Studies On The Construction Of Phage-displayed Candidate Combined Influenza A And B Vaccine And Its Immunogenicity

Influenza is a most important global respiratory infectious disease. Influenzavirus typically infects 10-20% of the total worldwide population during seasonalepidemics, resulting in 3-5 million cases of severe illness and 250,000-500,000 deathsper year. Vaccine is the most utility device for preventing influenza, at present.Influenza hemagglutinin and neuraminidase are large glycoprotein, well accessible onthe viral membrane and highly immunogenic. Therefore, they are good choices asvaccine antigens. All commercial human influenza vaccines presently availablecontain hemagglutinin or neuraminidase as their only or main viral antigen. Theantigenic variation of hemagglutinin (HA) and neuraminidase (NA) by mutations(drift) and gene re-assortment (shift), pose a major obstacle to control the viral diseaseby vaccination. Therefore, influenza infections can occur repeatedly throughout lifeand protection by vaccination requires annual administration with updated vaccines. Itis necessary to develop universal influenza vaccines based on invariable regions ofinfluenza virus, which can produce crossing protection against influenza infection.M2-protein is a tetrameric proton channel, which encoded by virus RNA,sparingly present on the influenza A virus, but abundant on the virus-infected cells.The extracellular domain of the influenza M2-protein (M2e), which is not subject todrift or shift, has remained nearly invariable since the 1918 Spanish flu to this day.M2e contains 24 amino acid residues, and several studies have demonstrated thatinfluenza vaccines based on M2e exhibited effective protective immunity in varioustypes of vaccine constructs. The influenza B virus BM2 protein, which contains 109amino acid residues, has been found to have ion channel activity and is expressed atthe infected-cell surface. Although BM2 has its own specific function in the life cycleof influenza B virus, BM2 is considered to be a functional counterpart to M2 ofinfluenza A virus, might be a candidate epitope of universal influenza B vaccines.Influenza virus does not transmit between mice, which is most commoninfluenza virus studies animal model. Guinea pigs are highly susceptible to infectionwith the unadapted influenza virus. Influenza virus can pass between guinea pigs by means of droplet spread and thereby guinea pigs can be a new animal model forinfluenza virus transmission studies. Phage vaccines make phages as vaccine vector,in which specific epitopes are displayed on the phage surface, which is easier tomanufacture and to purifiy than traditional vaccine, and safer than DNA vaccine.Intranal immunization is a newly developed mucosal route for administration whichcan induce local and systemic immune responses. Nose associated lymphoid tissue isthe inducing site of intranal immunization, including T&B cells, dendritic cells and Mcells. Chitosan microspheres are a new kind of delivery system, which can promoteimmune response to microsphere vaccine.In our study, we constructed recombination phage IFV/T7 by fusing M2e andBM2 polytone IFV to the T7 surface caspid accessory protein 10B. Positive IFV/T7was selected by PCR, and was prepared by formaldehyde inactivation. Guinea pigswere immunized with microsphere phage candidate vaccine using the dose of 10¹⁰ pfuseparately. And then, with the dose of 10³ pfu, H3N2,H1N1 and influenza B viruschallenged the guinea pigs both immunized and non-immunized. We detected thevirus titers in upstream respiratory tract (URT) and lungs for studying the protectionof candidate vaccine against influenza virus challenge.After three vaccinations, anti-M2e and anti-BM2 were detected separately byELISA, showed that recombination phage candidate vaccine has goodimmunogenicity. Virus titers in the respiratory tracts of virus-infected guinea pigsindicated, microsphere phage candidate vaccine can protected the entire respiratorytracts of guinea pigs against H3N2 and H1N1 challenge; candidate vaccine could alsoprotect lungs against influenza B virus' challenge, but not URT.We concluded that (1) the recombined phage candidate vaccine can elicithumoral immune responses; (2) Anti-M2e antibody induced by candidate vaccine canprotect the whole respiratory tracts against H3N2 and H1N1 infection; (3) Anti-BM2antibody induced by candidate vaccine can protect lungs, but not URT, againstinfluenza B virus infection.