Preparation And Application Of A Novel Compound Emulsion Adjuvant For Rabies Vaccine In Mice

Rabies is a worldwide viral zoonotic disease,which transmit from the animal to the human if bitten or scratched by disease animal.Once onset,the mortality rate is close to 100%.If rabies virus infection,it first plays replication and proliferation in local muscle tissue and then rapidly transmits the central nervous system along the intermittent body fluids around the peripheral nerves and the nerves.During the spreading process,the virus multiplies in the ganglion cells at a large degree.Because the central nervous system lacks specific immune response components such as neutralizing antibodies,complement proteins,and non-specific immune response components such as macrophages and inflammatory factors.Therefore,once the virus enters the central nervous system,even if the body produces high titters of neutralizing antibodies,it is without effective protection.The currently widely used non-adjuvant rabies vaccine often takes 14 DPI(day post inoculation)to achieve 100%positive conversion rate,while the aluminum adjuvant rabies vaccine used in the last century can achieve 100%positive conversion rate until 42 DPI.So the promising adjuvant should stimulate the body's rapid or long-lasting production of not only specific but also non-specific immune responses by using rabies vaccine.In this study,it used the rabies vaccine strain CTN-1-D15 by human embryonic lung diploid cells to prepare vaccine.Then,it employed the cell ELISA method to detect the anti-rabies virus antibody titter for screening the useful adjuvant components.Significantly,we found that the OD value of taxol group(using 100 ?g/mL)could reach 0.717 by 3 DPI with 1 injection,but the value of MF59 group could reach 0.945 by 14 DPI with 2 injections.These two groups had significant difference with non-adjuvant control groups by their own DPI,respectively.It mean that taxol is fastest and MF59 is highest efficient.Having regard to their cytotoxicity,sensitization,and short-term antibody peaks,here,we combined them together by special method to reduce their use concentration.On the other side,since coenzyme Q10 could stabilize the activity of succinate dehydrogenase(SDH)in cell mitochondria,which contribute to product inflammatory factors and active mitochondria driven macrophages,it is also an important adjuvant candidate organic component.In details,used squalene to extract coenzyme Q10 from cold suspension,then mixed each component according to the volume ratio(5.0%squalene;0.5%Tween;0.5%Siban)and dispersed them in succinic acid-sodium citrate buffer.And it was made an oil-in-water(O/W)nanoemulsion by a high-pressure homogenizer at 8,000 psi.Then diluted nanoemulsion with 0.01 M PBS.At last,added taxol(using 20 ?g/mL)in it.Because the solution is golden color during the preparation process,it named as Golden05 finally.In using,mixed the Golden05 and a quarter dose of rabies vaccine at radio of 1:1,then taken intramuscular injection(i.m.)for ICR mice at 0th,3rd,7th day,respectively.By monitoring the trend of neutralizing antibody titter in mouse serum by rapid fluorescence focus inhibition test(RFFIT)method,it found the mouse began to produce effective neutralizing protective antibody(0.83 IU/mL)from 5 DPI,which was under two times injection.Furthermore,the antibody was maintained at a high level till 3rd month.When challenge the immunized mice by rabies standard challenge virus strain(CVS-11),the rabies Golden05 adjuvant vaccine could provide 100%protection for these mice.If analyzed the potential immune mechanism of Golden05 adjuvant,it found that this new adjuvant can significantly improve the activity of SDH,which can help the expression of pro-inflammatory factors such as IFN-y and TNF,beside a large proliferation of CD8+T cells.That made plasma cell activation,and enables rapid and large-scale production of rabies virus neutralizing antibodies.Additional,the physicochemical indicators of Golden05 adjuvant and the results of acute toxicological indicators support the conclusion that this novel nanoemulsion adjuvant could be applied to rabies vaccine in the future,which inducted specific and non-specific protective immune responses rapidly.