Development And Evaluation Of Two Clades Of H5 Subtype Avian Influenza DNA Vaccines

H5N1 HPAIV can not only infect poutry and wild birds, it can also infect mammals like cat,tiger, stone marten, mink and humans.Since the first report of human infected H5N1 HPAIV in Hongkong,which cause 6 of 18 infected death. So far, of the 495 human H5N1cases recorded by the WHO by 21 April 2010, over 58.99% proved fatal. H5N1 viruses are widely considered to be the next influenza pandemic.Influenza vaccines are considered to form the main prophylactic measure against pandemic influenza.Compared to other vaccines, DNA vaccine shows its advantages.It can induce both humoral and cell-mediated immune responses, similar to an attennuated live virus vaccine,with the safety of a killed or subunit vaccine.It is easier to manufacture, store and transport.It can induces long-lasting protective immunity;It can also meet the need of large ammount in a short time and do not need to worry about the limitation of embyros; And it is easier to use and can boost many times and so on. It has reported that there are four types of DNA vaccine have been licensed for commercial use. It means the DNA vaccine arenas are slowly changing from research to the field use.In this study, we choose two H5N1 virus AH/06 (clade 2.3.4) and SX/06 (clade 7) as the donor of HAs inserted in plasmids pCAGGS and pVAX1. We succefssfully construct three DNA vaccines, named as pCAGGoAHHA, pVAXoAHHA and pCAGGSXHA.Then they are confirmed by immunoflurescence analysis of H5 AIV HA protein expression.To evaluate the immunogenicity and vaccine efficacy of pCAGGoAHHA in SPF chicken.Groups of chickens were immunlized with 100μg pCAGGoAHHA innoculated by intramuscular and electroportation, the result shows that it can induce high antibody tiers, and can completely protect from challenge with a lethal dose of AH/06.No virus shedding, clinical signs, or deaths were observed. The groups delivered by electroporation can be detected with antibody earlier and higher. Groups by electroporation can form completely protection as low as 10μg pCAGGoAHHA,while the minimize dose is 20μg without electroporation.During the 7 months'investigation, the HI antibody induced by 15μg pCAGGoAHHA with electroporation can keeps above 4Log2. It can reach 6.3 Log2 at the peak.After the challenge of heterologous viruses (BHG/05 andVN/06 ),the survival rate of the chicken immunized with 15μg pCAGGoAHHA delivered by electroporation is 6/9 and 6/10 respectively.To evaluate the efficacy of pCAGGSXHA, three-weeks-old chicken were inoculated with 10μg or 100μg pCAGGSXHA by electroporation.The second vaccination was given three weeks later,then challenged by SX/06 two weeks later.The result showed that both of the immunized groups can provide compelete protection. Two doses of 10μg pCAGGSXHA can provide complete protection two weeks after the second dose.During the 16 weeks invetigation, the HI antibody induced by 10μg pCAGGSXHA could reach 7Log2 5 weeks post the first vaccination and then gradually declined to 4 Log2.in 4 monthsWe also evaluated the immunogenicity and vaccine efficacy of pVAXoAHHA in mouse model as the candidate vaccine for the future influenza pandemic.Groups of BALB/c mice were immunized with 10μg or 100μg pVAXoAHHA and were boosted 4 weeks later.The results show that it can induce mice producing NT antibodies and can protect mice from death against the lethal challenge of AH/06 with one dose or two doses of vaccine. The HI and NT antibody increased sharply after the boost.It also proves that mice with two doses of 100μg pVAXoAHHA can afford reliable protection against lethal heterogenous virus BHG/05 and VN/06 challenge.Our results show that pCAGGoAHHA and pCAGGSXHA are safe and highly efficacious in chicken.pVAXoAHHA are effective in mouse modle challenged with either homologous or heterologous H5N1 virus, which provide a compelling rationale for further testing of H5N1 DNA vaccine in other mammal trials.It indicates that pCAGGoAHHA and pCAGGSXHA can be two candidate vaccines to control the AIV in poultry industry and that pVAXoAHHA might be a candidate vaccine for humman use.