The Development Of A Universal Influenza A Vaccine Based On NP And M1Protein Of Inlfuenza A Virus

Backgrounds:Influenza virus is enveloped RNA virus belong to the family of Orthomyxoviridae,which cause severely contagious respiratory disease. There are3types of influenzaviruses: A, B and C, according to the antigenicity of nucleoprotein (NP) and matrixprotein (M). Influenza A virus can be further divided into different subtypes based ontheir antigenicity differences of the viral surface proteins: hemagglutinin (HA) andneuraminidase (NA). So far16subtypes of HA (H1-H17) and9subtypes of NA (N1-N9)have been identified, so, in theory,153different combinations of these proteins arepossible. Influenza A viruses infect a wide range of hosts and have caused severalpandemics. In2009, a new influenza A (H1N1) virus spread rapidly in a short period oftime around the world and caused enormous economic burden of human society, whichhighlights the seriousness of influenza A virus infection once again.Vaccination remains the most effective measure for the prevention and control ofinfluenza. Currently, there are four kinds of licensed influenza vaccines: inactivatedwhole virus vaccine, split vaccine, subunit vaccine and live attenuated vaccine. Annualsurveillance data of epidemics are collected by WHO and used to recommend the strainfor vaccine production. The antigenic similarity between circulating strains and vaccinestrains can affect the efficacy of influenza vaccine. These licensed vaccines are effectivein preventing and curbing the spread of influenza epidemics when the strains present inthe vaccines are closely matched with the predicted epidemic strains, but when they aresuboptimal matched, vaccine benefit is lower. In addition, the mutation rate of the influenza viruses is very fast, the flu vaccine every year replacement caused a lot ofinconvenience to the preparation of the vaccine production. The recent experience withthe2009H1N1influenza virus demonstrates the need to develop novel vaccines as thepresently used vaccination programs showed a significantly delay in controlling thespread of new pandemics. Therefore, broadly cross-protective universal influenza Avaccines are highly expected to meet the demand of protection from seasonal and/orpandemic influenza infection.The targets of universal influenza vaccine research were mainly focused on theconservative fragment of HA, M2e and internal protein such as M1and NP. But theuniversal influenza vaccines based on M2e and HA have poor immunogenicity andcannot provide sufficient cross-protection. There has not yet approved universalinfluenza vaccines based on these two proteins. More and more research has started totarget the highly conserved influenza virus proteins, NP and M1. HSPs, like HSP60,HSP70and gp96, act directly on antigen-presenting cells (APC). It can act as molecularlinks in innate and adaptive immune responses. In this study, NP and M1, with heatshock protein60(HSP60) as intra-molecular adjuvant, were used to construct fusionproteins to evaluate the possibility as a universal influenza vaccine.Objective:The objective of this study is to develop a universal influenza A vaccine antigen basedon NP and M1. The vaccine antigen will be expressed in E.coli expression system. Afterpurification and identification of the antigen, we will preliminarily evaluate theefficiency of vaccine by animal experiments.Methods and Results:1. Immune-bioinformatics and molecular design of universal influenza vaccine. By online BLAST analysis, NP and M1sequence of A/Panama/2007/1999strain werechose as template to designed recombinant gene NP-M1, NP-M1-HSP60with linker(GGGPGGG). Basic physicochemical properties, hydrophilicity plot, flexible regions,antigenic index, surface probability plot and secondary structure of NP, M1, HSP60andrecombinant protein NP-M1, NP-M1-HSP60were analyzed by Protean and onlineprotein analysis system ExPASy. The results demonstrated the recombinant proteinNP-M1and NP-M1-HSP60, were with reasonable construct.2. Construction, expression, purification and identification of universal influenzavaccine. Recombinant expression plasmids pET28a-NP-M1-HSP60, pET28a-NP-M1and pET28a-HSP60, were constructed by genetic engineering methods and weretransform into Escherichia coli cells. At the beginning, inclusion body proteinsNP-M1-HSP60, NP-M1and HSP60were got after inducing with IPTG at37℃.Through the optimization of induction condition, partly dissoluble proteins wereexpressed and gained at26℃. Then, the recombinant proteins were purified by His Taqaffinity chromatograph. Purified proteins were characterized by SDS-PAGE andidentified by Western Blot. It was shown that we successfully constructed universalinfluenza vaccine expressed by E.coli expression system.3. Preliminary evaluation of immunogenicity and protection efficiency of universalinfluenza vaccines on BALB/c mice. Mice for each group were vaccinated intranasally(i.n.) with10μg NP-M1-HSP60, NP-M1or HSP60with oil-in-water emulsion SP01adjuvant in2weeks apart. SP01was added by mixing1:1v/v with the antigen. Byindirect ELISA, ELISPOT, the vaccine immunogenicity in BALB/c mice was evaluated.Results showed, immunization with NP-M1-HSP60could induce high level of IgG insera and sIgA in nasal and lung lavage, and balanced Th1/Th2immune responsessuggested by IgG subtype assays and IFN-γ, IL-4ELISPOT assays. Most importantly,vaccinated with NP-M1-HSP60could protect mice against A/Beijing/501/2009(H1N1)virus, PR8(H1N1) virus and A/ostrich/Suzhou/097/2003(H5N1) virus challenge, with obvious alleviation of histopathological changes in the challenged mice lungs andsignificant decrease of viral replication. These data suggested that the NP and M1baseduniversal influenza vaccine had great immunogenicity and cross-protection efficiency.Conclusion:In this study, a universal vaccine based on NP, M1and HSP60was efficiently expressedin Escherichia coli (E.coli), and the immunogenicity and immuno-protective efficacy ofthe universal influenza vaccine were evaluated in a mouse model. Results showed thatthe vaccine had good immunogenicity, could stimulate the body to produce humoral,cellular and local mucosal immune responses, and can produce effectivecross-protection. This provides theoretical basis and experimental data for in-depthstudy of the universal influenza A vaccines.