© Veterinary Sciences Tomorrow - 15 May 2001

DNA vaccines: new applications for veterinary medicine

Vinciane Dufour


Introduction
In 1990, Wolff and his colleagues were the first to report the successful expression of naked plasmid DNA in mouse muscle tissue [108]. A few years later it was reported that the injection of DNA encoding an antigenic protein of influenza virus conferred protective immunity in mice [97]. Many papers have described and reviewed the protective immunity induced by DNA against a large variety of viruses [20], bacteria [90] and protozoa [47] in murine models. DNA immunisation has also been investigated for the treatment of cancer [6] and autoimmune diseases [72]. Trials are now being developed for human DNA vaccines against several diseases such as AIDS and malaria [96, 104].

The administration of a simple plasmid can induce a broad spectrum of immune responses [54]. They include the activation of CD8+ T lymphocytes, implicated in host defense against intracellular pathogens via cytotoxic T lymphocytes (CTL), and CD4+ T lymphocytes, which secrete cytokines and play a role in B cell production of specific antibodies (Figure 1). Despite its potential impact on protective immunity, however, DNA vaccination is not always successful. Protective immunity depends mostly on the immunogenicity of a pathogen's antigen, but other factors such as the frequency and route of administration, the amount of DNA, the localisation of the plasmid coded-antigen (secreted, membrane-bound or cytoplasmic), and the age, health and species of the animals vaccinated, have also been shown to have an effect.

Principle and advantages
Characteristics of the expression vectors
The first vectors used for DNA vaccination were bacterial plasmids, developed originally for the in vitro expression of foreign protein in mammalian cells [28]. A plasmid contains a prokaryotic replication origin that allows high-yield production in bacteria, and an antibiotic resistance gene for selective growing. The transcriptional unit carries a viral promoter that may lead to constitutive expression in a wide variety of cells (Figure 1). The human cytomegalovirus immediate early (HCMVie) enhancer/promoter or the Rous sarcoma virus long terminal repeat (RSV-LTR) are often used. Antigen expression is increased when an untranslated region, with its splicing signals, follows the promoter. The sequence encoding the antigen is 3'-flanked by the transcript termination/polyadenylation (polyA) signal of the SV40 or of the bovine growth hormone (BGH). The plasmid pcDNA3 and its derivatives, which contain the HCMVie promoter and the BGH polyA, have often been used as vectors for DNA vaccines.


Figure 1. Principle of DNA vaccination.

 Production of a genetic vaccine involves the isolation of genes from a selected pathogen followed by their insertion into a mammalian expression plasmid. After large-scale production and purification steps, the DNA vaccine is usually delivered to animals by intramuscular (i.m.) injection.

 The mechanisms by which the antigen is produced (by myocytes, keratinocytes, MHC II-negative cells or professional antigen-presenting cells), processed and presented to the immune system are still disputed. However, following the processes of antigen production and processing, pathogen-derived peptides are presented by MHC I and MHC II molecules to induce cellular and humoral immune responses.

 Advantages
Production and storage
Antigen-expressing plasmids are amplified in bacterial cultures, making the production of DNA vaccines easy, rapid and economical. Bacterial production of plasmid DNA also avoids the risk of potential contamination with the viruses/proteins present in eukaryotic cell lines used to produce conventional vaccines. In addition, plasmid DNA molecules are well conserved and show greater stability between 4°C and 20°C than protein-based or live-attenuated vaccines. The injection of DNA, contrary to live attenuated vaccines, avoids the risk of reversion to a pathogenic phenotype.

Rapid development of vaccines against emerging diseases
Progress in biotechnology has enabled the relatively quick and easy manufacture of DNA vaccines. This type of vaccine development may provide a technological solution to the emergence of new viruses for which cell culture systems have not yet been developed. An antigen-coding sequence can be amplified by PCR, cloned in a vector and produced by bacterial culture for subsequent injection into domestic animals and herds.

Veterinary applications of DNA vaccines
DNA vaccines have many advantages over conventional vaccines, and offer an attractive approach to vaccination for veterinary species. The biggest handicap to the development of animal vaccines is the lack of available data on immune responses to pathogens that exclusively infect veterinary species. Poor availability of commercial immunological reagents (e.g. monoclonal antibodies, cytokines and lymphocytes) and the absence of references on many pathogens, make DNA vaccine trials more difficult in these species than in mice. Immunity in veterinary species is most often analysed by titration of the neutralising antibodies (not yet developed for all the above mentioned viruses), by the determination of antigen specific lymphoproliferation or, in some rare cases (pig and cat), by the determination of CTL activity. The protective immunity conferred by naked DNA can be determined by clinical parameters such as mortality, fever, weight loss and pathogen titration, if an infection model is available. Compared to murine models, the number of animals used in the DNA vaccination trials of larger veterinary species often limits the application of their results. Nevertheless, many studies in mice have shown that naked DNA vaccination induces a large spectrum of immune responses, so that this type of vaccine can readily be considered against pathogens that have not inspired large-scale immunological studies. Table 1 lists the DNA vaccination trials carried out in mammals (cattle, pig, sheep, dog, cat, horse, rabbit), birds (duck, chicken, turkey) and fish (Salmonidae) against their target pathogens. Clearly, DNA vaccination has already inspired great interest in the veterinary field.

Immune responses induced by DNA vaccines
Immune responses against viruses
Naked DNA vaccination usually induces a humoral immune response, characterised by the production of antigen specific antibodies. In general, the antibody level is very low to undetectable after the first DNA injection but increases both with the number of injections and the amount of injected DNA. Neutralising antibodies have been detected against the following viruses: bovine herpesvirus-1 [1] (BHV-1) [4, 10, 11, 17, 98, 99], equine herpesvirus-1 [81], bovine viral diarrhoea virus (BVDV) [39], canine distemper virus (CDV) [15], classical swine fever virus (CSFV) [2], cottontail rabbit papillomavirus (CRPV) [23], foot and mouth disease virus (FMDV) [5, 106], duck hepatitis B virus (DHBV) [76, 77, 94], infectious bursal disease virus (IBDV) [29], infectious hematopoietic necrosis virus (IHNV) [1, 8, 16, 93], influenza viruses [27, 51, 52, 64, 74], Japanese encephalitis virus (JEV) [53], porcine reproductive and respiratory syndrome virus (PRRS) [57, 71], pseudorabies virus (PRV) [26, 32, 34, 58, 66, 88, 99, 100, 101], rabies virus (RV) [67, 69], vesicular stomatitis virus (VSV) [13] and viral hemorrhagic septicemia virus (VHSV) [8].

The induction of an antigen specific lymphoproliferative response has been reported after one or more injections of DNA. Lymphoproliferation after recall by antigen in vitro has been found for BHV-1 [4, 11, 61, 98, 99], BVDV [39], African horsesickness virus (AHV) (after vaccination of one horse) [78], PRRSV [57, 71], PRV [32, 34, 100, 101] and CRPV [91, 36, 37, 38]. CTL activity has been clearly demonstrated against PRV in pigs after several injections of DNA [100], and in a horse vaccinated against AHV [78]. Cytokines produced by T helper 1 (Th1) cells have been detected in both cattle and pigs, and interferon (IFN)-g was found after in vitro antigen specific stimulation against BHV-1 [10, 11, 61, 98], PRRSV [57] and PRV [26]. Interleukin (IL)-4, produced by Th2 cells, and IL-2 have also been found in vitro in peripheral blood mononuclear cells (PBMC) from pigs vaccinated by a single injection of DNA against PRV [26].

Immune responses against bacteria
DNA vaccination technology seems suitable for the development of vaccines against intracellular bacteria, for which T cell-mediated immunity is required. However, reports in the literature of DNA vaccination trials are far from numerous. Antibodies have been detected after DNA vaccination against two intracellular bacteria: Corynebacterium pseudotuberculosis in sheep [14] and Clamydia psittaci in turkeys, for which seroconversion is not reliable [102, 103]. Low levels of antibody were also detected in the colostrum or serum of pigs after vaccination against the enterobacterium Escherichia coli K8ab; antibodies were detected in the serum of one pig and the colostrum of another [95]. The lymphoproliferative response against these bacteria was not determined.

Immune responses against protozoa
To date, three protozoa have been involved in the development of DNA vaccines: Cryptosporidium parvum, Theileria annulata and Eimeria acervulina. Dose-dependent levels of antibodies were produced in the colostrum and serum of sheep after DNA vaccination against C. parvum, and stronger seroconversion was obtained using intramammary compared to intramuscular (i.m.) administration of the vaccine [45]. DNA vaccination also induces antibodies to C. parvum in goats [82]. The antibody response after DNA vaccination of three-day-old chicken neonates against E. acervulina is also dose-dependent [89] and is detected after two i.m. injections of 100 µg of DNA. The subcutaneous injection of DNA vaccine did not provide effective antibody induction. No humoral response was obtained after three injections of DNA against the protozoan T. annulata in cattle [22].

Immune responses against other parasites
IgG1 antibodies as well as lymphoproliferative responses have been induced in cattle against Anaplasma marginale [3], and in sheep IgG1 antibodies were similarly the most common to be found against Taenia ovis [24, 25, 79, 80]. Vaccination against the tick Boophilus microplus by two injections of DNA only induced very low immune responses in sheep [21].

Clinical protection following challenge
Clinical protection against viruses
Clinical protection is defined as the improvement of clinical signs after DNA immunisation and subsequent challenge compared to unvaccinated animals. Partial or complete protection, depending on the pathogen, has been conferred by several injections of DNA against BHV-1 [4, 11, 21, 99], bovine respiratory syncytial virus [85, 86], BVDV [39], canine parvovirus [46], CRPV [23, 35, 38, 59, 91], avian [30, 51, 52, 74], equine [63] and swine [27, 64] influenza viruses, PRRSV [71], PRV [32, 34, 100, 101], RV [69], CSFV [2], Newcastle disease virus (NDV) [83], DHBV [94, 77], infectious hematopoietic necrosis virus [1, 16, 48, 93] and VHSV [62]. Partial clinical protection was obtained against FIV after DNA vaccination of cats with gp120 gene, though these results are open to discussion [18, 73]. The strong efficacy of DNA vaccines has been demonstrated in pigs and fish, after one DNA injection was able to elicit clinical protection against PRV [26,88] and fish rhabdoviruses [1, 16, 48, 93]. Despite the reduced duration of PRV shedding, however, primary infection was not inhibited by a single injection of DNA or other conventional vaccine. Low levels of neutralising antibodies have not prevented the induction of good clinical protection against CSFV [2] or PRV [26] in pigs, or against influenza virus [51, 52] in chickens.

Clinical protection against heterologous viral strains
The influenza viruses exhibit antigenic variations at the antibody-binding sites on the hemagglutinin (HA) molecules. This HA antigenic variation defines several viral subtypes and is the cause of the difficulty in developing effective vaccines. DNA vaccination trials against influenza virus were carried out to evaluate the efficacy of plasmid DNA coding for the HA of one viral subtype against that of a different viral subtype. The results showed that the vaccination of chickens with the gene encoding the H5 or H7 subtype of HA was fully protective against a homologous challenge, but ineffective against a different subtype, i.e. the H5 gene was not protective against the H7 subtype, and vice versa [52]. On the other hand, DNA vaccination was protective against highly virulent strains of the same viral subtype [51].

DNA vaccination trials carried out on Salmonidae fish showed that injection of DNA coding for the glycoproteins of VHSV was able to protect fish from viruses of different subtypes and from different isolates [62].

Clinical protection against bacteria
Complete protection was obtained against the intracellular bacterium C. psittaci, after two vaccinations by gene-gun (g.g.) in turkeys, and when turkeys were challenged with the homologous strain [103]. In sheep partial protection was obtained against another intracellular bacterium, C. pseudotuberculosis [14], that is two i.m. injections of a targeted plasmid were able to protect 70 % of the vaccinated animals. Protection here was defined by the absence of abscesses characteristic of caseous lymphadenitis.

Clinical protection against parasites
Partial clinical protection was obtained in cattle against the protozoan T. annulata, despite the absence of antibody in sera [22]. Two of the three vaccinated bovines were indeed protected from infection after DNA immunisation with two allelic forms of the major merozoite surface antigen. These data indicate that protective immunity is mediated by a cellular immune response. DNA vaccination of chickens against E. acervulina induced only partial protection (defined as a reduction in the production of oocysts in the faeces after oral infection) because oocysts were still present in the faeces regardless of the amount of DNA used for vaccination [89].

Immune response and protection in neonates
Vaccination of neonates provokes two important questions: Is the neonate's immune system sufficiently mature to elicit a response? Can offspring of an immune mother be immunised despite the high levels of passively transferred maternal antibodies to the antigen? In murine models, DNA vaccination of neonates confers immunity against viruses such as respiratory syncytial virus [65] and lymphocytic choriomeningitis virus (LCV) [40], showing that DNA immunisation can be effective in early life in the absence of maternal antibodies. The presence of maternal antibodies decreases or abolishes the antibody response in neonates when they are immunised with DNA coding for a structural protein such as glycoprotein of RV or HA of influenza virus [70, 105]. On the other hand, vaccination with DNA encoding an internal viral protein (e.g. nucleoprotein of influenza virus or of LCV) induces antibodies in neonates even in the presence of maternal antibodies [41, 70]. Thus in mouse neonates, the localisation of the DNA-expressed antigen, i.e. in the plasma membrane or within the cell, is an important parameter for DNA vaccine efficacy in the presence of maternal antibodies.

A study of DNA immunisation of lamb neonates was carried out against BHV-1 [98]. Three-day-old lambs vaccinated with DNA expressing a gB secreted form of BHV-1 developed both humoral and cellular immune responses three weeks after vaccination, thereby demonstrating the capacity of the ovine immune system to respond to DNA vaccination at this age. Lambs produced by hyperimmune ewes also responded in the same manner, showing that the maternal antibodies did not prevent development of the anti-BHV-1 immunity in lamb neonates. The clinical protection was not determined.

DNA vaccine efficacy against PRV was evaluated in one-day-old piglets. Those born from a hyperimmune sow did not develop any antibody at 10 or 16 weeks of age, following one or two injections of a gD plasmid [66, 58]. Maternal antibodies are still present at this stage of animal development and inhibit the antibody response in neonates. Piglets born from a seronegative sow and vaccinated twice with gD plasmid produced very low levels of neutralising antibodies at 16 weeks. However, these piglets were not protected following subsequent challenge [66].

Other studies have involved DNA immunisation of duckling neonates against DHBV. The vaccination of three-day-old ducklings induced an antibody response after only one injection of DNA [75] demonstrating, as in lambs, the capacity of the neonate immune system. Ducks vaccinated by four injections of DNA against DHBV produced a humoral response and transmitted this immunity to their offspring via the egg yolk [76]. Significant rates of IgY antibody were found both in the egg yolk and in the duckling serum. The ducklings were also protected against a viral infection. These results show that, in this model, DNA vaccination is effective both by passive transfer of maternal antibodies or by direct immunisation of duckling neonates.

DNA vaccination was evaluated in chicken neonates against IBDV. Four of seven chicken neonates born from a non-immune hen produced antibodies after immunisation with a plasmid encoding the polyprotein VP2-VP3-VP4 [29]. However the chickens were not protected against subsequent infection. The injection of a plasmid coding solely for VP2 did not induce an antibody response.

DNA vaccination of pregnant goats to the protozoan C. parvum induced clinical protection of the offspring, thereby indicating the efficient transfer of maternal antibodies [82].

Influence of the route of DNA delivery
Gene-gun, intradermal and intramuscular routes of delivery
The effect of the DNA delivery route on immunity and clinical protection has been analysed. In chickens, g.g. delivery of DNA vaccine to influenza virus is the most efficient delivery route for DNA immunisation [30]. In rabbits, g.g. delivery of the E1 and E2 genes of CRPV provides complete protection, whereas i.m. injections were not effective [37]. In turkeys, combined i.m. and intranasal routes induced equivalent protection against C. psittaci compared to g.g. delivery [103]. In pigs, i.m. injections of a DNA vaccine against CSFV provided higher levels of antibodies than the g.g. route [2], and intradermal (i.d.) injections of PRV plasmid induced a higher immune response than i.m. injections [101]. In dogs, i.m. injection of a DNA vaccination against RV resulted in a higher and more durable antibody response than that obtained by i.d. injection [67], whereas in cats, i.d. injections of the same plasmid elicited a higher frequency of seroconversion than i.m. administration. In sheep, the intramammary delivery of plasmid produced higher levels of anti-C. parvum antibody in both serum and colostrum than was measured after i.m. injection of the vaccine [45]. Thus, the efficacy of DNA vaccines varies not only according to the route of delivery, but also to the animal species and pathogen.

Delivery of DNA vaccines at mucosal sites
Only a few studies have reported DNA immunisation at mucosal sites. In pigs, DNA vaccination against influenza virus was more effective by g.g. administration on the inferior surface of the tongue than by i.d. injection [64]. Intravulvomucosal g.g. vaccination against BHV-1 induced higher humoral and cellular immune responses than vaccination through i.d. injection in cattle [61]. This stronger immunity correlated with higher protection following challenge, and higher antigen expression in the mucosa than in the skin. Mucous and seral IgA were only detected after challenge.

Gene-gun administrations at cutaneous and mucosal sites in horses provided a high frequency of seroconversion and total protection against equine influenza virus, whereas cutaneous g.g. administration only induced partial protection [63]. Although skin and mucosal delivery routes were the more efficient, no mucosal IgA response was produced prior to challenge and IgA was only detected in ponies that shed virus. All successful mucosal DNA vaccination trials reported to date have used g.g. administration of the vaccine, with the exception of DNA vaccination of chickens against influenza virus [30]. Immunisation against influenza virus with DNA drops administered by intratracheal route only provided protection in 30 % of the vaccinated chickens and was less efficient than g.g. delivery.

Encapsulation of plasmid DNA into liposomes
Liposomes are used to encapsulate DNA vaccines to facilitate cellular uptake of the plasmid. Compared to naked DNA that elicited partial protection against BVDV, i.m. vaccination with plasmid DNA encapsulated in cationic liposomes was found to be totally ineffective [39]. In chickens i.m. vaccination against NDV was evaluated using DNA mixed with two different transfection reagents. Surprisingly, the addition of Lipofectin increased antibody levels and protection, whereas the addition of LipofecAmine reduced antibody levels and clinical signs after challenge [83].

Comparison between DNA vaccines and antigen preparations
DNA vaccination of duck neonates against DHBV elicited a higher humoral immune response than a recombinant protein mixed with Freund's adjuvant [75]. In adult ducks, DNA vaccine and recombinant protein vaccine induced similar levels of antibodies [77]. In sheep, the cytotoxic T-lymphocyte antigen 4-fusion DNA vaccine against the intracellular bacteria C. pseudotuberculosis was as protective as a formalin-inactivated toxin vaccine [14]. In chickens, g.g. delivery of a DNA vaccine against influenza virus was as effective, if not more so, than a conventional inactivated whole virus vaccine [51]. In pigs, DNA vaccine against influenza A virus delivered at mucosal sites was as good as a commercial inactivated whole virus vaccine [64], despite the lower level of antibodies. DNA vaccination with a mixture of PRV expressing plasmids was also more efficient than an inactivated whole virus vaccine, but less efficient than a modified live virus vaccine [33]. On the other hand, a DNA vaccine composed of the full-length genome of foot and mouth disease was less efficient than an inactivated whole virus vaccine [106]. One report describes that DNA vaccine against the tick B. microplus was less efficient than a recombinant protein vaccine in sheep [21]. All these results show that DNA vaccines against viruses or intracellular bacteria are often as efficient as recombinant protein vaccines and inactivated whole vaccines.

Improvement of DNA vaccines
Co-administration of immunostimulating molecules coding genes
In addition to inducing both antigen specific cellular and humoral immunity, DNA vaccine technology is able to modulate the amplitude and nature of the immune response through co-delivery of plasmids expressing immunostimulating molecules [68]. Genes encoding co-stimulatory molecules involved in antigen binding, such as CD80, CD86 and B7.2, or encoding cytokines involved in the recruitment of antigen presenting cells, such as granulocyte macrophage-colony stimulating factor (GM-CSF) [109] and IL-12 [49, 87], are good candidates for the enhancement of immune responses to viruses. Several cytokine encoding genes, including IL-2, IL-4 and IFN-g, have been tested for their ability to amplify the immune response to genetic vaccines in mice.

The addition of genes encoding the cytokines GM-CSF, IFN-g, IL-12, IL-16 and IFN-a to DNA vaccines has been tested in animals. For example, the addition of porcine GM-CSF gene to a DNA vaccine against PRV increased clinical protection in pigs [88], by enhancing T cell, but not B cell, responses following infection [26]. In sheep a very low level of protection against B. microplus was conferred only in the presence of the GM-CSF gene and following a protein boost [21]. In rabbits immunised against CRPV the priming effect of the gene encoding for murine GM-CSF has been analysed by administering the gene three days before immunisation with the virus plasmid. The result was an increased effect of CRPV vaccination, from 50 % to 67% of rabbits resistant to the disease [59]. It can be concluded that whatever the species and the pathogen, the presence of the GM-CSF plasmid enhances protection, as it does in murine models.

The gene coding for IFN-g was added to a vaccine against feline immunodeficiency virus (FIV) [43, 44] composed of the whole viral DNA with the pol gene deleted. The addition of IFN-g enhanced clinical protection against FIV, by revealing a decreased viral load after infection compared to cats that had not received IFN-g. In contrast, the addition of the IFN-g gene in pigs reduced clinical protection against PRV [88], as did the addition of the IL-2 [88] and IFN-a genes [26].

Addition of feline IL-12 plasmids to a mini-circle [2]DNA vaccine against FIV decreased the viral load after infection compared to the same DNA vaccine without IL-12. The IL-12 gene increased CTL activity and reduced FIV seroconversion following challenge [7]. Like IL-12, the feline IL-16 gene greatly reduced viral load [60] and induced seroconversion following challenge [7].

Addition of CpG motifs
The bacterial DNA, of which DNA vaccines are composed, contains immune enhancers [55]. In contrast to vertebrate DNA, bacterial DNA is unmethylated and typically exhibits a near random distribution of CpG dinucleotides and flanking bases. The vertebrate immune system seems to have evolved an intracellular pattern recognition receptor to detect the presence of foreign CpG DNA. Therefore, the plasmid DNA used for vaccination contains both the specific immunogen sequence and CpG adjuvant motifs. These unmethylated CpG motifs are essential for DNA immunisation of mice [50, 84]. The addition of oligonucleotides containing CpG motifs to a protein vaccine has been shown to enhance immune responses to viruses or bacteria in mice [12], but results concerning an enhancement of DNA vaccine efficacy are uncommon and conflicting. It seems that addition of oligonucleotides containing CpG motifs to a DNA vaccine induces a dose-dependent reduction of the gene expression, due to competition between the oligonucleotide and plasmid DNA for cellular binding sites [107]. Moreover, CpG have immunostimulatory effects (S) or immunoneutralising effects (N) according to their nucleotide environment, which could explain the apparently conflicting results obtained after their addition. The currently proposed solution to improve DNA adjuvant effects is to add CpG-(S) motifs and to eliminate CpG-(N) motifs from the vector backbone [56].

The addition of a 15-mer oligonucleotide containing three putative CpG motifs to a DNA vaccine was evaluated against VSV in horses; the presence of CpG motifs had no effect on B cell immune response [13]. CpG motifs were also tested in cats in a DNA vaccine coding for gp140 of FIV. The addition of CpG motifs resulted in a lower viral load following challenge than a DNA vaccine, which solely encoded gp140 [60], but did not prevent seroconversion.

Injection of multigenic vaccines
DNA vaccination trials in domestic animals indicate that multigenic injections could be more effective than injections of only one plasmid. Mixtures of plasmids have often been used to obtain protective immunity, either to protect against several viral subtypes, for example influenza virus [52] and fish rhabdoviruses [8], or to increase protection against a single virus such as PRV [26, 88], IBDV [29] or CDV [15]. The efficacy of multigenic DNA vaccines against CRPV depends on the injected plasmids: the injection of several plasmids coding for early proteins (E1, E2, E6 and E7) was more effective than the injection of each plasmid separately [35, 38]. On the other hand, when rabbits were vaccinated with plasmids coding for L1 and L2 capsid proteins, efficacy was not improved (and was even slightly worse) than that obtained with a vaccine coding for L1 capsid protein only [23].

A mixture of plasmids coding for several antigens does not always result in protective immunity. This is the case of DNA vaccination against FMDV, for which the injection of three plasmids coding for three viral proteins was not protective in pigs [5].

Injection of modified viral DNA
As multigenic vaccines are still ineffective against certain viruses, modified viral DNA was evaluated for DNA vaccination against FIV in cats and FMDV in pigs. Vaccination against FIV using proviral DNA with the pol gene deleted induced CTL activity but no detectable antiviral antibodies. The DNA vaccine induced significant protection after challenge [43, 44]. However, the pol deleted-DNA vaccine was not efficient against a more virulent strain of FIV [42].

Modified FMDV DNA has been evaluated in pigs. The full-length viral DNA was modified by removing the receptor binding site sequence found in the capsid protein VP1 gene to prevent the virus from spreading between normal cells. The resulting infectious DNA was not lethal in mice and produced viral capsids [5, 106]. Injection of the full-length FMDV DNA induced neutralising antibody and protection in only 20 % of the vaccinated pigs. The same full length FMDV DNA mutated within the pol gene was less immunogenic and non-protective, indicating that the immunogenicity of the FMDV DNA vaccine results, in part, from amplification of the viral genome in host cells [106].

Improvement of the plasmid backbone
Search for more effective promoters
The HCMVie enhancer/promoter has a very broad spectrum of expression (mammals, birds and fish), and consequently has been universally used for DNA vaccination. Other promoters have been tested for DNA vaccination of cattle or chickens. DNA immunisation efficiency in chickens was evaluated for the chicken b-actin promoter (a non-viral avian specific promoter) and the HCMVie promoter. Both promoters induced equivalent rates of clinical protection against influenza virus [52], showing that the species-specific promoter was not better than the viral one. Vaccination of chickens against influenza virus resulted in slightly lower protection when a vector derived from retroviral sequences of avian leucosis virus was used, in which antigen expression is under the control of the LTR promoter [30]. In cattle, the viral RSV-LTR promoter induced a lower anti-bovine herpes virus-1 immune response than that obtained with the HCMVie promoter [99]. To date, the HCMVie promoter is still the best for DNA vaccination in veterinary species.

Targeting the immune system using cytotoxic T-lymphocyte antigen 4 (CTLA-4) fusion products
Immunising genes were fused with the CTLA-4 gene to target foreign antigen to sites of immune induction, thereby enhancing the response to a DNA vaccine [9]. CTLA4 present on activated T cells binds to B7-expressing cells including antigen-presenting cells. DNA vaccination of sheep using a phospholipase D-CTLA4 fusion product enhanced the speed, magnitude and longevity of the antibody response to C. pseudotuberculosis compared to that obtained without CTLA4 gene [14]. CTLA4 fusion also provided the best clinical protection.

Influence of the antigen localisation
The in vivo localisation of antigen is a significant parameter of DNA vaccine efficacy in bovine. Immunisation of cattle with a plasmid expressing a gD secreted form of BHV-1 was more efficient than a plasmid expressing the full-length gD membrane form [99], thereby showing that extracellular localisation induced higher immune responses than membrane localisation in this model.

Conclusion
DNA vaccination is a promising technology to prevent diseases in farm and companion animals. The commercial development of DNA vaccines against certain pathogens that use veterinary species as their specific host, such as BHV-1, RV and PRV, is completely feasible, and will be helped by investigations into the mechanism of action of DNA vaccines using murine models. Efforts must now be concentrated on improving immunity in animals, by searching for appropriate adjuvants (CpG and other immunostimulating molecules) and the optimal route of administration, targeting the expression plasmids. The cost of the vaccine must also be taken into account. Plasmid purification is still expensive and the amounts injected need to be reduced if DNA vaccination is to be considered in breeding systems. Similarly, administration routes like g.g. that involve the use of gold beads cannot be applied to intensive breeding systems.

Index of pathogens
African horsesickness virus (family Reoviridae, genus Orbivirus; list A [3]): AHS is a truly African disease and is endemic in the central tropical regions of the continent, from where it spreads regularly to Southern Africa. It is not contagious by direct contact and is transmitted by biological vectors, causing severe acute disease in its most common host, the horse, for which the mortality rate is 70-95 %. The acute respiratory form causes oedema of the lung and lymph nodes, hydropericardium and pleural effusion.

Anaplasma marginale (Ehrlichial genogroup II Rickettia; list B): A. marginale causes anaplasmosis hemoparasitic disease of livestock. It can be transmitted between cattle by ticks and via contaminated hypodermic needles. The parasite causes acute disease, which often results in death. Animals that survive are persistently infected and become life-long carriers, serving as a reservoir for transmission.

Boophilus microplus (tick): B. microplus preferentially infests cattle, but has a broad host range including deer and sheep. In cattle, protection against B. microplus seems to be correlated with levels of antibodies against the Bm86 tick antigen.

Bovine herpesvirus-1 (family Herpesviridae, subfamily Alphaherpesvirinae): BHV-1 is a common cause of several clinical syndromes in cattle, such as respiratory tract infection, conjunctivitis, and vulvovaginitis in females and balanoposthitis in males. Systemic infection can lead to abortion and meningoencephalitis.

Bovine respiratory syncytial virus (family Paramyxoviridae, genus Pneumovirus): BRSV is a respiratory pathogen in cattle inducing mild clinical signs, but cattle and lambs infected with BRSV are more susceptible to secondary respiratory infection.

Bovine viral diarrhoea virus type 1 (family Flaviviridae, genus Pestivirus): BVDV-1 is found in livestock worldwide and spreads mainly by contact between cattle. The clinical signs range from subclinical to the fulminating fatal condition called mucosal disease. Acute hemorrhagic forms of the disease associated with high mortality have also been described. However, most infections in the young calf are mild and go unrecognised clinically.

Canine distemper virus (family Paramyxoviridae, genus Morbillivirus): CDV causes highly infectious and frequently lethal disease in dogs and other carnivores. The virus is closely related to measles and rinderpest viruses. Despite vaccination, outbreaks of CDV have recently occurred in several countries.

Canine parvovirus (family Parvoviridae, genus Parvovirus): CPV causes severe enteritis in juvenile dogs and myocarditis in neonatal puppies.

Clamydia psittaci (intracellular gram-negative bacterium): C. psittaci causes respiratory symptoms in turkeys by infection of the mucosal epithelial cells and macrophages.

Classical swine fever virus (family Flaviviridae, genus Pestivirus, list A): Pigs and wild boar are the only natural reservoir of CSFV. The virus spreads by direct contact between animals and causes high mortality in pigs (100 % in young pigs) in much of Asia, Central and South America, and parts of Europe and Africa. The acute form of the disease induces fever, hemorrhagic lesions and cyanosis of the skin, dyspnoea, etc.

Corynebacterium pseudotuberculosis (gram-positive intracellular bacterium): C. pseudotuberculosis causes caseous lymphadenitis in sheep. The disease is believed to be transmitted via skin wounds or by aerosol infection, and is characterised by the formation of abscesses within the superficial lymph nodes.

Cottontail rabbit papillomavirus (family Papovaviridae): papillomavirus causes mucosal and cutaneous hyper-proliferative lesions and is linked with the appearance of tumours in man. CRPV infection in rabbits was chosen as an experimental model for the first step in the development of vaccines against human papillomavirus infections.

Cryptosporidium parvum (protozoan): C. parvum causes intestinal cryptosporidiosis, mostly in young animals and children. Oral administration of monoclonal antibodies or hyperimmune serum specific to the protozoan antigens can prevent infection in calves and man.

Duck hepatitis B virus (family Hepadnaviridae, genus Orthohepadnavirus): DHBV has been chosen in its natural host as a model for human hepatitis B virus studies. The two viruses are closely related with respect to genomic organisation, hepatotropism and replication.

Equine herpesvirus-1 (family Herpesviridae, subfamily Alphaherpesvirinae): EHV-1 causes globally endemic disease characterised by mild rhinitis, abortion and occasional neurological signs.

Escherichia coli K88ab (enterotoxigenic bacteria): E. coli is an important pathogen, causing diarrhoea, oedema and colisepticemia in piglets, and diarrhoea in weaners/postweaned piglets.

Feline immunodeficiency virus (family Retroviridae, genus Lentivirus): FIV was studied in its natural host to develop an effective vaccine against lentiviruses, including the human immunodeficiency virus. FIV infection has been shown to result in an immunodeficiency in cats similar to AIDS in man.

Foot and mouth disease virus (family Picornaviridae, genus Aphthovirus; list A): FMDV causes one of the most contagious animal diseases affecting bovines (cattle, zebus, domestic buffaloes, yaks), sheep, goats, swine, and all wild ruminants. The disease causes important economic losses, with low mortality rates in adult animals, but often high mortality in young animals due to myocarditis. Clinical signs in cattle include pyrexia, anorexia, shivering, and reduced milk production for two-three days. FMDV is endemic in parts of Asia, Africa, the Middle East and South America.

Infectious bursal disease virus (family Birnaviridae, genus Avibirnavirus; list B): IBDV causes a highly infectious disease that mainly affects young chickens. Severe acute disease is associated with high mortality, but a less acute/subclinical disease is common. This can lead to secondary problems such as lymphoid depletion of the bursa of Fabricius, and, if this occurs in the first 2 weeks of life, may result in significant depression of the humoral immune response.

Infectious hematopoietic necrosis virus (family Rhabdoviridae; list B): IHNV causes significant mortality among both wild and cultured fish, usually at the juvenile stages. The virus is responsible for the greatest loss of fish in aquaculture. IHNV is enzootic in salmonid species along the Pacific coast of North America.

Influenza A virus (family Orthomycoviridae, genus Influenzavirus; list A): IV is a highly infectious respiratory pathogen of birds and mammals (man, horses and pigs). All highly pathogenic isolates from birds have been influenza A viruses of subtypes H5 and H7. The recent appearance of the avian IV H5N1 subtype in man has demonstrated that an avian virus can cross the species barrier to cause severe disease in man. In birds, influenza virus is responsible for severe depression, reduced egg production, hemorrhage and sudden death (mortality can reach 100 %), etc.

Japanese encephalitis virus (family Flaviviridae, genus Flavivirus; list B): JEV causes encephalitis principally in horses, but also infects man, and causes abortions in pigs. Pigs act as amplifiers of the virus, and birds can also be involved in its spread. The virus is an important human pathogen, and is responsible for human deaths in southern and eastern Asia.

Newcastle disease virus (family Paramyxoviridae, genus Rubulavirus; list A): NDV infects many species of domestic and wild birds; chickens are the most susceptible poultry. NDV is transmitted directly by respiratory discharge, faeces, etc. It is endemic in many countries, but some European countries are free of the disease. Clinical signs are respiratory and/or nervous signs, partial or complete cessation of egg production and diarrhoea. Morbidity and mortality depend on the virulence of the strain.

Porcine reproductive and respiratory syndrome virus (family Arteriviridae; list B): PRRSV is found in swine farms worldwide. The virus induces reproductive failure such as late-term abortion in sows, respiratory syndromes and mortality in young pigs.

Pseudorabies virus (family Herpesviridae, subfamily Alphaherpesvirinae; list B): causes Aujeszky's disease, which is characterised by severe central nervous and respiratory symptoms in pigs. Inactivated and live attenuated vaccines are commercially available for vaccination. Both humoral and cellular immune responses are involved in the protection of pigs against PRV infection.

Rabies virus (family Rhabdoviridae, genus Lyssavirus; list B): RV causes encephalitis followed by eventual paralysis in different species and almost always results in death. Despite the availability of inactivated vaccines, the virus still causes significant animal losses and public health problems in many parts of the world.

Taenia ovis (parasitic tapeworm): T. ovis causes ovine cysticercosis at the metacestode stage. Studies have shown that vaccination with antigens from the oncosphere stage of the parasite can induce complete protection against infection.

Theileria annulata (tropical protozoan parasite; list B): T. annulata infects both wild and domestic bovidae throughout much of the world (some species infect small ruminants). They are transmitted by ticks during feeding, and have complex life cycles in both vertebrate and invertebrate hosts. The parasite first invades mononuclear leucocytes where it develops into microschizonts, then into free merozoites able to invade erythrocytes. Vaccination using attenuated schizonte-infected cell lines has been widely used for T. annulata.

Vesicular stomatitis virus (family Rhabdoviridae, genus Vesiculovirus; list A): VSV causes severe diseases in domestic (equidae, bovidae and suidae) and wild (white-tailed deer and small mammals in the tropics) hosts, but induces a low mortality. VSV is more frequent in the rainy season in tropical areas of the Americas, but is also registered during the dry season in some countries. Clinical signs are excessive salivation, blanched raised or broken vesicles of various sizes in the mouth, foot and teat lesions, therefore VSV can easily be confused with FMDV.

Viral hemorrhagic septicemia virus (family Rhabdoviridae; list B): VHSV is one of the causative agents of the most important diseases in cultured rainbow trout in Europe. Only limited success has been obtained with traditional vaccines based on killed or inactivated virus.

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