The Study On Immune Response Of Ebola Vaccine Specific Antigen And Vector

Difference in the fold increase in the neutralizing antibody water of the Ad5 vector and the specific serum Ig G antibody level of the Ebola virus envelope glycoprotein(EBOV-GP)after the booster immunization against the recombinant Ebola virus disease vaccine(Ad5-EBOV)clinical trial To carry out the immune response study of Ad5-EBOV specific antigen and carrier.Firstly,an Ad5 carrier serum Ig G antibody detection method suitable for SPF animal models was established.During the purification of Ad5,a single protein peak was found in front of the virus peak during gradient elution.It was identified by SDSPAGE,HPLC and Western Blot.The main component was Ad5 Hexon protein.The source 30 Q anion column and Sepharose 4FF molecular sieve two-step column chromatography can obtain Hexon protein with a purity greater than 95%.Ad5-Hexon was used as antigen to establish Ad5 binding antibody detection ELISA method.The detection method has a certain cross-reaction with other subtypes of adenoviruses such as Ad4 and Ad7,but it has good specificity for unrelated viruses;at the same time,the detection method has good stability,the intra-plate repeat variation coefficient is less than 10%,and between plates And the day-to-day repeat coefficient of variation is less than 15%.The detection method has a good correlation with the detection result of Ad5 carrier vaccine immunized mouse serum and the detection result of Ad5 neutralizing antibody.The application of this method to the detection of Ad5 binding antibody levels in the serum of mice immunized with Ad5 carrier vaccine fully demonstrated the Adantages of the method's speed,simplicity,high sensitivity and accuracy.The immune response of Ad5-EBOV specific antigen and carrier was evaluated on the Ad5 nasal immunized mouse model.After a single intramuscular injection of Ad5-EBOV into pre-existing immunized mice,the EBOV-GP specific Ig G antibody response was consistent with the Ad5 carrier Ig G antibody response,both of which reached a certain peak 2 weeks after immunization and were able to maintain stable More than 6 month.The effect of different booster immunization intervals on Ad5-EBOV immunization effect was further explored.The results showed that the EBOVGP-specific Ig G antibody response induced by Ad5-EBOV at different booster immunization intervals was consistent with the Ad5 vector-specific antibody response,and afterwards boosted immunity,the median fold increase of the two specific antibodies was 2.60 and 2.90 respectively.For EBOV-GP specific Ig G antibody response,when booster immunization was performed at intervals of 3 months and 6 months,the level of Ig G antibodies after booster immunization was slightly higher than that at intervals of 1 month and 2 months,but there was no significant difference.Long time intervals may result in higher levels of antibody specific to the target protein.Comparing the boosted immune response of pre-existing immunized mice with nonpre-existing immunized mice,the results showed that the effect of boosting immunization of mice without pre-existing immunization was significantly higher than that of pre-existing immunizing mice,suggesting that the Ad5 carrier antibody before boosting immunization hindered the enhancement of the boosting effect of pre-existing immunized mice.In view of the importance of memory B cells in the immune response of vaccines and the homogeneity of Ad5 vector and EBOV-GP specific memory B cell detection,the B-ELISpot test results can more reflect the specific immune response of Ad5 vector and EBOV-GP Similarities and differences.Ad5-Hexon and EBOV-GP specific BELISpot detection methods were established,and the optimal stimulation activation time of lymphocytes was 4 to 5 days.Using biotinylated Ad5-Hexon and EBOV-GP antigen for detection,the detection method has been optimized to make the detection spot of the specific antigen B-ELISpot clear and identifiable,and improve the detection specificity and reduce the detection background signal.Specific B-ELISpot detection was performed on mice within 4 weeks after immunization with a single intramuscular injection of Ad5-EOBV.The results showed that mice could detect EBOV-GP specific antibodies significantly higher than the control group within 1 week after immunization.The number of secretory cells increased gradually within 2 to 4 weeks after immunization,and the number of EBOV-GP specific antibody secreting cells was significantly higher than Ad5-Hexon.The specific B-ELISpot test was performed on mice that were boosted 4 weeks after the first intramuscular injection of Ad5-EBOV.The results showed that the number of EBOV-GP-specific memory B cells increased within 4 weeks after booster immunization.There was no significant difference from the previous one.The number of Ad5-Hexon-specific memory B cells suddenly increased 1 week after booster immunization,and decreased to slightly higher than the level before booster immunization 2 weeks after booster immunization.Although the number of EBOV-GP specific antibody secreting cells was significantly higher than Ad5-Hexon within 4 weeks after a single immunization,there was no significant difference in serum Ig G antibody levels between the two;at the same time,EBOV-GP specific antibody within 4 weeks after booster immunization,there was no significant difference in the number of secretory cells from Ad5-Hexon,but Ad5-Hexon-specific serum Ig G antibody levels were significantly higher than EBOVGP.This result shows that due to the different sensitivity of ELISA detection methods with different antigen-specific antibody,the detection results cannot directly comparable.At the same time,it also illustrates the importance of the B-ELISpot detection method in comparing the differences in the immune response between Ad5-EBOV specific antigen and carrier.The next work will focus on the detection of memory B cells in peripheral blood lymphocytes of Ad5-EBOV clinical trial subjects.Compared with the mouse model,the test results of the clinical test subjects will be more able to reflect the real Ad5-EBOV specific antigen and carrier immune response.