Construction And Protective Efficacy Of Two Swine Influenza Vaccine Candidates(H1N1)

Swine flu is an acute,hot and highly contagious respiratory disease,which is caused by Swine influenza virus(SIV).Due to the large number of subtypes and the high variation rate,swine influenza viruses are widely prevalent in pig farms around the world,including China,and caused a huge economic loss to the global pig industry.Vaccination is still the most effective way to prevent and control swine flu.Under the premise of ensuring the safety of the vaccine,in order to improve the protective effect of the swine influenza vaccine,a live virus vector vaccine r PRV-HA expressing the SIV HA gene and a baculovirus expression system expressing HA protein were prepared.And then,preliminary studies of the prime-boost immunization strategies of the two subunit vaccines were done,using the two vaccines alone or in combination of them.Through detecting the level of humoral immunity and cellular immunity in each group and pathological damage after challenge,weight loss,survival rate and other aspects,we compared the protective effect of different groups,and found the best immunization strategies,which paved the way for the follow-up study of swine influenza vaccine.The main contents of this study are as follows:1.Preparation of recombinant of variant pseudorabies virus r PRV-HA expressing SIV HA geneIn this study,recombinant PRV?TK?g I/g E was used as a vector and the recombinant virus r SMX?g I/g E?TK-opti HA expressing codon optimized H1N1 swine influenza virus HA gene was successfully constructed by traditional homologous recombination.The biological characteristics of the obtained recombinant virus were also studied,including the proliferation characteristics of the recombinant virus on a variety of cells,one-step growth curve,genetic stability,and the safety to mouse.The results showed that recombinant virus r SMX?g I/g E?TK-opti HA could stably express HA protein,and after optimization,the expression level of the target gene was significantly increased.The proliferation titers of the recombinant virus on ST,PK-15,Vero and BHK-21 cells were all higher than 107.0 TCID50/0.1m L,and the sensitivity to PK-15 cells was the highest.The one-step growth curve results showed that the proliferative properties of the recombinant virus were consistent with the parental strain.After continuous passaged for 20 generations,the recombinant virus still had a good genetic stability and was safe to mouse.2.Preparation of HA protein subunit vaccineIn this study,according to the instructions of Bac-to-Bac baculovirus expression system,the signal peptide and extracellular domain fragment of SIV HA gene were cloned into the transfer vector p Fast Bac HT B and the recombinant plasmid p Fast Bac HTB-HA was obtained.The correct recombinant plasmid p Fast Bac HTB-HA was transformed into E.coli DH10 Bac competent cells,and after three times of blue and white spot screening,the r Bacmid-HA containing recombinant HA gene was obtained.Subsequently,it was transfected into sf9 insect cells to obtain recombinant baculovirus r Ac MNPV-HA.After three passages,the sf9 cells was used to optimally express the recombinant protein.The results of SDS-PAGE and Western blotting showed that the recombinant protein could be expressed both in culture supernatants and cells,and the secretory expressions were all present in glycosylated form,while the expression in the cells was mostly in non-glycosylated form.It is worth mentioning that each form of recombinant proteins can combine with a high serum-free serum against pig SIV.3.Study on the immune effect of two swine influenza vaccine candidates(H1N1)Based on the successful construction of attenuated virus vector vaccine r SMX?g I/g E?TK-opti HA and HA protein subunit vaccine,four-week-old healthy female BLAB/c mice were randomly divided into 8 groups,of which 3 experiment groups and 5 control groups.10 in each group,and 5 in control group of non-immunized.Subcutaneous immunization with r SMX?TK?g I/g E-opti HA attenuated virus vaccine alone,HA protein subunit vaccine alone and the combined immunization of r SMX?TK?g I/g E-opti HA attenuated virus vaccine and HA protein subunit vaccine.(see Table 3-6 for details).Vaccinate 2 times with 4 weeks interval.After the first immunization,blood samples were collected at 0,2,4 and 6 weeks for detection of SIV and PRV ELISA antibodies,SIV HI antibody and cytokine IFN-?,respectively.After 4 weeks of booster immunization,the mice were intranasally attacked under ether anesthesia.The dose of challenge was 50 ?L H1N1(F9)per mouse with a LD50 of 102.38 TCID50.The weight of each mouse was weighed daily after the challenge,and the clinical symptoms and the death of the mice were observed.The results showed that r SMX?TK?g I/g E-opti HA attenuated virus vaccine alone,HA protein subunit vaccine alone and the combined immunization of two vaccines can induce specific humoral and cellular immune responses;r SMX?TK?g I/g E-opti HA had the best immunity.The results of challenge against SIV showed that the two vaccines alone and in combination had good protection for mice.The survival rates were 85.7%,85.7% and 71.4%,respectively.While the survival rate of PBS control group was only 42.9%.The individual immunization with r SMX?TK?g I/g E-opti HA was comparable to that of the two vaccines and was higher than that of the HA subunit vaccine alone.Therefore,it can be initially determined that individual immunization with r SMX?TK?g I/g E-opti HA has the best protective effect.