Optimization Of Nano-emulsion Adjuvant In Influenza H3N2and Research On Influencing Factors To Immune Response

Influenza viruses can cause acute respiratory infection in human and animals. It is highly infectious and spreads fast and easily leads to worldwide epidemic which are very dangerous to human health and social security. The influenza virus can be divided into three types of A, B, C. Type A virus mutate more easily. And it caused a large number of deaths and huge economic loss during the influenza pandemic in the world.At present, the most important means of preventing influenza is vaccination. Traditional influenza vaccines are produced with chick embryo as substrate. Due to the limitations of the production methods and the production process, It is difficult to provide enough vaccine to the crowd when facing influenza pandemic outbreak. Adjuvants are added to the vaccine to enhance the immunogenicity of antigen and significantly reduce the needs of vaccine antigen. we can not only deal with influenza pandemic outbreak but also reduce the manufacturing cost of the vaccine.Nanoemulsion is a kind of thermodynamically stable, transparent and homogeneous dispersion system composed of oil phase, aqueous phase, surfactant and co-surfactant. There are many researches on drug convey function, but little on vaccine adjuvant.Now, some kinds of nano-emulsion like MF59, AS03, AF03are approved as human use adjuvant to deal with influenza pandemic outbreak.Based on the formula carried out in the presious study, we change somewhere and form new nano-emusions. Optimization of water, surfactant,oil and vaccine carrying way help us obtain a safe, stable, effective formula. And we compare different particle size, different particle density or different carry way, to find out what is the direct factor that influence adjuvant effect.The first part:Optimization of water concentrationFormula A(Tween80, Span80,1,2-propylene glycol,7#white oil), Formula D(Tween20, Span80,1,2-propylene glycol,7#white oil). We do a premaily optimization on their Km value and surfactant+co-surfactant and oil phase ratio, and form stable emulsion that can be endless diluted.Then, we make nano-emulsion with differert water concentration (66.7%,80%,85.7%), and discrible their physical characters. Finally, ICR mouse are injected of emusion mixed with influenza vaccine, the serum are tested of HI titer, IgG, IgG1, IgG2a, IL-4, IFN-γ level, to seek out the influence factor of adjuvant effect and explain the mechnisem why nanoemulsion enhance immune response. It turns out that nano-emulsion with different water concentration show different adjuvant effect. The reason maybe that, water can dilute nano particles, so it deciede particle density, there must be a most suitable water concentration dilute the particles to the suitable density that make the best adjuvant effect. Take safety and effect of the three different water concentration into consideration,we choose85.7%as the most suitable water concentration to optimize surfactant and oil phase.The second part:Optimization of different vaccine carrying wayWe compared different vaccine carrying way, absorption (Way2)and package (Way4)way.Absorption way carry the antigen on the nano-particle surface, package way carry the antigen in the nuclear of the nano-particle. The other situation, such as antigen concentration,particle density, particle size, are the same.Finally, it shows differert immune response, absorption is much better than package way, or even the package way show suppressive effect. The mechanism needs further research. The third part:Optimization of surfactant and oil phaseNumbers of researchs establish that ratio or species of surfactant and oil phase deciede partical size and particle density. In the experiment, Span80is one of the compound surfactant, the compare adjuvant effect between Tween20, Tween80, EL emulsion. Four kinds of oil are consonsidered as follows:white oil, IPM, soybean, grasp seed oil. The result turn out that Tween20or Tween80in the emulsion play an important role, While IPM and white oil are better choice for oil phase. Take particle size into consideration for the main factor that cause different adjuvant effect, small particle size emulsion show good adjuvant effect, while some formula with particle larger than100nm are not effective or show immune inhibition. So particle size may be the main factor that influence adjuvant effect. Considering the stability, uniformity, safety and effectiveness, formula D(Tween80, Span80,1,2-propylene glycol,7#white oil=1:1:2:1),is the best.