| Objective:1.To understand the effect of HBsAg-positive maternal HBV DNA CpG islands methylation on replication of HB V and expression of the biologically related viral proteins in mothers,neonates,and infants whose the possible immune response.2.To explore the possible effect of HBsAg-positive maternal HBV DNA CpG islands methylation on the infantile immune response with hepatitis B vaccine,and to provide basis for improving the rates of infantile immune response with hepatitis B vaccine.Methods:1.Research subjects and epidemiological methodsA total of 399 HBsAg-positive pregnant women were recruited,who were undergoing prenatal examinations and delivered between June 2011 and July 2013 in the Obstetrical and Gynecological department of the Third People’s Hospital of Taiyuan in Shanxi Province,China.Three ml maternal peripheral blood within 24 hours before delivery and neonatal femoral venous blood within 24 hours after birth(prior to given the hepatitis B immunoglobulin and hepatitis B vaccine)were collected.Hepatitis B vaccines were given to infants according to the 0,1,and 6 month immunization schedule.The infants were followed up at 12±1 month of age,and three ml of femoral venous blood were collected.Sequencing requires the sufficient viral load,and under the condition that the volume of serum is sufficient to complete the experiment,the 111 HBsAg-positive mothers with HBV DNA≥106 IU/ml were selected as the research subjects.A nested case-control study was used to select 11 infants with non-/hypo-immune response of hepatitis B vaccine as the case group according to the defined standard of non-/hypo-immune response of hepatitis B vaccine,and completely randomly selected 40 infants with high-response of hepatitis B vaccine as the control group who completed the follow-up.Fifty-one mothers(including neonates and infants)were subjected to HBV DNA whole-genome amplification and genotyping.There are 48 cases of HBV C genotype in the fifty-one mothers.Therefore,forty-eight HBsAg-positive maternal samples of HBV C genotype were selected for further methylated and analyzed.2.Laboratory testing methodsA real-time PCR-TaqMan kit was used to detect the HBsAg-positive maternal PBMC HBV cccDNA according to the manufacturer’s instructions;Fluorescent quantitation polymerase chain reaction(FQ-PCR)was used to test maternal,neonatal and infantile HBV DNA in serum according to the manufacturer’s instructions;Electrochemiluminescence immunoassay(ECLIA)kits was used to detect maternal,neonatal and infantile HBV serological markers according to the manufacturer’ s instructions(HBsAg,HBeAg,anti-HBs,anti-HBe,anti-HBc).Infants with anti-HBs<10 mIU/ml or anti-HBs between 10 and 100 mIU/ml were defined as non-/hypo-immune response,anti-HBs>100 mIU/ml were defined as high-response.The ProcartaPlex multi factor analysis technique was used to detect maternal,neonatal and infantile cytokines in serum including interleukin-2(IL-2),interleukin-4(IL-4),interleukin-6(IL-6),interleukin-10(IL-10),interleukin-12(IL-12),interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),and interferon-α(IFN-α).The Ezup Column Virus DNA Purification Kit were used to extract the maternal HBV DNA according to the manufacturer’s instructions.The single and double strands were amplified,purified,transformed,cloned and sequenced,separately.The sequence was cut and spliced to the full length to obtain the whole-genome sequence of HBV DNA;BioEdit software,EditSeq software(DNA Star),and SeqMan software was used to edit and splice the gene of the sequence;Mega 6.0 software was used to analyze and genotyping the sequence;The online software "MethPrimer" was used to predict the distribution of CpG islands in HBV DNA;Bisulfite Sequence PCR(BSP)was used to operate the CpG islands methylation of HBV DNA;The online software“QUMA" was used to analyze the methylation of CpG islands.3.Statistical analysisSAS version 9.4 software was used to record,clear up and analyze the data.Data for continuous variables is presented as mean and standard deviation(SD)or as median and range[M(QR)],and differences between the data were analyzed by Student’s t test and Wilcoxon signed-rank test,respectively.The dichotomous data was compared by a Chisquare test or Fisher’s exact test.Spearman rank correlation analysis was used for correlation analysis.P<0.05 indicates that the difference between the data was statistically significant.GraphPad Prism 5 software was used for figure.Results:1.General demographic characteristics of mothers,neonates and infantsThe analysis of HBV whole-genome sequence showed that 48 of the 51 HBsAgpositive mothers were C genotype of HBV(94.12%).A total of 48 mothers with HBV C genotype were selected for methylation and analysis in this study.No obvious difference was observed in the general characteristics of the mothers and neonates between the two groups of mothers with HBV DNA methylation and non-methylation(P>0.05).No significant difference was observed in the weight and height of infants between the maternal HBV DNA methylation group and non-methylation group(P>0.05).2.The effect of HBsAg-positive maternal HBV DNA CpG island methylation on replication of HBV and expression of biologically related viral proteins in mothers and neonates2.1 The distribution of HBV DNA CpG islands in HBsAg-positive mothersA total of five CpG islands were detected in the HBV DNA of 48 HBsAg-positive mothers of HBV C genotype in this study,CpG island Ⅰ,island Ⅱ,and island Ⅲ which named as conventional CpG islands,CpG island Ⅳ and island Ⅴ which were discovered recently.There are four distributed types of CpG islands,which were included CpG island Ⅱ and island Ⅲ all of them.Only included CpG island Ⅱ and island Ⅲ accounted for 91.67%(44/48);included CpG island I,island Ⅱ,and island Ⅲ accounted for 4.17%(2/48);included CpG island Ⅱ,island Ⅲ,and island Ⅳ accounted for 2.08%(1/48),included CpG island Ⅱ,island Ⅲ,and island Ⅴ accounted for 2.08%(1/48).2.2 The methylation of HBV DNA CpG islands in HBsAg-positive mothersMethylation analysis results showed that the 30 cases of methylation in HBVDNACpG island Ⅱ or island Ⅲ of 48 mothers(83.33%),including the 39 cases of methylation in HBV DNA CpG island Ⅱ of 48 mothers(81.25%)and the 14 cases of methylation in HBV DNA CpG island Ⅲ of 48 mothers(29.17%);the 13 cases of methylation in HBV DNA CpG island Ⅱ and island Ⅲ of 48 mothers(27.08%),the 8 cases of non-methylation in HBV DNA CpG island Ⅱ or island Ⅲ of 48 mothers(16.67%).The methylation rate of HBVDNACpG island Ⅱ or island Ⅲ was 45.42%(109/240),including the methylation rate of CpG island Ⅱwas 33.33%(80/240)and CpG island Ⅲ was 12.08%(29/240).CG sites of CpG island Ⅱ or island Ⅲ are methylated with a methylation rate of 10.39%(1202/11564),including the methylation rate of CG sites in CpG island Ⅱ was 11.14%(981/8806)and CpG island Ⅲwas 7.90%(218/2758).2.3 The effect of HBsAg-positive maternal HBV DNA CpG island methylation on replication of HBV and expression of proteinThe levels of PBMC HBV cccDNA in the maternal HBV DNA CpG island Ⅱmethylation group was significantly lower than that in the CpG island Ⅱ non-methylation group(Z=2.02,P=0.043),the levels of anti-HBc in the maternal HBV DNA CpG islandⅡ methylation group was significantly higher than the CpG island Ⅱ non-methylation group(Z=-2.07,P=0.039).No obvious difference was observed in the levels of maternal PBMC HBV cccDNA and HBV DNA between the HBV DNA CpG island Ⅲ methylation group and the CpG island Ⅲ non-methylation group(P>0.05).We analyzed the effect of methylation of CG sites in HBsAg-positive maternal HBV DNA CpG islands Ⅱ and island Ⅲ which were located in the different regions of gene on replication of maternal HBV and expression of proteins.It was observed that the levels of PBMC HBV cccDNA was sigificantly lower when the methylation of the CG sites in the P region of the maternal HBV DNA CpG island Ⅱ(Z=2.02,P=0.043),the levels of antiHBc was sigificantly higher than non-methylation of the CG sites(Z=-2.07,P=0.039).No statistically significant difference was found in the levels of replication of HBV and expression of proteins when the methylation of the CG sites in the X region of the maternal HBV DNA CpG island Ⅱ(P>0.05).The levels of PBMC HBV cccDNA was sigificantly lower was found when the methylation of the CG sites in the P region of the maternal HBV DNA CpG island Ⅲ(Z=-1.99,P=0.046).It was found that the levels of anti-HBs was sigificantly higher when the methylation of the CG sites in the C region of the maternal HBV DNA CpG island Ⅲ(Z=2.85,P=0.004).2.4 The relationship between HBsAg-positive maternal HBV and Thl/Th2 cytokinesWe analyzed the correlations among maternal HBV DNA,PBMC HBV cccDNA in serum and serological markers and Th1 cytokines(IL-2,IL-12,IFN-γ,IFN-α,TNF-α)/Th2 cytokines(IL-4,IL-6,IL-10).The maternal PBMC HBV cccDNA was positively correlated with the levels of IL-6 in serum(rs=0.858,P<0.001).2.5 The effect of HBsAg-positive maternal HBV DNA CpG islands methylation on neonatal replication of HBV and expression of proteinsNo statistical difference was found in the levels of neonatal HBV DNA and serological markers in serum when methylation of maternal HBV DNA CpG island Ⅱ and island Ⅲ(P>0.05).It was not observed that the neonatal replication of HBV and expression of proteins in serum was sigificantly difference when the methylation of the CG sites in the P or X region of the maternal HBV DNA CpG island Ⅱ(P>0.05).It was not found the sigificantly difference that the levels of neonatal HBV DNA and serological markers in serum when the methylation of the CG sites in the P or C region of the maternal HBV DNA CpG island Ⅲ(P>0.05).2.6 The relationship between HBV and neonatal Th1/Th2 cytokinesThe maternal anti-HBc in the serum was positively correlated with the levels of neonatal IFN-γ in the serum(rs=0.385,P=0.012).The neonatal anti-HBc in the serum was positively correlated with the levels of neonatal IFN-α(rs=0.349,P=0.024).3.The effect of HBsAg-positive maternal HBV DNA CpG islands methylation on the infantile immune response of hepatitis B vaccine3.1 Analysis of impact factors on infantile non-/hypo-immune response with hepatitis B vaccineThere were infantile non-/hypo-immune response group and infantile high-response group according to infantile immune response with hepatitis B vaccine.Nine infants with non-/hypo-immune response to hepatitis B vaccine and thirty-nine infants with highresponse.The rate of non-/hypo-immune response was 18.75%.The distribution of the levels of neonatal HBeAg and anti-HBc in serum was statistically significant between the infantile non-/hypo-immune response group and infantile high-response group(P=0.001,P=0.038).3.2 Relationship between the distribution of HBsAg-positive maternal HBV DNA CpG islands and infantile non-/hypo-immune response with hepatitis B vaccineNo significant difference was found in the length and CG sites of the HBsAg-positive maternal HBV DNA CpG islands between the infantile non-/hypo-immune response group and infantile high-response group(P>0.05).3.3 Relationship between the methylation of HBsAg-positive maternal HBV DNA CpG islands and infantile non-/hypo-immune response with hepatitis B vaccineComparing the methylation rate of HBV DNA CpG islands(the number of methylate clones/the total number of clones)and the infantile immune response of hepatitis B vaccine,the single methylation rate of maternal HBV DNA CpG island Ⅱ in the infantile non-/hypoimmune response group was found significantly lower than the infantile high-response group(χ2=6.51,P=0.011).Comparing the methylation rate of CG sites in HBV DNA CpG islands(the number of methylate CG sites/the total number of CG sites)and the infantile immune response of hepatitis B vaccine,the single methylation rate of CG sites in HBV DNA CpG island Ⅱ in the infantile non-/hypo-immune response group was found significantly higher than the infantile high-response group(χ2=5.48,P=0.019).The methylation rate of CG sites in HBV DNA CpG island Ⅱ or island Ⅲ in the infantile non-/hypo-immune response group was found significantly higher than the infantile high-response group(χ2=31.39,P<0.001).No statistical difference was found in the methylation rate of HBsAg-positive maternal HBV DNA CpG island Ⅱ and island Ⅲ in different regions of gene(P>0.05).It was observed that the methylation rate of CG sites in the P region of HBV DNA CpG island Ⅲin the infantile non-/hypo-immune response group was statistically significant higher than that in the infantile high-response group(χ2=5.56,P=0.018).The single methylation rate of CG sites in the P region of HBV DNA CpG island Ⅱ in the infantile non-/hypo-immune response group was found significantly higher than the infantile high-response group(χ2=24.11,P<0.001).3.4 The effect of HBsAg-positive maternal HBV DNA CpG islands methylation on infantile replication of HBV and expression of proteinsNo statistical difference was found in the levels of infantile HBV DNA and serological markers when maternal HBV DNA CpG island Ⅱ or island Ⅲ methylation(P>0.05).It was not observed that the levels of infantile replication of HBV and expression of proteins in serum was sigificantly difference when the methylation of the CG sites in the P or X region of the maternal HBV DNA CpG island Ⅱ(P>0.05).It was not found that the levels of infantile HBV DNA and serological markers in serum was sigificantly difference when the methylation of the CG sites in the P or C region of the maternal HBV DNA CpG island Ⅲ(P>0.05).3.5 The relationship between HBV and infantile Th1/Th2 cytokinesThe maternal PBMC HBV cccDNA in serum was negatively correlated with the levels of infantile IL-4 in serum(rs=-0.431,P=0.012).The maternal anti-HBc in serum was negatively correlated with the levels of infantile IL-10 in serum(rs=-0.354,P=0.040).The neonatal HBeAg in serum was positively correlated with the levels of infantile IL-6 in serum(rs=0.344,P=0.047).The infantile HBeAg in serum was negatively correlated with the levels of TNF-α in serum(rs=-0.356,P=0.039).The infantile anti-HBc in serum was negatively correlated with the levels of IL-6 in serum(rs=-0.340,P=0.049).3.6 Relationship between the levels of maternal,neonatal and infantile Th1/Th2 cytokines and infantile non-/hypo-immune response with hepatitis B vaccineIt was observed that the levels of maternal IL-6 in serum in the infantile non-/hypoimmune response group was sigificantly higher than that in the infantile high-response group(Z=2.68,P=0.007).The levels of maternal IFN-α in serum in the infantile non-/hypoimmune response group was sigificantly higher than the infantile high-response group(Z=2.13,P=0.033).It was observed that the levels of infantile IL-10 in serum was sigificantly higher in the infantile non-/hypo-immune response group than the high-response group(Z=1.99,P=0.047).No statistical difference was found in the remaining Th1/Th2 cytokines between the two groups(P>0.05).Conclusion:1.HBsAg-positive maternal HBV DNA CpG islands methylation may affect replication of HBV and expression of biologically related viral proteinsThe CG sites of HBsAg-positive maternal HBV DNA CpG island Ⅱ methylation which in the P region may inhibit the transcription of PBMC HBV cccDNA and promote the expression of anti-HBc.The methylation of CG sites in HB sAg-positive maternal HBV DNA CpG island Ⅲ which in the P region may inhibit the transcription of PBMC HBV cccDNA,and which in the C region may promote the expression of anti-HBs.Since HBV DNA CpG island Ⅲ has not been found methylation separately in the infatile non-/hypo-immune response group,the specific mechanism of methylation in CpG island Ⅲ needs further study.2.HBV DNA CpG islands methylation of HBsAg-positive mothers may affect infantile immune response with hepatitis B vaccineIt is different that the distribution of HBV DNA CpG islands in HBsAg-positive mothers with HBV C genotype which mainly including CpG island Ⅱ and CpG island Ⅲ.The location and length of HBV DNA CpG islands were found slightly different.It was not observed that relationships between the distribution of HBV DNA CpG islands in HBsAgpositive mothers and the infantile immune response with hepatitis B vaccine.It is necessary for us to further research the relationship between the distribution of HBsAg-positive maternal HBV DNA CpG islands and infantile immune response with hepatitis B vaccine.Both the methylation rates of clones and CG sites in HBsAg-positive maternal HBV DNA CpG island Ⅱ were found higher than that in the CpG island Ⅲ.It is more likely that methylation of HBV DNA CpG island Ⅱ,even hypo-methylation of the CG sites in the P region may be detrimental to the occurrence of infantile non-/hypo-immune response with hepatitis B vaccine.There were more methylated CG sites which in the P region of maternal HBV DNA CpG island Ⅲ in the infatile non-/hypo-immune response group.Since the limitation of the sample size,the effect of maternal methylation of HBV DNA CpG islandⅢ on infantile immune response with hepatitis B vaccine is not yet clear.The levels of maternal PBMC HBV cccDNA in the infatile non-/hypo-immune response group are higher than that in the infatile high-response group.The levels of Th1/Th2 cytokines are may changed,the ability of immune response to act on HBV and antigens are may affected,leading to infatile non-/hypo-immune response with hepatitis B vaccine.The specific mechanism of Th1/Th2 cytokines immune response needs further research in vitro.In conclusion,the methylation of HBsAg-positive maternal HBV DNA CpG islands may inhibit the transcription of maternal HBV cccDNA and the replication of HBV DNA,affect the expression of HBV related proteins such as promote the expression of anti-HBc or anti-HB s,affect the assembly and secretion of Th1/Th2 cytokines and promote the immune response of mothers and children to an extent.It is too high for methylation to affect the normal immune response.The secretion of immunosuppressive factors such as infantile IL10 in serum are increased,and may increase the rate of infantile non-/hypo-immune response with hepatitis B vaccine.It is necessary for us to increase the sample size or experiments in vitro to further study and verificate the specific mechanism of the relationship between HBV DNA CpG islands methylation and infantile immune response. |
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