Immunogenicityofa Recombinant Adenovirus Co-expnessing The HA Gene Of H1N1and H3N2Subtype Swine Inlfuenza Virus

The swine flu is an acute infectious respiratory disease caused by the swine fluvirus with high morbidity and low mortality rate. It Often concurrents with theporcine reproductive and respiratory syndrome or porcine respiratory coronavirusdiseases clinically, making the condition worse and causing greater economic lossesto the pig industry.Besides, pigs make a great role in the influenza virus transmissionand epidemiological studies as a "mixer" between human influenza virus and avianinfluenza virus. SIV etiology and serological survey show subtype H1N1dnd H3N2have become advantage strains in China. Vaccination is an effective measure toprevent swine flu. There are still inadequacies of conventional inactivated vaccine onthe immune control of the disease, while there is a potential biological safety issuesabout attenuated vaccine, Therefore the development of new and effective vaccineremains be the prevention of influenza issues to be resolved.As a live virus vector, replication-defective adenovirus has a variety ofadvantages for example high efficiency of infection, security and large capacity offoreign genes. It has been widely used in gene therapy and genetic engineeringvaccine development in recent years.FMDV2A is the typical of self cut polypeptide, it has the advantages of Highshear efficiency, good balance of upstream and downstream gene expression andshort structure. And it provides a new way for divalent genetic vaccine.Multi-epitope vaccine is the vaccine which carries multiple epitopes targetantigens relates as well as complementary epitopes. It has the following advantagescompared with other vaccines: first it can overcome the genetic restriction of majorhistocompatibility complex (MHC) molecules, because one specific epitope only canbe binded by part of MHC molecule and be deliveried to T cells, generally singleepitope vaccines can not cause the expected immune response in all individuals, while careful combination of multi-epitope vaccines can be recognized and binded by avariety of genetic backgrounds MHC molecules resulting in efficient delivery; secondmulti-epitope vaccine can effectively cope with some of the adverse factors in thevariability of pathogenic microorganisms and the immune response; againmulti-epitope vaccine has the unique advantages in the induced cellular immune. Sothe multi-epitope vaccine has become one of tne research focus.In this study we successfully constructed the recombinant adenovirusrAd-H1-2A-H3that carries the H1N1and H3N2HA1, two another recombinantadenovirus separate express the H1N1HA1or H3N2HA1. In the process ofconstructed recombinant adenovirus, selected human type5adenovirus type5lack ofE1and E3for a vector, chose293T cells that could integrate adenovirus E1gene forpackaging cell line, by screening we obtained a recombinant adenovirus expressedHA gene of H1N1and H3N2, and another two recombinant adenovirus separateexpressed the H1N1HA1or H3N2HA1. Results of fluorescent、PCR and WesternBlot showed that the recombinant virus could transcribe and express target genescorrectly. Then we determined the TCID50of the recombinant adenovirus afteramplifying. We chose mice as an animal model for experimental immune study of therecombinant adenovirus, detecting of immune humoral immune response andchallenge protection in order to evaluate the immune effect.In the level of humoral immunity, ELISA results showed that on IgG antibodylevels Multi-epitope rAd-H1-2A-H3was significantly different with the singleexpression group rAd-H1and rAd-H3. After inoculation, recombinant adenovirusrAd-H1-2A-H3provided effective protection against the H1N1and H3N2SIV at thesame time, recombinant adenovirus rAd-H1and rAd-H3provided effective protectionagainst the H1N1or H3N2SIV respectively.Proved by animal immunization experiments in mice, the recombinantadenovirus co-expression and single expression all could produce a strong humoralimmune response. The results of this study laid the foundation for the development ofnew recombinant adenovirus influenza vaccine.