Construction And Immunogenicity Study Of Transgenic Eimeria Expressing IgY/IgG Fc Fragment Fused To HA1from H9N2Avian Influenza Virus

Currently, commercial vaccines against H9N2avian influenza virus (AIV) predominantly induce humoral response. Despite higher humoral immunity response, the H9N2vaccines became less effective and H9N2influenza virus continues to circulate in vaccinated chicken flocks and has caused sporadic disease outbreaks. In addition, studieshave shown that cellular immunity by CD8+T cells play an important role in controlling influenza infection due to the faster response of this arm of the immune system. This has accelerated to devise more effective vaccine against influenza virus. Mammal IgG Fc can be used as a molecular adjuvant to enhance humoral response and cellular response to fused protein. It is approved that Eimeria species is a promising vaccine vectors for the presentation of antigen proteins from other pathogens. Therefore, in the study, we used chicken Eimeria as vaccine vector expressing HA1antigen from H9N2influenza virus fused to chicken IgY Fc or mouse IgG Fc and studied their immunogenicity in chickens and mice.In this study, we first detected the immunological enhancement of chicken IgY Fc to protective antigen from H9N2influenza virus with fused protein in chickens. The results showed viral excretion from immunized chickens after challenge in HA1chFc group was significantlyreduced, indicating chicken IgY Fc can be used as a molecular adjuvant similar to mammal IgG Fc. Based on this, four lines of transgenic Eimeira mitis and transgenic Eimeira tenella were constructed for further immunogenicity study, expressing chicken IgY Fc fragment (Emi.chFc, Et.chFc), HA1from H9N2influenza virus(Emi.HA1, EtHA1), fuesd chicken IgY Fc or mouse IgG Fc to HA1from H9N2influenza virus (Emi.HA1chFc, Emi.HA1mFc, Et.HA1chFc, Et.HA1mFc), respectively.AA chickens were immunized by transgenic E. mitis or E. tenella expressing chicken IgY Fc to detect the immune response against Eimeria antigen. The results showed that chicken IgY Fc expressed by transgenic Eimeira can enhance the immunogenicity of Eimeria antigen. Although specific influenza virus immune response wasn’t induced by transgenic Emi.HAlchFc and Emi.HAl in chickens, transgenic Emi.HA10mFc can elicit stronger influenza virus-specific IgA antibody and reduce weight loss after challenge compared to Emi.HA1and wild type E. mitis.We also detected immune response induced by transgenic E. tenella through common immunization with inactive H9N2influenza virus The results showed that inactive H9N2vaccine with E. tenella oocyst can increase HI antibody titers to influenza virus, number of IFN-y-producing cells and reduce influenza virus load in cohabitation SPF chickens. Similarly, Balb/c mice were immunized with subcutaneously mixed inactive H9N2influenza virus vaccine with E. tenella oocysts. Inactive H9N2vaccine with E. tenella oocysts can increase Thl type immune response and HI antibody titers to influenza virus, weight loss after challenge with H9N2influenza virus was significantly reduced. However, there was no difference between transgenic E. tenella and wild type of E. tenella.Taken together, chicken IgY Fc expressed by Eimeria can significantly enhance immunogenicity of Eimeria antigen, but not influenza virus antigen in chickens. In mice, fused mouse IgG Fc to HA1from H9N2influenza virus expressed by E. mitis can mount specific-influenza virus IgA antibody and reduce weight loss after challenge. Inactive H9N2vaccine adjuvant mixed with E. tenella can increase influenza virus-specific immune response and E. tenella oocysts have potential to use a molecular adjuvant. Our findings are encouraging for the development of a eukaryotic vaccine vector using genetically modified apicomplexan parasites and E. tenella as a vaccine adjuvant.