Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene.

A recombinant porcine adenovirus expressing the classical swine fever virus (CSFV) gp55/E2 gene was administered to commercially available pigs via oral or subcutaneous routes and their susceptibility to oral and subcutaneous challenge with CSFV was determined. 100% of animals vaccinated and challenged subcutaneously were protected. In the groups of pigs vaccinated either orally or subcutaneously and then challenged orally, 60% of animals were protected. Before challenge, neutralising antibodies to CSFV were detected in 60% of pigs vaccinated subcutaneously, but in none of those given the vaccine orally. CSFV antigen was found in the spleens of surviving pigs that had been vaccinated orally. In contrast, subcutaneous vaccination was shown to preclude the presence of CSFV in the spleen of animals that survived challenge.

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. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2-3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge.

A prime-boost vaccination strategy using naked DNA followed by recombinant porcine adenovirus protects pigs from classical swine fever.

Weaned pigs (6-week-old) and 7-day-old pre-weaned piglets were vaccinated with naked plasmid DNA expressing the gp55/E2 gene from classical swine fever virus (CSFV). Both groups of pigs were then given a booster dose of recombinant porcine adenovirus expressing the gp55 gene (rPAV-gp55). Following challenge with CSFV, 100% of weaned pigs and 75% pre-weaned piglets were protected from disease. Weaned pigs given a single dose of rPAV-gp55 were also protected, but showed a slight increase in temperature immediately post-challenge. However, weaned animals given a DNA prime before rPAV-gp55 showed no fluctuation in body temperature following challenge and no pathology in spleen or lymph nodes upon post-mortem. In addition, no CSFV could be re-isolated from the rPAV vaccinated group and from only one pig in the prime-boost group following challenge, suggesting that both vaccination regimes have the potential to reduce or prevent virus shedding following experimental challenge.

The presence of cross-reactive antibodies to rabbit haemorrhagic disease virus in Australian wild rabbits prior to the escape of virus from quarantine.

Sera collected from Australian wild rabbits prior to the escape of rabbit haemorrhagic disease virus (RHDV) from Wardang Island were examined for RHDV antibodies using purified recombinant capsid protein VP60 expressed from baculovirus. A VP60-based indirect ELISA showed that 196 of 392 wild rabbit sera reacted (OD(450) >0.15) with VP60. Twenty sera (OD(450) ranging from 0.15-2.47), randomly chosen from the 196 positive sera, recognized the 64 kDa VP60 in Western blot analysis, indicating that the reactivity detected in ELISA is indeed specific to the VP60 antigen. In a separate study, sera of 23 rabbits from an RHD-free area after the escape of RHDV were tested by ELISA and 21 of the 23 rabbits were found to be positive. When these rabbits were challenged with a lethal dose of RHDV, 11 out of the 23 rabbits survived. The presence of RHDV-protective antibodies in some of these rabbits suggested that they had been exposed to a pre-existing non-virulent rabbit calicivirus closely related to RHDV. These results highlight the need to study the prevalence of, and to characterize, this viral agent in order to effectively control rabbit populations in Australia and New Zealand.

Protection of pigs against classical swine fever with DNA-delivered gp55.

Classical swine fever virus causes significant mortality and morbidity in commercial piggeries in many countries in Europe and Asia. The protective antigen, gp55, is highly conformation-dependent and thus killed virus or bacterially produced proteins are not protective. This report demonstrates that DNA vaccination with the gene encoding gp55 can provide protective immunity with inoculation of two doses of 25 microg DNA or a single shot of 200 microg. Furthermore, the DNA can be delivered intramuscularly or by a simple spring-loaded needleless inoculator. In addition it is shown that inoculation of the DNA at a single site conveys the same level of immunity as division of the dose between two sites.

Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever.

A recombinant porcine adenovirus (rPAV) with the gp55 (E2) gene from the classical swine fever virus (CSFV) 'Weybridge' strain inserted into the right hand end of the PAV serotype 3 (PAV3) genome was constructed. Expression of gp55 was directed by the major late promoter and tri-partite leader sequences located and cloned from PAV3. No compensatory deletions of PAV DNA sequences were made. Vaccination of outbred pigs with a single dose of the recombinant virus (rPAV-gp55) resulted in complete protection from lethal challenge with CSFV. No adverse clinical signs were observed in vaccinated animals following administration of rPAV-gp55 and following challenge, no clinical signs of CSF were observed prior to, or at, post mortem. The insert made into the rPAV increased the genome length to 106.8% of wild type and therefore exceeded the expected maximum insert size for a stable recombinant by almost 2%. Thus rPAV-gp55 contains the largest stable insertion made into a non-deleted Mastadeno virus recombinant so far reported.

Fine mapping of a C-terminal linear epitope highly conserved among the major envelope glycoprotein E2 (gp51 to gp54) of different pestiviruses.

Envelope glycoprotein E2 (gp51 to gp54) is the major neutralizing antigen of pestiviruses, which include classical swine fever virus (CSFV), bovine viral diarrhoea virus (BVDV), and border disease virus (BVD). Previous studies carried out using a panel of monoclonal antibodies raised against CSFV strain Brescia have revealed the existence of four antigenic domains, A to D, of the E2 protein, all of which are located at the N-terminal half of the molecule. Here we report the detailed mapping, using three complementary techniques, of a novel linear epitope located at the C-terminal part of the molecule, which reacted with a monoclonal antibody (4-9D4) as well as polyclonal animal sera. This epitope is highly conserved in the three different members of pestiviruses and hence can be used as a genus-specific diagnosis tool. The observation that this epitope is not accessible on the native virus surface, together with its C-terminal location, supports a recently proposed structural model, indicating that the C-terminal part of E2 is membrane-bound while the N-terminal half of the molecule is exposed on the virus surface.

Self-assembly, antigenicity, and immunogenicity of the rabbit haemorrhagic disease virus (Czechoslovakian strain V-351) capsid protein expressed in baculovirus.

Rabbit haemorrhagic disease virus (RHDV) capsid protein was expressed in a baculovirus system. Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in ELISA and Western blot with the antibodies raised against RHDV. Direct electron microscopy of the cell culture supernatant and the purified protein demonstrated that the capsid protein expressed in insect cells self-assembled to form empty virus-like particles (VLP) which are similar in size and morphology to that of native virus. These particles were immunoreactive with polyclonal anti-RHDV antibodies and with four monoclonal antibodies which recognise conformational epitopes of the virus. The results indicated that the VLPs were morphologically and antigenically indistinguishable from native virus. The recombinant VLPs induced high levels of RHDV-specific antibodies in rabbits and mice following immunisation. The immune response to the VLPs protected the rabbits following challenge with the virulent RHDV. In haemagglutination assays, the VLPs bound to human red blood cells similar to the native virus particles. The recombinant protein and or VLPs is suitable for the development of a rapid, sensitive and reliable test for detection of antibodies to RHDV and for use as a vaccine for domestic rabbits.

High level expression of the envelope glycoprotein (gp53) of bovine viral diarrhoea virus (Singer) and its potential use as diagnostic reagent.

A 1.74-kb cDNA fragment containing the gp53 coding region has been cloned from bovine viral diarrhoea virus (BVDV) strain Singer by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that gp53 of BVDV strains Singer, NADL and SD-1 shared extensive sequence homology at both the RNA (85-94%) and protein (82-91%) levels. Nineteen cysteine residues and five potential N-linked glycosylation sites were identified within the sequenced region, all of which were conserved. These observations suggest that although the homology at the nucleotide sequence level may vary, there was strong structural conservation among bovine viral diarrhoea virus envelope proteins. Full-length gp53 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). The N-terminal half of gp53 was also synthesised in E. coli as a 28-KDa recombinant protein using the T7 RNA polymerase-directed expression system. Both recombinant proteins were expressed at high levels (approximately 30-50 mg/l). The recombinant proteins were recognised in ELISA and Western blot analyses by polyclonal serum raised against a mixture of BVDV and classical swine fever virus (CSFV). Rabbit antiserum raised against the 28-kDa recombinant protein reacted with different BVDV strains in ELISA and immunofluorescent antibody test, but not with CSFV in the same tests. These results demonstrated that the bacterial recombinant proteins have similar immunological properties to that of the native viral protein and, in conjunction with its homologous antisera, can be useful as diagnostic reagents.