Study On Manganese Based PDNA Nanovaccine For H5N1 Avian Influenza And The Expanded Application Of Screening Bivalent Metal Ion Adjuvant

H5N1 is a highly pathogenic,highly infectious and highly mutable avian influenza virus that infects birds such as chickens,ducks and geese by infecting the respiratory system.A concentrated outbreak of H5N1 avian influenza in current large-scale farming systems would cause significant economic losses to the breeding industry and pose a major threat to public health.Vaccination is the most cost-effective intervention to prevent deaths and reduce the incidence of avian influenza.As a new type of vaccine,DNA vaccine is safer,easier to manufacture and store,and has the advantages of rapid and large production in the event of virus pandemic,so it is a new type of vaccine with great application prospects.However,the immunogenicity of DNA vaccine is inferior to that of attenuated or inactivated vaccine because DNA vaccine only intercepts the sequence of partial epitopes of virus.In addition,DNA is easily degraded in the tissue after transfection,and cannot pass through the cell membrane freely,resulting in less effective cell uptake and other factors also limit its widespread use.Improving the delivery system of DNA vaccine and adding appropriate adjuvants to enhance the host’s specific immune response to DNA vaccine can effectively solve the bottleneck problems in the application of DNA vaccine.In recent years,with the rapid development of nanotechnology,more and more nanomaterials have been used as DNA vaccine carriers.The nanocarriers were found to be able to protect DNA vaccines from degradation.At the same time,it has a good antigen reservoir effect and can release antigens slowly and continuously.Nanocarriers can increase antigen uptake by APCs to stimulate host immune response efficiently and sustainably,which is a promising method for DNA vaccine delivery.In the development of DNA vaccines,the role of adjuvants cannot be ignored.In2018,the adjuvant effect of Mn²⁺was developed.High intracellular Mn²⁺can activate c GAS-STING signal transduction pathway,which is considered as the bridge between innate immunity and adaptive immunity.Intracellular overload of Mn²⁺promote the maturation and migration of DCs.Mn²⁺can enhance the cytotoxic effect of killer T cells and natural killer cells（NK cells）.As a new metal ion adjuvant,Mn²⁺has attracted much attention in academia.In addition,there are a variety of bivalent metal ions in cells,such as Mg²⁺,Fe²⁺,Cu²⁺,Co²⁺,etc.These bivalent metal ions regulate different physiological functions in cells.Artificially overloading the cells with these bivalent metal ions may produce some special biological effects.Whether these bivalent metal ions have the same adjuvant effect as Mn²⁺deserves further study.Therefore,developing a general platform for screening bivalent metal ion adjuvants is of great significance for finding more bivalent metal ion adjuvants.In this study,we successfully constructed a manganous phosphate core with a water-based core structure,and efficiently loaded H5N1 plasmid DNA vaccine（pDNA HA Re11）into the water-based core.The surface of the liposome is covered with DOPA\DOTAP\DSPE-PEG2000 lipid nanoparticles（pDNA HA Re11@Nano Mn）.Lipid nanoparticles can efficiently deliver and protect pDNA vaccine,enhance phagocytosis and activation of antigen-presenting cells,and overload Mn²⁺in cells can activate type I interferon response,and ultimately enhance the activation of immune cells.The results show that pDNA HA Re11@Nano Mn can effectively deliver pDNA HA Re11 and activate both cellular and humoral immune responses of the host.Further,we use this preparation method of lipid nanoparticles to pack Co²⁺,Mg²⁺,and Ca²⁺.In vivo and in vitro experiments have confirmed that the preparation method of lipid nanoparticles can be expanded into a screening platform for bivalent metal ion adjuvant,so as to screen out more excellent metal ion adjuvants.The main research results are as follows:1.The optimal Mn/P molar ratio of pDNA HA Re11@Nano Mn was determined to be1:100 by particle size,potential and encapsulation rate.The hydration particle size was46.49±1.71 nm and the Zeta potential was 18.23±2.22 m V.pDNA HA Re11@Nano Mn has a good encapsulation rate（83.31%±1.84%）,and the content of Mn²⁺is 4.62±0.256 m M.In addition,pDNA HA Re11@Nano Mn has better storage stability and colloidal stability.In vitro experiments,pDNA HA Re11@Nano Mn can effectively protect pDNA HA Re11 from degradation by DNase I,so as to effectively improve the delivery efficiency of pDNA HA Re11.Moreover,the release of pDNA HA Re11@Nano Mn in vitro showed p H responsiveness.2.pDNA HA Re11@Nano Mn contains metal cations and cationic lipids with good buffer capacity,making pDNA HA Re11@Nano Mn p H responsive.Buffer capacity assay,red cell lysis assay and laser confocal assay confirmed that pDNA HA Re11@Nano Mn delivers pDNA HA Re11 into the cytoplasm through lysosome escape process for expression.Further,we verified that pDNA HA Re11@Nano Mn can activate BMDCs,promote its maturation and differentiation,and release cytokines,laying the foundation for further humoral and cellular immunity activation.3.pDNA HA Re11@Nano Mn has a good antigen repository effect,which can significantly increase the HI titer and specific antibody Ig G level in mouse serum.Compared with commercially available Al adjuvant,pDNA HA Re11@Nano Mn can activate both humoral and cellular immunity in mice.At the same time,pDNA HA Re11@Nano Mn has better safety in vivo application.4.Three kinds of lipid nanoparticles,pDNA HA Re11@Nano Co,pDNA HA Re11@Nano Mg and pDNA HA Re11@Nano Ca were prepared by pDNA HA Re11@Nano Mn preparation method.Three kinds of lipid nanoparticles with similar morphology,stability and the loading efficiency.In vivo and in vitro experiments confirmed that the preparation method of liposome can carry a variety of bivalent metal ions,in vivo and in vitro experiments confirmed that it can effectively verify whether bivalent metal ions have adjuvant effect,and compare their ability of immune activation.This method can be further developed as a screening platform for bivalent metal ion adjuvants.