RESUMO
The impending influenza virus pandemic requires global vaccination to prevent large-scale mortality and morbidity, but traditional influenza virus vaccine production is too slow for rapid responses. In this study, bacterial system has been developed for expression and purification of properly folded HA1 antigen as a rapid response to emerging pandemic strains. Here, a recombinant H5N1 [A/Indonesia/05/05] hemagglutinin globular domain, the synthesized HA1 [1-320 amino acids], was amplified and cloned into pET-28a bacterial expression vector. Then, his-tagged HA1 protein was expressed in Escherichia coli BL21 under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA chromatography. Migration size of protein was detected at 40 KDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight. Since most antigenic sites are in the HA1 domain of HA, using this domain of influenza virus as antigen is of great importance in vaccine development. The ability of the antibody stimulation against HA1 expressed in bacterial cells is also examined using enzyme-linked immunosorbent assay [ELISA] analysis. Upon immunization of rabbits, oligomeric HA1 elicited potent neutralizing antibodies and high levels of serum antibody binding to HA1. Our findings suggest that HA1-based vaccines can be produced efficiently in bacterial systems and can be easily upscaled in response to a pandemic influenza virus threat
RESUMO
Continuous antigenic variation of Influenza a viruses causes a major concern to develop Influenza vaccine. Conserved antigens are suitable candidates for vaccine production due to its non-requirement to match the designed strains with circulating strains. The M2 gene is conserved among Influenza a viruses and has potential to be considered as a universal vaccine. This study was designed to evaluate the effects of aqueous Echinacea purpurea extract on immunogenicity of DNA vaccine encoding M2 gene of Influenza virus. This interventional study was carried out on female BALB/c mice with 3-4 week age [250-300 gr]. Plasmid DNA encoding M2 gene [pcDNA-M2] of Influenza virus A/New Caledonia/20/99 [H1N1] was transformed into E.coli top10 f' and cultured in LB broth media. Large scale plasmid preparation was done and the concentration was measured by spectrophotometric method. Mice were divided into eight groups and immunized three times with fifteen days apart. Vaccine groups received inactivated Influenza virus or pcDNA-M2, alone or in combination with Echinacea extract. Control groups were injected pcDNA, Echinacea extract, and phosphate buffer. All animals were left to bleed before immunization and at 21 days after the last vaccination and specific anti-M2 antibodies were measured by indirect ELISA. Then the mice were intranasally challenged under an aesthesia with mouse-adapted PR8 Influenza virus and monitored for 3 weeks to evaluate the vaccine regimen efficacy in reduction of mortality rate compared to control groups. Data were analyzed using SPSS-16, One-way ANOVA and Kaplan-Meier tests. The highest specific immune response was obtained in mice received inactivated virus plus extract [P<0.05]. Immune responses in mice inoculated with pcDNA-M2 were significantly higher compared to all control groups mice [P<0.05]. In addition the specific immune responses in group inoculated with pcDNA-M2 and aqueous extract was higher compared to the group receiving only pcDNA-M2 [P<0.001]. The highest survival rate was observed in mice injected with inactivated virus or pcDNA-M2 plus extract. This study showed that pcDNA-M2 induced specific immunity and protected mice against lethal challenge with PR8 Influenza virus. Furthermore, application of Echinacea extract with M2 gene vaccine increased vaccine efficacy