Study On Recombinant BacuLovirus-Mediated Genetic Engineering Vaccine Of Influenza Virus A (H1N1)

Influenza is an acute infectious disease transmmited via respiratory tract that significantly threatening human health and the global economy. In April 2009, several human cases infected with a novel H1N1 influenza virus A were reported in Mexico and the united states and then spread rapidly to many other countries all around the world. The WHO raised the outbreak alert level to the pandemic phase 6. Vaccination is the most effective measure to prevent and control influenza and currently, the inactivated vaccines were widely used. Though providing highly effective protection, the vaccines have some shortcomings, including the long time period for preparation and the requirement of large quantity of chicken embryo. Moreover, allergic reactions to residual egg proteins in the vaccine components can occur in some individual. So researchers have been attempting to develop a more secure and efficient flu vaccine.The bacuLovirus vector has long been used as a tool for the expression of exogenous proteins in insect cell. However, several groups rencently reported that the bacuLovirus express system, when driven by some appropriate eukaryotic promoters, can efficiently transduct different types of mammalian cells and express foreign genes. Furthermore, Same gene in the control of different promoters, its expression level will be have big difference.the bacuLovirus modified via the glycoprotein of vesicuLar stomatitis virus (VSV-G) has a broadened host range and dramatically increases the transduction efficiency in mammalian cells.Based on this, the current study aims at new explorsation of the disgne of gene engineering vaccines of H1N1 Inluenza A virus. BacuLovirus with vesicuLar stomatitis virus glycoprotein was constructed and used as a vector to express proteins of influenza virus A(H1N1) (A/California/04/2009). The immune potency of constructed vaccines was estimated on mice model. The main research work is as following:1.Construction of recombinant bacuLovirus (BV-CMV-HA/M1/NA,BV-ie1-HA/M1/NA)We used white spot syndrome virus (WSSV) promoter iel promoter and CMV promoter to construct two recombinant bacuLoviruses with vesicuLar stomatitis virus glycoprotein displayed on the envelope surface that expressed three immunogenic proteins of H1N1 influenza A virus, the hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of influenza virus A(H1N1) (A/California/04/2009). The two bacuLovirus vaccines are named BV-CMV-HA/M1/NA and BV-iel-HA/M1/NA.2. Preparation of polyclonal antibodies and expression of recombinant bacuLovirus (BV-CMV-HA/M1/NA,BV-iel-HA/Ml/NA) in VitroOn the basis of HA sequence, primers were designed to amplify subunit tHAl of HA, and by using genetic engineering methods, prokaryotic expression plasmid pET28a-tHA1 was constructed and transformed into E.coli BL21 (DE3) competent cells. The recombinant protein in the form of inclusion body was obtained after IPTG induction. The target protein was cut from SDS-PAGE gel and was grinded with 1ml PBS per 1mg gel. A multiple-point hypodermic injection at the back was given to mice at a dose of 200ul. Another injection in the same way was given two weeks later and then a strengthening immunization was given every week and sera were collected for antibody detection. When the antibody fulfilled the requirement, blood was collected at the eye socket and sera were harvested in large quantity. Mice-derived polyclonal antibodies were obtained.Recombinant bacuLovirus (BV-CMV-HA/M1/NA,BV-ie1-HA/M1/NA) were transducted to BHK-21. IFA showed those two recombinant bacuLovirus exhibited high transduction efficiency and the expression level of HA protein was high in mammalian cells.3. The immune effects induced by the recombinant baculovirus (BV-CMV-HA/M1/NA,BV-ie1-HA/M1/NA) in a mouse modelTo investigate the immune effects of constructed bacuLovirus BV-CMV-HA/M1/NA and BV-ie1-HA/M1/NA, mice were intramuscuLarly injected at a dosage of 109 PFU per mouse, and inactivated vaccines of H1N1 influenza A virus was injected as control. The results indicated that the recombinant bacuLovirus (BV-CMV-HA/M1/NA, BV-ie1-HA/M1/NA) and inactivated vaccine all induced a stronger humoral immune response. In the aspect of celluLar immunity, the transcription and expression level of IFN-γwas tested and the resuLts showed that the recombinant vaccines BV-CMV-HA/M1/NA and BV-ie1-HA/M1/NA induced a stronger celluLar immune response than inactivated vaccines. Genenally, BV-CMV-HA/M1/NA and BV-ie1-HA/M1/NA have the equivalent immune effect.4. Preparation of mouse-adapted variants of H1N1 virus and Protective immunity induced by the recombinant bacuLovirus (BV-CMV-HA/M1/NA,BV-ie1-HA/M1/NA) in a mouse modelThe influenza A H1N1 virus (A/wuhan/10/2010) was passaged in the mouse lung for 7 generations, generating an mouse-lung-adapted influenza A H1N1 influenza strain lethal for mice.The resuLt of challenging indicated that all the three vaccines can induce an immune reponse and resisted the H1N1 influenza A virus at a lethal dosage, providing a foundation for the development of safe and effective vaccines against H1N1 influenza A virus.