| Although hepatitis B?HB?vaccine has been proven to be effective and safe in protecting people against HBV infection,following a standard three-dose vaccination regimen,approximately 5-10%of healthy adults fail to elicit protective anti-HBs titer??10 mIU/ml?and remain at risk for HBV infection.Subjects with anti-HBs titers of<10 m IU/ml 1-6 months after standard immunization schedule,who tested negative for HBsAg and anti-HBc,are defined as non-responders of HB vaccine.Various factors may influence the immune response to HB vaccine such as vaccine type and dosage.Host-related factors including old age,obesity,smoking and chronic infection and immunological tolerance have all been reported attributable to lack of response to HB vaccine.Human leukocyte antigens?HLA?which were involved in the antigen processing and presentation,polymorphisms on cytokines,cytokine receptors and Toll-like receptors were reported associated with the status of the HB vaccine-induced protective immune response.Several researches have verified that HLA-DRB1*07 was positively correlated with HB vaccine non-responsiveness.Single nucleotide polymorphism?SNP?in cytokines including IL-1??IL-2?IL-4?IL-10?IL-12 and related cytokine receptors were also reported be in connection with HB vaccine immune response.TT haplotype of SNP rs2243250 and rs2070874 in IL-4 gene had been found associating with the poor humoral response in some research.As a cytokine derived from Th2 cells,IL-4 could prompt the proliferation of B cell,modify T cell dependent of independent humoral response and immunoglobulin class switching.Because of the large number of alleles,the selection of target SNP among different studies is different.In some studies,the results were not consistent with each other because of difference in selection of participants or other factors.Immune deficiency such as dysfunction or insufficient amount of Th cells induced low cytokine secretion or hyporesponsiveness in B cells may also lead to the production of non-response to vaccine.It is reported that the amount of anti-HBs antibody-secreting B cells were lower in non-responders as compared with responders.After stimulated with CD40L,IL-2 and IL-4 in vitro,IgG anti-HBs secreting memory B cells from non-responders were found significantly decreased,which exhibited the defection of memory B cells and antigen specific plasma cell in non-responders.Dysfunction of DC may also result in the non-responsiveness to HB vaccine.As markers for maturation and activation in monocytes derived dendritic cell?moDC?,MHC II and CD83 were significantly decreased in non-responders.Low activation of moDC was bound to affect the presentation of antigen.Meanwhile,the ability of moDC to stimulate antigen-specific T cells was also lower than that of responders.These factors may lead to low reactivity to HB vaccines.Previous researches on the mechanism of HB vaccine non-response,aiming at exploring the genetic factors such as HLA and cytokine gene polymorphism,can find the genetic characteristics of non-responders,and help early predict the responsiveness of HB vaccine.For non-responders transcriptome expression studies,to analyze gene characteristics of non-responders from the whole genome level,combined with the research on the immune responses of non-responders,can find the genetic and immunological causes of HB vaccine non-responders,and provide a basis for the development of improved vaccine induced response of non-responders.In this study,we first enrolled 1134 participants with no history of hepatitis B vaccination or HBV infection after questionnaire and preliminary screening.After first 3 doses of hepatitis B vaccine immunization,104 non-responders were selected and vaccinated with another 3 doses of vaccine.At last,12 non-responders were double checked for HBV serum markers and HBV DNA,and 7 non-responders were enrolled for subsequent study,and 7 responders after first 3 doses of vaccination were selected for normal control.Anticoagulated blood samples were obtained pre-vaccination,3,7,28 d after the first dose,7 d after the second dose and 28 d after the last dose.We applied transcriptome microarray to investigate the dynamic mRNA expression in HB vaccine non-responders and responders pre-and post-vaccination.For reason that fewer sample cases were analyzed in this study,to avoid accidental factors,we set 5 time points to analyze the microarray data,which could verify the results of each time point.Human Th1/Th2&Chemokine Panel?20 plex?and Human Th9/Th17/Th22/Treg Cytokine Panel?7 plex?were used to measure cytokines in plasma samples of all participants.ELISPOT assay were applied to compare the antigen specific cytokine secretion in non-and normal responders.Part I:Analysis of transcriptome changes in HB vaccine non-responders1.Principle components analysis?PCA?of microarray dataPCA was needed for the evaluation of quality of samples determined by microarray.Whether the differentially expressed genes?DEGs?were induced by the biological differences or not,it was an important evaluation criteria.For the reason that participants in this study had no difference in race or nationality,no distinguished difference should be existed in the whole transcriptome of the samples,and little dispersion of the results may exist for the individual difference.Our results exhibited that data points with different colors were cross-existed in all samples from different time points and groups with no specific aggregation,which indicated that DEGs in microarray data from all samples were the real biological difference instead of sample quality.2.Gene expression of hepatitis B vaccine responders and non responders after immunizationIn order to obtain the transcriptome expression profile of non-responders and responders immunized with HB Vaccine,we use transcriptome chip for gene expression analysis.Compared to the day before vaccination?Day 0?,up-and down-regulated genes appeared most at the 3rd and 7th day after vaccination?Day 3 and Day7?.At the time point of 28 days after injection?Day 28?and 7 days after the second dose of immunization?Day 35?,the number of differentially expressed genes was significantly decreased,reflecting expression pattern of vaccine induced gene changes from active state in the early start stage to stable state after the formation of immune response.The fold change?FC?of DEGs showed higher in responders at Day 3 and Day 7,in which OLR1?IL1A and CCL20 were the three most up-regulated genes,and KCNJ15 was the most down-regulated gene.At the time point of Day 28 and Day 35,the change patterns of DEGs were similar but lower in FC than Day 3 and Day 7.DEGs expression patterns in non-responders were similar with responders.At Day 3 and Day 7,most up-regulated genes were IL1A,CCL20 and OLR1,which were identical with responders.At Day 28 and Day 35,EGR1,ZNF331 and ERMN showed most up-regulated and FC values were lower in most of the DEGs than Day 3and Day 7.3.Comparison and analysis of the signal pathway between the responders and non-respondersThe KEGG database was used to analyze the DEGs after immunization of hepatitis B vaccine for non-responders and responders by Pathway enrichment analysis.We mainly analyzed two time points of Day 3 and Day 7.On Day 3,up-regulated pathways such as“Salmonella infection”,“NF-kappa B signaling pathway”and“NOD-like receptor signaling pathway”expressed high enrichment score in both groups,which suggested that the response of the two groups was similar after immunization.But in the down-regulated pathway,there was a great difference between the two,and only one pathway of"Pertussis"was enriched in the two groups.On Day 7,“Cytokine-cytokine receptor interaction”,“Salmonella infection”,“NF-kappa B signaling pathway”and“NOD-like receptor signaling pathway”were enriched up-regulating in both groups.Among the non responders,two unique pathways"Apoptosis"and“Toll-like receptor signaling pathway”had higher enrichment score.In the down-regulated pathway,several disease processes involving pathways such as“Hepatitis B”,“Tuberculosis”,“Leishmaniasis”and“Toll-like receptor signaling pathway”in two groups were enriched,and“Phagosome”and“Alcoholism”pathways were enriched in non-responders.4.Significant transcriptomic changes in non-responders at 5 time points pre-and post revaccinationTranscriptome microarray analyses were performed to explore the differences in expression profile between the non-responder and responder group.Hierarchical clustering demonstrated significant changes in the transcriptome of PBMCs of the two groups at 5 different time points.In the microarray analysis,55 up-regulated and 15down-regulated genes were identified in PBMC in non-responders compared with responders before vaccination?FC?2.0 and P<0.05?.After the first dose of vaccination,28,68 and 45 genes were differentially expressed on the 3rd,7th and 28thh day,respectively,whereas 23 genes were up-regulated and 6 genes were down-regulated on the 7th day post the second dose of vaccination.Interaction of proteins from differentially expressed coding RNAs pre-and post vaccination exhibited a dense network trend,especially for immune related cytokines,like BPI?CAMP?CECAM8?DEFA4?LTF?SLPI?MMP8 and RNASE3.5.Continuous genetic characteristics revealed in non-respondersCompared with responders,a total of 11 up-regulated genes and 3down-regulated genes were identified in non-responders at all 5 time points.Up-regulated genes contained 9 coding RNAs,including BPI,CEACAM8,DEFA1B,DEFA4,FOLR3,LTF,MMP8,TCN1,TKTL1,and 2 non-coding RNAs,one of which named lnc-CEACAM8,participating in CEACAM8 expression and regulation process.Down-regulated genes involved 3 non-coding RNAs playing role in protein synthesis.These DEGs which constantly existed in the two groups provided a clue for the existence of fixed genetic differences that were independent of vaccination and might be the transcriptomic characteristics of HB vaccine non-responders.6.Gene ontology?GO?analysisIn search of the differentially expressed genes in Biological Process between the two groups,the GO enrichment analysis was performed with default settings using the web-based database for annotation,visualization,and integrated discovery?DAVID?.At the 5 time points pre-and post-vaccination,152,262,562,253 and 428 Go items were identified respectively.According to Enrichment P value,these Go items mainly include“defense response to bacterium",“antibacterial and antimicrobial humoral response”,“defense response to other organism”,“immune response”,“immune system process”,etc.7.Validation of DEGs expression levels using qRT-PCRTo validate the DEGs exhibited from the microarray,a subset of 9 up-regulated genes with continuous expression at all 5 time points were selected for further qRT-PCR validation and all samples from 28 days after first dose vaccination which were analyzed in microarray were conducted validation.The expression pattern of the selected genes concurred with the microarray data for 7 of 9 of the analyzed probesets?BPI,DEFA1B,DEFA4,FLOR3,LTF,MMP8,TKTL1?.The qRT-PCR results demonstrated that 8 genes were significantly up-regulated with the FC?2.0.Down-regulation was detected by PCR in two of the selected probesets?CEACAM8and TCN1?which were inconsistent with microarray result.Part II:Analysis of immune characteristics of non-responders and exploration of possible factors of non-response1.Dynamic cytokine signatures of non-responders and respondersTo determine whether non-responders induce detectable cytokine signatures in plasma which were different from responders,cytokine and chemokine panels which contained 27 key mediators of host immune responses were used to measure the expression of cytokines in plasma on days 3,7,28,35 after first dose of vaccination and 7th day post the second dose of vaccination.IP-10 concentration was significantly higher at day 28 than pre-vaccination,3rd and 7th day after vaccination in non-responders and in responders and it elevated to high level at day 3 after first dose vaccination but with no significance.IFN-?and IL-18 rose to the peak with significant difference at day 28 in both groups compared with other time points,but no significance was observed between non-responders and responders.TNF-?and IL-4 exhibited similar trends with IFN-?and IL-18.Among these,lower concentration of IL-27 was induced in non-responders than responders at all 4 time points,and significant difference was observed on the 3rd day after first dose of immunization.At the time point of 7th day post the second dose,CXCL12 was significantly higher in responders than non-responders.And they may probably act as the characteristic cytokine marker for responders.Other cytokines and chemokines exhibited no significance between the two groups.2.Difference of antigen specific cellular immune response in non-and normal respondersELISPOT assay was performed determine specific cytokine secretion after in vitro antigen stimulation in PBMC.PBMC from non-and normal responders at the 7thh days after the second dose of immunization were selected for the assay.With a final concentration of 40?g/ml antigen stimulation,the numbers of spot forming cells?Spot-forming cells,SFCs?of antigen specific IL-2 and IL-4 were significantly higher than that of non-responders in responders.No significant difference was observed between the antigen stimulation group and the non stimulation group in non-responders.While in responders,SFCs of IL-2 and IL-4 were significantly higer in stimulation group compared with non-stimulated group,indicating the decreased response ability to hepatitis B antigen in non-responders.4.Antibody responses to other pathogens or vaccines in HB vaccine non-and normal respondersHB vaccine non-responders could not produce antibodies against hepatitis B antigen,whether there is a problem in the process of antibody production and secretion.It can be verified by non-responders'antibody response to other pathogenic microorganisms or antigens.In this study,we didn't survey the history of HAV or HEV vaccine immunization,but we had excluded the acute HAV or HEV infection,so if the participants had a positive antibody determination,it may be past vaccination of these two vaccines or past exposure to HAV or HEV.We use the ELISA method to determine anti-HAV and anti-HEV IgG in non-responders and responders.Results showed that the anti-HAV positive rate was 100%and 86%in non-responders and responders,respectively,and no significant difference were observed between the two groups.There are two anti-HEV positive participants in non-responder group,while no positive participant was found in responder groups,which also had no significant difference between the two groups.This indicated that non-responders have normal function of antibody secretion stimulated with other pathogenic antigens,in conjunction with the phenomenon of declining in HBsAg specific IL-2 and IL-4 in non-responders,we speculated that deficiency in HBsAg presenting,activation of Th cells,polarization to Th2 cells and specific bonding with HBsAg in B cells resulted in the non-response to hepatitis B vaccine.4.Study on the factors influencing the response of hepatitis B vaccine4.1 The effect of deficiency in NK cells and mice age on the immune response induced by HB vaccineIn PBMC,lymphocytes,mononuclear cells and DC cells are included,and T and B lymphocytes in lymphocytes play an important role in the occurrence,development and effect of immunoreaction.Monocytes and DC cells play an important role in phagocytosis,treatment and presentation of antigen and inducing lymphocyte specific reaction.As T and B cells play a leading role in the occurrence of immune responses,their defects are bound to disrupt the production of immune responses.The role of NK cells in the immunoreaction of the immune response in the immune response induced by hepatitis B vaccine should be explored.In order to verify whether NK cell defects can cause the non-response of HB vaccine,we immunized Beige mice with2?g HB vaccine and C57BL/6 mice were set as control group.ELISPOT assay were conducted for the detection of antigen specific cytokines IFN-?,IL-2 and IL-4.We found no significant difference in the secretion of the above three cytokines in Beige and C57BL/6 mice at 7 days post immunization,and there was no significant difference in the anti-HBs titer at 7 and 28 days after immunization.The results showed that deficiency in NK cells had no effect on the immune response to HB vaccine.4.2 The effect of mice age on the immune response induced by HB vaccineThe maturity of the immune related cells also may lead to the non-response to HB vaccine.BALB/c mice with the age of 3d,1w,2w,4w were immunized with 1?g HB vaccine subcutaneously.Cellular and humor immune responses were determined at 7 and 28 days after immunization,respectively.The results showed that the secretion of IFN-?and IL-2 were significantly decreased in 3d and 2w-old mice compared with 4w-old mice.Anti-HBs titers were also lower in younger mice than mature mice?4w-old?,and the seroconversion rate in 4w-old mice was 100%,higher than the other younger mice.Thus younger mice with the immaturation of the immune system induced lower cellular and humoral immune responses to HB vaccine.In summary,this research exhibited the transcriptome expression profile in non-responders,including 7 mRNA up-regulated continuously at 5 time points pre-and post boost immunization,which showed the transcriptome characteristics of HB vaccine non-responders different from responders.As the characteristic cytokine of non-responders,concentrations of IL-27 and CXCL12 in plasma were significantly lower in non-responders which may one of the mechanism for the presence of nonresponse to hepatitis B.The secreting function of specific IL-2 and IL-4 was significantly decreased compared with the responders,showing declined function of T cells.At the same time,the function of producing antibodies against other antigens or pathogens showed normal in non-responders,which proved the dysfunction in immune system existing in the Th cells activation and antigen presentation.In mice,we found that the functional defect of NK cells did not affect the immune response against HB vaccine,while younger mice showed a low response to HB vaccine due to the immature development of immune system.Our results revealed the characteristics of non-responders in transcriptome expression and immune response,which provided a new research approach for clarifying the mechanism of non-responsiveness to HB vaccine and it provided preliminary experimental support for further development of new hepatitis B vaccine to improve the level of antibody response of non-responders. |
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