Construction and immunogenicity of a recombinant swinepox virus expressing a multi-epitope peptide for porcine reproductive and respiratory syndrome virus.

To characterize neutralizing mimotopes, phages were selected from a 12-mer phage display library using three anti-porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing monoclonal antibodies: (1) A1; (2) A2; and (3) A7. Of these, A2 and A7 recognize the mimotope, P2, which contains the SRHDHIH motif, which has conserved consensus sequences from amino acid positions 156 to 161 in the N-terminal ectodomain of GP3. The artificial multi-epitope gene, mp2, was designed by combining three repeats of the mimotope P2. The resulting sequence was inserted into the swinepox virus (SPV) genome to construct a recombinant swinepox virus (rSPV-mp2). The rSPV-mp2 was able to stably express the multi-epitope peptide, mP2, in vitro. The rSPV-mp2 immunized pigs exhibited a significantly shorter fever duration compared with the wtSPV treated group (P < 0.05). There was an enhanced humoral and cellular immune response, decreased number of PRRSV genomic copies, and a significant reduction in the gross lung pathology (P < 0.05) was observed following PRRSV infection in rSPV-mp2-immunized animals. The results suggest that the recombinant rSPV-mp2 provided pigs with significant protection against PRRSV infection.

Recombinant Swinepox Virus for Veterinary Vaccine Development.

Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses.

Immune responses and protective efficacy of a recombinant swinepox virus co-expressing HA1 genes of H3N2 and H1N1 swine influenza virus in mice and pigs.

The recombinant swine poxvirus rSPV/H3-2A-H1 co-expressing HA1 genes of H3N2 and H1N1 subtype SIV has been constructed and identified. Inoculations of rSPV/H3-2A-H1 yielded ELISA and neutralization antibodies against SIV H1N1 and H3N2, and elicited potent H1N1 and H3N2 SIV-specific INF-γ response from T-lymphocytes in mice and pigs in this study. Complete protection against SIV H1N1 or H3N2 challenge in pigs was observed.

Protection of guinea pigs by vaccination with a recombinant swinepox virus co-expressing HA1 genes of swine H1N1 and H3N2 influenza viruses.

Swine influenza (SI) is an acute respiratory infectious disease of swine caused by swine influenza virus (SIV). SIV is not only an important respiratory pathogen in pigs but also a potent threat to human health. Here, we report the construction of a recombinant swinepox virus (rSPV/H3-2A-H1) co-expressing hemagglutinin (HA1) of SIV subtypes H1N1 and H3N2. Immune responses and protection efficacy of the rSPV/H3-2A-H1 were evaluated in guinea pigs. Inoculation of rSPV/H3-2A-H1 yielded neutralizing antibodies against SIV H1N1 and H3N2. The IFN-γ and IL-4 concentrations in the supernatant of lymphocytes stimulated with purified SIV HA1 antigen were significantly higher (P < 0.01) than those of the control groups. Complete protection of guinea pigs against SIV H1N1 or H3N2 challenge was observed. No SIV shedding was detected from guinea pigs vaccinated with rSPV/H3-2A-H1 after challenge. Most importantly, the guinea pigs immunized with rSPV/H3-2A-H1 did not show gross and micrographic lung lesions. However, the control guinea pigs experienced distinct gross and micrographic lung lesions at 7 days post-challenge. Our data suggest that the recombinant swinepox virus encoding HA1 of SIV H1N1 and H3N2 might serve as a promising candidate vaccine for protection against SIV H1N1 and H3N2 infections.

First insights into the protective effects of a recombinant swinepox virus expressing truncated MRP of Streptococcus suis type 2 in mice.

To explore the potential of the swinepox virus (SPV) as vector for Streptococcus suis vaccines, a vector system was developed for the construction of a recombinant SPV carrying bacterial genes. Using this system, a recombinant virus expressing truncated muramidase-released protein (MRP) of S. suis type 2 (SS2), designated rSPV-MRP, was produced and identified by PCR, western blotting and immunofluorescence assays. The rSPV-MRP was found to be only slightly attenuated in PK-15 cells, when compared with the wild-type virus. After immunization intramuscularly with rSPV-MRP, SS2 inactive vaccine (positive control), wild-type SPV (negative control) and PBS (blank control) respectively, all CD1 mice were challenged with a lethal dose or a sublethal dose of SS2 highly virulent strain ZY05719. While SS2 inactive vaccine protected all mice, immunization with rSPV-MRP resulted in 60% survival and protected mice against a lethal dose of the highly virulent SS2 strain, compared with the negative control (P < 0.05). Our data indicate that animals immunized with rSPV-MRP had a significantly reduced bacterial burden in all organs examined, compared to negative controls and blank controls (P <0.05). Antibody titers of the rSPV-MRP-vaccinated group were significantly higher (P <0.001), when compared to negative controls and blank controls. Antibody titers were also significantly higher in the vaccinated group at all time points post-vaccination (P <0.001), compared with the positive controls. These initial results demonstrated that the rSPV-MRP provided mice with protection from systemic SS2 infection. If SPV recombinants have the potential as S. suis vaccines for the use in pigs has to be evaluated in further studies.

A novel vaccine against Streptococcus equi ssp. zooepidemicus infections: the recombinant swinepox virus expressing M-like protein.

To develop a safer, more immunogenic and efficacious vaccine against Streptococcus equi ssp. zooepidemicus (SEZ) infections, the gene of M-like protein (SzP) was placed under the strong vaccinia virus promoter P28 and then inserted into swinepox virus (SPV) genome. The recombinant swinepox virus (rSPV-szp) was isolated in a non-selective medium by the co-expression of Escherichia coli LacZ gene and verified by PCR, western blotting and immunofluorescence assays. To evaluate the immunogenicity of this rSPV-szp, ICR mice were immunized with the rSPV-szp, inactivated SEZ vaccine (positive control), wild type SPV (negative control), or PBS (challenge control). All mice were intraperitoneally challenged with 5 LD(50) of homogenous ATCC 35246 strain 14 days post-vaccination. The results showed that at least 70% mice in rSPV-szp-vaccinated group were protected against homogenous ATCC 35246 challenge, the survival rate was significantly higher compared with mice in the negative control group and the challenge control group (P<0.001). The antibody titers of the rSPV-szp-vaccinated group were significantly higher (P<0.05) than the other three groups. Passive immune protection assays showed that the hyperimmune sera against M-like protein could provide mice with complete protection against challenge of ATCC 35246. Semi-quantitative RT-PCR analysis showed a marked increased in levels of IL-4 and IFN-γ mRNA in immunized mice. The results suggested that the recombinant rSPV-szp provided mice with significant protection from the SEZ infections. It is a promising candidate for the vaccine development against SEZ infections.

Feline B7.1 and B7.2 proteins produced from swinepox virus vectors are natively processed and biologically active: potential for use as nonchemical adjuvants.

Costimulatory ligands, B7.1 and B7.2, have been incorporated into viral and DNA vectors as potential nonchemical adjuvants to enhance CTL and humoral immune responses against viral pathogens. In addition, soluble B7 proteins, minus their transmembrane and cytoplasmic domains, have been shown to block the down regulation of T-cell activation through blockade of B7/CTLA-4 interactions in mouse tumor models. Recently, we developed swinepox virus (SPV) vectors for delivery of feline leukemia antigens for vaccine use in cats [Winslow, B.J., Cochran, M.D., Holzenburg, A., Sun, J., Junker, D.E., Collisson, E.W., 2003. Replication and expression of a swinepox virus vector delivering feline leukemia virus Gag and Env to cell lines of swine and feline origin. Virus Res. 98, 1-15]. To explore the use of feline B7.1 and B7.2 ligands as nonchemical adjuvants, SPV vectors containing full-length feline B7.1 and B7.2 ligands were constructed and analyzed. Full-length feline B7.1 and B7.2 produced from SPV vectors were natively processed and costimulated Jurkat cells to produce IL-2, in vitro. In addition, we explored the feasibility of utilizing SPV as a novel expression vector to produce soluble forms of feline B7.1 (sB7.1) and B7.2 (sB7.2) in tissue culture. The transmembrane and cytoplasmic regions of the B7.1 and B7.2 genes were replaced with a poly-histidine tag and purified via a two-step chromatography procedure. Receptor binding and costimulation activity was measured. Although feline sB7.1-his and sB7.2-his proteins bound to the human homolog receptors, CTLA-4 and CD28, both soluble ligands possessed greater affinity for CTLA-4, compared to CD28. However, both retained the ability to partially block CD28-mediated costimulation in vitro. Results from these studies establish the use of SPV as a mammalian expression vector and suggest that full-length-vectored and purified soluble feline B7 ligands may be valuable, nonchemical immune-modulators.

Swinepox virus as a vaccine vector for swine pathogens.

Several small and large viruses (e.g., adenovirus, poxvirus, and herpesviruses) have been investigated as vaccine vectors. Each viral system has its advantages and disadvantages. One major advantage for viral vector vaccines is their ability to elicit a protective cell-mediated immunity as well as a humoral response to the antigen delivered by the vector. One major problem to using recombinant viruses as vaccines is the pathogenic potential of the parent virus. Therefore, it is important that along with the optimal expression of the foreign genes and ability to provide protection, the pathogenicity of the vector virus must be reduced during genetic manipulation without affecting its multiplication. The requirements to develop a viral vector, for example, swinepox virus, are a cell culture system that will support the growth of the virus, a suitable nonessential region(s) in the virus genome for insertion of foreign DNA so that virus replication is not affected, a foreign gene(s) that encodes for an immunogenic protein of a swine pathogen, strong transcriptional regulatory elements (promoters) necessary for optimal expression of the foreign genes, a procedure for delivering the foreign gene(s) into the nonessential locus, and a convenient method of distinguishing the recombinant viruses from the parent wild-type virus. Using this methodology, recombinant swinepox virus vaccines expressing pseudorabies virus antigens have been developed and shown to provide protection against challenge. These studies and evidence of local infection of the oral tract by swinepox virus indicate its potential as a recombinant vector for providing immunity against various swine pathogens including those that infect the respiratory and gastrointestinal tracts.

Evaluation of swinepox virus as a vaccine vector in pigs using an Aujeszky's disease (pseudorabies) virus gene insert coding for glycoproteins gp50 and gp63.

Pigs were vaccinated by scarification or intramuscular injection with a swinepox virus-Aujeszky's disease (pseudorabies) recombinant (rSPV-AD) constructed by inserting the linked Aujeszky's disease virus genes coding for glycoproteins gp50 and gp63, attached to a vaccinia virus p7.5 promoter, into the thymidine kinase gene of swinepox virus. By 21 days after vaccination, 90 and 100 per cent of the animals vaccinated by scarification or intramuscular injection, respectively, had developed serum neutralising antibodies to Aujeszky's disease virus. Upon challenge with virulent virus, significantly fewer vaccinated pigs developed clinical Aujeszky's disease, nasal shedding of challenge virus was markedly reduced, and the vaccinated groups of pigs maintained or gained weight during the week after challenge whereas the unvaccinated control group lost weight. No transmission of rSPV-AD to in-contact controls was detected during the three weeks before challenge. In a second experiment, serum neutralising antibodies to Aujeszky's disease virus persisted for 150 days after the pigs were vaccinated with rSPV-AD by scarification or intramuscular injection and all the pigs showed an anamnestic response when they were revaccinated.

A poxvirus of primates. II. Immunology.

OrTeCa poxvirus shares antigens with Yaba virus, as evidenced by two-way crossreactions in complement-fixation and neutralization tests. Antibodies produced by vaccinia and monkeypox viruses showed weak complement-fixation reactions with OrTeCa viral antigen, but neutralization reactions were negative and cross protection in vivo was not demonstrated. OrTeCa and Yaba virus antisera both contained complement-fixing and neutralizing antibodies against swinepox virus, but swinepox antiserum had no antibodies to any other poxvirus. Long-lasting immunity to the OrTeCa pox disease was produced in monkeys and man by spontaneous infection or vaccination with OrTeCa poxvirus. Complement-fixing antibody titers could be measured in convalescence, but tended to drop to low levels or disappear thereafter. In contrast, neutralizing antibody titers remained high for 3 years, and monkeys that were challenged with OrTeCa virus were reFractory to a second infection.