Characteristics Of Immune And Transcriptome Responses To Respiratory Syncytial Virus Infection In A Vaccination-challenge Mice Model

Respiratory Syncytial Virus(RSV)is a pathogen of severe respiratory tract infection in infants and elderly people.Most of RSV infected patients appear obvious symptoms of cough,low fever,and difficult breathing.Occasionally,a few severe patients died.RSV has threatened to global health security.Vaccination is an important strategy to prevent viral infection.So far,no safe and effecious RSV vaccine is available.In 1960s,the clinical trial of formalin-inactivated RSV vaccine(FI-RSV)indicated that vaccinated infants and young children appeared severe low respiratory tract disease,which was called as vaccine-enhanced disease(VED).Vaccinated individuals had the symptom of cough,fever,bronchitis and pneumonia,and a few of them died.For a long time,the reasons behind RSV vaccine-induced augmentation are not completely understood,and fear of augmentation is an important obstacle of vaccine development.To better understand the mechanisms of VED induced by RSV vaccines,we prepared ultraviolet-inactivated RSV vaccine(UV-RSV)and investigated lung pathological injury and dynamic immune responses in mice vaccinated with UV-RSV,and non-vaccinated mice were used as control.We analyzed differentially expressed genes and the dynamic regulation networks by using RNA-seq data.The results were as follows.1.We propagated RSV in Hep-2 cells,and purified RSV by sucrose density gradient ultracentrifugation.We prepared inactivated RSV vaccine(UV-RSV)by ultraviolet radiation.After prime and boost vaccination of BALB/c mice using UV-RSV,vaccinated mice were infected with RSV.A vaccination-challege mouse model was created to measure the specific immune responses and pulmonary pathological damage in vaccinated and non-vaccinated mice upon RSV infection.2.Virus titers,histopathological injury,T cells subsets and cytokines in lungs of vaccinated and non-vaccinated mice were investigated at 1,2,4,and 6 days post-infection(dpi)using RSV,respectively.Low viral replication were observed at 2 and 4dpi in vaccinated mice,and no RSV was determined at 6 dpi.For non-vaccinated mice(PBS),high level virus replication were measured at 1 dpi and maintained to 6 dpi.Histopathological analysis identified that obvious pulmonary histopathology injury was observed in vaccinated and non-vaccinated mice at 1 dpi.However,the lung histopathology injury alleviated in non-vaccinated mice,and pulmonay immunopathological damage was persistently aggravated in vaccinated mice with lymphocyte infiltration.Next,we prepared lung lymphocyte and performed cell surface and intracellular molecules staining,and measured T cells subsets by flow cytometry.The responses of CD25~+Foxp3~+CD4~+(Treg)maintained low from 1 to 6 dpi in vaccinated mice.In non-vaccinated mice,the responses of IL-4~+CD4~+(Th2)was in low from 1 to 6 dpi;the responses of IFN-?~+CD4~+T cells(Th1)was fluctuated from 1 to 6 dpi.The responses of Treg cells significantly increased at 4 and 6 dpi in non-vaccinated mice(p<0.01 and p<0.001).However,at 4 and 6 dpi by RSV,the responses of Th1 and Th2 were higher in vaccinated mice than non-vaccinated mice(p<0.05?p<0.001).IL-2,IL-4,IL-5,IL-10 and TGF-?concentrations significantly increased in lungs of vaccinated mice compared to non-vaccinated mice(p<0.05?p<0.001).On the contrary,the concentrations of IL-6 and IL-17 were significantly decreased in vaccinated mice compared to non-vaccinated mice from 1 to 6 dpi(p<0.05?p<0.001).However,the concentrations of IFN-?and TNF-?were significantly increased in 1 to 4 dpi(p<0.01?p<0.001)in vaccinated mice,and significantly decreased at 6 dpi,compared to non-vaccinated mice.We first reported dynamic level of CD4~+T cell subsets and several cytokines in lungs of both UV-RSV vaccinated and non-vaccinated mice subsquential RSV infection from 1 to 6 dpi.3.We performed Illumina RNA-seq of lungs of UV-RSV vaccinated or non-vaccinated mice at 1,2,4,6 dpi by RSV infection.Differentially expressed(DE)genes and transcriptome regulation networks were investigated using system biological methods based on RNA-seq data.Total 5582 DE genes from RNA-seq data were identified.Compared to non-vaccinated mice,the reduced number of DE genes was observed at 1,2 and 6 dpi and increased number of DE genes was observed at 4 dpi in vaccinated mice.Analysis of clustering and functional enrichment showed that DE genes were mainly involved in pathways of immune and inflammatory responses,cell adhesion,angiogenesis,and signal transduction.By applying DE genes to STRING and Bio-GRID database,we constructed time-serial PPI networks.By applying Cluster-ONE algorithm,eleven high influential modules(HMs)were recognized,and DE genes within 11 HMs were functionally related to cell propagation and growth,signal transduction,immune and inflammatory responses.The two representative high influenced modules HM5(functionally as collagen fibril organization,cell adhesion,ECM-receptor interaction)and HM8(functionally as MHC antigen presentation and immune response)were further characterized based on network analysis.Data showed that for UV-RSV vaccinated mice the closely regulatory correlation of DE genes was observed at late 4 and 6 infection;however,the tightly regulatory correlation of DE genes in HM8 existed at early RSV infection in vaccinated mice.The selected 8 genes were conformed by real-time RT-q PCR.Data showed that m RNA levels of selected 8 genes were consistent with the gene expression in transcriptome data,indicating the RNA-seq data is reliable,and the database can be used for future researches.These findings enhance revealed the understanding relationship of RSV VED and immune responses,which could be benefit to the development of novel RSV vaccines.