Studies Of Stability,Immunogenicity,and Particulate Adjuvant Design For Foot And Mouth Disease Virus Vaccine

Lots of efforts have been devoted to improving stability of antigen and design of safe and effective adjuvant for vaccine.Inactivated foot and mouth disease virus(iFMDV),also known as 146S,is extremely unstable and easily dissociated,leading to severe decrease in its immunogenicity.The stability of iFMDV in the most widely employed commercial ISA 206 emulsion adjuvant was reported even worse,meanwhile,the ISA 206 was not efficient in stimulation of cellular immunity.To address these problems,the present work proposed biocompatible cationic solid lipid nanoparticles(cSLN)and zinc ion-chelated chitosan particles(CP-PEI-Zn)as potential vaccine adjuvant for iFMDV,on the basis of in-depth study of effect of antigen stability on its in vivo immunoactivity.The new adjuvant and the novel antigen delivery pattern were favorable for structural integrity of iFMDV and effectively improved the efficiency of the immune response of the antigenThe main results and findings are as follows(1)The influence of iFMDV stability on its in vivo immune activity was investigated.Four different purification processes,including hydrophobic chromatography,PEG 6000 precipitation,hydrophobic chromatography combined with size exclusion chromatography,as well as hydrophobic chromatography combined with PEG 6000 precipitation were developed to purify iFMDV.The iFMDV purified from the fourth process showed the highest stability and induced strongest immune response in immuninzed mice.To make more accurate investigation on influence of antigen stability and its immunogenicity,the stability of iFMDV purified from the fourth process was manipulated by changing the buffer solution to ensure an identical antigen and inoculation dosage.Results showed that more stable iFMDV could activate bone marrow derived dendritic cells(BMDC)and stimulate the secretion of FMDV specific IgG,IgM antibodys more effectively,which further confirmed the positive correlation between iFMDV in vitro statality and its in vivo immunogenicity.(2)The cSLN was prepared by O/W emulsion method with matrix lipid compritol 888 ATO and modified by cationic lipid didodecyldimethylammonium bromide(DDAB).The cSLN with diamter about 250 nm has good dispersibility and positive surface zeta potential to load negatively charged iFMDV via electrostatic adsorption.It was found that the buffer with low ion strength could enhance antigen adsorption but accelerate the dissociation of iFMDV.Then the adsorption condition was optimized to protect the structure of iFMDV and strengthen the loading capacity of cSLN.In the buffer containing 10 mM PB and 0.075 M NaCl(pH 8.0),cSLN could adsorb 87.8%initially added antigen,whose loading capacity reached 70.2?g/mg.Loading iFMDV onto cSLN by electrostatic attraction did not destruct iFMDV particle structure as measured by high performance size exclusion chromatography(HP SEC)and transmission electron microscopy(TEM).The Tm related to iFMDV dissociation increased for 1.2? after loading on cSLN,indicating a good biocompatibility of cSLN for this unstable antigen.(3)The adjuvant effect of cSLN was evaluated by both celluar and animial experiments.The cSLN loaded iFMDV enhanced antigen uptake by 6-fold and activation of bone-marrow-derived dendritic cells(BMDCs)with augmented expression of CD86,CD40,and MHC I.In the animal trials,cSLN significantly enhanced both humoral and cellular immune response in immunized Balb/c mice,with higher FMDV specific IgG,IgG2a and IgG1 antibodys titers as well as more memory T cells than that induced by widely used ISA 206 emulsion adjuvant.(4)Based on the principle that the histidine(His)residuals rich in iFMDV could coordinatedly interact with transition metal ions,zinc chelated chitosan particles(CP-PEI-Zn)were prepared to deliver iFMDV via the coordination interaction between iFMDV and Zn2+.The iFMDV loaded on CP-PEI-Zn showed better thermal stability than that on CP-PEI,as revealed by a slightly higher transition temperature(Tm).After subcutaneous immunization in female Balb/C mice,antigens loaded on CP-PEI and CP-PEI-Zn all induced higher specific antibody titers,better activation of B lymphocytes,and more effector-memory T cells proliferation than the free antigen and iFMDV adjuvanted with ISA 206 emulsion did.Moreover,CP-PEI-Zn showed superior efficacy to CP-PEI in promoting the proliferation of effector-memory T cells and secretion of cytokines,indicating a more potent cellular immune response.In summary,the cationic solid lipid nanoparticles and chitosan particles facilitated both strong cellular and humoral immune responses and stabilized iFMDV after loading.In addition,they are biocompatible,easily preparaed and low-cost,reflecting their potential to be a promising adjuvant for the iFMDV vaccine.