The Regulation Effect Of TLR Ligands As Adjuvants In Respiratory Syncytial Virus Vaccine Enhanced Disease

Objective: Respiratory syncytial virus is the main pathogen of lowerrespiratory tract illness in infants and young children, elderly person, andimmunodeficiency individuals, which even cause death. However, due tovaccine safety issues, there has been no safe and effective vaccine approvedfor production. Pathogenesis of RSV vaccine enhanced immunpathology is thekey to research vaccine. TLR ligands as adjuvants can adjust the type ofimmune response induced by vaccines, and then reduce or inhibit vaccineenhanced immunopathology.In this study, we selected Pam Cys3(ligand of TLR-2), poly(I:C)(ligandof TLR-3), LPS(ligand of TLR-4), Cp G(ligand of TLR-9) as adjuvants, usingwild-type C57BL/6 mice with the FI-RSV vaccine immunized,FI-RSV+Al(OH)3 immunized My D88-/- C57BL/6 mice as a control group tostudy the regulation effect of TLR ligands as adjuvants in respiratory syncytialvirus Vaccine enhanced disease.Methods:1 RSV was propagated in Hep-2 cells,and viral titer(pfu) was measured.2 Preparation of FI-RSV and FI-control.3 Animals experiments3.1 The mice immune: C57BL/6 mice were vaccinated with FI-Control,FI-RSV, FI-RSV+Cp G2216, FI-RSV+Al(OH)3, FI-RSV+Pam Cys3, FI-RSV+LPS.My D88-/-C57BL/6 mice were vaccinated with FI-RSV+Al(OH)3.Controlgroup was injected with PBS.The mice were immunized every 14 days and 2times.3.2 The humoral immune responsesThe levels of antibody Ig G、Ig G1、Ig G2 a in serum were measured byELISA method within 14 days after the last immunization.3.3 4 weeks after last vaccination, mice of all groups were challenged withRSV except control group, the weight changes of 1 to 6 days after challengewere recorded.3.4 After challenged 5 days, all mice were killed. Samples were collected todo the related testing.3.4.1Infected lung histopathologySections of paraffin embedding lung tissues were stained by HE/PAS forhlistological analyses.3.4.2Smears which were used with the methods of Wright-Giemsa is made bybronchoalveolar lavage to observe the changes of inflammatory cells.3.4.3Virus titer in the lung tissue was detected to observ the protection of the vaccine.3.4.4The levels of neutralizing antibody in serum were measured.3.4.5 ELISA was used to examine IL-4、IL-6、IL-10、IL-13、IL-17、TNF-α、IFN-γ、IL-1β levels in lung lavage fluid.3.4.6 Real-time PCR was used to examine the expression of cytokines IL-5、IL-10、IL-13、IL-17、T-bet、Gata-3、ROR-γt、IFN-γ、RSV-N in lung tissue.Results:1 RSV was propagated in Hep-2 cells.The viral titer was 107.9 pfu/ml.2 FI-RSV vaccines and FI-control vaccines was prepared.3 The level of antibody Ig G、Ig G1、Ig G2 a in serum was measured by ELISA.3.1 Ig GControl group did not produce Ig G, Ig G antibody titer inFI-RSV+Al(OH)3, FI-RSV+Cp G group, FI-RSV+poly(I:C) group,FI-RSV+Pamcys3, FI-RSV+LPS group was significantly higher thanFI-control group(P<0.05), FI-RSV+Al(OH)3, FI-RSV+Cp G group,FI-RSV+poly(I:C) group, FI-RSV+Pamcys3 group, FI-RSV+LPS group wassignificantly higher than FI-RSV group(P<0.05), FI-RSV+LPS group wassignificantly higher than the adjuvant group(P<0.05).3.2 Ig G1Control group, FI-RSV+Pamcys3 group and FI-RSV+Al(OH)3My D88-/-group did not produce Ig G1, Ig G1 antibody titer in FI- RSV+Al(OH)3 groups,FI-RSV+poly(I:C) group, FI-RSV+Cp G group, FI-RSV+LPS group wassignificantly higher than FI-control group and the FI-RSV group(P<0.05).There was no significant difference between FI-control group and FI-RSVgroup(P>0.05).3.3Ig G2aControl group, FI-Control group, FI-RSV+Pamcys3 group andFI-RSV+Al(OH)3My D88-/- group did not produce Ig G2 a. There was nosignificant difference between FI-RSV+Al(OH)3 groups, FI-RSV+ poly(I:C)group, FI-RSV+Cp G group, FI-RSV+LPS group and FI-RSV group(P >0.05).3.4 Ig G1/Ig G2aCompared with FI-RSV group(0.96), the FI-RSV+LPS group’s(0.83)ratio of Ig G1/Ig G2 a reduced, and FI-RSV+Cp G group(1.1),FI-RSV+Al(OH)3 group(1.24), FI-RSV+poly(I:C) group’s(1.26) ratio ofIg G1/Ig G2 a increased.4 Neutralizing antibodiesControl group generated no neutralizing antibody. Neutralizing antibodytiter of FI-RSV group, FI-RSV+Al(OH)3 group, FI-RSV+poly(I:C) group,FI-RSV+Cp G group, FI-RSV+LPS group, FI-RSV+Pamcys3 produce wassignificantly higher than that of FI-control group(P<0.05), There was nosignificant difference between FI-RSV+Al(OH)3My D88-/- group andFI-control group(P>0.05).5 Weight changes of mice(5 days)Compared with control group, from the second day to the sixth day, apartfrom FI-RSV+Cp G group, other groups had no statistical difference(P>0.05).From the next day to the forth day, weight loss of FI-RSV+Cp G group wassignificantly lower than that of control group(P<0.05), the fifth day, the sixthday there was no significant difference between FI-RSV+Cp G group andcontrol group(P>0.05).6 The inflammatory cells in lung lavage fluid6.1 MacrophagesThe proportion of Macrophages in the lung lavage fluid of immunizedgroups was significantly lower than that of control group(P<0.05). That ofFI-RSV+Pamcys3 group, FI-RSV+poly(I:C) group, FI-RSV+LPS group wassignificantly higher than that of FI-RSV+Cp G group, FI-RSV+Al(OH)3groups, FI-RSV+Al(OH)3My D88-/- group, and FI-RSV group(P<0.05). Thatof FI-RSV+LPS group was significantly higher than That ofFI-RSV+Pamcys3 group, FI-RSV+poly(I:C) group, FI-control group(P<0.05).6.2 PMNThe proportion of PMN in the lung lavage fluid of immunized groups wassignificantly higher than that of control group(P<0.05). That of FI-RSV+LPSgroup was significantly lower than that of other groups except controlgroup(P<0.05). That of FI-RSV+Al(OH)3My D88-/- group was that ofsignificantly higher than other groups(P<0.05).6.3 LymphocytesThe proportion of Lymphocyte in the lung lavage fluid of immunizedgroups was significantly higher than that of control group(P<0.05). That ofFI-RSV+LPS group was significantly lower than that of FI-RSV group 、FI-RSV+Al(OH)3 group、FI-RSV+Pamcys3 group、FI-RSV+poly(I:C) group(P<0.05)。6.4The total number of cellsThe total number of cells in the lung lavage fluid of immunized groupswere significantly higher than that of control group(P<0.05). there was nosignificant difference between immunized groups(P>0.05).7 The result of histropathology7.1 PAS staining of lung biopsyCompared with control group,groups of RSV attacks had been varyingdegrees of mucus secretion. FI-RSV+Al(OH)3My D88-/- group,FI-RSV+Al(OH)3 group had large amount of mucus secretion. FI-RSV group,FI-RSV+Cp G group have less mucus secretion than FI-RSV+Al(OH)3 group,FI-control group, FI-RSV+poly(I:C) group, FI-RSV+Pam Cys3 group had onlya small amount of mucus secretion. FI-RSV+LPS group had only a smallamount of mucus secretion.7.2 HE staining of lung biopsyControl group, the alveolar walls structure was complete, only a small amountof inflammatory cells. FI-control group 、 FI-RSV+poly(I:C) group 、FI-RSV+Pam Cys3 group of mice ’ s lung pulmonary inflammatory cellsincreased, visible trace thickening of alveolar walls. FI-RSV group 、FI-RSV+Cp G group are more severity than FI-RSV+Pam Cys3 group.FI-RSV+Al(OH)3My D88-/- group、FI-RSV+Al(OH)3 group displayed a largenumber of inflammatory cells infiltration, alveolar walls was obviousthickening, a fusion between alveolar and edema. The alveolar walls structureof FI-RSV+LPS group was complete, no visible inflammatory cellsinfiltration.8 Lung homogenates titrationControl group detect no virus, Viral titers of FI-RSV+poly(I:C) group,FI-RSV+Pam Cys3 group, FI-RSV group, FI-RSV+Al(OH)3 group,FI-RSV+LPS group, FI-RSV+Cp G group was significantly lower than that ofFI-RSV+Al(OH)3My D88-/- group, FI-control group(P<0.05), There was nosignificant difference beteen FI-RSV+Al(OH)3My D88-/- group and FI-controlgroup(P>0.05).9 The results of ELISA9.1 IFN-γThe expression of IFN-γ in FI-RSV+poly(I:C) group, FI-RSV+Pam Cys3 group was significantly higher than that of control group, FI-RSV group,FI-RSV+Al(OH)3 groups, FI-RSV+LPS group and FI-RSV+Al(OH)3My D88-/-group(P<0.05). There was no significant difference between the rest of theimmunized groups(P>0.05).9.2 IL-4The expression of IL-4 in FI-control group, FI-RSV+Pam Cys3 group,FI-RSV+Cp G group, FI-RSV+poly(I:C) group, FI-RSV group wassignificantly higher than that of control group, FI-RSV+LPS group andFI-RSV+Al(OH)3My D88-/- group(P<0.05). FI-RSV+Pam Cys3 group wassignificantly higher than FI-RSV+Cp G group, FI-RSV+poly(I:C) group,FI-RSV+Al(OH)3 groups(P<0.05). There was no significant differencebetween other groups(P>0.05).9.3 IL-10The expression of IL-10 in FI-RSV group was significantly higher thanthat of control group and other immunized groups(P<0.05), FI-RSV+Cp Ggroup, FI-RSV+Pam Cys3 group was significantly higher than FI-RSV+LPSgroup, FI-RSV+Al(OH)3My D88-/- group(P<0.05).9.4 IL-13The expression of IL-13 in FI-RSV+Cp G group, FI-RSV+Pam Cys3 group,FI-RSV group was significantly higher than that of control group,FI-RSV+Al(OH)3 group, FI-RSV+LPS group, FI-RSV+Al(OH)3My D88-/-group(P<0.05). FI-RSV+Pam Cys3 group was significantly higher thanFI-control group, FI-RSV+poly(I:C) group(P<0.05), and there was nosignificant difference between the rest of immunized groups(P>0.05).9.5 IL-17The expression of IL-17 in RSV group was significantly higher than thatof control group and other immunized groups(P<0.05). FI-RSV+LPS group,FI-RSV+Al(OH)3My D88-/- group was significantly lower than FI-RSV+Cp Ggroup(P<0.05), and there was no significant difference between otnergroups(P>0.05).9.6 TNF-αThe expression of TNF-α in FI-control group, FI-RSV+poly(I:C) group,FI-RSV+Cp G group, FI-RSV+Pam Cys3 group was significantly higher thanthat of FI-RSV+Al(OH)3My D88-/- group and control group(P<0.05), and therewas no significant difference between otner groups(P>0.05).9.7 IL-1βThe expression of IL-1β in FI-RSV group was significantly higher thanthat of Control group and other immunized groups(P<0.05). FI-RSV+LPSgroup was significantly lower than FI-control group, FI-RSV+Cp G group andFI-RSV+Pam Cys3 group(P<0.05).9.8 IL-6The expression of IL-6 in FI-RSV+Pam Cys3 group was significantlyhigher than that of control group and other immunized groups(P<0.05).FI-RSV group was significantly higher than control group, FI-RSV+LPSgroup and FI-RSV+Al(OH)3My D88-/- group(P<0.05). There was nosignificant difference between otner groups(P>0.05).10 The results of RT-PCR10.1 T-betThe expression of T-bet m RNA in FI-RSV+Cp G group was significantlyhigher than that of control group and other immunized groups exceptFI-RSV+poly(I:C) group(P<0.05). FI-RSV+LPS group and control group wassignificantly lower than FI-control group, FI-RSV+Al(OH)3 group, FI-RSV+Pam Cys3 group, FI-RSV+poly(I:C) group, FI-RSV group(P<0.05). There wasno significant difference between FI-RSV+LPS group and control group(P>0.05).10.2 IFN-γThe expression of IFN-γ m RNA in FI-RSV+Al(OH)3 group wassignificantly higher than that of control group, FI-control group,FI-RSV+poly(I:C) group, FI-RSV+LPS group, FI-RSV+Al(OH)3My D88-/-group(P<0.05). There was no significant difference between otner groups(P>0.05).10.3 GATA-3The expression of GATA-3 m RNA in FI-RSV+Al(OH)3 group wassignificantly higher than that of control group and other immunized groupsexcept FI-RSV group(P<0.05). FI-control group, FI-RSV+LPS group,FI-RSV+Al(OH)3My D88-/- group was significantly lower than control groupand other immunized groups except FI-RSV+Cp G group. In addition,FI-RSV+poly(I:C) group, FI-RSV+Cp G group was significantly lower thanFI-RSV group(P<0.05).FI-RSV+LPS group(P>0.05). FI-control group, FI-RSV group,FI-RSV+Al(OH)3 group was significantly higher than FI-RSV+Cp G group,FI-RSV+poly(I:C) group(P<0.05).10.4 IL-5The expression of IL-5 m RNA in FI-RSV+Al(OH)3 group wassignificantly lower than that of control group and other immunizedgroups(P<0.05). FI-RSV+poly(I:C) group, FI-RSV+Cp G group wassignificantly higher than other groups except FI-RSV+Al(OH)3 group(P<0.05),and FI-RSV+Cp G group was significantly higher than FI-RSV+poly(I:C)group(P<0.05).10.5 IL-10The expression of IL-10 m RNA in FI-RSV+Al(OH)3My D88-/- group,FI-RSV+LPS group was significantly lower than that of control group andother immunized groups(P<0.05). FI-RSV+poly(I:C) group, FI-RSV+Al(OH)3group, FI-RSV+Pam Cys3 group was significantly lower than FI-control group,FI-RSV+Al(OH)3 group, FI-RSV+Cp G group(P<0.05).10.6 IL-13The expression of IL-13 m RNA in FI-RSV+Cp G group, FI-RSV+Pam Cys3 group, FI-RSV+poly(I:C) group was significantly higher than thatof control group and other immunized groups(P<0.05). FI-control group,FI-RSV+LPS group was significantly lower than FI-RSV groupFI-RSV+Al(OH)3 group(P<0.05).10.7 IL-17The expression of IL-17 m RNA in FI-RSV+LPS group was significantlylower than other groups except FI-RSV+ Pam Cys3 group andFI-RSV+Al(OH)3My D88-/- group(P<0.05). FI-RSV+Pam Cys3 group andFI-RSV+Al(OH)3My D88-/- group was significantly lower than FI-controlgroup, FI-RSV group, FI-RSV+Al(OH)3 group, FI-RSV+Cp G group andFI-RSV+poly(I:C) group(P<0.05).10.8 ROR-γtThe expression of ROR-γt m RNA in FI-RSV+LPS group,FI-RSV+Al(OH)3My D88-/- group was significantly lower than other groupsexcept FI-RSV+Cp G group and FI-RSV+poly(I:C) group(P<0.05). There wasno significant difference between FI-RSV+Al(OH)3My D88-/- group and10.9 RSV-NThe expression of RSV-N m RNA in FI-RSV+ Pam Cys3 group and coutrolgroup was significantly lower than other groups(P<0.05).FI-RSV+Al(OH)3My D88-/- group was significantly higher than otherimmunized groups except FI-control group(P<0.05). FI-RSV+Cp G group,FI-RSV+poly(I:C) group, FI-RSV+LPS group, FI-RSV+Al(OH)3 group wassignificantly lower than FI-control group and FI-RSV group(P<0.05).Conclusion:1 As adjuvant, the TLR ligands, which improved specific Ig G levelsignificantly, had no influence on the neutralizing antibody; In addition, theLPS can significantly improve the level of Ig G2 a and reduce the ratio ofIg G1/Ig G2 a. But no specific antibody was detected in My D88 deficiencygroup.2 The vaccine had a protective effect on the immune mice while TLRligands as adjuvant had no significant effect on it.3 LPS as adjuvant for FI-RSV inhibited the occurrence of RSV vaccineenhanced disease; My D88 deficiency group, poly(I:C) as TLR3 ligands andPam Cys3 as TLR2 ligands had certain inhibited effect on vaccine enhanceddisease.