ISSN 1995-0780, Nanotechnologies in Russia, 2016, Vol. 11, Nos. 3–4, pp. 227–236. © Pleiades Publishing, Ltd., 2016. Original Russian Text © T.V. Gasanova, N.V. Petukhova, P.A. Ivanov, 2016, published in Rossiiskie Nanotekhnologii, 2016, Vol. 11, Nos. 3–4.
Chimeric Particles of Tobacco Mosaic Virus as a Platform for the Development of Next-Generation Nanovaccines T. V. Gasanova, N. V. Petukhova, and P. A. Ivanov Department of Virology, Faculty of Biology, Lomonosov Moscow State University, Moscow, 119991 Russia e-mail:
[email protected] Received October 14, 2015; accepted for publication December 14, 2015
Abstract—The production of vaccines and other proteins in plants for medical purposes offers a number of advantages over other expression systems. The tobacco mosaic virus (TMV) is an appropriate model for the development of a variety of vectors, including those used for the assembly of chimeric particles carrying heterologous peptides on the surface and inducing an effective immune response. To overcome the problems arising during the assembly of such particles from recombinant subunits of the coat protein, peptide linkers, suppressed stop codons, and proteolytic sites are used. To date, it has been shown that TMV-based nanovaccines provide protection against the viruses of influenza A, foot-and-mouth disease, papilloma, and they are also able to overcome B-cell tolerance for cancer-cell suppression. Genetically modified TMV-based virions can accommodate the ions of various metals and act as affinity agents for protein purification. DOI: 10.1134/S1995078016020051
INTRODUCTION Currently, the technologies of protein and peptide expression for medical purposes in various systems, including heterologous systems, employing recombinant DNA are widely used. The major direction of research in this area is the development of high-quality, safe, and effective vaccines [42]. For example, a classical subunit vaccine should contain immunodominant protein components of the pathogen without the genetic material, thus losing the ability to replicate and overspread in the environment. Viral capsid proteins are good candidates for the development of such a vaccine, while in some cases they may form virus-like particles (VLPs) without nucleic acids. Usually VLPs are highly immunogenic and induce both humoral and cellular immune responses [11]. Biological systems, which are used to produce target proteins, should possess the ability to scale up the technology, as well as meet the requirements of biosafety, profitability, and production flexibility. Modern expression systems, so-called “biofactories,” are generally based on the use of animal, insect, yeast, and bacteria cell cultures [42], while most of the recombinant proteins are produced in animal cells or Escherichia coli [84]. The ability of using plants as “biofactories” has been intensively or highly investigated in the past twenty years. As is well known, plants have no pathogens common with human and animals. Under certain conditions, we can expect a high yield level and a low production cost, as well as the possibility of a so-called “edible vaccine” [62, 48, 12]. Moreover, an important
advantage of plants compared with the cells of bacteria and yeast is the posttranslational modification of proteins, which is often similar to the modification in animal cells [23]. The possibility of simultaneous expression of several transgenes in plants should be also noted [75]. Stable nuclear transformation (the integration of the target gene into nuclear DNA, followed by obtaining of transgenic plant) is successfully used for the production of antibodies, hormones, peptide inhibitors, and other proteins [17, 79]. In particular, this method enables us to accumulate the targeted proteins not only in tissues, but in the dry seeds of cereals, thus avoiding their degradation during long storage [29]. Cereals grow practically everywhere, but the long life cycle of some of them and potential risk of mixing with natural species or food crops limit the use of this method [58]. Plastid transformation (the insertion of the foreign sequence into chloroplast DNA) has its own advantages, including multiplicity of the foreign gene copies (up to 10000 per cell) and the relative simplicity of the target protein purification. Pollen does not contain chloroplasts; therefore, the risk of uncontrolled transgene transfer is absent under cross fertilization [55]. The transformation of chloroplasts is achieved by the bombardment of plant tissue with gold or tungsten microparticles with DNA immobilized on their surface or by the transfection of isolated protoplasts followed by the regeneration of the plants; the yield may be up to 70% of the total soluble protein [60, 6]. The risk of proteolytic activity, which leads to the loss of the product output, is high when using suspension cell
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cultures of plants similar to the expression in animal cells [13]; unfortunately, only a small number of cell lines described in details is known [10]. The alternative of stable transformation is the socalled transient expression, a fast and convenient way to obtain target proteins in plants. The key variation of such an expression is agroinfiltration—a technology of foreign gene delivery into a plant using culture of soil bacteria Agrobacterium tumefaciens [26, 33]. The method includes the injection into the leaves and other tissues of the plant using Agrobacterium suspension, which is able to transfer an extended (up to 150000 nucleotides) region of single-stranded T-DNA (T—transfer, a fragment of bacterial Ti plasmid) with incorporated target gene into the nucleus of a plant cell. Transcription factors of the host plant play a significant role in the integration process [9]. The high expression level of the target gene can be explained by the fact that the factor of RNA interference [14], especially systemic, is less pronounced than in transgenic plants. The synthesis of the target protein proceeds for 1 or 2 weeks and then, as a rule, extensive chlorosis occurs and necrosis begins in the inoculation area. Agroinfiltration is widely used for the fast and highlevel expression of biological drugs and pharmaceuticals [58]. FOREIGN GENE EXPRESSION USING PHYTOVIRAL VECTORS Genetic engineering and applied virology are linked historically and inextricably. For example, the first recombinant proteins were obtained using the virus SV40, containing the genes of bacteriophage λ [30]. Unique properties of viruses, such as simplicity of manipulation, effective amplification of the genome, the ability for site-specific recombination, high-level translation, and the compact and morphologically ordered structure of the particles enable one to use viruses not only in basic but also in applied research, including the development of powerful expression systems. Viruses that have a wide range of hosts (bacteriophages, animal retroviruses, invertebrate baculoviruses, plant viruses) became the basis for the development of vectors for heterologous protein expression [42]. A special role in the understanding of the fundamental processes of molecular biology was assigned to plant viruses; it is enough to mention the discovery of tobacco mosaic virus (TMV) in 1898 [8] and RNA interference or “gene silencing” [3, 14]. Genetic elements of plant viruses (promoters, terminators, translational enhancers, and other cis-regulatory sequences) are extensively used in so-called “green” biotechnology [42]. Phytoviruses have been used for foreign gene expression since the mid-1980s, and technological progress in this field has given a number of examples of effective vectors [26]. There are several basic ways for target sequence expression using the modification of
the viral genome. The first way involves the replacement of the viral gene, which is not essential for replication, by a heterologous gene [56, 70]. The second variant is target gene cloning under the control of one of the duplicated viral subgenomic promoters, i.e., the development of an additional open reading frame to obtain the product free of virus proteins [15, 28]. Thirdly, it is possible to develop modular viral vectors with the genome divided into two or more functional fragments of cDNA. For example, the first module may comprise a virus replicase, and the second module may carry the target gene and the 3'-untranslated region necessary for efficient replication. Thus, they can complement each other in vivo, for example, via site-specific recombination, which enables one to modify the expression system depending on the desired objective [25, 51]. Finally, the construction of fused genes, in which the target sequence is translationally linked to the viral coat protein, enables one to obtain chimeric viral particles with exposed foreign peptide on their surface [32, 65]. DNA encoding the genome of viral vectors can be transferred into the nucleus of the plant cell using the agroinfiltration method, followed by transcription in planta, which is significantly more efficient than transcription in vitro and the mechanical inoculation of the leaves [51, 81, 21, 63, 64]. TARGET PEPTIDES AND PROTEINS ON THE SURFACE OF CHIMERIC VIRAL PARTICLES It was found that antigenic peptides effectively interact with the immune system if they are presented in an ordered, multicopy, quasicrystalline state [65]. It is believed that virus-like structures can promote the proliferation of dendritic and other antigen-presenting cells, so the effectiveness of specific B- and T-cell immune response is greatly enhanced if the antigen is associated with a similar structure. The regular arrangement of epitopes on the surface of the chimeric virus particle also stimulates the proliferation of B-cells and, thus, the production of antibodies. The multiplicity of antigen copies on the surface of the virion or virus-like particle helps to overcome the immunotolerance and accumulation of autoreactive therapeutic antibodies, including anticancer ones [18, 73]. Hence, chimeric viral particles are currently used in plant biotechnology, primarily for the production of immunologically important peptides and proteins. During the process of cloning heterologous peptide sequences into the coat protein gene, as a rule, regions exposed under natural conditions on the outer surface of the virion should be used. Obviously, it can be implemented only for viruses with the structure of the particles described in details. These viruses include TMV, cowpea mosaic virus, potato virus X, tomato bushy stunt virus, plum pox virus (sharka), and others. The main requirement for the choice of the region for
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Fig. 1. Schematic representation of the TMV genome cloned into a binary plasmid and viral chimeric particle assembly carrying heterologous peptides on the surface. The regions of viral cDNA are shown in color and additional sequences are white rectangles. Legend: prom and term, a promoter and transcription terminator; MP and CP, genes of the movement protein and the coat protein; 3’NTR, 3’ untranslated region; and amber, suppressed stop codon, which separates the functional domains of helicase and RNA-dependent RNA polymerase. The left and right borders of transferred T-DNA are marked with triangles; there are two possible variants of their mutual orientation.
foreign sequence insertion into the genome of plant virus is the following: the modified coat protein should not interfere with the development of the infection and normal virion assembly. Moreover, it is necessary to consider the length and physical and chemical characteristics of the target peptide for its correct allocation on the surface of the chimeric virus particle [4, 40, 41, 66]. There are several strategies to overcome problems with the assembly of chimeric virions, which bear long and/or “difficult-to-express” peptides. Firstly, this is the insertion of a linker (additional sequence), for example, flexible (glycine-rich, GGGGSx3) or spiral (EAAAKx3) linker between the coat protein and fused peptide, wherein the target antigen is sterically remote from the outer surface of the virion [21, 81]. Secondly, the cloning of foreign sequence at the existing proteolytic site region or the introduction of an additional cleavage site for fused polypeptide between the insert and the coat protein can be used [16]. Thirdly, a suppressed stop codon can be included between the coat protein and foreign peptide, which enables us to obtain viral particles containing both native (90–95% of the total protein in the preparation) and mutant (5–10%) structural proteins [42]. Finally, it is possible to use a 2A peptide of the foot-andmouth disease virus or a similarly functioning sequences between the coat protein and the heterologous peptide, which provides a “ribosomal skip” of translation. As a result, the fused product is synthesized along with the wild type of coat protein [50, 59, 69]. Currently, the genomes of TMV, the cowpea mosaic virus, and potato virus X are the most widely used for the production of chimeric particles. NANOTECHNOLOGIES IN RUSSIA
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A plant virus for the development of an efficient vector and the subsequent production of chimeric virus particles should possess the following properties: fast and productive replication in the infected plant, simplicity of product purification, well-studied and stable genome. From this point of view, the TMV is one of the most suitable candidates for the expression of antigenic epitopes. The TMV particles themselves are excellent antigens; the virus has been studied as a model object in immunology for over 50 years [80]. It was proved that the occurrence of the viruses in the form of native particles is an important factor affecting their immunogenicity. The introduction of the preparation of the “free” coat protein of TMV or the particles reconstructed from it decreased the antibody titer and provided weaker “immune memory” in animals compared with equivalent doses of the native virus [46, 49]. TOBACCO MOSAIC VIRUS: ONE OF THE MOST IMPORTANT OBJECTS IN PLANT BIOTECHNOLOGY TMV forms a rigid rod-shaped structure with a length of about 300 nm and diameter of 18 nm consisting of approximately 2130 coat protein subunits (17.5 kDa) arranged in a swirling spiral at a pitch of 16.3 subunits per turn (Fig. 1). The coat protein of TMV is described in detail by X-ray analysis and capable of self-assembly, which can be only partial in the absence of genomic RNA. Furthermore, it is sufficiently resistant to pH and temperature changes. The protein consists of a bundle of four antiparallel α helices and contains an RNA-binding domain. Based on crystallographic data, it was concluded that each sub2016
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unit contains four sites suitable for cloning of target epitope: N-terminal and C-terminal regions, as well as two loops of polypeptide sequence exposed on the surface of virion (59–65 and 152–156 amino acid residues, a.a.) [5]. The plants infected with TMV produce significant amounts of the coat protein, the main part of which is included in the virions. The amount of the TMV coat protein may reach a level of 10–40% of the soluble protein in Nicotiana tabacum leaves 30 days after inoculation with TMV. The plants can also “generate” the coat protein with the fused peptide in large quantities when infected with recombinant virus. The density of epitopes on the surface of rod-shaped virion can provide a significant advantage over icosahedral viral particles. For example, in theory, more than 2000 copies of the epitope can be exposed on the surface of the TMV virion against 180 copies on a virus-like particle with symmetry T = 3 (HBcAg, core part of hepatitis B virus), or 420 copies on a particle with symmetry T = 7 (papillomavirus, [73]). Thus, the use of chimeric particles based on TMV is one of the most promising methods for the development of next-generation nanovaccines, examples of which will be discussed in detail below. METHODS OF GENETIC MODIFICATION OF TMV PARTICLES An effective way for modifying the genome of TMV to obtain chimeric particles is cloning of heterologous sequence into one of the loops located between Gly-154 and Ser-155 residues of the coat protein sequence. This method was used to obtain TMV virions carrying the following antigens: a peptide from mouse ZP3 (zona pellucida) protein (13 a.a., [18]), epitopes G5-24 (20 a.a.) of rabies virus and 5B19 (10 and 15 a.a.) of murine hepatitis B virus [4, 35], fragments of rabbit papillomavirus L2 protein (13 and 18 a.a.) [61], peptides of VP1 protein of foot-andmouth disease virus (11 and 14 a.a.) [31, 83], conserved epitope M2e of influenza virus A (23 a.a. [63, 64]), and a region of protein F of Pseudomonas aeruginosa outer membrane (14 a.a., [24, 74]). After the immunization of animals with chimeric particles, a specific immune response providing a protective effect in immunized animals infected with sublethal and lethal doses of corresponding pathogens was observed in all cases. The peptide expressed on the surface of a viral particle, if necessary, can be released from the virion that enables its accumulation in significant quantities. For example, neuropeptide nocistatin was obtain as a part of chimeric TMV virion, whose sequence was cloned into the loop Ser-154–Gly-155 with the introduction of specific proteolytic site sequence (IEGR) after serine residue. Thus, the peptide can be eliminated from the carrier under the treatment of chimeric virions extracted from plants with proteases, for example, Xa and trypsin [7, 43]. A num-
ber of studies have demonstrated that, an additional stop codon can be cloned in front of heterologous sequence, providing the translational deletion of C-terminal amino acids of the coat protein (Thr-153–Thr-158); at the same time, chimeric particle assembly was not impaired. Conversely, the size of the target insert can be increased in this way (up to 28 a.a.) [31]. It was also noted that the presence of cysteine residues in the cloned heterologous sequence prevents the correct and efficient assembly of chimeric TMV virions [40, 63]. TMV particles with 2 different variants of conservative epitope M2e of influenza A virus have been characterized in detail by various methods [63, 64]. It was shown that the content of recombinant coat protein in purified preparations reaches 90%. The ratio of antibodies specific to the epitope and the support in the serum of immunized mice may be estimated as 5 : 1, which is considerably higher than the values known from previous papers [18, 35]. Single amino acid substitutions in the consensus human sequence of M2e (alanine residues instead of serine residues at 17 and 19 positions) significantly affected the profiles of immunoglobulins IgG1/IgG2a (3.2/1 instead of 0.7/1). The nanovaccine based on TMVM2e-ala particles provided protection against five lethal doses of both homologous (A/PR/8/34) and heterologous (A/California/04/09) strains of influenza A virus, so it can be called “universal” [63]. Significant amounts of TMV particles carrying Leu-enkephalin pentapeptide directly on the C-terminus of the coat protein were obtained in protoplasts [77]. The N-terminus the coat protein of TMV was successfully used for the expression of the epitope of feline panleukopenia virus [67] (Fig. 2, table). If it is necessary to use suppressed stop codon (usually it is amber codon, UAG), it is placed between the C-terminus of the coat protein and the heterologous sequence. In addition to the wild-type protein, a protein fused with foreign peptide can be synthesized with some probability (5–10%) under the “skipping” of this codon. Accordingly, in this case, chimeric particles will consist of two proteins in a ratio of about 20 : 1. In this way TMV chimeric virions carrying the Trypsin-modulating oostatic factor (TMOF) of mosquitoes hormone (10 a.a., [7]) on their surface were obtained. In this context antigenic determinants of influenza virus (8 a.a.) and human immunodeficiency virus I (13 a.a.) [76], the poliovirus peptide of Sabin type (15 a.a. [22]), B-cell epitopes of malaria [78], and the angiotensin converting enzyme inhibitory peptide [27] should also be mentioned. In all the cases there was a high-enough level of recombinant protein accumulation in plants infected with a viral vector; for example, the yield of fused product was 1.3% of the total soluble protein under TMOF expression. It should be noted that when larvae of Heliothis virescens were fed with tobacco leaves infected with TMV-TMOF
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M2e, VP1, ZP3, L2, etc. Site III Fig. 2. Regions of TMV coat protein (CP), which are used for the exposition of antigens and proteins. Peptide linkers are indicated with broken lines. Sites II and III are loops of the CP located on the surface of TMV particles. Lys, reactive lysine; 6xHis, six histidine residues; M2e, VP1, ZP3, L2, A, TBP, target amino acid sequences, cloned into the CP gene (see also in the text and in the table).
virus, the termination of their further development due to the inhibition of trypsin and chymotrypsin in midgut was observed. Since TMV has a quite wide range of host plants, TMV-TMOF infection can be used as a method for crop protection against pests [7]. TMV chimeric virions can also serve as affinity agents for purification and separation systems of various proteins. For example, recombinant TMV carrying streptavidin-specific peptide (TLIAHPQ) on the surface was used for the purification of streptavidin from Streptomyces avidinii [57]. The particles containing a fragment of protein A from Staphylococcus aureus were successfully used for the isolation of antibodies from plant extracts [81]. CHEMICAL MODIFICATION OF TMV RECOMBINANT PARTICLES Some mutations of the TMV genome enable us to use viral particles for the attachment of peptides and proteins without the direct incorporation of the sequences into the coat protein gene. One of the variants is the insertion of additional reactive lysine (Lys+) at the N-terminus of the coat protein of TMV, which makes it possible, in particular, to biotinylate capsid. Further, such particles can bind to the target protein conjugated with streptavidin. The extended fragment (36 a.a.) of structural protein L2 of canine papillomavirus located on the surface of such particles was significantly more immunogenic in animal experiments when compared with “free” antigen [72]. The direct attachment of antigenic proteins to TMV-Lysine particles using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) NANOTECHNOLOGIES IN RUSSIA
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enables one to develop multivalent vaccines using two or more antigens. For example, the simultaneous immunization of mice with a mixture of TMV particles carrying OmpA, DnaK, and Tul4 proteins (electrophoretic mobility of 70, 47 and 17 kDa) of Francisella tularensis significantly improved the effectiveness of the protective response against tularemia [2]. Also, along with the main antigen, supporting epitopes, which are known to promote the efficient capture of the particles by the cells of immune system, may be attached to TMV particles using the N-terminus lysine, which minimizes the use of adjuvants under vaccination. Thus, a more efficient stimulation of cancer protection was observed when epitopes of cytotoxic T-lymphocyte associated with melanoma (p15e and Trp2) were coexpressed on the surface of TMV with the further immunization of animals compared with the use of the same peptides alone [53]. The determinant sites of tumor-specific immunoglobulins were expressed in tobacco plants using vectors based on TMV when designing a plant vaccine against nonHodgkin lymphoma (NHL). The vaccine has passed the first phase of clinical trials and showed a high immune response profile. The oral administration of a plant vaccine was first used in these studies [52, 54]. IMMOBILIZATION OF METALS ON THE SURFACE OF TMV RECOMBINANT PARTICLES It is known that some metals (gold, platinum, palladium, nickel, and cobalt) can be immobilized on the surface of various rod-shaped viral particles, including wild-type TMV [34, 39, 44]. The TMV2Cys vector, containing two cysteine residues on the surface of the 2016
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Proteins and peptides exposed on the surface of TMV particles Length of antigen and its location at the coat protein of TMV
Epitope peptide from mouse ZP3 (zona pellucida) protein
References
13 a.a., loop 154-156 a.a. protein 20 a.a. and 10, 15 a.a.,
Fitchen et al., 1995 Вendahmane et al., epitopes G5-24 of rabies virus and 5V19 of murine 1999; hepatitis B virus loop 154-156 a.a. Коо et al., 1999 fragments of rabbit papillomavirus L2 13 and 18 a.a., loop 154-156 a.a. Palmer et al., 2006 peptides of VP1 protein of foot-and-mouth disease virus 11 and 14 a.a., loop 154-156 a.a.. Wu et al., 2003 conserved epitope M2e of influenza virus A 23 a.a., loop 154-156 a.a. Petukhova et al., 2013, 2014 14 a.a., Gilleland et al., 2000; region of protein F of Pseudomonas aeruginosa outer membrane loop 154-156 a.a. Staczek et al., 2000 Leu-enkephalin pentapeptide 5 a.a., C-terminus Takamatsu et al., 1990 of the coat protein epitopes of feline panleukopenia virus 16 and 17 36 a.a., N-terminus Pogue et al., 2004 of the coat protein protein L2 of canine papillomavirus 36 a.a., N-terminus Smith et al., 2006 of the coat protein Trypsin-modulating oostatic factor (TMOF) 10 a.a., C-terminus Borovsky et al., 2006 of the coat protein poliovirus peptide of Sabin type 15 a.a., C-terminus Fujiama et al., 2006 of the coat protein antigenic determinants of influenza virus and human 8 and 13 a.a., C-terminus Sugiyama et al., 1995 immunodeficiency virus of the coat protein Tumor-associated carbohydrate antigens (TACAs) 19 a.a. C-terminus Yin et al., 2012 of the coat protein functional fragment of protein A from Staphylococcus aureus 133 a.a., C-terminus Werner et al., 2006 of the coat protein core neutralizing epitope (COE) of porcine 150 a.a., N-terminus Kang et al., 2004 epidemic diarrhea virus (PEDV) of the coat protein nocistatin peptide of odontoglossum ringspot virus (ORSV) 17 a.a., C-terminus Lim et al., 2002 of the coat protein F11, F14 epitopes of foot-and-mouth disease virus (FMDV) 11 and 14 a.a., C-terminus Jiang et al., 2006 of the coat protein melanoma epitope p15e 15 a.a., N-terminus McCormick et al., 2006 of the coat protein trastuzumab-binding peptides (TBP) 14 and 20 a.a., C-terminus Frolova et al., 2010 of the coat protein antigens of tularemia DnaK/OmpA/Tul4 Proteins of 70, 47, 17 kDa N-ter- Banik et al., 2015 minus of the coat protein
coat protein [38], was used in the experiments on binding with palladium under the presence or absence of an external chemical reducing agent [20]. It was shown using transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) that the rod-like particles may contain several layers of the metal; having a standard length of 300 nm, their diameter (30–40 nm) is much greater than the diameter of wild particles.
Another approach to VLP metallization was shown by Wnek et al. (2013) [82]. The viral vector based on TMV, carrying selective histidine hexapeptide (6xHis) at the C-terminus of the coat protein, which binds nickel ions, was obtained. Wild-type coat protein, unlike mutant protein, is able to form discs in vivo in the absence of genomic RNA. However, the presence of this peptide enables one to purify the modified coat protein by affinity chromatography and lowering of
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pH values to ≤7 in both cases assumes the initial stages of virion assembly. Further, VLPs containing 6xHis tags were coupled with gold particles and subjected to high temperature annealing on the substrate, followed by rapid cooling. Extended fine threadlike structures formed by gold crystals of 5 nm were obtained. Thus, one can speak about the emergence of a new technology for the development of nanowires for potential use in the electronic industry, minimizing the size of electronic devices and increasing the speed of information transfer. PRACTICAL ASPECTS: HOST PLANT SELECTION, PURIFICATION, AND STORAGE OF CHIMERIC PARTICLE PREPARATIONS Under greenhouse conditions and on practice grounds, Nicotiana benthamiana (Australian tobacco), which is sensitive to infection by many plant viruses, including TMV, is usually employed. This species is a suboptimal host, because in natural conditions, cold winter and droughty summer months are not used for growing and harvesting. It has been shown [73] that certain cultivated varieties of tobacco (Nicotiana tabacum) are highly sensitive to infection by wild-type TMV; however, the level of virus particle accumulation was significantly decreased after infection with a recombinant virus containing the foreign epitope on the surface of the coat protein. To solve the problems connected with the optimization of biomass production, the program of host plant improvement by the hybridization of different varieties of Nicotiana has been developed. In the framework of this program, the interspecies N. excelsiana hybrid was obtained by the hybridization of N. benthamiana and N. excelsior and patented [19, 71]. An important question is the strategy of vaccine purification, its subsequent stability, and the safety of its use in human and veterinary medicine. The introduction of the vaccine into practice is inextricably associated with the use of adjuvants and the methods of particle sterilization. It is necessary to select optimal conditions and storage forms for each vaccine candidate that must maintain its properties over a long period of time. Wild-type TMV virions may be stored at a concentration of 20 mg/mL for several years at 4°C without visible degradation and the formation of a precipitate. However, in the case of chimeric particles carrying foreign protein, the concentration under storage at 4°C may change significantly due to the aggregation of the particles in the solution. Taking into account that such aggregation may significantly affect the immunogenicity of the vaccine, this fact should be considered and, if possible, controlled. NANOTECHNOLOGIES IN RUSSIA
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It is necessary to carefully select the method of sterilization for the preparation, isolated from plants, under formulating the vaccine candidate based on TMV. The passage through the bacterial filters by the preparation may be accompanied with incomplete purification from certain pathogens, such as mycoplasma. In the case of UV irradiation, the mild spontaneous release of the coat protein from TMV particles sometimes occurs [36]. Gamma radiation can serve as an acceptable alternative [1]. The use of several inactivating agents (formaldehyde and beta-propiolactone) is limited due to their carcinogenicity and damages, which they can cause in the exposed antigen. Aziridine (BEI) functions via the alkylation of nucleic acids. TMV chimeric particles with ten different epitopes were tested after sterilization using BEI. In all cases the effective suppression of total microflora with the retention of immunogenicity for all preparations was observed [61]. Currently, the BEI sterilization procedure is widely used in the production of veterinary vaccines, but it does not cover human vaccines. The next stage is to test the stability of the vaccine preparation under long-term storage. It was shown that 98% of TMV chimeric particles in the preparation containing the peptide of canine parvovirus protein VP2 (13 a.a.), which was stored for 14 months at 4°C, remained stable. Similar results were obtained for TMV particles with a fragment of papillomavirus protein L2 (6 months at –20°C) [73]. Sterilizing TMV preparations carrying M2e epitope of influenza A virus through nitrocellulose filters (0.45 μM) enables one to store them at 4°C for at least 4–6 months [63]. CONCLUSIONS The studies on vaccines based on plant virus particles suggest the broad potential of these preparations for the prevention and treatment of various diseases [37]. Commercial benefits for the production of such vaccines depends primarily on solving the problems of toxicity, biosafety, and further improvement of profitability [68]. A plant platform based on TMV chimeric particles possesses all the necessary qualities for successful practical application: the stability of the vaccine preparation, pronounced immunogenicity, high yield and low price of the product, and available production system (including in regions with scarce natural resources) [73]. TMV particles possess adjuvant properties; repeated immunizations are highly effective, while the preliminary presence of antibodies against TMV in the organism does not hinder the enhancement of immune response [47]. Recently it has been shown that the presence of antibodies to wild-type TMV in human blood reduces the risk of development of Parkinson’s disease and other neurodegenerative diseases [45]. Thus, nanovaccines based on TMV particles can be considered multifunctional: the immune response against the target antigen is sup2016
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