Nucleic acid vaccines: research tool or commercial reality

Veterinary Immunology and Immunopathology 76 (2000) 1±23

Mini-Review

Nucleic acid vaccines: research tool or commercial reality Lorne A. Babiuk*, Shawn L. Babiuk, Bianca I. Loehr, Sylvia van Drunnen Littel-van den Hurk University of Saskatchewan, Veterinary Infectious Disease Organization VIDO, 120 Veterinary Road, Saskatoon, Sask., Canada S7N 5E3 Received 21 March 2000; received in revised form 3 May 2000; accepted 3 May 2000

Abstract Polynucleotide immunization has captured the imagination of numerous researchers and commercial companies around the world as a novel approach for inducing immunity in animals. Clearly, the `proof-of-principle' has been demonstrated both in rodents and various animal species. However, to date, no commercial veterinary vaccine has been developed, or to our knowledge, is in the licensing phase. The present review summarizes the types of pathogens and host species for which polynucleotide immunization has been tried. We have tried to identify possible barriers to commercialization of this technology and areas that need attention if this promising technology is ever to become a reality in the commercial arena. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Polynucleotide vaccines; Veterinary vaccines; Immunity

1. Introduction The ®rst report of successful transfection of cells in vivo following injection of puri®ed DNA was published in 1960 (Atanasiu, 1962). However, it was not until 30 years later when Wolff and colleagues demonstrated that a reporter gene could be expressed in vivo for months and the expressed protein retained biological activity (Wolff et al., 1990). This observation led to the conclusion that if a foreign protein could be expressed in vivo, then

*

Corresponding author. Tel.: ‡1-306-966-7475; fax: ‡1-306-966-7478. E-mail address: [email protected] (L.A. Babiuk). 0165-2427/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 2 7 ( 0 0 ) 0 0 1 9 8 - 7

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immunity should be generated to that protein. Based on these factors, it was concluded that this could be an excellent method to immunize individuals against speci®c proteins (Tang et al., 1992; Ulmer et al., 1993). This was proven to be the case initially in mice and shortly thereafter in cattle (Tang et al., 1992; Cox et al., 1993; Ulmer et al., 1993). These initial reports ushered in the era of polynucleotide immunization Ð also termed as a third generation of vaccination. (Dixon, 1995). Although polynucleotide immunization generally stimulates immunity to a single protein it is distinct from immunization of individuals with subunit vaccines, in that the vaccine consists only of DNA or RNA which is taken up by cells resulting in the expression of protein which stimulates the host immune response. Since the protein is produced intracellularly, both MHC-I and MHC-II pathways are stimulated, thereby more closely resembling a natural infection and a more balanced immune response. In addition to inducing a broad range of immune responses, it also generally induces immunity of longer duration and if delivered appropriately can also induce mucosal immune responses (Fynan et al., 2000; Ban et al., 1997; Danko et al., 1997; Darji et al., 1997; Jones et al., 1997; Kuklin et al., 1997; Jones et al., 1998; Sasaki et al., 1998; Ruitenberg et al., 1999). Finally, it is possible to tailor the immune response to either induce a strong cellular immune response or a strong humoral response depending on the route of delivery, the co-stimulatory molecules administered with the plasmid or by tailoring the gene in such a way to either produce secreted proteins or cell-associated proteins. Other advantages of DNA immunization include safety, with no evidence of injection site reactions which are often observed with subunit proteins mixed in adjuvants, and no risk of disease caused by the vaccine as may occur with live-attenuated vaccines. Recent studies have clearly demonstrated that neonates with a relatively immature immune system, can be immunized with polynucleotide-based vaccines with minimal interference from passive maternal antibody (Monteil et al., 1996; Hassett et al., 1997; Livingston et al., 1998; van Drunen Littel-van den Hurk et al., 1999; Lewis et al., 1999b). Similarly, since there is no replicating pathogen, these vaccines can be used in immunocompromised animals. The vaccines are extremely thermostable, thus, removing the need for maintaining a cold chain. This is an extremely important factor in veterinary medicine, especially in developing countries. Thermal stability coupled with the potential for low cost production and administration as well as incorporating multi-component vaccines in a single dose makes these vaccines potentially very economical for a variety of situations. Finally, these vaccines can also be used as marker vaccines in conjunction with differential diagnostics to monitor both vaccination and disease incidence within vaccinated populations (van Oirschot et al., 1996). It is partially because of the simplicity of the procedure that this technology has progressed rapidly from the initial studies in mice to clinical studies in animals and humans in a variety of infectious disease models as well as in cancer (Hoffman et al., 1997; Calarota et al., 1998; Wang et al., 1998; Restifo and Rosenberg, 1999). However, even though the ®eld has progressed so rapidly with over 600 published manuscripts in the short history of polynucleotide immunization, numerous obstacles still exist and must be overcome before this promising technology becomes commercially viable and replaces many of the conventional vaccines used today.

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The present review will describe the methods used to deliver polynucleotide (DNA or RNA) vaccines to various animal species, the potential applications to improve the technology as well as some of the barriers to its commercialization. 2. Factors in¯uencing immune responses 2.1. Routes of immunization Initially most of the reports of DNA immunization involved direct injection of the plasmids into various muscles. These early studies created a considerable amount of debate as to whether myocytes were being transfected and acting directly as antigen presenting cells or whether other cells were involved in induction of immune responses. The importance of antigen presenting cells (APCs) in induction of immunity was clearly demonstrated through conventional immunization and it was shown that dendritic cells were the most potent APC and possessed all the accessory molecules required for induction of immunity. Since myocytes lack many of the accessory molecules and MHCII expression required for ef®cient induction of immunity, various experiments were done to identify the role of myocytes in immune induction. As a result of these studies, it is being accepted that intramuscular injection may result in transfection of the myocytes which then may act as factories for antigen expression (Corr et al., 1996; Doe et al., 1996; Spier, 1996; Fu et al., 1997; Iwasaki et al., 1997a). However, in most cases the antigen is transferred to adjacent antigen presenting cells which are then responsible for initiating the immune response. Alternatively, the resident antigen presenting cells themselves are transfected and then migrate to the regional lymph node and initiate an immune response. Adoptive transfer of transfected dendritic cells has clearly shown that such transfected cells can induce a speci®c immune response in vivo supporting the contention that transfection of dendritic cells may be critical in induction of immunity following nucleic acid immunization (Condon et al., 1996; Germain, 1998). Further support for the role of antigen presenting cells (dendritic cells) or (Langerhans cells) in in¯uencing the level of immunity induced by DNA immunization is provided by the studies involving intradermal delivery of plasmids encoding speci®c antigens or reporter genes. For example, plasmids encoding green ¯uorescence protein have been observed in Langerhans cells present in lymph nodes following gene gun delivery (Condon et al., 1996). In most cases, the quantity of plasmid required to induce an immune response, if delivered intradermally, either by needle or a gene gun, is signi®cantly lower than that required to induce an immune response with the same plasmid following intramuscular delivery. One possible reason for this may be the high density of specialized dendritic cells in the skin, which may either, be transfected directly or pick up antigen from transfected local ®broblasts, and migrate to the regional lymph node to initiate the immune response. Further support for the role of dendritic cells in induction of immunity following DNA immunization is provided by the `Van Gogh experiments' of Dr. Stephan Johnson, where removal of the transfected ear shortly after immunization did not prevent the development of immunity (Barry and Johnston, 1997). Since dendritic cells rapidly

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migrate out of the ear following immunization, removal of any transfected ®broblast, which would then transfer antigen to the antigen presenting cells is of little consequence for the development of immune response. Based on these studies, improvement of the transfection ef®ciency and expression of antigen in dendritic cells should be one of the targets of research for improving the ef®cacy of DNA immunization. This will be especially important in large animals and humans where it is more dif®cult to induce immunity to most antigens than has been observed in mice. The reason for this reduced ef®cacy of immune reduction is presently unknown but it may be related to the transfection ef®ciency of cells in these animals. The observation that human adenoviral DNA is 100±1000 times more ef®cient in transfecting certain cells in vitro versus bovine adenovirus DNA, demonstrates that there are many factors which may in¯uence the transfection ef®ciency as well as the ability to express speci®c genes in different cells. Not only is the ef®ciency of transfection, and subsequently, the level of immunity in¯uenced by the route of immunization, but so is the type of immune response that is generated. Although it is dif®cult to generalize, since the type of immune response generated is in¯uenced by many factors including the gene construct, it is often observed that needle injection induces a strong Th1-like immune response, whereas gene gun delivery induces a stronger Th2 antibody isotype response. In most cases, both methods of delivery induced CTL responses. Although the predominance of Th1 antibody responses over Th2 antibody responses may be related to the quantity of DNA used by each of the different methods, it is possible to shift the antibody isotype response by altering the quantity of DNA used by either of these delivery methods (Feltquate et al., 1997; Leitner et al., 1997; Prayaga et al., 1997). 2.2. Presence of CpG motifs One possible reason for being able to shift the type of immune response generated by modulating the quantity of plasmid DNA used may be related to the presence of CpG motifs in the plasmid. Recent studies have clearly demonstrated that CpG motifs can stimulate a cytokine cascade that directs the immune response and in theory acts as an adjuvant (Klinman et al., 1998; Krieg et al., 1998). Since these CpG motifs favor the Th1 dominated cytokine pathways, increasing the concentration of DNA or CpG motifs in a vaccine should shift the response to a Th1-like response. Further support for this contention is that it is possible to alter a Th2-like response induced by subunit vaccines to a Th1-like response if CpG is incorporated as an adjuvant (Davis et al., 1999). Fig. 1 is a simpli®ed diagram demonstrating the multiple effects of CpG motifs on cells of the immune system. Brie¯y, by interacting with speci®c cell populations, CpG can stimulate B cells to proliferate, secreting IgG, IL-6 and IL-10 (Klinman et al., 1996). In addition, CpG motifs can directly activate monocyte/macrophages or dendritic cells (antigen presenting cells) to secrete interferon, IL-6, IL-12, GM-CSF, chemokines and TNFa. Similarly, CpG motifs either directly or through the cytokines produced by antigen presenting cells, stimulate NK cells to produce interferon g as well as increased killer activity. As a result of these activities, CpG motifs rapidly stimulate host defenses through the strong Th1-like cytokines which are induced, polarizing immune responses to a Th1-like response in both DNA vaccines as well as if used as an adjuvant with protein

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Fig. 1. Simpli®ed summary of CpG effects on leukocyte activity. A CpG motif is comprised of a CG ¯anked by different purines and pyrimidines as follows X1X2CGY1Y2 when X1 is a purine, X2 is not a C, Y1 not a G, and Y2 is a T. A synthetic olignonucleotide is most active if it contains two CpG motifs in a 10±20 base sequence. The spacing between each CpG will also in¯uence the activity.

vaccines (Weiner et al., 1997; Krieg et al., 1998). These studies clearly demonstrate the importance of CpG sequences in DNA vaccines. Further support for the role of CpG sequences in DNA vaccines are studies where CpG sequences have been removed from the plasmid resulting in extremely poor or no induction of immune responses (Sato et al., 1996). 3. Antigen presentation/immune modulation 3.1. Enhancing ef®cacy Modi®cation of the gene can often enhance or modulate the type of immune response generated following DNA immunization (Levy, 1993; Barry and Johnston, 1997; Chen et al., 1998). Using a bovine herpes virus gD as a model antigen we constructed three different forms of the gene for intramuscular immunization. The authentic form with a membrane anchor intact, or a version with a transmembrane anchor removed, induced rapid immune responses whereas a cytosolic form, lacking both a signal sequence and a transmembrane domain, induced immunity with a 2-week lag. However, by 12 weeks all animals had equivalent levels of immunity (Lewis et al., 1999a). Although all three constructs generated equivalent levels of immunity, although with different kinetics, the type of immune response was not the same, with the full-length and cytosolic gD constructs (intracellular antigens) exhibiting a predominance of IgG2a antibodies whereas animals immunized with the secreted form showed a predominance of IgG1. Similarly, other membrane anchored glycoproteins from parain¯uenza-3 and measles virus

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stimulated a predominance of IgG2a whereas the secreted form generated predominantly an IgG1 antibody response. Induction of a correct immune response is critical in all vaccination regimes. Indeed, induction of the wrong type of response can have devastating effects that lead to severe pathology. For example, with Leishmania or mycobacteria infections, immunization protocols that induce a Th1 response are protective whereas induction of a Th2 response leads to severe immunopathology, disease progression and death (Bretscher, 1992; Bretscher et al., 1992). Similarly, immunization with a formalin-killed respiratory syncytial virus (RSV) vaccine induces a Th2-like response with increased lung pathology and eosinophilia (Connors et al., 1994). Dissection of the response induced by the individual proteins of RSV reveal that glycoprotein (G) induces a Th2-like response whereas the fusion protein (F) induces a Th1-like response (Srikiatkhachorn et al., 1999). Interestingly, a DNA vaccine encoding the F protein could redirect an existing RSV Th2like response to a Th1-like response (Li et al., 1998). A plasmid encoding the amino terminal portion of hepatitis C nuclear protein, when it was constructed to secrete the protein failed to deviate the serum IgG isotype (Geissler et al., 1998). Thus, it is dif®cult to make generalized conclusions stating that a speci®c type of construct will induce a speci®c type of immune response. These studies clearly demonstrate that one needs to test the immune response of protein to ensure that the appropriate type of immune response is induced by the different constructs. The type of immune response induced by each construct may also be in¯uenced by the species. Unfortunately, there are not enough constructs tested to date in different species to draw any major conclusions regarding species effects. These studies clearly demonstrate the importance of understanding the antigen, form of antigen and presentation in induction of an immune response. In addition to modulating the kinetics and the type of immune response by modifying the gene encoding the protein, removal of the transmembrane domain may be required in some instances where the protein is toxic to the host cell (Barry and Johnston, 1997). Thus, if it is over expressed, it could prematurely kill the transfected cell and reduce the quantity of antigen available for immune stimulation. Truncating the protein to either remove these toxic sequences Ð if they are known Ð or reduce the intracellular concentration of the protein by releasing it into the extracellular milieu may be a great advantage in elevating immunity by DNA vaccines. This has been shown with bovine herpesvirus-1 gD, a potentially toxic protein. When the gene encoding the full-length gD is introduced intradermally into cattle, the immune response is signi®cantly lower than if the protein is secreted into the extracellular environment. Many viral genes have codon biases, which do not re¯ect the codon bias of the host cell they replicate in. By altering the codon bias, it has been possible to increase the level of expression of at least a number of genes tested (Schrijver et al., 1998). Thus, if a speci®c gene is dif®cult to express in vitro, this might be a strategy that should be employed. Furthermore, genes from many RNA viruses are expressed poorly in vectors that replicate in the nucleus. The reason for this is probably related to the fact that RNA viral genes contain potential splicing sites which result in modi®cation of mRNA as it is transported from the nucleus to the cytoplasm. By modifying the gene to remove these splice sites, it may be possible to increase the level of expression of the speci®c protein. Using a bovine adenovirus expression vector, we have signi®cantly increased the level of RNA viral gene

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expression in cells by either incorporating an intron or re-engineering the gene to remove potential splice sites (unpublished results). This may be one of the reasons that incorporation of introns into plasmids dramatically increases immune responses to many genes (Li et al., 1998). In addition to altering the form of antigen, it is also possible to add additional known helper epitopes to enhance immune responses to poorly immunogenic proteins (Ciernik et al., 1996; Thomson et al., 1996). Furthermore, by incorporating speci®c Th1 and Th2 helper epitopes it may be possible to simultaneously enhance and redirect the immune response (Kuhrober et al., 1997; Wild et al., 1998). It may also be possible to enhance the immunogenicity of poorly immunogenic epitopes by modifying their sequences. This can be achieved by screening peptide libraries derived from the original sequence (Wilson et al., 1999). This may be especially important in outbred species where genetic restriction may occur with some proteins. Since cytokines modulate both the kinetics and type of immune responses generated, some researchers have used cytokines to accelerate or redirect immune response with varying degrees of success (Maecker et al., 1997; Chow et al., 1998). Unfortunately, the in vivo half-life of cytokines is extremely short, requiring multiple administrations to show ef®cacy. Therefore, it appears logical to introduce genes encoding cytokines or chemokines that would produce the desired effect over an extended period of time. Thus, cytokine or chemokine genes can be introduced either as a separate plasmid or incorporated into the same plasmid that encodes the gene for the pathogen of interest (Irvine et al., 1996; Biragyn et al., 1999). The cytokine genes used most extensively include IL-2, IL-6, IL-7, IL-12, IFNg and GM-CSF. In these instances, these chemokines or cytokines augmented immune responses either by elevating the level of immunity, shifting the response towards the Th1 or Th2 response or in some cases increasing the number of animals responding to the immunization. Increasing the number of responders will be critical, especially in outbred populations where responses to a single antigen may need to be broadened. In addition to cytokine modulation of immune responses, incorporation of costimulatory molecules such as B7 may be critical to ensure that anergy does not occur, especially after stimulating T cells with minute quantities of antigen. Not only are costimulatory molecules important for induction of immunity, but by using genes encoding B7.2 and B7.1 it is becoming possible to dissect the differential roles of these two molecules in immune modulation, and hopefully provide us with a better understanding of immune regulation (Iwasaki et al., 1997b; Kim et al., 1997). Thus, DNA immunization is not only providing us better ways of stimulating protective immunity but also helping us understand immune regulation. This will be critical as we hope to develop novel methods to manipulate the host immune response in various situations (see Section 4). Possibly the simplest way of enhancing immunity to DNA vaccination is through the incorporation of unique CpG sequences into the plasmid. The discovery that certain nonmethylated palindromic DNA sequences containing CpG oligonucleotides, can activate innate immune responses by activating monocytes, and NK cells, dendritic cells and B cells to secrete cytokines in an antigen independent manner has enhanced our understanding of how DNA vaccines induce immune responses, in the presence of very minute quantities of antigen. Methylation or removal of these CpG sequences from a plasmid abrogates the ability of the plasmid to induce an immune response (Lipford et al.,

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1997). Thus, it is hypothesized that these sequences enhance immune response by acting as an adjuvant and assist in driving the immune response towards a Th1-like response (Krieg, 1996). Indeed, these observations have prompted the use of CpG as an adjuvant even with conventional or subunit protein vaccines (Weiner et al., 1997; Davis et al., 1999). In these instances, the incorporation of CpG with alum not only enhanced the magnitude of the immune response but also redirected it from a Th2-like to a Th1-like response. This observation is providing us with an addition to our armamentarium in enhancing vaccination of both humans and animals. Although the exact mechanism of CpG activation of the immune response is not completely clear, it does appear that interaction of CpG with speci®c intracellular proteins, activates speci®c signal transduction pathways required for ef®cient induction of immunity. For example, treating resting dendritic cells with CpGs activates them to become strong antigen presenting cells (Witmer-Pack et al., 1987; Heu¯er et al., 1988). Although it is possible to co-administer CpG with protein antigens or plasmid vaccines, recent studies suggest that it may be more effective to incorporate these sequences into a plasmid backbone because co-administration of CpG oligonucleotides with plasmid vaccines may reduce the ef®cacy of DNA vaccination (Weeratna et al., 1998). Whether this is due to competition or some other factor such as induction of cytokines which turns off promoters or reduced transfection of activated cells, is presently unknown. However, this is becoming a very active area of investigation. Similarly, we are not yet sure as to what role speci®c sequences surrounding the CpG motifs play in induction of immunity in different species. We predict that these parameters will become clearer in the near future. Thus, it should be possible to design speci®c CpG motifs and plasmids that optimally stimulate immune responses in each species of interest. Most of the work conducted till date has involved immunization with a non-replicating plasmid. Unfortunately, over 90% of the plasmid never enter the cell and are degraded within a few hours. Secondly, since each transfected cell probably contains only one copy of the plasmid, the level of protein production in the cell is extremely low. Thus, if it was possible to increase the transfection ef®ciency and simultaneously have the transfecting plasmid replicate in the cell to increase the level of intracellular expression it might be possible to dramatically increase the level of immunity. By using replicons derived from alphaviruses, such as Sindbis, Semliki forest virus and Venezuela equine encephalitis virus, investigators have not only developed methods to increase the copy number of RNA molecules in a cell, but also improve the transfection ef®ciency of these cells (Berglund et al., 1996; Tubulekas et al., 1997). By replacing the genes encoding the alpha virus structural proteins with a gene of interest for induction of immunity, it is possible to package the polynucleotide vaccine in a protective coat which enhances cellular uptake, thereby overcoming one of the greatest barriers to polynucleotide vaccination. Since the alpha virus replicons function in most mammalian cells, and more importantly, the replication occurs in the cytoplasm, thereby removing the problem of splicing associated with expression of bacterial or RNA viral genes in the nucleus of cells, it is possible to overcome another barrier which is often observed in DNA plasmid vaccines. These vectors have been shown to induce both humoral and cellular immunity after a single intramuscular injection, often of higher magnitude than has been observed with DNA-based vectors (Zhou et al., 1994; Herweijer et al., 1995;

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Dubensky et al., 1996; Hariharan et al., 1998). In addition to possibly increasing the quantity of antigen or ef®cacy of transfection, replicons may also induce interferon, which may act as an adjuvant to direct the immune response. Double-stranded RNA is well known to stimulate interferon and to function as an adjuvant for both cellular and humoral immune induction (Wright and Alder-Moore, 1985; Chelbi-Alix and Sripati, 1994). 4. Nucleic acid vaccines as research tools Most of the interest to date in polynucleotide immunization has been in developing safer and more effective vaccines. However, this technique also has many other applications which may prove to be as important or more important than vaccination per se. For example, monoclonal antibodies have become indispensable tools for diagnosis, puri®cation and characterization of proteins, including protein structure, epitope mapping and protein±ligand interactions. Most monoclonal antibodies available have been developed by conventional immunization protocols which are very good at inducing immune responses to highly immunogenic proteins or epitopes. Unfortunately, these protocols do not result in the induction of clones secreting antibodies to weakly reactive epitopes or to minor proteins present in a virus, bacterium or parasite. The same can be stated for host cellular proteins to which monoclonal antibodies are needed. Using DNA immunization, immune responses can be induced to these minor proteins or subdominant epitopes which can then be better characterized with regard to their role in pathogenesis. Although the era of automated DNA sequencing of many pathogens has resulted in the rapid identi®cation of the genes of these pathogens, it has not provided us with any functional information regarding the importance of these proteins in host pathogen interactions. With the expanded genomic base of numerous pathogens combined with functional genomics, it is hoped that we will be able to rapidly identify homologous genes/proteins from related pathogens that may have similar roles in pathogenesis. Unfortunately, for many pathogens we do not yet know which genes or proteins are important in induction of immune responses or in altering host pathogen interactions. Using an expression library combined with DNA immunization, as developed by Dr. Stephan Johnston, provides us with an excellent opportunity to identify putative protective proteins for the entire array of proteins encoded by the genes of each pathogen (Johnston and Barry, 1997). Even more importantly, it may demonstrate that proteins we did not even suspect would be protective may play an important role in pathogenesis. In this way, it may be possible to assemble a single plasmid or a series of plasmids that contain epitopes from a variety of important proteins thereby inducing the broadest range of responses rather than just using the most abundant protein present in the pathogen or the one that is best characterized. Thus, the expression library immunization (ELI) technology or second and third generations of this approach should be an excellent addition to proteomics, and functional genomics, as they relate to vaccine discovery and development. These techniques will be especially important for those pathogens that have been poorly characterized or for newly emerging pathogens.

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In addition to identifying various genes present in a pathogen that may induce protective responses, a similar approach can be used to identify genes which may induce deleterious immune responses and thereby enhance disease. Such an approach would be extremely bene®cial in situations where one was proposing to develop a live gene deleted vaccine or for use as a live vector. Deletion or modi®cation of a gene, especially if it is non-essential, would dramatically increase both the ef®cacy and safety of such livevectored vaccines.The ability to activate speci®c B or T cells (CD4‡ and CD8‡) is the foundation of vaccinology. In the two-signal hypothesis proposed by Bretscher and Cohn (Bretscher and Cohn, 1970), signal 1 is provided by the antigen but the second precise identify of the signal is presently unknown. Since both signals are required for induction and development of immunity, it may be possible to provide the ®rst signal by DNA immunization with a single MHC II peptide and simultaneously provide putative second signals such as cytokines or co-stimulatory molecules to identify the role of each in both initiating and driving the CD4‡ response. Similarly, B cells and CD8‡ cells also require second signals provided by CD4‡ T cells. Using DNA immunization, it should be possible to elucidate the molecular component required for help as described above. 5. DNA vaccines in veterinary species Although the majority of publications regarding DNA immunization have used rodent models, very few vaccines will be developed to speci®cally control infectious diseases in mice. This is especially the case when most of the vaccines are being developed or tested in mice against agents that do not naturally infect mice. Thus, the main goal is to test these vaccines against important pathogens in livestock and other animals. Table 1 summarizes the various animal species and pathogens of veterinary interest that have been used in nucleic acid vaccination to date. It is clear that numerous routes and delivery methods have been tried. Furthermore, these studies have supported those done in mice where intradermal delivery appears to be more effective than intramuscular delivery. Indeed, in large animals there appears to be an even greater enhancement of immune responsiveness following intradermal delivery compared to intramuscular delivery. Whether this is related to the reduced ef®cacy of uptake by myocytes in large animals following DNA vaccination or other factors remains to be determined. Certainly, the amount of muscle damage, which appears to be critical for the induction of an immune response appears to be different in larger animals than in mice. We deliver 1 mg of DNA in a 2 ml volume to the gluteal muscle of a calf because higher volumes would be very impractical. In both mice and large animals, it is clear that polynucleotide immunization can be achieved in neonatal animals either in the presence or absence of maternal antibody. This is a critical observation since immunization at birth will be extremely advantageous under various livestock management practices (Potter and Babiuk, 1993). 6. Delivery Presently, over 90% of all the plasmids delivered in vivo get degraded before they even enter the cell, let alone enter the nucleus and initiate transcription. Thus, the greatest

Table 1 Summary of DNA vaccines tested in animals of veterinary importancea Species

Sheep

Pigs

Routeb

Number of Delivery device immunisations

Serum antibodies

Proliferation CTL Protection Reference

Total Neutralizing

i.m. i.d., i.m.

5 2, 3

Needle Needle

‡ ‡

‡ ‡

nd ‡

nd nd

‡ ‡

BHV-1 gD

i.d. (neck)

2

‡

‡

nd

‡

BRSV G protein BRSV G protein

6 3

nd nd

nd nd

nd nd

‡ ‡

Schrijver et al. (1997) Schrijver et al. (1998)

BVDV E2

i.d.‡i.m. i.d., i.m.; i.d. (hind leg) i.m.

BioRad Helios1 ‡ gene gun Needle‡PigJet ‡ Needle, PigJet ‡

Cox et al. (1993) van Drunen Littel-van den Hurk et al. (1998) Braun et al. (1999)

3

Needle

‡

‡

‡

nd

‡

Harpin et al. (1999)

BHV-1 tgD

i.d.

2

Needle

‡

‡

‡

nd

nd

Boophilus microplus Bm86 II-1b, GM-CSF Corynebacterium pseudotuberculosis, phospholipase D Cryptosporidium parvum (surface antigen 15, 60 kD) Taenia ovis 45 W Theileria annulata Tams 1±1, 1±2 E. coli K88abfae G FMDV (entire genome) FMDV (entire genome), FMDV P1‡3C PRRS ORF 5 (gp5)

i.m.

2

Needle

‡

nd

‡

nd

‡

van Drunen Littel-van den Hurk et al. (1999) De Rose et al. (1999)

i.m.

2

Needle

‡

nd

nd

nd

‡

Chaplin et al. (1999)

nd

nd

nd

nd

Jenkins et al. (1995)

i.d., i.m. i.m.

3 3

Jetinjection ‡ (Ped-O-Jet, Stirn Industries) Needle ‡ Needle ÿ

nd nd

nd nd

nd nd

nd ‡

Rothel et al. (1997) d'Oliveira et al. (1997)

i.m. i.d., i.m. i.d., i.m.; i.d. (groin) i.d.‡i.m.

2 4 4, 2

‡ ‡ ‡

nd ‡ ‡

nd nd nd

nd nd nd

nd ‡ ‡

Turnes et al. (1999) Ward et al. (1997) Beard et al. (1999)

3

Needle Needle Needle; Accell1 gene gun Needle

‡

‡

‡

nd

‡

PRRS ORF 4,5,6,7

i.m.

4

Needle

‡

‡

‡

nd

nd

Pirzadeh and Dea (1998) Kwang et al. (1999)

i.m. (hind limb), 3 imam.

11

BHV-1 gD BHV-1 gD and tgD

L.A. Babiuk et al. / Veterinary Immunology and Immunopathology 76 (2000) 1±23

Cattle

Origin of gene

12

Table 1 (Continued ) Species

Origin of gene

Dogs, cats

Serum antibodies

1±2 3±4

‡ ‡

‡ ‡

nd ‡

nd nd

ÿ ‡

Monteil et al. (1996) Gerdts et al. (1997)

‡ ‡ ‡

‡ ‡ nd

nd nd nd

nd nd nd

‡  nd

Le Potier et al. (1997) Monteil et al. (1997) Garmendia et al. (1998) van Rooij et al. (1998)

Total Neutralizing

i.m. i.d., i.m.; i.d. (back)

PRV gD, plus virus PRV gD, plus virus PRV IE 180

i.m. i.m. i.d.‡i.m.

PRV gDtgBtgC

i.d., i.m.; i.d. (neck) i.d. i.d.‡i.m.

3

Needle; PigjetTM ‡

‡

‡

nd

‡

3 3

I.D.A.L.TM Needle

‡ ‡

‡ ‡

nd ‡

nd nd

‡ ‡

i.m.

1

Needle

‡

‡

nd

nd

‡

Accell1 gene gun Needle

‡

‡

nd

nd

‡

Gerdts et al. (1999) Haagmans et al. (1999) Somasundaram et al. (1999) Macklin et al. (1998)

‡

‡

‡

‡

‡

Romito et al. (1999)

PRV gB‡gDGMÿ CSFILÿ2, IFN-g Swine in¯uenza virus, HA (H1N1) NP African horse sickness virus VP2 Equine in¯uenza virus HA

Needle Needle; hypodermic injector: (I.D.A.L.TM) 1, plus virus Needle 1, plus virus Needle 3 Needle

Proliferation CTL Protection Reference

i.d. (several 2 sites) and tongue i.m. Several

i.d.‡mucosa (several sites each) CPV VP1 i.m. IL-2, IL-6, GM-CSF oral mucosa‡ i.d. (lumbar) Rabies virus glycoprotein i.m. Rabies virus glycoprotein i.d., i.m.; scari®cation

3

Powder-JectXR gene gun

‡

‡

nd

nd

‡

Lunn et al. (1999)

2

Needle Accell1 gene gun Needle

‡ nd

‡ nd

nd nd

nd nd

‡ nd

Jiang et al. (1998) Keller et al. (1996)

‡

‡

nd

nd

‡

Perrin et al. (1999)

Needle; MonoVacc vaccinator

‡

‡

nd

nd

nd

Osorio et al. (1999)

2±4 2

L.A. Babiuk et al. / Veterinary Immunology and Immunopathology 76 (2000) 1±23

Dogs

Number of Delivery device immunisations

PRV gD PRV gD/gI, gC

PRV gB‡gC‡gD/IgE PRV gD

Horses

Routeb

Cats

i.m. i.m.

2 2

Needle Needle

‡ ÿ

ÿ nd

nd nd

nd nd

‡ ÿ

FIVdeltaRT, IFN g FIVdeltaRT, IFN g

i.m. i.d., i.m.

3 3

Needle Needle

ÿ nd

ÿ nd

nd nd

‡ ‡

‡ ‡

Cuisinier et al. (1997) Richardson et al. (1997) Hosie et al. (1998) Flynn et al. (2000)

1

Needle

‡

‡

nd

nd

‡

Anderson et al. (1996)

1 1

Needle Needle

‡ ‡

nd nd

nd ‡

nd nd

nd nd

Kanellos et al. (1999a) Kanellos et al. (1999b)

IHNV NP and G protein i.m. Fish (rainbow trout) Gold®sh b-Gal i.d., i.m., i.p. b-GalGM-CSF i.m. Poultry Chicken in¯uenza H7 chicken Chicken in¯uenza H7 NDV F

i.m., i.v., i.p., 2 i.b., s.c.; i.o., i.t. i.v., i.p., s.c. 2 i.m. 1

Needle; drops

nd

nd

nd

nd

‡

Fynan et al. (2000)

Needle Needle

‡ ‡

‡ nd

nd nd

nd nd

‡ ‡

Robinson et al. (1993) Sakaguchi et al. (1996)

Poultry ducks Poultry turkey

DHBV pre S/S‡S protein Chlamydia psittaci MOMP Chlamydia psittaci MOMP

i.m.

3

Needle

‡

‡

nd

nd

‡

Triyatni et al. (1998)

i.d. (back)

2

‡

nd

nd

nd

‡

‡

nd

nd

nd

‡

Vanrompay et al. (1999a) Vanrompay et al. (1999b)

Rabbits

CRPV L1

i.c. (30 sites)

4

‡

‡

‡

nd

‡

Sundaram et al. (1997)

CRPV E1, E2, E6, E7

i.c. (back)

3

ÿ

nd

‡

nd

‡

Han et al. (1999)

CRPV E1, E2, E6, E7

i.m.

3

Biorad Helios1 gene gun Needle, drops, Biorad Helios1 gene gun Accell1 gene gun Biorad Helios1 gene gun Needle

ÿ

nd

‡

nd

ÿ

Han et al. (1999)

a

i.m., i.n., i.d. (abdominal)

BHV: bovine herpes virus, BRSV: bovine respiratory syncytial virus, BVDV: bovine viral diarrhea virus, CPV: canine papilloma virus, CRPV: cottontail rabbit papilloma virus, DHBV: duck hepatitis B virus, E. coli: Escherichia coli, EIV: equine in¯uenza virus, FIV: feline immunode®ciency virus, FMDV: foot-and-mouth disease virus, IHNV: infectious haematopoietic necrosis virus, NDV: newcastle disease virus, PRRSV: porcine reproductive and respiratory syndrome virus, PRV: pseudorabies virus, b-Gal: b-galactosidase, MOMP: major outer membrane protein, ORF: open reading frame, GM-CSF: granulocyte and macrophage colony stimulating factor, IFN: interferon, IL: interleukin. b i.b.: intrabusal, i.c.: intracutaneous, i.d.: intradermal, i.e.: intraepidermal, i.m.: intramuscular, i.n.: intranasal, i.o.: intraorbital, i.p.: intraperitoneal, i.t.:intratracheal, i.v.: intravenous, RT: reverse transcriptase, s.c.: subcutanous, nd: not determined.

L.A. Babiuk et al. / Veterinary Immunology and Immunopathology 76 (2000) 1±23

FIV gp120‡p10 FIV env

13

14

L.A. Babiuk et al. / Veterinary Immunology and Immunopathology 76 (2000) 1±23

challenge in polynucleotide immunization today is in developing ef®cient delivery systems. The delivery systems used today include (1) mechanical (microinjection by various types of needles, microneedles, pressure injection or particle bombardment); (2) electrical (electroporation, ionophoresis); and (3) chemical (liposomes, dendrimeres, various polymers). Each of these approaches has different advantages and disadvantages in ensuring that the multi-step process of DNA entry across the cell membrane, migration through the cytoplasm, entry into the nucleus Ð the site of transcription Ð and ®nally transcription and exit of mRNA into the cytoplasm where it is translated is as ef®cient as possible. Thus, there is a chance of degradation of nucleic acid at each stage of this multistep process. Since viruses have evolved excellent mechanisms for delivery of genes, not only to cells but to speci®c individual cells and even to speci®c sites within cells, such as the nucleus, it appears logical to try and emulate what nature has evolved over centuries. The replicon system based on alpha viruses is one of the most ef®cient systems developed to date. However, it is also possible to simulate viral targeting mechanisms by purely synthetic means. For example, many of the lipid based (liposomes) delivery systems protect the DNA like a viral envelope and if designed properly they have improved transfection (cellular entry) ef®ciency. Presently, many novel lipid formulations are being developed that are much more ef®cient at transfection in vivo than the original liposomes developed in 1987 (Lipofectin) to aid in vivo transfection (Felgner et al., 1987; Monkkonen and Urtti, 1998; Templeton and Lasic, 1999). By incorporating speci®c proteins or ligands into protein-based or lipid-based delivery systems that target speci®c cells, it should be possible to not only increase the ef®cacy of cellular uptake of plasmids but also to target speci®c cell populations. For example, ligands such as asialoorosomucoid (ASOR) or epidermal growth factor (EGF) can target speci®c asialoglycoprotein receptors or EGF receptor bearing cells, respectively (Wu et al., 1989; Schaffer and Lauffenburger, 1998). Similarly, the use of reovirus s 1 protein can target M cells of the Peyer's patch to induce mucosal immunity (Wu et al., 2000). If we could identify a speci®c ligand such as chemokine that interacts with receptors on dendritic cells, this might greatly enhance both uptake by the most ef®cient antigen presenting cells, thereby enhancing immune responses. Some studies have been conducted to target mucosal immune sites by incorporating DNA into various polymers. Biodegradable polymers such as poly (DL-lactide-coglycolide) protect the DNA, but also target it to M cells of the gastrointestinal tract (Jones et al., 1997; Ando et al., 1999). Furthermore, these co-polymers can be designed to release DNA over extended periods of time providing booster immunizations. Although these pulsatile or slow release delivery systems have been shown to be critical in protein immunization, it is not yet clear whether this would have an advantage in DNA immunization. The fact that these polymers are already approved by the US Food and Drug Administration makes them attractive delivery vehicles for DNA immunization. Similarly, alginate particles are also being used for this purpose (Aggarwal et al., 1999). However, even if the DNA enters cells it is not a guarantee that it will enter the nucleus, the site where transcription must occur. Transport from the cellular membrane to the nuclear membrane is critical if expression is to be achieved. Once again we can learn from years of viral evolution where viruses target their nucleic acid to the nucleus. Recently, it has been shown that combining a nuclear localization signal with a peptide

L.A. Babiuk et al. / Veterinary Immunology and Immunopathology 76 (2000) 1±23

15

nucleic acid (PNA) facilitated nuclear transport of transfected DNA. (Branden et al., 1999) Based on these studies more emphasis must be directed at understanding and exploiting nuclear targeting to ensure the most ef®cient transfection possible. Since the skin is one of the most promising immune induction sites, because of the high density of APCs in the dermal layer, many delivery systems have targeted the skin as a potential site for immunization. Unfortunately the skin, especially the stratum corneum, is a major barrier to ef®cient uptake of nucleic acids. However, recent reports have demonstrated that topical application of plasmids formulated in liposomes is feasible although quite inef®cient (Shi et al., 1999). To improve transcutaneous delivery, a number of mechanical devices have been developed. These include microinjection, particle bombardment and electrical pulses. Although microinjection is possible, this can only be done on a single cell making it totally impractical for immunization regimes. To overcome this labor-intensive hurdle, gold or tungsten particle bombardment has been developed and shown to be relatively ef®cient at carrying the DNA directly into the cell. This approach is more ef®cient than pressure injection of naked DNA since many particles penetrate cells delivering their `payload' into the cell. The disadvantage of such an approach is the minute quantity of DNA that can be coated onto the particles and the number of particles that can be injected in one shot. An additional disadvantage, depending on the type of animal being immunized, is that in some cases the DNA may shear from the particles as they penetrate through the keratinocytes. This is especially the case in large animals such as cattle, where transfection of the super®cial keratinocytes does not lead to ef®cient induction of immune responses. Furthermore, these particles must be maintained in a dry state to ensure that the DNA remains ®rmly attached to the particles. Although electroporation and ionophoresis have been demonstrated to deliver plasmids in vivo, improved devices will need to be developed to make these practical in livestock management systems (Zhang et al., 1996; Watkins et al., 1999). What is clear is that novel combinations of mechanical, electrical and chemical methods are being developed and it may be possible to actually combine a number of these delivery systems to improve ef®ciency of DNA immunization in the future. 7. Barriers to commercialization For product to progress from research to the marketplace, a large number of hurdles need to be overcome. These include: (1) cost of production; (2) ease of delivery; (3) ef®cacy; and (4) safety. Many of these factors are controlled by different regulatory groups with different concerns. For example, the recent hysteria initiated by various `green' advocacy groups regarding genetically modi®ed foods can keep the best products from the marketplace. Unfortunately, government regulators often can become extremely cautious when confronted with new technology and vocal resistance by a small minority and they may not always follow a science-based assessment approach, which should be the only criteria for regulation of products. Uncertainty about which regulatory parameters will be important or irrational concerns about ethical or societal aspects of the product, which are dif®cult to measure, may discourage companies from bringing

16

L.A. Babiuk et al. / Veterinary Immunology and Immunopathology 76 (2000) 1±23

products to the marketplace. As a result, regulatory agencies will lose credibility with manufacturers of products. They have already begun to lose credibility with the general population as a result of the recent contamination of poultry in livestock feed with benzodioxan and the bovine spongiform encephalopathy (BSE) crisis in Europe. The recent recalls of food due to contamination with Escherichia coli O157 and Listeria monocytogenes in hamburger and processed meat are other examples which groups can point to, to discredit the regulatory system to their advantage. Unfortunately, although the recalls may have nothing to do with DNA vaccines, they all raise the question of whether or not our foods are safe and whether countries have the appropriate safeguards in place. Although the chance of adverse effects on food safety by residual plasmid is extremely low it is impossible to prove a negative. Therefore, regulators must make a judgment on the level of risk these products pose and communicate these very clearly to society. These factors all add to the cost of registration of the product, which may discourage companies from registering what might be a better product than is presently available. A second major factor that adds to the cost of DNA vaccines is the multiple patents that will need to be assembled to ensure a product does not infringe on another company or organization's property. Many of the DNA vaccines require rights to genes encoding protective antigens, promoters, technology for delivery of the genes and access to DNA vaccine technology which are all patented. If these patents are held by numerous groups, it will require a company to pay royalties to a minimum of four or ®ve different organizations for a single vaccine. Such stacking royalties reduce the return to everyone. However, if any one of these organizations is unreasonable in their demands, the cost for the vaccines becomes prohibitive and a product may never be licensed for the bene®t of society. This has recently been referred to as the `tragedy of the commons' by Heller and Eisenberg (Heller and Eisenberg, 1998). Although it has not been an issue in many vaccines licensed to date, the potential is real in DNA vaccine development. A ®nal hurdle to commercialization of DNA vaccines is that of delivery. Thus, even if one can identify the ideal proteins and express them in vivo, the ef®ciency of expression is still low. Without being able to deliver the genes and express them at suf®cient levels, and at the correct site, the technology may not be commercially viable. For example, although moderate levels of immunity to equine in¯uenza were developed after approximately 20 shots with a gene gun in multiple sites, no producer will embrace such a labor-intensive approach for immunization (Lunn et al., 1999). Thus, a single userfriendly delivery system must be developed that induces the type of immune response required at the port of entry of the pathogen. It is our contention that more emphasis must now be placed on delivery and improving immune responses in target species. Clearly, the phenomenon of DNA vaccination has been demonstrated to work in mice. Our challenge now is to demonstrate that it can be developed economically for induction of immune responses under various management systems in livestock. Acknowledgements Work in the authors' laboratories is supported by the Medical Research Council and the Natural Sciences and Research Council of Canada. The authors thank Sherri Hueser and Michelle Balaski for excellent secretarial assistance.

L.A. Babiuk et al. / Veterinary Immunology and Immunopathology 76 (2000) 1±23

17

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Han, R., Cladel, N.M., Reed, C.A., Peng, X., Christensen, N.D., 1999. Protection of rabbits from viral challenge by gene gun-based intracutaneous vaccination with a combination of cottontail rabbit papillomavirus E1, E2, E6 and E7 genes. J. Virol. 8, 7039±7043. Han, R., Reed, C.A., Cladel, N.M., Christensen, N.D., 1999. Intramuscular injection of plasmid DNA encoding cottontail rabbit papillomavirus E1, E2, E6 and E7 induces T cell-mediated but not humoral immune responses in rabbits. Vaccine 17, 1558±1566. Hariharan, M.J., Driver, D.A., Townsend, K., et al., 1998. DNA immunization against herpes simplex virus: enhanced ef®cacy using a Sindbis virus-based vector. J. Virol. 72, 950±958. Harpin, S., Hurley, D.J., Mbikay, M., Talbot, B., Elazhary, Y., 1999. Vaccination of cattle with a DNA plasmid encoding the bovine viral diarrhoea virus major glycoprotein E2. J. Gen. Virol. 80, 3137±3144. Hassett, D.E., Zhang, J., Whitton, J.L., 1997. Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge. J. Virol. 71, 7881±7888. Heller, M.A., Eisenberg, R.S., 1998. Can patents deter innovation? The anticommons in biomedical research. Science 280, 698. Herweijer, H., Latendresse, J.S., Williams, P., et al., 1995. A plasmid-based self-amplifying Sindbis virus vector. Hum. Gene Ther. 6, 1161±1167. Heu¯er, C., Koch, F., Schuler, G., 1988. Granulocyte/macrophage colony-stimulating factor and interleukin. 1. Mediate the maturation of murine epidermal langerhans cells into potent immunostimulatory dendritic cells. J. Exp. Med. 167, 700±705. Hoffman, S.L., Doolan, D.L., Sedegah, M., et al., 1997. Strategy for development of a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use. Vaccine 15, 842±845. Hosie, M.J., Flynn, J.N., Rigby, M.A., Cannon, C., Dunsford, T., Mackay, N.A., Argyle, D., Willett, B.J., Miyazawa, T., Onions, D.E., Jarrett, O., Neil, J.C., 1998. DNA vaccination affords signi®cant protection against feline immunode®ciency virus infection without inducing detectable antiviral antibodies. J. Virol. 72, 7310±7319. Irvine, K.R., Rao, J.B., Rosenberg, S.A., Restifo, N.P., 1996. Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases. J. Immunol. 156, 238±245. Iwasaki, A., Stiernholm, B.J.N., Chan, A.K., Berinstein, N.L., Barber, B.H., 1997a. Enhanced CTL responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. J. Immunol. 158, 4591±4601. Iwasaki, A., Torres, C.A., Ohashi, P.S., Robinson, H.L., Barber, B.H., 1997b. The dominant role of bone marrow-derived cells in CTL induction following plasmid DNA immunization at different sites. J. Immunol. 159, 11±14. Jenkins, M., Kerr, D., Fayer, R., Wall, R., 1995. Serum and colostrum antibody responses induced by jet-injection of sheep with DNA encoding a Cryptosporidium parvum antigen. Vaccine 13, 1658± 1664. Jiang, W., Baker, H.J., Swango, L.J., Schorr, J., Self, M.J., Smith, B.F., 1998. Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus. Vaccine 16, 601±607. Johnston, S.A., Barry, M.A., 1997. Genetic to genomic vaccination. Vaccine 15, 808. Jones, D.H., Clegg, J.C., Farrar, G.H., 1998. Oral delivery of micro-encapsulated DNA vaccines. Dev. Biol. Standardization 92, 149±155. Jones, D.H., Corris, S., MacDonald, S., Clegg, J.C.S., Farrar, G.H., 1997. Poly(DL-lactide-co-glycolide)encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine 15, 814±817. Kanellos, T., Sylvester, I.D., Howard, C.R., Russell, P.H., 1999a. DNA is as effective as protein at inducing antibody in ®sh. Vaccine 17, 965±972. Kanellos, T.S., Sylvester, I.D., Butler, V.L., Ambali, A.G., Partidos, C.D., Hamblin, A.S., Russell, P.H., 1999b. Mammalian granulocyte-macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in ®sh. Immunology 96, 507±510. Keller, E.T., Burkholder, J.K., Shi, F., Pugh, T.D., McCabe, D., Malter, J.S., MacEwen, E.G., Yang, N.S., Ershler, W.B., 1996. In vivo particle-mediated cytokine gene transfer into canine oral mucosa and epidermis. Cancer Gene Ther. 3, 186±191.

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