Immunological Effect Of The M And S Gene DNA Vaccines Against Xinjiang Hemorrhagic Fever Virus And Its Glycoprotein Primary Antigenic Analysis

Crimean Congo hemorrhagic fever (CCHF) is a kind of tick-borne hemorrhagicfever infectious disease caused by CCHF virus, the case fatality rate is up to50%.Currently, Crimean-Congo hemorrhagic fever has become a major infectious hazardto human health distributing in more than30countries in the world. Scientists beganto conduct etiological research of CCHF (by the name of Xinjiang hemorrhagic fever,XHF) since1965in China, and several strains of XHFV have been isolated, however,there have not been effectively diagnostic methods and treatments of XHF. Thenucleoprotein (NP) encoded by S gene is a major antigen that induces the immunereaction of organism. Meanwhile, the glycoprotein (GP) encoded by M gene plays animportant role in process of XHFV infection and pathogenicity. Therefore,investigating the antigenicities and immunogenicity of XHFV NP and GP (Gn，Gc)would greatly promote the development of XHF diagnostic techniques and vaccinesin our country. The study successfully constructed recombinant DNA vaccines of Sand M genes from XHFV and evaluated their immune effects. Furthermore, thecorresponding16-mer peptide-coding genes of glycoprotein Gc fragment from XHFVYL004057strain, were constructed into prokaryotic expression vector pXXGST-1and conducted inducible expressionion by using biosynthetic peptide approach. Thepotential epitopes on Gc fragment were determined by Western blotting assay withpreparation of rabbit polyclonal antisera against XHFV glycoprotein. The studymainly obtained three following results.1. In order to screen the dominant linear epitopes on nucleoprotein (NP) of XHFV,we designed and obtained six truncated proteins NP (aa1-482), NP2(aa170-305),NP2-c (aa235-305), NP2-c-1(aa237-256), NP2-c-2(aa250-265), NP2-c-3(aa260-276)) by prokaryotic expression and purification. The antigenicspecificities of recombinant proteins were detected in indirect ELISA assay byusing sheep sera whose anti-CCHFV antibody had been determined by indirect immunofluorescence assay (IFA). According to the results, the protein fragmentswith good antigen specificity, high sensitivity and low false-positive rate weredetermined as compared to IFA results. As a result, the sensitivity, specificity andtotal coincidence rate of NP2protein are respectively73.3%,100%, and87.9%.Similarly, the sensitivity, specificity and total coincidence rate of NP2-c proteinare66.7%,94.4%, and81.8%, respectively. These findings indicated that theantigenicities of NP2and NP2-c were superior to others, suggesting the twoproteins can be used as candidate antigens for research and development ofXinjiang hemorrhagic fever diagnostic reagents.2. The genes of full length nucleoprotein NP (aa1-482) and NP2(aa170-305) ofYL04057strain, and glycoprotein (denominated as Gn, Gc) were solely or bychimeric constructed into eukaryotic expression vector PVAX1, respectively. Fiverecombinant plasmids pVAX1-NP, pVAX1-NP2, pVAX1-Gn, pVAX1-Gc,pVAX1-Gc-NP2were obtained. These plasmids were used to immunize Balb/cmice, and the immune responses were assessed by using combination methods ofT cell proliferation assay, cytokine content determination and ELISA; the pVAX1was used as control. The average antiserum titer of mice in pVAX-1/NP2groupwas about1:6400. The mice in pVAX1/NP2and pVAX1/Gc-NP2groups showedan obvious proliferation of spleen T lymphocyte compared with that of controlgroup (P<0.01), meanwhile, the two groups also exerted high level expression forIFN-γ reaching865.150and1727.205pg/mL, respectively, which were of highlysignificant difference from the control group (P<0.01). The IL-4expressionquantities of mice in pVAX1/NP2and pVAX1/Gn groups were19.11and20.07pg/mL respectively, which were both apparently higher than that in control group(9.35pg/mL). The pVAX1/NP2and pVAX1/Gc-NP2groups similarly showed thebest immune effect, which could be utilized as candidate vaccines against XHFV. 3. According to comprehensively analyzing a number of bioinformatics data ofamino acid sequence of Gc fragment of XHFV strain79121, including α-helix,β-sheet, turn, hydrophobicity, flexibility and linear B-cell epitopes, we designedten16-mer peptides which contain a number of prediction epitopes. Aftersynthesis, the encoding genes of16-mer peptides were constructed intoprokaryotic expression vector pXXGST-1. To prepare rabbit polyclonal antisera,the truncated Gc1and Gc2segment of the C-terminus of glycoprotein of XHFVstrain YL04057, were cloned into prokaryotic expression vector pET-32a togenerate a recombinant plasmid pET-32a-Gc1and pET-32a-Gc2. The plasmidswere transformed into E. coli BL21(DE3)．The fusion protein Gc1and Gc2wereinduced by IPTG，purified by Ni-NTA purification system，and analyzed bySDS-PAGE． To prepare the antiserum, New Zealand white rabbits wereimmunized with purified Gc1and Gc2protein. The antigenic features of16-merpeptides were identified by using Western blot with the rabbit polyclonal antibodyagainst Gc1and Gc2. Our results showed that6peptides of1016-mer peptidescan specifically bind with the antisera. These findings indicated that we haveacquired6peptides (Gc116-131, Gc228-243, Gc362-377, Gc370-385, Gc505-520,Gc513-528) with potential antigenic epitopes. It is the first time that we haveidentificated epitope motifs of the6peptides of XHFV glycoprotein, which theiramino acid sequences are representative and extremely conservative (reach morethan81.25%) as compared to those of other XHFV strains. These findingselucidated very important information for development of the XHFV detection kitand multi-epitope peptide vaccines.