Introduction: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. Material and Methods: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. Results: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. Conclusion: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Proper conformational arrangement of the E2 molecules of bovine viral diarrhoea-mucosal disease virus (BVD-MDV) is crucial to obtain an effective recombinant vaccine candidate against the disease. In this study, we characterised a new molecule composed of two distinct sequences of the E2 glycoprotein of BVD-MDV and the Fc fragment of human immunoglobulin (BVDE2Fc). The chimaeric protein was expressed in mammalian cell lines of different species by adenoviral transduction and purified by immobilised metal-affinity chromatography. The N-glycans were profiled by HPLC, and the BVDE2Fc immunogenicity was assessed in male mice. The antigen-antibody reactions were evaluated by ELISA. The MDBK cell line was selected from among five for the final production of BVDE2Fc. After purification to over 90%, the N-glycan profile showed neutral and complex oligosaccharides. The mouse immunisation induced a strong humoral response, which produced antibodies able to attach to conformational epitopes on E2 molecules, while the Fc fragment barely contributed to the immune response. Additionally, BVDE2Fc attached to antibodies from bovine sera positive to distinct BVD-MDV subtypes, whereas the loss of BVDE2Fc structure during the deglycosylation process considerably diminished those interactions. These results demonstrate that the structure of E2 molecules arranged in tandem and attached to an Fc fragment could represent a viable design for future vaccine candidates against BVD-MD.

A study was conducted to determine whether subcomponent proteins (previously identified as BCSP20, BCSP3l, and BCSP45, and the corresponding recombinant proteins rBCSP20, rBCSP31, and rBCSP45) that were recovered from the cell surface of Brucella abortus strain 19 were immunogenic and protective for mice when compared with Brucella cell surface protein (BCSP) and with a proteinase K-treated lipopolysaccharide (PKLPS) extracted from B abortus strain 2308. Protection was evaluated after challenge exposure with a virulent culture of B abortus strain 2308, using CD-1 or BALB/c mice or both inoculated with vaccines of various combinations and concentrations, with and without PKLPS or BCSP. Protection was assessed by enumeration of splenic colony-forming units, reduced mean splenic weight relative to controls, and the relative serologic responses (immune response) in an ELISA. The general results indicate that BCSP, PKLPS, BCSP20, and BCSP31 are immunogenic or protective or both. Protectiveness was not observed for each of the recombinant proteins; however, results from the combined recombinant protein vaccine study suggest the immunogenicity of the recombinant proteins. The apparent immune-inducing properties of BCSP20 and BCSP3l are thought to be attributable to the presence of an immunogenic and protective BCSP fraction (possibly lipopolysaccharide) still associated. Serologic results support our conclusion that each of the recombinant protein vaccines did not induce a protective response comparable to that of BCSP or PKLPS, even when the subcomponents were combined. Although the results suggest that the subcomponents of BCSP apparently induced partial protection, they are thought to be only a part of the antigens contained in BCSP that influence the serologic response. Our findings may serve as an experimental model to determine the mechanisms involved in the protective responses induced by Brucella antigens.

The objective of this study was to evaluate a new recombinant chimeric vaccine against porcine reproductive and respiratory syndrome virus (PRRSV). The subunit vaccine, PRRSFREE, from Reber Genetics, Taiwan, Republic of China, is based on a plasmid containing a detoxified Pseudomonas exotoxin carrying open reading frame (ORF) 7, 1b, and 5 and 6 chimeric subunits of types 1 and 2 PRRSV. Pigs were injected intramuscularly with 2.0 mL of the vaccine at 21 and 42 d of age, according to the manufacturer's recommendation. At the age of 63 d the pigs were inoculated intranasally with either type 1 or type 2 PRRSV. Regardless of the genotype of the challenging PRRSV, the vaccinated challenged pigs had significantly lower (P < 0.05) mean rectal temperature, respiratory score, lung lesion score, and amount of PRRSV antigen within areas of interstitial pneumonia, along with overall lower levels of viremia due to type 1 or type 2 PRRSV compared with the unvaccinated challenged pigs. The vaccinated challenged pigs also had significantly higher (P < 0.05) numbers of interferon-γ secreting cells compared with the unvaccinated challenged pigs. This study demonstrated that the new vaccine provides protection against respiratory disease from heterologous types 1 and 2 PRRSV challenge in growing pigs.

The Cooper isolate of bovine herpesvirus 1 (BHV1) was used to produce a thymidine kinase-negative (TK-) recombinant by insertion of a beta-galactosidase (bgal) expression cassette into the TK coding region. The recombinant virus (TK- bgal+) was tested for abortifacient activity in cattle by inoculation of 5 pregnant heifers at 25 to 29 weeks gestation. Five additional heifers were inoculated with the Cooper TK-positive (TK+) virus to serve as controls. After inoculation, both groups of heifers developed similar febrile responses and neutralizing antibody titers. Virus was isolated from blood of all heifers during the first postinoculation (PI) week, and isolation frequencies were similar for both groups. In contrast, whereas virus was isolated from many of the nasal and vaginal swab specimens of heifers inoculated with TK+ virus, only rare virus isolations were made from the heifers given TK- bgal+ virus. All heifers inoculated with TK+ virus aborted between PI days 19 and 35. The finding of characteristic microscopic lesions and viral antigen in fetal tissues indicated that the abortions were caused by BHV-1 infection. Virus was isolated from 3 fetuses, and all isolates were TK+. Two heifers inoculated with TK- bgal+ virus aborted at PI days 25 and 39. Fetal tissues had typical BHV-1 microscopic lesions and viral antigen. Virus was isolated from blood of both fetuses, and the isolates were TK- bgal+. Results of this study indicate that inactivation of the TK gene reduces, but does not eliminate, the abortifacient activity of BHV-1.

The vaccine efficacy of a genetically engineered deletion mutant strain of pseudorabies virus, strain 783, was compared with that of the conventionally attenuated Bartha strain. Strain 783 has deletions in the genes coding for glycoprotein I and thymidine kinase. In experiment 1, which had a 3-month interval between vaccination and challenge exposure, strain 783 protected pigs significantly (P < 0.05) better against virulent virus challenge exposure than did the Bartha strain. The growth of pigs vaccinated with strain 783 was not arrested, whereas that of pigs vaccinated with the Bartha strain was arrested for 7 days. Of 8 pigs given strain 783, 4 were fully protected against challenge exposure; none of the pigs given strain Bartha was fully protected. In experiment 2, which had a 3-week interval between vaccination and challenge exposure, the growth of pigs vaccinated with strain 783 was arrested for 3.5 days, whereas that of pigs vaccinated with the Bartha strain was arrested for 6 days. In experiment 3, pigs with moderate titer of maternal antibodies were vaccinated twice IM or once intranasally with either strain 783 or Bartha and were challenge-exposed 3 months after vaccination. Pigs given strain 783 twice IM were significantly (P < 0.05) better protected than were the other pigs. They had growth arrest of only 6 days, compared with 9 days for pigs of other groups, and shed less virus after challenge exposure. Results of this study indicate that the vaccine based on the deletion mutant strain 783 is more efficacious than is the Bartha strain of pseudorabies virus.

The objective of this study was to compare the protection against challenge with porcine circovirus 2 (PCV2) induced by an experimental vaccine based on open reading frame (ORF) 2 of PCV2 DNA plus an adjuvant (aluminum hydroxide, cobalt oxide, or liposome) and a commercial PCV2 subunit vaccine. A total of 35 colostrum-fed, cross-bred, conventional piglets were randomly divided into 7 groups. The commercial vaccine was more efficacious against PCV2 challenge than the 4 experimental vaccines according to immunologic, virologic, and pathological outcomes. The pigs inoculated with the experimental vaccine containing the liposome adjuvant had significantly higher levels (P < 0.05) of neutralizing antibodies and interferon-γ-secreting cells, and significantly lower levels (P < 0.05) of PCV2 viremia than the pigs inoculated with the other experimental vaccines. The pigs inoculated with the experimental vaccines containing either the liposome adjuvant or the cobalt oxide adjuvant had significantly lower lymphoid lesion scores (P < 0.05) than the pigs in the group inoculated with the PCV2 DNA vaccine dissolved in phosphate-buffered saline. Liposome proved to be a potent adjuvant that efficiently enhanced both humoral and cellular immune responses induced by the PCV2 DNA vaccine.