Construction,Immunogenicity Evaluation And Adjuvant Study Of Recombinant NDV Vector Vaccines Of HP-PRRS

Highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS),also known as highly pathogenic porcine blue-ear disease,is an animal disease that is required to be reported to the World Organization for Animal Health(OIE).The pathogenic factor is the highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV).Since the first outbreak in China in 2006,the disease has caused huge economic losses to the pig industry.HP-PRRS is mainly characterized by respiratory diseases in various stages of pigs and reproductive disorders,such as premature delivery,abortion and stillbirth of pregnant sows,resulting in high mortality.In recent years,with the diversification of prevention and control methods for HP-PRRS,the epidemic is well under control.However,complex HP-PRRSV strains are still widespread,which poses a serious challenge to prevention and control.According to the national guidelines for the prevention and control of highly pathogenic blue ear disease(2017-2020),prevention is given priority.Further development of safe and effective vaccines against HP-PRRS will be conducive to the control of the disease,which can help achieve the ultimate goal of purification.Viral vector vaccine has become a hot research target for the prevention and control of HP-PRRS due to its immune advantages of virus carrier and exogenous proteins.Therefore,studies on construction,immunogenicity evaluation and adjuvant experiment of recombinant NDV vector vaccines of HP-PRRS were carried out in this paper.1.Construction and identification of HP-PRRS recombinant Newcastle disease virus candidate vaccinesNewcastle virus(NDV)is an excellent vaccine vector with a wide range of applications.Here,the main structural protein genes,ORF3 and ORF5 of HP-PRRSV GD strain,were amplified by RT-PCR.Based on the reverse genetic platform of NDV La Sota strain,exogenous fragments including ORF3-double,IRES,ORF5-double and ORF5-single were respectively connected into the NDV vector by homologous recombination technology.Two recombinant NDV infectious clones,p BRN-FL-ORF5 and p BRN-FL-ORF3-IRES-ORF5,were constructed.The recombinant plasmids,together with three helper plasmids,p BS-NP,p BS-P and p BS-L,were co-transfected into BSR cells by calcium phosphate.The cell culture supernatant was inoculated into 9-day-old SPF chicken embryo for virus rescue.Subsequently,the virus rescue efficiency was determined by hemagglutinin test.IFA and Western blot were used to determine the exogenous protein expressions of recombinant viruses.MDT,ICPI and IVPI were used to determine the virulence of recombinant viruses.The results of plasmid identification showed that two recombinant plasmids,p BRN-FL-ORF5 and p BRN-FL-ORF3-IRES-ORF5,were constructed respectively.The results of hemagglutination assay showed that two recombinant viruses,r La Sota-GP5 and r La Sota-GP3-GP5,were successfully rescued,and the titers of the recombinant viruses both reached the peak value after 72 hours of growth in chicken embryos(8.7 log EID50/m L).RT-PCR results showed that all exogenous fragments were successfully inserted into the NDV vector genome with good genetic stability.Besides,foreign proteins could be expressed effectively.According to the OIE evaluation criteria for NDV virulence,both the recombinant viruses were classified as the weak strains,consistent with the maternal virus.Overall,these results demonstrated that two recombinant viruses,r La Sota-GP5 and r La Sota-GP3-GP5,were successfully constructed and rescued,which can be used as candidate vaccines for subsequent experimental studies on animal immunogenicity and safety evaluation.2.Evaluation of pathogenicity and immune effect of recombinant NDV candidate vaccines in miceIn this part,mice were used as animal models to evaluate the pathogenicity and immune effect of recombinant candidate vaccines,namely r La Sota-GP5 and r La Sota-GP3-GP5.Mice were inoculated with two recombinant candidate vaccines via intramuscular injection and intracranial injection,respectively,and the changes of body weight were measured for two consecutive weeks.The main organs and tissues of mice in each group were collected for RT-PCR at 14 days post injection to identify the presence of virus residue.Mice were immunized by intramuscular injection to evaluate the immune effect at the humoral and cellular levels.The results of pathogenicity experiments showed that the changes of body weight in mice inoculated with two recombinant viruses were similar to those in PBS group.Moreover,no virus residue was detected in any of the main organs and tissues of mice.The results of immunogenicity evaluation showed that the recombinant candidate vaccine r La Sota-GP3-GP5 could stimulate mice to produce higher levels of specific neutralizing antibodies(12.64 ± 4.15)than r La Sota-GP5(9.10 ± 1.91).Furthermore,r La Sota-GP3-GP5 could significantly stimulate the proliferation of spleen lymphocytes in mice.These results indicated that none of the mice inoculated with two recombinant virus caused significant weight abnormalities and no tissue virus residues,and r La Sota-GP3-GP5 can stimulate mice to produce higher levels of humoral and cellular immune responses.3.Safety evaluation of recombinant NDV candidate vaccines in pigsIn this part,we evaluated the safety of the two recombinant NDV vaccines at gene levels,protein levels,virus efflux levels and clinical levels.After immunization,the samples of major organs and tissues,blood and excreta of pigs were collected regularly,and the replication cycle and viral excretion of recombinant vaccines were detected by q RT-PCR.Immunohistochemistry was used to analyze protein expressions in major organs and tissues.The growth activity of immunized pigs was monitored throughout the immunization cycle.Pathological changes were observed by preparing major histopathological sections.The results showed that there were no abnormal growth,diet or daily activities,and no obvious pathological changes were observed in the pathological sections of tissues and organs.What's more,the recombinant candidate vaccines had limited replication ability in pigs with no more than 10 days,and there was no possibility of virus efflux.Immunohistochemical results showed that protein expressions in all the main organs were negative.These results demonstrated that the two recombinant candidate vaccines have good safety in immunizied pigs.4.Evaluation of the immune effect of recombinant NDV candidate vaccines in pigsIn this part,the immune effect of recombinant candidate vaccines in pigs was evaluated in terms of humoral immunity(specific neutralizing antibody),cellular immunity(cytokines,lymphocyte proliferation and flow cytometry)and challenge experiment(temperature monitoring and virus residue).The results showed that the recombinant candidate vaccine r La Sota-GP3-GP5 had good immunogenicity and could stimulate pigs to produce higher levels of specific neutralizing antibodies(25.49 ± 2.65)and induce high concentrations of IFN-?(999.42 pg/m L)at 14 days post boost immunization,which was significantly better than r La Sota-GP5 in general.Compared with commercial vaccines(neutralizing antibodies: 16.93 ± 2.89,IFN-?: 450.49 pg/m L),the humoral and cellular immune response levels of the recombinant candidate vaccine r La Sota-GP3-GP5 were more prominent.What's more,r La Sota-GP3-GP5 could significantly reduce the viral load in blood and some organ and tissues of pigs after challenge.These results indicated that the recombinant candidate vaccine r La Sota-GP3-GP5 has prominent immunogenicity advantages and can be used as a technical reserve for HP-PRRS prevention and control.5.Preparation,screening and immunization enhancement evaluation of candidate adjuvantsIt has been confirmed that NDV played a negative regulatory role in antigen presentation,while adjuvants are considered to have corresponding antigen presentation regulatory effects.To further improve the immune effect of the recombinant vaccine r La Sota-GP3-GP5,this study prepared and screened compatible adjuvants with obvious advantages in NDV antigen presentation and immune enhancement effect in mice,and evaluated the combined immune enhancement effect of compatible adjuvants and recombinant candidate vaccine in pigs.By comparing the growth status and antigen uptake presentation ability of C57BL/6 and BALB/c mouse-derived DC,the DC source suitable for subsequent adjuvant experiments was screened out.Four TCM compatible ingredients(TCM 1-4)were set into three dose groups: high,medium and low.Two kinds of compatible adjuvants were prepared based on the commercial adjuvant(ISA 206)and TCM adjuvant(TCM-Main),respectively.The homogeneous stability of commercial compatible adjuvants and the levels of antigen uptake and presentation of TCM compatible adjuvants were evaluated respectively.The immune effects of the two kinds of compatible adjuvants were tested in mice before the dominant adjuvants were screened out.Furthermore,the immune enhancement effect was verified in pigs.The results showed that BALB/c and C57BL/6 mouse-derived DC had similar differentiation morphology,NDV antigen uptake and presentation ability.The commercial compatible adjuvants had good stability and uniform particle size.B-4 TCM compatible adjuvant(high dose)could significantly enhance the uptake and presentation ability of NDV antigen by DC.The immune evaluation results of the two kinds of adjuvants in mice showed that B-4 TCM compatible adjuvant(high dose)could stimulate the production of higher levels of specific antibodies and IFN-?.In pig immunization experiments,the recombinant vaccine supplemented with B-4 TCM compatible adjuvant(high dose)had more prominent immune enhancement effects,which could reduce the viral load in some organs and tissues of immunized pigs.These results indicated that B-4 TCM compatible adjuvant can enhance the immune effects of recombinant candidate vaccine in pigs,which can facilitate further development and optimization of TCM adjuvants of pig vaccines.