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DOI: 10.2478/nanca-2014-0001 Nanocarriers 2014; volume 1, 10–45
Review article
Open Access
Priyanka Prabhu and Vandana Patravale*
Potential of Nanocarriers in Antigen Delivery:
The Path to Successful Vaccine Delivery
Abstract: Vaccination has indubitably made noteworthy
contribution to global health. Recent years have witnessed
the employment of subunit antigens rather than inactivated
or live attenuated vaccines, owing to the superior safety of
the former. The intrinsic weak immunogenicity of subunit
antigens makes it imperative to formulate them with an
adjuvant. Presently, the armamentarium of approved
vaccine adjuvants is very poor. Nanocarriers hold great
promise for successful vaccine delivery owing to their
versatility, excellent cellular uptake properties, capacity
to protect antigen, amenability to targeting, and ability to
offer prolonged antigen presentation. All these attributes
ultimately endow nanocarriers with immense potential to
achieve needle-free vaccine delivery, reduce the number
of vaccinations, attain dose sparing of antigen, and lead
to stronger immune response generation. Nanocarriers
can be explored in manifold ways to accomplish
targeted antigen delivery to antigen presenting cells.
They can be formulated to contain both antigen and
immunostimulant molecules, and they can be engineered
from specific materials to achieve antigen presentation
through the desired pathway to stimulate a particular
arm of the immune response. This review discusses the
basics of immune response generation, mechanisms of
adjuvanticity by nanocarriers, parameters influencing
their adjuvanticity, and finally describes the incredible
opportunities offered by a gamut of nanocarriers for
vaccine delivery.
Keywords: Vaccine, nanoparticles, antigen, adjuvant,
dendritic cells, liposomes, chitosan, polymeric, calcium
phosphate, emulsions.
*Corresponding author: Vandana Patravale: Department of
Pharmaceutical Sciences and Technology, Institute of Chemical
Technology, Matunga, Mumbai-400019, India, Telephone no: 91-223361 2217, Fax no: 91-22-3361 1020, E-mail: [email protected]
Priyanka Prabhu: Department of Pharmaceutical Sciences
and Technology, Institute of Chemical Technology, Matunga,
Mumbai-400019, India
List of Abbreviations
APCs - Antigen Presenting Cells
AMVAD- Archaeal lipid Mucosal Vaccine Adjuvant and
Delivery
BRSV - Bovine Respiratory Synctial Virus
BSA
- Bovine Serum Albumin
BALT - Bronchus Associated Lymphoid Tissue
CMIS - Common Mucosal Immune System
CFA
- Complete Freund’s Adjuvant
CALT - Conjunctiva Associated Lymphoid tissue
DOTMA - N-[1-(2, 3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
GIT
- Gastrointestinal tract
GSK
- GlaxoSmithKline
GALT - Gut Associated Lymphoid Tissue
HSV- Herpes simplex virus
HIV
- Human Immunodeficiency Virus
HPV
- Human PapillomaVirus
ISCOMs - Immunostimulating complexes
ID
- Intradermal
IM
- Intramuscular
LPS
- Lipopolysaccharide
MHC - Major Histocompatibility Complex
MVP
- Major Vault Protein
MPLA - Monophosphoryl Lipid A
MALT - Mucosa Associated Lymphoid Tissues
MDP
- Muramyl dipeptide
NALT - Nasal Associated Lymphoid Tissue
NOD - Nucleotide-binding oligomerization domain
PAMPs - Pathogen Associated Molecular Patterns
PRRs - Pattern Recognition Receptors
PAMAM - Polyamidoamine
PCPP - Poly [di(carboxylatophenoxy)phosphazene]
PCEP - Poly
[di(sodiumcarboxylatoethylphenoxy)
phosphazene]
PEI - Polyethyleneimine
PLA
- Poly Lactic Acid
PLGA - Poly Lactic-co-glycolide
RSV
- Respiratory Syncytial Virus
SWCT - Single walled carbon nanotubes
© 2014 Priyanka Prabhu, Vandana Patravale licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercialNoDerivs 3.0 License.
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SALT
SLN
SC
TMC
TLRs
TDB
TNF
VLPs
Nanocarriers for vaccine delivery - Skin Associated Lymphoid Tissue
- Solid Lipid Nanoparticles
- Subcutaneous
- N-trimethyl chitosan
- Toll-like receptors
- Trehalose 6’6-dibehenate
- Tumour Necrosis Factor
- Virus like particles
1 Introduction
The main aim of vaccination is to incite a strong pathogenspecific immune response to confer life-long protection
against subsequent exposure to the pathogen [1]. An ideal
vaccine must be efficacious, non-toxic, cost-effective,
and amenable to large scale manufacture [2]. Since
its inception, vaccination has played a pivotal role in
reducing the morbidity and mortality of several debilitating
infections [3]. Eradication of small pox and significant
reduction in the global burden of polio, tetanus, pertussis,
mumps, rubella, diphtheria, yellow fever, and measles
[4] exemplify the incredible impact of vaccines on global
healthcare. Despite these impressive outcomes, there are a
few diseases for which effective vaccines are unavailable,
11
including AIDS, malaria, and tuberculosis [3]. As far
as vaccine delivery is concerned, there are three major
hurdles. Firstly, the majority of vaccines suffer from poor
thermal stability, necessitating cold storage, which poses
a hurdle for vaccine storage in developing nations [5].
Secondly, most vaccines require multiple administrations
resulting in poor patient compliance [6]. Thirdly, due to the
low immunogenicity of antigens administered via the noninvasive mucosal route of administration, most vaccines
are intended for parenteral administration. This not only
results in poor patient compliance but is also unable to
elicit mucosal immunity, eliciting only a systemic antibody
response, which is often insufficient to tackle pathogens
that employ mucosal surfaces for ingress into the host or
which result in disease at mucosal surfaces [7]. Additionally,
most vaccines only generate a humoral immune response,
which only deals with extracellular pathogens, whereas
cell-mediated immunity needs to be induced in order to
tackle intracellular pathogens [8].
There are three different types of vaccines, shown in
Table 1 [4,9-11].
Vaccines may be administered by three routes:
parenteral (invasive), mucosal (non-invasive), and
topical/transdermal (non-invasive) routes. Figure 1
Table 1: Types of vaccines [4,9,10,11].
Type
Key features
Killed/
Inactivated
Include microbes inactivated
by application of heat / chemical treatment [9]
Live attenuated
Advantages
Drawbacks
Examples
Not able to multiply and enter into
host cells [9]
Require multiple doses to elicit an
adequate immune response [9]
Prepared by repeated passage Ability to multiply within
Probability of causing disease in
of pathogen in culture to
the host and closely mimic
immunocompromised individuals
render microbe non-pathoge- natural infection [9]
and their probable reversion to
nic [9]
Single administration results virulence [9]
in development of a strong
humoral and cell-mediated
immune response [9]
Subunit vaccines Include portions of pathogenic microbes (toxins, proteins,
polysaccharides, or recombinant proteins) [9,10]
Possess superior safety
to live vaccines and lower
antigen competition due
to small number of defined
components [11]
Amenable to targeting to
specific sites where immunity is required [11]
Vaccinated animals can be
distinguished from infected
animals [11]
Their cost effective production is possible through
recombinant DNA technology
[11]
Inherent poor immunogenicity [9]
Low uptake by antigen presenting
cells (APCs) [9]
In vivo instability [9]
Parenteral Polio
vaccine, Influenza,
and Hepatitis A vaccines [9]
Small pox, oral polio
vaccine, measles,
mumps, rubella, and
Bacillus Calmette
Guerin (BCG)
[4,9]
Tetanus, Diphtheria,
and Human Papilloma
virus (HPV)
[9]
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12 Priyanka Prabhu and Vandana Patravale
Figure 1: Comparison of mucosal v/s parenteral immunization [12].
compares the key features of mucosal v/s parenteral
immunization [12].
1.1 Parenteral vaccination
Parenteral routes commonly employed for vaccination
purposes include the intramuscular (IM), subcutaneous
(SC), and intradermal (ID) routes.
1.1.1 Intramuscular route
The IM route is a very popular route for routine vaccination.
Cellular and humoral immunization achieved through
this route is comparable to that achieved after SC and
ID administration. Myoblasts are antigen-presenting
cells (APCs) found in the muscle tissue responsible for
immune response generation after IM administration.
However, due to the limited number of dendritic cells in
the muscle in comparison to the skin, in order to elicit an
effective immune response it is necessary to administer
higher doses of antigen by the IM route in comparison
to ID immunization [2]. Myocytes do not express major
histocompatibility complex (MHC) class II molecules
and are devoid of costimulatory compounds, rendering
them incapable of directly activating T cells [13]. IM
immunization results in a predominant Th2 response
unless boosters are given subsequently [2]. Examples
of vaccines which are currently administered via the IM
route include hepatitis A, hepatitis B, diphtheria toxoid,
inactivated polio vaccine, pertussis, human papilloma
virus (HPV), rabies vaccine, Streptococcus pneumonia,
and tetanus toxoid [10].
1.1.2 Subcutaneous route
The SC route is beneficial for antigen delivery owing to
the drainage of antigen from the injection site to lymph
nodes which house the immunocompetent cells [14].
Particles in the micrometer as well as nanometer size
range have been explored by the subcutaneous route
for vaccine adjuvant purposes [15,16]. The delivery of
carriers administered via the SC route to the lymph nodes
is governed by their size. Particles greater than 1000 nm
stay at the site of injection until they are broken down
into particles of a smaller size. They are neither able
to gain entry into the lymphatics directly nor are they
phagocytosed easily. Particles less than 100 nm in size
are able to enter the lymphatic capillaries via the gaps
between lymphatic endothelial cells. Particles between
100-1000 nm undergo phagocytosis by APCs, such as
dendritic cells, followed by passage into lymphatic
capillaries [14]. Benefits associated with the SC route
include lower clearance and longer persistence at the
site of administration resulting in prolonged antigen
presentation to the immune system [17]. Examples of
vaccines which are currently administered via the SC
route include the anthrax vaccine, Haemophilus influenza
type b vaccine, inactivated polio vaccine, Japanese
encephalitis, measles, mumps, rubella, chicken pox, S.
pneumonia, and yellow fever [10].
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Nanocarriers for vaccine delivery 1.1.3 Intradermal route
13
Mucosal routes explored for vaccination include nasal,
oral, pulmonary, sublingual, ocular, rectal, and the
vaginal route. The presence of mucus, poor antigen uptake
through the mucosa into immunocompetent tissue,
antigen degradation, and inability of the mucosal tissue
to respond to non-replicating antigens leads to failure
of mucosal immunization to generate a potent immune
response [19].
in soluble form is known to elicit a systemic immune
response because it can traverse the nasal epithelium
and gain access to the cervical lymph nodes, resulting in
IgG production. On the other hand, antigen administered
in particulate form is uptaken by M cells to produce IgA
antibody [12]. Based on the physiological environment
in the nasal cavity, an ideal intranasal vaccine delivery
system should have a number of attributes, as outlined
in Table 2 [21]. A major drawback to nasal vaccination
is the poor retention of vaccine formulation in the nasal
cavity due to nasal mucociliary clearance, which results
in removal of the vaccine formulation within 15 minutes
of administration, thus limiting vaccine uptake into the
nasal mucosa.Mucoadhesive substances, both in soluble
and particulate form, nanogels, and in situ gelling
systems have been utilized to augment the nasal retention
time of antigens and enhance the immune response [22].
Other drawbacks associated with the nasal route are the
probable delivery into the brain via the olfactory region,
and problems due to asthama, respiratory syndromes,
and allergy [21].
1.2.1 Nasal route
1.2.2 Oral route
The nasal route is well suited for the induction of
immunity against antigens which enter the body through
inhalation (e.g. influenza). The leaky characteristic of
the nasal epithelium can result in entry of the antigen
into the underlying blood vessels, cervical lymph nodes
and lymphocytes to elicit a potent immune response
[20]. In case of nasal vaccination, immune response
generation occurs through nasal-associated lymphoid
tissue (NALT). NALT includes M cells, lymphoid follicles,
dendritic cells, and goblet cells. Antigen administered
The oral route is the easiest and the most preferred route
for vaccination from a patient compliance perspective.
Gut-associated lymphoid tissue (GALT) is involved in the
generation of an immune response after oral vaccination.
GALT is comprised of the Peyer’s patches, where M cells
process the antigen and carry it to APCs such as dendritic
cells and macrophages. Oral vaccination induces an
immune response in the small intestine, ascending
colon, and distant tissues such as mammary glands and
salivary glands owing to the common mucosal immune
This route offers immense potential for successful
immunization owing to the localization of the most potent
APCs (dendritic cells) and other immune cells in the
dermis. The US Center for Disease Control and Prevention
has approved two vaccines for ID vaccination, namely the
Mycobacterium bovis tuberculosis vaccine, better known
as the Bacille Calmette Guerin (BCG), and the Vaccinia
smallpox vaccination [18].
1.2 Mucosal vaccination
Table 2: Attributes of an ideal intranasal vaccine delivery system [21].
Attribute
Benefit
Nanoparticulate system (less than 250 nm)
Enhanced cellular uptake by APC
Hydrophilic surface
Avoid aggregation and allow transport of discrete particles
Preferably contain targeting moiety
Receptor binding to M cells of nasal mucosa or APC
Afford protection to antigen against degradation
Dose sparing of antigen and enhanced vaccine efficacy
Exhibit prolonged antigen release into APC in nasal tissue
Reduce number of vaccinations
Comprise of immunostimulant molecule and antigen
Non-toxic and non-irritant to nasal tissue
Simultaneous delivery of immunostimulant molecule and antigen to
same APC for enhanced immune response generation
Safety
Preferably mucoadhesive
Prolonged nasal retention
Simple to fabricate
Easy public availability
Amenable to large scale production
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14 Priyanka Prabhu and Vandana Patravale
system (CMIS). But oral vaccination lacks the ability to
elicit an immune response in the female genital tract
and the tonsils. Administration of antigen in particulate
form enhances uptake by the Peyer’s patches [12].
To date, theoral polio vaccine is the most efficient
oral vaccine [22]. Other orally administered vaccines
include rotavirus, typhoid fever, and cholera vaccine
[10]. A major snag associated with oral vaccination is
the degradation of antigen in the harsh environment of
the GI tract comprising low pH and enzymes [12]. Oral
immunization is also associated with antigen tolerance.
The immune system has evolved to dampen systemic
immune responses against many substances which
pass through the gastrointestinal tract (GIT) in huge
quantities. As a result, larger doses of antigen have to be
administered via the oral route than the parenteral route
[8].
The nasal route has some benefits over the oral
route [12]. These include the superior permeability of the
nasal mucosa in comparison to the gastro-intestinal (GI)
mucosa, presence of dendritic cells in the NALT which
are reputedly the most potent APCs, rapid and stronger
immune response generation than the oral route, ability
of the NALT to retain immunological memory resulting
in a faster immune response on second exposure, ability
to elicit cytotoxic T lymphocyte stimulation to deal with
intracellular pathogens, lower dose of antigen required
due to absence of dilution of antigen in nasal fluids, low
enzymatic action, no exposure to low pH, and potent
immune response in the respiratory and genital tracts due
to the CMIS [21].
1.2.3 Pulmonary route
The respiratory tract is well equipped with
immunocompetent cells to tackle the pathogens which
commonly enter the body through the respiratory
tract. Human lungs are endowed with a huge surface
area, are extensively vascularised, and contain APCs
such as dendritic cells and alveolar macrophages [22].
Pulmonary vaccines are administered to the alveoli
using an aerosol [12]. Bronchus-associated lymphoid
tissue (BALT) is present in the lungs, and is able to
generate not only a systemic immune response but also
a mucosal immune response in both the respiratory
tract and the distal mucosal tissues. An example of a
vaccine delivered by the pulmonary route is the live
Newcastle disease vaccine for poultry. To date, there
is no commercially available vaccine for pulmonary
administration in humans [22].
1.2.4 Sublingual route
Sublingual immunotherapy is utilized to treat type I
allergy, which involves desensitization of the patient by
sublingual allergen delivery [12]. The route offers numerous
benefits over the oral route due to the absence of low pH
and comparatively lower enzymatic activity. Both live and
inactivated influenza virus, when administered through
the sublingual route, have been demonstrated to elicit
an immune response and protect mice against influenza
infection. Also, sublingual administration of inactivated
viruses has been shown to produce secretory IgA antibody
and cytotoxic T lymphocyte response in areas. The
sublingual route could prove to be useful in children and
infants owing to its simple administration [22].
1.2.5 Ocular route
This route could be utilized to achieve successful
immunization against microbes infecting the ocular
tissue. Both and systemic immune responses are induced
due to the conjunctiva-associated lymphoid tissue (CALT).
The route does not suffer from the drawbacks of the GI
environment, such as low pH, or of the nasal route, such
as antigen entry into the brain [12].
1.2.6 Rectal route
This route could be used as a substitute to oral
immunization owing to its ability to generate a potent
immune response in the intestine and other distant parts
without subjecting the antigen to hostile conditions of low
pH and proteolytic enzymes. The chief shortcomings of
the rectal route are poor patient acceptability and removal
of the vaccine after administration [12].
1.2.7 Vaginal route
This route would be ideal for conferring mucosal immunity
in the female genital tract against sexually transmitted
diseases such as the human immunodeficiency virus (HIV),
HPV, and Herpes simplex virus (HSV). A major disadvantage
associated with this route for immunization is the
dependence of the immune response on hormonal control
and the variation according to the stages of the menstrual
cycle. Reports suggest that nasal immunization results in
a more effective immune response in the vaginal mucosa
compared to vaginal immunization due to the CMIS [12].
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1.3 Topical/transdermal vaccination
This route offers several beneficial attributes for
vaccination. Firstly, it is non-invasive. Secondly, it prevents
exposure of the antigen to low pH or proteolytic enzyme
activity [23]. Thirdly, it is inherently endowed with more
immunocompetent cells than the muscles or SC tissues
because of its close contact with the outer environment
[22]. Also, it drains the antigen into the lymph nodes
for effective immune response generation [24]. Topical
immunization reduces the variation associated with the
IM and SC route depending on the site of injection, depth
of injection, local blood supply, and movement of the body
[23]. Skin-associated lymphoid tissue (SALT) is involved
in the induction of immunity after topical immunization.
SALT comprises the Langerhans cells and keratinocytes
present in the epidermis, dendritic cells and mast cells
present in the dermis, and B and T lymphocytes present in
the draining lymph nodes [25]. Langerhans cells are potent
APCs which take up antigen, process it, and present it to
naive T cells in the draining lymph nodes. Keratinocytes in
the epidermis produce pro-inflammatory cytokines, such
as IL-1 and tumour necrosis factor-α (TNF-α), on encounter
with danger signals resulting in the activation, maturation
and movement of Langerhans cells and drawing of effector
cells at the point of inflammation [26].
A major hindrance to topical vaccine delivery is
the barrier property of the skin due to the stratum
corneum. Only antigens with high lipophilicity
and a molecular mass of less than 500 Da can
passively traverse the stratum corneum [25]. Hence,
it is imperative to employ permeation enhancement
approaches for effective topical delivery of vaccines.
These may include physical approaches or formulation
of suitable delivery vehicles to deliver antigen across
the formidable skin barrier.
2 Basics of immune response
generation
Complete discussion of the human immune system in all
its complexity is too vast and beyond the scope of this
review. However, the following section will describe in
a nutshell, the functioning of the immune system, and
introduce the reader to the commonly used terminologies
and phenomena in immunology. This will afford a better
understanding of the immune response modulation
shown by vaccine adjuvants and eventually guide
researchers to design efficient vaccine adjuvants to elicit
the desired immune response.
Nanocarriers for vaccine delivery 15
2.1 Defence mechanisms of the human
immune system
The human body is endowed with the ability to defend itself
against pathogens encountered within the environment.
The human immune system consists of primary lymphoid
organs and secondary lymphoid organs. Primary lymphoid
organs consist of the bone marrow and the thymus.
These are the sites of generation of B lymphocytes and
T lymphocytes respectively. Secondary lymphoid organs
include the spleen, lymph nodes, skin, adenoids, tonsils,
and themucosa-associated lymphoid tissues (MALT), and
GALT (Peyer’s patches). Initiation of an adaptive immune
response takes place in the secondary lymphoid organs
[18].
The human immune system comprises three different
types of defence mechanisms.
1. Skin, ciliated epithelial cells and other mucous
membranes of the body which form an external
barrier and serve as the first line of defence against
invading pathogens; and enzymes and other chemical
secretions such as stomach acid [27].
2. Innate immune system. The innate immune system
constitutes the first line of protection against
pathogens which have entered the human body,
and comprises macrophages and dendritic cells [27].
Migratory dendritic cells exist in the periphery and
on activation by antigen migration to the lymphoid
tissues. Such migratory dendritic cells are present
in the skin, lungs, liver, kidneys, and intestinal tract
[2]. The innate immune system also has mobile cells
such as neutrophils, monocytes, and eosinophils
which tour throughout the body via blood and lymph
screening for pathogens [27,28]. A rapid immune
response can be induced against the pathogen in
question owing to the ability of the cells of the innate
immune system to be immediately recruited to the
site of infection. Cells of the innate immune system
are endowed with a set of receptors called “pattern
recognition receptors (PRRs)” which identify a number
of molecular structures unique to pathogens of a
particular class. These structures are called pathogenassociated molecular patterns (PAMPs) [27]. There are
different types of PRRs, including toll-like receptors
(TLRs), C-type lectin receptors, nucleotide-binding
oligomerization domain (NOD) receptors, NOD-like
receptors, and retinoic acid-inducible gene 1(RIG-1-like
helicases) [29]. TLRs are the most extensively explored
PRRs for immunomodulation. TLRs can be categorized
according to their localization and the PAMPs they
recognize. Different TLRs recognize an array of
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16 Priyanka Prabhu and Vandana Patravale
Table 3: List of endogenous and exogenous ligands for various TLRs [2, 27, 30].
TLR
Endogenous ligand
Exogenous ligand
Effect
TLR1
Triacyl lipoproteins
Microbial peptidoglycans
Stimulate inflammatory cytokine secretion
TLR2
Unknown
TLR3
mRNA
Gram positive peptidoglycan, lipoproteins, viral glyco- Stimulate inflammatory cytokine secretion
proteins, glycolipids
dsRNA, siRNA
Production of Type I interferons
TLR4
TLR5
Fibrinogen, hyaluronic
acid, defensin 2
Unknown
TLR6
Unknown
Lipopolysaccharide, RSV fusion protein, virus envelop Production of Type I interferons
protein
Flagellin
Present in intestinal and lung epithelia,
plays a key role in mucosal immune response generation
Lipopeptides
Stimulate inflammatory cytokine secretion
TLR7/8
Unknown
ssRNA, imiquimod, resiquimod, imidazoquinoline
TLR9
Chromatin complex
CpG DNA
TLR10
Unknown
Unknown
molecular motifs unique to pathogens. Table 3 shows
different TLRs with their ligands and the effect seen
on their activation [2, 27, 30]. TLRs 1, 2, 4, 5 and 6 are
chiefly expressed on the surface of the cell membrane
and recognize products of bacterial origin, whereas
TLRs 3, 7, 8, and 9 are found inside the intracellular
compartments and identify virus-derived products
and nucleic acids. The innate immune system is not
only capable of sensing infection through direct PRRmediated pathogen identification, but also the effects
of an infection via recognition of the danger signals or
stress signals released by infected cells. These signals
include uric acid or ATP, which are released during
cell lysis owing to infection. The innate immune
system exerts its activity through phagocytosis. All
the cells of the innate immune system, irrespective of
whether they are resident or moving, show efficient
phagocytosing ability. Once the innate immune cell
encounters a pathogen, it engulfs it and traps it within
an intracellular vesicular structure, followed by its
destruction by digestive enzymes or reactive oxygen
species [27].
3. Adaptive immune system. Pathogens manifesting a
high mutagenic rate may eventually evade the innate
immune system owing to the narrow diversity of
PRRs. Intracellular multiplication of microbes such as
viruses and parasites further makes their elimination
challenging. The adaptive immune system is a highly
advanced system developed by the body to tackle
highly mutagenic and intracellularly replicating
pathogens [27]. The innate immune response differs
from the adaptive immune response in that the former
responds immediately (minutes to a few hours) to
Anti-viral action
invading pathogens, whereas the adaptive immune
system takes longer (days to weeks) to respond. The
innate immune response is short-lived owing to the
numerous feedback regulation mechanisms which
intervene to curb any harm to tissues from the potent
non-specific effects of the innate immune system.
Innate immune responses are also devoid of memory
effects, and as a result do not show a faster and
stronger immune response on second exposure to the
same antigen [31]. Adaptive immune responses are of
two types: humoral and cellular. The humoral arm
consists of B cells which produce antibodies leading
to humoral immunity. The cellular arm consists of
CD4+ T lymphocytes and CD8+ T lymphocytes [32].
2.2 Stages in immune response generation
There are three key steps involved in the generation of
an immune response: antigen uptake by APCs, antigen
presentation by APCs, and antigenic elimination by the
effector cells of the adaptive immune system.
2.2.1 Antigen uptake by APCs
Antigen may take three different pathways postvaccination. Firstly, the antigen may enter the blood where
it will be exposed to plasmacytoid dendritic cells, blood
resident monocytes, B lymphocytes, splenic macrophages
or splenic dendritic cells. Secondly, the soluble antigen
may enter the lymphatic capillaries and vessels and
be carried through lymphatic fluid to the lymph nodes
where it would encounter lymph node-resident dendritic
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cells. Lastly, antigens which neither enter the blood
nor lymph may interact with the APCs at the site of
administration/injection. These include Langerhans
cells, dermal dendritic cells, and immune cells lining
the gut and respiratory mucosa, depending on the route
of administration [18]. Both macrophages and dendritic
cells are capable of phagocytosis, however, macrophages
phagocytose larger particles, 500-2000 nm in size, such
as bacteria, whereas dendritic cells phagocytose smaller
particles, 20-100 nm in size, such as viruses [33]. Only
dendritic cells are capable of activation of naive helper T
cells. Dendritic cells act as sentinels in peripheral tissues
and monitor the environment for pathogens. They exist
in an immature state characterized by potent endocytic
potential. These immature dendritic cells take up antigen,
process it, and present it on their surface in association
with MHC class II molecules followed by their migration
to lymphoid organs where they activate naive helper T
cells through a combination of antigenic, cytokine, and
co-stimulatory molecules. [27]. Uptake of virus sized
nanocarriers (20-200 nm) occurs via receptor-mediated
endocytosis, whereas particles > 0.5 µm in size are taken
up by phagocytosis [29]. Macropinocytosis is involved in
the uptake of small nanoparticles (50 nm) [34]. Antigen
uptake by dendritic cells results in their transformation
from an immature state adept at capturing antigen to
a mature state. Mature dendritic cells show increased
surface expression of co-stimulatory molecules such
as CD40, CD80, and CD86, which induce activation of
T cells. These dendritic cells then migrate to the T cells
to co-deliver the antigenic stimulus and co-stimulatory
signals to T cells resulting in their activation to eventually
mount a strong antigen-specific immune response [27]. A
single mature dendritic cell has the ability to activate 1001000 T cells [35].
2.2.2 Antigen presentation by APCs and elimination of
antigen by the effector cells of the adaptive immune
system
Dendritic cells serve as a valuable link between the innate
and adaptive arms of the human immune system. After
uptake of antigen, they process the antigen and present
it to the adaptive immune system comprising T cells and
B cells. Once antigens are internalized by APCs through
phagocytosis or endocytosis, they are degraded within the
endosome to shorter peptides which are then presented
in the context of MHC class II molecules. There are two
types of MHC molecules: MHC class I and MHC class II
molecules. MHC class II molecules are expressed only
Nanocarriers for vaccine delivery 17
by the cells of the immune system, unlike MHC class I
molecules, which all nucleated cells of the human body
display on their surface. When the intracellular proteins
undergo degradation as a part of the normal cell quality
control process, some are not completely degraded,
resulting in the formation of short chain peptides.
These peptides are delivered from the cytoplasm into
the endoplasmic reticulum where they are bound by
transmembrane presenting molecules encoded by the
major histocompatibility complex (MHC) gene. These
molecules consist of two chains which undergo folding
to form a cleft in which the peptide rests. These peptidebound MHC-encoded molecules are then transported
to the cell surface for peptide (antigen) presentation.
Lymphocytes are the key players in the adaptive immune
response, and are present in the lymph, blood, and spleen
and lymph nodes. B cells and T cells differ with respect to
their origin, their ability to recognize pathogens, and their
manner of destruction of pathogen. B cells originate in
the bone marrow, while T cells originate in the thymus. B
cells are capable of recognizing the pathogen directly with
the help of B cell surface receptors. Each B cell expresses
a number of copies of a unique antibody specific for a
particular antigen as a cell surface receptor. This results
in the production of B cells which are able to react with
a single antigen. Once the B cell recognizes a specific
antigen along with the presence of auxiliary cells and
signals, it undergoes activation to divide into plasma
cells and memory B cells. The majority of plasma cells
return to the bone marrow and generate huge quantities
of soluble antibody which are released in the blood and
lymph. Antibodies produced by B lymphocytes take care
of the pathogen within body fluids, however, due to
their large molecular size, antibodies are unable to cross
the plasma membrane and hence are ineffective against
intracellularly multiplicating pathogens. T lymphocytes
recognize antigens through MHC class I/II molecule
presentation. Unlike B lymphocytes, T cell receptors are
unable to directly recognize pathogens; they require
the pathogen/antigen to be presented in association
with MHC molecules. There are two distinct types of
T lymphocytes: CD4+ and CD8+ T lymphocytes. CD4+ T
lymphocytes express CD4+ markers on their surface and
recognize antigen loaded onto MHC class II molecules.
CD8+ T lymphocytes express CD8+ markers on their surface
and recognize antigen loaded onto MHC class I molecules.
Peptides of endogenous origin are generally presented in
the context of MHC class I molecules, whereas peptides of
exogenous origin are presented in the context of MHC class
II molecules. However, certain APCs such as dendritic
cells, macrophages, and B lymphocytes are capable
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18 Priyanka Prabhu and Vandana Patravale
of “cross-presentation”. Cross-presentation is defined
as the ability of the endocytosed antigenic material to
escape from the endosome and enter the cytoplasm for
presentation in the context of MHC class I molecules.
T cell surface receptors exhibit some peculiar features
as regards to their mode of antigen recognition. Firstly,
they are unable to react with soluble antigens, they can
only destroy the antigen presented to them in association
with MHC class I/II molecules. Secondly, they can mainly
only react to proteins. CD8+ T lymphocytes, after their
recognition of antigen/MHC class I complex, proliferate
and differentiate into CD8+ cytotoxic T lymphocytes that
secrete a pore forming protein called perforin. This in
turn causes the release of proteases into the cytoplasm
of the infected cells. These proteases initiate apoptotic
responses leading to the deathof the antigen expressing
cell. Antigen-specific T lymphocytes also secrete cytokines
such as interferon-γ (IFN-γ) and TNF-α, which interfere
with multiplication of viruses and inhibit their growth
in the cell. CD4+ T lymphocytes on activation by mature
dendritic cells differentiate into antigen-specific CD4+ T
cells and CD4+ helper T cells; the latter play a pivotal role
in the initiation and regulation of an effective immune
response. Regulation of immune responses by CD4+ helper
T cells occurs through the activity of soluble mediators
called cytokines secreted by activated T cells.
There are different subsets of helper T cells. Th1 cells
secrete mainly IFN-γ, and TNF-α. Th 2 cells secrete IL-4,
IL-5, IL-10, and IL-13. Th1 cells thus mount an effective
immune response against viruses and other intracellular
pathogens. Th2 cells activate eosinophils and mastocytes
and help to tackle the extracellular pathogens [27].
Commonly, Th2 cells result in an antibody-mediated
immune response and the Th1 cells lead to a strong cellmediated immune response [32]. Cytokines produced by
Th1 cells lead to the production of IgG2a antibody whereas
cytokines produced by Th2 cells enhance production of
IgG1 antibody [36]. The third subset is follicular helper T
cells which exist in close association with B lymphocytes
in follicles of lymphoid organs. Follicular helper T cells
produce IL-21 and augment increased production of
antigen-specific antibodies by B cells. The fourth subset
is Th17 cells which serve to regulate the local immune
responses against gut and lung pathogens. Th17 cells
secrete IL-17 and IL-22.
Dendritic cells themselves are able to produce certain
cytokines and thereby influence the differentiation of
naive CD4+ T cells into a particular subset of helper/
effector cells. For example, IL-12 produced by dendritic
cells results in the differentiation of naive CD4+ T cells
into Th1 cells. IL-6 is involved in the differentiation into
follicular helper T cells and Th17 cells. Th1 and Th2
cells possess the ability to inhibit each other’s function
[27]. IL-10 is reported to hamper the development of Th1
cells, and IFN-γ is known to avert the activation of Th2
cells [35]. Antibody production by B lymphocytes and
production of cytotoxic CD8+ T lymphocytes can occur
even in the absence of CD4+ helper T cells. However, this
results in the generation of low affinity antibodies. Also,
the antibody response is short-lived and does not elicit a
memory response on subsequent exposure to the same
antigen. A stronger antigen-specific secondary immune
response is only elicited when B cells are activated in a
T cell dependent manner. T cell dependent humoral
responses require simultaneous activation of both B and T
cells. Once an antigenic protein is injected into the human
body, the primary immune response is slow and involves
low affinity IgM antibodies. Subsequent exposure to the
same antigen results in the induction of a faster and a
stronger immune response involving higher affinity IgG
antibodies. Antigen-specific helper T cells play a pivotal
role in providing B cells with the necessary stimuli to give
rise to IgG antibodies of high affinity [27]. Memory B cell
responses exhibit different features from the primary B cell
responses with the former being generated more rapidly,
resulting in higher antibody levels predominantly of the
IgG, IgA, and IgE isotypes, and generating antibodies
of higher affinity [37]. T cell activation results in their
proliferation and differentiation into effector T cells. 90%
of effector T cells are not long lived, and after destroying
antigen they are removed from the body by apoptosis. The
remaining 10%, however, become memory T cells and are
retained for several years or even for a lifetime. There are
two types of memory T cells: central memory T cells and
effector memory T cells. Central memory T cells reside in
the lymphoid organs and require time for activation to
produce large levels of IL-2 and differentiate into effector
memory T cells, whereas effector memory T cells are
present at the peripheral sites and exhibit rapid activation
on subsequent exposure to antigen [32].
Figure 2 shows aschematic representation of the steps
involved in the generation of an immune response [29].
3 What are Vaccine Adjuvants?
A vaccine adjuvant can be defined as any substance or
a mixture of two or more substances or approaches to
enhance the nature, magnitude, and longevity of the
antigen/vaccine specific immune response stimulated
by the vaccine as compared to the antigen/vaccine alone
without causing any significant immunomodulation or
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Nanocarriers for vaccine delivery 19
Figure 2: Schematic representation of steps involved in the generation of immune response ([29], modified).
toxicity by its own [36, 38]. The selection of an appropriate
adjuvant for a vaccine is governed by a number of
factors including type of antigen, whether it is being
co-administered with adjuvant or co-formulated with
adjuvant, the proposed route of vaccination, vaccination
schedule, and the nature of the desired immune response
[38].
There are two types of vaccine adjuvants based on
the mechanism of adjuvanticity: immunostimulants/
immunopotentiators, and particulate vaccine delivery
systems. Immunostimulants/immunopotentiators directly
activate the immune system through their interaction
with specific receptors of the innate immune system.
These include TLR agonists, NOD like receptor agonists,
etc. [39]. Figure 3 depicts the different immunostimulants
[40]. Particulate vaccine delivery systems, on the other
hand, act by a number of mechanisms to enhance the
immunogenicity of the antigen. Several mechanisms
through which they act as adjuvants include:
1. Enhanced APC uptake. Particulate delivery systems
have similar dimensions to pathogenic microbes,
which our immune system has evolved to battle. This
results in their enhanced uptake by APCs which are
sentinels for pathogens [38].
2. Co-formulation of antigen, immunostimulant, and
targeting ligand. Particulate delivery systems offer an
opportunity to co-deliver antigen, immunostimulant,
and targeting ligand together in one system to the
same APC, which has dual benefits. Firstly, it results in
3.
4.
5.
6.
7.
effective activation of the APCs for a stronger adaptive
immune response generation. Secondly, it avoids the
toxic effects of the immunostimulant by restricting its
systemic uptake [38].
Multivalent/multimeric presentation of antigen.
Particulate systems like dendrimers and liposomes
can also be designed so as to display multivalent
copies of the antigen on their surface which closely
mimics natural pathogens thereby generating a
strong immune response [41].
Stability enhancement of vaccine/antigen. Particulate
delivery systems, due to encapsulation or covalent
association with antigen/vaccine, serve to protect
the antigen/vaccine from pH or enzyme based
degradation [9].
Allow mucosal vaccine delivery. Particulate delivery
systems serve to enhance the transport of antigens
through the mucosal tissue which otherwise show
poor uptake through mucosa [9].
Targeting to APC. Delivery systems for vaccine delivery
can be designed in such a way so as to achieve targeted
delivery of antigen to APCs for maximum uptake using
low doses of antigen [42].
Ability to trigger the cell-mediated arm of the immune
response. It is generally seen that administration of
antigen in soluble form leads to the induction of a
humoral response whereas administration of antigen
in a particulate carrier mimics a natural infection and
is able to induce a cell-mediated immune response [9].
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20 Priyanka Prabhu and Vandana Patravale
Figure 3: Summary of immunostimulant molecules [40].
8. Dose sparing effect. Vaccine delivery vehicles may
reduce the dose of antigen required to elicit an
immune response due to protection of antigen against
degradation, due to enhanced mucosal transport, or
due to targeted delivery to APCs [9].
9. Enhance patient compliance. Vaccine delivery systems
possess the ability to provide controlled release of the
antigen resulting in prolonged presentation to the
immune system thus mimicking natural infection.
This reduces the number of vaccinations required,
thereby increasing patient compliance [9].
Particulate vaccine delivery systems include micronsized (> 1000 nm) and nanosized systems (1 to 1000 nm).
Nanoparticles offer an advantage over microparticles
as vaccine delivery systems owing to their better uptake
through APCs due to their similar size to invading
pathogens, and the capacity of some nanocarriers such
as liposomes, archaeosomes, niosomes, and dendrimers
to offer multivalent immunostimulant and antigen
presentation resulting in simulation of viral invasion [12].
3.1 Need for vaccine adjuvants
The need for vaccine adjuvants stems from the fact that the
recent years have witnessed a paradigm shift from using
inactivated/live attenuated vaccines to subunit antigens.
These subunit antigens show poor immunogenicity owing
to their inability to activate APCs and their limited half
life in the body [9]. Secondly, although the clinical use of
vaccine adjuvants can be traced back about 90 years, only
a handful has been approved. In addition, the approved
adjuvants are all for parenteral administration and licensed
for a particular antigen by a single specified route only.
Multiple reasons are responsible for this phenomenon.
Firstly, prophylactic vaccines are administered to
huge populations of healthy persons emphasizing the
paramount requirement of safety. Secondly, approval of
vaccine adjuvants by regulatory agencies entails separate
registration of a given antigen/adjuvant combination for
a particular route of administration [22]. Thirdly, in vitro
APC uptake and activation studies, and immune response
generation in rodent animal models in vivo, which are
utilized to gauge the efficacy and safety of novel proposed
vaccine adjuvants do not completely predict or translate
into effective immune responses in humans for a number
of reasons. Expression patterns of PRRs differ between
humans and mice [31]. TLR9 expression occurs in different
subsets of dendritic cells in mice and in humans. Also,
TLR4 expression is substantial in mouse B cells whereas
TLR4 expression is absent in human B cells during normal
conditions [43]. Non-human primates offer a better option
to study the mechanism of adjuvanticity and safety since
they show greater resemblance to humans than mice as
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regards to PRR expression and dendritic cell subsets. A
major drawback associated with the use of non-human
primates is their availability and huge maintenanceassociated expenses [31]. Alum has enjoyed unparalleled
monopoly as the only FDA approved vaccine adjuvant
for almost eight decades [1]. However, there are some
serious drawbacks associated with alum. It induces a
biased humoral immune response, rendering it useless for
vaccination against intracellular infections where there is
an urgent need for cellular immunity [4]. Additionally,
alum cannot be freeze dried since it results in destruction
of the colloidal structure, and it is ineffective for mucosal
vaccination [29]. Alum is also ineffective in inducing
immunity against small peptides and vaccines such as
influenza and typhoid fever vaccines [19], and is known
to cause local adverse effects such as granuloma at the
site of injection [42]. However, the last twenty years have
witnessed the approval of a few novel vaccine adjuvants.
MF59 is a vaccine adjuvant approved in Europe in the
Fluad® influenza vaccine by Novartis. It is an oil-inwater emulsion (160 nm) containing 5% squalene, 0.5%
polysorbate 80, and 0.5% sorbitan trioleate. AS03™ by
GlaxoSmithKline (GSK) is another approved emulsion
based adjuvant (150 nm) employed in the Pandemrix™
influenza vaccine. It contains squalene, tocopherol,
and Tween 80 [39,44]. AS04 by GSK is another approved
adjuvant containing alum and monophosphoryl lipid
A (MPLA). It is used in two licensed vaccines made by
GSK, namely, Fendrix® for hepatitis B and Cervarix® for
cervical cancer caused by HPV [45].
Nanocarriers for vaccine delivery 21
Salient features of an ideal vaccine adjuvant [18,42]
are summarized in Figure 4.
3.2 Parameters impacting immune response
of nanocarriers
The various factors influencing the immune response
elicited by nanocarriers are discussed below.
3.2.1 Particle size
Particle size of the antigen delivery system plays a
pivotal role in its uptake by the APCs at the site of action
or by the lymphatic vessels to reach the lymph nodes.
Particles with a size of less than 1000 nm are apt for
uptake by macrophages and dendritic cells. The impact
of antigen delivery system size on the resultant immune
response generated also depends on the route employed.
Microparticles elicit effective immunity after oral and
nasal administration. Their larger size probably facilitates
their uptake into the GALT and NALT for greater mucosal
immunity. Particles in the size range of 20-50 nm are
suitable for transport through lymphatic vessels and
lymph nodes [8]. Particles of 500 nm or smaller are ideal for
dentritic cell or macrophage uptake, 20-200 nm particles
are mostly uptaken via endocytosis resulting in a Th1 type
immune response, whereas particles greater than 500 nm
in size are uptaken via phagocytosis or macropinocytosis
resulting in a humoral immune response [46].
Figure 4: Salient features of an ideal vaccine adjuvant [18,42].
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22 Priyanka Prabhu and Vandana Patravale
However, study results comparing the immune
responses elicited by nanoparticles and microparticles
are contradictory. Some reports support the use of
nanoparticles to elicit a stronger immune response
whereas some studies report the opposite. Kanchan et al.
demonstrated that hepatitis B surface antigen loaded poly
lactic acid (PLA) microparticles (2-8 µm) showed higher
antibody levels than hepatitis B surface antigen-loaded
PLA nanoparticles (200-600 nm). The microparticles
elicited a Th2 type response whereas the nanoparticles
elicited a Th1 type immune response [47]. Jung et al.
compared the immune response of tetanus toxoid
adsorbed onto sulfobutylated poly(vinyl alcohol)-graftpoly lactic-co-glycolide (PLGA) particles and reported that
particles of 100 and 500 nm resulted in greater antibody
levels as compared to the particles greater than 1000
nm by the oral and intranasal route [48]. Caputo et al.
demonstrated using HIV Tat protein that adsorption
onto cationic polymeric nanoparticles (220 or 630 nm)
elicited a strong cell-mediated immune response and a
poor antibody based immune response than the protein
adsorbed onto microparticles (2-8 µm) [49].
3.2.2 Surface characteristics
Surface characteristics such as shape, hydrophobicity, and
surface charge are reported to influence phagocytic uptake
by APCs. Positive charge and cylindrical or spherical shape
enhance more efficient phagocytic uptake than negatively
charged and disk shaped particles. Also, hydrophobic
surfaces are readily opsonised by endogenous serum
proteins and in the interstitial fluid at the site of injection
resulting in augmented uptake by APCs [38].
3.2.3 Route of administration
Administration of the same vaccine delivery system of
a particular particle size via different administration
routes results in different types of immune responses
which can be attributed to the different types of APCs
and different numbers present at various sites in the
body. Intraperitoneal immunization results in uptake by
macrophages present in the peritoneal cavity whereas ID
immunization leads to greater uptake by the dendritic
cells. Different APCs vary in their ability to process antigen
and produce immunity [50].
3.2.4 Type of antigen
Bovine serum albumin (BSA), ovalbumin, tetanus
toxoid, hepatitis B surface antigen, and HIV Tat protein
are the most widely used antigens to evaluate potential
vaccine adjuvants. Different antigens vary with respect
to their endotoxin content, inherent immunogenicity,
and purity as a result of which a given adjuvant may elicit
a different immune response depending on the antigen
[50]. A good example of this is a report highlighting
the difference in immune response obtained with
ovalbumin and Bacillus anthracis protective antigen.
Conjugation of ovalbumin onto lecithin solid lipid
nanoparticles (SLN) showed greater antibody levels
than those obtained with ovalbumin and aluminium
hydroxide. Conjugation of Bacillus anthracis protective
antigen onto the lecithin SLN showed similar immune
response to that of alum adjuvanted Bacillus anthracis
protective antigen [46].
3.2.5 Antigen release
Release of antigen is governed by the manner in which
it is loaded onto or associated with the nanocarrier.
Antigens can be covalently attached to nanocarriers or
physically adsorbed onto the surface via electrostatic
interactions or entrapped/encapsulated within the core of
the nanocarrier. Adsorption onto the nanocarrier surface
results in burst release of antigen or premature release
of antigen before uptake by the APCs. Covalent coupling
requires enzymatic activity to break the linkage and release
the free antigen. Encapsulation or entrapment involves
dependence on the degradation of the matrix material in
order to achieve antigen release. This leads to prolonged
or controlled release of antigen [50]. However, the ideal
method of loading would be to have some antigen on the
surface which would be released immediately to initiate
the immune response, in addition to sustained release
of the entrapped antigen over a period of time to help to
boost the immune response, thus eliminating the need for
multiple booster administrations and improving patient
compliance. Constant antigen delivery commonly results
in the generation of an efficient immune response as a
persistent encounter with the antigen permits sufficient
affinity maturation and antibody isotype switching, both
of which play a key role in generation of a strong antigenspecific immune response [51].
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3.2.6 Presence of immunostimulants
Inclusion of immunostimulants along with nanocarriers
further enhances the immune response compared to
nanocarriers alone [39].
3.3 Upcoming strategies for successful
vaccine delivery
This section highlights some upcoming approaches
which are employed to further enhance adjuvanticity of
nanocarriers.
3.3.1 Targeted delivery to APCs
Various ligands may be employed for the targeted delivery
of antigen-loaded nanoparticles to the dendritic cells or
other APCs. Nanocarriers decorated or functionalized with
mannose, fucose, or N-acetylglucosamine can be used
to interact with the lectin-like receptors (e.g. mannose
receptors, DC-SIGN, and DEC-205) present on dendritic
cell surfaces. Phosphatidyl serine may be included
while fabricating liposomes to promote interaction with
phosphatidyl serine receptors on monocytes. Monoclonal
antibodies against DC-SIGN or CD11c receptors may also
be employed to target dendritic cells [29, 52]. DC-SIGN is
mainly expressed on dendritic cells at mucosal areas, skin,
and lymph nodes [53]. Receptors employed for dendritic
cell targeting are chosen based on the following criteria.
Firstly, the receptor must show preferential expression on
dendritic cells compared to other cells. Secondly, antigen
delivered to the dendritic cell via the receptor should be
internalized, processed and presented in association with
both MHC class I and II molecules to elicit a balanced
humoral and cellular immune response. Thirdly, the
receptor binding should induce immunostimulatory
signals essential for dendritic cell maturation and
migration in order to avoid tolerance and to efficiently
activate naive T cells [52].
3.3.2 Use of intelligent particulate carriers to achieve
cytosolic delivery of antigen
Strategies which may be employed in order to achieve
cytosolic antigen delivery include use of endosome
disrupting agents in order to allow antigen delivery to
the cytosol where MHC class I presentation commences
[54]. There are six different types of intelligent carriers,
which disrupt the endosome in different ways. The first
Nanocarriers for vaccine delivery 23
type of carriers is pH responsive: when they reach the
acidic endosome (pH 5.9-6), they release the antigen and
disturb the endocytic vacuole resulting in antigen delivery
to cytosol. The second type includes use of fusogenic
components in the liposomal membrane. Sendai virus
proteins, when included in liposomes, confer a fusogenic
property to the carrier resulting in fusion with endosomes
to deliver antigen into the cytosol. The third type involves
use of cell penetrating peptides for delivery to cytosol [55].
Octaarginine, when included in liposomes containing
ovalbumin, led to more efficient cytotoxic T cell responses
than cationic liposomes and pH responsive liposomes [56].
The fourth type involves use of pore forming substances
which directly perforate the endosomal membrane and
reach the cytoplasm. Pore forming proteins which have
been used to deliver antigen into the cytosol of dendritic
cells are porins from Shigella dysenteriae and Listeriolysin
O from Listeria monocytogenes [55]. The fifth type includes
bubble liposomes. Perfluoropropane gas is included
inside the liposomes which, on exposure to ultrasound,
are able to disrupt the cell membrane and deliver antigen
into cytoplasm [55]. Exposure of dendritic cells to bubble
liposomes and ultrasound resulted in 3 fold higher IL-2
production compared to ovalbumin solution alone or
ovalbumin solution plus ultrasound [57]. The sixth type
includes use of virosomes and virus-like particles (VLPs)
[55]. Virosomes are liposomes containing glycoproteins
extracted from isolated viruses and inserted into the
lipidic membrane bilayer [58]. Glycoproteins include
hemagglutinin and neuraminidase [39]. The first subunit
of hemagglutinin helps in anchoring to the sialic acid
residues present on the APC surface, and the second
subunit assists fusion of the endocytosed virosome with
the endosome leading to delivery of the antigen into the
cytoplasm [59]. Virosomes are unilamellar and spherical,
with a size of 150 nm [60]. Virosome based vaccines
approved in Europe include Epaxal ® (hepatitis A) and
Inflexal® V (influenza) [58]. The vaccine adjuvant activity
of virosomes may be attributed to number of mechanisms.
Their virus particle mimicking structure results in
repetitive antigen presentation to B cells. They also
provide a depot effect for sustained antigen presentation
[60]. Additionally, virosomes also promote crosspresentation of antigen. They undergo uptake by APCs
via receptor-mediated endocytosis and subsequently fuse
with the acidified endosomes to release their antigen load
into the cytosol [29]. VLPs are composed of viral structural
proteins which self-assemble to form VLPs. They act as
PAMPs and show potent B and T cell stimulation owing
to the repetitive presentation of antigenic epitopes on
their surface. Commercially available VLP based vaccines
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24 Priyanka Prabhu and Vandana Patravale
include Gardasil® and Cervarix ® [58]. VLPs show antigen
cross-presentation owing to their fusogenic capability
resulting in antigen delivery to cytosol for effective MHC
class I presentation leading to CD8+ T cell stimulation [58].
3.3.3 Combination of immunostimulants and particulate
delivery systems
Another promising strategy is to formulate well known
imunostimulants within particulate carriers to achieve a
potentiated immune response. This technique also helps
to eliminate the untoward effects seen with administration
of free immunostimulant [43]. Also, one can reduce the
amount of immunostimulant needed thus reducing its
toxic effects. GSK has developed a technology called
Adjuvant Systems based on the combination of traditional
particulate adjuvants such as aluminium salts, liposomes,
oil-in-water emulsions with immunostimulants such
as MPLA and QS-21. MPLA, a strong TLR4 agonist, is
obtained from the lipopolysaccharide present in the
cell wall of Salmonella minnesota R595 strain and is
detoxified by mild hydrolysis. QS-21 is obtained from the
bark of the tree Quillaja saponaria, and induces cytotoxic
CD8+ immune responses. A drawback associated with
QS-21 is the haemolytic activity of the molecule, which
formulation in particulate system helps to overcome.
AS04 is composed of MPLA adsorbed onto aluminium
hydroxide or aluminium phosphate. AS02 is composed of
MPLA and QS-21 formulated in an oil-in-water emulsion to
induce both humoral and cellular immunity. The RTS, S/
AS02 vaccine for malaria is currently undergoing clinical
trials. AS01 is composed of QS-21 and MPLA formulated
in liposomes to induce a strong CD8+ T cell response.
The RTS,S/AS01 malaria vaccine is currently undergoing
Phase III clinical trials [61].
The following sections will summarize the various
nanocarriers explored for vaccine delivery. Figure 5
shows the various nanocarriers used to date for vaccine
delivery.
Figure 5: List of nanocarriers explored for vaccine delivery.
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4 Polymeric Nanoparticles
Polymeric nanocarriers have garnered considerable
attention for vaccine delivery over the years owing to their
potential for providing continuous as well as pulsatile
antigen release by modifying the polymer composition and
nanoparticulate structure. This feature may enable single
shot vaccination, however, this hypothesis is yet to be
established clinically [58]. Polymers explored for vaccine
delivery can be biodegradable or non-biodegradable.
Polymers from both natural (starch, gelatin, alginate) and
synthetic (PLGA, Polyanhydride) sources have been used.
The most extensively investigated biodegradable polymers
for vaccine delivery include polyesters (PLA, PLGA and
polyanhydrides) [62]. Natural polymers offer the beneficial
attributes of biocompatibility, low cost, and high aqueous
solubility compared to their synthetic counterparts.
Drawbacks associated with natural polymers include their
poor hydrophobicity, batch to batch inconsistency, and
occurrence of unwanted impurities. Synthetic polymers,
on the other hand, are amenable to reproducible
production and can be designed to achieve the desired
rate of degradation, molecular weight, and co-polymer
composition. But one major shortcoming of synthetic
polymers is their solubility, commonly being soluble only
in organic solvents, hampering the antigenicity of the
antigen [63].
This section summarizes a number of polymers which
have been explored for both parenteral and mucosal
vaccination.
4.1 Chitosan nanoparticles
Chitosan is a co-polymer of glucosamine and
N-acetylglucosamine prepared by the deacetylation
of chitin derived from insect skeleton [64]. Chitosan is
one of the most intensively explored biomaterials in
the field of vaccine delivery, both as an adjuvant and as
a delivery platform [65]. The vaccine adjuvant activity
of chitosan-based nanoparticles has been evaluated
by several different routes such as nasal, SC, oral, IM,
transcutaneous, and ID routes. This can be attributed to
its peculiar properties which make it a promising material
for vaccine delivery.It is non-toxic, biocompatible, and
cost effective [66]. It is an FDA approved biomaterial [67].
Chitosan easily forms nanoparticles possessing high
loading capacity for several proteins and other antigens
[66]. Chitosan nanoparticles can be fabricated without
exposure of the antigen to hostile conditions of heat or use
of organic solvents [65]. They are spontaneously prepared
using a precipitation/coacervation technique with the aid
Nanocarriers for vaccine delivery 25
of tripolyphosphate as a precipitating agent [67]. Chitosan
has been found to augment both cell-mediated and
humoral immune responses. It is mucoadhesive thereby
rendering it useful for mucosal vaccination. It also acts
as a penetration enhancer by opening the tight junctions
of epithelial cells present in the oral and nasal mucosae;
thereby increasing the paracellular absorption of antigen
[65]. Chitosan has been shown to possess macrophage
activation ability and shows cytokine induction [66]. It
also activates dendritic cells [65]. Activation of dendritic
cells occurs through binding to TLR4 and mannose
receptors [68]. Its positive charge confers it with superior
uptake by APCs due to ionic interactions with the
negatively charged cell membrane. The positive charge
also allows electrostatic binding with negatively charged
antigens and DNA for vaccine delivery [65]. Chitosan
based nanoparticles possess higher stability than other
vaccine delivery platforms such as liposomes and ISCOMs
[7]. A chief downside associated with the use of chitosan
is its poor solubility at physiological pH, as only the
protonated form of chitosan in which it is present at acidic
pH is able to act as a permeation enhancer. In order to
surmount this problem, a partially quaternized derivative
of chitosan known as N-trimethyl chitosan (TMC) chloride
is synthesized which possesses excellent solubility over
a broad pH range [69]. The immune response obtained
with chitosan depends on the degree of deacetylation and
molecular weight of chitosan [7].
Dzung et al. used chitosan of 30 kDa and 300 kDa to
formulate chitosan nanoparticles of around 80 nm and
106 nm respectively loaded with A/H1N1 influenza antigen
for SC vaccination. High molecular weight chitosan
nanoparticles showed 3-fold higher antibody levels
than the alum-adsorbed antigen, 2-fold higher antibody
levels than the low molecular weight chitosan (30 kDa),
and 100-fold higher antibody levels than plain antigen
administered alone [70]. Prego et al. developed chitosan
nanoparticles (160-200 nm) using an ionic gelation
method. The nanoparticles showed susitained release of
antigen and IM immunization in mice showed antibody
levels which were 9-fold greater than those obtained by
alum adjuvant [71].
Slutter
et al.
developed
ovalbumin-TMC
nanoconjugates using thiol chemistry for nasal
immunization. The aim was to improve transport across
the nasal mucosa using smaller nanoconjugates (28 nm)
in comparison to the larger TMC nanoparticles (300 nm).
The nanoconjugates demonstrated superior penetration
through a lung carcinoma cell monolayer and also elicited
higher ovalbumin-specific IgG and nasal IgA levels in
mice compared to ovalbumin-loaded N-trimethyl chitosan
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26 Priyanka Prabhu and Vandana Patravale
nanoparticles. The nanoconjugates elicited a mixed Th1/
Th2 response (rather than a biased Th1 or biased Th2
response) than ovalbumin-loaded N-trimethyl chitosan
nanoparticles [72]. Khatri et al. have investigated the
use of chitosan nanoparticles (337 nm) for nasal DNA
vaccination using DNA encoding hepatitis B surface
antigen. The nanoparticles induced serum antibody
levels which were greater than the clinically protective
levels. Nasal administration of nanoparticles resulted in
generation of mucosal IgA antibody generation in nasal,
salivary, and vaginal secretions which was not seen with
IM administration of naked DNA and alum adsorbed
hepatitis B surface antigen [73]. Sayin et al. have developed
nanocomplexes of TMC and mono-N-carboxymethyl
chitosan (283 nm) by simple electrostatic interactions for
nasal delivery of tetanus toxoid and compared their efficacy
with mono-N-carboxymethyl chitosan nanoparticles
and TMC nanoparticles. Intranasal immunization of the
nanocomplexes resulted in higher levels of both IgG1
and IgG2a compared to the plain antigen solution given
nasally or subcutaneously. Also, intranasal immunization
of the nanocomplexes led to a stronger immune response
compared to nanoparticles fabricated from chitosan,
mono-N-carboxymethyl chitosan, and TMC alone [74].
Bal et al. have also demonstrated the ability of
ovalbumin- TMC nanoconjugates to elicit higher antibody
levels and a greater number of ovalbumin positive
dendritic cells in the lymph nodes after transcutaneous
immunization compared to ovalbumin-loaded TMC
nanoparticles. After ID immunization, there was no
significant difference between the immune responses
generated by ovalbumin-TMC nanoconjugates and
ovalbumin-loaded TMC nanoparticles. This indicates
that the size of the nanocarrier plays an important
role for permeation through skin in transcutaneous
immunization, whereas in case of ID immunization, the
skin barrier is not involved for nanocarrier transport and
hence both nanoconjugates and nanoparticles perform
equally well [75].
Tafaghodi et al. coated hepatitis B surface antigen
with chitosan and TMC separately. Nasal as well as IM
immunization in mice with both the nanoparticles induced
higher serum antibody levels than those obtained with
intramuscularly administered alum adsorbed hepatitis
B surface antigen. Nasal and IM immunization in mice
with both nanoparticles also generated a cellular immune
response, whereas the alum adsorbed hepatitis B surface
antigen failed to do so. Nasal immunization also resulted in
induction of secretory IgA in nasal and vaginal secretions,
the latter would be valuable in curbing sexually transmitted
hepatitis B infection [76]. Balet al. showed that the
immune response obtained with chitosan nanoparticles
differs depending on the adjuvant used and the route
of administration. They developed TMC nanoparticles
containing both ovalbumin and an immunostimulant and
studied their immunostimulatory efficacy on nasal and
ID vaccination. Five immunostimulants were employed:
lipopolysaccharide (LPS), PAM3CSK4, CpG DNA, muramyl
dipeptide (MDP), and cholera toxin B subunit. Detectable
sIgA levels were not generated following ID vaccination
of any of the nanoparticles. Nasal vaccination with
nanoparticles containing ovalbumin/MDP or ovalbumin/
LPS resulted in higher serum IgG and IgA levels in nasal
secretions compared to non-adjuvanted TMC-ovalbumin
nanoparticles. Nasal vaccination was unable to induce
significant IgG2a levels with any of the nanoparticles.
ID vaccination with the nanoparticles containing
ovalbumin/CpG or ovalbumin/LPS demonstrated
higher serum IgG levels than non-adjuvanted TMCovalbumin nanoparticles. ID vaccination with CpG
adjuvanted ovalbumin TMC nanoparticles resulted in
induction of IgG2a levels. The differences observed in
the immune responses could be explained on the basis
of the expression of the PRRs such as TLRs and NODlike receptors depending on the dendritic cell subset
and localization. MDP, which showed efficacy on nasal
administration, binds to NOD 2, which along with other
NOD like receptors is found in the dendritic cells in the
nose. Also, the PRRs for LPS and CpG (both of which
proved to be most effective on ID vaccination), namely
TLR4 and TLR9, are present on keratinocytes, dendritic
cells in the dermis, and Langerhans cells [77]. Zhao et al.
entrapped a lentogenic live virus vaccine for Newcastle
disease virus in chitosan nanoparticles. The nanoparticles
were 371 nm in size. Oral and nasal vaccination with these
nanoparticles conferred 100% protection in chickens
after challenge with a highly virulent strain of Newcastle
disease virus, whereas the inactivated Newcastle disease
vaccine and live virus vaccine could not [78].
4.2 PLGA
PLGA is a biodegradable polymer approved by the FDA.
These polymers are aliphatic polyesters containing
different ratios of lactic acid and glycolic acid. PLGA
undergoes degradation by bulk erosion, during which
water diffuses faster into the polymeric matrix in
comparison to the rate of covalent bond breakage. In
this case, polymer degrades throughout the matrix until
a particular molecular weight is attained at which the
degradation products are tiny enough to be dissolved. The
polymeric structure is highly hydrated and porous at this
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point, leading to release of entrapped moiety. As a result,
there is a lag period for release of large molecular weight
substances from PLGA matrix. Degradation by bulk erosion
is beneficial for vaccine delivery since it does not allow
premature release of antigen or immunostimulant before
the nanoparticles have reached the APCs, thus limiting
the entry of immunostimulant and antigen into systemic
circulation. After uptake of PLGA nanoparticles by APCs,
endosome internalization of nanoparticles occurs, the
acidic environment of which expedites the degradation
of PLGA to release antigen or immunostimulant. The rate
of PLGA degradation is governed by its molecular weight,
hydrophilicity, and crystallinity. PLGA withsmaller
molecular weight and greater hydrophilicity tends to
degrade faster. Hydrophilicity can be adjusted by adjusting
the ratio of monomers: lactic acid: glycolic acid. Glycolic
acid is more hydrophilic, thus its usage in higher quantities
leads to greater hydrophilicity and faster degradation. The
more crystalline the polymer, the slower the degradation.
Crystallinity is governed by stereochemistry, wherein,
L-lactic acid is crystalline, the use of which results in slower
degradation. D,L-lactic acid is always employed to obtain a
uniform dispersion of the antigen in the polymeric matrix.
PLGA withlactic acid: glycolic acid in the ratio 50:50 is
least crystalline, resulting in rapid degradation with a
half life of about two weeks. Another important advantage
of PLGA nanoparticles for vaccine delivery is their ability
to cause cross-presentation of antigen resulting in both
humoral and cell-mediated immunity [79]. Shen et al.
have demonstrated this phenomenon using ovalbuminloaded PLGA nanoparticles. The nanoparticles induced T
cell IL-2 secretion in bone marrow derived dendritic cells
at 1000 times lower concentration compared to antigen
administered in the soluble form, and 10 times lower
compared to antigen-coated latex beads. The nanoparticles
showed sustained antigen presentation via the MHC class
I pathway for 72 hours [80]. PLGA hydrolyses into lactic
and glycolic acid, both of which are biocompatible and
easily bio-transformed by the body [81].
A grave concern associated with the use of PLGA
nanoparticles for vaccine delivery is the creation of an
acidic microenvironment on degradation, which may
endanger the integrity and immunogenic potential of the
vaccine [62]. Additionally, the use of organic solvents in
the generation of PLGA nanoparticles may destroy the
immunogenicity of the antigen [82]. PLGA nanoparticles
have been formulated with a variety of antigens for both
parenteral and mucosal routes. In certain cases, both
antigen and immunostimulant have been co-formulated
in PLGA nanoparticles in order to achieve a potent
antigen-specific immune response. Also, targeted delivery
Nanocarriers for vaccine delivery 27
of PLGA nanoparticles to dendritic cells has been achieved
resulting in potent immune activation.
Bharali et al. fabricated methoxypolyethylene glycolPLGA nanoparticles containing recombinant hepatitis B
surface antigen which, on intraperitoneal immunization
in mice, showed a higher and faster antibody generation
compared to plain antigen [83]. Muttil et al. formulated
recombinant hepatitis B surface antigen in PLGA/
PEG nanoparticles and spray dried the formulation.
Pulmonary immunization in guinea pigs elicited systemic
immunity comparable to parenteral immunization and
mucosal immune response in the lungs [84]. Elamanchili
et al. co-formulated the cancer associated antigen MUC1
mucin peptide (BLP25) and MPLA in PLGA nanoparticles
and showed better stimulation of naive T cells compared
to soluble antigen and MPLA [85]. Raghuwanshi et al.
fabricated PLGA based nanoparticles for targeted
delivery to dendritic cells. These were made by blending
recombinant fusion protein with biotinylated PEG-PLGA
nanoparticles containing ovalbumin. The recombinant
protein contained strepatividin bound to single chain
antibody against DEC-205 receptors found on dendritic
cells. In vivo immunization in mice using the targeted
delivery system in combination with a dendritic cell
maturation agent also showed an enhanced immune
response against ovalbumin. Co-administration of
dendritic cell maturation agent is necessary as DEC-205
targeting in its absence leads to antigen-specific tolerance
[86]. Hamdy et al. developed mannan conjugated PLGA
nanoparticles for targeted dendritic cell delivery via
mannose receptors. The nanoparticles showed increased
CD4+ and CD8+ T cell immune response against ovalbumin
in comparison to their non-targeted counterparts.
Mannan, in addition to being a targeting ligand, is also
reported to possess dendritic cell activation ability
possibly due to TLR agonism. The nature of mannan –
oxidized or reduced has been shown to guide the type of
immune response -CD4+ or CD8+. Oxidized mannan causes
endosomal escape of antigen into the cytoplasm resulting
in a CD8+ response, whereas reduced mannan stays in the
endosome, is degraded by the lysosomal enzymes and
results in a CD4+ response [87].
4.3 PLA
Mattheolabakis et al. formulated ovalbumin-containing
PLA nanoparticles (150 nm size) using the double
emulsion technique for transcutaneous immunization.
The nanoparticles demonstrated equivalent antibody
generation to ovalbumin in solution; however, the cytokine
production (IL-2, IFN-γ) was enhanced by administration of
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28 Priyanka Prabhu and Vandana Patravale
ovalbumin in nanoparticles along with cholera toxin [88].
Jain et al. synthesized block co-polymer of PEG-PLA (PEGPLA-PEG) and fabricated its nanoparticles for mucosal
delivery of hepatitis B surface antigen. The aim was to
avoid the creation of an acidic milieu on degradation of
PLA by making it more hydrophilic. The block copolymers
assemble in water to give rise to a core shell structure with
the shell made of PEG and the core made of PLA. The system
demonstrated a triphasic release pattern beneficial for
vaccine delivery. The nanoparticles showed comparable
humoral immune response after single administration as
the alum adsorbed hepatitis B surface antigen did after
boosters. Additionally, the PEG-PLA-PEG nanoparticles
also produced a mucosal immune response not only in the
nasal mucosa but also in salivary, intestinal, and vaginal
areas which was superior to that obtained after the
administration of PLA nanoparticles. Also, the immune
response generated was a mixed Th1/Th2 response [89].
Florindo etal fabricated PLA nanospheres encapsulating
recombinant Streptococcus equi. M-like protein and S.
equi protein extract for IM immunization in horses. The
research group also incorporated immunostimulant
molecules such as spermine, oleic acid, alginate and
glycol-chitosan. The nanoparticles demonstrated a more
effective humoral immune response than soluble protein
alone or mixed with CpG. Nanospheres made using glycol
chitosan showed a mixed Th1/Th2 response shown by the
generation of high IgG1 and IgG2a titers and induction
of IL-2 and IFN-γ levels. The nanospheres made using
glycol chitosan had a positive surface charge which is
known to enhance uptake by APCs [90]. Pavot et al.
studied the immunostimulating potential of Nod1 and
Nod2 ligands encapsulated within PLA nanoparticles. The
nanoparticles showed excellent uptake by dendritic cells
in vitro and showed upregulation of surface molecules and
pro-inflammatory cytokine generation. Co-administration
of nanoparticle encapsulated Nod ligands and PLA
nanoparticles (200 nm) adsorbed with Gag p24 HIV-1
antigen subcutaneously in mice showed a 100-fold higher
antibody generation as compared to alum adsorbed
antigen [91].
4.4 Polyanhydride nanoparticles
Ulery et al. developed biodegradable polyanhydride
nanoparticles (200 nm) for single shot intranasal
vaccination against pneumonic plague. Yersinia pestis,
the pathogen responsible for pneumonic plague, enters
the human body through the respiratory tract, and hence
intranasal vaccination, which can induce both local
mucosal and systemic immunity, would be desirable. The
developed nanoparticles induced higher F1-V specific
antibody titers, the IgG1 antibodies showed higher avidity
for F1-V, and the antibody response remained upto 23 weeks
after vaccination in comparison to recombinant protein
F1-V alone or MPLA mixed with F1-V. The nanoparticles
were able to confer long term protection in mice against a
lethal Yersinia pestis challenge [92].
Salman et al. developed mucoadhesive polyanhydride
nanoparticles coated with mannose or Salmonella
enteritidis derived flagellin for oral vaccination.
Polyanhydride nanoparticles coated with either of the
ligands (300-400 nm) were found to generate higher IgG1
and IgG2a antibody responses compared to the plain
ovalbumin-loaded polyanhydride nanoparticles after
single oral and SC administration. Oral vaccination of
both mannosylated and flagellin coated nanoparticles
showed higher intestinal secretory IgA antibody
generation compared to SC administration. Mannose
binds to mannose binding lectins (C-type lectin receptors)
present on the surface of dendritic cells and other gut
lymphoid cells enhancing uptake of the nanoparticles by
APCs. The flagellin coating serves a dual purpose of acting
as a TLR5 ligand and as a mucoadhesive. TLR-5 agonist
binding leads to maturation and activation of dendritic
cells resulting in a stronger immune response [93].
4.5 Poly (γ-glutamic acid)
Poly(γ-glutamic acid) is a bacterial capsular exopolymer
synthesized by particular strains of Bacillus natto. Poly(γglutamic acid) undergoes degradation in the human body
by γ- glutamyl transpeptidase. Poly(γ-glutamic acid)
nanoparticles show uptake by dendritic cells followed
by localization in lysosomal regions. Poly(γ-glutamic
acid) induces dendritic cell maturation as evidenced
by production of cytokines such as IL-12, TNF-α, and
upregulation of maturation markers such as CD40, CD80,
and CD86. Poly(γ-glutamic acid) nanoparticles induce
dendritic cell maturation via the MyD88-mediated NF-κB
signalling pathway [94].
SC vaccination with the influenza virus hemagglutinin
generates only a virus-specific antibody response but
fails to generate cellular immune responses, which are
needed to tackle the intracellular virus burden. Okamoto
et al. developed hemagglutinin-loaded polymeric
nanoparticles made from poly(γ-glutamic acid)-graftL-phenylalanine copolymer for vaccination against
influenza virus. SC immunization in mice resulted in
augmented virus-specific antibody and cellular immune
response generation compared to hemagglutinin alone
and hemagglutinin with alum and induced protection in
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mice against lethal influenza virus challenge [95]. Okamoto
et al. have also demonstrated the potential of nasally
administered influenza hemagglutinin along with poly(γglutamic acid) nanoparticles to produce cross-protection
in mice against influenza virus. Intranasal immunization
with A/New Caledonia 20/99 hemagglutinin vaccine plus
poly(γ-glutamic acid) nanoparticles resulted in effective
antibody and cell-mediated immune responses against both
A/New Caledonia 20/99 virus strain and A/PR/8/34 strain.
Intranasal immunization with a blend of hemagglutinin
and poly(γ-glutamic acid) nanoparticles resulted in
enhanced cytokine production (IL-4, IL-6, IFN-γ) compared
to hemagglutinin alone or hemagglutinin administered with
polyinosinic-polycytidylic acid (poly (I:C)) [94]. Wang et al.
developed poly(γ-glutamic acid) nanoparticles containing
ovalbumin. A good uptake of nanoparticles by dendritic cells
was seen, accompanied by induction of their maturation.
The physical mixture of poly(γ-glutamic acid) nanoparticles
and ovalbumin also led to dendritic cell maturation. They
also encapsulated HIV type-1 p24 in poly(γ-glutamic acid)
nanoparticles. SC immunization with these nanoparticles
demonstrated activation of antigen-specific IFN-γ secreting
cells in spleen and led to serum antibody production. The
serum antibody levels were comparable to those obtained
with Complete Freund’s Adjuvant (CFA) [96].
Uto et al. developed ovalbumin-containing poly(γglutamic acid) nanoparticles. The nanoparticles resulted
in production of both IgG1 and IgG2a antibodies in sera,
antigen-specific IFN-γ levels in splenocyte, and cytotoxic T
lymphocyte activation. Immunization with poly(γ-glutamic
acid) nanoparticles containing CD8+ T cell epitope peptide
of Listeria monocytogenes induced protection against a
lethal challenge [97]. Uto et al. in another study showed that
ovalbumin-containing poly(γ-glutamic acid) nanoparticles
could produce greater CD8+T cell proliferation than
ovalbumin administered with CFA. Also, the nanoparticles
were devoid of any local side effects on SC injection, which
is a major drawback associated with CFA [98]. Uto et al. have
also demonstrated that after SC immunization of ovalbumincontaining poly(γ-glutamic acid) nanoparticles in mice, the
nanoparticles were taken up by dendritic cells followed by
their maturation and migration to regional lymph nodes and
effective antigen presentation via the MHC class I pathway
resulting in potent activation of ovalbumin-specific CD8+
T cells compared to plain ovalbumin and alum adsorbed
ovalbumin [99].
4.6 Polyphosphazene
Polyphosphazenes are synthetic polymers comprising a
backbone of alternating phosphorus and nitrogen atoms, with
Nanocarriers for vaccine delivery 29
organic side groups tethered onto each phosphorus atom.
Polyphosphazenes can be altered so as to contain ionic groups
resulting in water solubility, and will undergo degradation
via hydrolysis. Poly[di(carboxylatophenoxy)phosphazene]
(PCPP) is the most extensively explored polyphosphazene
for vaccine delivery [100]. Polyphosphazene forms noncovalent complexes with antigens resulting in improved
stability of antigen and multimeric antigen presentation
[101]. PCPP adjuvanted vaccines have been evaluated
in clinical trials where they were shown to be safe and
efficacious [102]. An aqueous formulation of PCPP when
used in combination with influenza antigens showed 10-fold
higher antibody levels than plain influenza antigen. PCPP
has demonstrated good adjuvant activity with a plethora of
other antigens including tetanus toxoid, hepatitis B surface
antigen, HSV type 2 glycoprotein D, cholera, bovine serum
albumin, and porcine serum albumin. IM immunization
of rhesus monkeys with recombinant HIV-1 and PCPP led
to the induction of long-lasting antibody levels for upto 43
weeks. A new polyphosphazene, poly[di(sodiumcarboxylat
oethylphenoxy)phosphazene] (PCEP), when administered
subcutaneously in mice demonstrated superior adjuvant
activity to PCPP. Eng et al. administered influenza antigen
alongwith both soluble PCPP and PCEP and showed that
PCEP induced a stronger Th1 type response. Both PCPP
and PCEP induced IgG and IgA antibodies in lung and
vaginal washes. Another important advantage was the
ability of PCPP and PCEP to induce long-lasting immunity
for upto 8 weeks after single intranasal immunization
[100]. Mapletoft et al. delivered bovine respiratory synctial
virus (BRSV) co-formulated in combination with CpG
oligodeoxynucleotide and polyphosphazene by the nasal
route. The formulation induced both mucosal and systemic
immune responses in mice and resulted in decreased viral
replication on challenge [101]. Kovacs-Nolan et al. mixed
indolicidin and CpG oligodeoxynucleotide 1826 with
polyphosphazene and showed an enhanced ovalbuminspecific antibody response and cell mediated immune
response on SC immunization. Indolicidin is a peptide
containing 13 amino acids, obtained from cytoplasmic
granules present in bovine neutrophils, and is known to
stimulate chemokine IL-8 expression in bronchial epithelial
cells [103]. Kovacs-Nolan et al. also used the combination
of indolicidin and CpG oligodeoxynucleotide 1826 with
polyphosphazene to induce immunity against BRSV infection
in mice after SC immunization. The combination of adjuvants
not only induced high virus neutralizing antibody levels but
also resulted in enhanced IFN-γ secretion and protected mice
against subsequent BRSV challenge [104]. Andrianov et al.
demonstrated enhanced thermal stability and a dose sparing
effect of PCPP on avian influenza antigen. Improved thermal
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30 Priyanka Prabhu and Vandana Patravale
stability would be a boon for vaccine stocking in case of
pandemic episodes and it would decrease the dependence
on costly cold chain facilities for storage and distribution.
PCPP resulted in a four-fold higher half life of the vaccine at
40o C. Also, IM immunization of the PCPP adjuvanted vaccine
conferred 100% protection in ferrets at a 10-fold lower antigen
dose than the non-adjuvanted vaccine [102]. Andrianov et al.
also have formulated microneedles for ID immunization
using PCPP. PCPP possesses ideal properties for use as film
forming agent in microneedle technology owing to its high
viscosity, water solubility, antigen stabilizing ability, and
acceptability for human use. Unlike carboxymethylcellulose,
PCPP does not require surfactants for fabrication of coated
microneedles. PCPP-hepatitis B surface antigen coated
microneedles containing 10 µg antigen when administered
once ID elicited 10 times higher antibody levels than IM
administered microneedles containing 20 µg antigen, 3-fold
higher antibody levels than plain antigen administered IM,
and 100-fold higher antibody levels than ID administration
of non-adjuvanted antigen [105]. These studies all exemplify
the immense potential of PCPP as a vaccine adjuvant owing
to its biodegradability, efficacy and safety. It can certainly
be utilized to formulate nanoparticles to accomodate both
antigen and immunostimulant to further augment the
immune response elicited.
4.7 Polystyrene
Polystyrene is categorized as biocompatible by the
FDA. Minigo et al. fabricated polystyrene nanoparticles
for DNA vaccine delivery using poly-L-lysine as a
cationic linker. Poly-L-lysine served to enhance bonding
between negatively charged DNA (encoding for chicken
egg ovalbumin) and polystyrene nanoparticles. ID
vaccination in C57BL/6 mice demonstrated ovalbuminspecific antibody generation, cellular immune response
production, and reduced tumour formation post challenge
with ovalbumin expressing tumour. Nanoparticles of 50
nm were found to generate the most effective immune
response. Additionally, polystyrene nanoparticles and
DNA admixed without cationic linker were unable to
induce immunity, indicating that the enhanced immune
response was not just due to dose sparing of DNA (by
avoiding its degradation) [106].
4.8 Polypropylene sulphide (PPS)
Stano et al. fabricated PPS nanoparticles (50 nm)
and conjugated them with thiolated ovalbumin for
nasal vaccination. The disulfide linkage connecting
the antigen and the nanoparticle is cleaved in the
reductive environment of the phagosome or endosome
to release the antigen. The nanocarriers demonstrated
permeation through nasal mucosa and were transported
through the M cells followed by uptake into APC in
NALT. Humoral immune responses in the airways and
cytotoxic T lymphocyte responses in spleen and lung
were generated. Flagellin was also conjugated as a
TLR agonist, which led to the generation of mucosal
immunity in vaginal and rectal mucosae due to CMIS
[107]. Hirusoe et al. have demonstrated that the
reduction labile linkage (disulfide) between the antigen
and PPS nanoparticle is beneficial for successful
presentation of exogenous antigen via the MHC class I
pathway since it led to effective CD8+ T cell stimulation
both in vitro and in vivo compared to the presence of a
non-reductive linkage (vinyl sulfone) [108].
4.9 Gantrez AN
Gantrez AN is a copolymer composed of methyl vinyl ether
and maleic anhydride and can simply react with amino
groups. Gomez et al. fabricated Gantrez nanoparticles
containing ovalbumin in encapsulated or coated form.
The nanoparticles exhibited superior immune response
after single ID administration compared to free ovalbumin
and alum adsorbed ovalbumin. Gantrez nanoparticles
containing encapsulated ovalbumin and a low amount
of the cross linker 1,3-diaminopropane elicited a
predominant Th1 response [109]. Salman et al. developed
ovalbumin-containing
thiamine-coated
Gantrez
nanoparticles to target Peyer’s patches for effective oral
vaccination. Thiamine is known to interact with particular
receptors in the intestine. Thiamine-coated nanoparticles
were prepared by the reaction between the amino groups
of thiamine and the anhydride group of Gantrez ® AN.
Oral vaccination with the thiamine-coated nanoparticles
effectively induced both systemic immunity (IgG1 and
IgG 2a), and secretory antibody IgA (4 titers higher) in
comparison with uncoated nanoparticles [110].
4.10 Polyethyleneimine (PEI)
Chen et al. utilized PEI based nanocarriers for cross
presentation of ovalbumin. Ovalbumin was bound to
PEI through electrostatic interactions. PEI shows a well
established “proton sponge effect” wherein the nonprotonated amine groups of PEI undergo protonation in
the acidic pH of endosomes, resulting in osmotic swelling
and bursting of endosomes. As a result, ovalbumin is
released from the endosome into the cytosol for MHC class
I presentation. The nanoparticles were shown to present
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ovalbumin via the MHC class I pathway in mouse bone
marrow derived dendritic cells, and these dendritic cells
were found to stimulate IL-2 secretion in RF33.70 cells [111].
4.11 Dendrimers
Dendrimers are defined as synthetic and extensively
branched macromolecules composed of an inner core
surrounded by many branches attached to it similar
to a tree. Dendrimers are fabricated using polymers
such as polyethylenimine, polyamidoamine (PAMAM),
poly-(N-isopropylacrylamide), poly-(L-glutamic acid),
polypropyleneimine, and polyethylene glycol [12].
PAMAM dendrimers are spherical monodisperse polymers
with ahighly branched and symmetric structure. Their
commercially available form has activated functional
groups, thus permitting the attachment of different
antigens, targeting ligands through simple chemical
reactions. Dendrimers with 3 or 4 generations are ideal
with regards to size and multivalency for DC-SIGN
targeting [53].
Sheng et al. developed mannosylated dendrimers
to achieve avid binding of an antigen delivery system
to mannose receptors on dendritic cells. Mannosylated
PAMAM dendrimer was linked to ovalbumin by
controlled chemical synthesis. ID immunization in
mice showed higher production of ovalbumin-specific
CD4+ T cell and CD8+ T cell responses and higher serum
IgG levels compared to plain ovalbumin. Delivery of
ovalbumin using mannosylated dendrimer showed
cross-presentation of ovalbumin in vivo. Immunized
mice, when subcutaneously challenged with B16ovalbumin tumours, demonstrated late onset, retarded
tumour growth and superior survival compared to mice
immunized with plain ovalbumin [112]. Zaman et al.
developed a polyacrylate ester dendrimer for intranasal
delivery of J14 peptide to induce immunity against
infection due to Streptococcus pyogenes. J14 peptide was
chemically tethered onto the polyacrylate ester based
dendrimer to form a self-assembling nano-sized (21 nm)
vaccine delivery platform. Intranasal vaccination in mice
showed production of J14 specific serum IgG antibody
[113].
5 Lipidic nanocarriers
Lipidic nanocarriers are biocompatible and hence
preferred over polymeric nanocarriers for vaccine delivery.
This section highlights the various lipidic nanocarriers
that have been explored for antigen delivery.
Nanocarriers for vaccine delivery 31
5.1 Liposomes
Liposomes are vesicular structures composed of an aqueous
core surrounded by a lipid bilayer. Liposomes offer several
beneficial features for vaccine delivery. The bilayered
character of liposomes affords inclusion of both hydrophilic
and hydrophobic constituents, and they offer protection to
the antigen against enzymatic degradation. Additionally,
their composition, charge and size could be altered so as
to modulate their pharmacokinetic properties and their
particulate character results in good uptake by APCs. Notably,
they are biocompatible and biodegradable [114]. However,
they also possess some drawbacks including the expense of
raw materials, susceptibility of phospholipids to oxidative
breakdown, and requirement for special storage conditions
[12]. Liposomes have also been explored to enhance the
immunostimulant activity of several immunostimulant
molecules such as TLR ligands. Incorporation of MPLA into
cationic liposomes further enhanced its immunostimulant
potential. CAF01 is a promising cationic liposome-based
adjuvant. It has a size of 500 nm and a charge of + 50 mV.
It comprises dimethyldioctadecyl ammonium bromide
and trehalose 6’6-dibehenate (TDB) in a 5:1 ratio. TDB is
a synthetic analogue of mycobacterial cord factor, with a
lower toxicity, and is an agonist of C-type lectin receptor.
CAF01 systems have been explored for vaccination against
malaria, tuberculosis, and chlamydia. CAF01 liposomes
are undergoing clinical trials for tuberculosis vaccine. The
CAF01 system has been shown to generate both humoral
and cellular immune responses. CAF01 has also been
explored in nasal influenza vaccination. Incorporation of
TLR3 ligand poly(I:C) in CAF01 has also been studied. The
CAF01 system allows both entrapment and adsorption of
antigens. It can be lyophilized and reconstituted without
hampering its immunogenic character, and can also
be subjected to sterile filtration and γ-radiation based
sterilization [114].
Liposomes have also been investigated for nasal
vaccination. Bioadhesive liposomes have been prepared
in order to extend the nasal retention time of liposomes.
Chitosan adsorption to the surface of liposomes not only
confers bioadhesive properties to the liposome but also
induces a positive surface charge, enhancing uptake by
nasal mucosa [115]. Chiou et al. fabricated a mixture of
liposomes containing inactivated avian influenza virus
and xanthan gum or tremella gum. The bioadhesive
liposomes elicited a superior immune response in
intranasally immunized chickens as compared to nonbioadhesive liposomes. Apart from their mucoadhesive
property, xanthan gum and tremella are also reported to
possess some immunostimulant function [116].
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32 Priyanka Prabhu and Vandana Patravale
A number of factors are known to significantly impact
the nature and intensity of the immune response elicited
by liposomes, and are listed below [117].
Method of antigen association: Antigen can either be
physically or chemically associated with the liposome.
One can encapsulate the antigen inside the liposome,
covalently conjugate the antigen with liposome either
before or after vesicle formation, or adsorb the antigen
onto the surface via non-covalent interactions. Surface
association of the antigen results in an enhanced antibodymediated response since B cell receptors can recognize the
antigen on the surface of liposomes. Covalent conjugation
of antigen to lipid or liposomes has been shown to enhance
MHC class I presentation and subsequently an enhanced
cellular immune response.
Lipid
composition:
Some
lipids
show
immunostimulant activity. Incorporation of these
lipids would enhance the vaccine adjuvant potential of
liposomes. A number of these lipids are described. Alltrans retinoic acid, which is a metabolite of vitamin A,
has been shown to possess immunomodulatory activity
via retinoic acid receptor agonism. Lauric acid has been
shown to stimulate cytokine secretion and expression
of costimulatory molecules on dendritic cells via TLR4
signalling. Palmitic acid has been shown to activate
NLRP3-ASC inflammasome. Lysophosphatidylcholine
is known to enhance CD86 expression, chemokine
secretion, and upregulation of MHC class II molecules.
DiC14-amidine, which is a cationic transfection agent, has
been shown to be a TLR4 agonist.
Charge: Cationic charge favours uptake by APCs
owing to the negatively charged cell membrane.
Bilayer fluidity: The more rigid the lipidic bilayer, the
greater the immunostimulant activity of the liposome. As
a result, lipids with higher gel-liquid crystal transition
temperature show greater immunogenicity compared to
those with lower gel-liquid crystal transition temperature.
Size: 250-700 nm vesicles may result in a stronger Th1
response.
Lamellarity: Multilamellar vesicles may result in an
increased Th2 response.
Fusogenicity: Inclusion of certain fusogenic lipids
(dioleyl phosphatidyl ethanolamine) in the liposomal
bilayer confers onto the liposome the property of fusing
with the plasma membrane or the endosomal membrane.
Fusion with the plasma membrane aids in enhanced
uptake of liposomes by the APCs, while fusion with the
endosomal membrane results in cross presentation of
antigen. Fusogenic liposomes are extremely beneficial
in delivery of DNA vaccines wherein it is imperative to
release the vaccine cargo in the cytosol.
Addition of immunostimulant: Addition of
immunostimulant molecules such as TLR ligands, and
NLR ligands enhances the immunogenic potential of
liposomes.
Tiwari et al. developed in situ gelling liposomes (719
nm) containing hepatitis B surface antigen for nasal
vaccination. Liposomes were prepared by hydration of
the lipid film with antigen solution containing polyacrylic
acid. Polyacrylic acid shows pH dependent gelling and
mucoadhesive properties, which leads to prolonged
retention in the nasal cavity and enhanced mucosal
uptake. The unentrapped polyacrylic acid containing
antigen solution was used as continuous phase for
suspending the gel core liposomes before administration.
Intranasal immunization of these liposomes resulted in
potent systemic and mucosal antibody responses as well
as cellular immune response. IM administration of alum
adsorbed antigen failed to generate both mucosal IgA
and cellular immune responses. The liposomes showed
a triphasic release behaviour in which the first phase
involved burst release of antigens present on the surface
of the hydrogel. The second phase involved release of
the antigen via diffusion from the gel and liposomes. The
third phase involved release of antigen from polyacrylic
acid gel inside the liposomes, followed by its diffusion
through phospholipid bilayer [118]. Tiwari et al. in another
study formulated transmission-blocking malaria antigen
Pfs25 in gel core liposomes fabricated from polyacrylic
acid. IM immunization resulted in higher serum antibody
levels than plain liposomes and alum adsorbed antigen.
Also, the co-administration of CpG oligodeoxynucelotide
in liposomes led to a more potent immune response
compared to plain gel core liposomes. In conclusion,
gel core liposomes offer great potential for single-shot
vaccination, which is desirable from a patient compliance
perspective [119]. Copland et al. developed mannosylated
liposomes (260 nm) containing tetanus toxoid and studied
the effect of mannosylation on uptake, maturation,
and induction of T cell proliferation by dendritic cells.
Mannosylated liposomes showed the highest expression
of CD80, CD86, MHC class II molecules, and dendritic
cell maturation marker CD83, and the greatest T cell
proliferation. This was followed by plain liposomes, then
plain antigen solution [120].
Yuba et al. fabricated liposomes encapsulating
ovalbumin thatwere functionalized with succinylated
poly (glycidol) and 3-methylglutarylated poly (glycidol).
Functionalization with these polymers having carboxylic
groups endowed the liposomes with the ability to fuse
with the endosomes and release the antigen load into
the cytosol for MHC class I presentation thereby eliciting
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a strong cell mediated immune response. Liposomes
modified with 3-methylglutarylated poly (glycidol)
showed greater pH responsive fusogenic potential owing
to the more hydrophobic nature of the polymer. Intranasal
immunization led to superior cell-mediated immune
response compared to unmodified liposomes and cellular
immunity comparable to that of CFA [121].
5.2 Archaeosomes
Archaeosomes are vesicles enclosing lipid bilayer/s of total
polar lipids extracted from microbes of the class Archaea.
Archaeosomes exhibit greater uptake by macrophages
and other APCs compared to liposomes. Apart from their
uptake by APCs, they also promote recruitment and
activation of APCs and carry the antigen to both MHC class
I and MHC class II pathways for presentation of antigen to T
cells, resulting in induction of potent humoral and cellular
immune responses. Polar lipids obtained from Archaea
differ from the lipids obtained from Eukarya and Bacteria.
Phospholipids obtained from Eukarya and Bacteria
are generally composed of unbranched and commonly
unsaturated fatty acyl chains of different lengths, and
are tethered through ester linkages to the sn-1,2 glycerol
carbons of the glycerol backbone. Polar lipids of Archaea
are composed of regularly branched 5-carbon repeating
units, resulting in the formation of isopranoid chains
which are commonly saturated and tethered via ether
linkages to the sn-2,3 glycerol carbon atoms. Archaeosomes
offer various advantages as compared to conventional
liposomes. Due to the saturated character of the lipid
chains they can be fabricated and stored in the presence
of air/oxygen. They give rise to bilayer membranes with
greater stability and lower permeability than conventional
liposomes. They show 3 to 53 times greater uptake by
phagocytes than conventional liposomes. They are selfadjuvanting vaccine delivery systems and do not need
co-administration or co-formulation of immunostimulant
molecules, unlike liposomes. They are safe and exhibit
a good shelf life of more than 2 years [122]. Also, their
immunostimulatory properties are independent of the
inflammation in the host caused by infection [123].
Li et al. formulated archaeosomes composed of
polar lipid fraction E of Sulfobus acidocaldarius for
oral immunization with ovalbumin. The archaeosomes
demonstrated excellent stability in simulated gastric
fluids and intestinal fluids. The archaeosomes showed
better systemic IgG and mucosal IgA response, and
induced ovalbumin specific T cell proliferation than
conventional liposomes [124]. Higa et al. have fabricated
ultradeformable archaeosomes using total polar lipids
Nanocarriers for vaccine delivery 33
from Halorubrum tebenquichense. Sodium cholate and
soybean phosphatidylcholine were employed to make
ultradeformable archaeosomes. Topical application
of the developed ultradeformable archaeosomes
showed 10-100 times higher serum IgG levels than
ultradeformable liposomes applied topically [123]. Patel
et al. have developed a vaccine delivery platform using
archaeosomes for mucosal vaccination, called AMVAD
(Archaeal lipid mucosal vaccine adjuvant and delivery).
It is prepared by the interaction of archaeosomes sized
between 100-200 nm with multivalent cations such as
calcium. The difference between AMVAD and cochleates
is the absence of aqueous volume and multilamellar
nature of the latter. Antigen loading in the AMVAD
system may be carried out in two ways-either the antigen
entrapped archaeosomes may be interacted with calcium
or soluble antigen is included in the buffer and mixed
with preformed archaeosomes followed by blending with
cations. Intranasal immunization with the AMVAD system
showed good antigen-specific IgA antibody levels in the
nasal mucosa, vaginal mucosa, serum, faeces, and bile,
and potent serum IgG1 and IgG2a levels were produced.
The AMVAD system also demonstrated generation of CD8+
cytotoxic T lymphocyte response [125].
5.3 Niosomes
Non-ionic amphiphilic molecules self-assemble in water to
give rise to closed bilayer structures called niosomes. The
key advantages of niosomes include their biocompatibility,
superior stability, economical ingredients, nonimmunogenicity, and amenability to easy large scale
production, non-toxicity, and simple storage conditions.
Niosomes have been explored for vaccine delivery via
the SC, intraperitoneal, IM, oral, nasal, topical, and
transcutaneous routes [126]. Maheshwari et al. fabricated
niosomes containing hepatitis B surface antigen and
cholera toxin B as an immunostimulant for transcutaneous
vacccination. Cholera toxin B activated Langerhans
cells in the epidermis followed by antigen presentation
in the draining lymph node and generation of systemic
immunity. The co-administration of cholera toxin B with
hepatitis B surface antigen-loaded niosomes elicited both
Th1 and Th2 type immune responses [127]. Vyas et al.
developed niosomes composed of Span 85 and cholesterol
loaded with DNA encoding hepatitis B surface antigen
using the reverse-phase evaporation technique. Topical
application of these niosomes in Balb/c mice showed the
ability of niosomes to elicit equivalent systemic antibody
generation and cytokine levels (IL-2 and IFN-γ indicating
Th1 response) as IM recombinant hepatitis B surface
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34 Priyanka Prabhu and Vandana Patravale
antigen and topically applied liposomes. Immunization
of mice with recombinant hepatitis B surface antigen has
been shown to generate only a humoral response owing
to the presentation of exogenous antigen by the B cells
via the MHC class II pathway to the Th2 cells. However,
when DNA vaccine is given, a portion of the hepatitis B
surface antigen produced in vivo is presented by the B
cells via the MHC class II pathway to the Th2 cells and
the rest is lysed in the APCs and presented via the MHC
class I pathway to Th1 cells [128]. Rentel et al. developed
two different niosomes containing ovalbumin for peroral
administration, one composed of 70% stearate sucrose
ester and 30% palmitate sucrose ester, and the other
composed of 30% stearate sucrose ester and 70% palmitate
sucrose ester. The former, which was more hydrophobic
than the latter, was only effective in generating humoral
immunity. The observed results may be due to limited
uptake of the hydrophilic niosomes by the Peyer’s patches
resulting in poor immune response generation [129]. Jain
et al. developed mannose functionalized niosomes for
oral delivery of DNA encoding hepatitis B surface antigen.
Niosomes were coated with O-palmitoyl mannan to shield
them from bile salts and GIT enzymes and also to enhance
uptake by macrophages and dendritic cells present near
Peyer’s patches. The mannosylated niosomes elicited
both systemic and mucosal antibody responses and also
produced cellular immunity (high levels of IL-2 and IFN-γ)
on oral administration [130]. Cortesi et al. developed
niosomes (320 nm) by lipid film hydration, containing
secretory recombinant form of HSV type 1 glycoprotein
B. Intranasal immunization of these niosomes in mice
showed generation of both IgG 2a antibody in serum and
IFN- γ generation in CD4+ splenocytes, and also resulted in
90% protection from a vaginal challenge with HSV [131].
5.4 Emulsions
Emulsions are biphasic systems comprising an oily
phase and aqueous phase emulsified with the aid of
surface active agents. There are three types of emulsions
depending on the nature of the dispersed and continuous
phase. Oil-in-water emulsions contain oil in the dispersed
phase and water as the continuous phase, and water-inoil emulsions contain water in the dispersed phase and
oil as the continuous phase. Multiple emulsions contain
small droplets of the continuous phase dispersed in the
internal dispersed phase. These can be oil-in-water-in
oil or water-in-oil-in water. Emulsions have been used
for vaccine adjuvant purposes for a long time. The most
potent vaccine adjuvant, which is often considered as the
gold standard for vaccine adjuvants, is Freund’s Adjuvant.
CFA is a water-in-oil emulsion composed of mineral oil,
mannide monooleate and heat killed mycobacteria.
Incomplete Freund’s Adjuvant is devoid of the heat killed
mycobacteria. However, the ability of Freund’s Adjuvant to
cause granulomatous reaction at the injection site owing
to the non-metabolizing property of the oil precludes its
use in humans. MF59 is an oil-in-water emulsion that is
licensed for use in an influenza vaccine (Fluad®), and
contains squalene, Polysorbate 80, and sorbitan trioleate.
Humoral immunity is generally induced by water-in-oil
emulsions whereas oil-in-water or or water-in-oil-in-water
multiple emulsions lead to a cellular immune response.
The nature of the emulsion may also impact on the
longevity of the resulting immune response [132].
Shahiwala et al. fabricated water-in-oil-in-water
multiple emulsions containing ovalbumin and modified
them with chitosan. The emulsions produced higher
levels of both serum IgG and mucosal IgA than ovalbumin
solution. Oral immunization was more effective in inducing
both serum IgG and mucosal IgA levels compared to nasal
immunization. Intranasal immunization led to higher serum
IgG levels than mucosal IgA levels. Squalane oil employed
as the oily phase resulted in immunostimulatory effects,
and the presence of water in the outer continuous phase
provided a low viscosity solution for nasal instillation.
Chitosan was included with the aim of increasing the nasal
residence time of the formulation [133].
Bielinska et al. formulated a water-in-oil nanoemulsion
for nasal vaccination using Bacillus anthracis protective
antigen. The nanoemulsion was found to be noninflammatory to the nasal mucosa of animals and
conferred protection to guinea pigs against an ID challenge
with Bacillus anthracis spores. The nanoemulsion vaccine
only required single administration in guinea pigs and
twice in mice compared to the commercial vaccine which
requires six administrations over 18 months. The developed
nanoemulsion showed potential in overcoming the
drawbacks associated with the presently available anthrax
vaccine, which is invasive, requires multiple injections, and
is adjuvanted by alum leading to adverse effects [134].
5.5 Transfersomes
Transfersomes are ultra-deformable vesicles composed
of an aqueous core surrounded by a complex lipid
bilayer. Incorporation of membrane softeners endows
transfersomes with an ultra-flexible character permitting
them to squeeze across the intercellular lipid barrier of
the stratum corneum. They show enhanced permeation
through skin compared to traditional liposomes and
niosomes [135].
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Transfersomes have been explored for transcutaneous
delivery of several antigens. Mahor et al. developed cationic
transfersomes from cationic lipid N-[1-(2, 3-dioleyloxy)
propyl]-N,N,N-trimethylammonium chloride (DOTMA)
and sodium deoxycholate loaded with hepatitis B surface
antigen. Topical application of the cationic transfersomes
resulted in higher serum antibody levels and cytokine
levels (IL-2 AND IFN-γ) than topically applied naked
DNA, and equivalent antibody levels and cytokine
levels (IL-2 AND IFN-γ) as IM administered recombinant
hepatitis B surface antigen [136]. Gupta et al. fabricated
transfersomes containing tetanus toxoid. Topical
immunization resulted in comparable antibody levels to
those of IM administered tetanus toxoid [137]. Gupta et al.
in another study demonstrated that topical immunization
with tetanus toxoid-loaded transfersomes resulted in
generation of equivalent antibody levels to those of SC
administered alum adsorbed tetanus toxoid [138]. Xu
et al. developed transfersomes containing DNA encoding
respiratory syncytial virus (RSV) surface glycoproteins for
topical immunization in mice against RSV infection. IM
administration of DNA only resulted in serum antibody
induction; no mucosal IgA antibody was produced, nor
was virus-specific cellular immunity induced. Topical
immunization with DNA-loaded transfersomes resulted
in RSV-specific mucosal and systemic antibody responses
and a higher number of IFN-γ producing cells. Additionally,
topical immunization protected mice against RSV challenge
as evidenced by the lesser lung abnormalities seen in the
animals compared to IM immunization [139].
5.6 Bilosomes
Liposomes and niosomes show poor activity on oral
administration due to their instability in the GIT. Bile salts
cause dissolution and enzymatic breakdown. Inclusion
of bile salts in the membrane of these vesicles confers
stability to the vesicles against bile acids. Vesicles in
which bile salts are included are termed as bilosomes
[140]. Bilosomes possess greater chemical stability, GIT
stability, and require lower doses of antigens compared to
liposomes and niosomes. In addition, unlike liposomes,
there is no requirement for special storage conditions [12].
Arora et al. fabricated mannan functionalized bilosomes
for oral hepatitis surface B antigen vaccination. Mannan
coating not only stabilizes the vesicles in GIT but also
acts a targeting moiety for mannose receptors present
on dendritic cells and macrophages. Mannan coated
bilosomes showed higher antigen-specific IgG levels
and secretory IgA levels in intestinal, nasal, vaginal, and
salivary secretions compared to plain bilosomes [140].
Nanocarriers for vaccine delivery 35
Mann et al. fabricated tetanus toxoid-loaded bilosomes
for oral vaccination. The bilosomes showed both systemic
and mucosal immune responses [141]. Singh et al.
fabricated bilosomes loaded with BSA and conjugated
them with cholera toxin B subunit to increase affinity
for M cells present in Peyer’s patches. Single shot oral
immunization of these bilosomes demonstrated similar
immune responses to those obtained with parenteral
immunization of BSA emulsified with CFA. Also, unlike
CFA, the bilosomes were free from any side effects [142].
Shukla et al. developed bilosomes loaded with hepatitis
B surface antigen using the lipid film cast technique. Oral
immunization with these bilosomes showed generation of
both systemic IgG antibody and mucosal sIgA antibody in
saliva, vaginal, and intestinal secretions [143].
5.7 Immunostimulating complexes (ISCOMs)
ISCOMs are antigen-containing cage-like nanostructures
which are fabricated from phospholipids, cholesterol, and
Quil A saponin from Quillaja saponaria bark. They show a
spherical shape and are negatively charged with a size of
around 40 nm [29]. ISCOMs possess glucuronic acid on their
surface, thus conferring a negative charge and facilitating the
association of positively charged antigens via electrostatic
interactions [144]. They offer dual benefits as they resemble
viruses in terms of size and surface protein orientation
and also possess the strong immunostimulant potential of
Quillaja saponin [3]. Quil A saponin possesses haemolytic
properties, rendering it toxic for human use. Mixing it with
cholesterol, however, results in loss of this characteristic
[145]. ISCOMs devoid of antigen are called ISCOM matrices.
Hydrophobic antigen loading occurs via direct encapsulation
or anchoring with the lipidic portions whereas hydrophilic
antigens are loaded through special procedures. ISCOMs were
formerly used parenterally, but now have also been explored
successfully for oral and intranasal immunization. ISCOMs
undergo uptake by APCs via endocytosis and activate the APCs
[29]. ISCOMs enhance the expression of MHC class II molecules
and promote the secretion of pro-inflammatory cytokines
[144]. ISCOMs after their internalization and processing inside
the APCs are known to induce the production of IFN-γ and
IL-2 leading to the generation of a potent Th1 response [29]. A
commercially available ISCOM based veterinary vaccine used
for equine influenza is Equilis ®Prequenza by Merck Animal
Health [82,146].
5.8 Cochleates
Cochleates are solid particles containing large continuous
lipid bilayer sheets rolled up in a spiral structure. They are
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36 Priyanka Prabhu and Vandana Patravale
prepared by fusing small negatively charged unilamellar
liposomes in the presence of calcium. Cochleate
formation takes place owing to the hydrophobic lipid
bilayer sheets which fold up to minimize contact with
water. Cochleates possess higher stability than liposomes
against lipase, pH and temperature. Phospholipids
used to fabricate cochleates include phosphatidyl
serine,
dioleoylphosphatidylserine,
phosphatidic
acid, phosphatidylinositol, phosphatidyl glycerol,
phosphatidylcholine,
phosphatidylethanolamine,
diphosphatidylglycerol,
dioleoyl
phosphatidic
acid,
distearoyl
phosphatidylserine,
dimyristoyl
phosphatidylserine, and dipalmitoyl phosphatidylgycerol
[147]. Advantages of cochleates for vaccine delivery
include their biodegradability, biocompatibility, simple
fabrication, prolonged release of antigen, and ability to
protect the encochleated antigen from degradation, ability
to be lyophilized and stored post lyophilisation [148].
Kheiri et al. developed influenza membrane
glycoprotein-loaded cochleates for oral and IM
immunization. Both oral and IM immunization led to
high serum IgG levels, with the IM route giving higher
IgG levels. Oral immunization resulted in generation of
IgA in saliva, whereas IM administration only led to IgG
in saliva. Oral immunization also conferred protection
to mice against lung and tracheal infection post nasal
influenza challenge [149].
5.9 Solid Lipid Nanoparticles (SLN)
SLN are composed of a solid lipid matrix or a matrix
made up of a blend of solid lipids. SLN offer a number
of beneficial features for vaccine delivery, including
biocompatibility, simple manufacture, sustained release
of antigen, protection of antigen from degradation, and
amenability to ligand functionalization for targeted
delivery to APCs [150]. Mishra et al. developed hepatitis
B surface antigen-loaded mannose functionalized SLN
for SC vaccination. SLN were fabricated using the solvent
injection technique. They demonstrated lymph node
and spleen uptake and also elicited sustained antibody
production. Antibody titers obtained were equivalent
to those obtained after IM hepatitis B surface antigen
immunization [17].
5.10 Miscellaneous lipidic nanocarriers
Arias et al. developed yellow carnauba wax nanoparticles
onto which HIV-gp140 glycoprotein was adsorbed. The
nanoparticles resulted in superior T cell proliferation in
comparison to plain HIV gp-140. ID immunization resulted
in high levels of antigen-specific IgG antibody, and
intranasal immunization led to generation of serum and
vaginal IgG and IgA antibodies. The study is beneficial
for HIV vaccine design wherein it is necessary to activate
the mucosal immune response in the female genital tract
[151]. Sloat et al. developed lipidic nanoparticles (200
nm) made from glyceryl monostearate and lecithin. BSA
and Bacillus anthracis protective antigen were covalently
conjugated onto the surface of these nanoparticles for
SC vaccination. BSA conjugated nanoparticles showed
higher IgG and IgM antibody generation compared to
alum adsorbed antigen, and antibody levels comparable
to those obtained with Incomplete Freund’s Adjuvant.
Mice immunized with B. Anthracis protective antigen
conjugated nanoparticles showed protection against
lethal anthrax challenge [152]. Moon et al. developed
multilamellar lipidic vesicles composed of recombinant
Plasmodium vivax circumsporozoite antigen VMP001
encapsulated within the aqueous core in addition to
being attached to the outer membrane. SC immunization
with these vesicles and MPLA led to induction of high
antibody titres which persisted for more than a year at a
10-fold lower antigen dose compared to alum adsorbed
antigen and soluble antigen administered with MPLA. The
presence of antigen anchored onto the surface of vesicles
resembled the multivalent presentation of epitopes offered
by invading pathogens leading to B cell receptor crosslinking, germinal cell formation and increased expansion
of antigen-specific follicular helper T cells [153].
6 Inorganic nanocarriers
6.1 Silica nanoparticles
Porous silica nanoparticles are endowed with numerous
beneficial features for vaccine delivery. Firstly, their
large surface area and huge pore volume provides high
loading of vaccine. Secondly, the insoluble silica allows
slow release of antigen creating a depot phenomenon.
Thirdly, their rigid architecture provides protection to
entrapped antigen against degradation. Fourthly, they
show excellent chemical stability, biocompatibility,
less toxicity, and their pore diameter and particle size
can be adjusted. Wang et al. developed porous silica
nanoparticles containing BSA for oral vaccination. Their
results suggested dependence of the immune response
on the size and architecture of silica nanoparticles.
Nanoparticles of 430 nm and containing large pores
showed prolonged release of antigen due to increased
diffusional pathlength and induced both systemic and
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mucosal immunity more effectively compared to the
nanoparticles of 130 nm and 1-2 µm. Also, nanoparticles
of around 500 nm are taken up to a greater extent by M
cells and enterocytes, which may have contributed to the
superior adjuvanticity of the 430 nm nanoparticles. The
nanoparticles also induced a mixed Th1/Th2 response
[154]. Carvalho et al. developed mesoporous SB-15 silica
nanoparticles and compared their vaccine adjuvant
potential with alum and Incomplete Freund’s Adjuvant by
both IM and oral routes. BSA was encapsulated or adsorbed
onto SBA-15. SBA-15 nanoparticles with BSA generated
both Th1 and Th2 responses. SBA-15 particles are taken
up by macrophages by phagocytosis and then stimulate
production of IL-1β via NALP3 inflammasome activation
[155]. Guo et al. fabricated hollow mesoporous silica
nanoparticles containing porcine circovirus type 2 ORF2
protein for IM vaccination. Controlled release of protein
was seen from the nanoparticles over a 2 week period.
Also, the nanoparticles elicited higher antibody titres and
T lymphocyte proliferation compared to porcine circovirus
type 2 ORF2 protein administered alone [156]. Gordon
et al. fabricated temperature sensitive chitosan hydrogels
loaded with silica nanoparticles for SC ovalbumin
delivery. Thermoresponsive hydrogels are promising for
vaccine delivery as they are easy to inject in solution form
and gel in vivo at body temperature providing prolonged
delivery of antigen from the gel. Chitosan solutions gel
in response to pH, however, inclusion of polyol salts in
chitosan solutions results in temperature based gelation.
The study also compared the efficacy of Quil A adjuvant
and antigen delivered in silica nanoparticles loaded
within hydrogels with that of soluble ovalbumin and Quil
A delivered in chitosan hydrogels. The chitosan hydrogel
released 35% of soluble ovalbumin, whereas the chitosan
hydrogels loaded with silica nanoparticles containing
ovalbumin showed a release of 16% in 14 days. Chitosan
hydrogel loaded antigen irrespective of the form of antigen
(soluble v/s particulate), or the presence or absence of
Quil A as an adjuvant, generated both humoral and cellmediated immune responses. Silica nanoparticulate
based delivery of antigen and adjuvant in hydrogel proved
to be more effective in generating CD4+ T cell proliferation
in mice when compared to soluble antigen and adjuvant
in chitosan hydrogel [157].
6.2 Calcium Phosphate Nanoparticles
Inorganic nanoparticles possess certain benefits over
organic nanoparticles, including their lower propensity
to microbial degradation, superior resistance to bile salts
and lipases, non-toxicity, very good storage stability, low
Nanocarriers for vaccine delivery 37
cost, and ability to be fabricated at lower temperatures
[158]. Calcium phosphate has been utilized in Europe for
vaccines against tetanus and diphtheria antigens [159].
Calcium phosphate offers several beneficial features
for vaccine delivery, including good biocompatibility,
biodegradability, non-toxicity, easy manufacture,
economical, indigenous material present in bones and
teeth, and high affinity for antigens, DNA, and proteins
[158]. Calcium phosphate nanoparticles are taken up by
the cells and then dissolved in lysosomes; calcium ions
are then quickly removed from the cell [160].
Behera et al. developed calcium phosphate
nanoparticles (less than 200 nm) for parenteral
immunization in fish by adsorbing the S-layer protein
(a protein from Aeromonas hydrophila, a pathogen
infecting fish) as an antigen. The nanoparticles
demonstrated an adjuvant effect similar to that obtained
with antigen emulsified with Freund’s Incomplete
Adjuvant, but without the associated adverse effects
[158]. He et al. compared the vaccine adjuvant effect of
calcium phosphate nanoparticles with alum adjuvant
using HSV type 2 and Epstein-Barr virus as antigens.
Calcium phosphate nanoparticles were shown to be better
adjuvants than alum, with negligible inflammation at the
site of vaccination. Also, mice immunized with calcium
phosphate nanoparticles showed 100% protection against
intravaginal challenge with HSV type 2. Calcium phosphate
nanoparticles induced an IgG2a antibody response unlike
alum which only gives a Th2 type response [159]. He et al.
in another study have shown the efficacy of calcium
phosphate nanoparticles for mucosal immunization
against HSV type 2. Intranasal and intravaginal
administration of calcium phosphate nanoparticles
generated both mucosal and systemic immunity in
mice and enhanced their survival after viral challenge
[161]. Sokolova et al. fabricated calcium phosphate
nanoparticles containing TLR ligands: poly (I:C) and CpG,
and antigen hemagglutinin. The nanoparticles caused
dendritic cell activation as evidenced by the upregulation
of surface molecules and cytokine generation (IL-12, IL-2,
TNF-α), and also stimulated T cell proliferation [160].
6.3 Gold nanoparticles
Gold nanoparticles offer some attractive features for
vaccine delivery. Their size can be simply controlled from
around 1 nm to 100 nm or more. They also allow anchoring
of peptide antigens or nucleic acids onto their surface
using simple chemistry. They are biocompatible and safe
[162]. Gold nanoparticles have been explored in clinical
trials for hepatitis B and malaria vaccines. DNA vaccines
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38 Priyanka Prabhu and Vandana Patravale
were coated onto gold nanoparticles and delivered to the
skin epidermis using a gene gun [44].
Barhate et al. developed chitosan modified gold
nanoparticles (40 nm) loaded with tetanus toxoid and
Quillaja saponaria extract. The nanoparticles were able to
shield tetanus toxoid from gastric degradation in vitro. Oral
vaccination in mice demonstrated 28 times and 6.5 times
higher IgG immune response compared to tetanus toxoid
administered alone and with Quillaja saponaria extract
respectively. Induction of IgA antibody was also observed
in faeces and intestinal washes. Quillaja saponaria extract
is reported to induce dendritic cell maturation through
interaction with the TLR 4 on dendritic cells. Additionally,
chitosan, being mucoadhesive, enhances uptake by the
M cells of Peyer’s patches [163]. Bastus et al. conjugated
gold nanoparticles to amyloid growth inhibitory peptide
and sweet arrow peptide. Bone marrow macrophages
showed uptake of these nanoparticle conjugated peptides
although they are otherwise unable to uptake the peptide
alone. Uptake occurred via TLR-4. Macrophage activation
occurred and pro-inflammatory cytokine production (IL-6,
TNF-α, IL-1 β) was also stimulated [164].
6.4 Silver Nanoparticles
Jazayeri et al. developed silver nanoparticles (12 nm) for
oral DNA vaccination using green technology. The DNA
used was plasmid DNA encoding the hemagglutinin gene
of the avian influenza virus. Single shot oral immunization
in chickens resulted in the induction of both antibody
and cellular immune responses and increased cytokine
levels. Also, the silver nanoparticles were found to be noncytotoxic [165].
7 Miscellaneous nanocarriers
7.1 Carbon Nanoparticles
Wang et al. fabricated carbon nanoparticles (470 nm)
with larger pores than conventional mesoporous carbon
particles for BSA antigen delivery. Mesoporous carbon
particles offer several advantages for vaccine delivery
due to their chemical stability and biocompatibility. The
idea was to increase the antigen loading of conventional
mesoporous carbon particles which have a low pore size
of less than 10 nm. The researchers used a lower amount
of carbon source (sucrose) and fabricated mesoporous
carbon nanoparticles with a pore size of 40-60 nm. These
carbon nanoparticles not only offer higher loading of
antigen, but also aid in prevention of antigen aggregation,
and offer simple loading of antigen by mixing with antigen
solution at 4 o C, thus eliminating the harsh conditions
of heat and organic solvents which may be used to load
antigen in other carriers. Also, the rigid architecture
of the nanostructure offers protection to antigen in the
hostile environment of the GIT. Oral immunization in
mice resulted in serum antibody levels almost equivalent
to those obtained with parenteral administration of BSA
in CFA and mucosal IgA antibodies in intestinal, vaginal,
and salivary secretions. The success of these nanoparticles
may also be a result of their hydrophobic nature and
optimal particle size which is known to enhance M cell
uptake in oral antigen delivery [166].
Carbon nanotubes are nanodimensional allotropes
of carbon. They may be single walled carbon nanotubes
(SWCT) or multiwalled carbon nanotubes, the latter
being of a larger thickness and greater length [12]. Carbon
nanotubes offer several advantages for vaccine delivery.
They are able to internalize into cells by a myriad of
mechanisms, their high aspect ratio may afford better
immunogenicity of the associated antigen, and their large
surface area aids in chemical modification. Villa et al.
conjugated Wilm’s tumour protein to solubilised SWCT.
The protein is upregulated in many cancers but suffers
from poor binding affinity to MHC class II molecules.
Peptide conjugated SWCTs showed good uptake by
dendritic cells and macrophages in vitro. SC immunization
of these carbon nanotubes, along with the adjuvant
TiterMax®, resulted in induction of antigen-specific IgG
levels, however mere mixing of peptide and adjuvant
failed to generate significant antibodies [167].
7.2 Nanodecoy
Nanodecoy systems comprise a ceramic core and an
outer shell of carbohydrate. They offer many beneficial
features for vaccine delivery. They are biocompatible
and biodegradable, and their external hydrophilic
carbohydrate layer helps to retain the protein conformation
and prevents aggregation of the nanostructure. Goyal et al.
self-assembled hydroxyapatite and cellubiose followed
by coating with hepatitis B surface antigen to form
nanodecoy systems. SC immunization in mice showed
enhanced serum antibody generation and higher levels
of IFN-γ and IL-2 compared to alum adsorbed hepatitis B
surface antigen and plain hepatitis B surface antigen [168].
Goyal et al.in another study, fabricated aquasomes (200
nm) for BSA delivery. Self-assembly of the hydroxyapatite
core was enabled by a co-precipitation method followed
by coating with trehalose and cellubiose, BSA was then
adsorbed onto these systems. SC immunization in mice
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showed higher serum antibody levels compared to alum
adsorbed BSA and BSA alone. Aquasomes caused a mixed
Th1/Th2 response with a high ratio of IgG2a to IgG1.
Polyhydroxyl oligomers such as trehalose and cellubiose
which are epitaxially adsorbed onto the hydroxyapatite
core serve a dual purpose: firstly they act as ligands for
mannose receptors present on APCs, and secondly, they
stabilize the structure, both resulting in enhanced antigen
uptake and presentation [169].
7.3 Vaults
Vaults are defined as large ribonucleoproteins present
in eukaryotic cells such as dendritic cells, endometrium,
and lung. Vaults are barrel shaped, containing numerous
copies of three species of proteins and many copies of
small untranslated RNA. The major vault protein (MVP),
which is found in 96 copies per vault, is responsible for
the barrel shape of vaults. The MVP sequence is able to
encode all information required to achieve self-assembly
of protein giving rise to a recombinant vault nanoparticle
with bacilovirus system. Recombinant vaults possess
a huge internal cavity to house many immunogenic
proteins. The vault size mimics that of microbes, thus
facilitating their uptake by dendritic cells. Champion
et al. fabricated hollow vault nanocapsules encapsulating
major outer membrane protein of Chlamydia muridarum
for nasal vaccination. Vaults were found to activate
inflammasome and increase IL-β1 secretion without TLR
involvement. They were able to induce immunity without
causing inflammation (without causing production of proinflammatory cytokines), and reduce bacterial burden
in mice following vaginal challenge [170]. Kar et al.
formulated ovalbumin-containing vault nanocapsules
for SC vaccination. They resulted in potent ovalbuminspecific CD4+ memory T cell and CD8+ memory T cell
generation in vaccinated mice [171].
8 Conclusion
The burgeoning field of vaccine delivery has made
noteworthy advancements in recent years, fuelled by the
improved understanding of both innate and adaptive
immune mechanisms coupled with the development of a
myriad of novel nanoparticulate antigen delivery systems.
However, there are several unmet needs in vaccinology
including a paucity of adjuvants to mediate the cellular
arm of the immune response and lack of effective adjuvants
for non-invasive vaccination. Also, development and
approval of novel vaccine adjuvants is a time-consuming
Nanocarriers for vaccine delivery 39
process. Adjuvants cannot be registered alone; they must be
registered as an adjuvant/antigen combination. Apart from
the cardinal attributes of efficacy, safety, and stability, it is
necessary for vaccine adjuvants to be prepared on a large
scale in a simple and cost effective way to facilitate mass
vaccination especially in the developing countries of the
world. There is also a need for humanized animal models to
closely mimic the human immune system while evaluating
potential vaccine adjuvants. Nanocarriers possess immense
potential in successful vaccine delivery due to their ability
to protect the antigen, provide its controlled release and
target it specifically to the APCs. Nanocarriers such as
mucoadhesive nanoparticles and vesicular nanocarriers also
have the ability to afford mucosal and topical vaccination
respectively. Some issues in nanovaccinology still need to
be studied in depth including the uptake mechanisms of
nanocarriers through APCs, their uptake in other parts of
the body apart from APCs and the resulting effects, and their
elimination from the body, amongst other studies.
Future development in vaccine adjuvants would
involve combination of immunostimulants with traditional
delivery vehicles to achieve potent vaccine adjuvanticity
and minimize the systemic toxicity associated with
immunostimulant molecules. Also, vaccine delivery
systems utilizing core materials such as chitosan which
are endowed with inherent immunostimulant potential
or self-adjuvanting systems such as archaeosomes or
ISCOMs should be pursued. In conclusion, there are
countless opportunities concealed within the field of
nanovaccinology, which could pave the way for successful
vaccines in the market, driven by collaborations between
formulation scientists, immunologists, clinical research
scientists, and regulatory authorities.
Acknowledgements: The authors wish to thank
University Grants Commission for fellowship.
Conflict of interest: The authors have no conflict of interest.
Received: June 30, 2013; Accepted: December 16, 2013.
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44 Priyanka Prabhu and Vandana Patravale
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