Recombinant canarypox virus vaccine co-expressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep

We describe the development and preliminary characterization of a recombinant canarypox virus vectored vaccine for protective immunization of ruminants against bluetongue virus (BTV) infection. Sheep (n=6) immunized with recombinant canarypox virus vector (BTV-CP) co-expressing synthetic genes encoding the two outer capsid proteins (VP2 and VP5) of BTV serotype 17 (BTV-17) developed high titers (40-160) of virus-specific neutralizing antibodies and were resistant to challenge with a field strain of BTV-17. In contrast, sheep (n=5) immunized with a commercial recombinant canarypox virus vector expressing the E and preM genes of West Nile virus were seronegative to BTV and developed pyrexia, lymphopenia, and extended, high-titered viremias following challenge exposure to the field strain of BTV-17. These data confirm that the BTV-CP vaccine may be useful for the protective immunization of ruminants against bluetongue, and it may avoid the problems inherent to live-attenuated (LA) BTV 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.     

Recombinant capripoxviruses expressing proteins of bluetongue virus: evaluation of immune responses and protection in small ruminants

The development of recombinant capripoxviruses for protective immunization of ruminants against bluetongue virus (BTV) infection is described. Sheep (n=11) and goats (n=4) were immunized with BTV recombinant capripoxviruses (BTV-Cpox) individually expressing four different genes encoding two capsid proteins (VP2 and VP7) and two non-structural proteins (NS1, NS3) of BTV serotype 2 (BTV-2). Seroconversion was observed against NS3, VP7 and VP2 in both species and a lymphoproliferation specific to BTV antigens was also demonstrated in goats. Finally, partial protection of sheep challenged 3 weeks after BTV-Cpox administration with a virulent strain of BTV-2, was observed.     

Application of Bluetongue Disabled Infectious Single Animal (DISA) vaccine for different serotypes by VP2 exchange or incorporation of chimeric VP2

Bluetongue is a disease of ruminants caused by the bluetongue virus (BTV). Bluetongue outbreaks can be controlled by vaccination, however, currently available vaccines have several drawbacks. Further, there are at least 26 BTV serotypes, with low cross protection. A next-generation vaccine based on live-attenuated BTV without expression of non-structural proteins NS3/NS3a, named Disabled Infectious Single Animal (DISA) vaccine, was recently developed for serotype 8 by exchange of the serotype determining outer capsid protein VP2. DISA vaccines are replicating vaccines but do not cause detectable viremia, and induce serotype specific protection. Here, we exchanged VP2 of laboratory strain BTV1 for VP2 of European serotypes 2, 4, 8 and 9 using reverse genetics, without observing large effects on virus growth. Exchange of VP2 from serotype 16 and 25 was however not possible. Therefore, chimeric VP2 proteins of BTV1 containing possible immunogenic regions of these serotypes were studied. BTV1, expressing 1/16 chimeric VP2 proteins was functional in virus replication in vitro and contained neutralizing epitopes of both serotype 1 and 16. For serotype 25 this approach failed. We combined VP2 exchange with the NS3/NS3a negative phenotype in BTV1 as previously described for serotype 8 DISA vaccine. DISA vaccine with 1/16 chimeric VP2 containing amino acid region 249–398 of serotype 16 raised antibodies in sheep neutralizing both BTV1 and BTV16. This suggests that DISA vaccine could be protective for both parental serotypes present in chimeric VP2. We here demonstrate the application of the BT DISA vaccine platform for several serotypes and further extend the application for serotypes that are unsuccessful in single VP2 exchange.     

Protective efficacy of Bluetongue virus-like and subvirus-like particles in sheep: presence of the serotype-specific VP2, independent of its geographic lineage, is essential for protection

There have been multiple separate outbreaks of Bluetongue (BT) disease of ruminants in Europe since 1998, often entering via the Mediterranean countries of Italy, Spain and Greece. BT is caused by an orbivirus, Bluetongue virus (BTV), a member of the family Reoviridae. BTV is a non-enveloped double-capsid virus, which encodes 7 structural proteins (VP1-VP7) and several non-structural proteins (NS1, NS2, NS3/3a and NS4) from ten double-stranded RNA segments of the genome. In this report, we have prepared BTV virus-like particles (VLPs, composed of VP2, VP3, VP5 and VP7) and sub-viral, inner core-like particles (CLPs, VP3 and VP7) using a recombinant baculovirus expression system. We compared the protective efficacy of VLPs and CLPs in sheep and investigated the importance of geographical lineages of BTV in the development of vaccines. The Greek crossbred Karagouniko sheep, which display mild to sub-clinical BT, were vaccinated with VLPs or CLPs of BTV-1, derived from western lineage and were challenged with virulent BTV-1 from an eastern lineage. All VLP-vaccinated animals developed a neutralising antibody response to BTV-1 from both lineages prior to challenge. Moreover, post-challenged animals had no clinical manifestation or viraemia and the challenged virus replication was completely inhibited. In contrast, CLP-vaccinated animals did not induce any neutralising antibody response but developed the group specific VP7 antibodies. CLPs also failed to prevent the clinical manifestation and virus replication, but in comparison to controls, the severity of disease manifestation and viraemia was mitigated. The data demonstrated that the outer capsid was essential for complete protection, while the geographical origin of the BTV was not critical for development of a serotype specific vaccine.     

Enhancement of VP6 immunogenicity and protective efficacy against rotavirus by VP2 in a genetic immunization

VP2/VP6 virus like particles (VLPs) are very effective in inducing protection against the rotavirus infection in animal models. Individually, VP6 can also induce protection. However, there is no information about the immunogenicity of VP2. The aim of this work was to evaluate the efficacy of DNA vaccines codifying for VP2 or VP6, alone or combined, to induce protection against the rotavirus infection. Murine rotavirus VP2 and VP6 genes were cloned into the pcDNA3 vector. Adult BALB/c mice were inoculated three times by intramuscular (i.m.) injections with 100 or 200 µg of pcDNA3-VP2 or pcDNA3-VP6 alone or co-administered with 100 µg of pcDNA3-VP2/100 µg of pcDNA3-VP6. Two weeks after the last inoculation, mice were challenged with the wild type murine rotavirus strain epizootic diarrhea of infant mice (EDIM_wt). We found that both plasmids, pcDNA3-VP2 and pcDNA3-VP6, were able to induce rotavirus-specific serum antibodies, but not intestinal rotavirus-specific IgA; only 200 µg of pcDNA3-VP6 induced 35% protection against the infection. A similar level of protection was found when mice were co-administered with 100 µg of pcDNA3-VP2/100 µg of pcDNA3-VP6 (1:1 ratio). However, the best protection (up to 58%) occurred when mice were inoculated with 10 µg of pcDNA3-VP2/100 µg of pcDNA3-VP6 (1:10 ratio). These results indicate that the DNA plasmid expressing VP6 is a better vaccine candidate that the one expressing VP2. However, when co-expressed, VP2 potentiates the immunogenicity and protective efficacy of VP6.     

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

Bluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)–matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n = 6) or with adjuvant alone (n = 6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes.     

Protection of chickens,with or without maternal antibodies,against IBDV infection by a recombinant IBDV-VP2 protein

The use of avian herpesviruses (Marek's disease virus, MDV) as vectors to express the capsid protein of infectious bursal disease virus (IBDV) was well established, and its protection against IBDV challenge has been evaluated previously. However, there is little data about rMDV1 expressing the VP2 protein of IBDV protecting SPF and commercial chickens against virulent IBDV (vIBDV) challenge. In this study, we constructed a stable rMDV1 expressing the VP2 protein of IBDV by inserting the coding sequence within the US10 gene of MDVl by homologous recombination and designated this as rMDVl-US10L, and evaluated effectiveness of the recombinant VP2 protein with SPF chickens and commercial chickens with maternal antibodies in vIBDV challenge. The results can be summarized as follows: (1) We constructed a rMDV1 expressing IBDV-VP2 under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10L]. (2) rMDV-VP2 protein was readily expressed and induced 53% protection against a vIBDV challenge in SPF chickens with 10³ PFU/chicken, whereas 10⁴ PFU induced 73% protection. (3) Vaccination of commercial chickens having maternal antibodies to rMDV1-VP2 induced 87% protection in vIBDV challenge, which was similar to results using the live vaccine, BJ87 IBDV strain, in commercial chickens. These results demonstrate that the VP2 antigen expressed in the MDV vector was an effective and stable vaccine in correlation with the vaccine efficacy against lethal IBDV challenge, and can provide a better protective effect that is likely to persist for the life of the chickens.     

Effects of different promoters on the protective efficacy of recombinant Marek’s disease virus type 1 expressing the VP2 gene of infectious bursal disease virus

The vaccine efficacy of recombinant viruses can be influenced by many factors. Accordingly, the activity of promoters has been one of the major factors affecting the antigen expression and protection rate. In the present study, two recombinant Marek’s disease virus type 1 (MDV1) vaccines containing the VP2 gene of infectious bursal disease virus (IBDV) under control of different promoters were generated from overlapping fosmid DNAs. The rMDV-Pec-VP2 virus containing the VP2 gene under control of the Pec promoter (CMV enhancer and chicken β-actin chimera promoter) demonstrated higher VP2 expression and stronger antibody response against IBDV in chickens than the rMDV-CMV-VP2 virus using the CMV promoter. After IBDV lethal challenge in specific-pathogen-free chickens, rMDV-Pec-VP2 provided complete protection against developing mortality, clinical signs, and the formation of bursal lesions, which was better than that provided by rMDV-CMV-VP2. Our findings indicate that the protective efficacy of the recombinant MDV1 vaccine against IBDV highly correlates with VP2 expression. This recombinant MDV1 vaccine expressing VP2 could have significant potential as a bivalent vaccine against both virulent IBDV and MDV infections in chickens.     

An experimental subunit vaccine based on Bluetongue virus 4 VP2 protein fused to an antigen-presenting cells single chain antibody elicits cellular and humoral immune responses in cattle,guinea pigs and IFNAR(−/−) mice

Bluetongue virus (BTV), the causative agent of bluetongue disease (BT) in domestic and wild ruminants, is worldwide distributed. A total of 27 serotypes have been described so far, and several outbreaks have been reported. Vaccination is critical for controlling the spread of BTV. In the last years, subunit vaccines, viral vector vaccines and reverse genetic-based vaccines have emerged as new alternatives to conventional ones. In this study, we developed an experimental subunit vaccine against BTV4, with the benefit of targeting the recombinant protein to antigen-presenting cells. The VP2 protein from an Argentine BTV4 isolate was expressed alone or fused to the antigen presenting cell homing (APCH) molecule, in the baculovirus insect cell expression system. The immunogenicity of both proteins was evaluated in guinea pigs and cattle. Titers of specific neutralizing antibodies in guinea pigs and cattle immunized with VP2 or APCH-VP2 were high and similar to those induced by a conventional inactivated vaccine. The immunogenicity of recombinant proteins was further studied in the IFNAR(−/−) mouse model where the fusion of VP2 to APCH enhanced the cellular immune response and the neutralizing activity induced by VP2.     

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.     

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA).BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine.Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses.Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.     

The effect of Quil A adjuvant on the course of experimental Fasciola hepatica infection in sheep

Fasciola hepatica infection causes significant clinical disease in ruminants. Current control methods, based on flukicidal drugs, are becoming less useful because of resistance in fluke populations. Vaccination would be a viable alternative, but as yet no vaccine to protect ruminants against liver fluke infection has been commercialised. Adjuvants can be used to enhance and promote protective immune responses by vaccines. In previous vaccination trials, we have observed a distinct adjuvant effect, or a degree of protection, in animals administered adjuvant alone in the absence of any specific F. hepatica antigen. Understanding this effect will be important for continuing efforts to develop vaccines effective against fasciolosis. This study investigated the effects of three adjuvants (Quil A, Freund's Incomplete and TiterMax Gold) on the course of experimental F. hepatica infection in 6-month-old sheep (n=33). At completion of the trial, all animals were necropsied to determine fluke burden and fluke weight. Quil A administration led to a significant reduction in faecal egg count (P<0.0001) and significantly higher parasite-specific serum antibody activity for all isotypes measured (P<0.01). This suggests that Quil A, which promotes a Th1 response, may be useful as an adjuvant in anti-Fasciola vaccines. Furthermore, it reinforces the results of our previous studies indicating that enhanced Th1 responsiveness to vaccine antigens is required to achieve protection against challenge by F. hepatica.     

Avian adenovirus CELO recombinants expressing VP2 of infectious bursal disease virus induce protection against bursal disease in chickens

To develop a CELO virus vector that can induce protection against infectious bursal disease, CELO viruses expressing the host-protective antigen VP2 of infectious bursal disease virus (IBDV) were constructed. In the engineered recombinants, the VP2 gene (the 441-first codons of the IBDA polyprotein) was placed under the control of the CMV promoter. Two positions in the CELO genome were chosen to insert the VP2 expression cassette. The recombinants were found apathogenic, when inoculated by different routes and even at high doses (up to 10(8) per animal). Chickens vaccinated oro-nasally with these different recombinants and challenged with very virulent IBDV were found to be poorly protected. In contrast, when inoculated with one or two (subcutaneous or intradermic) injections of CELOa-VP2, the chickens showed no clinical signs and no mortality after challenge. In the vaccinated chickens, the titers of neutralization antibody reached 7-9 values, showing that protection could be explained by the induction of a sufficient humoral response. After challenge, the weight ratio Bursa of Fabricius/body was about 2.5 per thousand, a value similar to that obtained with the commercial Bur706 vaccine. However, histological lesions in the Bursa of Fabricius were observed, showing that a complete protection was not totally achieved. Contact transmission was evidenced. Protection was also obtained when inoculation of CELOa-VP2 was carried out in ovo. Prime-boost strategies were also tested with the CELOa-VP2 vector used in association with the purified VP2 antigen, or DNA encoding VP2 or a CELO vector expressing chicken myeloid growth factor (cMGF). None of these regimens were shown to substantially increase the level of protection when compared to double CELOa-VP2 inoculations. These results indicate that CELO-based vectors are useful to safely induce a strong protective immunity against vvIBDV in chickens.     

A canine adenovirus type 2 vaccine vector confers protection against foot-and-mouth disease in guinea pigs

Vaccination is a key element in the control of foot-and-mouth disease (FMD). The majority of the antigenic sites that induce protective immune responses are localized on the FMD virus (FMDV) capsid that is formed by four virus-encoded structural proteins, VP1 to VP4. In the present study, recombinant canine adenovirus type 2 (CAV2)-based FMD vaccines, Cav-P1/3C R° and Cav-VP1 R°, respectively expressing the structural P1 precursor protein along with the non-structural 3C protein or expressing the structural VP1 protein of the FMDV strain O/FRA/1/2001, were evaluated as novel vaccines against FMD. A strong humoral immune response was elicited in guinea pigs (GP) following immunization with Cav-P1/3C R°, while administration of Cav-VP1 R° did not induce a satisfying antibody response in GP or mice. GP were then used as an experimental model for the determination of the protection afforded by the Cav-P1/3C R° vaccine against challenge with the FMDV strain O₁ Manisa/Turkey/1969. The Cav-P1/3C R° vaccine protected GP from generalized FMD to a similar extent as a high potency double-oil emulsion O₁ Manisa vaccine. The results of the present study show that CAV2-based vector vaccines can express immunogenic FMDV antigens and offer protection against generalized FMD in GP. This suggest that Cav-P1/3C R° FMDV vaccine may protect natural host species from FMD. In combination with an appropriate diagnostic test, the Cav-P1/3C R° FMDV vaccine may also serve as a marker vaccine to differentiate vaccinated from infected animals.     

Immunization of knock-out α/β interferon receptor mice against lethal bluetongue infection with a BoHV-4-based vector expressing BTV-8 VP2 antigen

New effective tools for vaccine strategies are necessary to limit the spread of bluetongue, an insect-transmitted viral disease of domestic and wild ruminants. In the present study, BoHV-4-based vector cloned as a bacterial artificial chromosome (BAC) was engineered to express the bluetongue virus (BTV) immune-dominant glycoprotein VP2 provided of a heterologous signal peptide to its amino terminal and a trans-membrane domain to its carboxyl terminal (IgK-VP2gDtm), to allow the VP2 expression targeting to the cell membrane fraction. Based on adult α/β interferon receptor knockout (IFNAR^(−/−)) mice, a newly generated bluetongue laboratory animal model, a pre-challenge experiment was performed to test BoHV-4 safety on such immune-compromised animal. BoHV-4 infected IFNAR^(−/−) mice did not show clinical signs even following the inoculation of BoHV-4 intra-cerebrally, although many areas of the brain got transduced. IFNAR^(−/−) mice intraperitoneally inoculated twice with BoHV-4-A-IgK-VP2gDtm at different time points developed serum neutralizing antibodies against BTV and showed a strongly reduced viremia and a longer survival time when challenged with a lethal dose of BTV-8. The data acquired in this pilot study validate BoHV-4-based vector as a safe and effective heterologous antigen carrier/producer for the formulation of enhanced recombinant immunogens for the vaccination against lethal bluetongue.     

Plant-produced Bluetongue chimaeric VLP vaccine candidates elicit serotype-specific immunity in sheep

Bluetongue (BT) is a hemorrhagic non-contagious, biting midge-transmitted disease of wild and domestic ruminants that is caused by bluetongue virus (BTV). Annual vaccination plays a pivotal role in BT disease control in endemic regions. Due to safety concerns of the current BTV multivalent live attenuated vaccine (LAV), a safe efficacious new generation subunit vaccine such as a plant-produced BT virus-like particle (VLP) vaccine is imperative. Previously, homogenous BTV serotype 8 (BTV-8) VLPs were successfully produced in Nicotiana benthamiana plants and provided protective immunity in sheep. In this study, combinations of BTV capsid proteins from more than one serotype were expressed and assembled to form chimaeric BTV-3 and BTV-4 VLPs in N. benthamiana plants. The assembled homogenous BTV-8, as well as chimaeric BTV-3 and chimaeric BTV-4 VLP serotypes, were confirmed by SDS-PAGE, Transmission Electron microscopy (TEM) and protein confirmation using liquid chromatography-mass spectrometry (LC-MS/MS) based peptide sequencing. As VP2 is the major determinant eliciting protective immunity, the percentage coverage and number of unique VP2 peptides detected in assembled chimaeric BT VLPs were used as a guide to assemble the most appropriate chimaeric combinations. Both plant-produced chimaeric BTV-3 and BTV-4 VLPs were able to induce long-lasting serotype-specific neutralizing antibodies equivalent to the monovalent LAV controls. Antibody levels remained high to the end of the trial. Combinations of homogenous and chimaeric BT VLPs have great potential as a safe, effective multivalent vaccine with the ability to distinguish between vaccinated and infected individuals (DIVA) due to the absence of non-structural proteins.     

DNA-mediated vaccination against infectious bursal disease in chickens.

The objective of the present study was to investigate the feasibility of a DNA vaccine to protect chickens against infectious bursal disease virus (IBDV) infection. A plasmid DNA carrying VP2, VP4, and VP3 genes of the standard challenge (STC) strain of IBDV was constructed and designated as pCR3.1-VP243-STC. One-day-old chickens were intramuscularly injected with the plasmid pCR3.1-VP243-STC once (group D1), twice (group D2), or three times (group D3) at weekly intervals. Chickens at 3 weeks old were orally inoculated with IBDV strain STC and observed for 10 days after challenge. Immunization twice (group D2) or three times (group D3) with the plasmid pCR3.1-VP243-STC conferred protection for 50-100 or 80-100% of chickens, respectively, as evidenced by the absence of clinical signs, mortality, and bursal atrophy. Although chickens vaccinated once (group D1) with the plasmid pCR3.1-VP243-STC did not have clinical signs, they exhibited varying degree of bursal atrophy after challenge. Enzyme-linked immunosorbent assay (ELISA) antibody titers in chickens protected by the plasmid pCR3.1-VP243-STC were significantly lower (P<0.05) than those not protected 10 days after challenge. IBDV antigen was not detected in the bursae of chickens that were protected by receiving the plasmid pCR3.1-VP243-STC twice or three times. The results indicate that the constructed plasmid pCR3.1-VP243-STC as a DNA vaccine provided efficacious protection for chickens against IBDV infection.     

Enhancement of VP1-specific immune responses and protection against EMCV-K challenge by co-delivery of IL-12 DNA with VP1 DNA vaccine

It has been reported that co-delivery of IL-12 DNA with a DNA vaccine further enhances antigen (Ag)-specific protective immunity in pathogenic challenge models. However, the enhancing effects of antibody by IL-12 have been controversial. To clarify this issue, we constructed an IL-12 expression vector, co-immunized IL-12 DNA with an encephalomyocarditis virus (EMCV)-D VP1 plasmid vaccine, and then evaluated immune modulatory effects and protection against lethal EMCV-K challenge. We observed that VP1-specific IgG production, as well as seroconversion rates, were significantly enhanced by IL-12 co-injection, indicating that IL-12 can enhance antibody responses in this model system. In particular, co-injection with VP1 plus IL-12 DNA into the same leg enhanced systemic Ag-specific IgG production to a significantly greater extent than either the separate leg injection of VP1 and IL-12 DNA or VP1 DNA vaccine alone. This suggests that local co-expression of IL-12 along with antigens is more important for enhanced antibody production. Furthermore, IgG2a isotype was significantly enhanced by IL-12 DNA co-injection, indicating a Th1 bias. In addition, co-delivery of IL-12 DNA was demonstrated to enhance VP1-specific Th cell proliferative responses. When animals were challenged with a lethal dose of EMCV-K, IL-12 DNA-co-immunized animals exhibited enhanced survival, as compared to VP1 DNA vaccine alone. These studies suggest that IL-12 plays an important role in increasing Ag-specific Th1 type antibody and cellular responses, resulting in enhanced protection against lethal EMCV-K challenge.     

Protective immune response in mice vaccinated with a recombinant adenovirus containing capsid precursor polypeptide P1, nonstructural protein 2A and 3C protease genes (P12A3C) of encephalomyocarditis virus

Encephalomyocarditis virus (EMCV) infection can cause acute myocarditis and sudden death in pre-weaned piglets as well as severe reproductive failure in sows. In this study, two recombinant adenoviruses containing capsid precursor polypeptide P1 alone (Ad-P1) and P1 plus nonstructural protein 2A and 3C protease coding regions (Ad-P12A3C) of EMCV were respectively constructed using replication-defective human adenovirus serotype 5 as vector, and their antibody responses and protective efficacies against a lethal EMCV challenge were evaluated in mice. Both Ad-P1 and Ad-P12A3C were confirmed to be capable of expressing VP1 protein in BHK21 cells by immunoperoxidase monolayer assay (IPMA). The results showed that mice vaccinated once or twice with Ad-P1 and Ad-P12A3C generated specific antibody response against VP1 protein of EMCV. Although Ad-P1 induced higher antibody titers, virus-neutralizing antibody response was considerably less (p<0.05), compared to that of Ad-P12A3C. Upon challenging with a virulent EMCV strain, Ad-P12A3C elicited efficacious protection (100% for both vaccination once and twice) in the vaccinated mice; whereas the mice immunized with Ad-P1 showed a lower protection (12.5% for vaccination once and 75% for twice). Our work suggests that the recombinant adenovirus (Ad-P12A3C) containing the capsid precursor polypeptide coding region (P1) plus nonstructural protein 2A and 3C protease genes have an excellent potential to be used as a vaccine that can provide sufficient protective efficacy against EMCV infection in animals.