| Background and AimsChronicity of hepatitis B virus(HBV)infection,as well as liver cirrhosis and primary hepatocellular carcinoma(HCC)based on the development of it,have seriously threatening people's health in China.China is a country in high incidence of hepatitis B,with about 120 million HBV carriers,among which children aged 5 or less account for 1.0%.Since more than 90%of perinatally infected infants will develop chronic infection eventually,only effective prevention in early stage can ultimately control HBV prevalence.Hepatitis B vaccine(HepB)is an effective measure to prevent and control hepatitis B.The advent and popularity of hepatitis B vaccine has significantly reduced the incidence of chronic hepatitis B,and even reduced liver cirrhosis and HCC caused by HBV in high incidence region of HBV gradually.Hepatitis B vaccination had been incorporated into immunization program management as early as 20 years ago in China and its protective effect has also been confirmed by a large number of applications recently.However,in use of vaccine,one problem is significant regional differences on coverage and immune effects of hepatitis B vaccination.Another problem which is more difficult to solve is immune failure,in other words,no response or weak response after immunization.Immune failure people can not be protected by hepatitis B vaccine effectively and will be in high risk of HBV infection.In this study,we investigated and discussed immunization compliance and success rate of hepatitis B vaccination of preschool children in our region,further analyzed correlation of immune cell subsets and immune effects,and discussed differences and related immune factors on hepatitis B vaccination responses in preschool children,providing basis for prevention and control of hepatitis B for local children.Materials and Methods1 Objects1965 cases of pre-school children from Lin'an,Zhejiang were enrolled in this study.Questionnaire of hepatitis B vaccination status was conducted on nursery or school children taking physical examination in this area from January 2015 to May 2016 using random sampling method.Cellular immune responses were detected in 813 cases.2 Methods(1)Questionnaire Questionnaire was designed including basic information,immunization history of hepatitis B vaccine,hepatitis B family history and other releted information of children enrolled.Face to face investigation was conducted for each child as well as their parents or guardians in accordance with uniform questionnaire.(2)Sample collection Informed consent was signature at the same time of field survey.2-5mL venous blood was collected from surveyed children and transported to the specified detection point(The First Affiliated Hospital of Zhejiang University)timely.The serum and PBMC were separated for hepatitis B serological markers and cellular immune response detection respectively.(3)Detection and criteria of HBV virological indicators HBsAg,anti-HBs,HBeAg,anti-HBe and anti-HBc were detected by micro-particle Elisa(MEIA).HBsAg?0.05 IU/mL,HBeAg(s/co)? 1.000,anti-HBc(s/co)? 1.000,anti-HBe(s/co)<1.000 and anti-HBs ? 10 IU/L were determined as positive(+).Effects of hepatitis B vaccine were defined as follows:Negative HBV serological markers and anti-HBs<10 IU/L were regarded as no response to vaccination;simple anti-HBs positive and 10 IU/L<anti-HBs<100 IU/L were regarded as low response to vaccination;simple anti-HBs positive and anti-HBs ? 100 IU/L were regarded as high response vaccination.(4)Detection of lymphocyte subsets from peripheral blood 100?l anticoagulant blood was taken into each flow cytometry tubeand CD3-PerCP-Cy5.5,CD4-APC and CD8-APC-Cy7 antibodies were added in each tube by 20 ?l.Isotype control was conducted at the same time.Samples and reagents were mixed and incubated in dark for 15 min at room temperature.Coulter QPREP apparatus(Beckman coulter company,US)was used to lyse red blood cells and fix cells.More than 1×104 cells were counted by a flow cytometer(Coulter Cytomics FC 500,Beckman Coulter Co.,US).Percentage of CD3+,CD4+ and CD8+ T cells in total lymphocytes were recorded respectively.Similarly,B lymphocyte subsets and Follicular Helper T cells in peripheral blood were detected through CD 19-APC-Cy7?CXCR5-FITC fluorescence antibody.(5)Detection of CD45RA and CD45RO cells Peripheral blood mononuclear cells(PBMC)were isolated using Ficoll-Hypaque density gradient centrifugation.100?l PBMCs were taken and added to each tube,followed by adding 20?l of monoclonal fluorescent antibody anti-CD45RO-PE,anti-CD45RA-FITC,anti-CD4-APC and anti-CD8-APC-Cy7 for each tube.PBMCs and antibodies were mixed thoroughly and incubated in dark for 15min.After washing,500?l PBS was added and the tubes were vibrated,being prepared for testing.Lymphocyte immune phenotypes of CD4+CD45RO+,CD4+CD45RA+,CD8+CD45RO+ and CD8+CD45RA+ were analyzed by lymphocyte gating using double-color immunofluorescence technique.10000 cells were grasped for each tube.The results were acquired and analyzed by EXP032(Beckman Coulter)Software.(6)Detection of Thl/Th2 cell related factors by ELISPOT Changes of Th1/Th2 cytokines(IFN-y,IL-2/IL-4,IL-5,IL-10)were tested using ELISPOT assay.Protocol was described as follows:Adjust the PBMC concentration to 1×106 cells/ml and add 100?l to each hole of antibody pre-coated ELISPOT plate.Add specific stimulation polypeptides with positive and negative control.Add 10%FCS medium and incubated at 37?,5%CO2 for 16-20 hours.After washing by PBS,add corresponding secondary antibody and incubate at room temperature in dark.Wash again and add enzyme marked avidin,incubating at room temperature in dark.Wash again and add color substrate,incubating at room temperature in dark for 20min.Add stop solution and count spot-forming cells(SFC)by ELISPOT counter after washing the plate.(7)Statistical analysis Related methods of SPSS 13.0 software were used in statistical analysis.Results1 Investigation of effectiveness on hepatitis B vaccination on preschool children(1)In this study,serum samples of 1965 cases aging from 0 to 8 years old collected,among which 1695 cases of data is complete and 1591 cases were vaccinated according to standard protocols.Hepatitis B vaccination rate was 93.86%and vaccination rates were similar between boys and girls(93.68%vs 94.07%).Vaccination rate was 95.34%(654/686)for children between 0 to 2 years old,93.36%(914/979)for children between 3 to 5 years old and 76.67%(23/30)for children between 6 to 8 years old.(2)For complete full course HepB vaccinator,the positive rate of anti-HBs was 79.30%(1249/1591).Positive rate of anti-HBs for children aged 0-2 years old was higher than that of children aged 3-5 years old(86.85%vs 72.98%),and the rate of children aged 3-5 years old was higher than that of children aged 6-8 years old(72.98%vs 60.87%).There was no significant difference in vaccine response rates between children of different ages and genders.(3)Anti-HBs seroconversion rate for children whose mothers were hepatitis B positive was low,while the rate for immunity strengthened children was significantly improved.Other HBV positive family members have no effect on children's response rate to vaccine.2.Changes of cellular immune response in children with low response and no response after HepB vaccine(1)Among 813 samples detected by flow cytometry,1 was HBsAg positive and 4 were anti-HBc positive.In the remaining 808 cases,serum anti-HBs titers were<10 IU/L(non-response group)in 193 cases,10-100 IU/L(low response group)in 345 cases,and ?100 IU/L(high response group)in 270 cases.(2)By detecting lymphocytes,CD3+/CD4+/CD8+ T cell subsets,CD4+CD45RO+ T cells?CD4+CD45RA+ T cells and percentage of CD8+CD45RO+ T cells?CD8+CD45RA+ T cells in peripheral blood in different response groups,we found that CD8+ T cells,CD4+ CD45RO+ memory T cells,CD8+CD45RA+ effector T cells and T follicular helper increased significantly in no response or low response group compared with high response group and the difference was statistically significant(P<0.05).Lymphocytes,CD4+T cells,CD4+CD45RA+ effector T cells in peripheral blood in no response or low response group were lower than that in high response group and the difference was statistically significant(P<0.05).There was no statistically significant difference in CD3+ T cells?CD 19+ B cells and CD8+CD45RO+T cells in peripheral blood between different groups.(3)From analysis of Th1/Th2 cell related cytokine expression between different response groups,we discovered that in comparison with high response group,Thl cytokines IL-2 and IFN-? expression were significantly increased in no response or low response group,while Th2 cytokines IL-4 and IL-10 expression were significantly decreased,with difference statistically significant(P<0.05).However,there was no significant difference in IL-5 expression between groups.ConclusionHepatitis B vaccination in this region is good on the whole with strong compliance.Effectiveness of vaccination decreased with age increasing,which was particularly significant between 2 to 5 years old.Vaccination response rates in children of different ages and genders were similar.Anti-HBs seroconversion rate for children whose mothers were hepatitis B positive was significantly improved after immunity strengthened,while other HBV positive family members have no effect on children's response rate to vaccine.Cellular immune results showed that lymphocytes,CD4+/CD8+T cell subsets,CD45RO+ memory T cells and CD45RA+ effector T cells,T follicular helper,and Th1/Th2 related cytokines in peripheral blood were significantly different between preschool children,which might be an internal factor to affect recombinant hepatitis B vaccine's immune effectiveness.In the future,it is able to assess the presence of HBV immune protection and provide a more comprehensive basis for evaluation of the effectiveness of vaccination from the memory level of cellular immunity even if anti-HBs cannot be detected. |
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