Study On The Immune Protective Evaluation Of The Universal Influenza Vaccine Based On NP-M2e Fusion Protein Of NM2e

Development of a universal influenza vaccine is important for the control and prevention of influenza pandemic. Over the past decades, universal influenza vaccine has made great progress. Previous studies have shown that influenza virus nucleoprotein (NP) and the matrix protein 2 ectodomain (M2e) are highly conserved among different subtypes of influenza A virus and are very promising universal influenza vaccine target antigen, various forms of universal vaccines based on the two antigens have demonstrated effective cross-protection in animal experiments. NP and M2e proteins purified from Escherichia coli (E. coli) expression system as universal vaccine candidates have became an important direction for its maturely fermentation, high-level expression and low cost. However, as the immunogenicity of this subunit vaccine is very weak and the efficacies of the NP-or M2e-based universal influenza vaccine could not be evaluated through the hemagglutination inhibition (HI) antibody titer as the seasonal influenza vaccine, there are still some crucial issues in the development and preclinical studies of universal vaccine, including the choice of target antigen and effective vaccine adjuvant, the establishment of a rational vaccine immune protection evaluation system, and the evaluation of immune protection of the universal influenza vaccine.Trying to resolve the above problems, this study was designed. Firstly, to determined the antigen and adjuvant that can be used in the universal influenza vaccine, we compared the protective immunity of NP protein and NP-M2e fusion protein of NM2e in BALB/c mice and investigated the influences and effects of the Al(OH)3, CpG, and Al(OH)3+CpG adjuvants in the NP and NM2e subunit vaccines. Secondly, as the hemagglutination inhibition (HI) antibody titer of 40 (HI=40) induced by seasonal influenza vaccine is an internationally vaccine effectiveness correlate of immune protection, we suggested to establish an immune protective evaluation system based on the seasonal influenza vaccine, that is, to evaluate the NP-and M2e-based universal influenza vaccine with the virus challenge dose that can be protected by the seasonal influenza vaccine of HI=40. Finally, based on this immune protective evaluation system, we evaluated the cross-protective efficacies, the immune protective lasting, the influences of the immune processes and the immune dose on the protective efficacy of the universal influenza vaccine based on NP and M2e. Main results were summarized as follows.1. Fusion protein of NM2e purified from E.coli expression system was used as a vaccine candidate for the development of universal influenza vaccine. Based on the protective efficacies comparison of the NP and NM2e proteins in BALB/c mice, we found that NP protein could only stimulate strong NP-specific humoral immune responses (106), while NM2e fusion protein could induce both NP- and M2e-specific humoral immune response (102). Adjuvants of Al(OH)3 and Al(OH)3+CpG improved the M2e-specific humoral immune responses dramatically, with the IgG antibody titer increased from 102 to 104, and also improve the cellular immune response, with the NP55.69 or M2e-specific specific IFN-? cellular immune responses are enhanced from <50 to 200SFC/106SMNC. According to the challenge experiment with 20LD50 PR, we found that the survival percent of NM2e protein immunized mice (27%) is better than that of the NP protein (13%) under same immunization dose of 10?g. Adjuvants of Al(OH)3, CpG and Al(OH)3+CpG improved the survival percents to 47%,27% and 83% respectively for NP protein immunization group and to 93%,40% and 100% respectively for the NM2e protein immunization group. The protective efficacy induced by the NM2e fusion protein (93%) is also better than that of the NP immunization group (47%) with the same adjuvant of Al(OH)3. As the adjuvant of Al(OH)3 is a safety and successful vaccine adjuvant for human, the E.coli expressed NM2e fusion protein was chose as vaccine antigen, and the Al(OH)3 adjuvanted NM2e was used as an universal influenza vaccine for future study.2. Established the immune protection evaluation system based on seasonal influenza vaccine. Five strains of influenza virus were chosen to be passed in the mouse lung and three strains of mouse-adapted influenza virus succeed obtained, that is, two influenza virus strain of A1-P5-E1(H1N1) and GD-P40-E1(H1N1), which were well-matched homologous with the current H1N1 seasonal influenza vaccine strain, one heterogonous subtype influenza virus of A3J-P5-E1(H3N2); according to the study of immunogenicity, antibody kinetics and the immune protective efficacies in mice, the protective efficacies of seasonal influenza vaccine were confirmed. Results demonstrated that 1.5?g HA of seasonal influenza split vaccine could induce the HI antibody titer of 40 after one dose in mice, this dose of HA could effectively protect mice against 1000LD50 of well-matched and homologous subtype of influenza virus strain, but hardly to protect mice against 3LD50 of poor-matched homologous or heterogonous subtype of influenza virus strain. As the influenza virus's lethal dose of ILD50 in mice is 103-104TCID50, which is 10 to 100 times of that in the human natural infection by droplets with the dose of 102 TCID50, we suggested established the evaluation system based on seasonal influenza vaccine to evaluate the immune protective efficacies, that is, immunized with 1.5?g HA of seasonal influenza vaccine, challenged by the well-matched homologous subtype, poor-matched homologous and heterogonous subtype of influenza virus strain, and the virus challenge doses could be ranged from 3 LD50 to 1000LD50.3. Evaluated the immune protection efficacy of the NM2e+Al(OH)3 universal influenza vaccine. According to the immune protective evaluation system based on seasonal influenza vaccine, we evaluated the cross-protective efficacies, the protective lasting, the immune procedure and the immune doses of the NM2e+Al(OH)3 universal influenza vaccine. The results of cross-protective studies indicated that, although the H1N1 seasonal influenza vaccine could protect mice against challenge by 100LD50 or 300LD5O of well-matched and homologous subtype of influenza virus strains of A1-P5-E1 (H1N1) and GD-P40-E1 (H1N1), it could not protect mice against challenge with 3LD50 of poor-matched and homologous or heterogonous subtype of influenza virus strain of PR8 (H1N1) or A3J-P5-E1 (H3N2). On the contrast, the NM2e+Al(OH)3 universal influenza vaccine could protect more than 50% immunized mice against the above virus challenge with three immunization doses of 2.5?g,5?g or 10?g, among which, the 10?g NM2e+Al(OH)3 immunization group showed the most effective, with survival percents of 100%,100%,50% and 67% against the challenge of 20LD50 of PR8,100LD50 of Al-P5-E1,300LD50 of GD-P40-E1, and 30LD5O of A3J-P5-E1 respectively, the above results demonstrated that the NM2e+Al(OH)3 universal influenza vaccine could induce well broad-spectrum protection against strains of influenza virus challenge with the challenge dose ranged from 3 LD50 to 100 LD50, what's more, the NM2e based influenza vaccine also showed a better protective efficacies against poor-matched homologous (A3J-P5-E1) or heterogonous (PR8) subtype of influenza virus strain than the seasonal influenza vaccine; To investigate the immune procedure and immune protective lasting, we studied the antibody kinetics of the NM2e+Al(OH)3 universal influenza vaccine after the one-dose, two-dose and three-dose immunization procedures, and chose the immune procedure of two-dose (0,14) and three-dose (0,14,28) to immunize mice with 10?gNM2e+Al(OH)3. The protective lasting demonstrated that survival percents induced by the above two-dose or three-dose immune procedures could reach 90%,70 and 50% when challenged with 20LD50 of PR8 at week 2, month 2 or month 6 after the last immunization, this results indicated that the protection lasting could be up to at least 6 months for the NM2e+Al(OH)3 universal influenza vaccine. To evaluate the cross-protective efficacies and the immunize doses range for the large-scale produced NM2e universal influenza vaccine, the large-scale produced NM2e protein were immunized with or without adjuvant under the doses of lO?g,30?g,90?g and 270?g, results showed that this large-scale produced NM2e vaccine's immunogenicity and protective efficacy were as good as the laboratory products with the immunization doses of 10?g,30?g and 90?g. The above results provide bases for the preclinical study of NM2e universal influenza vaccine.All the above results suggested that, prokaryotically expressed NM2e fusion protein adjuvanted with A1(OH)3 could be used for the development of universal influenza vaccine; Immune protective evaluation system based on seasonal influenza vaccine could be used to evaluate the protective efficacy in the preclinical study of universal influenza vaccine based on NP and M2e protein; All the immune procedure, immune doses and immune protective efficacy results of this study provided valuable foundation for the preclinical study of NM2e universal influenza vaccine.