Induction Of Anti-viral Protective Antibody Through DNA Vaccination

Part I Optimal Immunogen Design of DNA Vaccine Against H7Avian InfluenzaObjective:To improve the immunogenicity of H7subtype avian influenza vaccine by optimal DNA vaccine designMethods:Our study designed three forms of DNA vaccines encoding hemagglutinin from a lethal avian influenza strain A/Netherlands/219/2003(H7N7) which emerged in Netherlands in2003. One form expressed full-length HA under wild type leader sequence, the second one replaced the wild type leader sequence with tPA in full-length HA. the third one expressed truncated HA without transmembrane domain and cytoplasmic tail under tPA leader. Immunogenicity was examined in rabbits. Binding antibodies were detected by ELISA, and functional antibodies against autologous and heterologous H7influenza virus were examined using hemaglutination inhibition assay and pseudotyped virus based neutralization assay. Three2013H7N9infected patients’sera were collected and the neutralizing activity against heterologous H7influenza virus used as vaccine strain in this study was examinedResults:Three DNA vaccines induced high-level HA specific binding antibodies, DNA vaccine under tPA leader sequence is most immunogenic to induce functional antibodies against autologous and heterologous H7influenza virus, followed by DNA vaccine with wild type leader, truncated HA is least immunogenic. Meanwhile, Sera from three H7N9infected patients were cross-neutralizing against H7influenza vaccine strainConclusions:DNA vaccine under tPA leader sequence is most immunogenic to induce functional antibodies against H7subtype influenza virus, and cross-reacvitity of hemagglutinin between2003H7N7influenza virus and2013H7N9influenza virus can be detected. Part Ⅱ Induction of Broad Env Specific Antibodies against Key HIV-1Subtypes in China by DNA Prime-Protein Boost VaccinationObjective:to optimize the immunogen formula and vaccination regimen to induce broad antibody responses targeting key HIV-1virus subtypes in China.Methods:Three codon-optimized DNA vaccines coding for three consensus gp120antigens from subtpes ThB, BC, and AE were constructed based on bioinformatics analysis previously. They were used to produce three gp120-expressing DNA vaccines and three gp120proteins purified from transiently transfected293F cells. New Zealand White rabbits were immunized with three or four doses of DNA vaccines followed by two doses of protein vaccines by one of the following regimens: monovalent DNA prime followed by either monovalent or trivalent protein boost; trivalent DNA prime-trivalent protein boost with three priming DNA vaccines delivered at the same time or sequentially. Binding antibody targeting gp120and V1V2region were measured using ELISA, antibody reponse specificities to linear B cell epitope were profiled through JPT peptide microarray, and Nabs against17pseudotyped viruses from different subtypes were detected using pseudotyped virus system.Results:a. High-level gp120-specific serum IgG responses were elicited by all vaccination regimens; however, the specificity of sera elicited by the monovalent regimen was higher against autologous gp120than the other two gp120antigens. DNA prime-protein boost induced higher antibody level than protein alone. Polyvalent vaccination was superior to induce binding antibody than monovalent vaccination. Simultaneous polyvalent vaccination was superior to induce binding antibody than sequential vaccination. b. DNA prime-protein boost vaccination was more effective than the protein alone in improving the breadth of Nab. Polyvalent vaccination can induced broader Nab than monovalent vaccination.. Simultaneous delivery was more effective than sequential immunization with the same trivalent formulation in eliciting broader NAb.c. All vaccination regimens induced V1V2-specific antibody.d. Peptide Microarray showed all regimens induced antibody mainly targeting linear B cell eptiope in C1, V2, C2, V3, C4and C5regions of gp120Conclusions:DNA prime-protein boost strategy was superior to induce antibody response than protein alone, and polyvalent vaccination was more effective than monovalent vaccine in eliciting broader NAbs, and it is feasible to use the poryvalent Simultaneous delivery principle to develop regional polyvalent HIV vaccines in China. Part Ⅲ Enhanced Immunogenicity of Hepatitis B Subunit Vaccine by Combined w ith DNA Vaccine Encoding Hepatitis B Surface Antigen Middle ProteinObjective:To improve the immunogenicity of Hepatitis B subunit vaccine used in the rational vaccination program.Methods:Hepatitis B subunit vaccine was used to immunize BALB/c mice alone or combined with DNA vaccine encoding Hepatitis B surface antigen middle protein intramuscularly at week0,4,8. Combined vaccination which employed a fixed dose of DNA vaccine plus with subunit vaccine at a high dose or a low dose.was delivered simultaneously at the same site or delivered separately at non-overlapped sites. HBsAg-specific IgQ IgG2a, IgG1and the ratio of HBsAg-specific IgG2a/IgG1was measured by ELIS A.Results:DNA vaccine combined with low dose of Hepatitis B subunit vaccine induced significantly higher level of HBsAg-specific IgG and IgG2a than Hepatitis B subunit vaccine alone. There was no signficantly difference on the antibody levels induced by two combined vaccination regimens. Both combined vaccination and subunit vaccine alone induced Th2-dominated immune response. However combined vaccination was more effective to elicit Thl immune response compared with HB subunit vaccine alone.Conclusions:DNA vaccine combined with Hepatitis B subunit vaccine at an appropriate ratio is effective to enhance the immunogenicity of Hepatitis B subunit vaccine.