Invasive Escherichia coli vaccines expressing Brucella melitensis outer membrane proteins 31 or 16 or periplasmic protein BP26 confer protection in mice challenged with B. melitensis

Because of the serious economic and medical consequences of brucellosis, efforts are to prevent infection of domestic animals through vaccines. Many disadvantages are associated with the current Brucella melitensis Rev.1 vaccine prompting development of alternative vaccines and delivery. Escherichia coli (DH5α) was engineered to express a plasmid containing the inv gene from Yersinia pseudotuberculosis and the hly gene from Listeria monocytogenes. These recombinant invasive E. coli expressing B. melitensis outer membrane proteins (Omp31 or 16) or the periplasmic protein BP26 were evaluated for protection of mice against virulent B. melitensis. Importantly, these invasive E. coli vaccines induced significant protection against B. melitensis challenged mice. Invasive E. coli may be an ideal vaccine platform with natural adjuvant properties for application against B. melitensis since the E. coli delivery system is non-pathogenic and can deliver antigens to antigen-presenting cells promoting cellular immune responses.     

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.     

Immune responses and protection against Streptococcus pneumoniae elicited by recombinant Bordetella pertussis adenylate cyclase (CyaA) carrying fragments of pneumococcal surface protein A,PspA

Streptococcus pneumoniae is a common agent of important human diseases such as otitis media, pneumonia, meningitis and sepsis. Current available vaccines that target capsular polysaccharides induce protection against invasive disease and nasopharyngeal colonization in children, yet their efficacy is limited to the serotypes included in the formulations. The virulence factor Pneumococcal Surface Protein A (PspA) interacts with host immune system and helps the bacteria to evade phagocytosis. Due to its essential role in virulence, PspA is an important vaccine candidate. Here we have tested a delivery system based on the adenylate cyclase toxin of Bordetella pertussis (CyaA) to induce immune responses against PspA in mice. CyaA was engineered to express fragments of the N-terminal region of PspAs from clades 2 and 4 (A2 and A4) and the resulting proteins were used in immunization experiments in mice. The recombinant CyaA-A2 and CyaA-A4 proteins were able to induce high levels of anti-PspA antibodies that reacted with pneumococcal strains expressing either PspA2 or PspA4. Moreover, reactivity of the antibodies against pneumococcal strains that express PspAs from clades 3 and 5 (PspA3 and PspA5) was also observed. A formulation containing CyaA-A2 and CyaA-A4 was able to protect mice against invasive pneumococcal challenges with isolates that express PspA2, PspA4 or PspA5. Moreover, a CyaA-A2-A4 fusion protein induced antibodies at similar levels and with similar reactivity as the formulation containing both proteins, and protected mice against the invasive challenge. Our results indicate that CyaA-PspA proteins are good candidates to induce broad protection against pneumococcal isolates.     

Oral delivery of a DNA vaccine against tuberculosis using operator-repressor titration in a Salmonella enterica vector

Attenuated Salmonella enterica offers a vaccine delivery route that has the benefits of enhanced immunogenicity and oral delivery. The majority of immunization studies have been conducted to deliver recombinant proteins, expressed from a gene that is either chromosomally integrated or carried on a low- or medium-copy number plasmid. There are, however, an increasing number of reports demonstrating the delivery of DNA vaccines, but the high-copy number plasmids that are preferentially used for this application are unstable in Salmonella. Here, we use the Operator-Repressor Titration (ORT) plasmid maintenance system in Salmonella enterica serovar Typhimurium to deliver a high-copy number plasmid expressing the Mycobacterium tuberculosis gene mpt64 to mice. MPT64 expression was detected in phagocytes using immunofluorescence microscopy following Salmonella-mediated delivery of the DNA vaccine. The indicative CD8+ responses measured by antigen-specific IFN-γ were higher from the live bacterial vector than from injected plasmid DNA, and a reduction in the pulmonary bacterial load was seen following an aerogenic challenge. This illustrates the potential of live attenuated Salmonella as oral tuberculosis vaccine vectors.     

Small animal jet injection technique results in enhanced immunogenicity of hantavirus DNA vaccines

DNA vaccine evaluation in small animals is hampered by low immunogenicity when the vaccines are delivered using a needle and syringe. To overcome this technical hurdle we tested the possibility that a device developed for human intradermal medicine delivery might be adapted to successfully deliver a DNA vaccine to small animals. Disposable syringe jet injection (DSJI) does not currently exist for small animals. However, a commercialized, human intradermal device used to to administer medicines to the human dermis in a 0.1 mL volume was evaluated in Syrian hamsters. Here, we found that hantavirus DNA vaccines administered to hamsters using DSJI were substantially more immunogenic than the same vaccines delivered by needle/syringe or particle mediated epidermal delivery (gene gun) vaccination. By adjusting how the device was used we could deliver vaccine to either subcutaneous tissues, or through the skin into the muscle. RNA and/or antigen expression was detected in epidermal, subepidermal and fibroblast cells. We directly compared six optimized and non-optimized hantavirus DNA vaccines in hamsters. Optimization, including codon-usage and mRNA stability, did not necessarily result in increased immunogenicity for all vaccines tested; however, optimization of the Andes virus (ANDV) DNA vaccine protected vaccinated hamsters from lethal disease. This is the first time active vaccination with an ANDV DNA vaccine has shown protective efficacy in the hamster model. The adaptation of a human intradermal jet injection device for use as a method of subcutaneous and intramuscular jet injection of DNA vaccines will advance the development of nucleic acid based medical countermeasures for diseases modeled in hamsters.     

Enhancement of live vaccines by co-delivery of immune modulating proteins

Vaccines are very effective in providing protection against many infectious diseases. However, it has proven difficult to develop highly efficacious vaccines against some pathogens and so there is a continuing need to improve vaccine technologies. The first successful and widely used vaccines were based on attenuated pathogens (e.g., laboratory passaged Pasteurella multocida to vaccinate against fowl cholera) or closely related non-pathogenic organisms (e.g., cowpox to vaccinate against smallpox). Subsequently, live vaccines, either attenuated pathogens or non-pathogenic microorganisms modified to deliver heterologous antigens, have been successfully used to induce protective immune responses against many pathogens. Unlike conventional killed and subunit vaccines, live vaccines can deliver antigens to mucosal surfaces in a similar manner and context as the natural infection and hence can often produce a more appropriate and protective immune response. Despite these advantages, there is still a need to improve the immunogenicity of some live vaccines. The efficacy of injectable killed and subunit vaccines is usually enhanced using adjuvants such mineral salts, oils, and saponin, but such adjuvants cannot be used with live vaccines. Instead, live vaccines can be engineered to produce immunomodulatory molecules that can stimulate the immune system to induce more robust and long-lasting adaptive immune responses. This review focuses on research that has been undertaken to engineer live vaccines to produce immunomodulatory molecules that act as adjuvants to increase immunogenicity. Adjuvant strategies with varying mechanisms of action (inflammatory, antibody-mediated, cell-mediated) and delivery modes (oral, intramuscular, intranasal) have been investigated, with varying degrees of success. The goal of such research is to define adjuvant strategies that can be adapted to enhance live vaccine efficacy by triggering strong innate and adaptive immune responses and produce vaccines against a wider range of pathogens.     

Cytotoxic T cell polyepitope vaccines delivered by ISCOMs

CD8 β cytotoxic T lymphocyte (CTL) polyepitope or polytope vaccines have traditionally been delivered using recombinant vector or DNA based delivery modalities. Here we show the delivery of polytope vaccines in the form of either synthetic polypeptides or recombinant polytope proteins by ImmunoStimulatory COMplexes (ISCOMs®). Induction of multiple protective CTL responses by these polytope-ISCOM formulations were comparable to viral vector or DNA based delivery modalities as assessed by IFNγ ELISpot, chromium release and viral challenge assays. Measurement of CTL responses specific for the different epitopes revealed immunodominance patterns, which were largely independent of the vaccine vector or the order of the epitopes in the polytope. ISCOMs thus emerge as a viable human delivery modality for protein-based polytope vaccines.     

Vaccination with the glycoprotein D gene of pseudorabies virus delivered by nonpathogenic Escherichia coli elicits protective immune responses

Attenuated intracellular bacteria, such as Salmonella and Shigella, have been exploited to act as gene delivery vectors. In this study, we report that nonpathogenic, live Escherichia coli can be used for the delivery of DNA vaccines in vivo, leading to generation of immune responses against plasmid-encoded foreign antigens. The pseudorabies virus (PrV) DNA vaccine carrying the glycoprotein D (gD) gene delivered by E. coli was able to induce protective immune responses in mice against a lethal PrV challenge. Co-delivery of E. coli carrying plasmid DNA encoding prothymosin alpha enhanced cellular immune responses to the PrV DNA vaccine delivered by E. coli. Our results suggest that nonpathogenic E. coli may be used as a vector for DNA vaccines in veterinary uses.     

In vivo electroporation of skeletal muscles increases the efficacy of Japanese encephalitis virus DNA vaccine

DNA vaccines can induce protective immunity against subsequent viral challenge. However, for some DNA vaccines to be effective when administered intramuscularly, cardiotoxin pretreatment is necessary. In this study, we used the technique of in vivo electroporation to facilitate DNA delivery and elicit an immune response without the use of cardiotoxin. Intramuscular delivery of DNA (pE) encoding the Japanese encephalitis virus (JEV) envelope protein-induced anti-E antibodies only when the injected muscles were pretreated with cardiotoxin. In vivo electrotransfer of pE eliminated the need for cardiotoxin pretreatment and produced higher antibody titer than that induced by conventional intramuscular injection. Moreover, the induced immunity also conferred protection against lethal viral challenge. Interestingly, like intramuscular immunization, in vivo electroporation immunization with plasmid pE generated anti-envelope antibodies that were predominantly of the immunoglobulin G2a (IgG2a) isotype. These results suggest that in vivo electroporation can be used as an efficient gene delivery system for DNA vaccines to provide efficient protection against viral infection.     

Recombinant attenuated bacteria for the delivery of subunit vaccines

Using attenuated intracellular bacteria as carriers, we have developed two different approaches for the delivery of subunit vaccines encoding heterologous antigens. The first system is based on the direct secretion of the heterologous antigens in Gram-negative bacteria via the hemolysin secretion system of Escherichia coli into either phagosome or cytosol of infected cells. The second approach is based on the transport of eukaryotic antigen expression vectors by intracellular bacteria like Listeria and Salmonella into the host cell and here, preferably, into the cytosolic compartment. After release of the plasmid DNA from the bacteria, the plasmid-encoded antigens can be expressed directly by the host cell. Finally, we combined both types of subunit vaccines in one live vector - we equipped Salmonella strains with a phagosomal escape function by utilization of the hemolysin secretion system and used this recombinant vaccine strain for the delivery of a eukaryotic antigen expression vector into the cytosol of macrophages.     

Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction

Within a few years of its emergence in the late 1980s, the PRRS virus had spread globally to become the foremost infectious disease concern for the pork industry. Since 1994, modified live-attenuated vaccines against porcine reproductive and respiratory syndrome virus (PRRSV-MLV) have been widely used, but have failed to provide complete protection against emerging and heterologous field strains of the virus. Moreover, like many other MLVs, PRRSV-MLVs have safety concerns including vertical and horizontal transmission of the vaccine virus and several documented incidences of reversion to virulence. Thus, the development of efficacious inactivated vaccines is warranted for the control and eradication of PRRS. Since the early 1990s, researchers have been attempting to develop inactivated PRRSV vaccines, but most of the candidates have failed to elicit protective immunity even against homologous virus challenge. Recent research findings relating to both inactivated and subunit candidate PRRSV vaccines have shown promise, but they need to be pursued further to improve their heterologous efficacy and cost-effectiveness before considering commercialization. In this comprehensive review, we provide information on attempts to develop PRRSV inactivated and subunit vaccines. These includes various virus inactivation strategies, adjuvants, nanoparticle-based vaccine delivery systems, DNA vaccines, and recombinant subunit vaccines produced using baculovirus, plant, and replication-deficient viruses as vector vaccines. Finally, future directions for the development of innovative non-infectious PRRSV vaccines are suggested. Undoubtedly there remains a need for novel PRRSV vaccine strategies targeted to deliver cross-protective, non-infectious vaccines for the control and eradication of PRRS.     

Immunogenicity of next-generation HPV vaccines in non-human primates: Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine

Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. HPVs are oncogenic small double-stranded DNA viruses that are the primary causal agent of cervical cancer and other types of cancers, including in the anus, oropharynx, vagina, vulva, and penis. Prophylactic vaccination against HPV is an attractive strategy for preventing cervical cancer and some other types of cancers. However, there are few safe and effective vaccines against HPV infections. Current first-generation commercial HPV vaccines are expensive to produce and deliver.The goal of this study was to develop an alternate potent HPV recombinant L1-based vaccines by producing HPV virus-like particles into a vaccine that is currently used worldwide. Live attenuated measles virus (MV) vaccines have a well-established safety and efficacy record, and recombinant MV (rMV) produced by reverse genetics may be useful for generating candidate HPV vaccines to meet the needs of the developing world.We studied in non-human primate rMV-vectored HPV vaccine in parallel with a classical alum adjuvant recombinant HPV16L1 and 18L1 protein vaccine produced in Pichia pastoris. A combined prime-boost approach using both vaccines was evaluated, as well as immune interference due to pre-existing immunity against the MV.The humoral immune response induced by the MV, Pichia-expressed vaccine, and their combination as priming and boosting approaches was found to elicit HPV16L1 and 18L1 specific total IgG and neutralizing antibody titres. Pre-existing antibodies against measles did not prevent the immune response against HPV16L1 and 18L1.     

The development of novel hepatitis B vaccines

Development of vaccines against viral hepatitis B has proceeded along four main lines. (1) Human plasma-derived vaccines are safe, effective and are now in general use. (2) Subunit polypeptide vaccines formulated in micelles have reached the stage of clinical trials. (3) Recombinant DNA vaccines have been produced in prokaryotic and eukaryotic cells, notably in yeast. The yeast-derived recombinant vaccines have proved safe and effective in extensive clinical trials, eliciting antibodies which in quantity and specificity are equal to those elicited by plasma-derived vaccine. DNA recombinant has also been applied to the development of hybrid and vaccinia virus vaccines which are capable of immunological ”priming”, and other hybrid virus vaccines are under development. (4) Finally, chemical synthesis has succeeded in producing small peptides which include specific epitopes eliciting antibody responses in experimental animals. Such chemically synthesized preparations offer a prospect of ultimately producing multivalent synthetic vaccines against several viruses, bacteria and protozoa.     

Cytoplasmic expression of a model antigen with M Cell-Targeting moiety in lactic acid bacteria and implication of the mechanism as a mucosal vaccine via oral route

Lactic acid bacteria (LAB) have been widely studied as mucosal vaccine delivery carriers against many infectious diseases for heterologous expression of protein antigens. There are three antigen expression strategies for LAB: cytoplasmic expression (CE), cell surface display (SD), and extracellular secretion (ES). Despite the generally higher protein expression level and many observations of antigen-specific immunogenicity in CE, its application as a mucosal vaccine has been overlooked relative to SD and ES because of the antigens enclosed by the LAB cell wall. We hypothesized that the antigens in CE could be released from the LAB into the intestinal lumen before host bacterial access to gut-associated lymphoid tissue (GALT), which could contribute to antigen-specific immune responses after oral administration. To elucidate this hypothesis, three recombinant Lactobacillus plantarum (LP) strains were constructed to produce a model antigen, BmpB, with or without an M cell-targeting moiety, and their immunogenicities were analyzed comparatively as oral vaccines in mouse model. The data indicated that the recombinant LPs producing BmpBs with different conformations could induce mucosal immunity differentially. This suggests that the cytoplasmic antigens in LAB could be released into the intestinal lumen, subsequently translocated through M cells, and stimulate the GALT to generate antigen-specific immune responses. Therefore, the CE strategy has great potential, especially in the application of oral LAB vaccines as well as SD and ES strategies. This research provides a better understanding of the mechanism for recombinant oral LAB vaccines and gives insight to the future design of LAB vaccines and oral delivery applications for useful therapeutic proteins.     

New strategies for combination vaccines based on the extended recombinant bacterial ghost system

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.     

Heterologous prime boost vaccination with DNA and recombinant modified vaccinia virus Ankara protects IFNAR mice against lethal bluetongue infection

Recent recombinant DNA technology has provided novel approaches to develop marker and safe vaccines against bluetongue virus (BTV). To develop new vaccination strategies against BTV infection we have engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP5 and VP7 proteins from BTV-4. IFNAR^(−/−) mice inoculated with DNA/rMVA-VP2, -VP5, -VP7 in an heterologous prime boost vaccination strategy generated significant levels of neutralizing antibodies against BTV-4 and they were completely protected against BTV-4 challenge. Interestingly, VP2 and VP7 proteins expressed in the DNA/rMVA vaccines induced a specific BTV T-cell response that might contribute to the protection of IFNAR^(−/−) mice against challenge with BTV-4. In addition, antibodies against VP2, VP5, and VP7, but not NS3 were detected in the sera of DNA/rMVA-VP2, -VP5, -VP7 immunized mice confirming the DIVA (differentiating infected from vaccinated animals) properties of this vaccine. Overall, our results show that the heterologous prime boost vaccination with DNA/rMVA expressing VP2, VP5, and VP7 proteins protects against BTV-4 infection.     

Homologous prime boosting based on intranasal delivery of non-pathogenic invasive Escherichia coli expressing MPT64, decreases Mycobacterium tuberculosis dissemination

Protein-subunit vaccines as boosting strategies against tuberculosis (TB) infection are currently in the pipeline of TB vaccine research. Their main limitation is represented by their poor immunogenicity, which makes it necessary to couple protein-subunits with adjuvant molecules. In this study, we employed replication-deficient invasive Escherichia coli strains to deliver Mycobacterium tuberculosis proteins to the cytoplasm of non-phagocytic eukaryotic cells using various priming and prime-boosting vaccination protocols. Our results demonstrate that intranasal administration of invasive E. coli expressing the M. tuberculosis protective antigen MPT64 to mice primed with a recombinant BCG strain over-expressing MPT64 on its surface, decrease bacterial burden in mice spleens. Our data suggest that replication-deficient invasive E. coli may represent a suitable platform for BCG/rBCG priming followed by homologous-boosting immunization strategies.     

Plasmid DNA and viral vector-based vaccines for the treatment of cancer

Plasmid DNA and viral vector-based cancer vaccines have many inherent features that make them promising cancer vaccine candidates. This review focuses on the use of plasmid DNA and viral vector vaccines to deliver tumour-specific antigens to induce a tumour-specific immune response. Examples of different antigen delivery systems that have been tested in recent clinical trials are summarised and advantages and disadvantages of a number of delivery systems and approaches are discussed. Finally, an outlook on how plasmid DNA and viral vectors might be developed further as cancer vaccines is provided.     

Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens

West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA–WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA–WNV vaccines in other preclinical models and use them as candidate vaccine in humans.     

DNA vaccination with VP2 gene of very virulent infectious bursal disease virus (vvIBDV) delivered by transgenic E. coli DH5alpha given orally confers protective immune responses in chickens

The efficacy of different doses of oral DNA vaccines carrying VP2 gene of vvIBDV delivered by E. coli DH5alpha was studied and compared with purified VP2 recombinant expression plasmid DNA vaccine injected intradermally and whole virus vaccine either from homologous virus or from commercial source. The recombinant plasmid pRc-VP2 was transformed in a non-pathogenic strain of E. coli, the DH5alpha and designated as EC/pRC-VP2. Oral immunization of maternal antibody free broiler chickens at 7 and 14-day-old with different dosages of EC/pRc-VP2 elicited specific humoral immune response as measured by ELISA. Protection in different groups was calculated through clinical signs, gross and histopathological lesions, bursa of Fabricius to body weight ratio, humoral and cellular immune responses and mortality in the chickens. Vaccination with EC/pRc-VP2 at the dose rate of 10(9)CFU per chicken conferred 95.4% protection of the chickens against the challenge with homologous virulent field strain of vvIBDV. Protection afforded by attenuated vero cell adapted UAF-06 strain of vvIBDV was comparable (94%) to that by EC/pRc-VP2 and pRc-VP2 vaccines, which was significantly higher (P<0.05) than the protection provided by a commercial attenuated IBDV stain D-78 vaccine (D-78 vaccine was used as positive control due to its frequent use in the field for vaccination of poultry chickens) and other control groups in the study. The results revealed that DNA vaccines against IBDV may be successfully done by adopting bacterial-vectored oral delivery system and vaccination with homologous vvIBDV (UAF-06) conferred significantly higher protection as compared with imported non-homologous commercial IBDV vaccine.