Antigen delivery by virus-like particles for immunotherapeutic vaccination.

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Therapeutic Delivery

Antigen delivery by virus-like particles for immunotherapeutic vaccination

Virus-like particles (VLPs) are an effective means of establishing both prophylactic and therapeutic immunity against their source virus or heterologous antigens. The particulate nature and repetitive structure of VLPs makes them ideal for stimulating potent immune responses. Epitopes delivered by VLPs can be presented on MHC-II for stimulation of a humoral immune response, or cross-presented onto MHC-I leading to cell-mediated immunity. VLPs as particulate subunit vaccine carriers are showing promise in preclinical and clinical trials for the treatment of many conditions including cancer, autoimmunity, allergies and addiction. Supporting the delivery of almost any form of antigenic material, VLPs are ideal candidate vectors for development of future vaccines.

Vaccines are traditionally designed for prophylactic use, stimulating the formation of preventative immunity against infection. While prophylactic vaccination remains essential for establishing herd immunity, it is generally ineffective in the clearance of preestablished disease. Recent advancements in therapeutic vaccination have demonstrated that it is possible to use vaccination to treat pre-existing conditions. Therapeutic vaccination involves active clearance of an infectious agent, infected cells or tumor cells. As the condition is already present, this often involves breaking immune tolerance or bypassing the mechanisms by which the disease has eluded the immune system. Designing therapeutic vaccines can therefore require a delicate balance between stimulating a potent targeted immune response and eliciting unintentional off-target hypersensitivity. Generally, this can be managed through careful selection of candidate antigen targets in combination with an appropriate vaccine scaffold. Virus-like particle (VLP) vaccines are a class of subunit vaccine formed from selfassembling virus-derived capsid proteins. The absence of genomic material from the source virus renders VLPs incapable of rep-

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lication or infection, with self-limiting vaccination kinetics and an impeccable safety profile. The capsid proteins in VLPs retain characteristic structural conformation of the native virus, retaining antigenic motifs with increased immunological relevance compared with inactivated virus vaccines. As the capsid proteins self-assemble into particles that are recognized and processed through similar pathways as native virions, VLP vaccines can also resemble a live attenuated virus without replicative or infectious capacity. VLPs also avoid the potential hazards posed by other virus-derived vaccines, such as incomplete inactivation or attenuated virus reversion. A natural by-product produced during the infection cycle of some viruses, VLPs were first identified and studied in patients infected with hepatitis B virus (HBV) [1] . The isolation of VLPs from patients infected with the native virus is an inefficient means of vaccine production. Patient serum purification also carries the risk of transmitting other infectious agents, surpassed by in vitro protein expression systems. Some examples of systems adapted for VLP production include bacteria, yeast, insect cell lines, mammalian cell lines, plants and cell-free systems. Recombinant expression of virus capsid proteins

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Farah Al-Barwani‡,1,2, Braeden Donaldson‡,1,2, Simon J Pelham‡,2, Sarah L Young2 & Vernon K Ward*,1 1 Department of Microbiology & Immunology, Otago School of Medical Sciences, Dunedin, New Zealand 2 Department of Pathology, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand *Author for correspondence: Tel.: +64 03 479 9028 [email protected] ‡ Authors contributed equally

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Review Al-Barwani, Donaldson, Pelham, Young & Ward

Key terms Virus-like particle: A particle formed from viral structural proteins, with no viral genomic material. Recombinant insertion: Genetic modification of the viral proteins in virus-like particles to contain foreign sequences. Chemical conjugation: Formation of a chemical bond between two molecules. Adjuvant: Immunostimulatory molecules capable of skewing immune responses towards either the humoral or cell-mediated arms of the immune system.

also enables the production of VLPs from viruses that cannot be routinely cultured, such as human norovirus or hepatitis C virus (HCV) [2,3] . Although most VLPs may be produced in several expression systems, each expression system has associated benefits and disadvantages [4] , such as ease of expression, scalability and production cost. Furthermore, recombinant capsid protein quaternary structural conformation can vary between systems owing to differences in posttranslational modifications, such as glycosylation and phosphorylation. Improper protein folding can impair VLP formation or significantly alter VLP immunogenicity, as some modifications are essential for eliciting a potent immune response. Despite differences in posttranslational modification, each protein expression system is compatible with large-scale good-manufacturing-practice production of vaccine-grade VLPs. This enables mass production of high quality VLP vaccines, many of which are already licensed for prophylactic vaccination, while others have moved into clinical trials for potential therapeutic applications. As with other protein-based vaccines, VLP vaccine stability is an important factor influencing clinical development. VLPs, like their native virus, differ in their ability to preserve capsid structure during long-term storage; however, many licensed VLP vaccines have proven to be highly stable [5–7] . This review covers VLP structure and function with a primary focus on therapeutic vaccination. VLP structure VLPs inherit their geometric structural symmetry from their source virus, consisting of a repetitive array of viral structural proteins. They can appear icosahedral, spherical or rod-like and vary in size depending on the properties of the native virus. VLPs can be produced with various levels of structural complexity, as illustrated in Figure 1 [4] . Most simple VLPs consist of a single layer formed from a single viral structural protein, such as HBV-VLP formed from HBV surface antigen [8] . Simultaneous expression of multiple viral structural proteins can produce VLPs with several dis-

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tinct capsid layers, such as rotavirus VLPs, which can have double or triple capsid layers depending on the combination of capsid proteins expressed [9,10] . VLPs produced from multiple strains of the same virus are termed mosaic VLPs. Vaccination with mosaic VLPs should theoretically confer protection against multiple virus strains, increasing vaccine efficiency against viruses with several infectious isotypes. Many viruses are encapsulated inside a phospholipid envelope derived from host cells. This feature can be translated into the VLP of these viruses, adding additional depth in complexity. Enveloped VLPs can consist of a capsid protein shell, coated within an envelope. For example, coexpression of HA, NA, M1 and M2 proteins results in the formation of influenza virus VLP encapsulated within a phospholipid envelope [11] . These VLPs have the characteristic HA and NA surface spikes of native influenza virus, and can be formed from multiple influenza strains. Enveloped VLPs can also be produced in the form of a virosome particle, with only membrane-anchored viral proteins supporting a lipid envelope particle (e.g., influenza A virosome) [12] . VLP vaccine design Recombinant insertion

As a proteinaceous nanoscale particle, VLPs can be used as versatile vaccination vectors. While they already harbor immunologically relevant antigenic motifs from their source virus, they can be manipulated to deliver heterologous antigens. VLPs can support a variety of modifications, such as recombinant insertion and chemical conjugation of molecules, including peptides, proteins, cell lysate, lipoproteins and carbohydrates. Modifications involving incorporation of heterologous antigens results in the formation of chimeric VLPs, capable of eliciting immunity against conditions other than those caused by the VLP source virus. The immune response is enhanced by the repetitive structure of VLPs, which may provide a viral tag to otherwise innocuous peptide sequences. The efficacy of VLPs as a vaccine scaffold relies on their particulate nature, which poses some limitations on the extent of potential modification. For example, rabbit hemorrhagic disease virus (RHDV) VLPs retain stability with recombinant insertions of less than 33 amino acids at the N-terminus of VP60 [13] , while polyomavirus VLPs can support up to 120 amino acids inserted into VP1 [14–17] . Polyomavirus VLPs also containing the nonstructural secondary capsid protein VP2 can even incorporate large truncated proteins [18] . VLPs formed from the HBV core antigen (HBcAg) have been used in a variety of commercial vaccines, such as Malariavax, targeting Plasmodium

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falciparum, the causative agent of malaria [19] , and the pan-influenza A vaccine ACAM-FLU A [20] . In order to avoid pre-existing immunity, VLPs derived from non-human viruses can also be used as a scaffold for human immune epitopes, such as VLPs derived from alfalfa mosaic virus [21] , Qβ bacteriophage [22] and RHDV [23,24] . Chemical conjugation

An effective means of avoiding the size limitations of recombinant insertion is chemical conjugation. There is a vast variety of potential conjugation candidates, limited by their effects on VLP stability, solubility and processing. The range of chemically functional motifs on amino acid residues in the capsid proteins of VLPs provide targets for conjugation, with potential modifications including activation of carboxylic acid, alkylation of sulfhydryl groups or acylation of amino groups. Some of the most common mechanisms of chemical conjugation to VLPs are illustrated in Figure 2 [25] . Alternative forms of conjugation include azide–alkyne click chemistry [26] and the formation of biotin–streptavidin complexes [27] . HeterobifuncA

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tional crosslinkers provide a nonspecific method of conjugation between proteins, which can result in aggregation of proteins in complex solutions. Selective conjugation with bioorthogonal crosslinkers targets chemical motifs that are absent in biological systems (e.g., phosphines), preventing conjugation and aggregation of nonspecific proteinaceous material. Cleavable linkers are another intriguing innovation amongst conjugation crosslinkers, with intracellular cleavage enabling release of conjugated material. This can enable the separation of the conjugated material away from the processing pathway of the VLP, which may promote efficient delivery of molecules such as toll-like receptor ligands and MHC-restricted peptide epitopes. While peptides can be conjugated to VLPs through specific amino acids, conjugation of adjuvants often requires modification of the adjuvant to include a chemical conjugation motif, such as a thiol group. Chemical conjugation provides additional benefits, such as surface exposure and avoiding interference with VLP formation; however, quantification can be difficult and nonconjugated material must be

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Chimeric VLP Figure 1. Virus-like protein structural forms. VLPs can be comprised of (A) a single viral structural protein, (B) multiple proteins in a single layer or (C) multiple layers , or (D) as a mosaic of proteins derived from several virus strains. Chimeric VLPs can be produced that harbor heterologous antigens inserted (E) internally, (F) externally or (G) chemically conjugated to the surface. Some VLPs are also encapsulated inside a phospholipid envelope (H). Further combinations of these features can be produced beyond those illustrated [4] . VLP: Virus-like particle.


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removed. Chemical conjugation may not be as efficient for delivery of peptides in comparison to recombinant insertion. Vaccination with chimeric RHDV VLP containing an H2kb-restricted peptide epitope from lymphocytic choreomeningitis virus gp33, through recombinant insertion or chemical conjugation, demonstrated that recombinant insertion induced a superior in vivo cytotoxic response [23] . The recombinant form of the chimeric VLP induced 50% tumor-free survival in mice grafted with Lewis’ lung carcinoma tumors after a single vaccination with no adjuvant, which was increased to 70% with a 4-week boost VLP vaccination [23] . The advantage of chemical conjugation is the delivery of large antigenic material, such as whole proteins, and mixed epitope complexes, such as tumor lysate. VLPs coupled with large antigens retain their particulate nature, with enhanced processing and induction of a potent immune response against conjugated material. For example, tumor lysate derived from the melanoma cell line Mel888 expressing MART-1 conjugated onto RHDV VLP improves the processing of lysate in dendritic cells, and enhances MART-1-specific CD8+ T-cell stimulation in comparison to lysate alone [28] . The potential Key terms Intraparticulate encapsulation: The capture of molecules such as nucleic acids inside the virus-likeparticle shell. Mucoadhesives: Molecules that adhere to and promote penetration of mucosal membranes.

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for chemical conjugation as an alternative to recombinant insertion further increases the breadth of VLP as a versatile vaccine vector. Intraparticulate encapsulation

VLPs are inherently devoid of genomic material from their source virus by definition, but not all VLPs are an entirely empty shell. Some virus capsid proteins have the ability to bind nucleic acids, which facilitates the encapsulation of the virus genome during replication. This property can be retained in VLPs, enabling the encapsulation of molecules such as oligonucleotides, plasmids and chemical polymers. Encapsulation is primarily limited by the volume of the internal cavity and the availability of charged amino acid motifs inside the VLP [29] . VLPs that retain the ability to encapsulate DNA have been explored as potential vectors for gene delivery. VLPs derived from the human polyomavirus John Cunningham (JC) virus VP1 capsid protein can encapsulate DNA plasmids up to 14 kb in size [30] . Plasmid loading is facilitated by osmotic shock of JC virus VLP, which increases permeability by temporarily disrupting the VLP structure [31] . Osmotic shock has been surpassed by direct plasmid loading during initial VLP formation in this expression system. This method was initially trialed with the JC virus VP1 capsid protein expressed in Escherichia coli encapsulating the pEGFP-N3 plasmid, followed by encapsulation of the pUMVC1-tk plasmid [32] . JC-virus VLP containing the pUMVC1-tk plasmid selectively targeted human colon carcinoma cells (COLO-320 HSR) grafted in nude



mice, with a significant reduction in tumor volume with subsequent administration of ganciclovir [32] . Chimeric VLPs can also gain the ability to encapsulate DNA plasmids by incorporating the DNA-binding motifs of other virus proteins. RHDV VLPs containing the DNA binding site from human papillomavirus type 16 (HPV-16) L1 (VP60Δ-L1BS) or L2 (VP60Δ-L2BS) through recombinant insertion at the N-terminus of the capsid protein were found to encapsulate the plasmid pCMV-β by in vitro VLP reassembly. Administration of VP60Δ-L1BS containing pCMV-β induced expression of β-galactosidase in a range of cell lines, including CaCo2, Cos-7, HuH-7 and R17 [33] . VLPs have also been used to induce expression of their own capsid protein, mimicking viral replication to enhance immunogenicity [34] . VLPs can also be similarly modified to encapsulate other molecules, such as fluorophores in cucumber mosaic virus VLP [35] , polymerase in rotavirus VLP [36] , enzymes in Qβ VLP [37] and up to 15 EGFP proteins in cowpea chlorotic mottle virus VLP [38] .

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Delivery mechanisms

VLP vaccines are compatible with a wide range of delivery mechanisms already utilized for administration and/or enhancement of commercial vaccines, with some common examples illustrated in Figure 3. Specific delivery mechanisms can be utilized to stimulate a particular type of immune response, such as aerosolization and inhalation of VLPs, which has been explored as a means of eliciting a mucosal immune response. Aerosol vaccination with Qβ VLPs containing peptides derived from the HIV coreceptor CCR5 induces anti-CCR5 IgA antibodies at the mucosal surface, which is absent with intramuscular injection alone [39] . Mucosal delivery has also been investigated for Norwalk VLPs using the mucoadhesive chitosan, which enhances penetration of mucosal membranes [40] . Establishment of a biocompatible depot may also be advantageous for some VLP vaccines by providing sustained vaccine release. PLGA forms biodegradable polymeric nanoparticles, which have

Respiratory tract delivery Aerosol delivery of vaccines increases the surface area of mucosa exposed, and facilitates targeted vaccination at mucosal surfaces Muco-adhesives Can enhance penetration of mucosal membrances, enhance stimulation or protect vaccine from enzymatic degradation

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Biocompatible depot Formation of a biocompatible depot enables sustained vaccine release, which boosts the immune response

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Figure 3. Delivery mechanisms for virus-like particle vaccination. Virus-like particle vaccines can be enhanced through a variety of mechanisms designed to improve delivery (nanopatch, aerosolization), elicit a specific type of immune response (mucoadhesives), release antigen over time (biocompatible depot) and increase stability (consumables).



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Key terms Antibody-dependent cell-mediated cytotoxicity: Lysis of target cells coated in antibodies through recognition of the Fc receptor, mediated by natural killer cells, γδ T cells, macrophages and neutrophils. Cross-presentation: Presentation of exogenous antigens on MHC-I, for stimulation of a cell-mediated immune response.

demonstrated sustained release of HBsAg VLP over a 14-day period [41] . A potential alternative to intramuscular injection has been developed that promises enhanced antigen delivery and recognition. The Nanopatch (Vaxxas) is mounted with a grid of microinjection tips that can deliver lyophilized or particulate vaccines directly to dermal and epidermal resident antigen-presenting cells (APCs). Nanopatch delivery has been explored for its potential applications to VLP vaccines, such as the HPV vaccine Gardasil [42] . Oral administration of VLP as a consumable vaccine has also been considered as an alternative delivery route. Consumption of tubers from transgenic potatoes expressing the HPV major capsid protein L1 was found to prime anti-L1 responses in 50% of mice, with anti-L1 antibody production observed in 12.5% [43] . VLP immunogenicity & response VLPs have the capacity to stimulate both the cellmediated and humoral arms of the immune system. A cell-mediated cytotoxic immune response is vital for the clearance of intracellular pathogens (e.g., viruses, bacteria) or abnormal cells, such as in cancer. By contrast, the humoral immune system is important for the production of antibodies, which can neutralize pathogens and circulating factors (e.g., toxins). Antigen processing and recognition is essential for activation of either arm of the immune system. This process is initiated by the internalization of target antigens by APCs with presentation of processed immune epitopes on MHCs. The repetitive capsid structure of VLPs can enhance internalization, processing and presentation of antigens by a variety of APCs, including B cells, macrophages and dendritic cells [44] . The route of administration and the physical properties of the individual VLP can alter the populations that preferentially internalize the particles. One of the major determinants of this is the size of the VLP, with particles greater than 200 nm in diameter requiring transport by APCs to the lymph node to enable presentation of antigen to effector cells [45] . APCs can internalize VLPs through a variety of mechanisms, such as phagocytosis, macropinocytosis and receptor-mediated endocytosis. For example,

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RHDV VLPs are internalized through clathrindependent macropinocytosis and phagocytosis [46] . Some VLPs also retain receptor ligands from their parent virus, such as influenza A VLPs that are capable of binding to sialic acids through hemagglutinin expressed on their surface. VLPs have also been modified to target specific receptors present on the surface of APCs. An example of this is the conjugation of superantigen to HBV VLP, which promotes internalization through MHC-II [47] . Humoral response

Stimulation of the humoral arm of the immune system involves recognition of native antigen by B cells, in addition to cytokine costimulation from T-helper 2 (TH2) cells. These TH2 cells are activated by recognition of epitopes presented on MHC-II by APCs. Antibodies produced by activated plasma cells can be generated against VLPs or antigenic material they carry. The capacity of VLPs to trigger robust antibody production is commonly utilized for prophylactic vaccination against their parent virus (e.g., HBV [8] , HPV [48] , influenza A [49]). VLPs are strong inducers of antibody production owing to their ability to drain into B-cell follicles [50] . Their repetitive structure triggers crosslinking of B-cell receptors, promoting internalization of VLPs and inducing antibody production. Qβ VLP can be transported to the germinal centre by naive B cells in a complement-dependent manner, independent of VLP-specific antibodies [50] . Delivery to the germinal centre is essential for B-cell activation and clonal selection. By contrast, Qβ VLPs delivered intranasally can be transported by B cells to the spleen in a B-cell receptor-dependent manner, independent of the complement receptor [51] . This demonstrates that different VLPs may be transported and processed through a variety of pathways within the immune system. Cells that express antigens derived from intracellular pathogens can also be targeted and eliminated through mechanisms such as antibodydependent cell-mediated cytotoxicity. This is a process where infected cells coated in pathogen-specific antibodies are selectively killed by Fc receptor-expressing cytotoxic or effector cells (e.g., natural killer [NK] cells, macrophages, γδ T cells) (Figure 4) . For example, in FcRγ-knockout mice vaccinated with influenza A VLPs, protection against influenza A infection is impaired despite normal virus-specific antibody production. This indicates that antibody-dependent cell-mediated cytotoxicity contributes significantly to the clearance of influenza A virus during infection in response to vaccination with influenza A VLPs.



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Figure 4. Antibody-dependent cell-mediated cytotoxicity. Antibody-dependent cell-mediated cytotoxicity can be facilitated through induction of apoptosis in target cells mediated by cytotoxic cells, such as NK cells and γδ T cells, and enhancement of phagocytosis by phagocytes including macrophages and neutrophils. FasL: Fas ligand; FcyRIIIa: Fc gamma receptor IIIa; NK: Natural killer.

Cell-mediated response

In addition to presentation on MHC-II, exogenous antigen can also be cross-presented onto MHC-I by some APC populations. CD8+ dendritic cells are the most efficient APCs for cross-presentation of antigen, utilizing pathways such as MHC-I receptor recycling, endosome-to-cytosol, endoplasmic reticulum endosome fusion and the CD74-dependent MHC-I shuttling pathways [52,53] , which are illustrated in Figure 5. Macrophages and B cells have also been implicated in cross-presentation of VLPs; although the involvement of B cells may be through cytokine-mediated stimulation of dendritic cells and macrophages, rather than direct antigen presentation [54] . Expression of immune epitopes on MHC-I is essential for stimulation of cytotoxic CD8+ T cells, in conjunction with sufficient costimulation. VLP vaccines can stimulate polyfunctional T cells to produce IFN-γ, TNF-α and IL-2 [55] , establishing a robust cytotoxic T lymphocyte (CTL) response and producing long-lived CD8+ memory T cells. Vaccine adjuvants

The addition of vaccine adjuvants can enhance and skew the immune system towards the most desirable response. Commonly used adjuvants include alum for stimulation of a humoral response, or imiquimod and unmethylated Type A CpG oligonucleotides for stimulation of a cell-mediated response. Some VLPs, such


as chimeric SV40 VLP, papaya mosaic virus and HIVVLP, have been reported as potentially self-adjuvanting; however, most VLP vaccines are enhanced by the addition of an adjuvant. Adjuvants can also be chemically conjugated to, encapsulated within or naturally associated with VLPs, enabling concurrent delivery directly to the APC that internalizes the VLP. This physical association mimics infection by the native virus through the inclusion of additional danger signals that provide costimulation of APCs. For example, the adjuvant α-galactosylceramide forms a composite particle when it associates with RHDV VLP, which induces increased activation of antigen-specific T cells [24] . In addition, encapsulation of Type B CpG within Qβ VLP can trigger B cells to undergo classswitching to produce antibody isotype variants, such as IgG1b, IgG1c and IgG2a [56] . Enveloped VLPs can also support the insertion of molecules containing a lipid residue or transmembrane motif. This interaction has been used to anchor adjuvants into the envelope, such as a recombinant membrane-bound flagellin in chimeric influenza VLP [57] , and glycosylphosphatidylinositol-anchored granulocyte-macrophage colonystimulating factor or CD40 ligand in chimeric simian immunodeficiency virus VLP [58] . VLP vaccines for cancer immunotherapy Oncogenesis and subsequent tumor formation is inherently due to the failure of the immune system


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Figure 5. Cross-presentation pathways in antigen-presenting cells. Antigen-presenting cells cross-present epitopes derived from exogenous antigen onto MHC-I through multiple pathways including: (A) gap junctions; (B) endosome to cytosol pathway; (C) ER–endosome fusion; (D) CD74-dependent pathway; (E) receptor recycling; and (F) exosomes. Epitopes derived from RHDV viruslike particles are known to be cross-presented through the receptor-recycling pathway [46] . ER: Endoplasmic reticulum; RHDV: Rabbit hemorrhagic disease virus. Adapted from [82] .

to control aberrant cell differentiation and proliferation. This phenomenon is theorized to follow a threephase process known as the immunoediting hypothesis, which includes tumor elimination, equilibrium and escape [59] . Recognition and elimination of tumor cells is facilitated by nonspecific cytotoxic immune cells, such as NK cells, NK T cells and γδ T cells (Figure 6A) . Immunotherapeutic vaccines that incorporate specific stimulatory molecules can enhance the effector function of these cells. For example, association of α-galactosylceramide with RHDV VLP enables robust stimulation of NK T cells [24] . Development of a tumor-specific immune response begins with the release of tumor antigen by nonspecific cytotoxicity and uptake of antigen by APCs (Figure 6B) . This process can be replicated through immunotherapeutic vaccination with VLPs containing tumor-associated antigens (TAAs). Robust tumor clearance is mediated by establishment of a cell-mediated immune response, with Th1 cell activation and proliferation of tumor-specific CD8+ T cells (Figure 6C & D) . The generation of an efficacious anti-tumor immune response can be impaired by the development of an immunosuppressive tumor microenvironment. This Key term Immunotherapy: Therapy involving the stimulation of an immune response, or treatment with immune products, such as antibodies.

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can occur gradually during the equilibrium phase due to diminished effector cell stimulation and the establishment of tumor-resident regulatory cells (e.g., CD4 T-regulatory cells, myeloid-derived suppressor cells). Selection and proliferation of a resistant subpopulation yields dysregulated tumor growth and escape of immunoedited tumors. Tumor immunotherapy is designed to overcome this tolerogenic environment, and stimulate reversion to the elimination phase. VLPs can deliver tumor antigens to the immune system through alternative pathways owing to their particulate nature and repetitive structure, overriding or bypassing tolerized pathways. VLP-based tumor vaccines may also be enhanced in combination with anti-tumor therapies that promote effector cell function, such as ablation of regulatory cells, blockade of immunoregulatory checkpoint pathways and renormalization of the tumor vasculature. Owing to the aggressive nature of the condition, current cancer treatment regimens rely primarily on combinations of surgical excision, radiation and chemotherapy; however, these treatments tend to be nonspecific and recurrence can occur. Immunotherapy is a promising alternative treatment for cancer that boasts generation of systemic and prolonged immunity. This may prevent tumor reestablishment and facilitate the clearance of metastases throughout the body. The success of these therapies relies on their ability to overcome tolerance and facilitate the activation and proliferation of autoreactive



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Figure 6. The immune response to cancer. (A) During the elimination phase of the cancer immunoediting hypothesis, tumor cells are recognized by nonspecific cytotoxic cells, such as NK cells, NK T cells and γδ T cells. (B) Antigenic material released by tumor cells can be phagocytized by antigen-presenting cells, such as dendritic cells, (C) while tumor antigen-laden antigen-presenting cells migrate to the lymph node to stimulate activation and proliferation of T cells, macrophages stimulated by IFN-γ production assist NK cells in tumor clearance. (D) Tumor antigen-specific T cells migrate to the tumor site, eliminating the remaining tumor cells. NK: Natural killer; Th: T helper. Adapted with permission from [59] © Nature Publishing Group (2002).



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Review Al-Barwani, Donaldson, Pelham, Young & Ward T lymphocytes. One issue faced by therapeutic cancer vaccine development is the identification of TAAs that can be targeted for the activation of tumor-specific immune cells capable of selectively targeting cancer. Tumors are also heterogeneous, with variable expression of TAAs throughout the tumor. Selective elimination based on a particular TAA may leave a resistant subpopulation to re-establish the tumor. In cancers such as melanoma and breast cancer, where ubiquitous target TAAs have been identified, preclinical and clinical studies have demonstrated promising results. Her-2/neu is a TAA overexpressed in some patients with metastatic breast cancer. An influenza virosomal formulated VLP vaccine containing three B-cell epitopes from the extracellular domain of Her-2/neu is currently under development [12] . Initial preclinical trials demonstrated significant Her-2/neu IgG antibody production and anti-tumor activity in mice. A Phase I clinical trial showed increased Her-2/neu-specific antibody production in eight out of ten patients, as well as a reduction in the number of T-regulatory cells [12] . This indicates that the Her-2/neu virosomal vaccine has the potential to initiate clinical benefits comparable to passive anti-Her-2 mAb therapy. Another defined TAA is the melanoma Melan-A/MART-1 peptide, which is expressed in melanocytes and can be overexpressed in melanoma. The VLP vaccine MelQβG10 consists of Melan-A/ Mart-1 conjugated to the exterior of Qβ VLP, and is loaded with a class A CpG [55] . In preclinical trials, this vaccine has demonstrated anti-tumor efficacy and stimulates the generation of IFN-g, TNF-α and IL-2-producing polyfunctional T cells. Recently, stage III or IV malignant melanoma patients who received the MelQβG10 vaccine subcutaneously with incomplete Freund’s adjuvant were found to have enhanced levels of Melan-A/Mart-1-specific T cells and effector memory T cells [55] . Many other promising VLP-based tumor immunotherapies are currently in preclinical studies. For instance, RHDV VLP expressing model tumor antigens has been shown to induce a CD8+ T-cell response causing delayed tumor growth and increased survival in murine subcutaneous tumor models [23,60] . Simian–human immunodeficiency virus VLP containing the pancreatic tumor antigen mesothelin is another promising vaccine candidate that has been shown to induce CD8+ T-cell response and reduce the number of regulatory T cells in an IL-6-dependent manner, leading to increased survival in an orthotopic pancreatic cancer mouse model [61] . Another promising VLP vaccine is the bursal disease virus VLP expressing a long C-terminal fragment of HPV-16 E7 protein. When delivered therapeutically, this vaccine can induce

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complete tumor clearance in humanized transgenic mice grafted with TC1/A2 tumors [62] . Although the two licensed HPV VLP vaccines, Gardasil (Merck) and Cervarix (GlaxoSmithKline), are extremely effective prophylactic vaccines against high-risk HPV and consequently HPV-induced cervical cancer, they do not work well therapeutically because they are designed to prevent infection rather than treat the condition. Even in the absence of a defined TAA, it may be possible to utilize VLPs in the therapeutic treatment of cancer in combination with a mixture of undefined epitopes, such as tumor lysate. This could theoretically enable the production of patient-specific personalized therapeutic vaccines for treatment of any form of cancer with VLPs. Other therapeutic VLP vaccines In addition to cancer, a variety of VLP are under development as potential therapeutic vaccines for conditions such as autoimmune disease, allergy, infectious diseases and addiction. Importantly, each condition requires stimulation of a particular immune response without exacerbating the disease. VLP provide a versatile vaccine scaffold that can be tailored to contain multiple antigens and adjuvants, enabling the stimulation of an immune response against even weakly immunogenic molecules such as auto-antigens. Although there are no therapeutic VLP vaccines currently commercially available, several are in clinical trials and many others have demonstrated promising results in preclinical studies. Many of these vaccines stimulate the production of antibodies through activation of the humoral immune system, with therapeutic effects mediated by processes such as opsonization, neutralization and receptor blockade. Some examples of therapeutic VLP vaccines in clinical trials are illustrated in Figure 7. Autoimmune diseases

During lymphocyte development in healthy individuals, the immune system becomes tolerized towards self-antigens. In autoimmune conditions, this tolerance becomes defective and the body mounts an immune response against self-antigens. This can involve the generation of autoreactive antibodies as well as an imbalance of proinflammatory cytokines that can lead to localized or systemic inflammation. Recently there has been success with anticytokine monoclonal antibody therapies in the treatment of autoimmune conditions, such as antiTNF-α for Crohn’s disease [63] and ulcerative colitis [64] , and anti-IL-2 receptor antibodies after kidney transplantation [65] . As cytokines are self-antigens, vaccines that aim to illicit anticytokine antibody production require the disruption of B-cell tolerance. Therefore, VLPs are an ideal candidate for the delivery of an anticytokine vaccine owing to their ability to facilitate the genera-



A Infected cell opsonization

B Cytokine neutralization

Review

C Receptor blockade

IL-1β Qβ VLPs

Her-2/neu influenza virolsome

Influenza VLP

Sm

oo

th

Plasma cell

Rough

gh

Rou gh

Rou

Sm

Sm

ooth

M2

oo

th

Antibodies h

ug

Ro

Rough

Her-2/neu IL-1β M2-expressing cell

Breast cancer cell

Figure 7. Examples of therapeutic virus-like particle vaccines. VLP vaccines stimulate the production of antibodies, which can mediate therapeutic effects. Examples include (A) opsonization of M2-expressing virally infected cells by stimulating production of M2-specific antibodies with influenza VLP [83] ; (B) neutralization of IL-1 with antibodies induced by IL-1β Qβ VLP vaccination [66] ; and (C) blocking the Her-2/neu receptor on breast cancer cells with antibodies induced by Her-2/neu influenza virosome vaccination [12] . VLP: Virus-like particle.

tion of B-cell responses against weakly immunogenic or tolerized antigens. Therapeutic VLP vaccines have been developed for a variety of autoimmune conditions. Autoimmune arthritis is a chronic inflammatory disorder that is characterized by the infiltration of immune cells into the synovial joints. This results in chronic inflammation and the degeneration of cartilage and bone tissue at the joints. IL-1 and IL-17 are two of a number of inflammatory cytokines that have been implicated in disease progression. In preclinical studies the administration of IL-1β (currently in Phase I/IIa clinical trials) or IL-17 Qβ VLP resulted to the generation of anticytokine antibodies that mediated disease suppression [66,67] . As these inflammatory cytokines are involved in many other inflammatory


conditions, IL-1β Qβ VLP and IL-17 Qβ VLP are also being assessed as a potential therapy for type II diabetes [68] and multiple sclerosis [67] , respectively. Systemic lupus erythematosus is another autoimmune disease that involves the development of chronic inflammation, characterized by production of inflammatory cytokines and autoantibodies. In patients with systemic lupus erythematosus, a reduced expression of miR-146a has been reported [69] . miR-146a is involved in the regulation of acute inflammation and has demonstrated potential therapeutic applications in systemic lupus erythematosus when delivered inside MS2 bacteriophage VLPs [70] . Following the administration of miR-146a MS2 VLP to lupus-prone mice, there was a reduction in the expression of autoantibodies and


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Table 1. Summary of virus-like particle vaccines included in this review. Source virus

Expression system

VLP platform

Vaccine antigen

Condition

Therapeutic Clinical or development prophylactic

Alfalfa mosaic Plant (Nicotiana virus tabacum)

AlMV CP

Rabies GP/NP

Rabies

Prophylactic Phase I

[21]

Cucumber mosaic virus

Plant (N. tabacum)

CMV CP

DNA encapsulation

–

–

[35]

Yeast (Saccharomyces cerevisiae)

HBsAg

HBsAg

HBV

Prophylactic Licensed (Engerix-B®)

HBV

Bacteria (Escherichia coli)

HBcAg

P.f. CSP

Malaria


[19]

Bacteria (E. coli)

HBcAg

Influenza A M2e Influenza


[20]

HCV

Insect cells (sf9)

HCV

HCV core, E1, E2 HCV and p7 proteins

Prophylactic Preclinical

Yeast (S. cereivsiae) HPV

HPV6/11/16/18 L1

HPV, cervical cancer Prophylactic Licensed (Gardasil®)

[48]

HPV

Insect cells (High five™)

HPV16/18 L1

HPV, cervical cancer Prophylactic Licensed (Cervarix®)

[48]

Mammalian (239TT) HPV-16 L1

Amyloid β

Alzheimer’s disease

[78]

Infectious Yeast (S. cereivsiae) VP2 Bursal disease virus

HPV-16 E7

HPV, cervical cancer Therapeutic

Insect cells (sf9)

Influenza virus

A/ Influenza California/04/09 (H1N1) HA, NA

Prophylactic Phase II

[49]

Influenza virus

Plant (transient Nicotiana benthamina)

Influenza virus

A/ Influenza Indonesia/05/05 (H5N1) HA

Prophylactic Phase II

[84]

Cell-free

Influenza virosome

Her-2/neu

Breast cancer

Therapeutic

Phase I

[12]

JC virus

Bacteria (E. coli)

JCV VP1

DNA encapsulation

–

–

Preclinical

[30]

Murine Insect cells (sf9) polyoma virus

MPyV VP1/ VP2/VP3

PSA

Prostate cancer

Prophylactic Preclinical

[14]

MS2

MS2 CP

miRNA-146a

Systemic lupus erthematosus

Therapeutic

[70]

Norwalk virus Insect cells (sf9)

NV

NV capsid protein

NV


[40]

Melan-A/MART- Melanoma 1

Therapeutic

Phase II/III

[55]

Angiotensin II

Hypertension

Therapeutic

Phase II

[76]

Qβ

Bacteria (E. coli)

Qβ

IL-1β

Autoimmune arthritis

Therapeutic

Phase II

[66]

TypeA CpG

Allergic rhinoconjunctivitis

Therapeutic

Phase II

[73]

Bacteria (E. coli)

HPV

Ref.

Preclinical

Prophylactic Preclinical Preclinical

Preclinical

[8]

[2]

[62]

AlMV: Alfalfa mosaic virus; CMV: Cucumber mosaic virus; CP: Capsid protein; GP: Glycoprotein; HBcAg: Hhepatitis B core antigen; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HPV: Human papilloma virus; IBDV: Infectious bursal disease virus; JCV: John Cunningham virus; MPyV: Murine polyomavirus; NP: Nucleocapsid protein; NV: Norwalk virus; P.f. CSP: Plasmodium falciparum circumsporozoite protein; PSA: Prostate specific antigen; RHDV: Rabbit hemorrhagic disease virus SLE: Systemic lupus erthymatosus.

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Review

Table 1. Summary of virus-like partice vaccines included in this review (cont.). Source virus

Expression system

VLP platform

Vaccine antigen

Condition

Therapeutic Clinical or development prophylactic

Amyloid β

Alzheimer’s disease

Therapeutic

Nicotine

Nicotine addiction

Therapeutic

Phase II

[81]

RHDV

Insect cells (sf21)

RHDV VP60

Melan-A/ MART-1

Melanoma

Therapeutic

Preclinical

[28]

Phase II

Ref.

[79]

AlMV: Alfalfa mosaic virus; CMV: Cucumber mosaic virus; CP: Capsid protein; GP: Glycoprotein; HBcAg: Hhepatitis B core antigen; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HPV: Human papilloma virus; IBDV: Infectious bursal disease virus; JCV: John Cunningham virus; MPyV: Murine polyomavirus; NP: Nucleocapsid protein; NV: Norwalk virus; P.f. CSP: Plasmodium falciparum circumsporozoite protein; PSA: Prostate specific antigen; RHDV: Rabbit hemorrhagic disease virus SLE: Systemic lupus erthymatosus.

proinflammatory cytokines, such as TNFα, IL-1β and IL-6 [70] . Allergies

As with autoimmunity, allergies occur when the body mounts an inappropriate immune response against a harmless antigen. Allergies involve a TH2-biased immune response, with increased production of TH2associated cytokines, such as IL-4, 5 and 13, and allergen-specific antibodies. One such condition is allergic rhinoconjunctivitis, caused by airborne allergens such as pollen and dust mites. Current treatments include administration of antihistamines and corticosteroids, which relieve symptoms but have no long-term therapeutic effects. Allergen-specific immunotherapy through administration of the causative allergen has been shown to be effective; however, side effects can include systemic allergic reactions [71] . CYT003-QβG10 is currently being tested as an immunotherapy for allergic rhinoconjunctivitis and allergy-induced asthma [72] . The therapy is composed of type A CpG encapsulated within Qβ VLP, and is allergen-independent. Type A CpG is a TLR9 agonist that can induce production of type I interferons, inducing a more balanced immune response that alleviates the TH2 bias present in allergic conditions [72] . Phase II trials have shown that this therapy can alleviate symptoms in both rhinoconjunctivitis and asthma [73] . Other conditions

Therapeutic VLP vaccines have also been considered as candidate treatments for a variety of other conditions, such as hypertension, Alzheimer’s disease and nicotine addiction. Hypertension is responsible for more than 45% of deaths caused by heart disease and 51% of deaths caused by stroke worldwide [74] . Although angiotensinconverting enzyme inhibitors or angiotensin II receptor blockers are effective therapies for hypertension [75] , they are dependent on patient adherence to the treatment regimen. An angiotensin II Qβ VLP vaccine was found to induce significant production of antiangiotensin II antibodies, relieving hypertension by reducing


blood pressure [76] . Although this vaccine is less effective than current hypertension drugs, the results show that this therapy has promise and it does not rely on patient compliance. Alzheimer’s disease is another condition that lacks an effective therapeutic vaccine. Recently, groups have tested a variety of potential therapies targeting amyloid-β, a protein that is deposited in the brains of affected patients and is thought to be directly responsible for disease pathogenesis. Although preclinical trials of a vaccine derived from 40–42 amino acids of amyloid-β demonstrated promising results, in subsequent clinical trials approximately 5% of patients developed autoimmune meningoencephalities [77] . HPV-16 and Qβ VLP containing B-cell epitopes from amyloid-β have been assessed as potential therapies for Alzheimer’s disease [78,79] . These VLPs induce the production of anti-amyloid-β antibodies, resulting in a reduction in protein aggregation. The Qβ VLP vaccine is currently in Phase II clinical trials. Smoking contributes to millions of deaths annually, associated with pulmonary disease, coronary heart disease and cancer. Nicotine is a highly addictive substance that drives the smoking habit, establishing a neurochemical dependency. Smoking cessation is the preferential treatment for preventing the development of potentially fatal smoking-related conditions later in life; however, it is common for smokers to relapse and continue smoking. A nicotine-containing Qβ VLP has been developed by Cytos Biotechnology, which stimulates the production of antinicotine antibodies [80] . This results in the neutralization of nicotine, preventing re-establishment of dependency when resuming smoking. In Phase II clinical trials, this vaccine demonstrated a significantly improved rate of smoking abstinence compared with a placebo [81] . Conclusion Immunotherapeutic vaccination provides the potential for manipulating the immune system to combat preexisting conditions. Standard subunit vaccination is mediated by antigenic molecules in the form of immu-


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Review Al-Barwani, Donaldson, Pelham, Young & Ward nogenic peptides or proteins. Delivery of these antigens is enhanced by incorporation into a particulate vaccine vector, which promotes uptake and processing by APCs. Amongst the breadth of nanoscale vaccine scaffolds available, VLPs have the advantage of retaining viral features that the immune system can recognize. These include the repetitive array of viral capsid proteins and native antigen conformation. VLPs are a versatile vaccine vector, which supports recombinant insertion and/ or surface conjugation of immune epitopes in the form of MHC-restricted peptides or unprocessed antigens. Currently there is a wide range of VLP-based therapeutic vaccines in clinical trials for treatment of conditions including cancer, autoimmune disease, Alzheimer’s disease and hypertension; and an even more extensive range of potential VLP vaccines are in preclinical studies. The examples highlighted in the review are summarized in Table 1. VLPs are a promising prospective vector for vaccination, facilitating the development of novel therapeutics.

Future perspective VLPs are a versatile vaccination vector with a wide range of potential applications. The burgeoning field of immunotherapeutic vaccination has only begun to explore the potential for immunological correction of disease. VLPs could theoretically deliver many forms of commercially available subunit vaccine simply through chemical conjugation onto the surface of preformed particles. Particle delivery is an effective means of increasing vaccine immunogenicity and nature has provided ample prospective particle vectors in the form of VLPs. VLP vaccines may also be compatible with other forms of immunotherapy, such as administration of immunomodulatory molecules or adoptive cell therapy. This review has covered a few of the many examples of therapeutic VLP vaccines in preclinical and clinical trials, with prospective results stimulating advancement and commercial availability. One of the most promising candidate applications is the use of VLPs in cancer immunotherapy. Although VLP

Executive summary Virus-like particle structure • Virus-like particles (VLPs) consist of a repetitive array of viral structural proteins. • They inherit their structural symmetry from their source virus. • VLPs vary in size and structural complexity, ranging from single protein to multiprotein and enveloped in a phospholipid membrane.

VLP vaccine design • VLP is a versatile vaccination vector that can vaccinate against the source virus or be utilized as a scaffold for other immune epitopes. • Immune epitopes can be recombinantly inserted into the viral structural proteins that form VLPs or chemically conjugated to the surface of preformed particles. • Some VLPs can encapsulate nucleic acids, and may have applications for gene delivery.

VLP immunogenicity & response • VLPs can stimulate both humoral and cell-mediated immunity. • When a particular type of immune response is desired, the addition of vaccine adjuvants or delivery mechanisms can skew the immunogenicity of VLPs.

VLP vaccines for cancer immunotherapy • Development of effective cancer immunotherapy is essential, as current treatments for cancer are nonspecific with potentially debilitating side effects. • Chimeric VLPs can induce immune responses against tolerized epitopes. • Delivery of tumor-associated antigens with VLPs can induce tumor-specific immune responses. • In the absence of known tumor-associated antigens, mixtures of undefined epitopes such as tumor lysate can be conjugated to VLPs, with theoretical applications for development of vaccines against many forms of cancer.

Other therapeutic VLP vaccines • VLPs are in development as potential therapeutic vaccines for various conditions. • Clinical trials have demonstrated that VLPs targeting inflammatory cytokines or their activation pathways can be utilized for therapeutic vaccination of autoimmune conditions. • Allergen-independent immunotherapy against allergic asthma and rhinoconjunctivitis is possible with VLP encapsulating type A CpG. • VLPs containing B-cell epitopes from amyloid-β have been shown to reduce protein aggregation in the brain of patients with Alzheimer’s disease. • Immunotherapy with VLPs containing nicotine promotes smoking abstinence.

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vaccines have only been developed for a small repertoire of cancer types so far, the identification of specific TAAs or the investigation of undefined epitope mixtures such as tumor lysate may enable VLP vaccines to be developed for a wide range of cancers. Recent advancements in immunotherapy herald a new age in the treatment of cancer, and VLP vaccines are at the forefront of progress.

Financial & competing interest disclosures

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Viruses and viruslike particles of eukaryotic algae.

Viruslike particles in peripheral nerve of the house cricket.

Taming the TCR: antigen-specific immunotherapeutic agents for autoimmune diseases.

Antigen-specific immunotherapeutic vaccine for experimental autoimmune myasthenia gravis.

Anti-cancer vaccination by transdermal delivery of antigen peptide-loaded nanogels via iontophoresis.

Visible and subvisible particles in the BCG immunotherapeutic product Immucyst®.

Assembly of viruslike particles by recombinant structural proteins of adeno-associated virus type 2 in insect cells.

Chemically Modified Human Serum Albumin Potently Blocks Entry of Ebola Pseudoviruses and Viruslike Particles.

Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins.

Beam delivery systems for charged particles.

Engineering nanolayered particles for modular drug delivery.

Nanostructured liquid-crystalline particles for drug delivery.

The hepatitis A virus polyprotein expressed by a recombinant vaccinia virus undergoes proteolytic processing and assembly into viruslike particles.

Development of viruslike particles in the crystal-containing nuclei of the midgut cells of tenebrio molitor.

Methods for Generating Hydrogel Particles for Protein Delivery.

Immunotherapeutic advancements for glioblastoma.

Immunotherapeutic approaches for Alzheimer's disease.

Particles from preformed polymers as carriers for drug delivery.

Novel antigen delivery systems.

Immunotherapeutic approaches for hepatocellular carcinoma.

Layer-by-layer assembled antisense DNA microsponge particles for efficient delivery of cancer therapeutics.

Alginate Particles as Platform for Drug Delivery by the Oral Route: State-of-the-Art.

Induction of high-titer neutralizing antibodies, using hybrid human immunodeficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant.

Prostate-specific antigen pox virus vaccination for recurrent prostate cancer.