Vaccine 31 (2013) 596–602
Contents lists available at SciVerse ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Review
Carrier molecules for use in veterinary vaccines
Volker Gerdts a,1 , George Mutwiri a,1 , James Richards b,1 , Sylvia van Drunen Littel-van den Hurk a,1 ,
Andrew A. Potter a,∗,1
a
b
Vaccine and Infectious Disease Organization – International Vaccine Centre, University of Saskatchewan, Saskatoon, SK, Canada
National Research Council of Canada, 100 Sussex Drive, Ottawa, ON, Canada
a r t i c l e
i n f o
Article history:
Received 5 October 2012
Received in revised form
16 November 2012
Accepted 25 November 2012
Available online 7 December 2012
Keywords:
Veterinary vaccine
Carrier
Conjugate
Nanoparticle
Microparticle
Virus-like particle
a b s t r a c t
The practice of immunization of animals and humans has been carried out for centuries and is generally
accepted as the most cost effective and sustainable method of infectious disease control. Over the past 20
years there have been significant changes in our ability to produce antigens by conventional extraction
and purification, recombinant DNA and synthesis. However, many of these products need to be combined
with carrier molecules to generate optimal immune responses. This review covers selected topics in the
development of carrier technologies for use in the veterinary vaccine field, including glycoconjugate and
peptide vaccines, microparticle and nanoparticle formulations, and finally virus-like particles.
Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glycoconjugate and peptide vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Delivery by synthetic and natural microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nanoparticles for antigen delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Virus-like particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Disclosure Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Vaccination of animals as an infectious disease control method
has been practiced for over a century with remarkable success. Prior
to the past two decades, most veterinary vaccines were either killed
products formulated with an oil-based adjuvant or live attenuated
vaccines. The field of biotechnology and molecular immunology
yielded rapid advancements starting in the 1980s, including the
∗ Corresponding author at: Vaccine and Infectious Disease Organization – International Vaccine Center, 120 Veterinary Road, Saskatoon, SK, Canada S7N 5E3.
Tel.: +1 306 966 7484; fax: +1 306 966 7478.
E-mail address: [email protected] (A.A. Potter).
1
All authors contributed equally to this manuscript.
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ability to produce subunit antigens in a cost effective fashion for the
veterinary market. Since that time, there has been an explosion in
the number of vaccines developed for use in production and companion animals as well as the types of killed products available;
the latter includes conventional protein and carbohydrate subunits, recombinant proteins, peptides, and more recently, nucleic
acid-based products. These antigens have a need for alternative
adjuvants and carrier molecules capable of stimulating the appropriate type of immunity at the appropriate site in the body, and
numerous technologies including protein conjugation partners,
microparticles, nanoparticles and virus like particles have seen use
in licensed veterinary and human vaccine products worldwide.
Vaccine targets are also changing, with non-infectious disease
targets representing a considerable growth area. These include control of fertility, behaviour and production by immunization against
0264-410X/$ – see front matter Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2012.11.067
V. Gerdts et al. / Vaccine 31 (2013) 596–602
597
Table 1
Overview of carrier technologies discussed in this review.
Carrier molecules
Types
Advantages
Disadvantages
Examples
Protein carrier
Proteins
Act as carrier molecules for oligo- or
polysaccharides. Can act as antigen and
elicit potent responses against carrier
Keyhole limpet hemcyanin
(KLH), ovalbumin (OVA),
hemolysins, AcrA of C.jejuni
Toxins, toxoids
Common carrier molecules for oligoand polysaccharides. Mostly toxoids,
which are safe to use, immunogenic
and very stable
Requires complicated coupling
procedures. Immunogenicity
may interfere with booster
immunizations
Often very reactogenic, may
cause adverse effects when
frequently used
Poly-lactidecoglycolide
(PLG)
Tested in a variety of species and with
a variety of antigens, effective for both
systemic and mucosal delivery. Have
been used in combination with a
variety of antigens including
DNA-plasmids, proteins and
carbohydrates
Alginate
Organic solvents not required for
assembly. Between 1 and 50 ␮m in
size. Fairly stable. Can be used
systemically and mucosally. Safe and
cost effective
Biodegradable polymer, easy assembly,
size can vary from 20 to 50 ␮m. No
organic solvents required. Water
soluble. Can be lyophilized. Side chains
can be added to influence the type of
immunity. Compatible with other
adjuvants. Safe and cost effective
Carriers for proteins, peptides and
nucleic acid. Liposomes may have
adjuvant properties. Multiple layers
that allow integration of other
adjuvants. Safe to use
Microparticles
Polyphosphazenes
Liposomes
Require use of organic
solvents. Not very stable, and
often not effective for mucosal
administration. No specific
targeting of immune cells or
lymphoid structures. Targeting
can be facilitated through
incorporation of specific
ligands
Assembly more complicated,
requires special equipment, no
targeting of specific immune
cells
Tetanus toxoid, diphtheria
toxoid, recombinant form CRM,
A. pleuropneumoniae exotoxins;
Mannheimia haemolytica
leukotoxin
Used for a variety of antigens
including DNA-plasmids,
proteins and carbohydrates
Use in combination with OVA,
B. abortus antigens, F. hepatica
antigens and others
Water soluble, some
formulations not very stable
following mucosal
administration
PCEP and PCPP, used with a
variety of antigens including
RSV, influenza, pertussis and
E. coli antigens
Not very cost effective.
Large-scale production can be
a limiting factor. Usually not
very stable. Have been rarely
used in animals
Rarely used in animals
Nanoparticles
Range in size from 1 to 1000 nm. Based
on lipids, polymers, surfactants and
carbohydrates, i.e. chitosan or alginate.
Safe to use, have the advantage of
increased uptake via mucosal surfaces.
Improve antigen uptake. Ligands can
be used to target specific immune cells
Assembly more complicated,
incorporation of antigens or
ligands sometimes
complicated due to small size.
Not suitable for all routes of
administration
Have been used with
enterotoxigenic E. coli, and
DNA-plasmid encoding
Newcastle virus antigens
VLPs
Can be used as vaccine itself or can act
as carrier for genetically-fused or
covalently linked antigens (chimeric
VLPs). During assembly additional
antigens can be incorporated. Easy
assembly, cost effective and safe. Can
be used to target specific immune cells
Assembly limited to specific
proteins only, in the case of
chimeric VLPs generation and
assembly more complicated.
Not very stable
Have been used with a variety
of antigens including HBV,
papillomavirus, rotavirus,
influenza, etc
hormones or hormone receptors. In addition, protein misfolding
targets such as prion diseases are of significant interest for the control of Bovine Spongiform Encephalopathy (BSE), Chronic Wasting
Disease (CWD) and scrapie, largely as a means of mitigating the
threat of transmission to humans or trade barriers. These types of
products require the use of peptide immunogens in many cases
and thus need to be conjugated to carrier molecules for optimal
immune responses.
For the purposes of this review, a “carrier” is defined as a
molecule which is either linked to a vaccine antigen by conjugation or encapsulation. Such carriers can have intrinsic adjuvant
activity, but it is not a requirement. We will focus on five areas;
glycoconjugate and peptide vaccines, microparticle and nanoparticle formulations, and finally virus like particles (summarized in
Table 1). It is recognized that this is not a comprehensive review of
carrier technologies in the veterinary field, but rather those which
either have had or will have a significant impact on animal health
and production.
2. Glycoconjugate and peptide vaccines
It is well established that carbohydrate-specific antibodies can
provide protection against pathogenic bacteria expressing surface
exposed capsule or lipopolysaccharide (LPS). This led to the early
development of polysaccharide vaccines against specific serotypes
of Streptococcus pneumoniae [1]. Indeed, polyvalent pneumococcal
polysaccharide vaccines have been commercially available since
the mid 1970s [2]. However, while polysaccharides elicit protective
immune responses in healthy adult populations, they are poorly
immunogenic in infants and the elderly which led to the development of protein-polysaccharide glycoconjugate vaccines [3]. Apart
from their inherent poor immunogenicity, polysaccharides when
used alone as vaccines typically elicit low affinity IgM antibodies
independent of T-cell help and, as a result, fail to generate boostable memory B-cell responses. By contrast, covalent coupling of
a polysaccharide antigen to a protein carrier yields a glycoconjugate that, when used to immunize mammals, elicits T-cell help for
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B cells that produce long-lasting IgG antibodies to carbohydrate
epitopes. Glycoconjugate vaccines against several clinically important bacterial pathogens based on capsular polysaccharides or LPS
O-polysaccharides are commercially available or currently under
development beginning with the first licensed polysaccharideprotein conjugate vaccine for Haemophilus influenzae type B (Hib)
in 1987 [4]. The HiB vaccine was the exemplar for this new generation of vaccines and it has essentially resulted in the eradication
of diseases caused by this pathogen in vaccinated communities [5].
In addition to HiB, glycoconjugate vaccines for preventing diseases
caused by S. pneumoniae and Neisseria meningitidis are commercially available.
Production of glycoconjugate vaccines requires covalent coupling of the oligo- or poly-saccharide to a protein carrier. The glycan
is either obtained from a bacterial source or by chemical synthesis. Carrier proteins are typically bacterial toxoids such as tetanus
and diptheria (most commonly as the recombinant form, CRM197 ),
although other carriers have been used. For example keyhole limpet
hemocyanin (KLH) has often been used in animal vaccine studies. Coupling of glycans to the carrier protein requires chemical
activation of the glycan and/or carrier which generally takes several steps and results in heterogenous products. Several procedures
have been developed to achieve this [6]. Even though glycoconjugate vaccines have enjoyed widespread use in humans, veterinary
applications have been limited due to the high cost of development.
In an early study, a conjugate vaccine approach was investigated
in an attempt to protect pigs against swine pleuropneumonia due
to Actinobacillus pleuropneumoniae. Using adipic acid dihydrazide
as the spacer, glycoconjugates were prepared by coupling either
the serotype 1 capsular polysaccharide or de-O-acylated LPS to A.
pleuropneumoniae haemolysin protein. Both anti-CP and anti-LPS
sera from vaccinated pigs were opsonic in a phagocytosis assay
suggesting their potential for providing protection as components
in a subunit swine vaccine [7]. In a more recent study, rabbits
vaccinated with CRM197 conjugates prepared from the conserved
LPS inner-core oligosaccharide from a Mannheimia haemolytica
serotype 1 losB mutant were shown to elicit immune sera that was
bactericidal to disease-causing, wild-type M. haemolytica strains
[8]. M. haemolytica is an important bacterial pathogen in bovine
respiratory disease complex that causes major economic losses
to the farming industry. In another study, BSA conjugates of B.
bronchiseptica O-specific polysaccharide (O-SP) injected as saline
solutions were shown to induce high levels of IgG antibodies in
mice [9]. B. bronchiseptica causes serious respiratory infections in a
variety of hosts, including kennel cough in dogs, atrophic rhinitis in
piglets, bronchopneumonia in rabbits and guinea pigs. Veterinary
vaccines against B. bronchiseptica are available but their efficacy is
limited. The conjugation methodologies described by Kubler-Kielb
et al. [9] could provide a cost effective approach for preparation of
vaccines for the companion animal market.
During the last 10 years it has been demonstrated that conjugates containing polysaccharides from pathogenic bacteria can
be produced in Escherichia coli by utilizing bacterial oligosyltransferase (OTase) systems that are capable of transferring oligoor polysaccharides from lipid carriers to proteins [10]. Bacterial
OTases involved in the transfer of both N- and O-glycosylation
has been described with the PglB N-glycosylation system from
Campylobacter jejuni being the most thoroughly studied [10].
Through the application of this approach, an efficient and reproducible fed-batch process for the in vivo production of shigellosis
glycoconjugate vaccine candidates that are composed of the
Shigella dysenteriae serotype 1 O-polysaccharide coupled to the
carrier proteins, AcrA of C. jejuni and exotoxin A of P. aeruginosa
(EPA) was recently reported [11]. In a related application, PglB was
employed to transfer the homopolymeric N-formalperosamine
O-polysaccharide of Yersinia enterocolitica O:9 to AcrA in a
transformed strain of Y. enterocolitica to investigate the immunogenicity of the bacterial engineered glycoconjugate as a brucellosis
vaccine by taking advantage of the fact that Brucella abortus and
Y. enerocolitica O:9 express identical LPS O-polysaccharides [12].
Glycoengineering of conjugates mediated by OTase systems could
significantly reduce costs of their production leading to a new
generation of vaccines for veterinary applications.
Glycans also make important targets on parasitic pathogens
which are known to display highly abundant, accessible and unique
glycan antigens on their surfaces during multiple developmental
stages [13]. Recently it was demonstrated by glycoarray analysis
that protein glycan epitopes from extracts of the gastro-intestinal
nematode, Haemochus contortus, elicits antibody induced protection in vaccinated lambs [14]. This approach is expected to facilitate
the discovery of glycan antigens for development of glycoprotein
vaccines. Advances in automated synthesis of carbohydrates are
also expected to open the door to well defined oligosaccharide
epitopes in a more cost-effective manner [15].
As in the human health field, toxoids typically used in the veterinary vaccine field include diphtheria and tetanus, although their
use is not widespread and is limited primarily to peptide antigens.
The Improvest® product (Pfizer Animal Health) is one of the most
widely used peptide conjugates and is composed of a Gonadotropin
Releasing Factor analogue conjugated to diphtheria toxoid [16,17].
This vaccine results in a temporary blockage of the production
of male sex hormones, including testosterone and androstenone,
which in turn leads to infertility and more importantly, a reduction
in boar taint through increased metabolism of skatole [17]. Conceptually similar applications of GnRH-based vaccines have been
studied using tetanus toxoid as a carrier for human cancer applications and both tetanus and diphtheria toxoids have been used
with success in rodent models [18]. The M. haemolytica leukotoxin,
a member of the RTX family of toxins [19,20], has also been used as
a carrier for a variety of peptide antigens, including GnRH [21–25].
This molecule is of interest as a carrier from a variety of perspectives, not the least of which RTX toxins are produced by a number
of veterinary pathogens and have been shown to be protective
antigens in each case [26–28]. Truncation of the amino terminal
portion of LktA in the absence of LktC results in the production
of a toxoid to which one can add peptides at either the aminoor carboxy-terminus [24,25,29]. This has been used to construct
chimeric proteins carrying multiple copies of a variety of peptides,
including GnRH, SRIF, VIP, and misfolded epitopes from the prion
protein PrP which are not present in the normal PrP molecule [30]
to name but a few. Due to the targeting capability of RTX toxins,
these chimeric proteins induce robust immune responses with a
relatively long duration of immunity following two immunizations
in both cattle and swine and can be adapted to virtually any species.
3. Delivery by synthetic and natural microparticles
Delivery by encapsulation into micro- or nanoparticles may protect and/or slowly release a vaccine antigen and enhance antigen
uptake by antigen presenting cells (APCs). Another advantage is
the potential for mucosal vaccine delivery. Depending on the size
particles are internalized by either phagocytosis (0.5–10 ␮m) or
endocytosis (<0.5 ␮m). Microparticles generally enhance induction of Th2-type, humoral immunity, while nanoparticles promote
Th1-biased, cell-mediated immune responses. A number of polymers including poly-lactide-coglycolide (PLG), alginate, starch and
polyphosphazene can be used to generate microparticles.
Alginate microparticles are promising for use in cattle, although
thus far have been tested with non-infectious disease antigens. Pig
serum albumin encapsulated in alginate microparticles of 1–50 ␮m
induced high IgG1 levels in serum, saliva and nasal secretions when
V. Gerdts et al. / Vaccine 31 (2013) 596–602
delivered intranasally, but not when given orally; however no IgA
was detected, and a weak lymphocyte proliferative response in
the peripheral blood mononuclear cells (PBMCs) [31]. In another
study oral administration of ovalbumin encapsulated in alginate
microspheres resulted in a mucosal immune response in the respiratory tract of cattle. In addition, calves primed subcutaneously
and boosted orally showed enhanced IgA, IgG1 and IgG2 responses
in the bronchoalveolar lavage fluid as well as higher serum IgG1
and IgG2 when compared to calves twice immunized orally [32].
In red deer, a B. abortus strain 19 was encapsulated into alginate
microspheres together with Fasciola hepatica vitelline protein B,
and administered orally or subcutaneously. Both humoral and cellmediated immune responses were induced in animals immunized
subcutaneously, while the orally immunized deer developed antibody responses. When challenged 1 year later, the orally vaccinated
deer had a lower bacterial tissue load compared to non-treated
controls [33]. This strategy was also found to be effective against
Brucellosis in elk [34].
While alginate MP can be generated under mild conditions, organic solvents need to be used to generate PLG MP,
which may lead to denaturation of antigens. Despite this, PLG
nano/microparticles have been used in a number of veterinary
species. An early study in pigs demonstrated that oral delivery
of E coli or fimbriae encapsulated into PLG microspheres did not
induce significant serum antibodies or protection from E. coli challenge [35]. In contrast, encapsulation of Staphylococcus aureus
polysaccharide conjugates in PLG microspheres induced stronger
opsonizing antibody responses to the polysaccharide, resulting in
more sustained enhancement of phagocytosis, in cows. In this study
a single dose of vaccine was administered parenterally [36]. In
sheep, Toxoplasma gondi tachoites encapsulated into PLG microand nanoparticles developed local and systemic IgA and cellmediated immune responses, including IFN-␥ production, when
delivered intranasally. After challenge with Toxoplasma oocysts the
vaccinated sheep showed an anamnestic response, and a reduction in the febrile responses, although they were not protected
from infection [37]. PLG nano/microparticles have also been used
to formulate a foot and mouth disease virus (FMDV) DNA vaccine. A plasmid encoding FMDV P1-2A3C3D and GM-CSF proteins,
when formulated in PLG and delivered parenterally, elicited T
cell responses and neutralizing antibodies in sheep. In contrast,
lipofectin-formulated plasmid induced higher antibody titres but
no significant T cell response, while naked plasmid did not induce
any responses. The immune responses induced by PLG-formulated
plasmid were protective against clinical disease and viraemia and
prevented a carrier state in four of the five sheep tested [38].
These and other studies suggest that in large animals PLG microparticles are effective when delivered parenterally, but still need to
be optimized for mucosal delivery. One of the approaches to achieve
this has been based on targeting microparticles to antigen presenting cells (APCs). In vitro studies have shown that ligands such as
wheat germ agglutinin, mannose-PEG3-NH2 and arginine-glycineaspartic acid may enhance uptake by macrophages [39]. However,
whether this strategy increases the efficacy of PLG microparticlemediated delivery in vivo still needs to be confirmed.
Polyphosphazenes poly[di(sodium carboxylatophenoxy)]polyphosphazene (PCPP) and poly[di(sodium carboxylatoethylphenoxy]-phosphazene (PCEP) are synthetic water-soluble polymers containing a backbone of alternating phosphorus and
nitrogen atoms. Polyphosphazenes have immunostimulatory
properties [40] and can be formulated into nano- or microparticles
[41,42] to facilitate mucosal delivery. Studies in large animals have
thus far been performed with model antigens as well as those from a
variety of infectious agents. We have shown that formulation of hen
egg lysozyme (HEL) with a derivative of PCPP resulted in ∼50-fold
improved IgG responses in cattle. Furthermore, co-formulation of
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HEL with CpG ODN, a host defense peptide (HDP) and PCPP further
enhanced IgG titres and induced the production of IFN-␥ secreting
T cells [43]. As this polymer has only been recently used as a vaccine
delivery system, it needs to be further tested in veterinary species.
Liposomes are comprised of natural biodegradable phospholipids and can be used as carriers for proteins, peptides or
nucleic acids. In addition, liposomes may have adjuvant properties, and contain immune modulators such as TLR ligands.
However, the manufacturing of liposomes at a large scale has been a
limiting factor for commercialisation. In veterinary species, liposomes have been rarely tested. A BVDV E2 encoding plasmid
induced improved immune responses in cattle when delivered
in liposomes in comparison to naked DNA [44]. Furthermore, a
piroplasm surface protein from Theileria sergenti incorporated in
a mannan-coated liposome elicited T cell proliferative responses in
all vaccinated calves [45].
4. Nanoparticles for antigen delivery
Nanoparticles (NP) are solid colloidal particles and those developed for use as antigen carriers range in size from 1 to 1000 nm
[46]. In general, antigens are either adsorbed on the surface of NP
or encapsulated inside the NP matrix. Nanoparticles can be primarily classified by the nature of the compounds used in their
fabrication such as lipids, polymers, surfactants and carbohydrates
such as chitosan or alginate [46]. Regardless of their nature, NP
can offer several advantages over other antigen delivery systems:
(i) NP can protect antigen against degradation in vitro and in vivo,
(ii) antigen release can be controlled, (iii) NP can be modified to
target certain immune cells, (iv) As NPs are comparable in dimensions to pathogens, they are efficiently taken up and internalized by
antigen presenting cells (APC), (v) NPs can co-deliver antigen and
adjuvant, ensuring that both cargoes are delivered to the same cell
or cellular compartment, and (vi) NPs may limit systemic distribution and thereby reducing dose and the probable side effects when
immunostimulatory adjuvants are included in the formulation.
The most important characteristic of NPs is their ability to
improve antigen uptake and presentation, resulting in enhancement of adaptive immune responses. In this regard, pulmonary
immunization of mice with ova-conjugated poly(propylene sulphide) [PPS] NPs with the adjuvant CpG promoted efficient
cross-presentation resulting in a 3-fold increase in ova-specific
CD8+ T cells and IFN-gamma production compared with soluble ova plus CpG ODN [47]. Furthermore, this enhanced response
was accompanied by a potent Th17 cytokine profile in CD4+ T
cells and memory CD8+ T cells [47]. Similarly, Ballester et al. [48]
demonstrated that PPS NPs containing the Mycobacterium tuberculosis antigen Ag85B and CpG led to enhanced antigen-specific Th1
responses and provided better protection against aerosal challenge
with M. tuberculosis [48].
Targeting of NPs to immune cells can further improve immune
responses. CpG is the ligand for TLR9 which is expressed by
immune cells. Activation of immune cells with CpG leads to induction of a variety of responses including production of cytokines
and chemokines which contributes to enhancement of immune
responses [49]. Mice immunized with PLGA NPs loaded with West
Nile (WN) antigen and surface modified with the TLR9 ligand CpG
(to improve targeting to immune cells) showed Th1-polarized antibody responses, a greater number of circulating effector T cells
and superior protection in a mouse model of WN virus encephalitis [50]. Interestingly, this NP delivery method minimizes the CpG
dose (0.5 ␮g) compared to the much higher doses used in similar
studies [50]. This is beneficial given the concerns with autoimmunity and potential lymphoid architectural damage from exposure
to high levels of CpG [51]. Reducing the dose of CpG required for
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biological activity would be highly desirable in large animals where
the high doses of CpG are not practical for economic reasons.
Tacken et al. [52] have showed that targeted delivery of TLR ligands to human and mouse DCs strongly enhances adjuvanticity. For
these studies, ligands for intracellular TLRs (TLR3 and TLR7/8) were
encapsulated in PLGA NPs and coated with anti-DC-SIGN antibodies recognizing DC-specific receptors. This targeted delivery of TLR
ligands to human DCs enhanced immune stimulatory cytokines and
antigen-specific activation of naive CD8+ T cells [52]. When used
in vivo, this formulation induced cytotoxic T lymphocyte responses
at 100-fold lower adjuvant dose, and reduced serum cytokine storm
often associated with toxicity after administration of soluble TLR
ligands [52].
It is apparent from the literature that there is interest in the NP
delivery of vaccine antigens in animals. There are a number of studies that have used antigens from pathogens of veterinary interest,
but most of these antigens have been tested in laboratory animals.
Immunization of mice with a swine influenza DNA vaccine encapsulated in chitosan NPs induced enhanced serum neutralization
antibodies compared to DNA vaccine alone [53]. Co-administration
of chitosan NPs entrapping plasmids encoding porcine IL-2 and
CpG ODN with bivalent vaccine against Pasteurella multocida and
classical swine fever resulted in improved immune responses and
protection against challenge in mice [54]. Chitosan NP loaded with
a plasmid carrying porcine IL-2 gene and paratyphoid vaccine
exhibited improved immune responses and protection against oral
Salmonella challenge [55]. FMDV DNA vaccine encapsulated in NPs
induced significant cell-mediated and humoral immune responses,
and protection against challenge with FMDV in guine pigs and
mice [56]. Similarly, intranasal delivery of PLGA NP loaded with
FMDV DNA vaccines encoding IL-6 enhanced protective immunity
to FMDV in mice [57]. While the studies in laboratory animals are
encouraging, it remains to be seen whether NPs delivery will be as
successful in target species.
Only a few studies have evaluated NP delivery systems in target veterinary species. Enterotoxigenic E. coli that express F4 (K88)
fimbriae on their surface (F4 + ETEC) are an important cause of diarrhoea in recently weaned pigs. The F4 fimbriae enable the bacteria
to bind to F4-specific receptors (F4R). Protection against F4 + ETEC
is provided by intestinal IgA antibodies against F4, which inhibit
adhesion and colonization of the bacteria. Ideally, piglets should
be orally vaccinated during the suckling period, often in the presence of F4-specific maternal antibodies and possibly other milk
factors which in milk, which can bind to F4 and interfere with
the stimulation of an immune response. It was reasoned that NPs
might circumvent these problems. Piglets immunized with F4 antigen plus NPs (F4 + NPs) induced an increased F4-specific serum
IgA and IgG response, and this response was boosted after challenge [58]. When immunized pigs were challenged with F4 + ETEC
at day 35, bacterial excretion was significantly reduced in the
F4 + NPs group compared to control group [58]. Surprisingly, encapsulation of the F4 in NPs did not perform as well as NPs mixed
with F4.
In chickens, DNA-chitosan NPs were shown to improve DNA
vaccine-elicited immunity against Newcastle Disease virus (NCDV)
[59]. Encapsulation of pCAGG-ChIL2 plasmid DNA containing
chicken IL-2 (ChIL-2) gene into chitosan NPs (CNPs) and administered to chickens simultaneously with a DNA vaccine against NCDV
significantly increased hemaglutination inhibition titres and serum
IFN-gamma levels compared to chickens immunized with NCDV
DNA vaccine alone [59]. Chickens immunized via the respiratory
route with chitosan-DNA NPs, which carried the gene coding for
the major flagellar protein, FlaA, of C. jejuni produced significantly
increased levels of serum anti-C. jejuni IgG and intestinal mucosal
IgA; and reduced bacterial load by 2–3 logs in the intestines of
chickens [60]. Thus, respiratory delivery of Chitosan-DNA vaccine
NPs successfully induced effective immune responses and may be
a promising vaccine candidate against C. jejuni infection.
Administration of PLGA NPs containing a model antigen (TNPLPH) and an immunostimulant (beta-glycan) in salmon resulted
in increased gene expression of the pro-inflammatory markers
TNF-alpha, IL-1beta, IL-8 and C3a [61]. NPs alone were able to modestly up-regulate pro-inflammatory markers. Subsequent studies
demonstrated that PLGA NPs were capable of improving humoral
antigen-specific immune responses in salmon [62].
5. Virus-like particles
Another vaccine carrier technology is based on virus-like particles (VLPs). These particles typically range from 20 to 100 nm
and consist of one or more recombinant proteins that form
either icosahedral- or rod-like structures through self-assembly
(reviewed in [63]). VLPs have been developed for a number of viral
and bacterial diseases and offer the advantage of delivering the
vaccine antigen in a particulate structure, thereby increasing the
immunogenicity of the vaccine [64]. Both, the conformation of the
antigen as well as the density on the particle surface are important
factors for generating strong immune responses against the antigen. VLPs can be expressed in a wide variety of expression systems
including yeast, E. coli, bacteriophages, baculovirus or mammalian
cells. VLPs can be used either as vaccine itself or as carrier for genetically fused (chimeric), incorporated or covalently linked antigens.
Typically, VLPs have a high safety profile, they are very stable and
can be produced at large scale and they can be combined with other
vaccine formulations.
Over the last three decades VLPs have been developed for a number of human and animal diseases. The first VLP-based recombinant
HBV vaccine was approved by the US Food and Drug Administration in 1986, followed in 2006 by the quadrivalent HPV VLP-based
vaccine GardasilTM (MERCK & Co. Inc.) and CervarixisTM (GlaxoSmithKline) in 2009. Several other VLP-based vaccines are currently
in (pre)clinical development including hepatitis B surface antigen
VLPs (HBsAg-VLP [65]), human immunodeficiency virus 1 VLPs
[66,67], dengue virus VLPs [68], norovirus VLPs [69]; rotavirus,
and influenza A VLPs [70,71]. For veterinary applications, VLPs are
in development for bovine rotavirus, bluetongue, Newcastle disease and avian influenza. While all of these examples are used as
vaccines themselves, VLPs have also been developed to act as carriers for foreign proteins or epitopes. These chimeric VLPs carry
foreign proteins or epitopes on their surface, which allows for optimal presentation to the immune systems, thereby ensuring better
immunity to both the carrier itself and the foreign epitope. Examples of chimeric VLPs include bovine rotavirus virus protein 6 (VP6),
which forms VLPs that are highly immunogenic and already confer protection against challenge infection [72,73]. However, using
the VP4 and VP7, other antigens can be covalently linked to the
VP6 particles and used for immunization [73]. Similarly, hepatitis
B core antigen VLPs (HBcAg VLPs; [74,75]) were shown to act as a
carrier for the influenza M2 protein (M2-HBcAg [76], the envelope
domain III of dengue virus type 2 (Arora et al., 2012), or malaria Band T-cell epitopes [77]. A chimeric human papillomavirus 16 VLP
with a chimeric L1-E7 was successfully tested in a murine model of
cervical cancer and multiple L1 proteins were evaluated in a bovine
papillomavirus VLP containing type 1,2, and 4 L1 genes (23).
Typically, immunization with VLP induces rapid and strong antibody responses. Similar to viruses and bacteria multiple copies
of the vaccine antigens are displayed in a highly repetitive and
ordered, quasi crystalline-structure [78]. It was shown that when
expressed on the surface, these antigens could cross-link the B
cell receptor resulting in activation of the B cell and subsequent
induction of T-independent IgM-responses [79]. Furthermore, this
V. Gerdts et al. / Vaccine 31 (2013) 596–602
enables interaction with the complement system resulting in
increased phagocytosis [80] and increased uptake by dendritic cells
and subsequent cross-presentation of the antigen. Lenz et al. (2001)
showed that cross-presentation of particulate antigens was more
effective than presentations of soluble antigens [81]. However, the
induction of T cell responses overall is still not as effective as those
induced by live vaccines. To overcome this, several strategies have
been tested to enhance their immunogenicity including combination with adjuvants such as CpG ODN [82] and single-stranded
RNA [83]. Interestingly, some VLPs demonstrate adjuvant activity
on their own, for example the papillomavirus L1-VLPs can directly
activate dendritic cells [81]. Also, delivery via the mucosal surfaces
increased vaccine efficacy [84].
Disclosure Statement
None of the authors declare a conflict of interest.
Acknowledgment
This manuscript is published with the permission of the Director
of VIDO as manuscript series #655.
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