[Construction of a bicistronic eukaryotic vector expressing M1 and NP genes of influenza virus]

In this study, two conserved genes [M1 and NP] of influenza virus were expressed in a bicistronic vector in order to develop a universal gene based vaccine. Plasmids M1-pIRES2-EGFP, pIRES2-NP were constructed by cloning the PCR products of M1 and NP genes which were amplified from the A/Peurto Rico/8/34 [H1N1] influenza virus strain into the plasmid expression vector pIRES2-EGFP, respectively. For construction of M1-pIRES2-NP bicistronic plasmid, M1 gene was extracted from M1-pIRES-EGFP plasmid and subcloned into pIRES2-NP construct. Finally, simultaneous expression of both genes was assessed by transient transfection of bicistronic plasmid into BHK-21 cell lines and subsequent immunofluorescence staining. The results of enzymatic double digestions on the constructed plasmids and sequencing demonstrated the success of cloning processes of above mentioned genes. Correct expression of these genes was confirmed by M1-pIRES2-NP plasmid expression in BHK-21 cell lines confirmed by immunofluoresence microscopy. Simultaneous expression of influenza M1 and NP genes from a bicistronic plasmid containing "IRES" sequence is achievable

Effect of LIGHT adjuvant on kinetics of T-cell responses induced by HSV-1 DNA Immunization

Studies on efficacy of various vaccines that prevent or reduce the primary and recurrent HSV-1 infection have demonstrated the importance of cellular immunity for protection against the infection. We previously used DNA vaccination to induce cellular immunity against HSV-1 infection in mice. The aim of our study was to evaluate the effect of LIGHT; a member of TNF super family, on the kinetic of CTL response induced by HSV-1 glycoprotein B based DNA vaccine. Using a granzyme B ELISA for detection and analysis of CD8+ T cells, CTL activity was determined in the spleen of BALB/c mice at various time points after primary and booster dose of vaccination. The kinetics of CTL response to primary and secondary HSV-1 infection and DNA vaccination were compared to those induced by DNA vaccination in combination with LIGHT adjuvant in the present study. In primary and secondary immunization, the CTL activity in the HSV injected group peaked 7 days and 12 hours post immunization, respectively. After 5 days, LIGHT could neither accelerate the CTL response compared to DNA vaccination alone nor could enhance the CTL activity in the primary and the first peak of memory response, the amount of granzyme B induced by the LIGHT containing vaccine was significantly higher than that induced by the vaccine without the adjuvant. Although LIGHT enhances the cellular response in the booster dose of vaccination, it does not accelerate the CTL response