| West Nile virus (WNV) within the Flavivirus genus of the family Flaviviridae, cause human severe diseases ranging from West Nile fever to West Nile encephalitis or meningitis. Since its introduction into the United States in 1999, West Nile virus has spread across North America and more than 3000 cases and 300 deaths reported every year. Over the last few years, many geographical areas have affected by this virus, posing an serious threat to public health all over the world. But until now, there is no one effective vaccine available for human use. DNA vaccine is an important vaccine modality for developing West Nile virus vaccine. But by far DNA vaccines have not been proved sufficiently immunogenic and lack of appropriate vector. Replicon may be one of important strategies to address these concerns of DNA vaccine.Replicon is known as a self-replicating subgenome stemed from corresponding virus. It is a potential ideal tool for exploring the viral replication and developing novel vaccines. The system lack the major part of the genes encoding the structural proteins, consequently can replicate autonomously like virus but are not packaged into viral particles in cells. So the safe subgenome Replicon can be delivered directly as RNA-based modality or DNA-based modality. Recently, most of WNV replicons were reported as RNA-based, whereas these replicons constructed based on SP6 or T7 promoter have the disadvantage in manipulating and keeping stability, DNA-modality replicon based on CMV promoter can be delivered into cells directly without in vitro transcription. Moreover, the immune reponse to WNV replicon is poorly defined.In this study, a series of DNA-based WNV replicons were constructed using the technology of reverse genetics, based on the full-length cDNA clone of WNV Chin-01 strain. Then the replication of replicons were identified by expressing reporter genes. Furthermore, constructed WNV replicons were used to immunize mice, and their immunogenicity were evaluated from cellular and humoral immune responses. This work may serve as a useful platform for the development of WNV vaccine.1. Construction and identification of DNA-based WNV repliconsThe vector of plasmid pWNIIrep-REN-IB having CMV promoter was chosen to construct DNA-based WNV replicon. The 3′-halves of WNV genome was ligated into the vector;Next, the bilateral sequence of′3-halves were modified respectively using an overlap extension PCR. In order to prepare an functional replicon, the non-coding regions, the sequence encoding the first 99 aa of C and the last 26 aa of E, and non-structural sequence were all retained. Meanwhile, the ribozyme sequence of Hepatitis D virus (HDVr) and the polyadenylic acid sequence of Simian virus 40 (SV40) were introduced downstream′o f 3-halves. Eventually, the WNV replicon pWNrep was obtained and confirmed correctly by DNA enzyme digest and sequencing. Based on this, the bicistrons strategy was adopted to construct two WNV replicons (pWNrep/eGFP and pWNrep/Rluc) possessing different reporter genes. The reporter genes (eGFP and Rluc) were inserted into the deletion region of structural genes, respectively. In addition, the replication-deficient replicon pWNrep/Rluc△NS5 was constructed to serve as a control.Furthermore, the constructed replicons were transfected into BHK21 cells for evaluating the replication of replicons. Specific GFP could be expressed in pWNrep/eGFP transfected cells, and be observed until 60h post-transfection. The cells transfected with pWNrep/Rluc and pWNrep/Rluc△NS5 were lysed and the Rluc activity was assayed. At 24h post-transfection, the first peak appeared, and the activity increased continually from 36h to 60h. But that in pWNrep/Rluc△NS5 transfected cells decreased quickly. The replicon RNA in transfected cells were further detected by RT-PCR, indicated specific 3'UTR fragment only founded in pWNrep/Rluc transfected cells. All results demonstrated that the DNA-based WNV replicons could express exogenous genes and replicate itself well. This may set a foundation for following observation of replicon immunogenicity.2. Immunogenicity of DNA-based WNV repliconReplicon plasmid pWNrep were used to immunize female BAL B/c mice (4 weeks old) for three times, intramuscularly, at a 2 week interval. The empty vector pVec and PBS (100μl) were immunized as control. Sera IgG titers were measured with IFA, and the result was that the sera antibody from pWNrep-immunized group with titers 1:160, could specificly recognize NS1 protein of WNV, while that from control group could not be detected. The anti-WNV neutralizing antibody was also evaluated by PRNT. The sera neutralizing titers in pWNrep group exhibit an increasing process depended on immunize times. The titers were 1:19 at the first week post primary immunization and rise to 1:47 at the fifth week. And the titers (1:55) were significally higher than that of sera in control group (1:11) at 13 weeks post primary immunization. This result told us that that immunization with WNV replicon can induce humoral immune response in mice and elicit neutralizing antibody against WNV infection. The cellular response was also evaluated with splenocyte proliferation and IFN-γassay. After stimulated with NS1 antigen, splenocytes from pWNrep group proliferated significantly and SI(8.9±0.7) is almost three times as high as that of pVec grous and four times as that of PBS group(p<0.01). IFN-γconcentration from the pWNrep group was (99.0±18.8)pg/ml, also higher than the control group (p<0.05), The result indicated the DNA replicon could activate T cells memory response and the signal pathway for releasing cytokines, induce high cellular response in mice.In this study, DNA-based WNV replicons were constructed successfully. These replicons not only replicated well, but also could induce robust humoral and cellular response in mice. Our results offered the theoretical foundation for developing WNV DNA vaccine and provided reference methods for replicons research on other virus. |
PDF Full Text Request