Development Of A Novel Universal Influenza Vaccine With CD40L

Influenza virus is a serious pathogen which could spread quickly.Universal influenza vaccine had been studied for many years. Conservative unitsor sites were found out in some proteins such as HA2, M2and NP.Hemagglutinin A2locates at the C-terminal protein of hemagglutinin which candrive HA1anchoring to the cellular or virul membrane. Previous studies showedthat HA2was one important protective antigen and can be a candidate ofInfluenza universal vaccine.Objective: To construct a novel influenza universal vaccine, conservativeantigen was selected. CD40L and optimized codon were used to enhance theimmunigenicity of the vaccine. Method:In this study, we constructed one vaccine candidate which involvedboth HA2and CD40L gene, and expressed a fusion protein by Adenovirusesvectors. The HA2will have more opportunity to target and activate B cells orAPCs by the help of CD40L. To promote CD40L forming native conformation,T4bacteriophage fibritin trimerization motif was inserted into the recombinant.Four control recombinants HA2, SHA2, SHA2FCD40L, SFCD40L,HA2FCD40L in which one or more gene absence and S2HA2FCD40L withoptimized codon were also constructed by SOE-PCR and RT-PCR. The animalexperiment was conducted on three doses:107PFU/ml,108PFU/ml and109PFU/ml and envoled four groups, Ad-SHA2, Ad-SHA2FCD40L,Ad-S2HA2FCD40L and Adenovirus vector as control. The animal experimentwas conducted on three doses:107PFU/ml,108PFU/ml and109PFU/ml andenvoled four groups, Ad-SHA2, Ad-SHA2FCD40L, Ad-S2HA2FCD40L andAdenovirus vector as control.Result:After linked into shuttle vectors pShuttle-CMV, all the six reconbinantswere insered into the vector pAdxsi.The results of Western-blot showed that allthe six Adenoviruses could express the target proteins by either anti-HA serumor anti-CD40L monoclonal antibody. The fusion proteins were all located atcell memberane by Indirect Immunofluorescence Assay (IFA). The animalexperiment showed that the level of TNF-α, IFN-γ and IL-4showed dose-relatedincrease after one dose administration of any group of SHA2FCD40L orS2HA2FCD40L.but not enough to against the Influenza virus challenge. Afterthe second dose, the whole immunity to HA2was boosted and all the groups ofAd-S2HA2FCD40L showed highest immunigenicity among the groups. Theantibodies specific to influenza virus were also raised to a high level especially the IgG and IgA.Conclusion:Cross-protectivity of Ad-SHA2, Ad-SHA2FCD40L, andAd-S2HA2FCD40L were found respectly against the lethal challenge ofInfluenza virus PR8(H1N1) and H9N2. The survival rate increased greatlywhen CD40L gene had been inserted into the fusion protein. The optimizedcodon would be helpful to enhance the immunigenicity as well.