Electroporation improves the efficacy of DNA vaccines in large animals.

It is generally recognized that DNA vaccines are often less effective in large animals than in mice. One possible reason for this reduced effectiveness may be transfection efficiency and the low level of expression elicited by plasmid vectors in large animals. A possible way to improve plasmid gene expression in vivo is electroporation.

The immunogenicity and protective efficacy of bovine herpesvirus 1 glycoprotein D plus Emulsigen are increased by formulation with CpG oligodeoxynucleotides.

The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.

CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D.

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum.

Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility.

Porcine monomyeloid cell lines were established following transfection of primary porcine alveolar macrophage cultures with plasmid pSV3neo, carrying genes for neomycin resistance and SV40 large T antigen. The parental clone 3D4 exhibited a relatively rapid doubling time (25.5 h), high plating efficiency and mixed phenotype with respect to growth on a solid support.

Plasmid DNA induces increased lymphocyte trafficking: a specific role for CpG motifs.

Bacterial DNA, primarily through immunostimulatory cytosine-guanine (CpG) motifs, induces the secretion of cytokines and activates a variety of effector cells. We investigated the possibility that CpG motifs might also modulate immunosurveillance by altering cell trafficking through a regional lymph node. Intradermal injection of plasmid DNA induced rapid and prolonged increases in the number of lymphocytes collected in efferent lymph.

Induction of mucosal immune responses following enteric immunization with antigen delivered in alginate microspheres.

Oral immunization is the most effective way of inducing immune responses in the intestinal tract. Biodegradable microspheres have been used extensively for the delivery of antigens to the Peyer's patches (PPs) within the gut-associated lymphoid tissue (GALT). We evaluated various formulations of alginate microspheres for their capacity to induce mucosal immune responses in vivo.

Th1/Th2 biasing effects of vaccination in cattle as determined by real-time PCR.

Real-time polymerase chain reaction (PCR) is now becoming an accepted tool for measuring gene expression at the transcriptional level. In this study, a direct comparison between real-time PCR, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay was performed. When interferon-gamma (IFN-gamma) gene expression was assessed, both ELISA and ELISPOT data strongly correlated to results obtained by real-time PCR.

Mutational analysis of early region 4 of bovine adenovirus type 3.

The primary objective of characterizing bovine adenovirus type 3 (BAV3) in greater detail is to develop it as a vector for gene therapy and vaccination of humans and animals. A series of BAV3 early region 4 (E4) deletion-mutant viruses, containing deletions in individual E4 open reading frames (Orf) or combinations of Orfs, were generated by transfecting primary fetal bovine retinal cells with E4-modified genomic DNA. Each of these mutants was further analyzed for growth kinetics, viral DNA accumulation, and early-late protein synthesis.

Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5'-TCGTCGTT-3' motif with two 5'-GTCGTT-3' motifs were highly stimulatory in both assays.

Oral DNA vaccination in utero induces mucosal immunity and immune memory in the neonate.

Infectious diseases are responsible for a significant number of deaths during the first weeks of life. Some of the salient pathogens include HSV, HIV, hepatitis B virus, group B streptococcus, Haemophilus sp., and Chlamydia sp.

CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved.

Oligodinucleotides containing CpG motifs stimulate vertebrate immune cells in vitro, have proven efficacy in murine disease models and are currently being tested in human clinical trials as therapies for cancer, allergy, and infectious disease. As there are no known immunostimulatory motifs for veterinary species, the potential of CpG DNA as a veterinary pharmaceutical has not been investigated. Here, optimal CpG motifs for seven veterinary and three laboratory species are described.

Priming by DNA immunization augments T-cell responses induced by modified live bovine herpesvirus vaccine.

DNA vaccines have several advantages over conventional vaccines. One of the most important characteristics is the presentation of antigen via both MHC class I and class II receptors. Although this generally results in strong T-cell responses, antibody production and protection achieved by DNA immunization are unfortunately not always adequate.

Veterinary applications of DNA vaccines.

DNA immunization of livestock has proven to be more challenging than similar approaches in mice. To identify parameters, which could influence the magnitude and type of immune response generated by DNA immunization, we have assessed promoter strength, the role of introns, route of delivery as well as form of antigen. Our results indicate that all of these factors can have an impact on whether an immune response will occur or not, as well as influence the type of immune response generated.

Suppository-mediated DNA immunization induces mucosal immunity against bovine herpesvirus-1 in cattle.

Mucosal surfaces are the primary sites for the transmission of infectious agents including viruses, so effective vaccines generally should induce mucosal immunity. Furthermore, noninvasive delivery is desirable because of the ease of application, the high degree of patient compliance, and the improved safety for patients and clinicians due to the elimination of needles. Unfortunately, most of the conventional vaccines are parenterally administered and result in systemic rather than mucosal immunity.

Fusion of C3d molecule with bovine rotavirus VP7 or bovine herpesvirus type 1 glycoprotein D inhibits immune responses following DNA immunization.

The binding of the complement C3d molecule with receptors on B cells and/or follicular dendritic cells (FDCs) influences the induction of humoral immune responses. For example, C3d fused to an antigen has been shown to have a strong adjuvant effect on antibody production. We investigated the possibility that co-expression of antigen and C3d as a fusion protein could enhance antigen-specific immune responses, following plasmid immunization.

Bovine adenovirus type 3 E1B(small) protein is essential for growth in bovine fibroblast cells.

In order to study the function of bovine adenovirus type 3 (BAV-3) E1A and E1B(small) proteins, we constructed two mutants: (a) BAV102A carries an in-frame deletion in the coding region for the E1A protein (nt 831-1080); (b) BAV102B carries an insertion of triple stop codons in the E1B region (nt 1654, 178 bp downstream of the E1B(small) start codon), which stops the translation of the E1B(small) gene. BAV102A virus could grow to the wild-type BAV-3 titer in transformed cell line VIDO R2 (HAV-5 E1 transformed) cells, but no progeny virus could be found in fetal bovine retina cells (FBRC). RT-PCR and Western blot analysis showed that neither mRNA transcripts nor protein expression of early genes [E1B(small) and DNA binding protein (DBP)] could be detected in BAV102A infected FBRC.

Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.

Mucosal immunity plays an important role in preventing disease but the induction of protective mucosal immune responses remains a significant challenge. We describe a novel in vivo model to analyze the induction of multiple mucosal immune responses in the small intestine. A sterile segment of intestine ('intestinal-segment'; 2-3 m long) was surgically prepared in the jejunum of 4-6-month-old lambs.

Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2.

Infectious porcine circovirus type 2 (PCV2) was generated following transfection of a porcine retina cell line (VIDO R1) with cloned circovirus DNA. Expression of open reading frame 2 (ORF2) was detected at 24 h postinfection and onwards increasingly throughout the infection by Western blot analysis using ORF2 specific polyclonal antibody. Moreover, the ORF2 protein was also detected in purified PCV2 virus, indicating that ORF2 is a structural component of PCV2 viral capsid.

Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus.

Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels.

Identification of a mutant bovine herpesvirus-1 (BHV-1) in post-arrival outbreaks of IBR in feedlot calves and protection with conventional vaccination.

Outbreaks of infectious bovine rhinotracheitis (IBR) have recently been observed in vaccinated feedlot calves in Alberta a few months post-arrival. To investigate the cause of these outbreaks, lung and tracheal tissues were collected from calves that died of IBR during a post-arrival outbreak of disease. Bovine herpesvirus-1 (BHV-1), the causative agent of IBR, was isolated from 6 out of 15 tissues.

Immunization of livestock with DNA vaccines: current studies and future prospects.

Early studies using DNA immunization suggest the potential benefits of this form of immunization including: long-lived immunity, a broad spectrum of immune responses (both cell-mediated immunity and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using marine and cow models, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen.

121R protein from the E3 region of bovine adenovirus-3 inhibits cytolysis of mouse cells by human tumor necrosis factor.

Based on the DNA sequence and known mRNA structures, the early region 3 (E3) of bovine adenovirus (BAV)-3 has the potential to encode four proteins. One of them (121R) is produced as a 14.5-kD protein throughout infection.

Effects of intradermally administered plasmid deoxyribonucleic acid on ovine popliteal lymph node morphology.

In the last decade it has become apparent that bacterial deoxyribonucleic acid (DNA) is recognized as a "danger signal" by the mammalian immune system. To investigate this interaction, sheep were injected intradermally two centimeters distal to the lateral prominence of the fibular head with 400 microg of purified plasmid DNA. Over a 28-day period ultrasound measurements indicated a progressive increase in size of both plasmid and saline (controls) treated popliteal lymph nodes and at Day 30 macroscopic and histological measurements of the lymph nodes were determined.

Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein.

The entire coding region of open reading frame 2 (ORF2) of porcine circovirus 2 (PCV2) was linked to the 3'-end of the maltose-binding protein (MBP)-His(8)-tag gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-d-galactoside. MBP-His(8)-ORF2 was purified to homogeneity by immobilized metal affinity chromatography based on the interaction of the polyhistidine-tag with metal ions.