Adjuvants Alleviate FI-RSV Vaccine-enhanced Diseases By Regulating Lung Immune Memory

Objective:Respiratory syncytial virus?RSV?is one of the main causes of lower respiratory tract infections in infants and young children.Millions of children are infected with RSV every year.Among them,infants and young children under 1 year old account for more than 50%,which seriously harms the health of infants and young children.But so far,there is still no effective vaccine for marketing.After the inoculation of the formalin-inactivated RSV?FI-RSV?vaccine developed in the 1960s,a more serious inflammatory response,a pulmonary-enhanced disease,occurred when RSV was naturally infected.Previous studies in our laboratory have found that FI-RSV immunization of mice with Notch signaling inhibitor L685,458 and the ligand CpG of TLR-9 as combined adjuvants can significantly reduce lung inflammation.Themechanismbywhichadjuvantsmodulate vaccine-enhancing diseases is unclear.Immune memory is the theoretical basis for vaccine development.It is the main purpose of this experiment to study whether adjuvants can alleviate lung inflammation by regulating the type and intensity of immune memory responses.Methods:1 RSV was cultured on Vero cells for preparation of formalin inactivated vaccine?FI-RSV?;RSV was cultured on Hep-2 cells for attacking mice.2 Animal experiments2.1 Mice grouping,immunization and sampling:6-8 weeks female BALB/c mice were randomly divided into 5 groups:PBS,PBS/RSV,FI-RSV,FI-RSV+Al?OH?₃ and FI-RSV+L685,458+CpG groups.Intramuscular injection 3 times with a interval of two weeks.14 days after the last immunization,blood and lung single cells were taken.2.2 Humoral immunoassay:serum antibody IgG was detected by ELISA.2.3 Detection of total lung CD69⁺cells in mice by flow cytometry2.4 In vivo labeling analysis of lung T_RM cells in mice2.5 Flow cytometry detection of lung Trained DCs and Trained macrophages in mice2.5.1 The lung CD11c⁺CD69⁺cells were detected by flow cytometry.Then,CD11c⁺CD11b⁺cells and CD11c⁺CD103⁺cells were detected with CD69⁺cells as a gate.The lung F4/80⁺CD69⁺cells and F4/80⁺SiglecF⁺cells were detected.The CD69⁺cells were used as the gate to detect F4/80⁺SiglecF⁺cells,F4/80⁺SiglecF^—cells,F4/80⁺TLR4⁺cells,and F4/80⁺TLR4^—cells.2.5.2 Lung CD11c⁺cells in mice were sorted with magnetic beads,cultured in vitro,and stimulated 5 hours by LPS.The activated marker molecule TLR4⁺CD69⁺cells were detected by flow cytometry.The expression of cytokine TNF-?was detected by real-time quantitative PCR.The concentration of IL-6 in the cell supernatant was analyzed by ELISA.3.5.3 Lung F4/80⁺cells in mice were sorted with magnetic beads,cultured in vitro,and stimulated 5 hours by FI-RSV.The activated marker molecule TLR4⁺CD69⁺cells were detected by flow cytometry,and IL-1?in cell supernatant was detected by ELISA.2.6 The immunized mice were challenged with 50?l of 10^7.5 TCID50 of RSV,and Corresponding tests were performed 5 days after challenge.2.6.1 Airway hyperresponsiveness was measured.2.6.2 The mice were sacrificed and the alveolar lavage fluid was taken to detect Th1-,Th2-and Th17-type cytokines IFN-?,IL-5 and IL-17 by ELISA.Lung tissues were taken for pathological sections and stained with HE and PAS;IFN-?,IL-13,IL-17,IL-23,chemokines GRO-?and Eotaxin,RSV-N gene,Caspase-3,Caspase-8,FADD,TNF-?,RIPK3,RIP3 and MLKL related to death patterns in lung tissue was detected by real-time quantitative PCR;RIP3 protein expression in lung tissues was detected by Western blot.Virus in lung homogenate supernatant was titrated?TCID50?.Results:1 Serumspecific antibody IgG levels in miceThe IgG level of each vaccine immunized group was significantly higher than that in the PBS group?P<0.05?,and the IgG level of the FI-RSV+Al?OH?₃ group was lower than that in the FI-RSV group?P<0.05?.2 Mice lung CD69⁺cellsThe proportion of CD69⁺cells induced by each vaccine immunized group was significantly higher than that in PBS group?P<0.05?.3 FI-RSV+L685,458+CpG induces high levels of CD4⁺/CD8⁺CD69⁺T_RM?P<0.05?.4 Lung Trained DCs and Trained macrophages in mice4.1 Lung CD69⁺DCs in miceCD69⁺DCs was induced in FI-RSV and FI-RSV+Al?OH?₃ groups?P<0.05?,but not in FI-RSV+L685,458+CpG group?P<0.05?.CD69⁺DC mainly consisted of CD69⁺CD103⁺CD11c⁺cells?P<0.05?.There was no significant difference among the three groups?P>0.05?.4.2 CD69⁺macrophages in miceF4/80⁺CD69⁺TLR4⁺cells and F4/80⁺CD69⁺SiglecF⁺cells were induced in all vaccine immunization groups,while FI-RSV+L685,458+CpG induced high levelsofF4/80⁺CD69⁺TLR4⁺?P<0.05?.Highlevelsof F4/80⁺CD69⁺TLR4^—cells and F4/80⁺CD69⁺SigleF^—cells were induced in FI-RSV+Al?OH?₃ group?P<0.05?.High levels of F4/80⁺CD69⁺SiglecF⁺cells were induced in FI-RSV group?P<0.05?.4.3 Trained macrophage cellsAfter in vitro stimulation,FI-RSV+L685,458+CpG induced macrophages expressed high levels CD69 and TLR4 and IL-1??P<0.05?.4.4 Trained DC cellsAfter in vitro stimulation,FI-RSV+L685,458+CpG induced DCs expressed high levels of CD69 and TLR4,as well as cytokines TNF-?and IL-6?P<0.05?.5 pathological changes of lung tissue in miceInflammatory response and mucus secretion in FI-RSV+Al?OH?₃ group were higher than that in PBS/RSV group?P<0.05?,while inflammatory.6 The results of airway hyper-responsiveness test6.1 When the concentration of methacholine was 25 mg/ml,the airway resistance?RI?of FI-RSV+Al?OH?₃ group was higher than that in of PBS group?P<0.05?.6.2 When the concentration of methacholine was 50 mg/ml,the FI-RSV and the FI-RSV+Al?OH?₃ group were higher than the PBS group or the PBS/RSV group?P<0.05?.7 The virus titer of lung homogenate supernatantThe results of virus titration showed that there are no virus in PBS group,the serum titer of lung homogenate supernatant in the FI-RSV,FI-RSV+Al?OH?₃ and FI-RSV+L685,458+CpG group were significantly lower than that in PBS/RSV group?P<0.001?,but each group there was no statistical difference?P>0.05?.8 Detection of alveolar lavage fluid cytokines by ELISA8.1 IFN-?:The levels of IFN-?production in FI-RSV and FI-RSV+L685,458+CpG group were higher than that in FI-RSV+Al?OH?₃ group?P<0.05?.8.2 IL-5 and IL-17:The levels of IL-5 production and The levels of IL-17production in FI-RSV and FI-RSV+Al?OH?₃ group were significantly higher than those in PBS or PBS/RSV group?P<0.01?,The levels of IL-5 production and The levels of IL-17 production in FI-RSV+L685,458+CpG group were lower than those in FI-RSV+Al?OH?₃ group?P<0.05?.9 Expression of lung tissue mRNA9.1 Expression of RSV housekeeping gene RSV-N:The expression levels of RSV-NinFI-RSVgroup,FI-RSV+Al?OH?₃groupand FI-RSV+L685,458+CpG group were significantly lower than those in PBS/RSV group?P<0.001?,There was no significant difference in the expression levels of RSV-N between the other groups?P>0.05?.9.2 Expression levels of cytokine mRNA9.2.1 GRO-?,EOTAXIN and IL-17:The expression levels of GRO-?,EOTAXIN and IL-17 in FI-RSV+Al?OH?₃ group were significantly higher than those in PBS and PBS/RSV group?P<0.01?.The expression level of GRO-?,EOTAXIN and IL-17 in FI-RSV+L685,458+CpG group were lower than those in FI-RSV+Al?OH?₃ group?P<0.05?.9.2.2 IFN-?:The expression levels of IFN-?in PBS group,FI-RSV group and FI-RSV+Al?OH?₃ group were lower than that in PBS/RSV group?P<0.05?.The expression levels of IFN-?in FI-RSV+L685,458+CpG group was higher than that in FI-RSV+Al?OH?₃ group?P<0.01?.9.2.3 IL-13 and IL-23:The expression levels of IL-13 and IL-23 in FI-RSV group were higher than those in PBS/RSV group?P<0.05?,and the expression levels of IL-13 and IL-23 in FI-RSV+L685,458+CpG group was lower than those in FI-RSV group?P<0.05?.9.3 Expression of death-related proteins9.3.1 Caspase-3:The expression level of caspase-3 in PBS/RSV group and FI-RSV+L685,458+CpG group was higher than that in PBS group,FI-RSV or FI-RSV+Al?OH?₃ group?P<0.05?.The expression level of caspase-3 in FI-RSV+L685,458+CpG group was higher than that in PBS/RSV group?P<0.05?.9.3.2 Caspase-8 and FADD:The expression levels of caspase-8 and FADD in PBS/RSV group and FI-RSV+L685,458+CpG group were higher than those in FI-RSV,FI-RSV+Al?OH?₃ or PBS group?P<0.05?.9.3.3 TNFR1:The expression level of TNFR1 in FI-RSV+L685,458+CpG group was higher than that in other immune groups?P<0.05?.9.3.4 RIPK3 and RIP3:The expression levels of RIPK3 and RIP3 in FI-RSV group were higher than those in other immune groups?P<0.05?.9.3.5 MLKL:The expression level of MLKL in FI-RSV group was higher than that in FI-RSV+Al?OH?₃ group or FI-RSV+L685,458+CpG group?P<0.05?.10 Analysis of lung protein RIP3 expression by Western blotThe protein expression of RIP3 in FI-RSV group was higher than that in FI-RSV+Al?OH?₃ and FI-RSV+L685,458+CpG group.Conclusion:1 FI-RSV with L685,458 and CpG as combined adjuvants can induce high levels of lung T_RM,Trained DCs and Trained macrophages.2 FI-RSV+L685,458+CpG-induced lung immune memory may reduce inflammation by modulating the type of response following RSV infection.3 CD69 may be a marker of lung memory cells induced by FI-RSV vaccine.4 L685,458 and CPG combined adjuvants may promote the transformation of programmed necrosis to apoptosis of lung cells in mice immunized with FI-RSV after virus infection.