ORCID
Ningshao Xia http://orcid.org/0000-0003-0179-5266
References
Papers of special note have been highlighted as either of interest (•) or of
considerable interest (••) to readers.
1. Bergmann-Leitner ES, Leitner WW. Adjuvants in the driver’s seat:
how magnitude, type, fine specificity and longevity of immune
responses are driven by distinct classes of immune potentiators.
Vaccines (Basel). 2014 Apr 10;2(2):252–296.
EXPERT REVIEW OF VACCINES 11

2. Lin Y, Wang X, Huang X, et al. Calcium phosphate nanoparticles as
a new generation vaccine adjuvant. Expert Rev Vaccines. 2017;16
(9):895–906.
3. O’Hagan DT, MacKichan ML, Singh M. Recent developments in
adjuvants for vaccines against infectious diseases. Biomol Eng.
2001 Oct 15;18(3):69–85.
4. Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future
trends. Immunol Cell Biol. 2004 Oct;82(5):488–496.
5. Cordeiro AS, Alonso MJ, de la Fuente M. Nanoengineering of
vaccines using natural polysaccharides. Biotechnol Adv. 2015;33
(6):1279–1293.
• Overview of the development and application of polysacchar-
ide-based nanocarriers.
6. Bashiri S, Koirala P, Toth I, et al. Carbohydrate Immune Adjuvants
in Subunit Vaccines. Pharmaceutics. 2020 Oct 14;12(10):965.
7. Barclay T, Petrovsky N. In: Skwarczynski M, Toth I, editors. Micro and
Nanotechnology in Vaccine Development. New York (NY): William
Andrew Publishing; 2017. Chapter 7, Vaccine Adjuvant
Nanotechnologies; p. 127–147
8. Gheibi Hayat SM, Darroudi M. Nanovaccine: a novel approach in
immunization. J Cell Physiol. 2019 Aug;234(8):12530–12536.
9. Kelly HG, Kent SJ, Wheatley AK. Immunological basis for enhanced
immunity of nanoparticle vaccines. Expert Rev Vaccines. 2019
Mar;18(3):269–280.
10. Zhao L, Seth A, Wibowo N, et al. Nanoparticle vaccines. Vaccine.
2014 Jan 9;32(3):327–337.
11. Boons GJ. Liposomes modified by carbohydrate ligands can target
B cells for the treatment of B-cell lymphomas. Expert Rev Vaccines.
2010 Nov;9(11):1251–1256.
12. Petrovsky N, Cooper PD. Carbohydrate-based immune adjuvants.
Expert Rev Vaccines. 2011 Apr;10(4):523–537.
13. Li P, Wang F. Polysaccharides: candidates of promising vaccine
adjuvants. Drug Discov Ther. 2015 Apr;9(2):88–93.
14. Hu J, Qiu L, Wang X, et al. Carbohydrate-based vaccine adjuvants -
discovery and development. Expert Opin Drug Discov. 2015 Oct;10
(10):1133–1144.
15. McGeary RP, Olive C, Toth I. Lipid and carbohydrate based
adjuvant/carriers in immunology. J Pept Sci. 2003 Jul;9
(7):405–418.
16. Pifferi C, Fuentes R, Fernández-Tejada A. Natural and synthetic
carbohydrate-based vaccine adjuvants and their mechanisms of
action. Nat Rev Chem. 2021 Jan 25;1–20. DOI:10.1038/s41570-020-
00244-3.
•• A latest and highly relevant review that comprehensively sum-
marized the mechanisms of carbohydrates with immunostimu-
lating properties.
17. Huang YL, Wu CY. Carbohydrate-based vaccines: challenges
and opportunities. Expert Rev Vaccines. 2010 Nov;9
(11):1257–1274.
18. Dambuza IM, Brown GD. C-type lectins in immunity: recent
developments. Curr Opin Immunol. 2015 Feb;32:21–27.
19. Geijtenbeek TB, Gringhuis SI. C-type lectin receptors in the control
of T helper cell differentiation. Nat Rev Immunol. 2016 Jul;16
(7):433–448.
20. Lugade AA, Bharali DJ, Pradhan V, et al. Single low-dose
un-adjuvanted HBsAg nanoparticle vaccine elicits robust, durable
immunity. Nanomedicine. 2013 Oct;9(7):923–934.
21. Jin JW, Tang SQ, Rong MZ, et al. Synergistic effect of dual targeting
vaccine adjuvant with aminated beta-glucan and
CpG-oligodeoxynucleotides for both humoral and cellular immune
responses. Acta Biomater. 2018 Sep 15;78:211–223.
22. Bartheldyova E, Turanek Knotigova P, Zachova K, et al. N-Oxy
lipid-based click chemistry for orthogonal coupling of mannan
onto nanoliposomes prepared by microfluidic mixing: synthesis of
lipids, characterisation of mannan-coated nanoliposomes and
in vitro stimulation of dendritic cells. Carbohydr Polym. 2019 Mar
1;207:521–532.
23. Skene CD, Sutton P. Saponin-adjuvanted particulate vaccines for
clinical use. Methods. 2006 Sep;40(1):53–59.
24. Shrestha A, Kishen A. Antibacterial nanoparticles in endodontics: a
review. J Endod. 2016 Oct;42(10):1417–1426.
25. Elieh-Ali-Komi D, Hamblin MR. Chitin and Chitosan: production and
application of versatile biomedical nanomaterials. Int J Adv Res.
2016 Mar;4(3):411–427.
26. Marradi M, Di Gianvincenzo P, Enriquez-Navas PM, et al. Gold
nanoparticles coated with oligomannosides of HIV-1 glycoprotein
gp120 mimic the carbohydrate epitope of antibody 2G12. J Mol
Biol. 2011 Jul 29;410(5):798–810.
27. Lebre F, Pedroso de Lima MC, Lavelle EC, et al. Mechanistic study of
the adjuvant effect of chitosan-aluminum nanoparticles.
Int J Pharm. 2018 Dec 1;552(1–2):7–15.
28. Moran HBT, Turley JL, Andersson M, et al. Immunomodulatory
properties of chitosan polymers. Biomaterials. 2018 Nov;184:1–9.
29. Carroll EC, Jin L, Mori A, et al. The vaccine adjuvant Chitosan
promotes cellular immunity via DNA sensor cGAS-STING-
dependent induction of Type I interferons. Immunity. 2016 Mar
15;44(3):597–608.
•• This research clearly revealed the molecular mechanism of
polysaccharide chitosan to induce cellular immune response,
which could serve as basis for rational design of chitosan-
containing adjuvants.
30. Bueter CL, Lee CK, Rathinam VA, et al. Chitosan but not chitin
activates the inflammasome by a mechanism dependent upon
phagocytosis. J Biol Chem. 2011 Oct 14;286(41):35447–35455.
31. Cui Z, Mumper RJ. Chitosan-based nanoparticles for topical genetic
immunization. J Control Release. 2001 Aug 10;75(3):409–419.
32. Pawar D, Jaganathan KS. Mucoadhesive glycol chitosan nanoparti-
cles for intranasal delivery of hepatitis B vaccine: enhancement of
mucosal and systemic immune response. Drug Deliv. 2016;23
(1):185–194.
33. Kaloti M, Bohidar HB. Kinetics of coacervation transition versus
nanoparticle formation in chitosan-sodium tripolyphosphate
solutions. Colloids Surf B Biointerfaces. 2010 Nov 1;81(1):165–173.
34. Ghalavand M, Saadati M, Ahmadi A, et al. Immunological evalua-
tion of chitosan nanoparticles loaded with tetanus toxoid. Bratisl
Lek Listy. 2018;119(2):71–74.
35. Garcia-Vello P, Speciale I, Chiodo F, et al. Carbohydrate-based
adjuvants. Drug Discov Today Technol. 2020 Dec;35-36:57–68.
• A highly relevant review published recently comprehensively
summarized the applications of carbohydrate-based
adjuvants.
36. Sharma S, Mukkur TK, Benson HA, et al. Enhanced immune
response against pertussis toxoid by IgA-loaded
chitosan-dextran sulfate nanoparticles. J Pharm Sci. 2012
Jan;101(1):233–244.
37. Wang YQ, Fan QZ, Liu Y, et al. Improving adjuvanticity of quater-
nized chitosan-based microgels for H5N1 split vaccine by tailoring
the particle properties to achieve antigen dose sparing effect.
Int J Pharm. 2016 Dec 30;515(1–2):84–93.
38. Belalia R, Grelier S, Benaissa M, et al. New bioactive biomaterials
based on quaternized chitosan. J Agric Food Chem. 2008 Mar 12;56
(5):1582–1588.
39. Mourya VK, Inamdar NN. Trimethyl chitosan and its applications in
drug delivery. J Mater Sci Mater Med. 2009 May;20(5):1057–1079.
40. Chase C, Kaushik RS. Mucosal immune system of cattle: all immune
responses begin here. Vet Clin North Am Large Anim Pract. 2019
Nov;35(3):431–451.
41. Zhang S, Huang S, Lu L, et al. Curdlan sulfate-O-linked quaternized
chitosan nanoparticles: potential adjuvants to improve the immu-
nogenicity of exogenous antigens via intranasal vaccination.
Int J Nanomedicine. 2018;13:2377–2394.
42. Trapani A, Sitterberg J, Bakowsky U, et al. The potential of glycol
chitosan nanoparticles as carrier for low water soluble drugs.
Int J Pharm. 2009 Jun 22;375(1–2):97–106.
43. Jesus S, Soares E, Borchard G, et al. Adjuvant activity of poly-ε-
caprolactone/Chitosan nanoparticles characterized by mast cell
activation and IFN-γ and IL-17 production. Mol Pharm. 2018 Jan
2;15(1):72–82.
12 X. ZHANG ET AL.

44. Rose F, Wern JE, Gavins F, et al. A strong adjuvant based on
glycol-chitosan-coated lipid-polymer hybrid nanoparticles potenti-
ates mucosal immune responses against the recombinant
Chlamydia trachomatis fusion antigen CTH522. J Control Release.
2018 Feb 10;271:88–97.
45. Tsoni SV, Brown GD. Beta-Glucans and dectin-1. Ann N Y Acad Sci.
2008 Nov;1143(1):45–60.
46. Hwang J, Lee K, Gilad AA, et al. Synthesis of beta-glucan nanopar-
ticles for the delivery of single strand DNA. Biotechnol Bioprocess
Eng. 2018;23(2):144–149.
47. Zhang M, Kim JA, Huang AY. Optimizing tumor microenvironment
for cancer immunotherapy: beta-glucan-based nanoparticles. Front
Immunol. 2018;9:341.
48. Kobiyama K, Temizoz B, Kanuma T, et al. Species-dependent role of
type I IFNs and IL-12 in the CTL response induced by humanized
CpG complexed with beta-glucan. Eur J Immunol. 2016 May;46
(5):1142–1151.
49. Brown GD, Taylor PR, Reid DM, et al. Dectin-1 is a major
beta-glucan receptor on macrophages. J Exp Med. 2002 Aug
5;196(3):407–412.
50. Khan NS, Kasperkovitz PV, Timmons AK, et al. Dectin-1 controls
TLR9 trafficking to phagosomes containing beta-1,3 glucan.
J Immunol. 2016 Mar 1;196(5):2249–2261.
51. Miyamoto N, Mochizuki S, Fujii S, et al. Adjuvant Activity Enhanced
by Cross-Linked CpG-Oligonucleotides in beta-Glucan Nanogel and
Its Antitumor Effect. Bioconjug Chem. 2017 Feb 15;28(2):565–573.
52. Lee DY, Nurunnabi M, Kang SH, et al. Oral gavage delivery of PR8
antigen with beta-glucan-conjugated GRGDS carrier to enhance
M-cell targeting ability and induce immunity. Biomacromolecules.
2017 Apr 10;18(4):1172–1179.
53. Evelyn Piyachat R, Evelyn Roopngam P. Effect of pleurotus
sajor-caju polysaccharide encapsulated in poly D, L lactide-co-
glycolide nanoparticles for HPV vaccine in murine model.
Nanomedicine. 2018;5(3):144–151.
54. Gonzalez PS, O’Prey J, Cardaci S, et al. Mannose impairs tumour
growth and enhances chemotherapy. Nature. 2018 Nov;563
(7733):719–723.
55. Zhang D, Chia C, Jiao X, et al. D-mannose induces regulatory T cells
and suppresses immunopathology. Nat Med. 2017 Sep;23
(9):1036–1045.
56. Robinson MJ, Sancho D, Slack EC, et al. Myeloid C-type lectins in
innate immunity. Nat Immunol. 2006 Dec;7(12):1258–1265.
57. Ip WK, Takahashi K, Ezekowitz RA, et al. Mannose-binding lectin
and innate immunity. Immunol Rev. 2009 Jul;230(1):9–21.
58. Geijtenbeek TB, Gringhuis SI. Signalling through C-type lectin
receptors: shaping immune responses. Nat Rev Immunol. 2009
Jul;9(7):465–479.
59. Kaur A, Jain S, Tiwary AK. Mannan-coated gelatin nanoparticles for
sustained and targeted delivery of didanosine: in vitro and in vivo
evaluation. Acta Pharm. 2008 Mar;58(1):61–74.
60. Kramer S, Langhanki J, Krumb M, et al. HPMA-based nanocarriers
for effective immune system stimulation. Macromol Biosci. 2019
Jun;19(6):e1800481.
61. Li Z, Xiong F, He J, et al. Surface-functionalized, pH-responsive poly
(lactic-co-glycolic acid)-based microparticles for intranasal vaccine
delivery: effect of surface modification with chitosan and mannan.
Eur J Pharm Biopharm. 2016 Dec;109:24–34.
62. Doores KJ, Kong L, Krumm SA, et al. Two classes of broadly neu-
tralizing antibodies within a single lineage directed to the
high-mannose patch of HIV envelope. J Virol. 2015 Jan 15;89
(2):1105–1118.
63. Shan M, Klasse PJ, Banerjee K, et al. HIV-1 gp120 mannoses induce
immunosuppressive responses from dendritic cells. PLoS Pathog.
2007 Nov;3(11):e169.
64. Daniels CN, Saunders KO. Antibody responses to the HIV-1 envel-
ope high mannose patch. Adv Immunol. 2019;143:11–73.
65. Toda S, Ishii N, Okada E, et al. HIV-1-specific cell-mediated immune
responses induced by DNA vaccination were enhanced by
mannan-coated liposomes and inhibited by anti-interferon-
gamma antibody. Immunology. 1997 Sep;92(1):111–117.
66. Glass JJ, Kent SJ, De Rose R. Enhancing dendritic cell activation and
HIV vaccine effectiveness through nanoparticle vaccination. Expert
Rev Vaccines. 2016 Jun;15(6):719–729.
67. Kensil CR, Wu JY, Soltysik S. Structural and immunological charac-
terization of the vaccine adjuvant QS-21. Pharm Biotechnol.
1995;6:525–541.
68. Cox JC, Sjölander A, Barr IG. ISCOMs and other saponin based
adjuvants. Adv Drug Deliv Rev. 1998 Jul 6;32(3):247–271.
69. Del Giudice G, Rappuoli R, Didierlaurent AM. Correlates of adjuvan-
ticity: a review on adjuvants in licensed vaccines. Semin Immunol.
2018 Oct;39:14–21.
70. Laurens MB. RTS,S/AS01 vaccine (Mosquirix™): an overview. Hum
Vaccin Immunother. 2020 Mar 3;16(3):480–489.
71. Press JB, Reynolds RC, May RD, et al. In: Atta-ur-Rahman, editor.
Studies in Natural Products Chemistry: Structure—function rela-
tionship of immunostimulating Saponins. Amsterdam
(Netherlands): Elsevier Academic Press; 2000. p. 131–174
72. Lorent JH, Quetin-Leclercq J, Mingeot-Leclercq MP. The amphiphilic
nature of saponins and their effects on artificial and biological
membranes and potential consequences for red blood and cancer
cells. Org Biomol Chem. 2014 Nov 28;12(44):8803–8822.
73. Kensil CR, Kammer R. QS-21: a water-soluble triterpene glycoside
adjuvant. Expert Opin Investig Drugs. 1998 Sep;7(9):1475–1482.
74. de Groot C, Müller-Goymann CC. Saponin interactions with model
membrane systems - Langmuir monolayer studies, hemolysis and
formation of ISCOMs. Planta Med. 2016 Dec;82(18):1496–1512.
75. Sun HX, Xie Y, Ye YP. ISCOMs and ISCOMATRIX. Vaccine. 2009 Jul
16;27(33):4388–4401.
76. Coccia M, Collignon C, Hervé C, et al. Cellular and molecular
synergy in AS01-adjuvanted vaccines results in an early IFNγ
response promoting vaccine immunogenicity. NPJ Vaccines.
2017;2(1):25.
77. Shah RA, Limmer AL, Nwannunu CE, et al. Shingrix for Herpes
Zoster: a review. Skin Therapy Lett. 2019 Jul;24(4):5–7.
78. Sjölander A, Drane D, Maraskovsky E, et al. Immune responses to
ISCOM formulations in animal and primate models. Vaccine. 2001
Mar 21;19(17–19):2661–2665.
79. Sanders MT, Brown LE, Deliyannis G, et al. ISCOM-based vaccines:
the second decade. Immunol Cell Biol. 2005 Apr;83(2):119–128.
80. Lövgren Bengtsson K, Morein B, Osterhaus AD. ISCOM technology-
based Matrix M™ adjuvant: success in future vaccines relies on
formulation. Expert Rev Vaccines. 2011 Apr;10(4):401–403.
81. Klein O, Davis ID, McArthur GA, et al. Low-dose cyclophosphamide
enhances antigen-specific CD4(+) T cell responses to NY-ESO-1/
ISCOMATRIX™ vaccine in patients with advanced melanoma.
Cancer Immunol Immunother. 2015 Apr;64(4):507–518.
• This study proved that a vaccine using saponin-containing
nanoparticulate adjuvants had potent effect on the treatment
of cancer.
82. Pedersen GK, Madhun AS, Breakwell L, et al. T-helper 1 cells elicited
by H5N1 vaccination predict seroprotection. J Infect Dis. 2012 Jul
15;206(2):158–166.
83. Pushko P, Tretyakova I. Influenza Virus Like Particles (VLPs): oppor-
tunities for H7N9 Vaccine Development. Viruses. 2020 May 8;12
(5):518.
84. Venkatraman N, Anagnostou N, Bliss C, et al. Safety and immuno-
genicity of heterologous prime-boost immunization with viral-
vectored malaria vaccines adjuvanted with Matrix-M™. Vaccine.
2017 Oct 27;35(45):6208–6217.
85. Dong Y, Dai T, Wei Y, et al. A systematic review of SARS-CoV-2
vaccine candidates. Signal Transduct Target Ther. 2020 Oct 13;5
(1):237.
86. Keech C, Albert G, Cho I, et al. Phase 1-2 Trial of a SARS-CoV-2
Recombinant Spike Protein Nanoparticle Vaccine. N Engl J Med.
2020 Sep 2;383(24):2320–2332.
•• A recent clinical trial evaluated safety and efficacy of
a promising vaccine against SARS-CoV-2 named NVX-
CoV2373 (Novavax), in which saponin-containing nanopar-
ticulate adjuvants contribute much to vaccine
effectiveness.
EXPERT REVIEW OF VACCINES 13

87. Mahase E. Covid-19: novavax vaccine efficacy is 86% against UK
variant and 60% against South African variant. BMJ. 2021;372:n296.
•• Latest clinical efficacy report for one of the most promising
vaccines against SARS-CoV-2.
88. Novavax confirms high levels of efficacy against original and var-
iant COVID-19 strains in United Kingdom and South Africa trials
2021 [cited 2021 May 04]. Available from: https://ir.novavax.com/
node/15661/pdf
89. Cibulski SP, Mourglia-Ettlin G, Teixeira TF, et al. Novel ISCOMs from
Quillaja brasiliensis saponins induce mucosal and systemic anti-
body production, T-cell responses and improved antigen uptake.
Vaccine. 2016 Feb 24;34(9):1162–1171.
90. Cibulski SP, Rivera-Patron M, Mourglia-Ettlin G, et al. Quillaja brasi-
liensis saponin-based nanoparticulate adjuvants are capable of
triggering early immune responses. Sci Rep. 2018 Sep 11;8
(1):13582.
91. Turmagambetova AS, Alexyuk PG, Bogoyavlenskiy AP, et al.
Adjuvant activity of saponins from Kazakhstani plants on the
immune responses to subunit influenza vaccine. Arch Virol. 2017
Dec;162(12):3817–3826.
92. Song X, Zang L, Hu S. Amplified immune response by
ginsenoside-based nanoparticles (ginsomes). Vaccine. 2009 Apr
14;27(17):2306–2311.
93. Tong YN, Yang LY, Yang Y, et al. An immunopotentiator, ophio-
pogonin D, encapsulated in a nanoemulsion as a robust adju-
vant to improve vaccine efficacy. Acta Biomater. 2018 Sep;1
(77):255–267.
94. Zhao JH, Zhang QB, Liu B, et al. Enhanced immunization via dis-
solving microneedle array-based delivery system incorporating
subunit vaccine and saponin adjuvant. Int J Nanomedicine.
2017;12:4763–4772.
95. Marciani DJ. Elucidating the mechanisms of action of
Saponin-derived adjuvants. Trends Pharmacol Sci. 2018 Jun;39
(6):573–585.
•• Most comprehensive review of the mechanism of action of
triterpene saponin, an active component of adjuvant AS01,
ISCOM, ISCOMATRIX and Matrix M.
96. Rhodes J, Chen H, Hall SR, et al. Therapeutic potentiation of the
immune system by costimulatory Schiff-base-forming drugs.
Nature. 1995 Sep 7;377(6544):71–75.
97. Soltysik S, Wu JY, Recchia J, et al. Structure/function studies of
QS-21 adjuvant: assessment of triterpene aldehyde and glucuro-
nic acid roles in adjuvant function. Vaccine. 1995;13
(15):1403–1410.
98. Pati R, Shevtsov M, Sonawane A. Nanoparticle vaccines against
infectious diseases. Front Immunol. 2018;9:2224.
99. Wang T, Zou M, Jiang H, et al. Synthesis of a novel kind of carbon
nanoparticle with large mesopores and macropores and its appli-
cation as an oral vaccine adjuvant. Eur J Pharm Sci. 2011 Dec 18;44
(5):653–659.
100. Wusiman A, Xu S, Ni H, et al. Immunomodulatory effects of Alhagi
honey polysaccharides encapsulated into PLGA nanoparticles.
Carbohydr Polym. 2019 May;1(211):217–226.
101. Dhakal S, Lu F, Ghimire S, et al. Corn-derived alpha-D-glucan
nanoparticles as adjuvant for intramuscular and intranasal immu-
nization in pigs. Nanomedicine. 2019 Feb;16:226–235.
102. Cummings RD. Stuck on sugars - how carbohydrates regulate cell
adhesion, recognition, and signaling. Glycoconj J. 2019 Aug;36
(4):241–257.
103. Mythreye K, Blobe GC. Proteoglycan signaling co-receptors: roles in
cell adhesion, migration and invasion. Cell Signal. 2009 Nov;21
(11):1548–1558.
104. Villar RF, Patel J, Weaver GC, et al. Reconstituted B cell receptor
signaling reveals carbohydrate-dependent mode of activation. Sci
Rep. 2016 Oct;31(6):36298.
105. Hakomori S. Carbohydrate-to-carbohydrate interaction, through
glycosynapse, as a basis of cell recognition and membrane
organization. Glycoconj J. 2004;21(3–4):125–137.
106. Xia Y, Wu J, Wei W, et al. Exploiting the pliability and lateral
mobility of Pickering emulsion for enhanced vaccination. Nat
Mater. 2018 Feb;17(2):187–194.
107. Desai KG. Chitosan nanoparticles prepared by ionotropic gelation:
an overview of recent advances. Crit Rev Ther Drug Carrier Syst.
2016;33(2):107–158.
108. Ratih DN, Sari NI, Santosa P, et al. Time-dependent effect of chit-
osan nanoparticles as final irrigation on the apical sealing ability
and push-out bond strength of root canal obturation. Int J Dent.
2020;2020:8887593.
109. Del Carpio-Perochena A, Bramante CM, Duarte MA, et al. Chelating
and antibacterial properties of chitosan nanoparticles on dentin.
Restor Dent Endod. 2015 Aug;40(3):195–201.
110. Holmgren J, Svennerholm AM. Vaccines against mucosal infections.
Curr Opin Immunol. 2012 Jun;24(3):343–353.
111. Kumar S, Anselmo AC, Banerjee A, et al. Shape and size-dependent
immune response to antigen-carrying nanoparticles. J Control
Release. 2015 Dec 28;220(Pt A):141–148.
112. Connolly A, Gagnon E. Electrostatic interactions: from immune
receptor assembly to signaling. Immunol Rev. 2019 Sep;291
(1):26–43.
113. Vogel FR, Sarver N. Nucleic acid vaccines. Clin Microbiol Rev. 1995
Jul;8(3):406–410.
114. Alfagih IM, Aldosari B, AlQuadeib B, et al. Nanoparticles as adju-
vants and nanodelivery systems for mRNA-based vaccines.
Pharmaceutics. 2020 Dec 30;13(1):45.
115. Jakab L. [Biological role of heterogeneous glycoprotein structures].
Orv Hetil. 2016 Jul;157(30):1185–1192.
116. Walls AC, Park YJ, Tortorici MA, et al. Structure, function, and antigeni-
city of the SARS-CoV-2 Spike glycoprotein. Cell. 2020 Apr 16;181
(2):281–292.e6.
117. Schulze IT. Effects of glycosylation on the properties and functions of
influenza virus hemagglutinin. J Infect Dis. 1997 Aug;176(Suppl 1):S24–
8.
118. Tate MD, Job ER, Deng YM, et al. Playing hide and seek: how
glycosylation of the influenza virus hemagglutinin can modulate
the immune response to infection. Viruses. 2014 Mar 14;6
(3):1294–1316.
14 X. ZHANG ET AL.