Study On New Genetic-engineering Vaccines Against Porcine Reproductive And Respiratory Syndrome Virus And Antiviral Development

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious disease of swine. It is characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs. Since it was first reported in the United States in 1987, PRRS has already been one of the most economically important diseases of the global swine industry. Vaccine inoculation is the main method to prevent and control this disease. Currently, the commercial vaccines to prevent PRRS are mainly attenuated and inactivated vaccines. A discrepancy of inadequate efficacy and safety concerns surrounds the modified live or killed vaccines. It has become a focus to develop safer and more effective and cheap vaccines to prevent and control PRRS. However, due to limited immunity protection in several experimental vaccines, the progress of new vaccines against PRRSV maintains slowly.Therefbre, in the present study, we explored the new vaccine design against PRRSV, and constructed suicidal DNA vaccines based on Semiliki Forest virus (SFV)-replicon and the recombinant viruses based on pseudorabies virus (PRV)-vector. Furthermore, the immune efficacy and protection of the candidate vaccines were investigated. The most research works were as following:1.2.1 Functional modification of native ORF5 geneThe ORF5-encoded major envelope glycoprotein (GP5) is one of the key immunogenic proteins of PRRSV and is the leading target for the development of the new generation of vaccines against PRRSV. However, weak and tardy neutralizing antibodies have been elicited in several developed experimental vaccines expressing native GP5 protein. In order to obtain the new perfected target genes for developing new vaccines against PRRSV, we modified ORF5 gene by inserting a Pan DR T-helper cell epitope (PADRE) between the neutralizing epitope and the decoy epitoe to minimize or eliminate the decoy effect of the non-neutralizing epitope. The invitro expression and immunogenicity of the modified ORF5 (ORF5m) was further evaluated using DNA vaccination. Western blot showed that ORF5m gene can express GP5m protein with about 25～26 kDa molecular mass, which reacted well with porcine polyclonal antiserum against PRRSV. IFA assay showed that the intracellular location of GP5m was equal to that of native GP5. The results of the mice immunization suggested that significantly enhanced neutralizing antibodies were elicited in mice immunized with the DNA construct expressing ORF5m gene compared with the native ORF5. Significantly increased levels of GP5-specific ELISA antibodies and T-cell proliferative activities were also observed. These results indicated that the high immunogenicity of ORF5m gene might facilitate the development of improved PRRS vaccines in the future.2.2.1 Formation of GP5/M heterodimer and influence on the intracellular localization and induced immune responsesThe two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed to investigate the biological characterization and immunological responses to co-expressing GP5 and M proteins. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells, and heterodimerization facilitated the transport of GP5 from endocytoplasmic reticulum (ER) to Golgi's body. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 4 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibodies could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies (≥1:8) at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in posttranslational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.3.2.1 Construction and immune effect of new DNA vaccines against PRRSVThe immunogenicity of general DNA vaccine pCI-ORF5m/ORF6 and suicidal DNA vaccine pSFV-ORF5m/ORF6 co-expressing ORF5m and ORF6 genes was investigated in mouse model. The results showed that all immunized mice developed neutralizing antibodies (≥1:8) at 4 weeks after primary immunization and achieved the peak at 8 weeks after primary immunization. The neutralizing antibodies level induced by co-expression of ORF5m and ORF6 genes was higher than that induced by co-expression of native ORF5 and ORF6 genes. And suicidal DNA vaccine also displayed the enhanced lymphocyte proliferative compared with general DNA vaccine. These results indicated that a suicidal DNA vaccine pSFV-ORF5m/ORF6 co-expressing ORF5m and ORF6 genes elicited the immunized animals to produce highest neutralizing antibodies and cellular immune responses, and is a new promising vaccine against PRRSV.4.2.1 The recombinant genetically-engineering vaccines against PRRSV based on PRV-vector (rPRV-PRRSV)A live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. The results of Western blot using anti-PRRSV hyperimmune sera also demonstrated correct expression of PRRSV genes in the four identified recombinant viruses. Furthermore, under non-reducing condition, GP5 or GP5m and M could form heterodimers in cells infected by rPRV-GP5-M or rPRV-GP5m-M. Mice immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, immune responses to PRRSV induced by these PRV recombinants showed distinctness, rPRV-GP5-M co-expressing GP5 and M proteins elicited higher neutralizing antibody levels than rPRV-GP5 expressing GP5 alone. In contrast, the recombinant viruses using ORF5m as the target protein including rPRV-GP5m and rPRV-GP5m-M displayed a stronger capacity eliciting neutralizing antibodies and lymphocyte proliferative responses. The highest level of PRRSV-specific immune responses was observed in mice immunized with rPRV-GP5m-M, in which 2 out of 6 mice produce 1:32 neutralizing antibodies level at 10 weeks after primary immunization and induced lymphocyte proliferative responses were significantly higher than the other PRV recombinants. These results indicated that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.5.2.1 Immunological protection induced by the new candidates vaccines against PRRSVTo evaluate sufficiently the immune efficacy and protection provided by two new candidates vaccines (suicidal DNA vaccine pSFV-ORF5rn/ORF6 and the recombinant rPRV-GP5m-M), specific humoral and cellular immunity and the capacity prevented the immunized animals from the virulent infection were investigated and compared with PRRSV killed vaccine in pig model. The recombinant rPRV-GP5m-M elicited comparative neutralizing antibodies and ELISA antibodies against PRV with the PRV parental virus. The piglets immunized with pSFV-ORF5m/ORF6 and rPRV-GP5m-M produced detectable PRRSV-specific neutralizing antibodies at 42d after primary immtmization. Subsequently, the immune responses were further enhanced gradually. But no neutralizing antibodies were detected in the group vaccinated with PRRSV killed vaccine before PRRSV challenge (63 days after primary immunization). After PRRSV challenge, piglets vaccinated with rPRV-GP5m-M and pSFV-ORF5rm/ORF6 developed rapidly increased neutralizing antibodies and reached 1: 128-1: 256 neutralizing antibodies at 17 and 21 dpc. In additional, vaccination with PRRSV killed vaccine produced very low specific lymphocyte proliferative responses before challenge, and it was not significantly increased by the subsequent infectious challenge. In contrast, rPRV-GP5m-M vaccination resulted in significantly proliferative responses at days 42 and 63 after primary immunization, and virus challenge resulted in a faster and higher improvement. Compared with the protection provided by killed vaccine, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M and pSFV-ORF5m/ORF6, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions.