Study On Immunogenicity Of The Polyvalent Recombinant Plasmids DNA Containing PrM-E Genes Of Dengue Viruses

Dengue viruses are an important member of the family Flaviviridae and exist as four antigenically distinct serotypes. These viruses cause human infections, which are manifested clinically by either mild DF, or more severe DHF/DSS. Recently with the increase of temperature and international communication, the incidence of DF and DHF is rising rapidly. DF/DHF has become the most severe public health problems in many tropical and subtropical areas.There are still no safe dengue vaccines currently available in clinic. Although traditional attenuated live vaccines have entered clinical trials for many years and the vaccines are considered as the most promising one, so much clinical data shows that the tetravalent vaccines can't simultaneously afford protective immune responses against four serotypes. Thus it is very important to develop new dengue vaccines.DNA vaccine is a new vaccine and appears in the 1990's. Compared with traditional vaccines, the vaccine can induce humoral and cellular immune responses, and have preponderances in simple preparation, no virulence reversion, combined immunization. Currently it has been used to construct vaccine candidates of JEV, HIV, influenza, hepatitis B, HSV and et al. Mice and monkeys, immunized with plasmids DNA containing of prM, E or NS1 genes of dengue viruses, produce immune responses and protect animals from viral challenge. Bicistronic expression vectors, containing internal ribosomal entry sites(IRES), can simultaneously express two different antigens. Experiments show that mice, immunized with the bicistronic expression plasmid containing HbsAg and HbcAg, can produce specific immune responses against the two antigens. It lays a foundation for develop bivalent and polyvalent DNA vaccines against dengue viruses.In view of above considerations, this study constructed bivalent and tetravalent recombinant plasmids containing prM-E genes of dengue viruses using bicistronic expression vector and observed their immunogenicity in mice.Firstly, we constructed the bicistronic recombinant plasmid DNA containing prM-E genes of dengue virus type 1 and 3. Viral RNAs were extracted from brains of suckling mice infected by dengue virus type 1 and 3 with Trizol reagent. And prM-E genes of dengue virus type 1 and 3 were amplified from extracted RNA by RT-PCR, thensequentially inserted into upstream and downstream of IRES of expression vector pIRES. To manifest that the constructed bicistronic recombinant plasmid pMEl-IRES-ME3 could express prM-E proteins of dengue viruses, pMEl-IRES-ME3 was electroporated into BHK21 cells. Results showed that specific nucleic acid fragments and proteins of dengue virus type 1 and 3 were detected in transfected cells at 24 hours post-transfection. The other bicistronic recombinant plasmid pME2-IRES-ME4, containing prM-E genes of dengue virus type 2 and 4, was constructed in the same manner. Successful construction of two bicistronic recombinant plasmids containing dengue virus type 1~4 laid a foundation for observing their immunogenicity in mice.To improve the immunogenicity of plasmids DNA, the immunization was carried out in combination with pUMCVl-mGM-CSF, containing GM-CSF gene derived from mouse, pMEl-IRES-ME3 and pME2-IRES-ME4. Mice were immunized intramuscularly, and electroporation was immediately performed at the same sites post-immunization to increase DNA uptake. The results suggested that specific antibodies against corresponding dengue viruses could be detected in mice immunized with recombinant plasmids at weeks 4 post-priming. And antibody titers were rising until weeks 14 post-priming. Neutralizing antibodies of serum from individual mouse at weeks 14 post-priming was measured. The results showed that neutralizing antibody titer of serum from mice immunized with recombinant plasmid was up to 1:32, and those immunized with pIRES were less than 1:8. To observe cellular immune responses of immunized mice, spleen cells were isolated from mice on days 47 and 62 post-priming. The splenocytes were stimulated with inactivated dengue viruse...