Development And Efficacy Evaluation Of Novel Nanoemulsion Adjuvant For Animals Based On FMD-VLPs Vaccine

Vaccination is the most effective and economical way to combat infectious diseases of human and animal origin.However,a safe and effective vaccine requires a good adjuvant.Compared with traditional emulsions,nanoemulsion has the advantages of high safety,excellent stability and good immune effect,which has become the key direction of veterinary vaccine adjuvant research.Novel vaccines,especially genetically engineered vaccines based on virus-like particles,have received much attention for their higher immunogenicity and safety.Genetically engineered vaccines,especially those based on virus-like particles(VLPs),have received widespread attention for their higher immunogenicity and safety.However,such subunit antigens are usually not sufficiently immunogenic to induce protective immunity,making it more important to combine them with effective adjuvants to ensure a strong immune response.At present,ISA206 adjuvant,which is more frequently used in FMD vaccines,is immunologically effective,but it has long been dependent on imports and has risks such as trade barriers,so it is particularly urgent to select suitable compounds and develop adjuvants that are both safe and effective against FMDV-VLPs vaccines.In this study,two novel nanoemulsion adjuvants were prepared by pseudo-ternary phase diagram method and phase transition method,and their physicochemical properties were characterized.The homemade adjuvants were selected and the vaccines were prepared by emulsifying FMD virus-like particles as antigens,and their biocompatibility and immune enhancement effects were investigated using experimental animals as models,in order to provide some ideas and basis for further in-depth research and application of vaccine adjuvants.(1)Preparation of Astragalus polysaccharide nanoemulsion and evaluation of its adjuvant effectAstragalus polysaccharide nanoemulsion(APSN)consists of white oil,Span80,Tween80,1,2-propanediol and astragalus polysaccharide solution.The average particle size was 85 nm,zeta potential was-15.6 m V,and polydispersity index(PDI)was 0.196 as measured by particle size analyzer.The microscopic morphology of APSN was observed by transmission electron microscopy,which showed a homogeneous sphere-like shape with a particle size ranging from 80-100 nm,which was consistent with the results of the particle size analyzer.APSN was stored at room temperature(25℃)and 37℃ for 180 days protected from light,there were no significant changes in microscopic morphology,particle size,zeta potential,PDI,p H,etc.this result show that the nanoemulsion has good stability,the above results indicate that APSN has good stability.After injecting a certain dose of APSN into the mice,there was no redness,swelling,hair loss,or ulcer at the injection site,and the body weight increased normally within 1 month.The mice ate and drank normally,moved freely,and were in good mental condition.The histopathological examination showed that there was no obvious pathological damage to the important organs,which indicated that the formulation of APSN was safe,less toxic and had good biosafety.APSN was used as an adjuvant to prepare FMD-VLPs vaccine,immunize mice,guinea pigs and pigs,its immunological enhancement effect was evaluated.The results showed that APSN could significantly increase the level of specific antibody and the secretion levels of Ig G1,Ig G2 a,IFN-γ and IL-1β in mice within 4 weeks.Meanwhile,specific and neutralizing antibodies were also significantly increased in guinea pigs within 4 weeks,and splenic lymphocyte proliferation was promoted.After immunization of guinea pigs with a single dose of APSNVLPs vaccine,the attack experiment was performed on 28 days,and the APSN-VLPs group achieved 100% protection,which was higher than the ISA206 control group.The levels of specific antibodies and neutralizing antibodies immunized by APSN-VLPs vaccine were also higher than those of the ISA206-VLPs vaccine control group,and the levels of cytokines IL-1β,IL-4 and IFN-γ were also higher than those of the ISA206-VLPs vaccine control group.(2)Preparation,safety evaluation and immune enhancement effect of new CpGcontaining nanoemulsion(SNA)SNA consists of Span85,Tween60,squalane,polyethylene glycol-400(PEG400)and CpG.At room temperature,freshly prepared SNA appeared as a yellow transparent emulsion;The microscopic morphology of SNA observed by transmission electron microscopy as uniformly sized circles;The average particle size of SNA was 95 nm;About 75% of the nanoemulsion particles were concentrated between 68 nm and 105 nm,with a very narrow particle size distribution;from TEM.In addition,we determined the PDI of nanoemulsion SNA with a value of 0.4,and the average potential was-25.76 m V.A series of experiments have demonstrated that SNA has good centrifugal resistance,thermal stability,long-term shelf stability and biocompatibility.A foot-and-mouth disease virus-like particle vaccine was prepared using SNA as an adjuvant,and experimental animals were immunized to evaluate the immune enhancement effect.Compared with the control group,NSA-VLPs could further increase cytokine secretion,promote lymphocyte proliferation,induce enhanced cellular and humoral immune responses after animal immunization,and provide better challenge protection against FMDV.The results of this study confirm that APSN and SNA,two novel nano-emulsion adjuvants,can significantly improve the immune response to FMDV,stimulate stronger cellular immune response,have a more comprehensive immune enhancement effect than ISA206 and are effective FMD virus-like particles vaccine adjuvants。APSN is expected to be an alternative adjuvant to ISA206.This study also provides experimental basis for the subsequent research and application of veterinary vaccine adjuvants.