DNA vaccines for biodefence

Advanced Drug Delivery Reviews 57 (2005) 1343 – 1361 www.elsevier.com/locate/addr

DNA vaccines for biodefence Helen S. Garmorya,T, Stuart D. Perkinsa, Robert J. Phillpottsa, Richard W. Titballa,b a Department of Biomedical Sciences, Defence Science and Technology Laboratory, Porton Down, Salisbury, SP4 0JQ, UK Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK

b

Received 14 April 2004; accepted 25 January 2005 Available online 13 April 2005

Abstract The advantages associated with DNA vaccines include the speed with which they may be constructed and produced at largescale, the ability to produce a broad spectrum of immune responses, and the ability for delivery using non-invasive means. In addition, DNA vaccines may be manipulated to express multiple antigens and may be tailored for the induction of appropriate immune responses. These advantages make DNA vaccination a promising approach for the development of vaccines for biodefence. In this review, the potential of DNA vaccines for biodefence is discussed. Crown Copyright D 2005 Published by Elsevier B.V. All rights reserved. Keywords: Immunity; Targeting; Co-stimulatory molecules; Delivery; Carriers; Regimens; Viruses; Bacteria

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Approaches to modulating immunity . . . . . . . . . . . . . . . 2.1. Enhancement of immunity by manipulation of the vaccine 2.2. Targeting approaches . . . . . . . . . . . . . . . . . . . 2.3. Co-stimulatory molecules . . . . . . . . . . . . . . . . . Approaches to delivery . . . . . . . . . . . . . . . . . . . . . . 3.1. Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Immunisation regimen . . . . . . . . . . . . . . . . . . . DNA vaccines for biodefence. . . . . . . . . . . . . . . . . . . 4.1. Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . .

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T Corresponding author. Tel.: +44 1980 614755; fax: +44 1980 614307. E-mail address: [email protected] (H.S. Garmory). 0169-409X/$ - see front matter. Crown Copyright D 2005 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2005.01.013

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5. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction There is a requirement to develop biodefence vaccines against microorganisms that may be misused for bioterrorism (BT) or biowarfare (BW) purposes. These microorganisms include viruses, e.g., smallpox [1] and bacteria, e.g., anthrax [2], as well as toxins produced by bacteria, e.g., botulinum toxin [3]. There are no licensed vaccines for some of the diseases caused by these organisms and for others, improved next-generation vaccines are needed. In the past, some vaccines have been produced by inactivation of whole bacterial or viral cells. However, the high incidence of adverse effects associated with such vaccines makes them generally unsuitable for modern day use. Other licensed vaccines generally consist of recombinant proteins (e.g., hepatitis B, tetanus), live attenuated bacteria (e.g., typhoid, tuberculosis) or live attenuated viruses (e.g., measles, mumps, rubella). There are both advantages and disadvantages associated with each of these technologies, as outlined in Table 1. For example, recombinant proteins are often very effective against diseases requiring a predominantly Th2-type (humoral) immune response, but they are relatively ineffective at inducing cytotoxic T lymphocytes (CTLs). For many pathogens associated with BT or BW, the protective immune mechanisms are not fully defined and it is considered likely that both neutralising antibodies and cellular immune responses may

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contribute to protection against infection. In comparison, live attenuated bacteria and viruses, in general, are usually able to stimulate the appropriate type of immune response required to protect against the particular infection, but there is a possibility of reversion of the microorganisms to virulence. Vaccination with plasmid DNA has several advantages compared to these traditional formulations that are applicable to the development of vaccines for biodefence (see Table 1). DNA vaccines, also known as dgeneticT, dnucleic acidT or dpolynucleotideT vaccines, deliver genes encoding protein antigens into host cells, enabling antigen production to occur in vivo [4]. Consequently, both strong cellular and humoral immune responses may be induced. In addition, the ability to genetically manipulate DNA offers the advantage of vaccines designed to produce co-stimulatory molecules, or that are able to target protein production to specific cell compartments in order to modulate the specificity of the immune response. It is also possible to create multivalent DNA vaccines that may be able to stimulate immunity against a range of pathogens. The speed with which genetic manipulation may be carried out offers the possibility of rapid production of biodefence DNA vaccines. In comparison, the production of defined, live attenuated mutants for vaccine purposes is significantly more complicated and time-consuming. A further advantage of DNA over protein vaccines is

Table 1 General comparison of traditional vaccine technologies with DNA vaccination Recombinant protein

Live attenuated bacteria/virus

DNA vaccine

humoral

cellular and humoral

cellular and humoral

specific to protein

specific to encoded protein

requires refrigeration

possible cross-reactivity to other microorganisms possibility of reversion to virulence and side effects may require refrigeration

expensive to produce

expensive to produce virus

Predominant immune response Specificity of immune response Safety of vaccine

considered safe

Stability of vaccine Cost of production

considered safe potentially stable at room temperature relatively inexpensive

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the relative ease of storage, allowing retention of potency. Ease of storage and DNA delivery by needlefree technology represent distinct advantages for a biodefence vaccine, which may need to be mass administered, possibly in the field. In this review progress in developing DNA vaccines and prospects for their use in biodefence will be discussed.

2. Approaches to modulating immunity Immunisation with plasmid DNA induces both cellular and humoral immune responses. The mechanism of DNA vaccination has been reviewed by Sharma and Khuller [5]. Generally, in DNA vaccination, direct parenteral injection of the plasmid DNA results in the in vivo synthesis of the encoded protein with post-translational modification similar to those that occur during natural infection. DNA-transfected antigen presenting cells (APCs) synthesise protein endogenously, mimicking viral or intracellular bacterial infection, and present the heterologous antigen by

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class I MHC. This leads to the induction of CTLs (Fig. 1). Endogenous protein is taken up by APCs and presented by class II MHC, thereby inducing both CTLs and T helper (Th) cells. The exact mechanisms by which these immune responses are generated are not yet fully elucidated. It is considered possible that heterologous antigen produced in non-APCs such as myocytes (following intramuscular injection of DNA) may be shuttled to APCs for presentation, in the crosspriming method of immune induction. It is believed that the mechanisms by which gene gun-mediated DNA immunisation occurs is different to that occurring following parenteral injection of DNA. However, with this knowledge of the mechanism of parenteral DNA vaccination, the ability to genetically engineer the plasmid DNA allows the vaccine to be modified to modulate or enhance the immune responses generated. For example, the induction of apoptosis in host cells transfected with the DNA vaccines has been shown to enhance immune responses. Since APCs scavenge apoptotic bodies, DNA that is present in apoptotic cells may be transferred to APCs and

Th Exported protein may be taken up by APC. Subsequent processing via class II MHC

CTL Processing and presentation on the cell surface via class I MHC mRNA

Fig. 1. Description of basic mechanisms for induction of T cell responses. Generally, DNA-transfected cells may either (i) process encoded proteins and present peptides in association with class I MHC molecules, or (ii) release the protein for uptake by professional antigen presenting cells (APCs) using its endolysosomal pathway. These APCs degrade the protein into peptides and present the protein in association with class II MHC molecules. Specific T helper cells recognise antigen presented via class II MHC, subsequently inducing cytokines. Cytotoxic T lymphocytes are activated following recognition of antigen presented class I MHC.

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subsequently be expressed within the APCs [6,7]. Other reports have suggested that the prevention of apoptosis may enhance DNA vaccine efficacy [8]. The molecules used in each case affect different types of cells and it may be possible to devise strategies that take advantage of both pathways [9]. 2.1. Enhancement of immunity by manipulation of the vaccine vector The basic requirements for the backbone of a plasmid DNA vector are: (i) a eukaryotic promoter for expression in mammalian cells; (ii) a cloning site downstream of the promoter for insertion of heterologous genes; (iii) a polyadenylation sequence to provide stabilisation of mRNA transcripts; (iv) a selectable marker, most commonly bacterial antibiotic resistance genes; (v) a bacterial origin of replication, most commonly the Escherichia coli ColE1 origin of replication, since it provides large copy numbers in bacteria allowing high yields on purification. A schematic diagram of a basic plasmid for parenteral DNA vaccination is shown in Fig. 2. It has been proposed that the translational initiating sequence

( 6GCCA/GCCAUGG+4), known as the dKozakT sequence, should be included around the initiator codon [10] for optimal translational efficiency of the expressed genes. The inclusion of a Kozak consensus sequence may be an important consideration in the design of DNA vaccines. In recent years it has become clear that optimising the level of gene expression in DNA vaccines may improve their potency [11]. Several studies have evaluated the strength of promoter/enhancers or other transcriptional elements in DNA vaccines. The most commonly used promoter, from human cytomegalovirus (HCMV), is the major HCMV immediate early enhancer–promoter (known as the CMV promoter). The HCMV or other cytomegalovirus (CMV) promoters have often been shown to produce the highest level of transgene expression in various tissues, when compared with other promoters [12–21]. For example, in one study a plasmid expressing the influenza hemagglutinin protein under the regulation of a CMV promoter was compared to similar plasmids using the Rous sarcoma virus (RSV), simian virus 40 (SV40), and chicken actin promoters [18]. In poultry inoculated with the plasmids, the CMV promoter

Fig. 2. Schematic diagram of a basic DNA vaccine vector for parenteral DNA immunisation. A plasmid DNA vector will normally contain a eukaryotic promoter, intron and polyadenylation sequence to express a heterologous antigen, and a prokaryotic origin of replication and antibiotic selection marker to allow production of the plasmid in E. coli. The DNA vector may also be manipulated to contain eukaryotic signal or targeting sequences, CpG motifs or co-stimulatory molecules to modulate or enhance the immune response induced by heterologous antigen expression.

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induced the greatest antibody response and provided the greatest survival against influenza challenge. Alternatives to the CMV promoter include tissuespecific promoters such as the desmin promoter/ enhancer [22] and the creatine kinase promoter [23,24]; both specific to muscle cells, and the metallothionein and 1,24-vitaminD(3)(OH)(2) dehydroxylase promoters, both of which are specific to keratinocytes [25]. The inclusion of introns further increases the amount of antigen expression and the activity of naive T cells [26,27]. Since mammalian codon usage is generally different from that of microorganisms [28], the level of gene expression of prokaryotic genes in mammalian cells may be limited. Therefore, another strategy for improving gene expression involves optimising codon usage of the DNA-encoded gene for mammalian cells. A number of studies have reported that increased immune responses may be obtained by DNA vaccination with a heterologous gene sequence with optimised codon usage [29–34]. For example, Deml et al. [32] evaluated a DNA vaccine containing a synthetic human immunodeficiency virus (HIV)-1 gag gene using codons optimised to that of highly expressed mammalian genes. Mice intramuscularly immunised with the DNA construct showed increased Th1-type immune responses compared to a control plasmid containing non-codon optimised gag. Another means of enhancing the immune response to parenteral DNA vaccines is through the inclusion of certain sequences that have an adjuvant effect. Unlike mammalian DNA, bacterial DNA has potent immunostimulatory properties. These immunostimulatory properties have been attributed to unmethylated CpG dinucleotides flanked by two 5V purines and two 3V pyrimidines [35,36]. Such CpG dinucleotides are found at approximately 1 in 16 frequency in most bacterial DNA, whereas in vertebrates they are suppressed to approximately 1 in 64 frequency, are methylated, and are usually flanked by immuneneutralising base contexts [35]. Pattern recognition receptors are believed to distinguish bacterial DNA from self-DNA by specifically binding to unmethylated CpG dinucleotides in particular base contexts. CpG motifs present in bacterial plasmid DNA contribute to the immunogenicity of DNA vaccines [37]. In vitro studies showed that DNA plasmids induced production of the same cytokines as those stimulated

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by bacterial DNA. In vivo, the immunogenicity of an intramuscularly administered DNA vaccine was significantly reduced by methylation of the CpG motifs but was significantly increased by co-administering exogenous CpG-containing DNA [37]. DNA vaccines contain many CpG motifs that, overall, induce a Th1like pattern of cytokine production [37] and are thought to account for the strong CTL responses frequently seen following parenteral DNA vaccination [38]. The effect of various CpG motifs on the immune responses to DNA vaccines has been studied. For example the CpG motifs present in plasmid pUC19 significantly improved the antibody response stimulated by a DNA vaccine for dengue type 2 [39]. In another study it was not possible to augment the responses to DNA vaccines by co-administering CpG oligodeoxynucleotides (ODN); the CpG motifs had to be incorporated into the DNA backbone of the plasmid [40]. Although the inclusion of CpG sequences in the plasmid DNA has been shown to be important when the DNA vaccine is delivered parenterally to the extracellular space by injection, it has been shown to be unimportant for DNA accelerated directly into cells on gold particles [41]. In general, these studies suggest that the incorporation of unmethylated CpG into parenteral DNA vaccines is another strategy that may enhance immunogenicity. However, some CpG motifs may have immunosuppressive effects. Yamamoto et al. [42] found that CpG motifs can inhibit lymphocyte stimulation. Furthermore, it is known that the motifs that are optimally stimulatory in human and murine cells differ [43]. Thus it may be necessary to optimise the CpG content for specific DNA vaccines by increasing the number of stimulatory motifs and decreasing the number of suppressive motifs. 2.2. Targeting approaches Strategies used to modify the immune responses to DNA vaccines include the addition of heterologous genes encoding localisation/secretory signals or ligand fusions to direct the antigen to sites appropriate for immune induction. A number of studies have shown that higher antigen-specific IgG titres were induced when the expressed antigen was secreted rather than localised on the cell membrane or within the cell [44–50]. From these studies, there is also

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evidence that secreted antigens induce a higher IgG1/ IgG2a ratio in mice (suggesting a Th2 bias) than cellassociated antigens [48–50]. However, in a separate study, secreted and intracellular antigen induced comparable levels of antigen-specific IFN-g on in vitro stimulation [47]. Thus, the exact role of antigen location in biasing immune responses is unclear. The cellular location of heterologous antigen may play a role in modulation of immune responses, although the role may depend upon the antigen and/or model system used. In other studies, plasmid DNA vaccines encoding antigen–ligand fusions have enhanced the immune response induced by directing antigen to the appropriate locations for presentation [51–57]. For example, a novel fusion of the translocation domain of Pseudomonas aeruginosa exotoxin A with a model tumor antigen significantly increased the immunogenicity of the DNA vaccine, probably by enhancing the presentation of class I antigen to CD8+ T cells [55]. Similarly HBV antigen was fused to an IgG Fc fragment, to direct antigen to APC’s, resulting in enhanced HBV-specific humoral and cellular immune responses [56]. Fusion of sequences that encode protein transduction domains (PTDs) such as VP22 [58–61] and the HIV-tat protein [62] have also enhanced immune responses to DNA vaccines. PTDs are thought to be capable of translocation from cell to cell [63], although recent studies have suggested that the mechanism by which PTDs enhance immune responses may not be through intercellular spread [64–68]. Another fusion strategy involves fusing the gene encoding the heterologous antigen to the gene encoding ubiquitin, thereby accelerating cytoplasmic degradation of the antigen by targeting it to proteasomes and improving class I antigen presentation [69]. To enhance class II presentation of antigen, targeting of antigens to endosomal or lysosomal compartments has been achieved using the lysosomal associated membrane protein [70,71]. For weak immunogens, the level of T cell help has been increased by fusing activating sequences to antigenencoding sequences [72]. This approach has proved highly effective against B-cell tumors [73] and is currently in clinical trial for patients with follicular lymphoma. Alternatively, the use of an M cell ligand, reovirus protein final sigma1, to direct the DNA

vaccine to mucosal inductive tissues also induced antigen-specific IgG and mucosal IgA [54]. Therefore a wide variety of strategies, based on modification of the encoded heterologous antigen, may be attempted to both direct and enhance DNAmediated immunity. 2.3. Co-stimulatory molecules The co-expression of stimulatory molecules and cytokines may also modulate or enhance immune responses. For example, plasmid DNA expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) may attract infiltrates, including dendritic cells injected into the muscles of mice [74]. Furthermore, the co-injection of a plasmid expressing GMCSF with a plasmid expressing the Plasmodium yoelii circumsporozoite protein (CSP) induced enhanced immune responses against CSP [74]. Other cytokines that have been encoded within plasmid DNA vectors for their effects on humoral and cellular immunity include IL-2, IL-4, IL-12, IFN-g [75]. It is generally thought that co-delivery of GM-CSF and IL-2 improves antibody responses and T cell proliferation [76]. IL-12 co-delivery results in increased CTL activity (a Th1-type immune response), whilst IL-4 co-delivery improves antibody responses and results in decreased CTLs (a Th2-type immune response). Thus, the type of immune response to a DNAencoded antigen may be modified by co-delivery of stimulatory molecules.

3. Approaches to delivery The way in which a DNA vaccine is delivered may have an effect on the type and magnitude of immune response generated. Specifically, the method and route of inoculation is likely to influence the immunogenicity of the vaccine. The most commonly studied routes for administration of DNA vaccines have been intramuscular, and to a lesser extent, epidermal. Injection via a variety of other routes has also been studied, including intradermal, subcutaneous, intravenous, intraperitoneal, oral, vaginal, and intranasal [75,77]. DNA vaccines are normally injected in distilled water or saline, or occasionally sucrose. In some cases a facilitator, such as bupivicaine, designed

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to enhance DNA uptake has also been included [78]. Typically, 10–100 Ag DNA is required to elicit a response in mice by the intramuscular route. However, the intramuscular injection regime is often not sufficiently potent for successful vaccination. Carriers for delivery of DNA and mixed modality approaches to vaccination are under investigation to increase the potency of next-generation DNA vaccines. 3.1. Carriers It is believed that the majority of DNA injected intramuscularly is degraded by extracellular deoxyribonucleases [79]. Carrier-mediated delivery may provide protection from degradation by introducing DNA directly into target cells. For example, the gene gun delivery system involves the gas-driven acceleration of DNA-coated gold particles directly into the epidermis of the skin [80]. This direct injection of DNA into the cytosol of host cells often results in higher levels of antigen-specific stimulated immunity than those achieved by intramuscular DNA immunisation [38,81–88]. Gene gun-mediated DNA immunisation is far more efficient than needle-based immunisation, usually requiring only 0.1–1.0 Ag DNA to induce immunity in mice [75]. This is 100to 1000-fold less DNA than required for intramuscular injection. It is thought that the gene gun-mediated delivery system may induce immunity by a different mechanism from intramuscular inoculation, since a different type of immune response is frequently stimulated. In general, intramuscular immunisation with large amounts of DNA results in the generation of Th1type immune responses, characterised by predominantly IgG2a in mice. Conversely, gene gun-based epidermal immunisation with small amounts of DNA elicits a predominantly Th2-type immune response, characterised by predominantly IgG1 in mice [89]. Intradermal injection of DNA has been reported to induce both Th1- and Th2-type immune responses. It is not clear why these different immunisation methods stimulate different types of immune response. One possibility is that immunisation with larger amounts of DNA may steer the immune system towards a Th1type response as a result of the increased CpG content [38]. Alternatively, the delivery of DNA to extracellular or intracellular space, for intramuscular injec-

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tion and gene gun inoculation, respectively, may induce different intracellular signals and lead to different cytokine production. Whatever the reason for the differences in immune induction, it is possible to modify the bias in the immune responses elicited by the immunisation method. For example, IgG1 and mixed IgG1/IgG2-responses have been induced by gene gun immunisation by altering the immunisation regimen [90]. Alternatively, a Th2-type immune response induced by gene gun-mediated immunisation may be switched to a Th1-type response by codelivering the genes encoding IL-2, IL-7 or IL-12 [91] or CpG motifs [92]. An alternative system to deliver DNA directly to target cells is through encapsulation inside or onto the surface of liposomes [93,94] or microspheres [95– 100]. Chen et al. [101] induced intestinal IgA and protective immunity against virulent rotavirus by oral immunisation of mice with a microencapsulated rotavirus DNA vaccine. In addition, cochleate/DNA formulations induce strong humoral and cellular immune responses following parenteral or oral administration [102]. Cochleates are rigid spiral bilayers of anionic phospholipids that are thought to fuse with the cell membrane to deliver DNA into the cytosol. These delivery systems offer the advantages of DNA vaccine delivery to mucosal surfaces and directly to the cells appropriate for antigen expression. The use of attenuated intracellular bacteria as carriers has similar advantages. Attenuated strains of invasive Shigella flexneri [103,104], Listeria monocytogenes [105], E. coli [106] and S. enterica var. Typhimurium [107] and var. Typhi [108] have all been studied as carriers of heterologous plasmid DNA. When these bacteria are phagocytosed by antigen presenting cells the attenuated strains undergo lysis, releasing the DNA into the host cells for antigen presentation. Most studies to date have focused on the use of Salmonella for this purpose [109]. The use of carriers for DNA vaccines has various advantages for biodefence purposes. The carriers often bias the immune responses induced in mice to the DNA-encoded antigen (see Table 2) and, although it is likely that multiple factors influence the polarisation of the immune response induced, a carrier may be chosen for its likelihood of producing the type of immune response required for optimal protection against a BW or BT agent. Gene gun-based DNA

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Table 2 General murine immune responses to DNA vaccines delivered with carriers Carrier

Inoculation site

Predominant immune response

Gene gun Encapsulation Live attenuated bacteria

skin mucosa mucosa

Th2 Th1 Th1 and Th2

vaccines may be delivered to the skin, and encapsulated or bacteria-based DNA vaccines may be delivered to mucosal surfaces either intranasally or orally. These are non-invasive routes leading to the possibility of self-administration and therefore greater ease of mass vaccination. Furthermore, administration of the vaccines to mucosal surfaces may promote mucosal immunity, which may be important in protecting against microorganisms in BW aerosols. 3.2. Immunisation regimen The efficacy of DNA vaccines may also be influenced by the immunisation regimen used. Multiple immunisations may be necessary to maximise the induction of the immune responses to DNA vaccines, although the optimal number and frequency of inoculations is as yet unknown. However, a promising strategy has been to devise a regimen that combines DNA immunisation with other forms of immunisation, in so-called heterologous prime-boost regimens [110,111]. The order of immunisation in a heterologous prime-boost regimen may be important. Using the antigenically simpler vector for priming and the more complex vector for boosting usually provides the most effective regimen [90]. Thus, this approach usually involves using immunisation protocols that combine DNA vaccine priming followed by alternative methods of boosting. For example, recombinant protein [112] and recombinant vaccinia virus [113] have been separately examined. In one study, heterologous prime-boosting yielded full protection in the malaria infection-model when DNA vaccination was followed by recombinant vaccinia virus administration, although homologous boosting, i.e. repeated DNA vaccine delivery, was ineffective [113]. The potential of the prime-boost approach for us in humans has recently been demonstrated in a clinical trial [114]. Human volunteers given a malaria DNA

vaccine either intramuscularly or epidermally, followed by intradermal administration of recombinant vaccinia virus, developed immune responses 5- to 10fold higher than volunteers given either immunogen alone, and were partially protected against sporozoite challenge. Whichever DNA vaccine delivery system is used, it may be beneficial to use heterologous boosting to improve the immune responses produced. Furthermore, the use of such prime-boost protocols may offer the advantage of providing a more balanced immune response by switching from Th2-type (associated with DNA vaccine delivery) towards Th1-type (associated with protein delivery).

4. DNA vaccines for biodefence Biodefence vaccines against viruses, bacteria and bacterial toxins may need to be rapidly produced. Given that the genomes of several of these agents have been or are currently being sequenced, it should be possible to identify the genes encoding candidate protective subunits [115]. These genes could be cloned into DNA vaccine vectors with relative speed. In an emergency, DNA vaccine production may be rapidly scaled up. Since pre-exposure immunisation of humans may not be feasible, the ability to rapidly produce large quantities of biodefence vaccines is of fundamental importance. As described here, DNA vaccines are easily genetically manipulated to induce appropriate immune responses, including the mucosal immunity likely to be important in protection against weaponised, aerosolised microorganisms. Here, approaches that might be applied to producing DNAbased biodefence vaccines against viruses and bacteria are described, with examples from the literature for the pathogens considered most likely to be weaponised. 4.1. Viruses DNA vaccines protect in animal models against many different viruses, some of which have actually been developed as, or have the potential for development as, biological weapons. The likelihood of certain viruses being used a bioweapons depends on a number of factors including pathogenicity, ease of production and dissemination, transmission from person-to-person and availability of medical counter-

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measures. The US CDC has prioritised work on potential BW agents by categories [116]. For viruses such as poxvirus (e.g., smallpox) and filoviruses (Ebola and Marburg), which are identified in category A (highest priority), and alphaviruses (e.g., Venezuelan Equine Encephalitis Virus), which are category B, DNA vaccines have been constructed and partially evaluated. Other viruses may be considered as potential BW weapons, though their threat has been assessed as a lower priority (category C). These include Hantaviruses, tick-borne encephalitis viruses, yellow fever and highly pathogenic emerging viruses e.g., Nipah virus. DNA vaccines against the category A arenaviruses, such as Lassa Fever and Machupo, have not been developed. However, studies in which intramuscularly delivered DNA vaccines expressing the nucleoprotein of the less pathogenic arenavirus, lymphocytic choriomeningitis virus, showed partial protection against lethal challenge in mice [117]. This suggests that a similar strategy may prove successful against the arenaviruses considered to be a BW threat. Poxviruses have two infectious forms, the intracellular mature virion (IMV) and the extracellular enveloped virion (EEV). A number of proteins associated with these forms have been identified and tested for immunogenicity and protective efficacy in mice. The A33R and B5R proteins (proteins from the EEV form) both protect mice against a lethal i.n. challenge with the IHD-J strain of vaccinia when expressed from a DNA vaccine and delivered by the intramuscular route [118]. However, gene gun immunisation with DNA vaccines expressing either the L1R gene (from the IMV form) or the A33R gene (EEV, delivered simultaneously on different gold particles) resulted in greater protection than when either immunogen was used alone [119]. Similarly, the combination of DNA vaccines expressing the A33R and B5R proteins (EEV) and the L1R and A27L proteins (IMV) resulted in improved, complete protection of mice in the poxvirus challenge model following gene gun administration. This combination of vaccines was also gene gun administered to rhesus monkeys and antibodies were detected against three of the four antigens, although protection was not assessed [120]. Combining DNA vaccines in this way is an attractive approach and, in this case, allows both of the infectious IMV and EEV forms of

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poxvirus to be targeted. This controlled mixing of antigens may reduce the impact of immunodominance, which may be seen when numerous antigens are included in a single DNA vaccine vector [121,122]. These poxvirus proteins elicit neutralising antibodies, although their role in cell-mediated immunity is unknown. The DNA vaccine approach offers the potential of introducing other proteins, such a T cell epitopes, into the regimen and targeting them appropriately. The induction of neutralising antibody may also be important in protection against filovirus infections. DNA vaccines have been prepared against the Ebola and Marburg viruses based upon the transmembrane glycoprotein (GP) that is known to elicit neutralising antibody. DNA expressing the GP protein of Marburg, when delivered by gene gun, protected 50% of cynomolgus monkeys against a lethal challenge with virulent virus [123]. Similarly, a DNA vaccine expressing the GP protein of Ebola virus protects against Ebola virus challenge in mice. In addition, immunisation with DNA expressing the Ebola nucleocapsid protein (NP) is effective against Ebola challenge in rodent models [124]. As few as two gene gun immunisations of either of these DNA vaccines conferred complete protection against challenge in the mouse model. Though promising, the inability of the model to predict efficacy in primates suggests caution [123]. Alphaviruses such as Venezuelan Equine Encephalitis Virus (VEEV) may cause disease in humans and although usually transmitted by mosquito bite, are highly infectious by the aerosol route. DNA vaccines encoding a truncated structural protein region from the 26S mRNA (E3-E2-6K) elicit antibody responses in mice [84] (Fig. 3). Furthermore, a DNA vaccine encoding the 26S cDNA representing the 26S mRNA protected 80% of vaccinated mice against aerosol challenge following gene gun administration [123]. DNA vaccines expressing immunogenic glycoproteins or nucleoproteins also protect in small animal models against some of the lower categorised agents e.g., St. Louis encephalitis virus [125], Central European tickborne encephalitis [126], and Seoul virus (Hantaviridae) [127]. The ultimate vaccine against BW viruses would provide simultaneous protection against a number of these agents. However, the combination and admin-

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Group mean IgG to VEE E2 (mg/ml)

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14 12 10 8 6 4 2 0

i.m. (2 x 50 µg)

i.d. (2 x 50 µg)

g-g (2 x 4 µg)

Route of administration Fig. 3. Immune response to a DNA vaccine expressing Venezuelan Encephalitis Virus E2 following delivery via different routes. Mice receive two injections with 50 Ag DNA via the intramuscular (i.m.) or intradermal (i.d.) routes, or two inoculations with 4 Ag DNA using the gene gun (g-g), and serum samples were assayed by ELISA. Data taken from Bennett et al. [84].

istration of the vaccines would need to be carefully optimised. The coating of a combination of poxvirus DNA vaccines onto the same gold particles for gene gun-based administration leads to interference and the absence of an antibody response [119]. This interference was later suggested to be at the level of translation [120] and serves as a reminder that this issue must be considered when attempting to mix DNA vaccines. Encouragingly, poxvirus DNA vaccines on different gold particles may be mixed together in the same gene-gun cartridge without disadvantage [120]. Further evidence that administration of numerous vaccines at the same time may be effective is demonstrated by the gene gun delivery of anthrax, Ebola, Marburg and VEEV DNA vaccines to guinea pigs [123] although these vaccines were not mixed prior to administration. DNA vaccines that are used in combination against various agents must also be demonstrated to be effective against different strains of the same virus. For example, although the Marburg GP DNA vaccine delivered by the gene gun to guinea pigs induced protective responses to both the Musoke and Ravn strains, other unpublished data suggests incomplete cross-protection to heterologous challenge. This led the authors to suggest inclusion of two or more genes from various isolates within a combination vaccine [123]. Similar problems may arise due to the multiple serogroups of VEEV that are pathogenic for humans.

DNA vaccines allow the inclusion of genes from different strains of a virus in the same vaccine, offering a possible solution to the problem of the need to protect against multiple serotypes. Should there be a need to deliver DNA vaccines sequentially, there is no generic immune response to the DNA molecule. Vector-specific immune responses are commonly encountered with live vaccine vectors, and may inhibit the immunogenicity of subsequent vaccinations. This reduction in efficacy would not occur with repeated DNA vaccinations and would therefore not limit their repeated use. 4.2. Bacteria DNA vaccine vectors have already been shown to have potential for immunisation against Yersinia pestis [84,88,128–130] and Bacillus anthracis [123,128,131–134]. In the case of Y. pestis, DNA vaccines expressing the known major protective antigens, the V antigen [135] and the F1 antigen [136], have been generated. We have published a number of studies involving V antigen. Firstly, evaluation of a DNA vaccine expressing V antigen as a fusion to glutathione-S-transferase showed that specific antibody responses could be induced in mice inoculated with this vaccine [84,128]. Furthermore, the antibody responses were greater when vaccine delivery was by the gene gun compared to intra-

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muscularly administered DNA vaccine based on the full-length F1 antigen induced a poor antibody response when injected in mice [130]. The DNA vaccine expressing the modified F1 antigen was able to provide protection against 4000 LD50s of virulent Y. pestis when delivered using the gene gun. The difference seen between the DNA vaccines expressing different forms of F1 antigen may reflect differences in the cellular processing of the expressed proteins [129]. Together, the demonstration of protection afforded against Y. pestis by DNA vaccines expressing the V and F1 antigens shows promise for the further development of a DNA vaccine against plague. Since a human case of plague caused by an F1 antigennegative strain of Y. pestis has been reported [138], an improved DNA vaccine against plague should ideally induce protective immune responses against both the F1 antigen and V antigen. DNA vaccine vectors expressing the B. anthracis protective antigen (PA) or lethal factor (LF) have been produced. PA is the central component of the tripartite toxin secreted by B. anthracis that also includes LF and oedema factor (EF), and is the main component of the current human vaccine against anthrax. DNA vaccines expressing PA and given to mice by intramuscular injection [133] or gene gun administration [134] protect against anthrax toxin challenge. Similarly, gene

muscular inoculation [84]. More recently, we have produced a second DNA vaccine expressing V antigen alone under the control of an optimised promoter [88]. Immunisation studies with this DNA vaccine confirmed that gene gun immunisation induced greater V antigen-specific antibody responses than intramuscular injection and also demonstrated that the immune responses induced in mice by gene gun immunisation could provide protection against virulent Y. pestis (Fig. 4). Unlike intramuscularly immunised mice (which developed Th1-type immunity), gene gunimmunised mice had a Th2-type biased immune response as indicated by the induction of predominantly IgG1 against V antigen. Such a response is considered likely to be important in providing protection against plague [137]. Using a dprime and boostT strategy, administering the DNA vaccine followed by recombinant V antigen could further increase the protection achieved. Recently, DNA vaccines expressing different forms of the Y. pestis F1 capsular antigen have also been produced [129]. Immunisation of mice with a DNA vaccine expressing the F1 antigen, without its putative signal peptide, was more efficient at inducing F1specific antibodies than a DNA vaccine expressing full-length F1. This finding is in agreement with the results from an earlier study in which an intra-

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Days post-challenge Fig. 4. Protection against plague afforded by DNA vaccines expressing the Y. pestis V antigen administered via intramuscular or gene gun routes. Mice primed with a DNA vaccine expressing V antigen delivered by intramuscular injection (i.m.) or gene gun administration (g-g) and boosted with purified recombinant V antigen (+P) were challenged with 64 LD50s of virulent Y. pestis. Data taken from Garmory et al. [88].

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gun administration of a DNA vaccine expressing LF also protected mice against anthrax toxin challenge [134]. In all cases, the DNA vaccines stimulated predominantly Th2-type immune responses, indicated by predominantly IgG1 in mice. Since it has been shown that immunisation of guinea pigs with adjuvanted recombinant PA induces a predominantly IgG1 response [139] and provides good levels of protection against an aerosol challenge of B. anthracis [140], it is likely that a Th2-type immune response is important in protection against anthrax. More recently, protective immunity against B. anthracis spores afforded by a PA DNA vaccine was demonstrated in rabbits [123]. Since the rabbit model is the model of choice for predicting anthrax vaccine efficacy in humans, these findings show promise for the development of a DNA-based anthrax vaccine for human use. DNA vaccines against Y. pestis and B. anthracis are required to stimulate a Th2-type biased immune response, since it is known that antibody is important in clearing these infections. The use of the gene gun has proved successful in stimulating Th2-type immune responses against both V antigen and PA in mice [84,88,129,134]. Another approach that may be used to bias the immune response to Th2-type is secretion of the expressed antigen (see Section 2.2). For a number of other bacteria for which biodefence vaccines are required, it may be necessary to stimulate cellular immunity. This may be important for providing protection against intracellular bacteria such as Francisella tularensis (the causative agent of tularemia) [141], for example. To date, there are no reports of DNA vaccines against these organisms, reflecting the lack of known protective antigens. However, the DNA vaccination strategies that may be suitable for immunisation against these intracellular pathogens can be considered by evaluating the approaches taken in DNA vaccination against other intracellular bacteria e.g., Mycobacteria spp. A comparison of different delivery systems for the mycobacterial hsp65 protective antigen in mice showed that little immunity was induced by purified hsp65 protein in Freund’s incomplete adjuvant [142]. In comparison, DNA vaccines, liposome-entrapped hsp65 protein, or antigen presenting cells expressing hsp65 were able to induce protective immunity similar to that of the live Bacillus Calmette Guerin (BCG) vaccine. The delivery systems allowing endogenous

protein expression elicited a high level of cellular immunity. The immunogenicity afforded by DNA vaccines expressing hsp65 may be improved by protein boosting [143]. One of the most promising strategies involves targeting the expressed antigen to cellular sites appropriate for the induction of cellmediated immunity. In a recent study, 10 different tuberculosis DNA vaccines that expressed mycobacterial proteins fused at the N-terminus to eukaryotic intracellular targeting sequences were evaluated [144]. These DNA vaccines, with fusions to the tissue plasminogen activator sequence or conjugated to ubiquitin, were able to induce cellular immune responses in intramuscularly vaccinated mice that were comparable to those in BCG-immunised mice. Another successful approach used to stimulate cellular immune responses has been the expression of costimulatory molecules such as IL-12 and IL-18 [145,146]. It is clear that DNA vaccination has emerged as a powerful approach to the development of new vaccines against tuberculosis. The strategies developed should be applicable to the development of DNA vaccines for biodefence against intracellular bacteria. It will also be necessary to produce DNA vaccines against bacterial toxins for biodefence. DNA vaccines have been evaluated against the botulinum neurotoxins (BoNTs) of Clostridium botulinum [147–149]. Two of these vaccines expressed non-toxic fragments of the type A BoNT [147,148] and one expressed a non-toxic fragment of the type F BoNT [149]. There are seven serologically distinct BoNTs, classified A– G, that are produced by the corresponding C. botulinum types A–G. Of these, BoNT types A, B, E and F are most associated with human disease. The DNA vaccine against BoNT type F was most effective of those described to date, providing 90% protection against 104 MLD BoNT type F in mice following two intramuscular immunisations with 100 Ag DNA [149]. This DNA vaccine incorporated the gene encoding BoNT type F Hc fragment fused to a signal peptide to allow secretion of the expressed protein. In comparison, a similar DNA vaccine that did not allow secretion of the expressed BoNT type F Hc fragment induced lower levels of Hc-specific IgG (Fig. 5) and provided a lower level of protection against toxin challenge. Since protection against BoNT is antibodymediated, secretion of the expressed antigen is an

IgG concentration (ng/ml)

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1400 1200

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Fig. 5. Effect of antigen secretion on immune response to a DNA vaccine expressing BoNT FHc. Mice were intramuscularly inoculated with DNA vaccines expressing (and secreting or nonsecreting) the botulinum toxin type F Hc fragment (BoNT FHc), or with purified FHc protein. BoNT FHc-specific IgG was measured by ELISA of pooled serum samples taken from the mice. Data taken from Bennett et al. [149].

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produced vaccines that may stimulate all types of immune responses. Human clinical trials have demonstrated the safety and immunogenicity of such vaccines (reviewed by [150]). From these it is apparent that although DNA vaccines are able to induce cellular immune responses they may induce relatively poor humoral immune responses [150]. This suggests that their current use should be directed towards those pathogens for which cellular immunity is considered important in the prevention of disease, e.g., F. tularensis. Since it is also possible to produce DNA vaccines expressing multiple epitopes [153], the development of a DNA vaccine expressing protective epitopes from multiple BW agents presents a very attractive target for those involved in biodefence.

6. Conclusions obvious strategy for stimulating the required immune response.

5. Future prospects There are technical hurdles limiting the progress of DNA vaccines, the major barrier being their lack of potency in humans following intramuscular administration [150]. In order to improve the efficacy of DNA vaccines, gene gun-like technologies and dprime and boostT regimes are being evaluated. In addition, although DNA vaccines have been well tolerated in numerous animal studies, there are still safety concerns associated with human use. One of these is that DNA vaccines could integrate into the human chromosome, although assays carried out to address this concern have found no evidence of integration [151]. Another concern is the development of autoimmunity, since mice in one study developed antiDNA antibodies following immunisation with DNA vaccines [152]. In this study, however, anti-DNA antibodies were at low levels and transient, and no symptoms of autoimmunity were observed. These issues need to be fully addressed before DNA vaccination is considered acceptable for routine use in humans. However, if these technical and regulatory barriers can be overcome, DNA vaccination offers great promise for the rapid development of stable, easily

The advantages of DNA vaccines are that they may be rapidly produced for widespread use and are able to produce a broad spectrum of immune responses following non-invasive delivery. These attributes are of particular value for the development of vaccines for biodefence, where the rapid production of vaccines that stimulate potent cell-mediated immunity may be required. The ability to genetically manipulate the DNA means that not only may further protective antigens be added with ease, but that vaccines may be tailored for induction of the appropriate immune responses. Clinical trials have suggested that DNA vaccines may be safe and immunogenic in humans and appropriate scale production methods have been developed by the pharmaceutical industry [154]. Although regulatory issues associated with licensing remain to be addressed, DNA vaccines hold great promise for our future protection against biological attack.

References [1] Whitley R.J., Smallpox: a potential agent of bioterrorism, Antivir. Res. 57 (2003) 7 – 12. [2] Pile J.C., Malone J.D., Eitzen E.M., Friedlander A.M., Anthrax as a potential biological warfare agent, Arch. Intern. Med. 158 (1998) 429 – 434. [3] Sotos J.G., Botulinum toxin in biowarfare, JAMA 285 (2001) 2716.

1356

H.S. Garmory et al. / Advanced Drug Delivery Reviews 57 (2005) 1343–1361

[4] Davis H.L., Plasmid DNA expression systems for the purpose of immunization, Curr. Opin. Biotechnol. 8 (1997) 635 – 646. [5] Sharma A.K., Khuller G.K., DNA vaccines: future strategies and relevance to intracellular pathogens, Immunol. Cell Biol. 79 (2001) 537 – 546. [6] Chattergoon M.A., Kim J.J., Yang J.S., Robinson T.M., Lee D.J., Dentchev T., Wilson D.M., Ayyavoo V., Weiner D.B., Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis, Nat. Biotechnol. 18 (2000) 974 – 979. [7] Sasaki S., Amara R.R., Oran A.E., Smith J.M., Robinson H.L., Apoptosis-mediated enhancement of DNA-raised immune responses by mutant caspases, Nat. Biotechnol. 19 (2001) 543 – 547. [8] Kim T.W., Hung C.F., Ling M., Juang J., He L., Hardwick J.M., Kumar S., Wu T.C., Enhancing DNA vaccine potency by coadministration of DNA encoding antiapoptotic proteins, J. Clin. Invest. 112 (2003) 109 – 117. [9] Leitner W.W., Restifo N.P., DNA vaccines and apoptosis: to kill or not to kill? J. Clin. Invest. 112 (2003) 22 – 24. [10] Kozak M., At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells, J. Mol. Biol. 196 (1987) 947 – 950. [11] Garmory H.S., Brown K.A., Titball R.W., DNA vaccines: improving expression of antigens, Genet. Vaccines Ther. 1 (2003) 2. [12] Cheng L., Ziegelhoffer P.R., Yang N.S., In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 4455 – 4459. [13] Manthorpe M., Cornefert-Jensen F., Hartikka J., Felgner J., Rundell A., Margalith M., Dwarki V., Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice, Hum. Gene Ther. 4 (1993) 419 – 431. [14] Lee A.H., Suh Y.S., Sung J.H., Yang S.H., Sung Y.C., Comparison of various expression plasmids for the induction of immune response by DNA immunization, Mol. Cells 7 (1997) 495 – 501. [15] Loirat D., Li Z., Mancini M., Tiollais P., Paulin D., Michel M.L., Muscle-specific expression of hepatitis B surface antigen: no effect on DNA-raised immune responses, Virology 260 (1999) 74 – 83. [16] Galvin T.A., Muller J., Khan A.S., Effect of different promoters on immune responses elicited by HIV-1 gag/env multigenic DNA vaccine in Macaca mulatta and Macaca nemestrina, Vaccine 18 (2000) 2566 – 2583. [17] Tucker C., Endo M., Hirono I., Aoki T., Assessment of DNA vaccine potential for juvenile Japanese flounder Paralichthys olivaceus, through the introduction of reporter genes by particle bombardment and histopathology, Vaccine 19 (2000) 801 – 809. [18] Suarez D.L., Schultz-Cherry S., The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model, Avian Dis. 44 (2000) 861 – 868.

[19] Watts A.M., Bright R.K., Kennedy R.C., DNA cancer vaccination strategies target SV40 large tumour antigen in a murine experimental metastasis model, Dev. Biol. (Basel) 104 (2000) 143 – 147. [20] Xu Z.L., Mizuguchi H., Ishii-Watabe A., Uchida E., Mayumi T., Hayakawa T., Optimization of transcriptional regulatory elements for constructing plasmid vectors, Gene 272 (2001) 149 – 156. [21] Garapin A., Ma L., Pescher P., Lagranderie M., Marchal G., Mixed immune response induced in rodents by two naked DNA genes coding for mycobacterial glycosylated proteins, Vaccine 19 (2001) 2830 – 2841. [22] Kwissa M., von Kampen K., Zurbriggen R., Gluck R., Reimann J., Schirmbeck R., Efficient vaccination by intradermal or intramuscular inoculation of plasmid DNA expressing hepatitis B surface antigen under desmin promoter/enhancer control, Vaccine 18 (2000) 2337 – 2344. [23] Bartlett R.J., Secore S.L., Singer J.T., Bodo M., Sharma K., Ricordi C., Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo, Cell Transplant. 5 (1996) 411 – 419. [24] Gebhard J.R., Zhu J., Cao X., Minnick J., Araneo B.A., DNA immunization utilizing a herpes simplex virus type 2 myogenic DNA vaccine protects mice from mortality and prevents genital herpes, Vaccine 18 (2000) 1837 – 1846. [25] Itai K., Sawamura D., Meng X., Hashimoto I., Keratinocyte gene therapy: inducible promoters and in vivo control of transgene expression, Clin. Exp. Dermatol. 26 (2001) 531 – 535. [26] Rush C., Mitchell T., Garside P., Efficient priming of CD4+ and CD8+ T cells by DNA vaccination depends on appropriate targeting of sufficient levels of immunologically relevant antigen to appropriate processing pathways, J. Immunol. 169 (2002) 4951 – 4960. [27] Chapman B.S., Thayer R.M., Vincent K.A., Haigwood N.L., Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells, Nucleic Acids Res. 19 (1991) 3979 – 3986. [28] Ikemura T., Codon usage and tRNA content in unicellular and multicellular organisms, Mol. Biol. Evol. 2 (1985) 13 – 34. [29] Uchijima M., Yoshida A., Nagata T., Koide Y., Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class I-restricted T cell responses against an intracellular bacterium, J. Immunol. 161 (1998) 5594 – 5599. [30] Andre S., Seed B., Eberle J., Schraut W., Bultmann A., Haas J., Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage, J. Virol. 72 (1998) 1497 – 1503. [31] Vinner L., Nielsen H.V., Bryder K., Corbet S., Nielsen C., Fomsgaard A., Gene gun DNA vaccination with Revindependent synthetic HIV-1 gp160 envelope gene using mammalian codons, Vaccine 17 (1999) 2166 – 2175. [32] Deml L., Bojak A., Steck S., Graf M., Wild J., Schirmbeck R., Wolf H., Wagner R., Multiple effects of codon usage

H.S. Garmory et al. / Advanced Drug Delivery Reviews 57 (2005) 1343–1361

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein, J. Virol. 75 (2001) 10991 – 11001. Narum D.L., Kumar S., Rogers W.O., Fuhrmann S.R., Liang H., Oakley M., Taye A., Sim B.K., Hoffman S.L., Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice, Infect. Immun. 69 (2001) 7250 – 7253. Stratford R., Douce G., Zhang-Barber L., Fairweather N., Eskola J., Dougan G., Influence of codon usage on the immunogenicity of a DNA vaccine against tetanus, Vaccine 19 (2000) 810 – 815. Krieg A.M., Yi A.K., Matson S., Waldschmidt T.J., Bishop G.A., Teasdale R., Koretzky G.A., Klinman D.M., CpG motifs in bacterial DNA trigger direct B-cell activation, Nature 374 (1995) 546 – 549. Klinman D.M., Yi A.K., Beaucage S.L., Conover J., Krieg A.M., CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 2879 – 2883. Klinman D.M., Yamshchikov G., Ishigatsubo Y., Contribution of CpG motifs to the immunogenicity of DNA vaccines, J. Immunol. 158 (1997) 3635 – 3639. Krieg A.M., Yi A.K., Schorr J., Davis H.L., The role of CpG dinucleotides in DNA vaccines, Trends Microbiol. 6 (1998) 23 – 27. Porter K.R., Kochel T.J., Wu S.J., Raviprakash K., Phillips I., Hayes C.G., Protective efficacy of a dengue 2 DNA vaccine in mice and the effect of CpG immuno-stimulatory motifs on antibody responses, Arch. Virol. 143 (1998) 997 – 1003. Weeratna R., Brazolot Millan C.L., Krieg A.M., Davis H.L., Reduction of antigen expression from DNA vaccines by coadministered oligodeoxynucleotides, Antisense Nucleic Acid Drug Dev. 8 (1998) 351 – 356. Payne L.G., Fuller D.H., Haynes J.R., Particle-mediated DNA vaccination of mice, monkeys and men: looking beyond the dogma, Curr. Opin. Mol. Ther. 4 (2002) 459 – 466. Yamamoto S., Yamamoto T., Shimada S., Kuramoto E., Yano O., Kataoka T., Tokunaga T., DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth, Microbiol. Immunol. 36 (1992) 983 – 997. Hartmann G., Krieg A.M., Mechanism and function of a newly identified CpG DNA motif in human primary B cells, J. Immunol. 164 (2000) 944 – 953. Locher C.P., Witt S.A., Ashlock B.M., Levy J.A., Enhancement of antibody responses to an HIV-2 DNA envelope vaccine using an expression vector containing a constitutive transport element, DNA Cell Biol. 21 (2002) 581 – 586. Svanholm C., Bandholtz L., Lobell A., Wigzell H., Enhancement of antibody responses by DNA immunization using expression vectors mediating efficient antigen secretion, J. Immunol. Methods 228 (1999) 121 – 130.

1357

[46] Sbai H., Schneider J., Hill A.V., Whalen R.G., Role of transfection in the priming of cytotoxic T-cells by DNAmediated immunization, Vaccine 20 (2002) 3137 – 3147. [47] Inchauspe G., Vitvitski L., Major M.E., Jung G., Spengler U., Maisonnas M., Trepo C., Plasmid DNA expressing a secreted or a nonsecreted form of hepatitis C virus nucleocapsid: comparative studies of antibody and T-helper responses following genetic immunization, DNA Cell Biol. 16 (1997) 185 – 195. [48] Lewis P.J., Cox G.J., van Drunen Littel-van den Hurk S., Babiuk L.A., Polynucleotide vaccines in animals: enhancing and modulating responses, Vaccine 15 (1997) 861 – 864. [49] Boyle J.S., Koniaras C., Lew A.M., Influence of cellular location of expressed antigen on the efficacy of DNA vaccination: cytotoxic T lymphocyte and antibody responses are suboptimal when antigen is cytoplasmic after intramuscular DNA immunization, Int. Immunol. 9 (1997) 1897 – 1906. [50] Rice J., King C.A., Spellerberg M.B., Fairweather N., Stevenson F.K., Manipulation of pathogen-derived genes to influence antigen presentation via DNA vaccines, Vaccine 17 (1999) 3030 – 3038. [51] Boyle J.S., Brady J.L., Lew A.M., Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction, Nature 392 (1998) 408 – 411. [52] Aris A., Feliu J.X., Knight A., Coutelle C., Villaverde A., Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression, Biotechnol. Bioeng. 68 (2000) 689 – 696. [53] Deliyannis G., Boyle J.S., Brady J.L., Brown L.E., Lew A.M., A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 6676 – 6680. [54] Wu Y., Wang X., Csencsits K.L., Haddad A., Walters N., Pascual D.W., M cell-targeted DNA vaccination, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 9318 – 9323. [55] Hung C.F., Cheng W.F., Hsu K.F., Chai C.Y., He L., Ling M., Wu T.C., Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen, Cancer Res. 61 (2001) 3698 – 3703. [56] You Z., Huang X., Hester J., Toh H.C., Chen S.Y., Targeting dendritic cells to enhance DNA vaccine potency, Cancer Res. 61 (2001) 3704 – 3711. [57] De Marco F., Hallez S., Brulet J.M., Gesche F., Marzano P., Flamini S., Marcante M.L., Venuti A., DNA vaccines against HPV-16 E7-expressing tumour cells, Anticancer Res. 23 (2003) 1449 – 1454. [58] Hung C.F., Cheng W.F., Chai C.Y., Hsu K.F., He L., Ling M., Wu T.C., Improving vaccine potency through intercellular spreading and enhanced MHC class I presentation of antigen, J. Immunol. 166 (2001) 5733 – 5740. [59] Oliveira S.C., Harms J.S., Afonso R.R., Splitter G.A., A genetic immunization adjuvant system based on BVP22antigen fusion, Hum. Gene Ther. 12 (2001) 1353 – 1359. [60] Michel N., Osen W., Gissmann L., Schumacher T.N., Zentgraf H., Muller M., Enhanced immunogenicity of HPV

1358

[61]

[62]

[63]

[64]

[65]

[66] [67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

H.S. Garmory et al. / Advanced Drug Delivery Reviews 57 (2005) 1343–1361 16 E7 fusion proteins in DNA vaccination, Virology 294 (2002) 47 – 59. Hung C.F., He L., Juang J., Lin T.J., Ling M., Wu T.C., Improving DNA vaccine potency by linking Marek’s disease virus type 1 VP22 to an antigen, J. Virol. 76 (2002) 2676 – 2682. Leifert J.A., Lindencrona J.A., Charo J., Whitton J.L., Enhancing T cell activation and antiviral protection by introducing the HIV-1 protein transduction domain into a DNA vaccine, Hum. Gene Ther. 12 (2001) 1881 – 1892. Elliott G., O’Hare P., Intercellular trafficking and protein delivery by a herpesvirus structural protein, Cell 88 (1997) 223 – 233. Leifert J.A., Whitton J.L., bTranslocatory proteinsQ and bprotein transduction domainsQ: a critical analysis of their biological effects and the underlying mechanisms, Molec. Ther. 8 (2003) 13 – 20. Falnes P.O., Wesche J., Olsnes S., Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment, Biochemistry 40 (2001) 4349 – 4358. Lundberg M., Johansson M., Is VP22 nuclear homing an artifact? Nat. Biotechnol. 19 (2001) 713 – 714. Lundberg M., Johansson M., Positively charged DNAbinding proteins cause apparent cell membrane translocation, Biochem. Biophys. Res. Commun. 291 (2002) 367 – 371. Lundberg M., Wikstrom S., Johansson M., Cell surface adherence and endocytosis of protein transduction domains, Molec. Ther. 8 (2003) 143 – 150. Tobery T.W., Siliciano R.F., Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization, J. Exp. Med. 185 (1997) 909 – 920. Ji H., Wang T.L., Chen C.H., Pai S.I., Hung C.F., Lin K.Y., Kurman R.J., Pardoll D.M., Wu T.C., Targeting human papillomavirus type 16 E7 to the endosomal/lysosomal compartment enhances the antitumor immunity of DNA vaccines against murine human papillomavirus type 16 E7expressing tumors, Hum. Gene Ther. 10 (1999) 2727 – 2740. Wu T.C., Guarnieri F.G., Staveley-O’Carroll K.F., Viscidi R.P., Levitsky H.I., Hedrick L., Cho K.R., August J.T., Pardoll D.M., Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 11671 – 11675. Zhu D., Rice J., Savelyeva N., Stevenson F.K., DNA fusion vaccines against B-cell tumors, Trends Mol. Med. 7 (2001) 566 – 572. King C.A., Spellerberg M.B., Zhu D., Rice J., Sahota S.S., Thompsett A.R., Hamblin T.J., Radl J., Stevenson F.K., DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma, Nat. Med. 4 (1998) 1281 – 1286. Haddad D., Ramprakash J., Sedegah M., Charoenvit Y., Baumgartner R., Kumar S., Hoffman S.L., Weiss W.R., Plasmid vaccine expressing granulocyte-macrophage colonystimulating factor attracts infiltrates including immature

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

dendritic cells into injected muscles, J. Immunol. 165 (2000) 3772 – 3781. Gurunathan S., Klinman D.M., Seder R.A., DNA vaccines: immunology, application, and optimization*, Annu. Rev. Immunol. 18 (2000) 927 – 974. Pan C.H., Chen H.W., Tao M.H., Modulation of immune responses to DNA vaccines by codelivery of cytokine genes, J. Formos. Med. Assoc. 98 (1999) 722 – 729. McCluskie M.J., Brazolot Millan C.L., Gramzinski R.A., Robinson H.L., Santoro J.C., Fuller J.T., Widera G., Haynes J.R., Purcell R.H., Davis H.L., Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates, Mol. Med. 5 (1999) 287 – 300. Pachuk C.J., Ciccarelli R.B., Samuel M., Bayer M.E., Troutman R.D., Zurawski D.V., Schauer J.I., Higgins T.J., Weiner D.B., Sosnoski D.M., Zurawski V.R., Satishchandran C., Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine, Biochim. Biophys. Acta 1468 (2000) 20 – 30. Kawabata K., Takakura Y., Hashida M., The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake, Pharm. Res. 12 (1995) 825 – 830. Tang D.C., DeVit M., Johnston S.A., Genetic immunization is a simple method for eliciting an immune response, Nature 356 (1992) 152 – 154. Webster R.G., Fynan E.F., Santoro J.C., Robinson H., Protection of ferrets against influenza challenge with a DNA vaccine to the haemagglutinin, Vaccine 12 (1994) 1495 – 1498. Chinsangaram J., Beard C., Mason P.W., Zellner M.K., Ward G., Grubman M.J., Antibody response in mice inoculated with DNA expressing foot-and-mouth disease virus capsid proteins, J. Virol. 72 (1998) 4454 – 4457. Colombage G., Hall R., Pavy M., Lobigs M., DNA-based and alphavirus-vectored immunisation with prM and E proteins elicits long-lived and protective immunity against the flavivirus, Murray Valley encephalitis virus, Virology 250 (1998) 151 – 163. Bennett A.M., Phillpotts R.J., Perkins S.D., Jacobs S.C., Williamson E.D., Gene gun mediated vaccination is superior to manual delivery for immunisation with DNA vaccines expressing protective antigens from Yersinia pestis or Venezuelan Equine Encephalitis virus, Vaccine 18 (1999) 588 – 596. Asakura Y., Lundholm P., Kjerrstrom A., Benthin R., Lucht E., Fukushima J., Schwartz S., Okuda K., Wahren B., Hinkula J., DNA-plasmids of HIV-1 induce systemic and mucosal immune responses, Biol. Chem. 380 (1999) 375 – 379. Belperron A.A., Feltquate D., Fox B.A., Horii T., Bzik D.J., Immune responses induced by gene gun or intramuscular injection of DNA vaccines that express immunogenic regions of the serine repeat antigen from Plasmodium falciparum, Infect. Immun. 67 (1999) 5163 – 5169. Yoshida A., Nagata T., Uchijima M., Higashi T., Koide Y., Advantage of gene gun-mediated over intramuscular inocu-

H.S. Garmory et al. / Advanced Drug Delivery Reviews 57 (2005) 1343–1361

[88]

[89] [90]

[91]

[92]

[93]

[94] [95]

[96]

[97]

[98]

[99]

[100]

[101]

lation of plasmid DNA vaccine in reproducible induction of specific immune responses, Vaccine 18 (2000) 1725 – 1729. Garmory H.S., Freeman D., Brown K.A., Titball R.W., Protection against plague afforded by immunisation with DNA vaccines optimised for expression of the Yersinia pestis V antigen, Vaccine 22 (2004) 947 – 957. Barry M.A., Johnston S.A., Biological features of genetic immunization, Vaccine 15 (1997) 788 – 791. Leitner W.W., Ying H., Restifo N.P., DNA and RNA-based vaccines: principles, progress and prospects, Vaccine 18 (1999) 765 – 777. Prayaga S.K., Ford M.J., Haynes J.R., Manipulation of HIV-1 gp120-specific immune responses elicited via gene gunbased DNA immunization, Vaccine 15 (1997) 1349 – 1352. Schirmbeck R., Reimann J., Modulation of gene-gunmediated Th2 immunity to hepatitis B surface antigen by bacterial CpG motifs or IL-12, Intervirology 44 (2001) 115 – 123. Bramwell V.W., Eyles J.E., Somavarapu S., Alpar H.O., Liposome/DNA complexes coated with biodegradable PLA improve immune responses to plasmid encoding hepatitis B surface antigen, Immunology 106 (2002) 412 – 418. Gregoriadis G., Saffie R., de Souza J.B., Liposome-mediated DNA vaccination, FEBS Lett. 402 (1997) 107 – 110. Denis-Mize K.S., Dupuis M., MacKichan M.L., Singh M., Doe B., O’Hagan D., Ulmer J.B., Donnelly J.J., McDonald D.M., Ott G., Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells, Gene Ther. 7 (2000) 2105 – 2112. Singh M., Briones M., Ott G., O’Hagan D., Cationic microparticles: a potent delivery system for DNA vaccines, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 811 – 816. Jones D.H., Clegg J.C., Farrar G.H., Oral delivery of microencapsulated DNA vaccines, Dev. Biol. Stand. 92 (1998) 149 – 155. McKeever U., Barman S., Hao T., Chambers P., Song S., Lunsford L., Hsu Y.Y., Roy K., Hedley M.L., Protective immune responses elicited in mice by immunization with formulations of poly(lactide-co-glycolide) microparticles, Vaccine 20 (2002) 1524 – 1531. O’Hagan D., Singh M., Ugozzoli M., Wild C., Barnett S., Chen M., Schaefer M., Doe B., Otten G.R., Ulmer J.B., Induction of potent immune responses by cationic microparticles with adsorbed human immunodeficiency virus DNA vaccines, J. Virol. 75 (2001) 9037 – 9043. Luo Y., O’Hagan D., Zhou H., Singh M., Ulmer J., Reisfeld R.A., James Primus F., Xiang R., Plasmid DNA encoding human carcinoembryonic antigen (CEA) adsorbed onto cationic microparticles induces protective immunity against colon cancer in CEA-transgenic mice, Vaccine 21 (2003) 1938 – 1947. Chen S.C., Jones D.H., Fynan E.F., Farrar G.H., Clegg J.C., Greenberg H.B., Herrmann J.E., Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles, J. Virol. 72 (1998) 5757 – 5761.

1359

[102] Mannino R.J., Canki M., Feketeova E., Scolpino A.J., Wang Z., Zhang F., Kheiri M.T., Gould-Fogerite S., Targeting immune response induction with cochleate and liposomebased vaccines, Adv. Drug Deliv. Rev. 32 (1998) 273 – 287. [103] Sizemore D.R., Branstrom A.A., Sadoff J.C., Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization, Science 270 (1995) 299 – 302. [104] Sizemore D.R., Branstrom A.A., Sadoff J.C., Attenuated bacteria as a DNA delivery vehicle for DNA-mediated immunization, Vaccine 15 (1997) 804 – 807. [105] Dietrich G., Bubert A., Gentschev I., Sokolovic Z., Simm A., Catic A., Kaufmann S.H., Hess J., Szalay A.A., Goebel W., Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes, Nat. Biotechnol. 16 (1998) 181 – 185. [106] Grillot-Courvalin C., Goussard S., Courvalin P., Bacteria as gene delivery vectors for mammalian cells, Curr. Opin. Biotechnol. 10 (1999) 477 – 481. [107] Darji A., Guzman C.A., Gerstel B., Wachholz P., Timmis K.N., Wehland J., Chakraborty T., Weiss S., Oral somatic transgene vaccination using attenuated S. typhimurium, Cell 91 (1997) 765 – 775. [108] Pasetti M.F., Anderson R.J., Noriega F.R., Levine M.M., Sztein M.B., Attenuated deltaguaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses, Clin. Immunol. 92 (1999) 76 – 89. [109] Garmory H.S., Brown K.A., Titball R.W., Salmonella vaccines for use in humans: present and future perspectives, FEMS Microbiol. Rev. 26 (2002) 339 – 353. [110] Hill A.V., Reece W., Gothard P., Moorthy V., Roberts M., Flanagan K., Plebanski M., Hannan C., Hu J.T., Anderson R., Degano P., Schneider J., Prieur E., Sheu E., Gilbert S.C., DNA-based vaccines for malaria: a heterologous primeboost immunisation strategy, Dev. Biol. (Basel) 104 (2000) 171 – 179. [111] Devico A.L., Fouts T.R., Shata M.T., Kamin-Lewis R., Lewis G.K., Hone D.M., Development of an oral prime-boost strategy to elicit broadly neutralizing antibodies against HIV1, Vaccine 20 (2002) 1968 – 1974. [112] Haddad D., Liljeqvist S., Stahl S., Hansson M., Perlmann P., Ahlborg N., Berzins K., Characterization of antibody responses to a Plasmodium falciparum blood-stage antigen induced by a DNA prime/protein boost immunization protocol, Scand. J. Immunol. 49 (1999) 506 – 514. [113] Schneider J., Gilbert S.C., Blanchard T.J., Hanke T., Robson K.J., Hannan C.M., Becker M., Sinden R., Smith G.L., Hill A.V., Enhanced immunogenicity for CD8+ T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara, Nat. Med. 4 (1998) 397 – 402. [114] McConkey S.J., Reece W.H., Moorthy V.S., Webster D., Dunachie S., Butcher G., Vuola J.M., Blanchard T.J., Gothard P., Watkins K., Hannan C.M., Everaere S., Brown K., Kester K.E., Cummings J., Williams J., Heppner D.G., Pathan A., Flanagan K., Arulanantham N., Roberts M.T., Roy

1360

[115]

[116] [117]

[118]

[119]

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127]

H.S. Garmory et al. / Advanced Drug Delivery Reviews 57 (2005) 1343–1361 M., Smith G.L., Schneider J., Peto T., Sinden R.E., Gilbert S.C., Hill A.V., Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans, Nat. Med. 9 (2003) 729 – 735. Titball R.W., Williamson E.D., Vaccine development for potential bioterrorism agents, Curr. Drug Targets Infect. Disord. 3 (2003) 255 – 262. Lane H.C., Montagne J.L., Fauci A.S., Bioterrorism: a clear and present danger, Nat. Med. 7 (2001) 1271 – 1273. Yokoyama M., Zhang J., Whitton J.L., DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection, J. Virol. 69 (1995) 2684 – 2688. Galmiche M.C., Goenaga J., Wittek R., Rindisbacher L., Neutralizing and protective antibodies directed against vaccinia virus envelope antigens, Virology 254 (1999) 71 – 80. Hooper J.W., Custer D.M., Schmaljohn C.S., Schmaljohn A.L., DNA vaccination with vaccinia virus L1R and A33R genes protects mice against a lethal poxvirus challenge, Virology 266 (2000) 329 – 339. Hooper J.W., Custer D.M., Thompson E., Four-gene-combination DNA vaccine protects mice against a lethal vaccinia virus challenge and elicits appropriate antibody responses in nonhuman primates, Virology 306 (2003) 181 – 195. Rice J., Elliott T., Buchan S., Stevenson F.K., DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines, J. Immunol. 167 (2001) 1558 – 1565. Le T.T., Drane D., Malliaros J., Cox J.C., Rothel L., Pearse M., Woodberry T., Gardner J., Suhrbier A., Cytotoxic T cell polyepitope vaccines delivered by ISCOMs, Vaccine 19 (2001) 4669 – 4675. Riemenschneider J., Garrison A., Geisbert J., Jahrling P., Hevey M., Negley D., Schmaljohn A., Lee J., Hart M.K., Vanderzanden L., Custer D., Bray M., Ruff A., Ivins B., Bassett A., Rossi C., Schmaljohn C., Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus, Vaccine 21 (2003) 4071 – 4080. Vanderzanden L., Bray M., Fuller D., Roberts T., Custer D., Spik K., Jahrling P., Huggins J., Schmaljohn A., Schmaljohn C., DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge, Virology 246 (1998) 134 – 144. Phillpotts R.J., Venugopal K., Brooks T., Immunisation with DNA polynucleotides protects mice against lethal challenge with St. Louis encephalitis virus, Arch. Virol. 141 (1996) 743 – 749. Aberle J.H., Aberle S.W., Allison S.L., Stiasny K., Ecker M., Mandl C.W., Berger R., Heinz F.X., A DNA immunization model study with constructs expressing the tick-borne encephalitis virus envelope protein E in different physical forms, J. Immunol. 163 (1999) 6756 – 6761. Kamrud K.I., Hooper J.W., Elgh F., Schmaljohn C.S., Comparison of the protective efficacy of naked DNA, DNA-based Sindbis replicon, and packaged Sindbis replicon vectors expressing Hantavirus structural genes in hamsters, Virology 263 (1999) 209 – 219.

[128] Williamson E.D., Bennett A.M., Perkins S.D., Beedham R.J., Miller J., Baillie L.W., Co-immunisation with a plasmid DNA cocktail primes mice against anthrax and plague, Vaccine 20 (2002) 2933 – 2941. [129] Grosfeld H., Cohen S., Bino T., Flashner Y., Ber R., Mamroud E., Kronman C., Shafferman A., Velan B., Effective protective immunity to Yersinia pestis infection conferred by DNA vaccine coding for derivatives of the F1 capsular antigen, Infect. Immun. 71 (2003) 374 – 383. [130] Brandler P., Saikh K.U., Heath D., Friedlander A., Ulrich R.G., Weak anamnestic responses of inbred mice to Yersinia F1 genetic vaccine are overcome by boosting with F1 polypeptide while outbred mice remain nonresponsive, J. Immunol. 161 (1998) 4195 – 4200. [131] Gaur R., Gupta P.K., Banerjea A.C., Singh Y., Effect of nasal immunization with protective antigen of Bacillus anthracis on protective immune response against anthrax toxin, Vaccine 20 (2002) 2836 – 2839. [132] Tucker S.N., Lin K., Stevens S., Scollay R., Bennett M.J., Olson D.C., Systemic and mucosal antibody responses following retroductal gene transfer to the salivary gland, Molec. Ther. 8 (2003) 392 – 399. [133] Gu M.L., Leppla S.H., Klinman D.M., Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen, Vaccine 17 (1999) 340 – 344. [134] Price B.M., Liner A.L., Park S., Leppla S.H., Mateczun A., Galloway D.R., Protection against anthrax lethal toxin challenge by genetic immunisation with a plasmid encoding the lethal factor protein, Infect. Immun. (2001) 4509 – 4515. [135] Leary S.E., Williamson E.D., Griffin K.F., Russell P., Eley S.M., Titball R.W., Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague, Infect. Immun. 63 (1995) 2854 – 2858. [136] Andrews G.P., Heath D.G., Anderson Jr. G.W., Welkos S.L., Friedlander A.M., Fraction 1 capsular antigen (F1) purification from Yersinia pestis CO92 and from an Escherichia coli recombinant strain and efficacy against lethal plague challenge, Infect. Immun. 64 (1996) 2180 – 2187. [137] Williamson E.D., Vesey P.M., Gillhespy K.J., Eley S.M., Green M., Titball R.W., An IgG1 titre to the F1 and V antigens correlates with protection against plague in the mouse model, Clin. Exp. Immunol. 116 (1999) 107 – 114. [138] Winter C.C., Cherry W.B., Moody M.D., An unusual strain of Pasteurella pestis isolated from a fatal human case of plague, Bull. World Health Organ 23 (1960) 408 – 409. [139] McBride B.W., Mogg A., Telfer J.L., Lever M.S., Miller J., Turnbull P.C., Baillie L., Protective efficacy of a recombinant protective antigen against Bacillus anthracis challenge and assessment of immunological markers, Vaccine 16 (1998) 810 – 817. [140] Miller J., McBride B.W., Manchee R.J., Moore P., Baillie L.W., Production and purification of recombinant protective antigen and protective efficacy against Bacillus anthracis, Lett. Appl. Microbiol. 26 (1998) 56 – 60. [141] Altman G.B., Tularemia. A pathogen in nature and a biological weapon, AAOHN J. 50 (2002) 373 – 377 (quiz 378–379).

H.S. Garmory et al. / Advanced Drug Delivery Reviews 57 (2005) 1343–1361 [142] Lima K.M., Bonato V.L., Faccioli L.H., Brandao I.T., dos Santos S.A., Coelho-Castelo A.A., Leao S.C., Silva C.L., Comparison of different delivery systems of vaccination for the induction of protection against tuberculosis in mice, Vaccine 19 (2001) 3518 – 3525. [143] Vordermeier H.M., Lowrie D.B., Hewinson R.G., Improved immunogenicity of DNA vaccination with mycobacterial HSP65 against bovine tuberculosis by protein boosting, Vet. Microbiol. 93 (2003) 349 – 359. [144] Delogu G., Li A., Repique C., Collins F., Morris S.L., DNA vaccine combinations expressing either tissue plasminogen activator signal sequence fusion proteins or ubiquitinconjugated antigens induce sustained protective immunity in a mouse model of pulmonary tuberculosis, Infect. Immun. 70 (2002) 292 – 302. [145] Triccas J.A., Sun L., Palendira U., Britton W.J., Comparative affects of plasmid-encoded interleukin 12 and interleukin 18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis, Immunol. Cell Biol. 80 (2002) 346 – 350. [146] Martin E., Kamath A.T., Briscoe H., Britton W.J., The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette-Guerin) in protecting against systemic Mycobacterim avium infection, Immunology 109 (2003) 308 – 314. [147] Shyu R.H., Shaio M.F., Tang S.S., Shyu H.F., Lee C.F., Tsai M.H., Smith J.E., Huang H.H., Wey J.J., Huang J.L., Chang

[148]

[149]

[150]

[151]

[152] [153]

[154]

1361

H.H., DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice, J. Biomed. Sci. 7 (2000) 51 – 57. Clayton J., Middlebrook J.L., Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A, Vaccine 18 (2000) 1855 – 1862. Bennett A.M., Perkins S.D., Holley J.L., DNA vaccination protects against botulinum neurotoxin type F, Vaccine 21 (2003) 3110 – 3117. Donnelly J., Berry K., Ulmer J.B., Technical and regulatory hurdles for DNA vaccines, Int. J. Parasitol. 33 (2003) 457 – 467. Ledwith B.J., Manam S., Troilo P.J., Barnum A.B., Pauley C.J., Griffiths T.G. 2nd, Harper L.B., Schock H.B., Zhang H., Faris J.E., Way P.A., Beare C.M., Bagdon W.J., Nichols W.W., Plasmid DNA vaccines: assay for integration into host genomic DNA, Dev. Biol. (Basel) 104 (2000) 33 – 43. Griffiths E., Assuring the safety and efficacy of DNA vaccines, Ann. N.Y. Acad. Sci. 772 (1995) 164 – 169. Livingston B.D., Newman M., Crimi C., McKinney D., Chesnut R., Sette A., Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines, Vaccine 19 (2001) 4652 – 4660. Ferreira G.N., Monteiro G.A., Prazeres D.M., Cabral J.M., Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications, Trends Biotechnol. 18 (2000) 380 – 388.