The Study Of Influenza A Virus Universal Virus Like Particles Vaccine And The Evaluation Of Universal Antibodies

Influenza A virus(IAV)is a capsular,segmented,negative-strand RNA virus belonging to Orthomyxoviridae family,influenza virus.Influenza A virus(IAV)consists of eight gene segments,each of which encodes at least one protein.To date,IAV encodes 13 viral proteins.Based on the antigenic differences between the viral surface proteins hemagglutinin(HA)and neuraminidase(NA),IAV can be divided into 18 HA and 11 NA subtypes.In recent years,the number of viruses of the influenza A virus that crosses the barrier of animal-human host species has increased,posing a serious threat to human health.Currently,influenza virus vaccines are an effective means of preventing influenza viruses,especially for seasonal influenza.However,the main antigen hemagglutinin(HA)of influenza virus is susceptible to gene mutation.When the vaccine strain does not match the current epidemic strain,the original influenza virus vaccine will lose the protective effect on the newly emerged epidemic strain,increasing the risk of the influenza virus spreading in humans.Therefore,the research on the influenza A virus universal virus like particles vaccine and universal antibodies are of great significance for coping with the epidemic of influenza virus after antigen drift variation in humans and animals.1.The universal influenza virus vaccine has several research directions,including the conserved stalk domain of HA,the extracellular domain of M2 ion channel(M2e),internal protein nuclear protein NP and matrix protein M1.We selected the highly pathogenic avian influenza H5N1 A/meerkat/Shanghai/SH-1/2012(SH-1;clade 2.3.2.1;group 1)hemagglutinin stem protein(HLHA),nuclear protein(NP),matrix protein(M1)and influenza A virus fusion protein 5M2e(2 human sources M2e,1 pig source M2e and 2 avian sources M2e),and the fusion protein HL5M2e(5M2e chimeric into HLHA to replace the position of the HA head)as target antigen genes,constructed and prepared AIV VLPs by insect-baculovirus expression system,and carried out immunogenicity studies.Mice were immunized by the intranasal route,the results of mouse challenge experiments showed that the three AIV VLPs showed a protection rate of 80%-90%for BALB/c mice.All data show that HL5M2e AIV VLPs are the most immunogenic.Therefore,the development of a universal AIV vaccine based on HL5M2e AIV VLPs has potential development space.2.AIV first invades the mucosal tissues of the host’s nasal cavity,trachea,lungs,etc.Therefore,we consider that the immunization vaccine should be based on mucosal immunity that can induce mice,so that mice can obtain enough mucosal immune antibody slgA to make the first line of defense.There,based on HL5M2e AIV VLPs,we rescued the recombinant baculoviruses rpFastBac-Dual-2GPI-CCL28 and rpFastBac-Dual-2GPI-GM-CSF.We prepared chimeric AIV cVLPs(CCL28 cVLPs、GM-CSF cVLPs and CCL28/GM-CSF cVLPs).Comparing to AIV virus-like particles HL5M2e VLPs,when encountering the homologous A/meerkat/Shanghai/SH-1/2012(H5N1;SH-1;clade 2.3.2.1;group 1)(100%)and heterologous mouse-adapted A/Changchun/01/2009(H1N1;group 1)(50%-60%),mouse-adapted A/baikal teal/Shanghai/SH-89/2013(H3N2;group 2)(40-50%),and mouse-adapted A/Lesser White-fronted goose/HuNan/412/2010(H7N7;group 2)(10-20%)influenza viruses all showed certain attack protection activities.3.We constructed and expressed three universal humanized antibodies F10,H98 and H40 of influenza A virus.Then,we identified their hemagglutination inhibitory activity and microneutralization assays.The results show that humanized mAb F10 produces a higher neutralizing titer and a broader neutralization range than the other two humanized mAbs,indicating that the humanized mAb F10 can be used as a basic antibody for constructing a random mutant antibody library,which lays a laboratory foundation for the subsequent antibody screening work.