Study On Thermo-stable Sugar Glass Vaccine Against PRRS

Porcine reproductive and respiratory syndrome(PRRS)is caused by porcine reproductive and respiratory syndrome virus(PRRSV)which causes swine fever,anorexia,pregnant sows late miscarriage,premature birth,stillbirth,weak and mummified fetus,and the characteristic of disordered breathing in pigs of all ages(especially piglets),which is a highly infectious diseases,leading to significant harm on the pig industry,it is the most common method to inject attenuated live vaccines to prevent PRRSV.At present,vaccine production abroad mainly by freeze-drying technology,can be stored at 2～8 ℃ for 1.5-2 years,it must rely on the ’cold chain’ system during vaccine using,transportation and storage.In this study,wu use a new foam vacuum drying method,make the PRRS resistant sugar glass vaccines base on trehalose,in order to obtain higher heat storage properties,can get rid of the "cold chain" rely on high-quality vaccines.First study the heat protection recipe ingredients univariate analysis by selecting trehalose,sucrose,tryptone,EDTA,gelatin,etc.,and choose 37 ℃ anti-aging test and the residual moisture as screening index,so as to can it obtain the better thermostability in the room temperature porcine reproductive and respiratory syndrome attenuated live vaccine;then make the optimization of morphology structure,glass transition temperature and other process parameters optimization;final study the shelf time and immune effects to establish the laboratory preparation process,provide technical support for industrial production.1.Screening of heat protect formulations of the foam dried vaccineTo choose a well heat-resistante protective agent,we selecte sucrose,trehalose,gelatin,tryptone and other ingredients,use single factor design to add and compate the ingredients.Prepare vacuum foam vaccine in the same vacuum drying process.By 37℃ anti-aging test,first discover T28 foam dried vaccine preserve 10 d and the TCID50 loss is below 0.5 Lg,this is superior to other formulations.Then continue ptimizing and get T28C-1,T28C-5,T28C-6,T28C-8 formulations,the foam dried vaccine prepared in 37 ℃ for 10 d and loss of TCID50 is 0.43 Lg,0.30 Lg,0.44 Lg and 0.38 Lg respectively.2.The optimization of foam dried vaccine manufacture programFoam drying process affect the physical properties of the prepared vaccine,it also affect the potency loss and heat resistance of the vaccine.We adjuste the process on the basis of the original foam drying process on Newcastle,and get T28 formulation which has a good result,it could form stable foam and maintain the low viral titer loss;further adjust the dispensing volume,separator temperature and vacuum descent rate to prepare the foam dried vaccine,get T28C heat vaccine and compare the foam physical properties and thermal activity of vaccine in different forms,then find there is little virus TCID50 difference with diffient foam formation.In the same batch foam dried vaccines,vaccines with foam full of more than 1/2 bottle high and no stick wall phenomenon have the virus titer of 6.7 + 0.08 Lg TCID50;vaccines with irregular shape foam and have stick wall phenomenon have the virus titer of 6.5 + 0.11 Lg TCID50;vaccines with foam not fully expanded and gather at the bottom have the virus titer of 6.6 + 0.07 Lg TCID50.3.The physical and chemical properties of foam dried vaccineWe make several batches foam dried PRRS vaccine in laboratory use the best formulation and preparation process get across the first two chapters,T28C series resistant protective vaccine have the dense milky white foam,with osteoporosis,uniform height(up to 1/2～2/3 bottle height),big surface area which conducive to evaporation.Scanning electron microscope image shows the vaccine microscopically.Determinate the traits,residual moisture,anti-aging tests refer to《People’s Republic of China veterinary biologics regulations》,T28C-1,T28C-5,T28C-6 and T28C-8 formulation have residual water of 2.9%,2.86%,2.94%,2.88%,binding to anti-aging test results of 37 ℃ 10 d,T28C-1,T28C-5,T28C-6 and T28C-8 foam dried vaccine kept in 37 ℃ lOd and loss of virus values are 0.43 Lg,0.30 Lg,0.44 Lg,0.38 Lg,we can pick out T28C-5,T28C-6 as the best formulation.At the same time we determine the glass transition temperature(Tg)of T28C-5 and T28C-6 formulation,combine with the virus in liquid state are-33.68 ℃,-33.82℃,foam dried vaccine are 56.1 ℃,57.45 ℃,that 12℃ high than freeze-dried vaccine.4.Quality of foam dried vaccineIn order to detect the heat resistance of dried vaccine trials foam,we place the vaccine in the environmental of 4 ℃,25 ℃,37 ℃ and room temperature,test the virus content and residual moisture to determine the preservation conditions and time;at the same time,we do the immunological assay In vivo to evaluate the immune effect of the vaccine.The results show that T28C-1,T28C-8 formulation get viral titer loss more than 1.0 Lg after store at 37 ℃ 80 d,25 ℃ 120 d,the natural environment 120 d,respectively.The T28C-5 and T28C-6 foam dried vaccine formulations store at 37 ℃ 4 months,the loss of viral titer below 1.0 Lg;stored at 25 ℃ 150 days,the viral titer loss remains below 0.8Lg;save at the natural environment 150 d,(through summer,autumn,winter transformation,the maximum temperature and the minimum temperature have difference of more than 30 ℃),viral titer loss remains below 0.6 Lg.Final get T28C-5 formulations have significant heat resistance.Results of in vivo test show that foam dried vaccine preserve at 37 ℃,25 ℃and 4 ℃ three months and then immunize pigs,the blood is determined by ELISA after immunizating two weeks,three weeks,four weeks.Antibody levels were detected in a high positive level,and foam dried vaccine saves at 4 ℃ can reach the same level of antibody levels 4 weeks after immunization lyophilized vaccine.