Development Of Lung Adaptation X31(H3N2) Influenza Virus In Mice With High Lethality And Evaluation Novel Universal Influenza A Vaccine Based On NM2e Formulated With Nanoparticles Adjuvant

Objective To establish mouse-adaptation strain of influenza A virus X31?H3N2?with high pathogenicity in BALB/c mice,and to analyze the potential molecular mechanism of adaptation of X31;and to evaluate the immune protection of a novel influenza nanoparticle vaccine NM2e+DP in BALB/c mice.Methods 1.The adaptation of influenza A virus X31 in BALB/c mice:BALB/c mice were infected with influenza A virus X31?H3N2?intranasally and mouse adaptation strain with high pathogenicity was established by serial passages in the lung of BALB/c mice,the molecular mechanism of adaptation were analyzed by next generation sequencing.2.BALB/c mice were immunized with novel influenza vaccine NM2e+DP intramuscularly or intranasally,we also immunized mice in parallel with NM2e and NM2e+Al?OH?₃+CpG vaccines.Then humoral and cellular immune responses were analyzed by ELISA and ELISPOT respectively;mice were challenged with homologous influenza A virus X31?H3N2?and heterologous influenza A virus PR8?H1N1?intranasally on the 14th day post the last immunization,and then weight alterations and survivals were recorded daily for 2 weeks.And the protective effects of different adjuvant vaccines were compared.Results1.Based on the low-pathogenic H3N2 influenza A virus X31 prototype strain,high pathogenic mouse lung-adapted strain X31_MA was obtained after 10 consecutive passages in the lungs of BALB/c mice.And the virus titer in lung homogenate was significantly increased after adaptation(TCID50 titer from P1 generation was below10/ml,while P10 generation was increased to 10^3.5/ml),and all mice infected with X31 P1 virus survived,while mice infected with P5 and P10 virus appear death at 6days post-infection,and all mice died on the 8th day post-infection.X31_MA-infected mice appear death at 3 days post-infection,and all died at 6 days post-infection.Whole-genome sequencing revealed that there were 4 amino acid mutations in the mouse lung-adapted strain X31MA compared with the prototype strain X31.Among them,hemagglutinin HA was mutated at 3 amino acid positions,and PB2 had 1 amino acid site mutation.).2.The novel influenza nanoparticle vaccine NM2e+DP immunized intramuscularly induced NP and M2e-specific humoral immune responses in BALB/c mice with IgG titer of 1:10⁶ and 1:10⁴ respectively,and induced cellular immune responses against NP_55-69 and peptide pool of M2e;immunization with NM2e+DP intramuscularly protected 100%and 90%of mice against the lethal challenge of 20LD50 homologous X31 and heterologous PR8;immunization of NM2e+DP intramuscularly induced stronger protection than intranasally.Conclusions The H3N2 highly pathogenic mouse lung adaptation strain X31MA was successfully obtained,and the amino acid mutations in HA and PB2 proteins were closely related to the lung adaptation of H3N2 in mice.When immunizing BALB/c mice by intramuscular injection,the novel nanoparticle adjuvant DP enhances the immunogenicity and protective effect of NM2e fusion protein,and its potency is comparable to that of Al?OH?₃ CpG,which induces stronger immune protection against homogeneous H3N2(X31_MA)or heterogenous H1N1?PR8?influenza challenge;the immunity and protective effect induced by intramuscular injection is better than intranasal immunization.