| Objective: Respiratory syncytial virus(RSV) is a single-stranded RNA virus, belonging to pneumonia virus in the paramyxovirus family. Epidemiological investigation showed that RSV is one of the most important pathogen of lower respiratory tract infection in infants, harming the health of infants seriously. In the worldwide, millions of children are infected with RSV every year and more than 50% infants are under one year. Repeated infection often occurs, even leading to death. RSV is also an important cause of high morbidity and mortality in immunodeficiency adults and the elderly people. But there is no effective vaccine now. Formalin inactivated vaccines(FI-RSV) that developed in the 1960 s, resulted in vaccine enhanced immunopathology in the lungs of the vaccinized children. The World Health Organization has listed RSV vaccine as one of the preferential developed vaccines.Notch signaling is a highly conservative signaling pathways, regulating the differentiation of the tissues and cells through the interaction between adjacent cells. It is an important signaling pathway guiding a cell toward its final destiny. Almost all cells differentiation, proliferation and apoptosis are closely related to Notch signaling, so it affects a series of physiological and pathological process. Notch signaling plays an important role in the process of activation and differentiation of innate and adaptive immune cells. Four Notch receptors(Notch1-4) and five ligands(Delta1, 3, 4 and Jagged1, 2) have been identified in mammals. Notch signaling may regulate vaccine enhanced immunopathology by affecting the activity of the APC and lymphocytes stimulated by RSV vaccines. Cp G, a ligand of TLR9, can activate all kinds of immune cells by antivating the NF-κB-dependent pathway. It has wide immunoregulatory effects on immune cells, especially in promoting Th1 response and enhancing CTL activity by stimulating dendritic cellss(DCs) to generate IFN-γ, IFN-α/β, IL-12, IL-18, etc. This study investigated the role of Notch signaling and Cp G on FI-RSV vaccine enhanced immunopathology by inhibiting the Notch signaling of mice, with Cp G2216 and Notch inhibitor as adjuvants.Methods: 1 RSV was propagated in Hep-2 cells, and viral titer(pfu) was measured. 2 Preparation of FI-RSV, FI-RSV+Al(OH)3 and FI- control. 3 Animal experiments 3.1 Immunization of mice6-9 week old female C57Bl/6 mice were randomly divided into 9 groups(6 mice/group):①Control group, no treatment; ②FI-control group; ③FI-RSV group; ④FI-RSV+Al(OH)3group; ⑤FI-RSV+Cp G group; ⑥FI-control+DMSO group; ⑦FI-RSV+L-685,458 group ⑧FI-RSV+Al(OH)3+L-685,458 group;⑨FI-RSV+Cp G+L-685,458 group, The mice were immunized 2 times on day 0 and 28, respectively. 3.2 The humoral immune responsesThe levels of antibody Ig G, Ig G1 and Ig G2 a in serum were measured by ELISA method on day 10 after the last immunization. 3.3 28 days after last vaccination, mice of all groups were challenged with 100μl 106.0 pfu/ml RSV except control group, the weight changes of 1 to 5 days after challenge were recorded. 3.4 After challenged 5 days, all mice were killed.Samples were collected to do the related testing. 3.5 Infected lung histopathologySections of paraffin embedding lung tissues were stained by HE/PAS for hlistological analyses.Real-time PCR was used to examine the expression of RSV-N gene in the lung tissue to determine the replication of the virus in mice; RNA was extracted and real-time PCR was used to examine cell factors in lung tissue after infection. smears is made by bronchoalveolar lavage lavage to observe the changes of inflammatory cells; virus titer in the lung tissue was detected to observ the protection of the vaccine; ELISA was used to examine TNF-α, IFN-γ, IL-1β, IL-4, IL-10, IL-6, IL-13, IL-17 levels in lung tissues.Results: 1 RSV was propagated in Hep-2 cells.The viral titer is 107.86pfu/ml. 2 FI-RSV vaccines, FI-RSV+Al(OH)3 vaccines and FI-control vaccines were successfully prepared. 3 Measured results of antibody Ig G and neutralizing antibody in mice serumThe level of Ig G, Ig G1 and Ig G2 a antibody titer in each immunized group and that in control group showed significant difference(P<0.05). No significant difference was observed among FI-control group and FI-control+DMSO group.No significant difference was observed in each immunized group of Ig G and Ig G1 antibody titer. Ig G2 a antibody titer in FI-RSV+Cp G+L-685,458 group is significantly higher than other immunized groups(P<0.05). The ratio of Ig G1/Ig G2 a of FI-RSV+Cp G+L-685,458 group(1.02) was significantly lower than other immunized groups(P<0.05). The ratio of FI-RSV+Al(OH) 3+L-685,458 group(1.44) was higher than other immunized groups and no significant difference was observed among the other groups.Neutralizing antibody titer in each immunized group is significantly higher than that in FI-control and FI-control+DMSO groups(P<0.05). no significant difference was observed among FI-control and FI-control+DMSO groups. No significant difference was observed between FI-RSV+Cp G and FI-RSV+Cp G+L-685,458 group. 4 Weight changes of mice after challenged by RSVCompared with control group, there is no significant difference in body weight changes of FI-RSV+Cp G+L-685,458 group at any time point(P>0.05), while various degrees are observed in other immune groups at any time point. The weight of FI-RSV+Cp G group and FI-RSV+L-685,458 group reduced to peak on the following day after infecion and was significantly lower than that of control group(P<0.05). The weight of FI-RSV group, FI-RSV+Al(OH)3 group and FI-RSV+Al(OH)3+L-685,458 group respectively reduced to peak at day 1, 3 and 5 after infection and significantly lower than that of control group(P<0.05). In addition, compared with other immune groups, the weight of FI-RSV+Cp G group reduced most and significantly lower than that of other groups at day 1, 2, 3 after affection(P<0.05). There is no difference between FI-RSV+L-685,458 group and FI-RSV group at any time point(P>0.05). The weight of FI-RSV+Al(OH)3+L-685,458 group is significantly higher than that of FI-RSV+Al(OH)3 group at day 1, 2. There is no difference between FI-RSV+Al(OH)3 group and FI-RSV group at any time point. 5 Staining of lung biopsy 5.1 PAS staining of lung biopsy: compared with the Control group, varying degrees of mucus secretion were observed in FI-control group, FI-RSV group, FI-control+DMSO group, FI-RSV+Al(OH)3 and FI-RSV+Al(OH)3+L-685,458 group by RSV attacks. Abudant of mucus secretion presented in FI-RSV+Al(OH)3 group and FI-RSV+Al(OH)3+L-685,458 group, while mucus secretion in FI-RSV+Cp G and FI-RSV+Cp G+L-685,458 group was not obvious. 5.2 HE staining of lung biopsyIn Control group, alveolar walls of mice were complete, no edema and only a small amount of inflammatory cells. Inflammatory cells around the brunchus, blood vessels and interstitial tissue increased and alveolar walls thickened mildly in FI-control group, FI-control+DMSO group and FI-RSV+Cp G+L-685,458 group. After RSV attack, inflammatory cells infiltration, the alveolar walls obvious thickening and a fusion between alveolar and edema were observed in FI-RSV+Cp G group, FI-RSV+Al(OH)3 group, FI-RSV+Al(OH)3+L-685,458 group and FI-RSV+L-685,458 group. Petechiae appeared occasionally, most of which were lymphocytes, mononuclear macrophages, neutrophils and eosinophilic granulocyte can also observed. In FI-RSV+Cp G+L-685,458 group, lung pulmonary inflammatory cells didn’t increase, alveolar walls of mice were omplete, only a small amount of inflammatory cells appeared around the brunchus and blood vessels. 6 The RT-PCR detection of cytokines 6.1 Detecting the expression levle of RSV housekeeping gene of RSV-N in the lung tissueA significantly higher expression amount of RSV-N of lung tissue in FI-control group, FI-control+DMSO and FI-RSV group were observed than that in other immunized group(P<0.05); In addition, the amount of RSV-N expression in FI-RSV+Cp G+L-685,458 was significantly lower than that in FI-RSV+Cp G group and FI-RSV+L-685,458 group. 6.2.1 IFN-γThe m RNA expressions of IFN-γ in FI-RSV+Cp G, FI-RSV and FI-RSV+Al(OH)3 group were significantly higher than those of the other groups(P<0.05). 6.2.2 IL-4The m RNA expressions of IL-4 in control group and the FI-RSV+Al(OH)3+L-685,458 group were significantly higher than those of the other groups(P<0.05). There was no significant difference in other groups(P>0.05).6.2.3 IL-5The m RNA expressions of IL-5 in FI-RSV+Cp G+L-685,458 group was significantly lower than that of FI-RSV+Cp G group, that of FI-RSV+Al(OH)3 groups and FI-RSV+Al(OH)3+L-685,458 group(P<0.05). 6.2.4 IL-10The m RNA expressions of IL-10 in FI-control group, FI-RSV group and the FI-RSV+Al(OH)3+L-685,458 group was significantly higher than those of the other groups(P<0.05). 6.2.5 IL-13The m RNA expressions of IL-13 in each immunized group were significantly higher than those of Control group(P<0.05). 6.2.6 IL-1βThe m RNA expressions of IL-1β in FI-control group and FI-RSV+Cp G group were significantly higher than those of the other groups(P<0.05); The m RNA expressions of IL-1β in FI-RSV+Cp G+L-685,458 group were significantly lower than those of FI-RSV group and FI-RSV+L-685,458 group(P<0.05). 6.2.7 IL-17The m RNA expressions of IL-17 in FI-RSV+Cp G group were significantly higher than those of the other groups excpt for FI-control group and FI-RSV group(P<0.05). The m RNA expressions of IL-17 in FI-RSV group were significantly higher than those of FI-RSV+Al(OH)3+L-685,458 group, FI-RSV+L-685,458 group and FI-RSV+Cp G+L-685,458 group(P<0.05). 6.2.8 TNF-αThe m RNA expressions of TNF-α in FI-RSV+Al(OH)3+L-685,458 group and FI-RSV+Al(OH)3 group were significantly higher than those of the other groups(P<0.05). In addition, The m RNA expressions of TNF-α in FI-RSV+Cp G+L-685,458 group were significantly lower than those of FI-RSV group and FI-RSV+Cp G+L-685,458 group(P<0.05). 6.2.9 T-betThe m RNA expressions of T-bet in FI-RSV+Cp G group were significantly higher than those of the other groups(P<0.05); The m RNA expressions of T-bet in FI-control+DMSO group were significantly higher than those of the Control group(P<0.05). 6.2.10 ROR-γtThe m RNA expressions of ROR-γt in FI-RSV+Cp G group were significantly higher than those of the other groups(P<0.05). The m RNA expressions of ROR-γt in FI-RSV+Cp G+L-685,458 group were significantly lower than those of the FI-RSV group and FI-RSV+L-685,458 group(P<0.05). 6.2.11 GATA-3The m RNA expressions of GATA-3 in FI-RSV+Al(OH)3+L-685,458 group and FI-RSV+Al(OH)3 group were significantly higher than those of the other groups(P<0.05). The m RNA expressions of GATA-3 in FI-RSV+Cp G+L-685,458 were significantly lower than those of FI-RSV group and FI-RSV+Cp G group(P<0.05). 7 The inflammatory cells in lung lavage fluid 7.1 The total number of cells in the lung lavage fluid in each immunized group was significantly higher than that of Control(P<0.05). The total number of cells in FI-RSV+Cp G group was higher than that of the other groups(P<0.05). The total number of cells in FI-RSV+Cp G+L-685, 458 group was significantly lower than that of other groups(P<0.05), except for FI-control and FI-RSV group. 7.2 The proportion of macrophages in the lung lavage fluid of each immunized group was significantly lower than that of control group(P<0.05). The proportion of macrophages in the lung lavage fluid of FI-RSV+Cp G+L-685,458 group was significantly higher than that of other groups(P<0.05), except for FI-RSV+L-685,458 group. 7.3 The proportion of lymphocytes in the lung lavage fluid of each immunized group was higher than that of control group(P<0.05). The proportion of lymphocytes in the lung lavage fluid of FI-RSV+Cp G+L-685,458 group was significantly lower than that of the other groups(P<0.05), except for FI-RSV+Al(OH)3+L-685,458 group and FI-RSV+L-685,458 group. 7.4 The proportion of neutrophils in the lung lavage fluid of each groups was higher than that of control group(P<0.05). The proportion of neutrophils in the lung lavage fluid of FI-RSV+Cp G+L-685,458 group was significantly lower than that of the FI-RSV group, FI-RSV+Cp G group, FI-RSV+Al(OH)3 group and FI-RSV+Al(OH)3+L-685,458 group(P<0.05). 8 The virus titers in lungNo virus were detected in Control group. A certain virus titers were detected in each immunized group. The virus titer of each immunized group were significantly lower than that of FI-control group and FI-control+DMSO group(P<0.05). There is no significant difference in FI-control+DMSO group and FI-control had(P>0.05). 9 The results of ELISA9.1 IFN-γThe expressions of IFN-γ in each immunized group were significantly higher than those of Control group(P<0.05). The expressions of IFN-γ of FI-RSV+Al(OH)3+L-685,458 group, FI-RSV+Al(OH)3 groups, FI-RSV+Cp G+L-685,458 group and FI-RSV+L-685,458 group were significantly lower than that of FI-control group(P<0.05). In addition, The expressions of IFN-γ of FI-RSV+Cp G+L-685,458 group was significantly lower than those of FI-RSV group and FI-RSV+Cp G group(P<0.05). 9.2 IL-1βThe expression of IL-1β in the FI-RSV group was significantly higher than that of other groups; The expression of IL-1β of FI-RSV+Al(OH)3+L-685,458 group was significantly lower than that of FI-RSV+Cp G+L-685,458 group(P<0.05). The expression of IL-1β of FI-RSV+Cp G+L-685,458 group was significantly lower than that of FI-RSV+Cp G group(P<0.05). 9.3 IL-4The expressions of IL-4 in each immunized group were significantly higher than those of Control group(P<0.05). The expressions of IL-4 of FI-RSV+Cp G+L-685,458 group was significantly lower than those of FI-RSV group and FI-RSV+Cp G group(P<0.05). 9.4 IL-6The expression of IL-6 in each immunized group was significantly higher than that of Control group(P<0.05), except for FI-RSV+Al(OH)3+L-685,458 group and FI-RSV+Cp G+L-685,458 group. 9.5 IL-10The expression of IL-10 in each group was significantly higher than that of Control group(P<0.05), except for FI-RSV+Al(OH)3+L-685,458 group and FI-RSV+L-685,458 group. In addition, the expression of IL-10 of FI-RSV+Cp G+L-685,458 group was significantly lower than that of FI-RSV group and FI-RSV+Cp G group(P<0.05). 9.6 IL-13The expression of IL-13 in each group was significantly higher than that of Control group(P<0.05), except for FI-control group, FI-RSV+Al(OH)3 group and FI-RSV+Al(OH)3+L-685,458 group. 9.7 IL-17The expressions of IL-17 in the FI-RSV group and FI-RSV+Cp G group were significantly higher than those of the Control group, FI-control group, FI-control+DMSO group and FI-RSV+Al(OH)3+L-685,458 group(P<0.05).9.8 TNF-αThe expressions of TNF-α in each group were significantly higher than those of Control group(P<0.05), except for FI-RSV+L-685,458 group and FI-RSV+Al(OH)3+L-685,458 group. The expressions of TNF-α of FI-RSV+L-685,458 group were significantly lower than those of FI-control group, FI-RSV group, FI-RSV+Cp G group and FI-control+DMSO group(P<0.05). In addition, The expressions of TNF-α of FI-RSV+Cp G+L-685,458 group were significantly lower than those of FI-RSV group and FI-RSV+Cp G group(P<0.05).Conclusions:1 Cp G combined with L-685,458 as adjuvants has no significant effection on the titers of Ig G antibody and neutralizing antibody. However, it increases significantly the titer of Ig G2 a, makes the ratio of Ig G1/Ig G2 a more balanced.2 Cp G combined with L-685,458 as adjuvants has no significant effection on the protective effect of the vaccine, that is to say, it doesn’t reduce the protective effect of the vaccine.3 Cp G combined with L-685,458 as adjuvants could significantly reduce the immunopathological damage to the lung. FI-RSV+Cp G+L-685,458 group have no enhanced diseases by RSV challenge. Therefore, it was ideal adjuvants for FI-RSV vaccine.4 The possible mechanism by which Cp G combined with L-685,458 as adjuvant regulates the immune response to vaccine: the cross talking between Notch signaling and TLR signaling regulates the balance of inflammatory response in the lung through regulating the balance of Th1, Th2 and Th17 response. 9.1 IFN-γ... |
PDF Full Text Request