Construction And Immune Responses Of The Tandemed Gene Of Infectious Bursal Disease Virus VP2and Chicken Complement C3D

Infectious bursal disease (IBD) virus (IBDV) is the etiological agent of "Gumboro diseases". Although firstly observed about40years ago, this disease continues to pose an important threat to the commerce. Infections caused by IBDV may exacerbate infections with other etiologic agents and reduce the chicken’s ability to respond to vaccination. Lymphoid cells in the bursa of Fabricius (BF) are the target cells of IBDV. Between3and6weeks after hatching, when the BF reaches maximum development, chickens are highly susceptible to the virus. Infection results in lymphoid depletion and the final destruction of the bursa as the predominant feature of the pathogenesis of IBD. IBDV is highly infectious and very resistant to inactivation. Despite strict hygienic measures, vaccination is inevitable under high infection pressure and mandatory to protect chickens against infection during the first weeks after hatch. Europe has experienced the emergence of "very virulent"(vv)strains of IBDV. The segmented nature of vvIBDV genome might have allowed the appearance of new strains in which a complete genome segment had been introduced from until now unknown sources. The occurrence of variants causes immune failure. It has also to be taken into consideration that vvIBDV will break through immunity provided by highly attenuated vaccine strains. On the other side, it is well known that less attenuated strains may cause lesions in the BF and immunosuppression even in vaccinated birds. With so many disadvantages associated with the currently available live attenuated vaccines against IBDV infection, the search for a new approach to improve the vaccine or to produce new vaccines is warranted. DNA vaccines have been developed for IBDV. However, developments of DNA vaccines were limited because of low protein expression level in vivo and weak immune response elicited. Chicken complement C3d and PEI can be applied as a adjuvant which can dramatically enhance the antibody responses to the target antigens. This study evaluated the effects of C3d on the immunogenicity of IBDV VP2expressed by a DNA vaccine conjugation of target antigen to multiple copies of C3d and that of PEI on DNA vaccine. ExpⅠ:Construction of the tandemed gene of vp2of infectious bursal disease virus and chicken complement c3d, and its expression in eukaryotic cellsTo enhance the immune efficacy of the DNA vaccine against the challenge of IBDV, the chicken complement C3d gene adding the sequence of (G4S)2at5’ end was synthesized and the tandemed one to three copies of C3d was obtained using isoschizomer construction technique. The recombinant plasmid pcDNA-1～3C3d was constructed by1～3C3d gene inserting into pcDNA3.1vector. The recombinant plasmid pcDNA-VP2-C3d, pcDNA-VP2-2C3d, pcDNA-VP2-3C3d was achieved by VP2gene inserting into the upstream of the C3d sequence in the plasmid of pcDNA-C3d, pcDNA-2C3d, pcDNA-3C3d, then transfected into BHK21cells. The recombinant VP2-C3d, VP2-2C3d, VP2-3C3d proteins were detectable by Western blot test and indirect immunofluorescence assay (IFA). Western blot showed that the bands with expected size were observed including a84ku C3d-VP2band, a124ku2C3d-VP2band, a162ku3C3d-VP2band. BHK21cells transiently transfected with the plasmids were confirmed and stained positive against IBDV antibody using the indirect immunofluorescence assay (IFA). Our research laid foundation for the development of a novel vaccine against the challenge of IBDV by using chicken C3d as a molecular adjuvant to enhance the immunogenicity of VP2gene.ExpII:The effects of C3d on the immunologic function of IBDV VP2expressed by DNA vaccineThis study evaluated the effects of C3d on the immunologic function of IBDV VP2expressed by DNA vaccine. Six groups of2week SPF chicken were respectively injected with100ug of plasmid pcDNA-VP2, pcDNA-VP2-C3d, pcDNA-VP2-2C3d, pcDNA-VP2-3C3d and empty vector pcDNA3.1and boosted with same amount of plasmids at week4.4week after primary immunization, mice were challenged by the intramuscularly route with100TCID50of BC6/85stain. Survivals were assessed for2weeks after the virus challenge. Sera were collected at1,2,3,4week after primary inoculation to detect VP2-specific ELISA antibodies and neutralizing antibodies against IBDV. At week4the anti-VP2antibody titers for chicken vaccinated with pcDNA-VP2, pcDNA-C3d-VP2, pcDNA-VP2-2C3d, pcDNA-VP2-3C3d plasmids were respectively1:1440,1:2532,1:3254and1:4652, pcDNA-VP2-3C3d plasmids produced anti-VP2antibody titers significantly higher anti-VP2antibody titers compared to chicken vaccinated with only VP2expressing plasmid (p<0.05). At week4, the neutralizing titers for chicken vaccinated with pcDNA-VP2, pcDNA-C3d-VP2, pcDNA-VP2-2C3d, pcDNA-VP2-3C3d plasmids were respectively1:650,1:900,1:1690and1:2408, while the empty vector inoculation did not yield any neutralizing antibody. The neutralizing titer of pcDNA-VP2-3C3d plasmid is the highest of chicken vaccinated with various plasmids. The relative affinity of antibodies was measured by ELIS A using thiocyanate elution. The relative affinity of serum of chicken vaccinated with pcDNA-VP2-3C3d plasmid was highest of chicken vaccinated with various plasmids. Lymphocyte proliferative responses for five chickens each group were analyzed at week4after the primery immunization. The stimulation index of chicken injected with pcDNA-VP2-3C3d expressing plasmid was highest among DNA immunized groups. This study demonstrated that chicken C3d gene fused to VP2gene significantly enhanced the humoral immunity and Cellular immunity induced by DNA vaccine encoding VP2fusion complement C3d. pcDNA-VP2-3C3d plasmid can induce the best protection against IBDV challenge by VP2expressed by DNA vaccination.ExpⅢ:The immunologic function of polyethylenimine-mediated pcDNA-VP2-3C3dPolyethylenimines (PEIs) are synthetic, charged polymers which function as transfection reagents based on their ability to compact DNA into complexes. Polyethylenimine is a polysaccharide and has been demonstrated as a potential gene encapsulation and delivery system. It can condense DNA to form PEI/DNA complexes, which can be transfected into mammalian cells for gene expression. PEI complexe of nucleic acids has been extended towards the in vivo delivery of DNA. This study evaluated the immunologic function of PEI-mediated pcDNA-VP2-3C3d. Experimental groups i.e VP2-3C3d-4, VP2-3C3d-6, VP2-3C3d-8, VP2-3C3d-10were designed and the groups of2week SPF chicken were respectively injected with complexe of100μg of pcDNA-VP2-3C3d and branched PEI at N/P=4,6,8,10in a final volume of400μl and boosted with same amount of complexes at week4. Four weeks after primary immunization, chicken were challenged by the intramuscularly route with100TCID50of BC6/85stain. The pcDNA-VP2-3C3d and control groups were also designed. Sera were collected at1,2,3,4week after primary inoculation to detect VP2-specific ELISA antibodies and neutralizing antibodies against IBDV. Analysis of antibody titers and the neutralizing titers indicated that immunization with PEI/pcDNA-VP2-3C3d at N/P=6or8had significantly higher titers of anti-VP2antibodies compared to serum from chicken vaccinated with pcDNA-VP2-3C3d; PEI/pcDNA-VP2-3C3d at N/P=4induced the same titers of anti-VP2antibodies than pcDNA-VP2-3C3d; PEI/pcDNA-VP2-3C3d at N/P=10induced lower titers of anti-VP2antibodies than pcDNA-VP2-3C3d (p<0.5). Survival rate of3C3d-VP2-4,3C3d-VP2-6,3C3d-VP2-8,3C3d-VP2-10, pcDNA-VP2-3C3d were90.0%,93.33%,70.0%,67.7%respectively.3C3d-VP2-6,3C3d-VP2-8groups had significantly higher Survival rate than pcDNA-VP2-3C3d group(p<0.5). The result demonstrated that PEI enhanced antibody responses to protective immunity against IBDV. PEI/pcDNA-VP2-3C3d may represent an effective candidate vaccine against IBDV.