Preliminary Study On Universal A Influenza Vaccine With Hepatitis B Virus Core Particle As Vector

【Backgrounds】Influenza is a severely contagious respiratory disease caused by influenza virus which belongs to the family of Orthomyxoviridae. Influenza virus is classified into three types based on the antigenicity of their nucleoprotein (NP) and matrix protein (M). Influenza A viruses have caused four pandemics during the last century. The pandemic of 1918 killed as many as 50 million people worldwide. The pandemic of influenza A (H1N1)2009 virus, characterized by its rapid transmission and large scale epidemics, has a potential to cause fatal infection and exert high economic burden to humans.Inoculation remains the most effective approach for influenza prophylaxis and control. The inactivated vaccine, split vaccine and subunit vaccines are currently used in humans. These vaccines are effective to the homosubtypes of influenza virus, but have little cross protection to the different subtype of influenza virus. In addition, the genome of influenza virus is vulnerable to mutate. This phenomenon makes it more difficult to produce vaccines to counter the newly mutated influenza virus annually. WHO will recommend the component of influenza virus annually based on the changing of influenza virus.However, there will be less efficient if there is any inaccuracy in predicting the influenza virus strains. It brings a latent threat of abrupt pandemic of influenza. Therefore, it is greatly significant to develop one universal influenza vaccines which could provide broad-spectrum protection for controlling the abrupt pandemic caused by mutated influenza virus, building the immunologic barrier, block the epidemics of influenza and abase the perniciousness of influenza pandemics.Matrix protein (M)2 is an Influenza A membrane protein with an extracellular domain of 24 amino acid residues, which is strongly conserved among influenza A virus strains, and its use as a vaccine antigen can induce special protective antibody. NP is one of influenza nucleoproteins, and its sequence is highly conserved. On the surface of NP, there are many CTL epitopes which can induce cellular immunity. Accordingly, the highly conserved M2e and CTL epitopes on NP are thought to be excellent universal influenza vaccine antigen targest. However, the small molecules of M2e and NP epitopes are and weak antigens and vulnerable to degradation, which only induce poor immunity response in body and hardly induce enough immunoprotection. Therefore, using adjuvant or virus protein constructs as vector to elevate immunogenicity has been a hot topic in development of universal influenza vaccine. Hepatitis B virus core protein (HBc) is capable of constructing into VLP and inducing specific humoral and cellular immunity targeting to the exogenous epitopes. Recently, more successful results gained in the application of HBc particles. The satisfied effectiveness was observed in clinical with HBc as the vector in vaccination with the hepatitis B surface antigen, E7 of human papilloma virus (HPV), and CPS of Streptococcus B antigens. Therefore, it is promising and practical to develop universal influenza epitop vaccine with Hepatitis B virus core protein as vector.In animal experiments, the immune response and cross-protection effect of candidate universal influenza vaccine were preliminary evaluated. By ELISA, FACS, ELISPOT, the vaccine immune response effect in Balb/C mice were evaluated. Results showed, special humoral immunity and cellular immunity against different subtypes influenza virus were induced through both intranasal and intraperitoneal vaccination. And intranasal vaccination could induce higher level mucosa local immune response. This demonstrated that intranasal vaccination which more like to natural immunity could significantly enhance protection effect of influenza vaccine. Meanwhile, challenge experiments results showed, mice mortality in vaccine group was distinguishably lower than control group. It indicated that our universal influenza virus vaccine with hepatitis B virus core (HBc) particle as a vector could induce fine cross-protection immunity.【Objective】Our objective is to develop a universal A influenza vaccine antigen based on M2e with Hepatitis B virus core protein as vector. The vaccine antigen will be generated in Baculovirus and Escherichia coli expression system. After purification and identification of antigen, we will preliminarily evaluate the efficiency of vaccine by animal experiments. This research lays a foundation for developing a safe and effective universal A influenza vaccine.【Methods and Results】1. Construction, expression, purification and identification of universal influenza vaccine by Bac-to-Bac baculovirus insect cell expression system 3M2e-HBc-pFastBacHTA recombinant plasmid was constructed by genetic engineering methods. Then 3M2e-HBc-pFastBacHTA plasmid was transformed into DH10Bac cells. Through homologous recombination, we got the 3M2e-HBc-Bacmid recombinant bacmid. Then, the isolated Bacmic DNA was transfected into sf9 nsect cells. By immunofluorescence, the recombinant protein was detected in insect cells infected by recombinant baculovirus. Immunological activity was detected by WB assay. 3M2e-HBc recombinant protein was purified by affinity chromatograph. Electron microscope images displayed that 3M2e-HBc gene correctly expressed and assembled to form VLP in Bac-to-Bac baculovirus expression system.2. Construction, expression, purification and identification of universal influenza vaccine by Escherichia coli expression system3M2e-NP-HBc-pET21a recombinant expression plasmid was constructed by genetic engineering methods and was transform into Escherichia coli cells. At the beginning, inclusion body protein was got after inducing with IPTG. However, undertaking denaturation and renaturation procedure, the state of VLP formed by inclusion body protein was bad. Through the optimization of induction condition, partly dissoluble protein was expressed and gained. Then, 3M2e-NP-HBc recombinant protein was purified by affinity chromatograph and gel chromatograph. Western blot result indicated the purified protein had immunogenicity. Electron microscope images displayed that, the dissoluble 3M2e-NP-HBc recombinant protein could correctly assembled to form VLP in Escherichia coli cells. The size of VLP was approximately 30nm.It was shown that we successfully construct universal influenza vaccine expressed by Escherichia coli expression system.3. Evaluation of protective efficiency of universal influenza vaccines on Balb/C mice. In animal experiments, the immune response and cross-protection effect of candidate universal influenza vaccine were preliminary evaluated. By ELISA, FCM, ELISPOT, the vaccine immune response effect in Balb/C mice were evaluated. Results showed, special humoral immunity and cellular immunity against different subtypes influenza virus were induced through both intranasal and intraperitoneal vaccination. And intranasal vaccination could induce higher level mucosa local immune response. This demonstrated that intranasal vaccination which more like to natural immunity could significantly enhance protection effect of influenza vaccine. Meanwhile, challenge experiments results showed, mice mortality in vaccine group was distinguishably lower than control group. It indicated that our universal influenza virus vaccine with hepatitis B virus core (HBc) particle as a vector could induce fine cross-protection immunity.【Conclusion】In this study, we constructed two universal influenza virus vaccines with hepatitis B virus core (HBc) particle as a vector by Bac-to-Bac baculovirus insect cell expression system and Escherichia coli procaryotic expression system. By animal experiments, we evaluated the vaccine which was expressed in Escherichia coli. It showed that our influenza vaccine had fine immunegenicity. This vaccine induced system immune response and mucosa local immune response, and provided effective cross-protection. This research provided a base theory for the further development of universal influenza vaccine.