Study On DNA Vaccine Against Hantavirus

Hantavirus is the causative agent of the most severe form of a rodent -borne disease known as hemorrhagic fever with renal syndrome ( HFRS). The widespread distribution of hantaviruses the large number of HFRS cases reported each year and no specific drugs in curing the disease, clearly indicate the need for effective vaccines. Several approaches have been used to develop candidate hantavirus vaccines, including cell - culture or rodent - brain - derived inactivated virus vaccine or protein subunit vaccines expressed in insect cells as well as most recently naked DNA - based vaccines. Nucleic acid immunization is an important vaccination strategy that has many characters desirable for an i-deal vaccine, such as induction of broad immune responses (humoral and cellular) , long - lasting immunity, simple and cheap production. This technique regarded as one of the most promising approaches for future vaccine development is being explored as a vaccination strategy against a variety of infectious diseases, therefore, the study on DNA vaccine against hantavirus has tremendous practical importance. It is well established that nucleiocapsid protein (NP) is highly conserved and antigenically cross - reactive between HTNV and SEOV. The cell - mediated immunity induced by NP is believed to be important for clearing a hantavirus infection, thus NP has become one of the most important candidate immunogens. For these reasons, NP was chosen to design a DNA vaccine against hantavirus. Previous reports have shown that intramuscularly injected DNA vaccine in human has failed to generate vigorous immune responses, which has been a bottleneck of DNA vaccine application to human. Now, the research on DNA vaccines has moved to its second phase with the emphasis on improving immunogenicity and efficacy. A new form of vaccination, co - administration DNA constructs that contains the gene of the candidate antigen and cyto-kine gene, is under intensive investigation. This technique has the potential todrive immune responses in a particular direction through the co - delivery of genes for immunologically important molecules such as cytokines and co - stimulatory molecules. However, there has been no study on the modulatory effect of using the murine IL -12 gene or DNA containing an unmethylated CpG motifs or B7 - 1 costimulation molecules as a molecular adjuvant to enhance to potency of DNA vaccine in an HTNV infection. Based on these previous evidence, in the present study we examined the effect of cytokine gene adjuvant pcIL - 12, CpG motifs as well as B7 - 1 to modulate the level of immune response generated by a DNA vaccine based on the S gene encoding NP against hantavirus infection in mice.MethodsWe construct the eukaryotic expression vector pTARGET - hanS by cloning S gene segment obtained by PCR into eukaryotic expression vector pTARGET . After identified by enzyme analysis and sequencing, the recombinant expression vector pTARGET - hanS was transfected into Vero - E6 cells by electroporation and the transient expression of Hantaan virus nucleocapsid protein was detected by indirect immunofluorescence assay (IFA). As follows, the 1. 3kb HTNV S gene fragment removed from the pTARGET - hanS ( digested with EcoR I / Sal I ) was ligated into linearized pcDNA3. 1 + ( digested with EcoR I /Xho I ) and pCA14 ( digested with EcoR I / Sal I ) respectively to generate the pcD-NA3.1 +S and pCA14 -S constructs. After verified by enzyme analysis, Bgl II was used to removed S gene fragment containing CMV from pCA14 - S to pcD-NA3. 1 + B7 to construct co - expression vector pcDNA3.1 + S + B7, which was verified by enzyme analysis. pcDNA3. 1 + S(ISS) was constructed as same as pcDNA3.1 +S constructed strategy, except that the S segment containing CpG motif was obtained by PCR using specific primers. All plasmid DNA was purified from transformed Escherichia Coli TGI, adjusted to a final concentration of 1mg/ml in the sterile PBS and stored at -20C until used. DNA concentration was measured by absorbance at 260nm. The OD260/OD280 ra...