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Recent advances in peptide-based subunit nanovaccines.
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Abstract
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Vaccination is the most efficient way to protect humans against pathogens. Peptide-
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based vaccines offer several advantages over classical vaccines, which utilized whole
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organisms or proteins. However, peptides alone are not immunogenic and need a delivery
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system which can boost their recognition by the immune system. In recent years,
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nanotechnology-based approaches have become one of the most promising strategies in
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peptide vaccine delivery. This review summarizes knowledge on peptide vaccines and
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nanotechnology-based approaches for their delivery. The recently reported nano-sized
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delivery platforms for peptide antigens are reviewed, including nanoparticles composed of
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polymers, peptides, lipids, inorganic materials and nanotubes. The future prospects for
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peptide-based nanovaccines are discussed.
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Keywords
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Adjuvant, peptide vaccine, vaccine delivery, nanoparticles, polymer, lipids, self-assembly,
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macromolecules, dendrimers, nanotechnology
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Introduction
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The introduction of a vaccine for human treatment was one of the most
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revolutionizing discoveries in health care. While Edward Jenner and Louis Pasteur are
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considered as the fathers of vaccination, the first vaccination attempt reaches back hundreds
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years, when the first small pox inoculations were applied in China. Considering that until the
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18th century smallpox caused about 10% of global mortalities in Europe, the success of
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vaccine against this disease can be only compared with the introduction of penicillin.
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Vaccinology has changed greatly since its early development but the classical vaccine
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strategy based on attenuated or inactivated pathogens is still used. Problems associated with
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conventional vaccines include the risk of infection, especially in the case of immune
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compromised humans, difficulties and impurities associated with the production of pathogens
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in vitro, and instability of the biological material. Therefore, there is increasing interest in
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development of vaccines which use only minimal components from pathogens. Such vaccines
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can be based on recombinant proteins or even minimal fragments carrying immunological
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information from this protein, namely peptide epitopes.
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Vaccine efficacy is largely dependent on its biochemical composition, which
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predominantly includes antigen and immunostimulator (adjuvant). However, recently it has
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been shown that morphological properties and particle size of the antigen/adjuvant system
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play a major role in a vaccine’s ability to induce the desired immune responses. Therefore,
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development of nanovaccines has been growing extensively in recent years [1-4].
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Nanomaterials, which are usually defined as structures that have at least one size of 1-100 nm
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dimension (according to American Chemistry Council-Nanotechnology Panel), have started
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to be widely used for vaccine development. Such materials can be composed of polymers,
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lipids, peptides, or inorganic constituents. This review summarizes the latest advances (with
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special focus on the last five years) in delivery of peptide-based vaccines using nanomaterials
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as carriers, as well as self-assembly delivery systems which are produced by self-organization
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of appropriately modified peptide antigens. Most of the historical data as well as the study on
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the use of nanoadjuvants such as Iscomatrix and MF59 have been reviewed elsewhere [1, 3,
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5-9]. In this review, following the common understanding existing in the published literature,
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we are defining nanovaccine as immunogenic nanomaterial including any particles with sizes
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that do not exceed 1 micrometer.
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Figure 1. Simplified diagram of the immune response to nanoparticles (or pathogens).
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Antigen presenting cells (APCs) are major components of innate immunity. APCs recognize
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uptake by the endocytosis or phagocytosis process and display antigen. The antigen then is
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presented to the adaptive immune system and with the help of T-helper cells, appropriate
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humoral or cellular responses are induced.
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Immune response
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Vaccines are designed to induce an adaptive immune response; cellular and/or
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humoral responses. In general, antigen presenting cells (APCs), including dendritic cells
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(DCs), are parts of an innate immune system and are positioned at the first line of
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pathogen/vaccine recognition. Antigen can be recognized by DCs localized in peripheral
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tissue and then transported to the lymph nodes or can travel independently to lymph nodes
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where they are taken up by lymph node-resident DCs. DCs stimulate T-cells to respond to the
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antigen by sensing immunogens usually through pattern recognition receptors (PRRs) which
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recognize pathogen components. Examples of PRRs are Toll like receptors (TLRs) 1 to 13
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[10] and mannose receptors [11]. The TLR family of receptors recognize a variety of
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bacterial and viral molecules including free DNA, lipoprotein, lipopolysaccharide, flagellin,
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etc [12]. Following recognition by PRRs on DCs, pathogen/antigen is taken up. The
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mechanism of uptake is size-dependent (e.g. nanoparticles (<150 nm) are usually taken up by
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clathrin-mediated endocytosis, while microparticles are taken up by phagocytosis) which
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partially explains size-dependent immunogenicity of particles [9]. The antigen is processed
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inside the APCs, and loaded onto major histocompatibility complex (MHC) class-1 or MHC
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class-2 (Figure 1). Exogenous particles, toxins, or pathogens are usually endocytosed or
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phagocytosed and processed into small antigens which are loaded inside vesicles on MHC
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class-2 molecules. MHC class-2 presentation leads to activation of T-helper cells which
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further stimulate antibody production or cellular immunity. The MHC-1 pathway, required
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for production of cellular immunity, is activated through the processing of endogenous
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antigen presented in the cytosol. However, the production of immune responses through
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vaccination requires induction of the MHC-1 pathway through exogenous antigen. This
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process, known as cross-presentation, includes uptake, processing and presentation via MHC
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class-1 molecules of external antigen. It is not well understood but generally it is believed
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that exogenous antigen is transported via phagocytosis to the cytosol where it can be
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processed in the usual manner for endogenous antigens [13]. However, direct delivery of
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antigen to the cytosol (e.g. with the help of fusogenic liposome) or endosomal escape of
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antigen (e.g. in a virus-like manner) cannot be ruled out for some antigen delivery platforms.
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Finally, alongside antigen presentation, signaling protein (cytokines) production is stimulated
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and adaptive immunity is induced with the help of T-helper cells. Recognition of antigen on
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MHC class-1 by T-helper cells subtype 1 is primarily responsible for activating and
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regulating the development of cytotoxic T-lymphocytes (CTLs, CD8+ T-cells). T-helper cells
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subtype 2 (Th2) favor humoral response (B-cell activation and antibody production).
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Humoral immune responses are usually targeted to extracellular or intracellular pathogens
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during or before infection. For example, a vaccine against human papilloma virus (HPV) was
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developed using virus major capsid protein and thus targeting the virus in the pre-infection
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stage [14]. Cellular immune responses are responsible for destroying already infected or
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abnormal human cells. Therefore, in vaccine development this type of immunity is needed to
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be induced against intracellular pathogen or tumors.
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Peptide-based subunit vaccine
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A peptide-based subunit vaccine is defined as a vaccine which contains only the
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peptide component, derived mainly from bacterial, viral or parasite protein, necessary to
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stimulate appropriate immune responses [15]. Its minimalistic composition is associated with
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several benefits over the use of whole pathogenic microorganisms or protein. However,
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removal of vast numbers of components typical for a pathogen (known also as “danger
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signal”) brings significant reduction in vaccine efficacy and additional additives are required
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to counteract this problem [16].
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The major advantages of peptide-based vaccines are as follows:
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1.
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the use of microorganisms;
2.
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they are non-infectious: cannot revert to virulent state, their production does not include
some pathogens are problematic to culture (e.g. sporozoites for malaria vaccines), and a
subunit-based vaccine (including peptide) might be the only solution in such cases;
3.
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they do not possess redundant components, which significantly reduce the risk of allergic
or autoimmune responses;
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they can be designed (customized) to recognize certain pathogen-associated targets;
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they might be especially useful for development of anticancer vaccines in cases where
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whole protein cannot be used due to its similarity to endogenous human protein or
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carcinogenic properties;
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they can include several peptide epitopes targeting different stages in the life cycle or
subtypes of a pathogen;
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7.
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they can be easily produced, using solid phase peptide synthesis (SPPS), in a pure state,
in a highly reproducible manner, economically and in large scale; and
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are generally water-soluble, stable under storage conditions even at room temperature,
and can be freeze dried.
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The major disadvantages of peptide-based vaccines are as follows:
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1.
they require the use of an immunostimulant (adjuvant) to trigger the desired immune
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response. Currently available experimental adjuvants suffer from side-toxicity, while
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commercially available (safe for human) adjuvants are mostly limited to aluminum
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derivatives that have limited potency in stimulating humoral immune responses and are
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not effective at inducing cellular immunity; and
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they often lack a T-helper epitope that needs to be incorporated for optimal vaccine
efficacy.
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Thus, in the peptide-based vaccine significant reduction of side effects and production
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difficulties has been made at the cost of general vaccine efficacy (Figure 2). Finally, it is
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necessary to take into account that protein-based vaccination can be similar or even more
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valuable depending on the circumstances. Development of peptide vaccines is usually
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considered in situations where the recombinant protein-based approach is unproductive. More
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information on development of peptides as vaccine components can be found in recent
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reviews [12, 15-17].
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Figure 2. Vaccines progression - from whole pathogen to nanoparticles. Antigens and their
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properties; (A) whole pathogen, (B) protein, (C) peptide, and (D) nanoparticles incorporating
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peptide epitopes (peptides can be both presented on particle surface and/or encapsulated) .
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Nanotechnology
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The nanotechnology-based approach is considered to be one of the most advantageous
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for development of peptide-based vaccines. Nano-sized vaccines are produced based on
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nanomaterials with properties as described in the introduction. Such nanoparticles can be
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built from inert (non-immunogenic) material, in/on which antigen is incorporated or from
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appropriately modified antigen, which can self-assemble to form nanoparticles [18, 19].
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Additional immunostimulant or PRR-targeting moieties can be incorporated in their structure.
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The major advantages of nanovaccines include:
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1. enhanced uptake by APCs:
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size driven uptake (usually smaller particle are more easily uptaken and therefore are
more immunogenic)
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cationic particles are more effectively uptaken into macrophages and DCs (due to the
attraction to negatively charged APC cell membranes);
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2. larger particles can form a depot effect, that is, they retain the antigen at the injection site
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and in this manner increase the time of vaccine exposure to the immune cells (however, it
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is necessary to indicate that the depot effect is usually associated with micro rather than
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nanoparticles);
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3. particulate vaccines can potentially cross-present antigen (via MHC class-1). Antigen
cross-presentation is especially important to induce CD8+ T-cell immune responses;
4. particles might be covered by multiple copies of the same peptide antigen, mimicking
natural pathogen antigen recurrence;
5. antigens formulated into particles are also at least partially protected against enzymatic
degradation, which is an important issue for highly susceptible peptide antigens;
6. small nanoparticles can easy travel to lymph nodes (without participation of peripheral
DCs), and the nodes are the fighting core of the human immune system;
7. immunological properties of nanoparticles can be altered by changing their size, surface
charge, hydrophobicity, shape, etc.
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Polymer-based nanoparticles
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A polymer-based drug delivery system is one of the most dynamically growing fields
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of research. Taking into account that the first polymeric drugs have been approved for human
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treatment[20], this class of compounds have started to become very attractive from a
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commercial point of view. Polymeric nanoparticles are usually stable in vivo but also may
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have biodegradable properties; can protect incorporated antigen from metabolism and
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elimination; their size, charge and hydrophobicity can be easily altered; and they usually have
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low or no toxicity [21]. They can be used to form a depot effect to improve vaccine efficacy
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via elongated exposure/release of antigen at the site of vaccine injection. Such factors as the
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speed of polymer biodegradation and its shelf-life, rate of antigen release, loading capacity
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and antigen stability during this loading can be controlled through the choice of polymer and
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process of antigen incorporation.
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The pioneering study in the use of polymer nanoparticles for peptide vaccine delivery
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was performed by Plebanski and co-workers [22]. They showed that polystyrene
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nanoparticles loaded with ovalbumin (OVA) derived peptide epitopes induced immune
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responses in a size-dependent manner without the need of additional stimulation with an
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adjuvant. Among tested particles with a variety of sizes (20, 40, 100, 200, 500, 1000 and
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2000 nm), 40 nm particles induced the strongest cellular and humoral immunity. Covalent
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linkage of the peptide was necessary for particle efficacy and therefore nanoparticles served
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as the delivery system with self-adjuvanting properties rather than as a classical adjuvant, that
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is, a physical mixture of polystyrene beads and the epitope was not effective. The induction
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of stronger immune responses by 40-50 nm nanoparticles was later correlated with
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preferential uptake of these nanoparticles by DCs [23]. It has been also shown using
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polyhydroxylated nanoparticles of different sizes, that small nanoparticles (25 nm) are
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capable of trafficking to lymph nodes by themselves and therefore induce stronger immune
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responses than their larger counterparts [24, 25].
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One of the most commonly used biodegradable polymer for drug delivery is poly(D,L-
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lactic-co-glycolide) (PLGA) [26]. This polymer is often used as a first choice for polymeric
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vaccine delivery systems mainly due to its excellent safety profile and established use in
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commercial products for controlled delivery of peptide-based drugs [27]. Zhang et al. loaded
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PLGA nanoparticles (80 ± 27 nm) prepared using the double emulsion method with tumor
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associated peptide antigens (hgp10025-33 or TRP2180-188) [28]. The nanoparticles were
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efficiently uptaken by murine DCs and induced stronger cellular immune responses in the
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mouse model than the peptides mixed with Freund’s adjuvant. Both complete Freund’s
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adjuvant (CFA) and incomplete Freund’s adjuvant (IFA) are commonly used as the “gold”
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standard for stimulation of immune responses against peptide-based antigens; however, they
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are not allowed for human use (particularly because CFA has shown high toxicity).
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Nanoparticles formulated with TRP2180-188 were able to significantly reduce tumor growth in
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mice following prophylactic subcutaneous immunization (mice were immunized trice prior to
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a melanoma cells injection). Similarly, PLGA nanoparticles with a diameter of 215 and 330
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nm loaded with tumor associated peptide antigen were able to stimulate cellular immunity
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[29, 30]. To improve the efficacy of PLGA nanoparticles, several additives to the basic
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nanoparticle formulation were tested. One of the approaches was designed to target human
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follicle-associated epithelium derived M-cells, which are responsible of internalizing luminal
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antigen and delivering it to lymphoid tissue [31]. Peptides targeting M-cells were conjugated
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to PLGA nanoparticles and subsequently showed improved transport of antigen-loaded
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nanoparticles across the intestinal mucosal barrier [32]. In other studies, Messmer and co-
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workers conjugated DCs inducing peptide (Hp91) to PLGA and demonstrated that this
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construct formulated into particles (~ 200 nm) activated both human and mouse DCs more
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efficiently than peptide alone [33]. Lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine) coated
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PLGA nanoparticles (with diameters of 100 nm but smaller particles were also observed by
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TEM) were studied [34]. Interestingly, a mixture of nanoparticles incorporating several tumor
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associated antigens showed reduced stimulation of T-cells (assessed by IFN-γ production) but
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improved prophylactic antitumor effect in mice when compared to any other nanoparticle-
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bearing single antigen. It was suggested that improved antitumor efficacy was related to
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reduction of the risk of tumor escape as the host immune system attacked multiple targets
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simultaneously. PLGA nanoparticles have been recently used to generate immune responses
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against tetanus and diphtheria toxoid and universal memory T-cell helper peptide, active in
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vitro in human and in vivo in non-human primates, was developed [35]. PLGA nanoparticles
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were also tested as a peptide-based vaccine candidate against Chlamydia trachomatis [36].
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Chitosan is a chitin derived natural cationic polymer with adjuvanting properties [19].
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It is recognized by cell surface receptors including macrophage mannose receptors and TLR-
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2 [37]. Jackson and co-workers studied chitosan-based nano- and microparticles for delivery
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of luteinizing hormone-releasing hormone (LHRH) as a peptide antigen [38]. They
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demonstrated that antigen was mostly localized on the surface of chitosan particles.
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Confirming previous observations with polyhydroxylated nanoparticles, the nanoparticles (~
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200 nm) travelled from the injection site to the draining lymph nodes faster than
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microparticles (~ 2 µm). However, no significant difference in antibody production was
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observed for both types of particles after subcutaneous immunization in mice. Another
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commonly used polymer for drug delivery is poly glutamic acid (PGA) which is
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biodegradable, highly water-soluble, non-toxic and non-immunogenic [39]. Tumor specific
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peptide antigen (EphA2 peptide), conjugated to PGA nanoparticles grafted with phenyl
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alanine (246 ± 88 nm), demonstrated activity against liver tumor similar to that of the peptide
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mixed with toxic CFA (which induced liver damage), but did not show any toxic side-effects
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[40].
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Recently nano-self-assembling strategies are receiving growing recognition in
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biomedical fields [18] and it has been suggested that self-assembling amphiphilic polymers
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might be useful systems for development of subunit vaccines [41]. To prove this concept,
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Toth and co-workers applied a non-toxic tert-butyl polyacrylate as an dendrimer core and
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chemically conjugated it with multiple copies of Group A Streptococcus (GAS) B-cell
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epitope [42]. The produced construct was self-assembled to form 20 nm nanoparticles, which
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were able to induce the desired helical conformation of attached peptides and elicit high
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levels of antigen-specific antibodies without the aid of an adjuvant. These nanoparticles were
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effective when administered via subcutaneous or intranasal routes and were also capable of in
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vitro opsonization of GAS [43]. Furthermore, it was proved that smaller nanoparticles (~ 20
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nm) were more immunogenic than larger ones (~ 500 nm) even after single immunization
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[44]. Interestingly, when cervical cancer associated peptide epitopes were conjugated to
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branched tert-butyl polyacrylate, nanoparticles as well as microparticles (depending on the
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peptide structures) were formed in water. When the same conjugates were formulated in PBS
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buffer all of them aggregated into large microparticles. Despite their large size, these particles
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were able to reduce tumor growth in a therapeutical setup (vaccine treated existing tumor)
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and even eradicate a model of cervical tumor in mice after a single immunization, without the
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help of any external adjuvant [45]. In another approach, tumor-associated MUC1 peptide as
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the B-cell epitope and a T-helper cell epitope, with or without a lipophilic unit (lauryl
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methacrylate) were assembled on poly(N-(2-hydroxypropyl)methacrylamide) to form linear
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polymeric amphiphiles with self-assembling properties [46]. The formed nanoparticles were
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able to induce strong humoral immune responses only when mixed with CFA, consistently
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with an older study, which used epitope polymerization technique based on the formation of
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linear polyacrylate [47].
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Lipid-based nanoparticles
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Lipid carriers have been studied extensively for vaccine delivery and liposomes are
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one of the most widely used lipid-based vaccine delivery vehicles [48, 49]. Surprisingly,
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liposomes have been rarely used for peptide-based nanovaccine delivery. In a recent study,
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multiepitope peptides from the rat HER2/neu oncogene were incorporated into liposome-
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polycations with CpG oligonucleotides adjuvant (LPD) nanoparticles (~150 nm) [50]. Lead
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liposomal formulation (with p5 peptide) was able to completely protect mice in a
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prophylactic TUBO tumor model (overexpressing the rHER2/neu protein) challenge. In
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another approach, highly conserved influenza-derived peptides were encapsulated into
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liposomes (30 – 100 nm) with monophosphoryl lipid A (MPL) and trehalose 6,6’-dimycolate
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as adjuvants [51]. While the peptides alone were practically non-effective, a liposomal
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formulation was able to induce protective immune responses after intranasal administration
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against a lethal influenza challenge in mice. The immune responses were T-cell dependent
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with macrophages playing a major role (rather than DCs) in response induction.
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Unfortunately, both the above liposomal strategies required the use of an adjuvant in the
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formulation. A more popular lipid-based strategy used lipidation of peptide antigens to form
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amphiphiles, which were self-assembled into nanoparticles. During study on the conserved
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peptide epitope-based vaccine against GAS, it was demonstrated that the balance between
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hydrophilic and hydrophobic properties of individual segments of such lipopeptides was
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responsible for the size of formed particles and the more polar peptide epitopes attached to
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the lipid core produced smaller nanoparticles [52]. In this approach the lipid peptide core
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(LCP) strategy was used, in which unnatural lipidic amino acids (amino acids with long
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aliphatic side chains) were conjugated via the branching moiety (based on polylysine,
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carbohydrate, etc) to the desired peptide epitopes [53, 54]. In the LCP, lipid moieties served
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as a hydrophobic core to allow self-assembly and act as a self-adjuvanting moiety with TLR-
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2 agonist properties [54]. When multiple copies of GAS-derived B-cell epitopes (J14) were
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incorporated into LCP constructs, large nanoparticles were formed (200-1000 nm) that
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induced rather moderate B-cell response in comparison to the CFA-based control [55]. In
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contrast, an LCP construct possessing modified J14 epitope (dJ14i), when self-assembled into
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small nanoparticles (15-20 nm), was able to induce the same level of anti-dJ14i IgG titers as
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the peptide formed with CFA when administered subcutaneously in mice [56]. However,
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heterogeneous size distribution of nanoparticles with no clear size-dependent immune
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responses were also reported for a variety of LCP-based vaccine candidates [57]. Robinson
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and coworkers used lipopeptides to form self-assembled homogenous nanoparticles (20-25
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nm) which were able to induce strong humoral immunity with or without the use of CFA [58,
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59]. They also demonstrated that DCs used multiple endocytic routes even for uptake of
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small nanoparticles. While the above particles were taken up mainly by macropinocytosis,
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clathrin independent uptake was also observed [60].
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Self-assembled peptide
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The ability of certain peptides to self-assemble into particlse or fibrils is a well-known
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phenomenon and peptide self-assembly has been used for biomedical purposes [61]. Peptide
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self-assembled nanomaterials are biologically compatible, multifunctional, multivalent, well-
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chemically defined, usually low or non-toxic and the position of attachment of an antigen can
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be well controlled. Collier and co-workers have been intensively studying a vaccine delivery
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system based on β-sheet forming Q11 peptide. Several different peptide epitopes were
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conjugated to this peptide and self-assembled into fibrils (5-15 nm thick) [62-65]. They
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observed strong humoral responses in mice when OVA peptide epitopes were covalently
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bonded to Q11, however, a relatively large quantity of immunogen was required to induce
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production of high antibody titers (0.3 mg per injection) [65]. They demonstrated that two
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conjugates, incorporating Q11, linked with two single malaria-related peptide antigens can be
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co-assembled together to produce an immune response without help of adjuvant through the
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MyD88 pathway but without participation of TLR-2 and TLR-5 [64]. The fibres induced
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immune responses with the help of CD4+ cells, were non-toxic and did not induce
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inflammation [63]. When OVA-derived CTL epitope was conjugated to Q11, the formed
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fibrils elicited robust CD8+ T-cell responses [62]. Toth and co-workers demonstrated that
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such peptide antigen-bound fibrils can be formed upon request from non-fibrilizing
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precursors using an isopeptide strategy. Stable in solid form, O-acyl isopeptide (ester isomer
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of original peptide) showed high aqueous solubility and released native peptide through
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physiological pH-triggered O-N acyl migration reaction with simultaneous fibril formation.
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They claimed that this strategy can overcome potential problems related to over-aggregation,
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precipitation, and changes in other properties during storage of fibril-based vaccines [66].
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Burkhard and co-workers previously demonstrated that peptides which possess coil-
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coil conformation were able to aggregate and upon conjugation with malaria peptide epitope
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form nanoparticles (~25 nm). These nanoparticles induced protective immunity in mice in a
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malaria challenge experiment [67]. Recently, they incorporated into their delivery system
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tumor targeting moiety (bombesin) and formed nanoparticles (33-36 nm) [68]. While these
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particles did not demonstrate tumor targeting properties, their spleen uptake was significantly
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increased, proportionally to the increasing level of the bombesin in the particles. As spleen is
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a primary organ of the immune system, it was suggested that such particles can be used for
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design of vaccine candidates with improved efficacy. When this delivery system incorporated
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CD8+ epitope from Toxoplasma gondii, it was able to self-assemble into ∼38 nm
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nanoparticles and induce strong cellular immunity (assessed by IFN-γ production) [69]. The
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nanoparticle was also able to reduce T. gondii parasite burden in vivo.
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Inorganic nanoparticles and nanotubes
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Nanoparticles built from inorganic material such as a gold or ferric oxide have
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recently become attractive drug delivery vehicles [70]. They have unique physicochemical
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properties such as porous structures, facile surface functionalization with a variety of ligands,
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and their size and shape can be controlled. Interestingly, commercially available alum
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adjuvant (which can form inorganic nanoparticles) was found to be safe and an effective
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immunostimulant for whole pathogen or protein-based vaccine delivery; however, its
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adjuvanting properties are generally too mild for stimulation of immune responses against
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peptide antigen [71]. To overcome alum pure immunostimulatory activity, Neutra and
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coworkers conjugated peptide epitopes derived from HIV-1 gp120 glycoprotein to the
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aluminum oxide nanoparticles (~350 nm). These particles were able to stimulate a moderate
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antibody response after intraperitoneal injection; however, they failed to stimulate mucosal
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immunity [72, 73]. Further study was discontinued. Huang and co-workers used foot-and-
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mouth disease virus associated peptide antigen conjugated to several gold
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nanoparticles with sizes ranging from 2 to 50 nm (2, 5, 8, 12, 17, 37, 50 nm) [74]. The
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highest antibody titers were observed for mice immunized with 8 nm nanoparticles,
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while the 37 and 50 nm were ineffective. Generally, 2-17 nm particles induce strong
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humoral response. The highest spleen uptake was observed for nanoparticles with size
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12 nm while uptake was also high for particles of size 8-50 nm. Larger particles, which
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are more easily endocytosed, were absorbed at the injection site and therefore their
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concentration in the circulation (blood) was low. As size-dependant spleen uptake of
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nanoparticles was similar to their efficacy profile, it was suggested that the ability of particles
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to travel and accumulate in the spleen was crucial to induce immunity. Baneyx and co-
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workers applied calcium phosphate to form peptide antigen-coated nanoparticles (50-70 nm)
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which showed the ability to induce humoral immunity in mice [75]. In another study, calcium
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carbonate nanoparticles were coated with polylysine and polyglutamic acid based on opposite
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charge attraction [76]. During coating process, OVA and influenza peptide epitope were also
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incorporated to form nanoparticles with diameter of ~ 250 nm and ~150 nm, respectively.
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These nanoparticles were able to induce both humoral and cellular immunity after a single
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injection in mice without the help of an adjuvant. Importantly, no immune responses to the
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matrix components were detected.
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The recent discoveries of carbon nanotubes as a drug delivery agent [77, 78] triggered
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interest in developing this nanomaterial for vaccine delivery purposes [79, 80]. Early attempts
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have shown that peptides conjugated to nanotubes were able to induce high titres of antibody
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when CFA was used as an adjuvant [81, 82]. More recently, Villa et al. demonstrated that
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peptide derived from Wilm's tumor protein conjugated to nanotubes of high length variability
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could be rapidly internalized into APCs, and induced humoral immunity; however, external
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adjuvant was still necessary for nanotube efficacy [83]. These data suggested that carbon
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nanotubes are a rather poor immunostimulator for peptide-based vaccines.
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Conclusion
Nanomaterial-based approaches for peptide vaccine development are clearly of high
415
importance in current vaccine delivery research. Particle size of these peptide vaccines plays
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an important role for their immunostimulatory properties. Interestingly, size-dependent
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activity is not as consistent as can be expected with different groups reporting different
418
optimal size for vaccine formulation. This phenomenon can be explained by the differences
419
in measurement techniques which are often determining diverse sizes of the same particles
420
(e.g. dynamic light scattering (DLS) measure hydrodynamic size, while transmission electron
421
microscopy (TEM) is showing the size of nanoparticles after drying and only the part which
422
efficiently absorbs light is visible). Particle size distribution also can vary significantly and
423
the immune system does not produce responses against single particle size but always against
424
a whole range of sizes present in the vaccine formulation. For simplicity however, dominant
425
size or “peak” size is usually reported. In addition, particles may differ not only in size but
426
also in (a) antigen loading; (b) level of antigen absorption on the surface against
427
encapsulated, and different loading methods may incorporate antigen into/onto nanoparticles
428
in different ways; (c) the nature of the composition material; (d) the ability of the antigen to
429
be released from nanoparticles; and (e) the level of its protection against biodegradation.
430
Moreover, dose and dosing frequencies differ between studies and the route of administration
431
can have major impact on vaccine efficacy. However, the message from most of the current
432
studies is clear; size plays an important role in vaccine efficacy. Smaller particles are more
433
immunogenic due to their easier uptake by DCs and their efficient transport in the lymphatic
434
system; however, large particles including microparticles can form a stable depot and in this
435
manner induce strong immune responses as well.
436
The antigen is often chemically conjugated into/onto nanoparticles, or the particles are
437
formed from self-assembled antigen-carrier conjugates; such stable composition ensures
438
delivery of the adjuvanting moieties and antigen to the same APCs. This limits systemic
439
distribution of adjuvant and its concentration required to boost immunity, therefore limiting
440
toxicity of the vaccine. Moreover, vaccines are not administered in a repetitive manner to the
441
host; therefore, the risk of excessive accumulation in the body of even relatively stable
442
particles is low. Nevertheless, nanoparticle-based formulations should undergo strict quality
443
control and such factors like reproducibility of formulation, storage related aggregation, and
444
surface charge changes need to be carefully monitored during production, storage and
445
transportation. This cost is warranted as in return, safe vaccines can be developed and the use
446
of classical adjuvant with their toxic side-effects can be omitted.
447
448
449
450
Future Perspectives
451
progress in nanotechnological approaches for vaccine delivery should overcome many of
452
existing obstacles. Especially, vaccine efficiency can be greatly improved and toxicity
453
reduced using an adjuvant-free nanovaccine strategy. In addition, the only immune adjuvant
454
commonly used for humans (alum) is not able to stimulate cellular immunity. Stimulation of
455
cellular immune responses has been found crucial for development of vaccines against
456
cancer, malaria, HIV and other intracellular pathogens. Thus, the ability of nanoparticles to
457
induce cellular immunity against incorporated peptide antigens would be of special interest in
458
the field of vaccine development. There are several examples of peptide-based vaccines in
459
clinical trials (e.g. vaccines against GAS or therapeutic vaccines against cervical cancer).
460
Thus, the prospect for commercial success of peptide-based vaccines is substantial and the
461
use of nanotechnology-based approaches can only increase this chance. In addition, many
462
current peptide vaccine delivery platforms have not been analyzed for their ability to form
463
particles, their size dependent immunity, and the influence of morphological properties on
464
their efficacy, but in the near future such analyses are expected to become standard in
465
peptide-based vaccine development. It is not anticipated that just one single size will be
466
found to be optimal for all vaccine deliveries; rather each delivery system and antigen will
467
have its unique optimal size and other properties (such as charge, shape etc.) and therefore,
There is no example of a peptide-based vaccine in the market so far; however, recent
468
each system will need to be optimized separately. Moreover, the use of a mixture of different
469
sizes might be advantageous in some cases (e.g, for the same antigen the stable depot with be
470
formed with large particles while at the same time small nanoparticles will be used to target
471
the antigen to lymph nodes). In future development, size-dependent toxicity needs to be
472
studied in more detail. Some recent reports have shown that very small cationic particles can
473
have significant toxicity. Thus, vaccine candidates, especially those with broad size
474
distribution might not be as safe as currently claimed. Even in such cases, the immune system
475
is expected to clear those nanoparticles before they can harm the human body. As vaccination
476
remains associated with some toxicity, approaches based on single immunization are
477
particularly advantageous and, as has been shown in this review, such immunization schedule
478
becomes possible with the help of nanoparticles. The use of fully biodegradable carriers is
479
also recommended. In future development, the cost of vaccine production needs to be taken
480
into account as well. For example, approaches for neglected tropical diseases, which are
481
slightly less affective but significantly cheaper, should be endorsed. Finally, in the near future
482
it is expected that nanoparticle-based formulations will not be limited to antigens and
483
immunostimulating moieties but additional functional elements will be incorporated (such as
484
targeting moieties, stabilizing coatings, or mucosal adhesive functionalities).
485
486
Executive Summary
487
Peptide-based subunit vaccine
488

489
490
effects.

491
492
The use of only minimal immunogenic component allows reduction of undesirable side-
Removal of danger signal reduces peptide-based vaccine immunogenicity; therefore,
external adjuvants or special delivery systems have to be used for vaccine efficacy.

Peptide-based vaccine can be relatively easy customized, produced, stored and
493
transported.
494
The nanotechnology
495

496
497
vaccine immunogenicity.

498
499
500
It has been widely accepted that the size of antigen particles plays an important role in
The use of nanoparticles can stimulate better antigen uptake by APCs, protect antigen
from degradation and elimination, and induce antigen cross-presentation to CTLs.

Nanoparticles can be engineered to contain multiple peptide epitopes, self-adjuvanting
moieties and targeting moieties.
501

502
Nanoparticles may mimic natural pathogen through size and display of multiple copies of
surface antigens.
503
Polymer-based nanoparticles
504

Peptide-based antigen can be encapsulated or attached on the surface of polymeric
505
nanoparticles
506
nanoparticles.
507

508
509
while
polymer-peptide
conjugates
can
be
self-assembled
into
Most of the data suggests that small polymer-based nanoparticles (20-50 nm) induce
optimal immune responses.

510
Poly(D,L-lactic-co-glycolide), chitosan, and acrylates are the most commonly used
polymeric carries for peptide vaccines delivery.
511
Lipid-based nanoparticles
512

Lipids have a natural tendency to self-assemble and might be recognized by TLRs.
513

Lipidation of peptides forms amphiphiles which are often able to self-assemble into
514
nanoparticles with self-adjuvanting properties and capacity to induce strong immune
515
responses.
516

517
Size-dependant immunogenicity of lipid-based peptide vaccine has not yet been
comprehensively studied.
518
Self-assembled peptides
519

520
521
Self-assembly properties of certain peptides can be used to form nanoparticle or
nanofibril structures.

522
Self-assembled peptides are fully biodegradable, biocompatible and can induce both
cellular and humoral immune responses without help of an adjuvant.
523
Inorganic nanoparticles and nanotubes
524

525
526
Peptide antigens can be conjugated to inorganic nanoparticles and induce size-dependent
immune responses.

While some studies have suggested optimal efficacy for small nanoparticles (2-17 nm),
527
larger nanoparticles are also effective in inducing immune responses without help of an
528
adjuvant.
529
530
531
532
533

Carbon nanotubes can serve as carriers for peptide-based vaccines but the use of an
adjuvant is still required for their efficacy.
Financial & competing interest disclosures
534
This work was supported by the National Health and Medical Research Council (NHMRC),
535
Australia. The authors have no relevant affiliations or financial involvement with any
536
organizations or entity with a financial interest in or financial conflict with the subject matter
537
or material discussed in the manuscript apart from those disclosed. The contents are solely
538
the responsibility of authors and do not necessarily represent the official views of the
539
NHMRC.
540
541
542
References
543
Papers of special note have been highlighted as:
544
 of interest
545
 of considerable interest
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