Advances in Animal and Veterinary Sciences

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Advances in Animal and Veterinary Sciences 1 (6): 164 – 177

Progress in DNA Vaccinology against Bacterial Diseases– An Update

Thangalazhy Gopakumar Sumithra, Vinod Kumar Chaturvedi^*, Ajay Kumar Rai, Sunita S Chougule, Lekshmi S Rajan, Siju S Jacob, Susan Cherian

Indian Veterinary Research Institute, Izatnagar –243122, Uttar Pradesh, India

*Corresponding author:[email protected]

ARTICLE CITATION: Sumithra TG, Chaturvedi VK, Rai AK, Chougule SS, S Rajan LS, Jacob SS, Cherian S (2013). Progress in DNA vaccinology against bacterial diseases– an update. Adv. Anim. Vet. Sci. 1 (6): 164 – 177.
Received: 2013–09–15, Revised: 2013–09–20, Accepted: 2013–09–21
The electronic version of this article is the complete one and can be found online at ( http://nexusacademicpublishers.com/table_contents_detail/4/111/html ) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

ABSTRACT

DNA vaccines are the emerging promising approach to protect humans and animals from various infections. However, their application to bacterial infections has been scarcely advanced compared to viral DNA vaccines and, till date no licensed bacterial DNA vaccines are available. The various limitations of currently available bacterial vaccines, dangers due to the surge of antibiotic resistant bacteria, a decreased rate of discovery and development of new antibiotics, lack of efficient vaccines against many bacteria and threat of bioterrorism necessitate development of newer technology such as DNA vaccines against bacterial pathogens. Advancements in vector and antigen design, improved formulations and delivery devices/methods, inclusion of adjuvants and prime boosting strategy have greatly enhanced the immunogenicity of many DNA vaccines. This improved performance has spurred a renewed interest in bacterial DNA platform, which is reflected by the numerous ongoing experiments. Because of the strong cell–mediated immunity they can induce, DNA vaccinology is a promising method even against intracellular bacteria. DNA immunization studies have been conducted to combat various significant bacterial diseases such as anthrax, brucellosis, mycobacterial infections, tetanus, leptospirosis, borreliosis, staphylococcosis, mycoplasmosis, caseous lymphadenitis Pseudomonas aeruginosa infetctions, Rhodococcus equi pneumonia, Escherichia coli infection, chlamydiosis, typhoid, yersiniosis, Helicobacter pylori infection, streptococcal pneumonia and dental caries. The results of these experiments are quite encouraging which indicates that bacterial DNA vaccines are likely to become a reality in the near future. The present review provides updated information about DNA vaccinology and second generation DNA vaccine optimization strategies against the important bacterial pathogens, along with the concerns and future prospects that will help to improve their potency in order to achieve better outcomes in future clinical trials.

INTRODUCTION

Immunization indisputably is the most effective intervention in the medical history to control and prevent infectious diseases. A number of dreadful diseases which have previously overwhelmed medical and veterinary field such as small pox, polio, rinderpest and diphtheria, have now become the diseases of past due to the wide spread use of efficient vaccines (Ghanem et al., 2013). However, a number of diseases have not yet been conquered by vaccines and millions of animals, and humans die each year from infectious diseases for which there is no effective vaccine (Curtiss, 2011). Additionally, there are many concerns associated with the use of first generation (vaccines based on attenuated and killed forms of microorganisms) and second generation vaccines (vaccines using defined natural or recombinant protein or proteinaceous components of whole organisms) (Mateen and Irshad, 2011) which are summarized in Table 1. These limitations continue to drive the need for developing novel technologies that offer easier production, administration and better protection without any adverse effects. DNA vaccines based on plasmid offer such an opportunity and are emerging as a promising system against many infectious diseases (Dhama et al., 2008) and such plasmid based vaccines are considered as the third generation vaccines (Tuteja, 1999).

Table 1: Limitations of the first and second generation vaccines

DNA vaccines are basically recombinant bacterial plasmids which normally contain two units: first, the antigen expression unit composed of strong eukaryotic promoter, antigen–encoding gene and transcription termination/polyadenylation sequences to stabilize mRNA transcripts; second, the production unit composed of bacterial origin of replication allowing growth and amplification in bacteria and selectable marker, such as antibiotic resistance gene to facilitate the selection of transformed bacteria (Kumar et al., 2013). The basic steps in the construction of DNA vaccines of the first generation (Jumba, 2010) are shown in Figure 1. The purpose of the present article is to provide an updated summary on DNA vaccinology against bacterial diseases that help the scientific community to avail all the scattered data in a more concise format.

Figure 1: The basic steps in the construction of the first generation DNA vaccines

Background
The concept of DNA vaccine was evolved by Wolf et al. (1990) when they demonstrated that direct transfer of recombinant bacterial plasmid encoding beta galactosidase into mouse muscle induced expression of the protein within muscle cells. Subsequently, Tang et al. (1992) gave the first report that introducing a protein encoding gene directly into the skin of mice by propelling DNA–coated gold microprojectiles could elicit antibody responses against the delivered antigen.

One year later, three more papers were published regarding the ability of DNA vectors to drive both humoral and cellular immune responses against pathogens or tumour antigens in–vivo (Ulmer et al., 1993; Fynan et al., 1993; Wang et al., 1993). Altogether these reports provided evidence to the scientific community that this simple technique could be developed to create immunity against proteins (Liu, 2011). Afterwards considering the potential advantages of this immunization strategy (Table 2) several experimental trials and safety evaluation of various DNA vaccines have been conducted (Ferraro et al., 2011). Currently, there are 4 licensed DNA vaccines which are against infectious haematopoietic necrosis of salmon in Canada, West Nile fever of horses in USA, melanoma of dogs in USA and DNA vaccine encoding growth hormone releasing hormone for swine in Australia (Williams, 2013)

Table 2: Advantages of DNA immunization strategy

Mechanism of Protection by DNA Vaccines
When a DNA vaccine is injected into the host it enters host cells (transfection) and antigenic protein is expressed endogenously. There are three possibilities for the transfection such as direct transfection of antigen presenting cells especially Dendritic Cells (DCs), direct transfection of somatic cells and cross–priming. Antigen presentation mediated by MHC–I and MHC–II pathways is then followed (Gurunathan et al., 2000; Dunham, 2002) resulting in the induction of both cellular and humoral immune response against the antigen (Shedlock and Weiner, 2000). So, DNA vaccines are considered to be the best tool to prevent various infectious diseases especially against intracellular pathogens (Moreno and Timon, 2004). In addition to the antigen, unmethylated CpG motifs present in the plasmid backbone that are recognized by TLR9 can also cause stimulation of DCs which in turn promote strong cell mediated immune response (Klinman et al., 1997).

Figure 2: The optimization strategies for second generation DNA vaccines

Second Generation DNA Vaccines
Early in the clinical trials of DNA vaccines it was found that these are well–tolerated & safe vaccines without induction of autoimmunity and tolerance. There was both humoral and cell mediated immune responses but, the potency was found to be disappointing (Liu, 2003). As a result varieties of approaches are now under evaluation to increase the potency of DNA vaccine whilst still retaining their attractive features. These optimization strategies (Figure 2; Table 3) lead to the development of second generation DNA vaccines (Donnelly et al., 2005; Kutzler and Weiner, 2008).

Table 3: Optimization strategies for improving the potency of DNA vaccines

Progress in DNA Vaccinology against Bacterial Diseases
There are significant advances in the development of DNA vaccines against viral pathogens, while their application to bacterial infections has been scarcely advanced and till to date no licensed bacterial DNA vaccines are available. The reasons for the less advancement in DNA vaccinology against bacterial diseases may be the successful performance of many conventional vaccines and antibiotics against bacterial infections. However, the clear cut advantages of DNA vaccines over other vaccines (Table 2), the dangers due to the surge of antibiotic resistant bacteria, lack of efficient vaccines against many bacteria and the threat of bioterrorism necessitate development of DNA vaccines against bacterial pathogens. Moreover, DNA vaccination is a promising method o fight against intracellular bacteria since it can efficiently induce a cellular immune response (Nagata and Koide, 2010). As complete genome sequences of many bacteria have been recently decoded, researchers can now select the appropriate antigens and design the specific DNA vaccine construct (Ingolotti et al., 2010). Although there are some specific concerns in developing DNA constructs against bacteria such as difficulties in folding, transport and post–translational modifications of prokaryotic proteins in eukayotic cells leading to unwanted effects after immunization (Reyes–Sandoval and Ertl, 2001) and limitation in designing DNA construct against capsular polysachharide antigens (Tizard, 2012), DNA vaccine technology has been applied against different types of bacteria which can be summarized as follows

Bacillus anthracis
B. anthracis is a spore–forming, Gram–positive bacterium that causes a fulminating disease called anthrax in mammalian livestock and humans. The currently available anthrax vaccines in both animals and human are far from ideal. The need for a well defined vaccine that can stimulate both humoral (essential for toxin neutralization and protection) and cellular arms of adaptive immunity (for the clearance of encapsulated B. anthracis) (Glomski et al., 2007) and that can be prepared without handling the dangerous pathogen and increasing threat of bioterrorism etc drive the scientists to develop DNA vaccine against anthrax. The work towards anti–anthrax DNA vaccine mainly included demonstration of anti–PA immune response in mice, rats and rabbits (Gu et al., 1999; Luxembourg et al., 2008). However, reports of successful protection of hosts through the administration of PA–encoding DNA alone have been limited (Riemenschneider et al., 2003; Midha and Bhatnagar, 2009). Therefore, some investigators (Price et al., 2001b; Hahn et al., 2006; Zhang et al., 2008) later on tried DNA constructs encoding other vaccine candidates of anthrax bacilli (lethal factor, spore antigens or vegetative cell antigens) along with PA encoding construct and found that co–administration of these vaccine candidates could generate synergestic effect. In the attempt to further enhance the intensity of immune response to B. anthracis several groups have attempted many second generation DNA vaccination strategies which are depicted in

Brucellosis
Brucellosis, an infectious disease affecting livestock and humans by different species of Brucella remains endemic in many developing countries, where it undermines animal health and productivity, causing important economic losses. In human it is a potentially life–threatening multisystem disease. Unfortunately, the current vaccines are not ideal because of their limited efficacy and potential to cause disease in humans (Singha et al., 2008). Consequently, numerous attempts are made to develop efficient DNA vaccines against brucellosis which are briefly outlined below. It was Kurar and Splitter (1997) who started DNA based immunization studies of brucellosis. They used plasmid expressing ribosomal protein L7/L12, to immunize mice and found that the protection induced after 28 days of immunization against challenge was equivalent to that induced by live B. abortus strain 19 vaccine. The further DNA vaccination trials of B. abortus included immune response studies against plasmid encoding Cu, Zn superoxide dismutase (Onate et al., 2003; Singha et al., 2008), plasmid encoding GroEL heat–shock gene (Leclero et al., 2002), plasmid encoding bacterioferritin or P39 gene (Al–Mariri et al., 2001), divalent fusion DNA vaccine encoding L7/L12 and Omp16 (Luo et al., 2006) and a combined DNA vaccine encoding three antigens BCSP31, SOD, and L7/L12 (Yu et al., 2007). Some second generation DNA vaccine optimization strategies have also been attempted (Table 4). In case of B. melitensis, DNA vaccine encoding omp31 (Cassattaro et al., 2005; Doosti et al., 2009) and DNA vaccine encoding bp26 and trigger factor (Yang et al., 2005) had been tried. Of these DNA vaccine encoding bp26 and Tf was also found to induce immune response in bison (Clapp et al., 2011).

Table 4: Optimization strategies used in second generation bacterial DNA vaccines

Mycobacterial Infections
The increasing number of infection cases and lower efficacy of BCG vaccine in controlling pulmonary infection, emergence of multidrug–resistant strains and co–infection with HIV–1 are the major impetus for developing novel vaccine strategy such as DNA vaccine against Mycobacterium (Nagata and Koide, 2013). Because of the strong cell–mediated and humoral immunity they can induce, DNA vaccines were rapidly considered for use against Mycobacterium which are facultative intracellular pathogens and a considerable number of preclinical studies on the subject have been published in recent years (Table 5).In 1996, Tascon et al. and Huygen et al. were the first to report on the value of naked DNA vaccination against TB after using DNA vaccine encoding 65–kDa heat shock protein from M. leprae and 32–kDa mycolyl transferase or Ag85A from M. tuberculosis, respectively. Afterwards large numbers of studies were conducted and several these DNA vaccines conferred significant protection against TB in mice.

Although to date none of the vaccines have been assessed in human results of trials in rodents have demonstrated the potential of mycobacterial DNA vaccines in larger animals and humans. Also, immunization protocols combining the optimization strategies for second generation DNA vaccines especially potent priming capacity of plasmid DNA with subsequent boosting with BCG are particularly promising for future applications (Jiang et al., 2013).

Table 5: Antigens targeted in Mycobacterial DNA vaccines

Clostridium Tetani
Anderson et al. (1996) used plasmid encoding the non–toxic C–terminal domain of tetanus toxin to immunize BALB/c mice and found that DNA immunization induced a Th1–like response. In contrast, immunization with tetanus toxoid or a polypeptide of fragment C induced a Th2–like response. The serum immunoglobulin response following DNA immunization was sufficient to protect 100% of mice from lethal challenge with tetanus toxin. However, the level of protection conferred by DNA immunization was lesser than that achieved with conventional toxoid or a polypeptide of fragment C. The results of Saikh et al. (1998) also suggested that polypeptide or toxoid vaccines are preferable to plasmid–based vaccination for control of tetanus so that there were no further studies in DNA vaccination against tetanus.

Leptospirosis
Leptospirosis, a zoonosis caused by bacteria of the genus Leptospira, is an important emerging infectious disease worldwide. Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit only serovar specific immunity and unsatisfactory immunological memory. The advantages of DNA vaccines over the inactivated vaccines and requirement of a broad spectrum anti–leptospira vaccine that induces long–lasting memory led to the development of different DNA constructs against leptospirosis. Dai (1998) reported that DNA vaccination with the sero–reactive P68 antigen protected 77% of vaccinated guinea pigs from death and 85% from pulmonary diffuse hemorrhage, following challenge with L. interrogans. Another study with DNA vaccine expressing Hap–1/LipL32 of L. interrogans serovar Autumnalis or Grippotyphosa, used in gerbils showed cross–protection against challenge with Canicola (Branger et al., 2005). Similarly, many works (You et al., 1999; Dai et al., 2000; Wang et al., 2002; Dai et al. 2003) has suggested that DNA vaccines based on endoflagellar (flaB2) gene can be successful vaccines against Leptospira as CpG motif found within the gene could give additional immune–stimulatory property. Likewise, Faisal et al. (2008) showed that immunization with LigA DNA vaccine could provide significant protection against challenge with virulent L. interrogans serovar Pomona. Very recently, Forster et al. (2013) showed that LigBrep DNA vaccine is a promising candidate against leptospirosis. All these observations suggest that use of DNA constructs encoding various immunogenic leptospiral proteins can be a promising approach for protection against leptospirosis.

Borrelia burgdorferi
Although lyme borreliosis caused by Borrelia spp. is the most prevalent arthropod–borne disease in the Western world, no vaccines are currently available to prevent the disease (Yin et al., 2009). The role of outer surface protein genes (ospA and ospC) of B. burgdorferi to elicit protective immune responses when administered as DNA vaccines has been explored (Luke et al., 1997; Wallich et al., 2001; Scheiblhofer et al., 2003), the results of which shows that DNA vaccines against borreliosis need more extensive investigations.

Staphylococcus aureus
S. aureus is one of the five most common causes of nosocomial infections and can cause a range of illnesses from minor skin infections to many life–threatening diseases. Ohwada et al. (1999) used DNA encoding mecA to immunize mice against methicillin–resistant S. aureus infection and showed that the vaccination could produce 0.4 log reduction in kidney CFU compared to control animals. Similarly, Senna et al. (2003) showed that vaccination of mice with plasmid encoding PBP2a protein could reduce the bacterial load of kidneys 1000 times less compared to the non–immunized mice. DNA vaccine comprising of clfA or fibronectin binding protein gene was also shown to induce sufficient protection against S. aureus infection (Shkreta et al., 2004; Nour El–Din et al., 2006). Castagliuolo et al. (2006) showed that intranasal immunization with a DNA vaccine mixture encoding four adhesins of S. aureus (fibrinogen binding protein Efb, fibronectin–binding protein A (FnbpA), clumping factor A (ClfA) and collagen–binding protein (Cna) could trigger significant levels of specific serum and mucosal Ig that inhibited S. aureus adhesion to cow mammary gland epithelial cells in–vitro. Later, Gaudreau et al. 2007) showed that multi gene plasmids (encoding Clfa, FnbpA and the enzyme Sortase) could induce better immune response compared to mixture of the individual plasmids. Very recently, Dai et al. (2013) demonstrated that novel DNA vaccine encoding M. tuberculosis secreted antigen Ag85A fused with influenza A virus HA2 protein could provide protection against both influenza and secondary infection with S. aureus. Thus, DNA immunization against the most important virulence factors, adhesins of S. aureus has been proved as valuable tool to prevent infections in lab animals warranting further studies in target animals.

Mycoplasmosis
Mycoplasma, the smallest self–replicating life–forms are responsible for a variety of diseases in humans, domestic animals, insects, and plants. The various antigens targeted in DNA vaccine studies against Mycoplasmoses included a repeat region of P97 adhesin (Chen et al., 2006), heat shock protein (P42) (Chen et al., 2003), P36, P46, NrdF, P97 and P97R1 (Chen et al., 2008) of M. hyopneumoniae, carboxy terminal region of p1 gene of M. pneumoniae (Zhu et al., 2012; 2013) and P48 of M. agalactiae (Chessa et al., 2008). Very recently, Galli et al. (2012) proved that P46 is a promising candidate for DNA vaccine against M. hyopneumoniae. It was also shown that the licensed DNA vaccine encoding growth hormone delivered before specific vaccination could enhance protection against M. hyopneumoniae (Thacker et al., 2006).

Corynebacterium Pseudotuberculosis
C. pseudotuberculosis, a facultative intracellular bacterium, is the etiological agent of caseous lymphadenitis which is a chronic and contagious disease of sheep and goats worldwide. Although various strategies have been tested to develop vaccine against C. pseudotuberculosis, the search continues for identification of an effective and safe vaccine. Chaplin et al. (1999) vaccinated sheep with DNA encoding genetically detoxified phospholipase D, and obtained good protection against experimental challenge. When Costa et al. (2011) immunized mice with DNA vaccine encoding hsp60 of C. pseudotuberculosis, there was significant humoral immune response but immunization did not confer protective immunity. So improvement of the DNA construct of Chaplin et al. (1999) by adopting second generation DNA vaccine optimization strategies or new vaccines encoding alternative antigens should be targeted in future trials.

Pseudomonas aeruginosa
P. aeruginosa was identified as the fifth most frequently isolated nosocomial pathogen and the second most common cause of nosocomial pneumonia. The increasing numbers of antibiotic resistant P. aeruginosa further necessitate the need to develop suitable immunization strategies against this pathogen. In a study evaluating the immunoprotective potential of a DNA vaccine encoding oprF of P. aeruginosa in mouse model of chronic pulmonary infection Price et al. (2001a) found that there was a significant reductions in the presence of severe macroscopic lesions, as well as in the number of bacteria present in the lungs, of immunized mice. DNA vaccine encoding a fusion protein comprising oprF and another outer membrane protein OprL was later found to be more protective (Price et al., 2002). Denis–Mize et al. (2000) and Shiau (2000) found that upon immunization of mice with DNA encoding the coding sequence of non–toxic mutant form of P. aeruginosa exotoxin A there was a strong serum immunoglobulin response and vaccinated mice were completely protected from the lethal effect of intraperitoneal injection with wild–type Exotoxin A. Saha et al. (2006) showed that immunization with DNA vaccine targeting a fusion of outer membrane proteins (OprF/OprI), a protein regulating type III secretion system (PcrV), or an appendage (PilA) produced the strongest immune response and protection against pulmonary infection caused by P. aeruginosa.

Rhodococcus equi
R. equi remains as significant bacterial pathogen causing severe pyogranulomatous pneumonia in foals. There is no effective vaccine currently available for the prevention of R. equi pneumonia in which the protective immunity is largely based on cell–mediated immune response. DNA vaccine encoding VapA virulence protein of R. equi has been found to be able to induce specific IgG antibody response and Th–1 response in foals (Vanniasinkam et al., 2005). Subsequently, Phumoonna et al. (2008) demonstrated that a chimeric vapA/groEL2 DNA vaccine enhanced the clearance of R. equi in aerosol challenged mice.

Escherichia coli
Infection with E. coli O157:H7 causes bloody diarrhea and hemolytic uremic syndrome with renal failure that can be deadly dangerous. The search for an effective vaccine also includes some immunization trials with different DNA constructs. Caprioli et al. (2005) described anti–Stx2 DNA vaccines encoding either the B subunit or a fusion protein between the B subunit and the first N–terminal amino acid of the A1 subunit of shiga toxin elicited Stx–specific immune responses. Later, Bentancor et al. (2009) developed DNA construct encoding both Stx2 A2 and B subunit and found that this could induce specific humoral responses and could confer in–vitro as well as in–vivo Stx2 neutralization activity. Recently Shariati et al. (2012) used a triplet synthetic gene (eit) designed from three genes (espA, eae and tir) and found that this DNA vaccine could induce protective immunity either alone or in combination with purified antigens to reduce EHEC infection. DNA vaccines against other E. coli such as DNA vaccine encoding K88 fimbrial protein (Cho et al., 2004), CFA/I fimbrial adhesin (Alves et al., 1998), faeG adhesin gene (Turnes et al. 1999) of enterotoxigenic E. coli and adhesin of enteroaggregative E. coli (Bouzari et al. 2010) were also reported to elicit satisfactory protection.

Chlamydiosis
They are obligate, intracellular, Gram–negative bacteria that can produce a variety of diseases in humans and animals. As DNA immunization can induce both humoral and cellular immune responses which are especially suited to fight against intracellular bacteria these represent an opportunity for researchers to explore a novel method of vaccination against these pathogens (Ling et al., 2011). The genes tested included MOMP, pgp3, ORF–5 for C. trachomatis (Donati et al., 2003; Li et al., 2008), MOMP, variable domains of MOMP and CTP synthetase for C. muridarum (Zhang et al., 1997; 1999; Pal et al., 1999; Dongji et al., 2000), Omp2, panel of ORF, Hsp60, MOMP for C. pneumoniae (Svanholm et al., 2000; Penttilä et al., 2001), MOMP for C. psittaci (Vanrompay et al., 1999) and DnaK (Hsp70) and ompA for C. abortus (Hechard et al., 2002; 2003; Ou et al., 2013). Of these MOMP was proved as the most important antigen, however, use of additional chlamydial antigen genes and evaluation of different optimization strategies is necessary in future trials to improve the degree of protection.

Salmonella Typhi
Despite advances in technology and public health strategies, typhoid fever remains as a major cause of morbidity in the developing countries. Surprisingly, there was only one report of DNA vaccination against this disease. When Lopez–Macias et al. (1995) immunized BALB/c mice with DNA expressing the Outer Membrane Protein C Porin of Salmonella Typhi there was a serum IgG response specific to the protein.

Yersiniosis
DNA immunization trials have been attempted against two Yersinia Spp namely Y. pestis and Y. enterocolitica. The maximum trials were against pneumonic plague, a highly lethal and contagious disease caused by Y. pestis. DNA vaccine encoding V antigen (Bennett et al., 1999; Wang et al., 2004a; Garmory et al., 2004), F1 capsular antigen (Grosfeld et al., 2003) or both (Yamanaka et al., 2008; 2009) were shown to be protective against pneumonic plague. In case of Y. enterocolitica Noll et al. (1999) successfully immunized mice with HSP60 DNA vaccine which were then found to be protected from challenge with lethal dose of Y. enterocolitica administered either intravenously or orally.

Helicobacter Pylori
H. pylori a Gram–negative microaerophilic spirochete classified as a class I carcinogen, has infected half of the world’s human population. The high prevalence of infection, emergence of antibiotic resistant strains, its role in pathogenesis of gastritis, peptic ulcer, MALTomas and adenocarcinomas and difficulty and high cost of treatment make it an important target for DNA vaccination. Different DNA immunization trials using heat shock Protein A or B (Todoroki et al., 2000), urease B (Hatzifoti et al., 2006; Sun et al., 2006; Xu et al., 2007) outer inflammatory protein A (Chen et al., 2012) and Lpp20 antigen (Yu et al. 2010) of H. pylori demonstrated that DNA immunization can be as a productive and economic novel method against H. pylori in humans.

Streptococcosis
S. pneumoniae is a leading cause of morbidity and mortality in both developing and developed countries. The disadvantages of current polysaccharide or licensed conjugate vaccines and requirement of cell mediated immunity for protection directed the scientists to focus on DNA vaccine against S. pneumoniae. Lesinskia et al. (2001) demonstrated that DNA vaccine encoding peptide mimic of S. pneumoniae serotype–4 capsular polysaccharide could induce specific anti–carbohydrate antibodies in Balb/c mice. In the same year Miyaji et al. showed that PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines could induce humoral and cellular immune responses against S. pneumoniae. Subsequently, Ferreira et al. (2006, 2010) reported that immunization with a plasmid expressing PspA could protect mice from lethal pneumococcal septicaemia. Another Streptococcus, S. iniae is an important fish pathogen with a broad host range that includes both marine and freshwater fish species. Very recently, Sun et al. (2012) demonstrated that DNA vaccines based on sagF, G, and I, especially when they are formulated as multivalent vaccines, were highly efficacious against S. iniae infection.

Dental caries is a widespread infectious disease, of which the principal causative agent is S. mutans. The production of a safe and cost–effective dental caries vaccine which can block tooth colonization and plaque buildup by S. mutans has been a high priority in dental research (Shi et al., 2012). Fan et al. (2002) showed that DNA vaccine encoding cell–surface protein antigen (PAc) of S. mutans could induce protective anti–caries immune responses. Then, Jia et al. (2006) constructed a fusion anti–caries DNA vaccine encoding GLU fragment of S. mutans gtfB gene and A–P fragment of PAc gene and found that this vaccine reduced the levels of dental caries caused by S. mutans in gnotobiotic animals. However, the protective effect against S. sobrinus infection was weaker (Xu et al., 2007). Thereafter, Niu et al. (2009) showed that addition of DNA vaccine encoding catalytic fragment of S. sobrinus gtf–I gene to the above vaccine could provide better protection against caries. Some second generation DNA vaccine optimization strategies has also been tested against dental caries (Table 4) Thus, the idea of anti dental caries DNA vaccine seems to be attractive, but the full potential of DNA vaccines has not yet been fully realized which has to be achieved in future studies.

Concerns and Future Prospects
Despite the numerous advantages some issues have been raised with regard to DNA vaccines. First, integration of injected vaccine DNA might occur in the genome of the host cell which may result in insertional mutagenesis, activation of oncogenes or inactivation of tumor suppressor genes (Martin et al., 1999). Second, repeated injections can induce autoimmunity (Mor et al., 1997). Finally, the antibiotic resistance gene carried on vector plasmid can introduce that resistance property in immunized animals or humans when that plasmid is used for vaccination. However, exhaustive research has found little evidence of integration, and the risk for integration appears to be significantly lower than that associated with naturally occurring mutations (Ledwith et al., 2000; Wang et al., 2004b; Sheets et al., 2006). Studies have also shown that evidence for the changes in clinical markers of autoimmunity have not been reported and early human studies did not detect any increase in anti–DNA antibodies, therefore, systemic autoimmunity is unlikely to result from DNA vaccination (MacGregor et al., 1998; Tavel et al., 2007; Klug et al., 2012). For avoiding the antibiotic resistance two precautions should be kept during the selection of vector plasmid. First, the antibiotic resistance genes contained by vaccine plasmids are driven by bacterial origin of replication (not mammalian one) and are therefore expressed only in bacteria, not in host cells. Second, the antibiotic resistance employed does not involve antibiotics commonly used to treat human infections (Mateen and Irshad, 2011). Overall, multiple studies have reported that DNA platform is well tolerated and have an enviable safety record.

DNA vaccines are emerging as a promising new approach to protect humans and animals from various infections (Dhama et al., 2008; Nagata and Koide, 2010; Liu et al., 2011; Ghanem et al., 2013; Kumar et al., 2013). However, their application to bacterial infections has been scarcely advanced compared to viral DNA vaccines and, till to date no licensed bacterial DNA vaccines are available. On the other hand, a great deal of progress has been made in bacterial DNA vaccinology which indicates that these are likely to become a reality in the nearby future. The significant obstacle to the successful development of DNA vaccines has been the low efficacy in the induction of immune response. The recent sequencing of the complete genomes of many pathogenic bacteria will help in the identification of novel antigen candidates for DNA vaccination (Ingolotti et al., 2010). Another potentially interesting study would be to implement an expression library immunization and to screen it in vitro, as well as in vivo, for its protective effect. Such a novel approach will be very useful for the rapid identification of protective genes especially for microorganisms that are difficult to grow or attenuate, such as Chlamydiaceae (Leclercq et al., 2003).

It is now well known that DNA vaccination efficiency also depends on delivery method, dose of immunization and challenge, adjuvants as well as the species and strain of the animal used for immunization study. Therefore, as the protective effect of a given antigen can differ depending on the immunization protocol, it seems important to first test the same antigen with different immunization protocols before reaching a definitive conclusion regarding its protective effect. Similarly, the strain and age of the mice can also influence the outcome of experimental study. In addition, DNA vaccination is often more efficient in mice than in large animals and humans which are the target species. So the promising DNA vaccines from various experiments should be tested in animal models that closely mimic the definite host of that disease. But the high animal cost and the limited number of immunological reagents makes trials on target species more difficult. So efforts should also be made to reduce the cost of DNA vaccination to make it commercially viable for use in higher animals and humans. Advancements in vector and antigen design, improved formulations and deliver devices/methods, inclusion of adjuvants and prime boosting strategy have greatly enhanced the immunogenicity of second–generation DNA vaccines. This improved performance has spurred a renewed interest in DNA platform, which is reflected by the numerous ongoing experiments on bacterial DNA vaccines. However, the various optimization strategies of second generation DNA vaccines should be evaluated on the proven antigens of different bacteria to further improve their performance.

CONCLUSION

Many attempts have been made in the last decade to develop a DNA vaccine against many bacterial diseases which are shortly reviewed in the present literature. The results of these vaccination trials are quite encouraging which strongly suggests that DNA immunization can represent an efficient method to fight against many bacterial infections especially against intracellular bacteria in the nearby future itself. But, before becoming a reality, it must still be subjected to further experiments, including the various second generation DNA vaccine optimization strategies.

REFERENCES

Al–Mariri A, Tibor A, Lestrate P, Mertens P, De Bolle X and Letesson JJ (2002). Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen. Infect. Immun. 70(4): 1915–1923.
http://dx.doi.org/10.1128/IAI.70.4.1915-1923.2002
PMid:11895955 PMCid:PMC127831

Al–Mariri A, Tibor A, Mertens P, De Bolle X, Michel P, Godfroid J, Walravens K and Letesson JJ (2001). Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella spp. Infect. Immun. 69(10): 6264–6270.
http://dx.doi.org/10.1128/IAI.69.10.6264-6270.2001
PMid:11553569 PMCid:PMC98760

Alves AM, Lasaro MO, Almeida DF and Ferreira LC (1998). Immunoglobulin G subclass responses in mice immunized with plasmid DNA encoding the CFA/I fimbria of enterotoxigenic Escherichia coli. Immunol. Lett. 62(3): 145–149.
http://dx.doi.org/10.1016/S0165-2478(98)00035-2

Anderson R, Gao XM, Papakonstantinopoulou A, Roberts M and Dougan G (1996). Immune response in mice following immunization with DNA encoding fragment C of tetanus toxin. Infect. Immun. 64(8): 3168–3173.
PMid:8757849 PMCid:PMC174203

Baldwin SL, D'Souza C, Roberts AD, Kelly BP, Frank AA, Liu MA, Ulmer JB, Huygen K, Mc Murray and Orme IM (1998). Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect. Immun. 66: 2951–2959.
PMid:9596772 PMCid:PMC108294

Bellet JS and Prose NS (2005). Skin complications of Bacillus Calmette–Guerin immunization. Curr. Opin. Infect. Dis. 18(2): 97–100.
http://dx.doi.org/10.1097/01.qco.0000160895.97362.4f
PMid:15735410

Bennett AM, Phillpotts RJ, Perkins SD, Jacobs SC, Williamson ED (1999). Gene gun mediated vaccination is superior to manual delivery for immunisation with DNA vaccines expressing protective antigens from Yersinia pestis or Venezuelan Equine Encephalitis virus. Vaccine 18(7–8): 588–596.
http://dx.doi.org/10.1016/S0264-410X(99)00317-5

Bentancor LV, Bilen M, Fernández Brando RJ, Ramos MV, Ferreira LCS, Ghiringhelli PD and Palermo MS (2009). A DNA vaccine encoding the enterohemorragic Escherichia coli shiga–like toxin 2 A2 and B subunits confers protective immunity to shiga toxin Challenge in the murine model. Clin. Vaccine Immunol. 16(5): 712–718.
http://dx.doi.org/10.1128/CVI.00328-08
PMid:19176691 PMCid:PMC2681575

Bouzari S, Dashti A, Jafari A and Oloomi M (2010). Immune response against adhesins of enteroaggregative Escherichia coli immunized by three different vaccination strategies (DNA/DNA, protein/protein, and DNA/protein) in mice. Comp. Immunol. Microbiol. Infect. Dis. 33(3): 215–225.
http://dx.doi.org/10.1016/j.cimid.2008.10.002
PMid:19022502

Branger C, Chatrenet B, Gauvrit A, Aviat F, Aubert A, Bach JM and Andre–Fontaine G (2005). Protection against Leptospora interrogans Sensu Lato challenge by DNA immunization with the gene encoding haemolysin associated protein 1. Infect. Immun. 73(7): 4062–4069.
http://dx.doi.org/10.1128/IAI.73.7.4062-4069.2005
PMid:15972494 PMCid:PMC1168576

Brown TH, David J, Acosta–Ramirez E, Moore JM, Lee S, Zhong G, Hancock RE, Xing Z, Halperin SA and Wang J (2012). Comparison of immune responses and protective efficacy of intranasal prime–boost immunization regimens using adenovirus–based and CpG/HH2 adjuvanted–subunit vaccines against genital Chlamydia muridarum infection. Vaccine 30(2):350–360.
http://dx.doi.org/10.1016/j.vaccine.2011.10.086
PMid:22075089

Brun P, Zumbo A, Castagliuolo I, Delogu G, Manfrin F, Sali M, Fadda G, Grillot–Courvalin C, Palù G and Manganelli R (2008). Intranasal delivery of DNA encoding antigens of Mycobacterium tuberculosis by non–pathogenic invasive Escherichia coli. Vaccine 26(16): 1934–1941.
http://dx.doi.org/10.1016/j.vaccine.2008.02.023
PMid:18342411

Cai H, Yu DH, Tian X and Zhu YX. (2005). Co–administration of interleukin 2 plasmid DNA with combined DNA vaccines significantly enhances the protective efficacy against Mycobacterium tuberculosis. DNA Cell Biol. 24 (10): 605–613.
http://dx.doi.org/10.1089/dna.2005.24.605
PMid:16225391

Caprioli A, Morabito S, Brugere H and Oswald E (2005). Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission. Vet. Res. 36: 289–311.
http://dx.doi.org/10.1051/vetres:2005002
PMid:15845227

Cassataro J, Veliskovisky CA, Barrera S, Estein SM, Bruno L, Bowden R, Pasquevich KA, Fossati CA and Iambartolomei GH (2005). A DNA vaccine coding for the outer membrane protein 31 confers protection in mice against B. meltensis and B. ovis infection by eliciting a specific cytotoxic response. Infect. Immun. 73(10): 6537–6546.
http://dx.doi.org/10.1128/IAI.73.10.6537-6546.2005
PMid:16177328 PMCid:PMC1230944

Castagliuolo I, Piccinini R, Beggiao E, Pal G, Mengoli C, Ditadi F, Vicenzoni G and Zecconi A (2006). Mucosal genetic immunization against four adhesins protects against Staphylococcus aureus–induced mastitis in mice. Vaccine 24(20): 4393–4402.
http://dx.doi.org/10.1016/j.vaccine.2006.02.055
PMid:16580097

Chambers MA, Vordermeier HM, Whelan A, Commander N, Tascon R, Lowrie D and Gewinson R (2000). Vaccination of mice and cattle with plasmid DNA encoding the Mycobacterium bovis antigen MPB83. Clin. Infect. Dis. 30(3): S283–S287.
http://dx.doi.org/10.1086/313875
PMid:10875801

Chambers MA, Williams a, Hatch G, Gavier–Widén D, Hall G, Huygen K, Lowrie D, Marsh PD and Hewinson RG (2002). Vaccination of guinea pigs with DNA encoding the mycobacterial antigen MPB83 influences pulmonary pathology but not hematogenous spread following aerogenic infection with Mycobacterium bovis. Infect. Immun.70: 2159–2165.
http://dx.doi.org/10.1128/IAI.70.4.2159-2165.2002
PMid:11895982 PMCid:PMC127856

Changhong S, Hai Z, Limei W, Jiaze A, Li X, Tingfen Z, Zhikai X and Yong Z (2009). Therapeutic efficacy of a tuberculosis DNA vaccine encoding heat shock protein 65 of Mycobacterium tuberculosis and the human interleukin 2 fusion gene. Tuberculosis 89(1): 54–61.
http://dx.doi.org/10.1016/j.tube.2008.09.005
PMid:19056317

Chaplin PJ, De Rose R, Boyle JS, McWaters P, Kelly J, Tennent JM, Lew AM and Scheerlinck JY (1999). Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in Sheep. Infect. Immun. 67(12): 6434–6438.
PMid:10569760 PMCid:PMC97052

Chen AY, Fry SR, Forbes–Faulkner J, Daggard G and Mukkur TK (2006). Evaluation of the immunogenicity of the P97R1 adhesin of Mycoplasma hyopneumoniae as a mucosal vaccine in mice. J. Med. Microbiol. 55: 923–929.
http://dx.doi.org/10.1099/jmm.0.46088-0
PMid:16772421

Chen J, Lin L, Li N and She F (2012). Enhancement of Helicobacter pylori outer inflammatory protein DNA vaccine efficacy by co–delivery of interleukin–2 and B subunit heat–labile toxin gene encoded plasmids. Microbiol. Immunol. 56(2): 85–92.
http://dx.doi.org/10.1111/j.1348-0421.2011.00409.x
PMid:22150716

Chen YL, Wang SN, Yang WJ, Chen YJ, Lin HH and Shiuan D (2003). Expression and immunogenicity of Mycoplasma hyopneumoniae heat shock protein antigen P42 by DNA vaccination. Infect. Immun. 71(3): 1155–1160.
http://dx.doi.org/10.1128/IAI.71.3.1155-1160.2003
PMid:12595427 PMCid:PMC148838

Chen AY, Fry SR, Daggard GE and Mukkur TK (2008). Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice. Vaccine 26(34): 4372–4378.
http://dx.doi.org/10.1016/j.vaccine.2008.06.005
PMid:18602730

Chessa B, Pittau M, Puricelli M, Zobba R, Coradduzza E, Dall'ara P, Rosati S, Poli G and Alberti A (2009). Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice. Res. Vet. Sci. 86: 414–420.
http://dx.doi.org/10.1016/j.rvsc.2008.09.010
PMid:19007952

Cho SH, Loewen PC and Marquardt RR (2004). A plasmid DNA encoding chicken interleukin–6 and Escherichia coli K88 fimbrial protein FaeG stimulates the production of anti–K88 fimbrial antibodies in chickens. Poult. Sci. 83(12): 1973–1978
http://dx.doi.org/10.1093/ps/83.12.1973
PMid:15615009

Clapp B, Walters N, Thornburg T, Hoyt T, Yang X and Pascual DW (2011). DNA vaccination of bison to brucellar antigens elicits elevated antibody and IFN–γ responses. J. Wildl. Dis. 47(3): 501–510.
http://dx.doi.org/10.7589/0090-3558-47.3.501
PMid:21719815

Coler RN, Skeiky YA, Vedvick T, Bement T, Ovendale P, Campos–Neto A, Alderson M and Reed SG (1998). Molecular cloning and immunological reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis. J. Immunol. 161: 2356–2364.
PMid:9725231

Costa MP, McCulloch JA, Almeida SS, Dorella FA, Fonseca CT, Oliveira DM, Teixeira MFS, Laskowska E, Lipinska B, Meyer R, Portela RW, Oliveira SC, Miyoshi A and Azevedo V (2011). Molecular characterization of the Corynebacterium pseudotuberculosis hsp60–hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice. BMC Res. Notes 4: 243.
http://dx.doi.org/10.1186/1756-0500-4-243
PMid:21774825 PMCid:PMC3158118

Coulter LJ, Sinclair MC, Livingstone M, Maley SW, Buxton D and Longbottom D (2002). Validation of DNA vaccine constructs for Chlamydophila abortus protection studies. Res. Vet. Sci. 72: 24. http://dx.doi.org/10.1016/S0034-5288(02)90069-X

Curtiss R (2011). The impact of vaccines and vaccinations: challenges and opportunities for modelers. Math. Biosci. Eng. 8(1): 77–93.
http://dx.doi.org/10.3934/mbe.2011.8.77
PMid:21361401

Dai B, You Z, Chen Z, Yan H and Fang Z (2000). Protection against leptospirosis by immunization with plasmid DNA encoding 33 kDa endoflagellin of L. interrogans serovar lai. Chin. Med. Sci. J. 15: 14–19.
PMid:12899392

Dai B, You Z, He P, Wang M and Wang Y (2003). Analysis of cpG motif in endoflagellar gene and expression vector of Leptospiral DNA vaccine. Sichuan Da Xue Xue Bao Yi Xue Ban 34: 1–4.
PMid:15600164

Dai J, Pei D, Wang B, Kuang Y, Ren L, Cao K, Zuo B, Shao J, Li S, Jiang Z, Li H and Li M (2013). A novel DNA vaccine expressing the Ag85A–HA2 fusion protein provides protection against influenza A virus and Staphylococcus aureus. Virol. J. 10: 40–52.
http://dx.doi.org/10.1186/1743-422X-10-40
PMid:23369570 PMCid:PMC3598506

Dai B (1998). Immunoprotection in guinea pigs using DNA recombinant plasmid rpDJt and expressed protein P68 in L. interrogans serovar . Hua Xi Yi Ke Da Xue Xue Bao 29(3): 248–251.
PMid:10684084

Delogu G and Brennan MJ (2001). Comparative immune response to PE and PE_PGRS antigens of Mycobacterium tuberculosis. Infect. Immun. 69: 5606–5611.
http://dx.doi.org/10.1128/IAI.69.9.5606-5611.2001
PMid:11500435 PMCid:PMC98675

Denis–Mize KS, Prize BM, Baker NR and Galloway DR (2000). Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A. FEMS Immunol. Med. Microbiol. 27(2): 147–154. http://dx.doi.org/10.1111/j.1574-695X.2000.tb01425.x
PMid:10640610

Dhama K, Mahendran M, Gupta PK and Rai A (2008). DNA Vaccines and their applications in veterinary practice: current perspectives. Vet. Res. Commun. 32(5): 341–356.
http://dx.doi.org/10.1007/s11259-008-9040-3
PMid:18425596

Dillon DC, Alderson MR, Day CH, Lewinsohn DM, Coler R, Bement T, Campos–Neto A, Skeiky YAW, Orme IM, Roberts A, Steen S, Dalemans W, Badaro R and Reed SG (1999). Molecular characterization and human T–cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family. Infect. Immun. 67: 2941–2950.
PMid:10338503 PMCid:PMC96604

Donati M, Sambri V, Comanducci M, Di Leo K, Storni E, Giacani L, Ratti G and Cevenini R (2003). DNA immunization with pgp3 gene of Chlamydia trachomatis inhibits the spread of chlamydial infection from the lower to the upper genital tract in C3H/HeN mice. Vaccine 21: 1089–1093.
http://dx.doi.org/10.1016/S0264-410X(02)00631-X

Dong–Ji Z, Yang X, Shen C, Lu H, Murdin A and Brunham RC (2000). Priming with Chlamydia trachomatis major outer membrane protein (MOMP) DNA followed by MOMP ISCOM boosting enhances protection and is associated with increased immunoglobulin A and Th1 cellular immune responses. Infect. Immun. 68: 3074–3078.
http://dx.doi.org/10.1128/IAI.68.6.3074-3078.2000
PMid:10816446 PMCid:PMC97534

Donnelly JJ, Ulmer JB, Shiver JW and Liu MA (1997). DNA vaccines. Annu. Rev. Immunol. 15: 617–648.
http://dx.doi.org/10.1016/S0264-410X(96)00268-X

Donnelly JJ, Wahren B and Liu MA (2005). DNA vaccines: progress and challenges. J. Immunol. 175(2): 633–639.
http://dx.doi.org/10.4049/jimmunol.175.2.633
PMid:16002657

Doosti A, Dehkordi PG, Javadi GR, Sardari S and Shokrgozar MA (2009). DNA vaccine encoding the omp31 gene of Brucella melitensis induces protective immunity in BALB/c Mice. Res. J. Biol. Sci. 4(1): 126–131.

D'Souza S, Rosseels V, Denis O, Tanghe A, De Smet N, Jurion F, Palfliet K, Castiglioni N, Vanonckelen A, Wheeler C and Huygen K. (2002). Improved tuberculosis DNA vaccines by formulation in cationic lipids. Infect. Immun. 70(7): 3681–3688.
http://dx.doi.org/10.1128/IAI.70.7.3681-3688.2002
PMid:12065510 PMCid:PMC128113

Dunham SP (2002). The application of nucleic acid vaccines in veterinary medicine. Res. Vet. Sci. 73: 9–16.
http://dx.doi.org/10.1016/S0034-5288(02)00032-2

Erb KJ, Kirman J, Woodfield L, Wilson T, Collins DM, Watson JD and LeGros G (1998). Identification of potential CD8+ T cell epitopes of the 19 kD and AhpC proteins from Mycobacterium tuberculosis: no evidence for CD8+ T cell priming against the identified peptides after DNA vaccination of mice. Vaccine 16: 692–697.
http://dx.doi.org/10.1016/S0264-410X(97)00253-3

Faisal SM, Yan W, Chen CS, Palaniappan RUM, McDonough and Chang YF (2008). Evaluation of protective immunity of Leptospira immunoglobulin like protein A (LigA) DNA vaccine against challenge in hamsters. Vaccine 26(2): 277–287.
http://dx.doi.org/10.1016/j.vaccine.2007.10.029
PMid:18055070

Fan MW, Bian Z, Peng ZX, Zhong Y, Chen Z, Peng B and Jia R (2002). A DNA vaccine encoding a cell–surface protein antigen of Streptococcus mutans protects gnotobiotic rats from caries. J. Dent. Res. 81(11): 784–787.
http://dx.doi.org/10.1177/154405910208101112
PMid:12407095

Fan X, Gao Q and Fu R (2007). DNA vaccine encoding ESAT–6 enhances the protective efficacy of BCG against Mycobacterium tuberculosis infection in mice. Scand. J. Immunol. 66(5): 523–528.
http://dx.doi.org/10.1111/j.1365-3083.2007.02006.x
PMid:17916110

Ferraro B, Morrow MP, Hutnick NA, Shin TH, Lucke and Weiner DB (2011). Clinical Applications of DNA Vaccines: Current Progress. Clin. Infect. Dis. 53(3): 296–302.
http://dx.doi.org/10.1093/cid/cir334
PMid:21765081 PMCid:PMC3202319

Ferreira G, Monteiro G, Prazeres D and Cabral J (2000). Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. Trends Biotechnol. 18: 380–388.
http://dx.doi.org/10.1016/S0167-7799(00)01475-X

Ferreira DM, Miyaji EN, Oliveira ML, Darrieux M, Araas AP, Ho PL and Leite LC (2006). DNA vaccines expressing pneumococcal surface protein A (PspA) elicit protection levels comparable to recombinant protein. J. Med. Microbiol. 55: 375–378.
http://dx.doi.org/10.1099/jmm.0.46217-0
PMid:16533983

Ferreira DM, Oliveira MLS, Moreno AT, Ho PL, Briles DE and Miyaji EN (2010). Protection against nasal colonization with Streptococcus pneumoniae by parenteral immunization with a DNA vaccine encoding PspA (Pneumococcal surface protein A). Microb. Pathogenesis 48(6): 205–213.
http://dx.doi.org/10.1016/j.micpath.2010.02.009
PMid:20206678

Forster KM, Hartwig DD, Seixas FK, Bacelo KL, Amaral M, Hartleben CP and Dellagostin OA (2013). A conserved region of leptospiral immunoglobulin–like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis. Clin. Vaccine Immunol. 20(5): 725–731.
http://dx.doi.org/10.1128/CVI.00601-12
PMid:23486420 PMCid:PMC3647749

Fynan EF, Robinson HL and Webster RG (1993). Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine. DNA Cell Biol. 12: 785–789.
http://dx.doi.org/10.1089/dna.1993.12.785
PMid:8216849

Galli V, Simionatto S, Marchioro SB, Fisch A, Gomes CK, Conceiçao FR and Dellagostin OA (2012). Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines. Vaccine 31(1): 135–140.
http://dx.doi.org/10.1016/j.vaccine.2012.10.088
PMid:23137841

Galloway DR and Baillie L (2004). DNA vaccines against anthrax. Expert. Opin. Biol. Ther. 4(10): 1661–1667.
http://dx.doi.org/10.1517/14712598.4.10.1661
PMid:15461577

Garmory HS, Freeman D, Brown KA and Titball RW (2004). Protection against plague afforded by immunisation with DNA vaccines optimised for expression of the Yersinia pestis V antigen. Vaccine 22(8): 947–957.
http://dx.doi.org/10.1016/j.vaccine.2003.09.020
PMid:15161071

Gaudreau M, Lacasse P and Talbot BG (2007). Protective immune responses to a multi–gene DNA vaccine against Staphylococcus aureus. Vaccine 25: 814–824.
http://dx.doi.org/10.1016/j.vaccine.2006.09.043
PMid:17027124

Ghanem A, Healey R and Adly FG (2013). Current trends in separation of plasmid DNA vaccines: A review. Anal. Chim. Acta. 760: 1–15.
http://dx.doi.org/10.1016/j.aca.2012.11.006
PMid:23265728

Glomski IJ, Corre JP, Mock M and Goossens PL (2007). Noncapsulated toxinogenic Bacillus anthracis presents a specific growth and dissemination pattern in naive and protective antigen–immune mice. Infect. Immun. 75(10): 4754–4761.
http://dx.doi.org/10.1128/IAI.00575-07
PMid:17635863 PMCid:PMC2044546

Grosfeld H, Cohen S, Bino T, Flashner Y, Ber R, Mamroud E, Kronman C, Shafferman A and Velan B (2003). Effective Protective Immunity to Yersinia pestis Infection Conferred by DNA Vaccine Coding for Derivatives of the F1 Capsular Antigen. Infect. Immun. 71(1): 374–383.
http://dx.doi.org/10.1128/IAI.71.1.374-383.2003
PMid:12496187 PMCid:PMC143422

Gu ML, Leppla SH and Klinman DM (1999). Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen. Vaccine 17: 340–344.
http://dx.doi.org/10.1016/S0264-410X(98)00210-2

Gurunathan S, Klinman DM and Seder RA (2000). DNA vaccines: immunology, application and optimization. Ann. Rev. Immunol. 18: 927–974.
http://dx.doi.org/10.1146/annurev.immunol.18.1.927
PMid:10837079

Gurunathan S, Wu CY, Freidag BL and Seder RA (2000). DNA vaccines: a key for inducing long–term cellular immunity. Curr. Opin. Immunol. 12(4): 442–447.
http://dx.doi.org/10.1016/S0952-7915(00)00118-7

Haghighi HR and Prescott JF (2005). Assessment in mice of vapA–DNA vaccination against Rhodococcus equi infection. Vet. Immunol. Immunopathol. 104(3–4): 215–225.
http://dx.doi.org/10.1016/j.vetimm.2004.12.006
PMid:15734542

Hahn UK, Boehm R and Beyer W (2006). DNA vaccination against anthrax in mice–combination of anti–spore and anti–toxin components. Vaccine 24: 4569–4571.
http://dx.doi.org/10.1016/j.vaccine.2005.08.031
PMid:16157424

Han TK and Dao ML (2007). Enhancement of salivary IgA response to a DNA vaccine against Streptococcus mutans wall–associated protein A in mice by plasmid–based adjuvants. J. Med. Microbiol. 56: 675–680.
http://dx.doi.org/10.1099/jmm.0.47020-0
PMid:17446293

Hanif SN, Al–Attiyah R and Mustafa AS (2010). DNA vaccine constructs expressing Mycobacterium tuberculosis specific genes induce immune responses. Scand. J. Immunol. 72(5): 408–415.
http://dx.doi.org/10.1111/j.1365-3083.2010.02452.x
PMid:21039735

Hatzifoti C, Roussel Y, Harris AG, Wren BW, Morrow JW and Bajaj–Elliott M (2006). Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori. Helicobacter 11(2): 113–122.
http://dx.doi.org/10.1111/j.1523-5378.2006.00385.x
PMid:16579841

Hechard C and Grepinet O (2004). DNA vaccination against Chlamydiaceae: current status and perspectives. Vet. Res. 35: 149–161.
http://dx.doi.org/10.1051/vetres:2004007
PMid:15099493

Hechard C, Grepinet O and Rodolakis A (2002). Protection evaluation against Chlamydophila abortus challenge by DNA vaccination with a dnaK–encoding plasmid in pregnant and non–pregnant mice. Vet. Res. 33: 313–326.
http://dx.doi.org/10.1051/vetres:2002019
PMid:12056482

Hechard C, Grepinet O and Rodolakis A (2003). Evaluation of protection against Chlamydophila abortus challenge after DNA immunization with the major outer–membrane protein–encoding gene in pregnant and non–pregnant mice. J. Med. Microbiol. 52: 35–40.
http://dx.doi.org/10.1099/jmm.0.04983-0
PMid:12488563

Hermanson G, Whitlow V, Parker S, Tonsky K, Rusalov D, Ferrari M, Lalor P, Komai M, Mere R, Bell R, Brenneman K, Mateczun A, Evans T, Kaslow D, Galloway D and Hoart P (2004). A cationic lipid–formulated plasmid DNA vaccine confers sustained antibody–mediated protection against aerosolized anthrax spores. Proc. Natl. Acad. Sci. U.S.A. 101: 13601–13606.
http://dx.doi.org/10.1073/pnas.0405557101
PMid:15342913 PMCid:PMC518760

Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, Deck RR, DeWitt CM, Orme IM, Baldwin S, D'Souza C, Drowart A, Lozes E, Vandenbussche P, Van Vooren JP, Liu MA and Ulmer JB (1996). Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat. Med. 2: 893–898.
http://dx.doi.org/10.1038/nm0896-893
PMid:8705859

Ingolotti M, Kawalekar O, Shedlock DJ, Muthumani K and Weiner DB (2010). DNA vaccines for targeting bacterial infections. Expert Rev Vaccines. 9(7): 747–763.
http://dx.doi.org/10.1586/erv.10.57
PMid:20624048 PMCid:PMC2962930

Jia R, Guo JH, Fan MW, Bian Z, Chen Z, Fan B, Yu F and Xu QA (2006). Immunogenicity of CTLA4 fusion anti–caries DNA vaccine in rabbits and monkeys. Vaccine 24: 5192–5200.
http://dx.doi.org/10.1016/j.vaccine.2006.03.090
PMid:16675075

Jiang Q, Zhang J, Chen X, Xia M, Lu Y, Qiu W, Feng G, Zhao D, Li Y, He F and Pen G (2013). A novel recombinant DNA vaccine encoding Mycobacterium tuberculosis ESAT–6 and FL protects against Mycobacterium tuberculosis challenge in mice. J. Biomed. Res. 27: 1–15.
http://dx.doi.org/10.1155/2013/493019
http://dx.doi.org/10.1155/2013/656391
http://dx.doi.org/10.7555/JBR.27.20120114
http://dx.doi.org/10.1155/2013/681234
http://dx.doi.org/10.7555/JBR.27.20120077
http://dx.doi.org/10.1155/2013/683768
http://dx.doi.org/10.1155/2013/287019

Jumba M (2010). Genetically Modified Organisms: The Mystery Unraveled. Trafford Publishers. p. 190.

Kamath AT, Feng CG, MacDonald M, Briscoe H and Britton WJ (1999). Differential protective efficacy of DNA vaccines expressing secreted proteins of M. tuberculosis. Infect. Immun. 67: 1702–1707.
PMid:10085007 PMCid:PMC96517

Kindt TJ, Goldsby RA, Osborne BA and Kuby J (2007). Kuby Immunology. W. H. Freeman Publishers. p. 574

Klinman DM, Yamshchikov G and Ishigatsubo Y. (1997). Contribution of CpG motifs to the immunogenicity of DNA vaccines. J Immunol. 158(8): 3635–3639.
PMid:9103425

Klug B, Reinhardt J and Robertson J (2012). Current status of regulations for DNA vaccines. Gene Vaccines 2: 285–295.
http://dx.doi.org/10.1007/978-3-7091-0439-2_14

Kumar U, Kumar S, Varghese S, Chamoli R and Barthwal P (2013). DNA Vaccine: A modern biotechnological approach towards human welfare and clinical trials. Int. J. Res. Biomed. Biotech. 3(1): 17–20.
http://dx.doi.org/10.1016/j.vaccine.2013.09.010
http://dx.doi.org/10.1016/j.vaccine.2013.05.006
http://dx.doi.org/10.1016/j.vaccine.2013.06.067
PMid:23830975

Kurar E and Splitter GA (1997). Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response. Vaccine 15: 1851–1857.
http://dx.doi.org/10.1016/S0264-410X(97)00140-0

Kutzler and Weiner (2008). DNA Vaccines: Ready for Prime Time? Nat. Rev. Genet. 9: 1–13.

Lai WC, Bennett M, Johnston SA, Barry MA and Pakes SP (1995). Protection against Mycoplasma pulmonis infection by genetic vaccination. DNA Cell Biol. 14(7): 643–651.
http://dx.doi.org/10.1089/dna.1995.14.643
PMid:7626224

Leclercq S, Harms JS and Oliveira SC (2003). Enhanced efficacy of DNA vaccines against an intracellular bacterial pathogen by genetic adjuvants. Curr. Pharm. Biotechnol. 4: 99–107.
http://dx.doi.org/10.2174/1389201033489892
PMid:12678885

Leclerq S, Harms JS, Rosinha GM, Azevedo V and Oliveira SC (2002). Induction of a Th1–type of immune response but not protective immunity by intramuscular DNA immunisation with Brucella abortus GroEL heat–shock gene. J. Med. Microbiol. 51(1): 20–26.
PMid:11803949

Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG, Harper LB, Schock HB, Zhang H, Faris JE, Way PA, Beare CM, Bagdon WJ and Nichols WW. (2000). Plasmid DNA vaccines: assay for integration into host genomic DNA. Dev. Biol. 104: 33–43.

Lefevre P, Denis O, De Wit L, Tanghe A, Vandenbussche A, Content J and Huygen K (2000). Cloning of the gene encoding a 22–kDa cell surface antigen of Mycobacterium bovis BCG and analysis of its potential for DNA vaccination against tuberculosis. Infect. Immun. 68: 1040–1047.
http://dx.doi.org/10.1128/IAI.68.3.1040-1047.2000
PMid:10678905 PMCid:PMC97246

Lesinski GB, Smithsonb SL, Srivastava N, Chen D, Widera G and Westerink MA (2001). A DNA vaccine encoding a peptide mimic of Streptococcus pneumoniae serotype 4 capsular polysaccharide induces specific anti–carbohydrate antibodies in Balb/c mice. Vaccine 19(13–14): 1717–1726.
http://dx.doi.org/10.1016/S0264-410X(00)00397-2

Li Z, Howard A, Kelley C, Delogu G, Collins F and Morris S (1999). Immunogenicity of DNA vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences. Infect. Immun.67: 4780–4786.
PMid:10456931 PMCid:PMC96809

Li Z, Wang S, Wu Y, Zhong G and Chen D (2008). Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice. Sci. China C. Life Sci. 51(11): 973–980.
http://dx.doi.org/10.1007/s11427-008-0130-9
PMid:18989639

Ling Y, Liu W, Clark JR, March JB, Yang J and He C (2011). Protection of mice against Chlamydophila abortus infection with a bacteriophage–mediated DNA vaccine expressing the major outer membrane protein. Vet. Immunol. Immunopathol. 144 (3–4): 389–395.
http://dx.doi.org/10.1016/j.vetimm.2011.08.003
PMid:21872342

Liu MA (2003). DNA vaccines: a review. J. Intern. Med. 253(4): 402–410.
http://dx.doi.org/10.1016/S0264-410X(03)00030-6
http://dx.doi.org/10.1016/S0264-410X(03)00009-4
http://dx.doi.org/10.1016/S0264-410X(03)00172-5
http://dx.doi.org/10.1016/S0264-410X(02)00695-3

Liu TH, Oscherwitz J, Schnepp B, Jacobs J, Yu F, Cease KB and Johnson PR (2009). Genetic vaccines for anthrax based on recombinant adeno–associated virus vectors. Mol. Ther. 17: 373–379.
http://dx.doi.org/10.1038/mt.2008.242
PMid:19002162 PMCid:PMC2835057

Lopez Macias C, López–Hernández MA, González, CR, Isibasi A and Ortiz–Navarrete V. (1995). Induction of antibodies against Salmonella typhi OmpC porin by naked DNA immunization. Ann. N. Y. Acad. Sci.772: 285–288.
http://dx.doi.org/10.1111/j.1749-6632.1995.tb44761.x
PMid:8546410

Lowrie DB, Silva CL, Colston MJ, Ragno S and Tascon RE (1997). Protection against tuberculosis by a plasmid DNA vaccine. Vaccine15: 834–838.
http://dx.doi.org/10.1016/S0264-410X(97)00073-X

Lowrie DB, Tascon RE, Bonato VLD, Lima VMF, Faccioli LH, Stavropoulos E, Colston MJ, Hewinson RG, Moelling K and Silva CL (1999). Therapy of tuberculosis in mice by DNA vaccination. Nature 400: 269–271.
http://dx.doi.org/10.1038/22326
PMid:10421369

Lozes E, Huygen K, Content J, Denis O, Montgomery DL, Yawman AM, Vandenbussche P, Van Vooren JP, Drowart A, Ulmer JB and Liu MA (1997). Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine 15(8): 830–833.
http://dx.doi.org/10.1016/S0264-410X(96)00274-5

Luke CJ, Camer K and Liang X (1997). An OspA–based DNA vaccine protects mice against infection with Borrelia burgdorferi. J. Infect. Dis. 175(1): 91–97.
http://dx.doi.org/10.1093/infdis/175.1.91
PMid:8985201

Luo D, Ni B, Li P, Shi W, Zhang S, Han Y, Mao L, He Y, Wu Y and Wang X (2006). Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice. Infect. Immun. 74(5): 2734–2741.
http://dx.doi.org/10.1128/IAI.74.5.2734-2741.2006
PMid:16622210 PMCid:PMC1459688

Luxembourg A, Hannaman D, Nolan E, Ellefsen B, Nakamura G, Chau L, Tellez O, Little S and Bernard R (2008). Potentiation of an anthrax DNA vaccine with electroporation. Vaccine 26: 5216–5222.
http://dx.doi.org/10.1016/j.vaccine.2008.03.064
PMid:18462850

MacGregor RR, Boyer JD, Ugen KE, Lacy KE, Gluckman SJ, Bagarazzi ML, Chattergoon MA, Baine Y, Higgins TJ, Ciccarelli RB, Coney LR, Ginsberg RS and Weiner DB (1998). First human trial of a DNA–based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J. Infect. Dis. 178(1): 92–100.
http://dx.doi.org/10.1086/515613
PMid:9652427

Malin AS, Huygen K, Content J, Mackett M, Brandt L, Andersen P, Smith SM and Dockrell HM (2000). Vaccinia expression of Mycobacterium tuberculosis secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85. Microbes Infect. 2: 1677–1685.
http://dx.doi.org/10.1016/S1286-4579(00)01323-X

Manca C, Lyashchenko K, Wiker HG, Usai D, Colangeli R and Gennaro ML (1997). Molecular cloning, purification, and serological characterization of MPT63, a novel antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 65: 16–23.
PMid:8975887 PMCid:PMC174551

Martin EA, Kamath T, Triccas JA and Britton WJ (2000). Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35–kilodalton antigen is accompanied by induction of gamma interferon–secreting CD4+ T cells. Infect. Immun. 68: 3090–3096.
http://dx.doi.org/10.1128/IAI.68.6.3090-3096.2000
PMid:10816448 PMCid:PMC97536

Martin T, Parker SE, Hedstrom R, Le T, Hoffman SL, Norman J, Hobart P and Lew D (1999). Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection, Hum. Gene Ther. 10: 759–768.
http://dx.doi.org/10.1089/10430349950018517
PMid:10210143

Martin E. Roche PW, Triccas JA and Britton WJ (2001). DNA encoding a single mycobacterial antigen protects against leprosy infection. Vaccine 19: 1391–1396.
http://dx.doi.org/10.1016/S0264-410X(00)00374-1

Mateen I and Irshad S (2011). A review on DNA vaccines. J. Health Sci. 1(1): 1–7.
http://dx.doi.org/10.5923/j.health.20110101.01

Meerak J, Wanichwecharungruang SP and Palaga T (2013). Enhancement of immune response to a DNA vaccine against Mycobacterium tuberculosis Ag85B by incorporation of an autophagy inducing system. Vaccine 31(5): 784–790.
http://dx.doi.org/10.1016/j.vaccine.2012.11.075
PMid:23228812

Midha S and Bhatnagar R (2009). Anthrax protective antigen administered by DNA vaccination to distinct subcellular locations potentiates humoral and cellular immune responses. Eur. J. Immunol. 39: 159–177.
http://dx.doi.org/10.1002/eji.200838058
PMid:19130551

Mir FA, Kaufmann SHE and Eddine AN (2009). A Multicistronic DNA Vaccine Induces Significant Protection against Tuberculosis in Mice and Offers Flexibility in the Expressed Antigen Repertoire. Clin. Vaccine Immunol. 16(10): 1467–1475.
http://dx.doi.org/10.1128/CVI.00237-09
PMid:19656992 PMCid:PMC2756856

Miyaji EN, Dias WO, Gamberini M, Gebara VC, Schenkman RP, Wild J, Riedl P, Reimann J, Schirmbeck R and Leite LC (2001). PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae. Vaccine 20(5–6): 805–812.
http://dx.doi.org/10.1016/S0264-410X(01)00395-4

Mollenkopf HJ, Grode L, Mattow J, Stein M, Mann P, Knapp B, Ulmer J and Kaufmann SH (2004). Application of Mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime–Rv3407 DNA boost vaccination against tuberculosis. Infect. Immun. 72(11): 6471–6479.
http://dx.doi.org/10.1128/IAI.72.11.6471-6479.2004
PMid:15501778 PMCid:PMC523041

Mor G, Singla M, Steinberg AD, Hoffman SL, Okuda K and Klinman DM (1997). Do DNA vaccines induce autoimmune disease? Hum. Gene Ther. 8: 293–300.
http://dx.doi.org/10.1089/hum.1997.8.3-293
PMid:9048196

Moreno S and Timon M (2004). DNA vaccination: an immunological perspective. Inmunologia 23(1): 41–55.

Morris S, Kelley C, Howard A, Li Z and Collins F (2000). The immunogenicity of single and combination DNA vaccines against tuberculosis. Vaccine 18(20): 2155–2163.
http://dx.doi.org/10.1016/S0264-410X(99)00540-X

Munoz–Montesino C, Andrews E, Rivers R, Gonzalez–Smith A, Moraga–Cid G, Folch H, Cespedes S and Onate AA (2004). Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD–specific CD4+ and CD8+ T cells. Infect. Immun. 72(4): 2081–2087.
http://dx.doi.org/10.1128/IAI.72.4.2081-2087.2004
PMid:15039330 PMCid:PMC375181

Nagata T and Koide Y (2010). Induction of specific CD8+ T cells against intracellular bacteria by CD8+ T– cell–oriented immunization approaches. J. Biomed. Biotech. 2010: 764542. DOI: 10.1155/2010/764542.
http://dx.doi.org/10.1155/2010/764542

Nagata T and Koide Y (2013). Identification of T cell epitopes of Mycobacterium tuberculosis with biolistic DNA vaccination. Methods Mol. Biol. 940: 285–303.
PMid:23104350

Niu Y, Sun J, Fan M, Xu QA, Guo J, Jia R and Li Y (2009). Construction of a new fusion anti–caries DNA vaccine. J. Dent. Res. 88(5): 455–460.
http://dx.doi.org/10.1177/0022034509336727
PMid:19493890

Noll A, Bücheler N, Bohn E, Schirmbeck R, Reimann J and Autenrieth IB (1999). DNA immunization confers systemic, but not mucosal, protection against enteroinvasive bacteria. Eur. J. Immunol. 29(3): 986–996.
http://dx.doi.org/10.1002/(SICI)1521-4141(199903)29:03<986::AID-IMMU986>3.0.CO;2-9

Nour El–Din AN, Shkreta L, Talbot BG, Diarra MS and Lacasse P (2006). DNA immunization of dairy cows with the clumping factor A of Staphylococcus aureus. Vaccine 24: 1997–2006.
http://dx.doi.org/10.1016/j.vaccine.2005.11.033
PMid:16426711

Ohwada A, Sekiya M, Hanaki H, Arai KK, Nagaoka I, Hori S, Tominaga S, Hiramatsu K. and Fukuchi Y (1999). DNA vaccination by mecA sequence evokes an antibacterial immune response against methicillin–resistant Staphylococcus aureus. J. Antimicrob. Chemother. 44(6): 767–774.
http://dx.doi.org/10.1093/jac/44.6.767
PMid:10590277

Okada M, Kita Y, Nakajima T, Kanamaru N, Hashimoto S, Nagasava T, Kaneda Y, Yoshida S, Nishida Y, Nakatani H, Takao K, Kishigami C, Inoue Y, Matsumoto M, McMurray DN, Dela Cruz EC, Tan EV, Abalos RM, Burgos JA, Suanderson P and Sakatani M (2009). Novel prophylactic and therapeutic vaccine against tuberculosis. Vaccine 27(25–26): 3267–3270.
http://dx.doi.org/10.1016/j.vaccine.2009.01.064
PMid:19200841

Onate AA, Cespedes S, Cabrera A, Rivers R, Gonzalez A, Munoz C, Folch H and Andrews E (2003). A DNA vaccine encoding Cu, Zn Superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice. Infect. Immun. 71: 4857–4861.
http://dx.doi.org/10.1128/IAI.71.9.4857-4861.2003
PMid:12933826 PMCid:PMC187304

Ou C, Tian D, Ling Y, Pan Q, He Q, Eko FO and He C (2013). Evaluation of an ompA–based phage–mediated DNA vaccine against Chlamydia abortus in piglets. Int. Immunopharmacol. 16(4):505–510.
http://dx.doi.org/10.1016/j.intimp.2013.04.027
PMid:23669337

Pal S, Barnhart KM, Wei Q, Abai AM, Peterson EM and de la Maza LM (1999). Vaccination of mice with DNA plasmids coding for the Chlamydia trachomatis major outer membrane protein elicits an immune response but fails to protect against a genital challenge. Vaccine 17: 459–465.
http://dx.doi.org/10.1016/S0264-410X(98)00219-9

Pan Y, Cai H, Li SX, Tian X, Li T and Zhu YX (2003). Combined recombinant DNA vaccine results in significant protection against Mycobacterium tuberculosis. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao 35(1): 71–76.

Penttila T, Vuola JM, Puurula V, Anttila M, Sarvas M, Rautonen N, Mäkelä PH and Puolakkainen M (2001). Immunity to Chlamydia pneumoniae induced by vaccination with DNA vectors expressing a cytoplasmic protein (Hsp60) or outer membrane proteins (MOMP and Omp2). Vaccine 19: 1256–1265.
http://dx.doi.org/10.1016/S0264-410X(00)00237-1

Phumoonna T, Barton MD, Vanniasinkam T and Heuzenroeder MW (2008). Chimeric vapA/groEL2 DNA vaccines enhance clearance of Rhodococcus equi in aerosol challenged C3H/He mice. Vaccine 26(20): 2457–2465.
http://dx.doi.org/10.1016/j.vaccine.2008.03.015
PMid:18423949

Price BM, Barten Legutki J, Galloway DR, Von Specht BU, Gilleland LB, Gilleland HE Jr and Staczek J (2002). Enhancement of the protective efficacy of an oprF DNA vaccine against Pseudomonas aeruginosa. FEMS Immunol. Med. Microbiol. 33(2): 89–99.
http://dx.doi.org/10.1111/j.1574-695X.2002.tb00577.x
PMid:12052563

Price BM, Galloway DR, Baker NR, Gilleland LB, Staczek J and Gilleland HE Jr (2001a). Protection against Pseudomonas aeruginosa chronic lung infection in mice by genetic immunization against outer membrane protein F (OprF) of P. aeruginosa. Infect. Immune. 69(5): 3510–3515.
http://dx.doi.org/10.1128/IAI.69.5.3510-3515.2001
PMid:11292786 PMCid:PMC98322

Price BM, Liner AL, Park S, Leppla SH, Mateczun A and Galloway DR (2001b). Protection against anthrax lethal toxin challenge by genetic immunization with a plasmid encoding the lethal factor protein. Infect. Immun. 69: 4509–4515.
http://dx.doi.org/10.1128/IAI.69.7.4509-4515.2001
PMid:11401993 PMCid:PMC98526

Reed SG, Coler RN, Dalemans W, Tan EV, DeLa Cruz EC, Basaraba RJ, Orme IM, Skeiky YA, Alderson MR, Cowgill KD, Prieels JP, Abalos RM, Dubois MC, Cohen J, Mettens P and Lobet Y (2009). Defined tuberculosis vaccine, Mtb72F/ AS02A, evidence of protection in cynomolgus monkeys. Proc. Natl. Acad. Sci. USA 106(7): 2301–2306.
http://dx.doi.org/10.1073/pnas.0712077106
PMid:19188599 PMCid:PMC2650151

Reyes–Sandoval A and Ertl HC (2001). DNA vaccines. Curr. Mol. Med. 1(2): 217–243.
http://dx.doi.org/10.2174/1566524013363898
PMid:11899073

Riemenschneider J, Garrison A, Geisbert J, Jahrling P, Hevey M, Negley D, Schmaljohn A, Lee J, Hart MK, Vanderzanden L, Custer D, Bray M, Ruff A, Ivins B, Bassett A, Rossi C and Schmaljohn C (2003). Comparison of individual and combination DNA vaccines for B. anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine 21: 4071–4080.
http://dx.doi.org/10.1016/S0264-410X(03)00362-1

Roche PW, Neupane KD, Failbus SS, Kamath A and Britton WJ (2001). Vaccination with DNA of the Mycobacterium tuberculosis 85B antigen protects mouse foot pad against infection with M. leprae. Int. J. Lepr. Other Mycobact. Dis. 69: 93–98.
PMid:11757171

Rosada RS, de la Torre LG, Frantz FG, Trombone APF, Zarate–Blades CR, Fonseca DM, Souza PRM, Brandao IT, Masson AP, Soares EG, Ramos SG, Faccioli LH, Silva CL Santana MHA and Coelho–Castelo AAM (2008). Protection against tuberculosis by a single intranasal administration of DNA–hsp65 vaccine complexed with cationic liposomes. BMC Immunol. 9: 38.
http://dx.doi.org/10.1186/1471-2172-9-38
PMid:18647414 PMCid:PMC2500095

Saha S, Takeshita F, Sasaki S, Matsuda T, Tanaka T, Tozuka M, Takase K, Matsumoto T, Okuda K, Ishii N, Yamaguchi K, Klinman DM, Xin KQ and Okuda K (2006). Multivalent DNA vaccine protects mice against pulmonary infection caused by Pseudomonas aeruginosa. Vaccine 24(37–39): 6240–6249.
http://dx.doi.org/10.1016/j.vaccine.2006.05.077
PMid:16806598

Saikh KU, Sesno J, Brandler P and Ulrich RG (1998). Are DNA–based vaccines useful for protection against secreted bacterial toxins? Tetanus toxin test case. Vaccine 16(9–10): 1029–1038.
http://dx.doi.org/10.1016/S0264-410X(97)00280-6

Scheiblhofer S, Weiss R, Dürnberger H, Mostbock S, Breitenbach M, Livey I and Thalhamer J (2003). A DNA vaccine encoding the outer surface protein C from Borrelia burgdorferi is able to induce protective immune responses. Microbes Infect. 5(11): 939–946.
http://dx.doi.org/10.1016/S1286-4579(03)00182-5

Schirmbeck R and Leite LC (2001). PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae. Vaccine 20(5–6): 805–812.
PMid:11738744

Sechi LA, Gazouli M, Sieswerda LE, Molicotti P, Ahmed N, Ikonomopoulos J, Scanu AM, Paccagnini D and Zanetti S (2006) Relationship between Crohn's disease, infection with Mycobacterium avium subspecies paratuberculosis and SLC11A1 gene polymorphisms in Sardinian patients. World J. Gastroenterol. 12(44): 7161–7164.
PMid:17131479 PMCid:PMC4087778

Senna JP, Roth DM, Oliveira JS, Machado DC and Santos DS (2003). Protective immune response against methicillin resistant Staphylococcus aureus in a murine model using a DNA vaccine approach. Vaccine 21(19–20): 2661–2666.
http://dx.doi.org/10.1016/S0264-410X(02)00738-7

Shariati Mehr K, Mousavi SL, Rasooli I, Amani J and Rajabi M (2012). A DNA vaccine against Escherichia coli O157:H7. Iran Biomed. J. 16(3): 133–139.
PMid:23023214 PMCid:PMC3629931

Shedlock DJ and Weiner DB (2000). DNA vaccination: antigen presentation and the induction of immunity. J. Leukocyte Biol. 68: 793–806.
PMid:11129646

Sheets RL, Stein J, Manetz TS, Duffy C, Nason M, Andrews C, Kong WP, Nabel GJ and Gomez PL (2006). Biodistribution of DNA plasmid vaccines against HIV–1, Ebola, severe acute respiratory syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts. Toxicol. Sci. 91: 610–619.
http://dx.doi.org/10.1093/toxsci/kfj169
http://dx.doi.org/10.1093/toxsci/kfj170

Shi W, Li YH, Liu F, Yang JY, Zhou DH, Chen YQ, Zhang Y, Yang Y, He BX, Han C, Fan MW and Yan HM (2012). Flagellin Enhances Saliva IgA Response and Protection of Anti–caries DNA Vaccine. J. Dental Res. 91: 249–254.
http://dx.doi.org/10.1177/0022034511424283
PMid:22027714

Shiau JW, Tang TK, Shih TL, Tai C, Sung YY, Huang JL and Yang HL (2000). Mice immunized with DNA encoding a modified Pseudomonas aeruginosa exotoxin A develop protective immunity against exotoxin intoxication. Vaccine 19(910): 1106–1112.
http://dx.doi.org/10.1016/S0264-410X(00)00335-2

Shkreta L, Talbot BG, Diarra MS and Lacasse P (2004). Immune responses to a DNA/protein vaccination strategy against Staphylococcus aureus induced mastitis in dairy cows. Vaccine 23(1): 114–126.
http://dx.doi.org/10.1016/j.vaccine.2004.05.002
PMid:15519714

Singha H, Mallick AI, Jana C, Isore DP, Goswami TK, Srivastava SK, Azevedo VA, Chadhuri P and Owais M (2008). Escheriosomes entrapped DNA vaccine co–expressing Cu–Zn superoxide dismutase and IL–18 confers protection against Brucella abortus. Microbes Infect. 10(10–11): 1089–1096.
http://dx.doi.org/10.1016/j.micinf.2008.05.007
PMid:18602490

Stratford R, Douce G, Zhang–Barber L, Fairweather N, Eskola J and Dougan G (2000). Influence of codon usage on the immunogenicity of a DNA vaccine against tetanus. Vaccine 19(7–8): 810–815.
http://dx.doi.org/10.1016/S0264-410X(00)00246-2

Sun B, Li ZS, Tu ZX, Xu GM and Du YQ (2006). Construction of an oral recombinant DNA vaccine from H. pylori neutrophil activating protein and its immunogenicity. World J. Gastroenterol. 12(43): 7042–7046.
PMid:17109503 PMCid:PMC4087352

Sun Y, Hu YH, Liu CS and Sun L (2012). Construction and comparative study of monovalent and multivalent DNA vaccines against Streptococcus iniae. Fish Shellfish Immunol. 33(6): 1303–1310.
http://dx.doi.org/10.1016/j.fsi.2012.10.004
PMid:23063784

Svanholm C, Bandholtz L, Castanos–Velez E, Wigzell H and Rottenberg ME (2000). Protective DNA immunization against Chlamydia pneumoniae. Scand. J. Immunol. 51: 345–353.
http://dx.doi.org/10.1046/j.1365-3083.2000.00684.x
PMid:10736106

Tang DC, DeVit M and Johnston SA (1992). Genetic immunization is a simple method for eliciting an immune response. Nature 356: 152–154.
http://dx.doi.org/10.1038/356152a0
PMid:1545867

Tanghe A, Content J, Van Vooren JP, Portaels F and Huygen K (2001). Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer. Infect. Immun. 69: 5403–5411.
http://dx.doi.org/10.1128/IAI.69.9.5403-5411.2001
PMid:11500410 PMCid:PMC98650

Tanghe A, Lefèvre P, Denis O, D'Souza S, Braibant M, Lozes E, Singh M, Montgomery D, Content J and Huygen K (1999). Immunogenicity and protective efficacy of tuberculosis DNA vaccines encoding putative phosphate transport receptors. J. Immunol. 162: 1113–1119.
PMid:9916741

Tascon R, Colston M, Ragno S, Stavropoulos E, Gregory D and Lowrie DB (1996). Vaccination against tuberculosis by DNA injection. Nat. Med. 2: 888–892.
http://dx.doi.org/10.1038/nm0896-888
PMid:8705858

Tavel JA, Martin JE, Kelly GG, Enama ME, Shen JM, Gomez PL, Andrews CA, Koup RA, Bailer RT, Stein JA, Roederer M, Nabel GJ and Graham BS (2007). Safety and immunogenicity of a Gag–Pol candidate HIV–1 DNA vaccine administered by a needle–free device in HIV–1–seronegative subjects. J. Acquir. Immune Deﬁc. Syndr. 44: 601–605.
http://dx.doi.org/10.1097/QAI.0b013e3180417cb6
PMid:17325604 PMCid:PMC2365751

Teixeira FM, Teixeira HC, Ferreira AP, Rodrigues MF, Azevedo V, Macedo GC and Oliveira SC (2006). DNA vaccine using Mycobacterium bovis Ag85B antigen induces partial protection against experimental infection in BALB/c mice. Clin. Vaccine Immunol. 13(8): 930–935.
http://dx.doi.org/10.1128/CVI.00151-06
PMid:16893994 PMCid:PMC1539111

Thacker EL, Holtkamp DJ, Khan AS, Brown PA and Draghia–Akli R (2006). Plasmid–mediated growth hormone–releasing hormone efficacy in reducing disease associated with Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus infection. J. Anim. Sci. 84(3): 733–742.
PMid:16478966

Tian X, Cai H and Zhu YX (2005). Immunogenicity and protection of divalent DNA vaccine encoding antigens MPT83 and MPT64 of Mycobacterium tuberculosis. Zhonghua Yi Xue Za Zhi. 85(20): 1410–1413.
PMid:16029655

Tizard I (2012). Veterinary Immunology, 9th Edition. Saunders publishers. p. 568.

Todoroki I, Joh T, Watanabe K, Miyashita M, Seno K, Nomura T, Ohara H, Yokoyama Y, Tochikubo K and Itoh M (2000). Suppressive effects of DNA vaccines encoding heat shock protein on Helicobacter pylori induced gastritis in mice. Biochem. Biophys. Res. Commun. 277(1): 159–163.
http://dx.doi.org/10.1006/bbrc.2000.3632
PMid:11027657

Turner J, Rhoades ER, Keen M, Belisle JT, Frank AA and Orme IM (2000). Effective pre–exposure tuberculosis vaccines fail to protect when they are given in an immunotherapeutic mode. Infect. Immun. 68: 1706–1709.
http://dx.doi.org/10.1128/IAI.68.3.1706-1709.2000
PMid:10678993 PMCid:PMC97334

Turnes CG, Aleixo JA, Monteiro AV and Dellagostin OA (1999). DNA inoculation with a plasmid vector carrying the faeG adhesin gene of Escherichia coli K88ab induced immune responses in mice and pigs. Vaccine 17(15–16): 2089–2095.
http://dx.doi.org/10.1016/S0264-410X(98)00384-3

Tuteja R (1999). DNA vaccines: A ray of hope. Crit. Rev. Biochem. Mol. 34(1): 1–24.
http://dx.doi.org/10.1080/10409239991209165
PMid:10090468

Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, Gromkowski SH, Randall Deck R, DeWitt CM, Friedman A, Hawe LA, Leander KR, Martinez D, Perry HC, Shiver JW, Montgomery DL and Liu MA (1993). Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259: 1745–1749.
http://dx.doi.org/10.1126/science.8456302
PMid:8456302

Vanniasinkam T, Barton MD and Heuzenroeder MW (2005). Immune response to vaccines based upon the VapA protein of the horse pathogen Rhodococcus equi in a murine model. Int. J. Med. Microbiol. 294: 437–445.
http://dx.doi.org/10.1016/j.ijmm.2004.09.011
PMid:15715172

Vanrompay D, Cox E, Vandenbussche F, Volckaert G and Goddeeris B (1999). Protection of turkeys against Chlamydia psittaci challenge by gene gun–based DNA immunizations. Vaccine 17: 2628–2635.
http://dx.doi.org/10.1016/S0264-410X(99)00053-5

Velaz–Faircloth M, Cobb AJ, Horstman AL, Henry SC and Frothingham R (1999). Protection against Mycobacterium avium by DNA vaccines expressing mycobacterial antigens as fusion proteins with green fluorescent protein. Infect. Immun. 67: 4243–4250.
PMid:10417198 PMCid:PMC96731

Vordermeier HM, Cockle PJ, Whelan AO, Rhodes S, Chambers MA, Clifford D, Huygen K, Tascon R, Lowrie D, Colston MJ and Hewinson RG (2001). Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skin test. Vaccine 19: 1246–1255.
http://dx.doi.org/10.1016/S0264-410X(00)00238-3

Wallich R, Siebers A, Jahraus O, Brenner C, Stehle T and Simon MM (2001). DNA vaccines expressing a fusion product of outer surface proteins A and C from Borrelia burgdorferi induce protective antibodies suitable for prophylaxis but not for resolution of lyme disease. Infect. Immun. 69(4): 2130–2136.
http://dx.doi.org/10.1128/IAI.69.4.2130-2136.2001
PMid:11254567 PMCid:PMC98139

Wang B, Ugen K, Srikantan V, Agadjanyan M, Dang K, Refaeli Y, Sato A, Boyer J, Williams W and Weiner D (1993). Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90: 4156–4160.
http://dx.doi.org/10.1073/pnas.90.9.4156
PMid:8483929 PMCid:PMC46465

Wang LM, Bai YL, Shi CH, Gao H, Xue Y, Jiang H and Xu ZK (2008). Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of Mycobacterium heat shock protein 65 (Hsp65) with human interleukin–2 against Mycobacterium tuberculosis in BALB/c mice. APMIS 116(12): 1071–1081.
http://dx.doi.org/10.1111/j.1600-0463.2008.01095.x
PMid:19133010

Wang M, Dai B, You Z, Fang Z and Wang Y (2002). Construction of DNA vaccine including a chimeric gene encoding flagellin and outer membrane protein antigen from Leptospira interrogans serovar lai. Hua Xi Yike Daxue Xue Bao 33: 169–71.

Wang S, Hackett A, Jia N, Zhang C, Zhang L, Parker C, Zhou A, Li J, Cao WC, Huang Z, Li Y and Lu S (2011). Polyvalent DNA vaccines expressing HA antigens of H5N1 influenza viruses with an optimized leader sequence elicit cross–protective antibody responses. PLoS One 6(12): e28757.
http://dx.doi.org/10.1371/journal.pone.0028757
PMid:22205966 PMCid:PMC3244406

Wang S, Heilman D, Liu F, Giehl T, Joshi S, Huang X, Chou TH, Goguen J and Lu S (2004a). A DNA vaccine producing LcrV antigen in oligomers is effective in protecting mice from lethal mucosal challenge of plague. Vaccine 22(25–26): 3348–3357.
http://dx.doi.org/10.1016/j.vaccine.2004.02.036
PMid:15308359

Wang Z, Troilo PJ, Wang X, Griffiths TG, Pacchione SJ, Barnum AB, Harper LB, Pauley CJ, Niu Z, Denisova L, Follmer TT, Rizzuto G, Ciliberto G, Fattori E, Monica NL, Manam S and Ledwith BJ (2004b). Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Ther. 11: 711–72.
http://dx.doi.org/10.1038/sj.gt.3302213
PMid:14724672

Williams JA (2013). Vector design for improved DNA vaccine efficacy, safety and production. Vaccines 1: 225–249.
http://dx.doi.org/10.3390/vaccines1030225

Williamson ED, Beedham RJ, Bennett AM, Perkins SD, Miller J and Baillie LW (1999). Presentation of protective antigen to the mouse immune system: immune sequelae. J. Appl. Microbiol. 87: 315–317.
http://dx.doi.org/10.1046/j.1365-2672.1999.00901.x
PMid:10475979

Wolf JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A and Felgner PL (1990). Direct gene transfer into mouse muscle in vivo. Science 247: 1465–1468.
http://dx.doi.org/10.1126/science.1690918

Xu C, Li ZS, Du YQ, Gong YF, Yang H, Sun B and Jin J (2007). Construction of recombinant attenuated Salmonella Typhimurium DNA vaccine expressing H. pylori ureB and IL–2. World J. Gastroenterol. 3(6): 939–944.
http://dx.doi.org/10.3748/wjg.v13.i6.939
PMCid:PMC4065934

Xu QA, Yu F, Fan M, Bian Z, Guo J, Jia R, Chen Z, Peng B and Fan B (2005). Immunogenicity and protective efficacy of a targeted fusion DNA construct against dental caries. Caries Res. 39(5): 422–431.
http://dx.doi.org/10.1159/000086851
PMid:16110216

Yamanaka H, Hoyt T, Bowen R, Yang X, Crist K, Golden S, Maddaloni M and Pascual DW (2009). An IL–12 DNA vaccine co–expressing Yersinia pestis antigens protects against pneumonic plague. Vaccine 27(1): 80–87.
http://dx.doi.org/10.1016/j.vaccine.2008.10.021
PMid:18955097 PMCid:PMC2628569

Yamanaka H, Hoyt T, Yang X, Golden S, Bosio CM, Crist K, Becker T, Maddaloni M and Pascual DW (2008). A nasal interleukin–12 DNA vaccine coexpressing Yersinia pestis F1–V fusion protein confers protection against pneumonic plague. Infect. Immun. 76(10): 4564–4573.
http://dx.doi.org/10.1128/IAI.00581-08
PMid:18694965 PMCid:PMC2546814

Yang X, Hudson M, Walters N, Bargatze RF and Pascual DW (2005). Selection of protective epitopes for Brucella melitensis by DNA vaccination. Infect. Immun. 73: 7297–7303.
http://dx.doi.org/10.1128/IAI.73.11.7297-7303.2005
PMid:16239526 PMCid:PMC1273852

Yeremeev VV, Lyadova IV, Nikonenko BV, Apt AS, Abou–Zeid C, Inwald J and Young DB (2000). The 19–kD antigen and protective immunity in a murine model of tuberculosis. Clin. Exp. Immunol.120: 274–279.
http://dx.doi.org/10.1046/j.1365-2249.2000.01212.x
PMid:10792376 PMCid:PMC1905638

Yin RL, Li C, Yang ZT, Zhang YJ, Bai WL, Li X, Yin RH, Liu H, Liu S, Yang Q, Cao YG and Zhang NS (2009).Construction and immunogenicity of a DNA vaccine containing clumping factor A of Staphylococcus aureus and bovine IL18. Vet. Immunol. Immunopathol. 132(2–4): 270–274.
http://dx.doi.org/10.1016/j.vetimm.2009.05.012
PMid:19540000

You Z, Dai B, Chen Z, Yan H, Fang Z, Li S and Liu J (1999). Immunogenicity and immunoprotection of a Leptospiral DNA vaccine. Hua Xi Yi Da Xue Xue Bao 30 :128–132.

Yu DH, Hu XD and Cai H (2007). A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses. DNA Cell Biol. 26: 435–443.
http://dx.doi.org/10.1089/dna.2006.0552
PMid:17570767

Yu W, Zhang Y, Jing J and Liu Z (2010). Construction of Helicobacter pylori Lpp20–IL2 DNA vaccine and evaluation of its immunocompetence in C57BL/6 mice. Wei Sheng Wu Xue Bao. 50(4): 554–559.
PMid:20560362

Zhang DJ, Yang X, Berry J, Shen C, McClarty G and Brunham RC (1997). DNA vaccination with the major outer–membrane protein gene induces acquired immunity to Chlamydia trachomatis (mouse pneumonitis) infection. J. Infect. Dis. 176: 1035–1040.
http://dx.doi.org/10.1086/516545
PMid:9333163

Zhang DJ, Yang X, Shen C and Brunham RC (1999). Characterization of immune responses following intramuscular DNA immunization with the MOMP gene of Chlamydia trachomatis mouse pneumonitis strain. Immunology 96: 314–321.
http://dx.doi.org/10.1046/j.1365-2567.1999.00682.x
PMid:10233711 PMCid:PMC2326737

Zhang Y, Jianxia Q, Yu Z, Farhang F, Jenny H, Augustine LY and William DK (2008). Plasmid–based vaccination with candidate anthrax vaccine antigens induces durable type 1 and type 2 T–helper immune responses. Vaccine 26: 614–622.
http://dx.doi.org/10.1016/j.vaccine.2007.11.072
PMid:18166249

Zhu C, Wang S, Hu S, Yu M, Zeng Y, You X, Xiao J and Wu Y (2012). Protective efficacy of a Mycoplasma pneumoniae P1C DNA vaccine fused with the B subunit of Escherichia coli heat–labile enterotoxin. Can. J. Microbiol. 58(6): 802–810.
http://dx.doi.org/10.1139/w2012-051
PMid:22642685

Zhu C, Yu M, Gao S, Zeng Y, You X and Wu Y (2013). Protective immune responses induced by intranasal immunization with Mycoplasma pneumoniae P1C–IL–2 fusion DNA vaccine in mice. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 29(6): 585–588.
PMid:23746241

Zhu X, Venkataprasad N, Thangaraj HS, Hill M, Singh M, Ivanyi J and Vordermeier HM (1997). Functions and specificity of T cells following nucleic acid vaccination of mice against Mycobacterium tuberculosis infection. J. Immunol. 158(12): 5921–5926.
PMid:9190945

Advances in Animal and Veterinary Sciences

Advances in Animal and Veterinary Sciences

Special Issues

Membership/Association

Review Article

Progress in DNA Vaccinology against Bacterial Diseases– An Update

ABSTRACT

INTRODUCTION

CONCLUSION

REFERENCES