| Porcine reproductive and respiratory syndrome(PRRS),characterized by severe reproductive failure,respiratory disease and growth retardation in pigs,is one of the economically devastating infectious diseases to the pig industry worldwide.Main reasons for persistent epidemic of PRRS are variation,immunosuppression and immune escape of PRRSV.Vaccination is the predominant strategy for the prevention and control of PRRV infection.Currently,modified live-attenuated vaccines and killed virus vaccines are commercially available for prevention of PRRS.There is a controversy for the use of the commercial vaccines because of their inherent drawbacks.So,the focus and direction in the field of PRRS is the development of a new generation of vaccines,which was safer and more effective one.DNA vaccine is a new generation of vaccines,at the basis of gene recombinant technology and immunology,which can effectively prevent and treat diseases.Use of adjuvants can improve the immunogenicity of DNA vaccine.Along with the enhanced immune efficacy and protective immunity,there is a new breakthrogh for the research of PRRSV vaccine.In the study,,we utilized molecular adjuvant(PoIFN-?1 and PoGPX-1),delivery system NP adjuvant(BPEI/PLGA)and mucosal adjuvant(UEA-1/PLGA)to design a series of PRRSV DNA vaccine on the base of plasmid pcDNA3.1-SynORF5 in order to improve its immune efficacy.Four DNA vaccines(pcDNA3.1-PoIFN-?1-SynORF5,pcDNA3.1-GPX1-LSynORF5,BPEI/PLGA-pcDNA3.1-SynORF5,and UEA-1/PLGA-pcDNA3.1-SynORF5)were prepared and the immunogenicity were detected and evaluated in mice and pigs.This research provides a new insight for development of effective vaccine against PRRSV infection.The contents of this study contain five parts as following:1.Analysis of immunogenicity of PRRSV DNA vaccine improved by molecular adjuvant PoIFN-?1 in micePoIFN-?1 was amplified from PBMC by RT-PCR,then inserted into pcDNA3.1-SynORF5.Plasmid pcDNA3.1-PoIFN-?1-SynORF5 was transfected into Hela cellwhich was identified by restriction enzyme digestion and sequencing.Fusion protein with molecular size about 48 KDa was identified by Western blot performed at 48 h after transfection.Twenty-one Balb/c mice was randomly devided into three groups,and inoculated intramuscularly with pcDNA3.1,pcDNA3.1-SynORF5 and pcDNA3.1-PoIFN-?1-SynORF5.Serological tests,IFN-y assay and lymphocyte proliferation assay were performed in order to evaluate for plasmids' abilities to induce humoral and cellural responses.The results showed that the GP5-specific antibody titer in the group injected with pcDNA3.1-PoIFN-?1-SynORF5 was higher than those given pcDNA3.1-SynORF5 at 14,28 and 42 dpi,and at 42 dpi,the GP5-specific antibody titer was 1:2560,and the neutralizing antibody titer was 1:16.The lymphocyte stimulation index was significantly higher in groups immunized with pcDNA3.1-PoIFN-?1-SynORF5 than that injected with pcDNA3.1-SynORF5(P<0.05).The concentration of IFN-y in the mice immunized with pcDNA3.1-PoIFN-?1-SynORF5 is 395.8 pg/mL,which was higher than that given pcDNA3.1-SynORF5(297.8 pg/mL)(P<0.05).The results in this study suggested that PoIFN-?1 could enhance the immunogenicity of PRRSV DNA vaccine pcDNA3.1-SynORF5,is a promising molecular adjuvant.2.Analysis of immunogenicity of PRRSV DNA vaccine improved by molecular adjuvant GPX1 in miceGPX1 and LSynORF5 were amplified from pcDNA3.1-GPX1 and pcDNA3.1-SynORF5 by PCR,then they were inserted into pcDNA3.1 in sequence.Plasmid pcDNA3.1-GPX1-LSynORF5 was transfected into Vero cell which was identified by restriction enzyme digestion and sequencing.Fusion protein about 50 KDa with good immunogenicity was identified by Western blot performed at 48 h after transfection.Twenty-one Balb/c mice was randomly divided into three groups,and inoculated intramuscularly with pcDNA3.1,pcDNA3.1-SynORF5 and pcDNA3.1-GPX1-LSynORF5.Serological tests,IFN-y assay and lymphocyte proliferation assay were performed in order to evaluate for plasmids' abilities to induce humoral and cellural responses.The results showed that the GP5-specific antibody titer detected only in the group injected with pcDNA3.1-GPX1-LSynORF5,and it was also higher than those given pcDNA3.1-SynORF5 at 28 and 42 dpi(P<0.01 and P<0.05),which was reached to 1:5120 at 42 dpi,and the neutralizing antibody titer was 1:16.The lymphocyte stimulation index was significantly higher in groups immunized with pcDNA3.1-GPX1-LSynORF5(2.625)than that injected with pcDNA3.1-SynORF5(1.82)(P<0.05).The concentration of IFN-y in the mice immunized with pcDNA3.1-GPX1-LSynORF5 is 404.5 pg/mL,which was higher than that given pcDNA3.1-SynORF5(227.25 pg/mL)(P<0.01).The results highlighted that GPX1 was a promising molecular adjuvant in the prevention of PRRSV infection.3.Analysis of immunogenicity of PRRSV DNA vaccine improved by nanoparticles adjuvant BPEI/PLGA in micePLGA,BPEI/PLGA and SPEI/PLGA were prepared by a modified double emulsion solvent evaporation technique.PLGA-DNA,BPEI/PLGA-DNA and SPEI/PLGA-DNA were constructed by absorbed pcDNA3.1-SynORF5 to nanoparticles.The size of nanoparticles was a range of 100-600 nm.And the morphology of nanoparticles was spherical,with no surface discontinuity seen in scanning electron microscopy.Forty-nine Balb/c mice was randomly divided into seven groups,and delivered intranasally(i.n.)with pcDNA3.1,pcDNA3.1-SynORF5 and pcDNA3.1-PoIFN-?1-SynORF5.Serological tests,IFN-y assay and lymphocyte proliferation assay were performed in order to evaluate for the ability of nanoparticle vaccine to induce humoral and cellural responses.The results showed that the GP5-specific antibody detected only in the group injected with PLGA-DNA,the antibody titers were 1:5120 and 1:2160 induced by PLGA-DNA and BPEI/PLGA-DNA,which were both higher than those given pcDNA3.1-SynORF5 at 42 dpi(P<0.01 and P<0.05).However,the highest neutralizing antibody titer was observed in the group given BPEI/PLGA-DNA(1:19.43).Lymphocytes proliferation assay showed that BPEI/PLGA-DNA induced the most significant lymphocytes proliferation.The secretion of IFN-y by stimulated lymphocytes from the mice immunized with BPEI/PLGA-DNA is 678.9 pg/mL,which was markly higher than that given pcDNA3.1-SynORF5(268.5 pg/mL)(P<0.05).The results in this study suggested that PoIFN-?1 could enhance the immuno-genicity of PRRSV DNA vaccine pcDNA3.1-SynORP5,is a promising molecular adjuvant.The results of the study demonstrated that BPEI/PLGA nanoparticles served as a delivery system and adjuvant provides a new model for the development of vaccines against PRRSV.4.Analysis of immunogenicity of PRRSV DNA vaccine improved by mucosal adjuvant UEA-1/PLGA in miceIn the study,we successfully constructed lectinized nanopartcles which encapsulated pcDNA3.1-SynORF5 or GP5,which were UEA-1/PLGA-DNA or UEA-1/PLGA-GP5 nanoparticles,in while,PLGA-DNA and PLGA-GP5 were also prepared by a modified double emulsion solvent evaporation technique.Forty-nine Balb/c mice was randomly divided into seven groups,and delivered orally with PBS,pcDNA3.1-SynORF5,GP5,PLGA-DNA,PLGA-GP5,UEA-1/PLGA-DNA and UEA-1/PLGA-GP5.Serum sample were collected for detecting GP5-specific antibody titers at 14,28,42 dpi,and intestine for IgA antibody titers sampled at 42 dpi.Results:At 42 dpi,IgG antibody titer in group delivered UEA-1/PLGA-DNA,as high as 1:2560,was markly higher than that immunized UEA-1/PLGA-GP5(P<0.05)in while,IgA antibody titer in groups inoculated with UEA-1/PLGA-DNA or UEA-1/PLGA-GP5 were 1:960 or 1:1066,respectively,both were significantly enhanced compared to the groups given pcDNA3.1-SynORF5 or GP5(P<0.01).Ex vivo ligated ileal loop assay revealed lectinized nanoparticles conferred more antigens to M cell,leading a large part of antigen absorbed by M-cell,and then induced mucosal immunity:These results suggested that UEA-1/PLGA NP could potentially be used as a universally robust oral vaccine delivery system.5.Analysis of immunogenicity and protective efficacy of PRRSV DNA vaccine in pigsThirty-five 3-week old healthy pigs were randomly separated into seven groups with five in each group.They were confirmed negative for PRRSV by RT-PCR and ELISA.Group 1 to 5 were injected intramuscularly twice at two-week intervals with pcDNA3.1-PoIFN-?1-SynORF5,pcDNA3.1-GPX1-LSynORF5,BPEI/PLGA-DNA,UEA-1/PLGA-DNA and UEA-1/PLGA-PRRSV(PRRSV was inactived by UV),respectively.Group 6 was vaccinated with commercial attenuated vaccine JXA1-R.And Group 7 was infected with PBS and used as controls.Serum samples were collected from each pig at 14,28,35,42 and 49 days post immunization(dpi)detect specific anti-PRRSV antibodies.At 14 and 28 dpi,fresh PBMCs were collected from inoculated pigs for T lymphocyte proliferation,IFN-? release assay and T cell subset(CD3+CD4+and CD3+CD8+)assays.All groups were challenged at 28 dpi with PRRSV JSKM intramuscularly(2 mL)with an infectious titer of 1×105 TCID50/mL.The animals were monitored for a further 21 days,and rectal temperatures and clinical signs were observed daily.Blood was collected at 0,4,7,10,14 and 21 days post challenge(dpc)for viremia and antibody detection.At 7 and 14 dpc,fresh lymphocytes were separated from the peripheral blood of piglets to detect specific cell-mediated immune(CMI)responses.At the end of the experiment,all pigs were euthanized for pathological analysis.At 14,28,35,42 and49 dpi,the GP5-specific antibody titers induced by BPEI/PLGA-DNA were lower than that immunized by JXA1-R,among the sampling tjmes,at 28,35 and 49 dpi,the differences were non-significant,(P>0.05).The neutralizing antibodies were in the similar level among experimental groups and JXA1-R injected group.The results showed that all prepared vaccines could provide partly protection against PRRSV JSKM challenge in piglets.All of the adjuvants used in this study,BPEI/PLGA is the most efficient one that improve the most significant protective immunity induced by PRRSV DNA vaccine.In conclusion,molecular adjuvant(PoIFN-?1 and PoGPX-1),delivery system NP adjuvant(BPEI/PLGA)and mucosal adjuvant(UEA-1/PLGA)could enhance the humoral and cellural immune responses induced by pcDNA3.1-SynORF5,moreover,the prepared DNA vaccines could provide partial protection against PRRSV challenge.BPEI/PLGA-DNA could provide the most protective effct to PRRSV challenge.This study provides new strategies in development of more effective PRRSV vaccines. |
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