| Illness and death caused by infection with Seasonal Influenza Viruses are most likely to occur in the elderly and in people with chronic lung disease.Elderly people aged 60 and older are at higher risk of complications such as encephalitis,pneumonia and even exacerbation of underlying diseases associated with chronic heart and lung disease.Seasonal influenza spreading worldwide is caused by A/H1N1,A/H3N2 and type B influenza viruses.The influenza virus genome contains eight RNA fragments,two of which encode two envelopment proteins,namely,hemagglutinin(HA)and neuraminidase(NA).The commonly used antiviral drugs such as oseltamivir,zanamivir and peramivir on the market in China are all neuraminidase inhibitors.However,the safest long-term antiviral defense with the greatest public health benefit requires regular vaccination of all ages against seasonal influenza viruses,especially the elderly who are at high risk of infection.Immunogenicity and effectiveness of influenza virus vaccines were evaluated using the Hemagglutinin Inhibit Test(HAI),and the specific antibody evaluation criteria underestimated the benefits of influenza virus vaccines in the elderly population.Influenza virus vaccination in older populations has resulted in a reduction in influenza incidence,hospitalization rates,complications and mortality.However,seasonal influenza vaccines,which are updated and administered annually,are not as protective as expected.On the one hand,there was a mismatch between circulating wild strains and vaccine strains in the epidemic season.At the same time,the vaccination rate of the elderly population is low;At the other end of the spectrum,the immune response to the flu vaccine is reduced in the elderly population due to immune aging.At present,data from the National Bureau of Population Statistics shows that China is rapidly aging.As of January 2020,there were 253.88 million people aged 60 or above,accounting for18.1% of the country's total population.It is estimated that the proportion of the elderly population will reach 26% in 2030.This would make influenza virus infection an extremely heavy burden of disease among the elderly,but it could be mitigated or prevented by vaccination.However,the coverage rate of influenza virus vaccination among the elderly population in China is far below the target of 75% vaccination coverage set by the World Health Assembly in 2010,which is only 4%.Many factors contribute to the low vaccination rate among the elderly,including policy,personal economic level,education level and health awareness,etc.In addition,the levels of influenza virus specific antibodies in the elderly were lower than those in adults aged18 to 60 years,and memory B cells and longevity plasma cells were also significantly reduced.Immunosenescent has become a major challenge in the development of new or more effective influenza virus vaccines for the elderly aged 60 years and older.Immunosenescence is characterized by a decline in immune function and an increased risk of various infectious diseases.Therefore,understanding the immune aging older population immune inactivated tetravalent cracking of seasonal influenza virus vaccine(QIVs)immune mechanisms,in peripheral blood transcriptome,T cells,cytokines and immune globulin in QIVs dynamic characteristics before and after,help to found in the elderly population immunity how changes associated with age,sex,cause this kind of risk and of the weak the humoral immune response to influenza vaccine.In fact,it is now widely accepted that HAI titers measured after vaccination do not fully reflect vaccine protection in the elderly population.In addition,annual vaccination of elderly subjects with weak or no antibody response also showed improved protection against influenza,suggesting that cellular immune mechanisms may be important for protection in the elderly as well.At the earliest,in 2009,Querec and other researchers applied the methods of systems biology to the study of the mechanism of yellow fever virus vaccine,and derived the concept of systematic vaccinology.Since traditional vaccine research is based on humoral immune response,there is a lack of understanding of cellular immunity of vaccines.In addition,QIVs vaccine immune mechanism is networked,multidimensional,this study adopts the method of systems biology research,from multiple dimensions,with the help of high flux testing means and computer bioinformatics analysis associated with traditional vaccine studies the characteristics of the indicators,identify QIVs after vaccination in the older population establish effective immune protection of important biological molecules and signaling pathways,screening and vaccine effectiveness of hub genes,immune response and persistence,in order to look for alternative biomarkers of vaccine effectiveness evaluation,speed up vaccine clinical research progress.Due to the complexity of the immune mechanism of the body,the immune mechanism of QIVs in the elderly population will be analyzed from multiple dimensions.In this study,the transcriptome data of 16 elderly subjects with significant demographic and immunological differences were obtained using high-throughput sequencing RNA-seq,followed by pooled association analysis.In addition,different bioinformatics analysis methods were used to analyze the gender-specific expression of genes and signaling pathways in the QIVs immunization process of elderly women and elderly men by biologically Driven comparative cluster analysis.Then,through data-driven weighted Gene co-expression network analysis(WGCNA),the differentially expressed genes were clustered according to the expression patterns,and the clustered Gene sets were correlated with traits(demographic and immune response characteristics of subjects)to identify the key core genes influencing traits.In addition,fluorescent quantitative qRT-PCR was used to verify that the expression characteristics of hub genes were consistent with the transcriptome results.Given the transcriptome RNA-Seq from RNA molecule level only clarify elderly population immunity QIVs mechanism,to understand the dynamics of cell mediated immune QIVs characteristics,this research then uses high flux multicolor flow cytometry analyzed the demographic characters and QIVs immune response characteristics of the obvious 17 more than 60 elderly subjects of T cell subsets of peripheral blood specimens of PBMC detailed immune phenotype,and the results are different traits grouping comparison,It involves age,sex and QIVs-related Reactogenicity.Finally,we use multiple cytokines highthroughput detection technology is analyzed and older subjects in the peripheral blood plasma samples of cellular immunity and humoral immunity is representative of cytokine and immunoglobulin Ig,the same result are compared,and different traits grouping Related to age,Sex and QIVs-related reactogenicity.The dynamic characteristics of cytokine networks and major immunoglobulin Ig before and after QIVs immunization in elderly subjects with different traits were determined to understand the role of cytokines in cellular immunity.Part ?: Bioinformatics analysis of transcriptome data before and after QIVs immunization in elderly population obtained by RNA-Seq.1.Influence of gender factors on the immune effect of QIVs in the elderly populationThe aim of this study was to identify the sex bias associated with the immunogenicity of differentially expressed genes(DEGs)in the elderly population when administered with quadrivalent inactivated influenza vaccine(QIVs).The gene expression was analyzed in healthy adults aged 60 to 80 years before and after inoculation.The specific antibody titer HAI was detected by hemagglutination inhibition assay,and the correlation between different gene expression profiles and humoral immunity in the two sex groups was analyzed.In older women,DEGs involved in the type I interferon signaling pathway and classical pathway complement activation were upregulated within 3 days of influenza vaccination.At day 28,a bias pattern of immune responses in older men was shown to be associated with classical pathways that regulate protein processing and complement activation.A series of DEGs associated with different responses to QIVs inoculation in older women and men were identified by biometric driven clustering.Older women have a stronger immune response to QIVs,but the antibody declines rapidly after six months,while older men have the advantage of maintaining a long-lasting response.In addition,we have identified genes that may cause sex variations in influenza vaccination in older adults.Our findings underscore the importance of developing personalized seasonal influenza vaccines.2.Hub genes MCEMP1 and SPARC drive QIVs adverse immunoreaction events and maintain effective antibodiesAn in-depth understanding of potential candidate central genes may help produce safe and effective seasonal influenza immunity,as well as the development of personalized influenza vaccines for elderly populations at high risk of influenza virus infection.The purpose of this study was to identify potential central genes associated with the immune induction process of the 2018/19 season tetravalent inactivated influenza virus vaccine(QIVs)by weighted gene co-expression network analysis(WGCNA).A total of 13,345 genes were obtained from 63 whole blood samples of 16 elderly people,which were divided into 8 co-expression modules,two of which were significantly associated with vaccine-induced immune response.After functional enrichment analysis,the Hub gene subnetwork was constructed using vaccine-related immune genes under GO terms,and Hub gene identification and functional verification were carried out.MCEMP1 and SPARC were confirmed to be central genes affecting QIVs-induced immunity.Within 7 days after inoculation,the expression of MCEMP1 was negatively correlated with QIVs-related reactivity.CXCL8/IL-8 could inhibit the expression of MCEMP1,and Granzyme-B cytotoxic medium could increase the expression of MCEMP1.Meanwhile,SPARC expression increased the immune response to QIVs and contributed to sustained protective humoral antibody titers.These two genes can be used to predict QIVs-induced adverse reactions,the strength of the immune response,and the duration of humoral anti-influenza antibodies.This work provides clues for further research into the development of personalized QIVs that have an appropriate immune response and durable immunity to the upcoming seasonal influenza.Part ?: Distribution and kinetic characteristics of T lymphocytes before and after QIVS immunization in elderly population.The cellular immune damage caused by senescence is characterized by the degeneration of thymus gland and the decrease of T lymphocyte output in the immune system.However,a detailed study on the distribution characteristics of T lymphocyte subsets in peripheral blood before and after QIVs inoculation in the elderly population was lacking.The purpose of this study was to identify the distribution characteristics of T lymphocytes in the elderly population,and to compare the dynamic characteristics of T lymphocyte subsets in different traits and immune status groups(including age,sex,and QIVs-related adverse reactions).Among the 60 randomly selected elderly subjects in this study,the analysis of subjects' traits related to demographic baseline characteristics and pre-immune influenza virus antibody level,and 17 subjects with significant trait differences were selected to identify the characteristics of senescence phenotype of peripheral blood T lymphocytes in elderly population.The detailed distribution characteristics of T cell subsets in peripheral blood were detected and analyzed by 10-color high-throughput flow cytometry.The proportion of T cell subsets in the parents was calculated,and different traits and immune states were compared,including age,sex and QIVs-related adverse reactions.Demographic characteristics of the subjects were basically consistent with baseline characteristics,and the number of lymphocytes measured by blood routine tests was within the normal range.Comparing the distribution of T cell subsets by age groups,the CD8+PD1-CD57-T cell subsets were significantly higher in the M65 yrs Group of the elderly Group,while the CD8+PD1+CD57+T cell subsets were significantly lower than those of the young Group.CD8+CD27-CD28+T cell subsets were significantly higher in the elderly Group M65 yrs Group,but the frequency of CD27+CD28+/-T cells in the CD3+,CD4+ and CD8+T cell subsets were significantly increased in the two age groups after QIVs immunization.In both age groups,TCMs significantly decreased in CD3+,CD4+ and CD8+T cell subsets after QIVs immunization,while TNs significantly increased after QIVs immunization.The distribution differences of T cell subsets were compared according to gender groups.The proportion of total T(CD3+)and CD4+T cells in the elderly female subjects was higher than that in the elderly male subjects,and CD4+ showed significant gender difference in Day180 after immunity.The proportion of CD27+CD28+ T cells in the elderly female subjects was significantly higher than that in the elderly male subjects.There was little difference in the CD3+,CD4+ and CD8+T cell subsets among the elderly population with different traits and immune response characteristics.However,through more detailed analysis of depletion phenotype molecules(PD-1),senescence phenotype molecules(CD57),co-stimulatory molecules(CD27 and CD28),and T cell effector memory phenotype molecules(CD45RA and CCR7),it was found that there were significant differences between the elderly population with different traits and immune response characteristics before and after QIVs immunization.Part ?: The production and kinetic characteristics of major cytokines and immunoglobulin Ig in the QIVs immune response in the elderly population.As redundant secretory proteins,Cytokines and Chemokines play critical roles in cell growth,differentiation and activation.They regulate and determine the nature of immune response,and control the migration of immune cells and the arrangement of cells in immune organs.The type of cytokines initially produced in response to immune injury determines the occurrence of the immune response,and even the subsequent outcome of the immune response is characterized by cytotoxicity,humoral immunity,cell-mediated immunity,or allergy.Therefore,this study aims to confirm the level characteristics of major cytokines and immunoglobulins related to immune response in the elderly population,and compare the differences in the Ig kinetic characteristics of major cytokines and immunoglobulins related to different traits and immune status groups(including age,sex and QIVs-related adverse reactions).In this study,we use the high flux multiple cytokines detection technology,in view of the demographic characteristics and QIVs immune response characteristic obvious18 plasma samples after 60 years of age or older subjects,the quantitative detection of cellular immunity and humoral immunity is representative of cytokine and immunoglobulin Ig concentration,and the results are compared,and different traits grouping Related to age,sex and QIVs-related reactogenicity.Demographic characteristics of the subjects were basically consistent with baseline characteristics,and routine physical examination showed that the subjects were in good physical condition.By age Group comparison,IL-5 was significantly higher in the M65 yrs Group before immunization than that of the younger NM65 yrs Group,and significantly decreased after immunization.Granzyme-B was significantly higher in the younger NM65 yrs Group than in the older M65 yrs Group after immunization and significantly increased in both age groups after immunization.By gender Group comparison,IL-6 was significantly higher in Day 3 and Female Group than in Male Group,and then Day 28 was significantly decreased.At Day180 after immunization,IL-2 was significantly higher in the elderly Female Group than in the elderly Male Group.Before immunization,IL-12 secretion in the GR Group was significantly higher than that in the NGR Group.Both IL-18 and IFN-alpha were significantly higher expressed in the GR Group on Day 28 after QIVS immunization.In addition,the expression of Granzyme-B in GR Group was significantly higher than that in NGR Group on Day 03 after immunization.Many cytokines have both pro-inflammatory and anti-inflammatory potential,and which activity is observed depends on the presence of immune cells and the state of their response to the cytokines.There were significant age differences between the cytokine IL-5 associated with humoral immunity and granzyme-B associated with cytotoxicity.At the same time,the significantly higher expression of cytokines IL-6 and IL-2 after QIVs immunization demonstrated that the elderly female group had higher levels of influenza virus-specific cytotoxic CD8+T cells and CD4+ memory T cells. |
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