Preliminary Studies On The Novel Adjuvants Inactivated Vaccines Of H9N2 Subtype Avian Influenza Virus

Objective:For developing a new type of H9N2 subtype avian influenza vaccine,a strain of avian influenza virus in north china was isolated and identificated in this study.The proportion and immune pathway of the inactivated vaccine constructed with novel adjuvant and antigen of this isolated strain were confirmed by a series of experiments,and the efficacy of this vaccine were evaluated.Methods:In this investigation,a strain of H9N2 avian influenza virus from North China was isolated by the method of chicken embryo inoculation and identified with the methods of serology and RT-PCR.The SPF chickens were immunized intramuscularly with vaccine contained 5%,10%or 15%novel adjuvant,and challenged with H9N2 virus on 18 days post inoculation.Sera were collected from all animals prior to challenge,and samples on oropharyngeal swab were collected at 120h after challenge for virus ioslation.HI antibody titer and protective rate were compared in each group.The SPF chickens were immunized with the vaccine contained 10%novel adjuvant intranasally,subcutaneously or intramuscularly,and the SPF chickens were artificially infected with H9N2 virus followed by sera collection for HI antibody titer dection on 12 days and 30 days post inoculation Samples on oropharyngeal swabs collected at 120h after challenge were used for virus ioslation.Efficacy of commercial vaccines A,B,C and novel adjuvant vaccine were compared by vaccinating SPF chickens subcutaneously and intramuscularly.The SPF chickens were challenged with H9N2 virus followed by sera collection for HI antibody titer dection on 22 days post inoculation.Samples on oropharyngeal swabs collected at 120h after challenge were used for virus isolation;The SPF chickens were immunized with the novel adjuvant vaccine and oil adjuvant vaccine,and sera were collected from ali animals for HI antibody titer dection on day 7,day 14,day 21 and day 24 post inoculation The leve of SIgA in throat,trachea,tracheal fork and lung was detected by ELISA kits.Samples on oropharyngeal swabs were collected at 120h post infection were used for virus isolation.All the data were analyzed with Excel and SPSS software.Results:The hemagglutination characteristics of this isolated strain could be inhibited by H9 standard serum and H9(Dacheng)positive serum at a titer of 10-5,the EID50 of this isolated viru is 10-8.25/0.1mL.The isolated virus whose HA fragment was 1770bp and that of NA fragment was 1470bp belong to Y280-like H9N2 subtype avian influenza virus,and was named as A/chicken/Huabei/2015(H9N2).The optimal concentration of the novel adjuvant was 10%.Preliminary results indicated that immune efficacy of the novel adjuvant vaccine by intramuscular was better than any of the other immune routes.The efficacy of novel adjuvant vaccine was better than any of commercial vaccine in efficacy.After immunization,novel adjuvant vaccine group serum antibody titer and respiratory SIgA levels were higher than that of oil adjuvant group,and the difference of OD between immune group and control group was significant,the titer of SIgA in novel adjuvant vaccine group was slightly higher than oil adjuvant vaccine group.but the difference was not significant.The serum antibody titer and rising trend of the novel adjuvant vaccine group was higher and faster than the control group.Conclusion:The virus isolated belonged to Y280-like H9N2 subtype avian influenza virus,named as A/chicken/Huabei/2015(H9N2).The H9N2 avian influenza vaccine with the novel adjuvant produced a better immune effect.especially vaccinating by intramuscular route.The immune efficacy of H9N2 avian influenza vaccine of 10%novel adjuvant was better than that of other three commercial vaccine and oil adjuvant vaccine.After immunization with novel adjuvants inactivated vaccine,SIgA level and serum antibody titer were higher than that of oil adjuvant vaccine in SPF chickens.