Construction Of Two Influenza Universal Vaccine And Evaluation On Immune Efficiency

Swine influenza has been a commonly recognized disease of swine for more over 90 years.Despite considerable interest and research efforts over past 50 years,swine influenza continues to be an important economic issue in swine production and public healthy.In last century four pandemics were recorded: Spanish Flu(1918),Asia Flu(1957),Hongkong Flu(1968)and 2009 pandemic Flu(2009).1957 Flu,1968 Flu and 2009 Flu were involved with swine influenza.Since 1996 highly pathogenic avian influenza(HPAI)H5N1 virus has been found in the domestic geese in Guangdong,China,HPAI H5N1 virus has spread rapidly throughout China.In recent years the epidemic surveillance in China,Vietnam and Egypt showed that HPAI H5 viruses have adapted in pigs.HPAI H5N1 virus may acquire the human-to-human transmissibility though the adaptation in pigs and this will cause a big disaster because there is over 60% mortality among the human infection.Over ten years,hemagglutinin(HA)of HPAI H5N1 viruses has evolved into 10 clades in various host species.Among them,clade 2 is divided into 5 subclades 2.1,2.2,2.3,2.4,and 2.5,and clade 7 is divided into 2 subclades 7.1 and 7.2.High level of genetic and antigenic diversity has caused huge difficulty for the prevention of HPAI H5N1 influenza diseases.In the present study we selected an A/Thailand/1(KAN)-1/2004 H5N1 strain,whose HA sequence is the closest to the consensus sequence among diverse WHO recommended H5 vaccine strains analyzed,as immunogen.We primed BALB/c mice twice with DNA plasmid encoding H5 HA protein from this strain and boosted once with virus-like particles(VLP)from the same strain.After the last immunization,we tested the activation of CD8+T cells.The cellular immunization was evaluated based on the quantity of the active T cells and the level of the cytokine secretion.The neutralization titers of the immune sera against the same subtype and different subtype strains were evaluated by pseudotype-neutralization assay,hemagglutinin inhibition assay,microneutralization assay and plaque reduction assay.We also detected the binding breadth against the HA proteins from H1 to H16 influenza strains.Then we tested the protection of this immune strategy against the same and different subtype.The protective efficiency was evaluated by the weight and survival levels of the immune mice infected by the wild influenza virus.Meanwhile we used the viral load in the lung of the immune mice challenged to test the viral clearance level of the immune mice challenged.The results showed that this prime-boost regimen induces not only highly HA peptide-specific CD8 T cell responses but also broad antibody responses that cross-neutralize all reported clades and subclades of HPAI H5N1 viruses and the influenza strains from different subtypes.The binding assays showed that TH DDV sera bound the different proteins from H1 to H16 influenza subtypes.The mice by the active immunization or passive immunization of TH DDV strategy were protected against the viral infection from the similar antigenic strains,A/Cambodia/P0322095/2004 and A/Shen Zhen/406/2004,the middle diverse antigenic strains,A/common magpie/Hong Kong/5052/2007 and A/chicken/Shanxi/2/2006,and very diverse strains,A/chicken/Netherlands/14015526/2014.These immune mice were also partially protected by the influenza strains from different subtypes.Through the analysis of the antigenic epitopes we found that the main neutralization antibodies induced by this strategy targeted the HA head region,namely there is the conserved neutralization epitope in HA head.Then in swine animal model the broad neutralization antibodies were induced.Based on the above found,the main neutralization antibodies induced by this strategy targeted the HA head region,we developed a new immune strategy,epitope-focused vaccine design to improve the potency and breadth of the neutralization antibodies directed to HA head.TH DDV sera recognized the conserved neutralization epitope,but the antibodies against this epitope are not potency.To improve the antibody potency against this epitope.We transferred this epitope to the narrowly focused,but highly potent backbone and used the chimeras as immunogen to immunize the mice.The mice were primed twice by HA DNA expressing the chimera and boosted once by viral-like-particle including the chimeric HA protein.The results showed the chimeric HA elicits significantly more potent and broader neutralizing antibody responses than parental strains,which cross-protect mice from lethal challenges of diverse H5 as well as heterosubtypic H1,H9 and H3 viruses via both active and passive immunizations.Then in swine animal model this strategy also induced the broad neutralization antibodies.This study provided two new approaches to design the swine influenza universal vaccine;1)the combination of the selection immunogen whose sequence is close to the consensus sequence and DNA prime-VLP boost;2)epitope focus vaccine design based on the antigenic minimization.