Porcine Reproductive And Respiratory Syndrome Virus GP5 Research Articles

Vaccination of Plasmid DNA Encoding Somatostatin Gene Fused with GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Induces Anti-GP5 Antibodies and Promotes Growth Performance in Immunized Pigs

Abstract Somatostatin (SS) is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus (PRRSV). A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.

Enhanced immunogenicity of the modified GP5 of porcine reproductive and respiratory syndrome virus

The ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of the porcine reproductive and respiratory syndrome virus (PRRSV) and is the leading target for the development of the new generation of vaccines against PRRS. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing PRRSV GP5. More recent evidence has demonstrated a non-neutralizing decoy epitope upstream of the neutralizing epitope of GP5, which might prevent the development of a strong neutralizing antibody response against PRRSV. In the present study, we modified the ORF5 gene by inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the decoy epitope to minimize or eliminate the decoy effect of the non-neutralizing epitope. The immunogenicity of the modified GP5 was further evaluated using DNA vaccination. The results showed that significantly enhanced neutralizing antibodies were elicited in mice immunized with the DNA construct expressing the modified GP5 compared with the native GP5. Slightly increased levels of GP5-specific ELISA antibodies and T-cell proliferative activities were also observed. These results indicate that the high immunogenicity of the modified GP5 might facilitate the development of improved PRRS vaccines in the future.

Protective immunity induced by a recombinant pseudorabies virus expressing the GP5 of porcine reproductive and respiratory syndrome virus in piglets

Pseudorabies virus (PRV) has been developed as a vaccine vector for expressing foreign immunogens. Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), continues to be a major problem to the pork industry worldwide. Many vaccine strategies have been developed to control the disease but most of them turn out to be unsuccessful. The objective of this research was to explore the feasibility of PRV-based vector vaccine in protection against PRRSV. A live attenuated vaccine-based PRV recombinant expressing the envelope protein GP5 of PRRSV was generated using recombinant DNA techniques. The Bartha-K61-derived recombinant virus, named rPRV-GP5, was shown to express PRRSV GP5 efficiently. Sixteen healthy piglets were assigned to one of four groups (one to four, four pigs per group). Animals in Groups 1 and 2 were each inoculated intramuscularly and intranasally with 10 7.0 PFU of rPRV-GP5 and its parent Bartha-K61, respectively; Group 3 were vaccinated intramuscularly with one-dose of PRRS inactivated vaccine; Group 4 was served as non-vaccinated control. One month later, all animals were all challenged with 10 6.5 TCID 50 of virulent PRRSV CH-1a. All animals in Groups 1 and 3 remained clinically healthy before and after challenge, with only a short period of fever (no more than 41 °C and 3 days), mild and gradually improving lung and kidney lesions, and short-term viremia (2 and 3 week, respectively) in spite of no detectable anti-PRRSV antibody before challenge. On the other hand, all animals in the other two groups showed evident clinical signs with higher temperatures (more than 41 °C) after challenge, and severe lung, kidney and spleen lesions and extended viremia (4 weeks). The results indicate that the rPRV-GP5 is safe for vaccinates and able to confer significant protection against clinical disease and reduce pathogenic lesions induced by PRRSV challenge in vaccinated pigs.

Humoral immune response induced by oral administration of S. typhimurium containing a DNA vaccine against porcine reproductive and respiratory syndrome virus

The ORFs-encoded major envelope glycoprotein (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major structural proteins of this virus. In this report, we described the induction of a PRRSV GP5-specific immune response by oral vaccination of mice with eukaryotic expression vectors containing the GP5 gene of PRRSV (pcDNA3-GP5), delived by attenuated Salmonella typhimurium aroA. It demonstrated that oral administration of the transformants resulted in expression of the GP5 transcript in the intestinal epithelium. The level of serum neutralization antibodies to PRRSV was not significantly different between the mice immunized with the transformants and the naked plasmid DNA. But the neutralizing antibody titres in sera of the mice immunized with SL7207/pcDNA3-GP5' and pcDNA3-GP5' (resides 2-25 deletion mutant of GP5) were significantly lower than those immunized with the complete GP5 gene. These results show that oral inoculation of the transformants can induce humoral immune response to PRRSV. The signal peptide of the GP5 protein of PRRSV is associated with the neutralizing epitope of the protein. The attenuated S. typhimurium may be used as a delivery system for oral DNA vaccines containing PRRSV GP5 glycoprotein.

Identification and characterization of auto-anti-idiotypic antibodies specific for antibodies against porcine reproductive and respiratory syndrome virus envelope glycoprotein (GP5)

Auto-anti-idiotypic antibodies (Aab-2s) were detected from pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The Aab-2s were specific against the idiotypic antibodies (Ab-1s) to the envelope glycoprotein GP5 of PRRSV and were detected from serum samples collected between 21 and 98 days post-infection (DPI). Serological characterization indicated that the Aab-2s recognized the idiotype located within or near the antigen-combining sites of the anti-GP5 antibodies, which was shared by both mouse MAb anti-GP5 and swine polyclonal antibodies. The fact that the Aab-2 inhibited the anti-GP5 antibodies from binding to PRRSV and that they were detected at different time periods in pigs that cleared the infection prior to 98 DPI versus pigs in which virus was detected at 98 DPI suggests that Aab-2 antibodies may play a role in immunity to PRRSV infection.

Identification of Neutralizing and Nonneutralizing Epitopes in the Porcine Reproductive and Respiratory Syndrome Virus GP5 Ectodomain

After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain.

After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

Monoclonal antibodies against conformationally dependent epitopes on porcine reproductive and respiratory syndrome virus

Monoclonal antibodies (MAbs) against porcine reproductive and respiratory syndrome virus (PRRSV) were prepared and characterized. Four MAbs were developed from the mice immunized with the recombinant GP4 protein expressed in insect cells, and six MAbs were derived from the immunization with recombinant GP5 protein. All of the MAbs showed strong perinuclear fluorescence in PRRSV VR2385 infected cells by immunofluorescence staining. Among the MAbs to GP5 protein, one showed strong reactivity in ELISA and recognized a 26 kDa band of PRRSV in a western blot assay, while another showed neutralizing activity against the VR2385 isolate. Out of the four MAbs to GP4 protein, one showed mild reactivity in ELISA with detergent extracted antigen, but had no reactivity in a western-blot assay. The failure of MAb binding to detergent extracted antigen in ELISA or in western-blot analysis indicated that the MAbs were against conformationally dependent epitopes. Reactivity patterns of the MAbs with PRRSV field isolates tested by fixed-cell ELISA showed that there are antigenic variations in PRRSV GP4 and GP5 proteins. Development of these MAbs will benefit further studies on PRRSV structural proteins as well as in understanding their roles in PRRSV pathogenesis.

Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus.

The ORF5-encoded major envelope glycoprotein (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is one of the three major structural proteins of this virus. While some porcine convalescent sera and monoclonal antibodies directed against GP4 and GP5 have the capacity to neutralize the virus in vitro, the protein specificity of porcine neutralizing sera has not yet been established. DNA immunization with a plasmid encoding GP5 of PRRSV, under the control of a human cytomegalovirus promoter, induced anti-GP5-specific neutralizing antibodies in pigs and BALB/c mice. The GP5 protein specificity of neutralizing sera was confirmed by immunoblotting and ELISA. Peripheral blood mononuclear cells obtained from DNA-vaccinated pigs underwent blastogenic transformation in the presence of E. coli-expressed recombinant ORF5-encoded protein, indicating the specificity of the cellular immune response to GP5. Following a massive intratracheal challenge with the virulent IAF-Klop strain of PRRSV, DNA-vaccinated pigs were protected from generalized viraemia and the development of typical macroscopic lung lesions that were observed in unvaccinated, virus-challenged controls, as well as in pigs that were immunized with E. coli-expressed GST-ORF5 recombinant fusion protein. Interstitial pneumonitis and broncho-alveolitis were remarkably milder in DNA-vaccinated animals. These results suggest that the GP5 of PRRSV is a good candidate for a subunit recombinant-type vaccine.