| People of different ages can be infected influenza by virus for its numerous subtypes and spreading rapidly.Over the past century, the influenza virus caused several pandemic of flu, which induce tremendous loss of life and property. For the past few years, avian influenza virus began to infect humans that constantly broke the species barrier and caused deaths. Traditional flu vaccines only protect against specific subtypes of strains, but for avian H5, H7 and H9 subtypes of influenza, it provid little or no protection.To develop a influenza vaccine which not only can protect major subtypes of human influenza, but also can protect subtypes of avian influenza virus that cross immunized become an new research direction. With the develepment of genetic engineering vaccine, genetic engineering vaccine of influenza such as subunit influenza vaccines, DNA influenza vaccine, influenza vaccine virus-like particles, multi-epitope vaccine and virus vector influenza vaccine grew fast.Adenovirus vector vaccine possess many advantages, such as safety, high titer of recombinant virus, widespread host cells, integration of large segments of exogenous gene and ability of causing a strong cellular and humoral immune response, making it to a hot research.To obtain functional epitopes of influenza viruse, we need analysis, predict, screen and assay the major protective antigen epitope of influenza virus. We need selectively added linker between the epitopes in order to achieve efficient cleavage.In this study, we constructed multi epitope adenovirus vaccine named pacAd5-H5-EHB-H3-M2 which include HA gene of H5 subtype of avian influenza, HA1 gene of H3 subtype of human influenza, epitopes of H1, H7,H9 subtype and M2 gene.We also constructed single expression adenovirus vaccine named pacAd5-H5HA and pacAd5-H3HA1.RT-PCR,Consequence of Immunofiltration assay (IFA) and Western Blot indicated that antigenic of adenovirus vaccine was well. Purified the multiple epitopes and single expression of recombinant adenovirus by a adenovirus purification kit and calculated the titer of virus particles.Mouse were employed to evaluate the immunogenicity of recombinant adenovirus vaccine. We set different immunization doses and different immunization procedures to immunize mouse.The result showed that the multi-epitope recombinant adenovirus group pacAd5-H5-EHB-H3-M2 related ELISA serum antibody level was equal to the same dose of pacAd5-H5HA but significantly higher than the low dose group(P<0.05).IgG antibody level of H1/H7/H9 subtype-specific epitope of multi-epitope group was significantly higher than the single expression group (P<0.05). The antibody level of Single immunity and twice immunity of PacAd5-H3HA1 was not significant in same dose.Experiment of Lymphocyte Transformation and detection of IFN-y ELIspot showed that the cellular immunity level of pacAd5-H5-EHB-H3-M2 with multi-epitope was significantly higher than the single expression group(P<0.05). When subtype-specific antigen of H1/H7/H9 stimulated, the addition of epitope box significantly enhanced the cellular immune responses of multi-epitope recombinant adenovirus group.The SI index and the spot numbers of ELIspot of single expression group with two immunity was not significant(P>0.05) compared to single immunity,and high dose group of adenovirus was significantly higher than that low dose immunization group (P<0.05).The results of the animal experiment confirmed that multi-epitope recombinant vaccine group pacAd5-H5-EHB-H3-M2, single expression of adenovirus vaccine pacAd5-H5HA and pacAd5-H3HA1 all can produce strong specific humoral and cellular immune response.But pacAd5-H5-EHB-H3-M2 is in the highest levels of immune response and when antigen associated with epitope stimulated it showed off a significant advantage.Our study had made benefical exploration in human-avian co-prevention influenza vaccine. |
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