The Study On The Mechanism Of AddaVax Combined With Poly I:C To Enhance The Immune Response Of Influenza Vaccine

?Background?Despite the immunogenicity of influenza vaccines,the protective efficacy of existing influenza vaccines is limited during influenza pandemics and in immunocompromised population.Therefore,adjuvants are needed to enhance the human response to influenza vaccine,and it has become a new research trend to study novel combined adjuvants that could promote both Th1 and Th2 immune responses.Toll-like receptor agonists,which could promote Th1 immune responses,has been extensively studied as vaccine adjuvants.Oil-in-water adjuvants,which could promote Th2 immune response,could be used as influenza vaccine components.Therefore,this study investigated the immune enhancement mechanism of the combined oil-in-water adjuvant Adda Vax and the TLR agonist Poly I:C adjuvant(AP)on influenza vaccine and its application.?Methods?1.Enhancement effect of AP adjuvant on the humoral immune response of influenza vaccine.Advantages of AP adjuvant activity:monovalent H3N2 vaccine(5?g)combined with AP adjuvant and traditional aluminum adjuvant,the serum antibody titer of mice was detected 21 days after the primary immunization;Dose-dependent relationship:BALB/c mice and Wistar rats were administered with 3?6?9?15?g doses of monovalent H3N2vaccine combined with AP adjuvant,and the antibody titers were detected 21 days after the primary immunization;B cells response:BALB/c mice were immunized with monovalent H3N2 vaccine(5?g)compatible with AP adjuvant,4,7,14,21,28 days after the primary immunization,4,28 days after the booster immunization,Elispot test was used to detect HA-specific antibody-secreting cells,and HI test was used to detect antibody titer;Long-term antibody:BALB/c mice were immunized with monovalent H3N2 vaccine(5?g)combined with AP,the antibody titer was detected on the 21st,100th,and 270th day after the booster immunization completed;Immunogenicity of tetravalent split influenza vaccine(IIV4):IIV4 was combined with AP adjuvant for 0-21-day immunization schedule,and antibody titers for the four influenza subtype strains were detected on 21 and 100 day post the booster immunization.2.The relationship between immunogenicity and immune microenvironment.Mice were injected with monovalent H3N2 vaccine and AP adjuvant mix or separately at 1,24,and 48 hours at intervals,and the antibody titers were detected 21 days after the imminization;0,24,48,and 120 hours after the immunization of mice with monovalent H3N2 vaccine and AP adjuvant,the level of local cytokines was detected by Flow Cytometry;4 and 24 hours after AP adjuvant immunization,q RT-PCR was used to evaluate the levels of local chemokines and cytokines;The local macrophage response was detected two days after the immunization to evaluate the changes in immune microenvironment.3.DC cell-mediated immune response.Immature BMDCs from mouse bone marrow were isolated and cultured in vitro,after the stimulation with AP,Adda Vax,Poly I:C,the expression of mature molecules on the surface of BMDCs was detected by Flow Cytometry and the secretion of cytokines was detected by ELISA;Under the stimulation of Cy5.5-H3combined with AP,Adda Vax or Poly I:C,the uptake of Cy5.5-H3 by BMDCs was detected;BMDCs and immunized mouse spleen T cells were co-cultured,and was stimulated with H3N2 combined with AP,Adda Vax,and Poly I:C,then the level of secreted IL-4 and IFN-?was measured.4.Immune response in lymph nodes.Mice were injected with Cy5.5-H3 combined with AP,and two days after immunization,the location of Cy5.5-H3,B cells,T cells,macrophages and DC cells in draining lymph nodes was detected by immunohistochemistry;The changes of immune cells levels in the draining lymph nodes of mice were detected 0,24,and 60 hours after the immunization with the mixed monovalent H3N2 vaccine and AP adjuvant;The number of germinal centers in the draining lymph nodes was detected on 4,7,14,21,28 day after primary immunization and 4,14,28 days after booster immunization of mice with the mixed monovalent H3N2 vaccine and AP adjuvant.5.Immune response induced in spleen cells.On the 5th day after the booster immunization of mice with H3N2 and AP adjuvant,the mouse spleen lymphocytes were stimulated with H3N2 antigen for two days,and the cytokines secreted by the spleen cells were detected Flow Cytometry.At the same time,ELIspot was used to detect the level of IL-4 and IFN-?secreting T cells,and on the 5th day after the booster immunization,ELISA was used to detect the level of H3N2 HA-specific Ig G,Ig G1 and Ig G2a antibodies.6.Antigen metabolization.BALB/c mice were immunized with Cy5.5-H3 in combination with AP,0,2,5,7,9,11,13,and 15 days after the immunization,the metabolism of Cy5.5-H3 in mice was detected by vivo imaging.7.Tolerability evaluation of adjuvant vaccine.To evaluate the impact of high-dose adjuvants and vaccines on mice,BALB/c mice and Wistar rats received three high-dose AP or H3+AP,each injection two weeks apart,body weight and vital signs were monitored for7 consecutive days and on 14th days after each injection.On the 7th day after the third immunization,peripheral blood was taken for C-reactive protein level detection;Three days after the first immunization,the size and texture of the draining lymph nodes was measured and scored.The spleen was separated and weighed 5 days after the immunization for the the spleen index calculation.8.The protective effectiveness of adjuvant influenza vaccine.BALB/c mice were immunized with three subtypes monovalent influenza seasonal influenza vaccine respectively.On the 28th day after booster immunization,mice were infected with the matched lethal dose of influenza virus,and the efficacy of the vaccine combined with AP in vivo was evaluated by the body temperature,survival rate,lung tissue viral load,pathological changes and death of mice.?Results?1.AP adjuvant enhances the humoral immune response to monovalent H3N2 vaccine and IIV4.Advantages of AP adjuvant activity:compared with the traditional aluminum adjuvant,the antibody titer of mice immunized with H3+AP was significantly higher than those immunized with H3+Al(OH)₃;Dose-dependent relationship:the dose-dependent relationship was supported in the adjuvant-vaccine group and the vaccine group;B cells response:H3N2 combined with AP promotes the proliferation and differentiation of H3N2HA-specific antibody secreting cells,and promotes the secretion of H3N2 HA specific antibody,four days after the booster immunization,the antibody-secreting cells and antibody titers increased significantly;Long-term antibody:the HI antibody titer of mice immunized with monovalent H3N2 vaccine(5?g)combined with AP,Adda Vax,Poly I:C showed that the antibody titer was the highest at 21 days after the booster immunization,and the antibody titer decreased gradually with time increasing,but the antibody titer of the H3+AP group was always higher than that of the other groups at 21,100,200,and 270 days after the booster immunization,the antibody titer of the H3+AP group was still higher than40 on the 270 days after the booster immunization,which was higher than the threshold of antibody protection;Immunogenicity of IIV4:the HI titer and MN titer of four influenza strains of mice immunized with IIV4+AP were higher than those immunized with IIV4.2.The relationship of immunogenicity and immune microenvironment.There was no statistical difference of antibody titer between the mixed H3+AP and the separately injected H3N2 and AP(0h).The longer the injection interval of monovalent H3N2 vaccine and AP,the lower level of antibody titer induced;The local transient cytokine level of IL-5,IL-6,IFN-?,IL-4,IL-10,IL-12,IL-13,IL-17,and TGF-?induced by H3+AP higher than those induced by H3N2,the m RNA expression levels of IL-6,CCL2,CCL3,CXCL1,CXCL2,CXCL3,CXCL5,IFN-?,and IFN-?induced by AP increased compared to PBS;Two days after H3+AP immunization,the number of macrophages(M,M1,M2)in the injection site increased.3.DC cell-mediated immune response.AP promoted the expression of mature markers CD40,CD80,CD86 and the secretion of cytokine IL-12 of immature BMDCs;AP promotes the uptake of Cy5.5-H3 by BMDCs;H3+AP promotes the secretion and expression of IL-4 and IFN-?from the co-cultured BMDCs and spleen T cells.4.Immune response induced in draining lymph nodes.Two days after the immunization with Cy5.5-H3 or Cy5.5-H3+AP,Cy5.5 fluorescence could be detected in the center of draining lymph nodes in Cy5.5-H3+AP group,but not in H3N2 vaccine group;4,24 and 60hours after the immunization,the number of B cells,neutrophils,macrophages,and DC cells increased after the stimulation of H3+AP and H3N2 in draining lymph nodes.On the14th and 28th day,H3+AP promoted the number of germinal centers in the lymph nodes.5.Immune response induced in spleen cells.On the 5th day after the booster immunization,the expression of antigen-specific IL-1,IL-4,IL-5,IL-6,IL-10,IL-13,IFN-?in H3+AP group was higher than that in H3N2 group.The number of IL-4 secreting cells in the H3+Add group and the H3+AP group was similar,and both are higher than in the H3N2 group.The number of IFN-?secreting cells in the H3+AP group and H3+Poly I:C group was equivalent,which was higher than that in H3N2 group.Five days after the booster immunization,the Ig G antibody titer and Ig G2a/Ig G1 ratio of H3+AP group was higher than those of H3N2 group(P<0.05).6.Antigen metabolization in mice.The mice were injected with Cy5.5-H3 combined with Adda Vax,Poly I:C,AP,according to the animal live imaging,it is found that fluorescence of Cy5.5 in mice could be detected on the 15th day after the immunization of Cy5.5-H3 but not of Cy5.5-H3+Adda Vax,Cy5.5-H3+Poly I:C and Cy5.5-H3+AP.7.Tolerability evaluation of adjuvant vaccine.BALB/c mice and Wistar rats were immunized with 45?g H3N2,500?L AP,15?g H3+AP,45?g H3+AP,500?L PBS in two weeks apart.In BALB/c mice:there was statistical difference in weight change among those groups after the first and second immunization in 3 days,but not after the third immunization.Wistar rats:No statistical difference was found in the weight change among those the groups.CRP level:on the 7th day after the third immunization,there was no statistical difference of CRP level in BALB/c mice among those groups,the CRP level was higher in 15?g H3+AP group and the 45?g H3N2 group in Wistar rats.There was no statistical difference in spleen index and lymph node score among H3N2 group and H3N2combined adjuvant group.8.The challenge and protection experiment.The mice was immunized with three subtypes monovalent influenza vaccine respectively and challenged with the match lethal influenza virus,the weight of mice decreased for 5%in H1+AP,BV+AP,and BY+AP group on the 6th day,then the weight slowly increased with better vital signs and with less lung tissue inflammatory,lung tissue viral load was under the detection limit on the 6th day after virus challenge.?Conclusions?1.A new compound adjuvant(AP)of addavax and Poly I:C was prepared.AP adjuvant induced the expression of cytokines and chemokines in the local microenvironment,induced the proliferation and differentiation of local immune cells,promoted the maturation and antigens uptake of antigen-presenting cells(DCs),and then enhanced the immune response of peripheral immune organs such as draining lymph nodes and spleen,and induced more balanced cellular and humoral immunity,which plays an important role in enhancing the immune protection effect of influenza vaccine.2.AP adjuvant influenza vaccine was tolerated.3.The protective effect of influenza vaccine combined with AP was satisfied,which could be used as new adjuvanted influenza candidate vaccine that is safe and effective,laying a foundation for influenza vaccine research.