| With the development of vaccine industry,much research efforts have been devoted to impoving stability of vaccine antigens and design of safe and effective vaccine adjuvants.Inactivated foot and mouth disease virus(FMDV),which is the most effective vaccine antigen against highly contagious foot and mouth disease,is extremely unstable.The intact FMDV particle,also known as 146S,is easily dissociated during production,storage and application,leading to severve decrease in its immuniginicity.This work proposed new strategies for the stabilization and developed a new adjuvant design based on in depth analyses of the factors and mechanism affecting the structural integrity and the immunogenicity of the 146S particles.Transition metal ions and ionic liquids(ILs)were selected and examined for possible enhancement of the interaction between the inter-pentamers of virus capsids so that the particle disassociation of particle is prevented and its stability is improved.Furthermore,an ILs-based O/IL nanoemulsion was constructed and tested as a novel adjuvant for the FMDV vacccine.This O/IL nanoemulsion was also applied as a new adjuvant for an influenza virus vaccine.The results and novelty are as follows.(1)Transition metal ions Ni2+ and Cu2+ were found to bind specifically to 146S through a mechanism different from bingding of nontransition metal ions Ca2+.Combining with isothermal titration calorimetry,microscale thermophoresis and ICP-MS analyses,the thermodynamics and capacities of Ni2+,Cu2+ and Ca2+ binding to 146S were studied and discussed.All three metal ions could bind spontaneously to 146S through an enthalpy driving proces(?G?0,?H?0,?S?0),among which the Cu2+showed the highest binding affinity.The binding capacities of Ni2+and Cu2+on 146S capsids were about 10 times higher than Ca2+.These results suggested that the binding modes and amino acid types involved in binding of these two different types of metal ions on 146S might be different.(2)The binding of transition metal ions resulted in improvment in stability and immunogenicity of the 146S.The binding of Ni2+,Cu2+ or Ca2+ could all improve the thermostability of 146S,and binding of Cu2+was the most effective.The binding of Ni2+and Cu2+also improved the acid-resistant stability of 146S,while binding of Ca2+was not conducive.Aminal experiments indicated that the immunization of 146S bound with Ni2+or Cu2+ induced higher antibody titers in mice.The the affinities between 146S with two important cell surface receptors,integrin ?6 and heparin sulfate,was increased by 3 and 10 times,respectively,after binding of Cu2+.Based on the above results and combined with the characteristics of numerous histidine residues at inter-pentameric interface of FMDV,we speculate that "transition metal ion bridges" were formed to stabilize 146S by coordination inertactions between transition metal ions and adjacent histidines at the inter-pentameric interface of 146S.(3)Using choline-based ILs to stabilize the 146S by adjusting the microenvironmental proton intensity surrounding the particle.Choline-based ILs[Cho][Cl]and[Cho][SO4]were found to improve the transition temperature(Tm)related to 146S dissociation by 4?,while[Cho][H2PO4]decreased the thermostability of 146S.By analyzing the effects of different choline-based ILs on the microenvironmental pH value of 146S particle,[Cho][Cl]and[Cho][SO4]were found to stabilize the FMDVD 146S mainly by reducing microenvironmental proton intensity surrounding the particle,so that the protonation of histidines at the inter-pentameric interface of 146S was supressed.(4)An O/IL nanoemulsion was constructed and tested as adjuvant for FMDV vaccine to enhance the humoral immune response.By substituting the anion of choline-based ILs with nicotinic acid with biological activity,a[Cho][nicotinic](CANI)IL was constructed.With CANI sulution as aqueous phase,squalene as the oil phase,and the Tween 80 as the surfactant,a stable O/IL nanoemulsion with uniform size about 160 nm was successfully constructed.Compared with commercial emulsion adjuvant ISA-206,the O/IL emulsion could significantly improve the stability of 146S.Using the O/IL nanoemulsion as adjuvant of 146S vaccine,significantly enhanced humoral immune response was induced by subcutaneous immunization.The level of IgG titer was comparable to that using ISA-206 adjuvant.(5)O/IL nanoemulsion exhibited excellent adjuvant effects for H1N1 split influenza vaccine both by subcutaneous and nasal mucosal vaccinations.Using the O/IL nanoemulsion as adjuvant of H1N1,IgG titers significantly higher than that by using MF59 adjuvant were induced by by subcutaneous vaccination.When was used as nasal mucosa adjuvant,the O/IL nanoemulsion not only enhanced the mucosal immune response,but also the humoral and cellular immune responses.Compared with MF59 adjuvant,the O/IL nanoemulsion induced significantly higher level of sIgA and IgG titers,as well as higher IFN-y secretion.The excellent adjuvant effects of the O/IL nanoemulsion for mucosal immune was mainly benefited from prolonging the retention time of antigen in nasal cavity,promoting the proliferation of DC cells and CD4+T cells in nasal mucosa associated lymphoid tissue(NALT). |
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