Basic And Clinical Research On Prevention And Treatment Of Hepatitis B Virus And Cytomegalovirus Mother-to-child Transmission

Chapter One:Study on the prevention of mother-to-child transmission of hepatitis B virusPart I:Influence of feeding modes on risk of mother-to-child transmission of hepatitis B virusObjective:To investigate whether breastfeeding may add any risk for mother-to-child transmission of hepatitis B virus(HBV),and determine the influence of maternal hepatitis B e antigen(HBeAg)positivity during pregnancy on the immune response against HBV in infants.Methods:Totally 674 children born to women with hepatitis B surface antigen(HBsAg)positivity during pregnancy were included in the present follow-up study,with 491 breastfed and 183 formula-fed.All children had been vaccinated against hepatitis B after birth,but only 416(61.7%)received simultaneous use of hepatitis B immune globulin(HBIG).Of the 674 children,179(26.6%)were born to HBeAg-positive mothers.Blood samples were collected from each child at follow-up for testing HBV serologic markers.Results:Of the 674 children(368 males)at age of 1-7 years(mean,4.1 ± 1.7 years),17(2.5%)were HBsAg-positive,indicating chronic HBV infection.The HBsAg prevalence in breast-and formula-fed children was 1.6%(8/491)and 4,9%(9/183)respectively(?2 ? 4.603,P = 0.015);similarly,the rate of self-resolved infection,presented as anti-HBc-positive but HBsAg-negative,was 2.0%in breastfed children,lower than that(4.9%)in formula-fed children(P = 0.044).The difference was likely due to the higher rate of maternal HBeAg positivity in formula-fed group(formula-fed 52.5%vs.breastfed 16.9%,P<0.001).All the HBsAg-positive children were born to HBeAg-positive mothers,indicating that maternal HBeAg positivity may be an important risk factor for perinatal HBV transmission.Further comparison of HBV infection in children of the 179 HBeAg-positive mothers showed that the rate of chronic infection and self-resolved infection in 83 breastfed children were 9.6%and 6.0%respectively,each comparable to that of 96 formula-fed children(chronic infection 9.4%,P = 0.952 and self-resolved infection 8.3%,P = 0.553),while their mothers had similar HBV DNA levels(6.32 × 107 IU/ml vs.5.48 × 107 IU/ml,P =0.418).Logistic regression analysis further demonstrated that breastfeeding is not an independent risk factor for perinatal HBV infection.In addition,the overall prevalence of anti-HBs-positive in the 657 children(4.1 ± 1.7-year-old)without chronic HBV infection was 77.9%,with median anti-HBs level of 68.3 mIU/ml.The rate of anti-HBs-positive(78.5%vs.76.4%,P = 0.580)and antibody level(73.4 mIU/ml vs.56.8 mIU/ml,P = 0.963)were both comparable between breast-and formula-fed children.Similarly,the anti-HBs immune response in children born to women with maternal HBeAg positivity during pregnancy was not significantly different from that in children of HBeAg-negative mothers.Conclusions:After the recommend prophylaxis is implemented in infants of HBsAg-positive mothers,breastfeeding does not add any risk for the mother-to-child transmission of HBV.Therefore,regardless of maternal HBeAg carrier status,clinicians should encourage HBV carrier mothers to breastfeed their infants.In addition,although maternal HBeAg positivity is an important risk factor for perinatal HBV infection,it does not affect the immune response against HBV in infants.Part II:Effect of delivery modes on the risk of mother-to-child transmission of hepatitis B virusObjective:To investigate whether caesarean section for pregnant women with chronic hepatitis B virus(HBV)infection may reduce mother-to-child transmission of HBV in infants with combined immunoprophylaxis.Methods:Totally 674 children born to women with maternal hepatitis B surface antigen(HBsAg)positivity during pregnancy across Jiangsu Province,were followed-up at age of 1-7 years(mean,4.1 ± 1.7 years).Of the 674 children,357(53.0%)were delivered by caesarean section,and 317(47.0%)others were born by vaginal delivery.In addition,179(26.6%)were born to hepatitis B e antigen(HBeAg)-positive mothers.All the children had been vaccinated against hepatitis B after birth,and 416(61.7%)received simultaneous use of hepatitis B immune globulin(HBIG).Blood samples were collected from each child at follow-up for testing HBV serologic markers.We analyzed the use of HBIG and hepatitis B vaccine in infants,and further compared the HBV infection in children born by caesarean section or vaginal delivery.Results:Of the 674 children,17(2.5%)were HBsAg-positive,indicating chronic HBV infection;19(2.8%)were HBsAg-negative but anti-HBc-positive,suggestive of self-resolved infection.Of the 17 children with chronic infection,13(76.5%)were not administered with HBIG or received delayed hepatitis B vaccine,indicating that the perinatal HBV infection may be attribute to inappropriate immunoprophylaxis.The prevalence of HBsAg-positive in children born by caesarean section and vaginal delivery was 2.8%(10/357)and 2.2%(7/317),respectively;the two rates were not significantly different(P = 0.624).Similarly,the rate of self-resolved infection was 2.2%in children delivered by caesarean section,comparable to that(3.5%)in children born by vaginal delivery(P = 0.336).We further analyzed the HBV infection in children of the 179 HBeAg-positive mothers,including 96 children born by caesarean section and 83 born by vaginal delivery.The rate of chronic and self-resolved infection in children delivered by caesarean section was 10.4%and 4.2%respectively,each comparable to that of children born by vaginal delivery(chronic infection 8.4%,P = 0.652 and self-resolved infection 10.8%,P = 0.086),while their mothers had similar HBV DNA levels(6.18 × 107 vs.5.99 × 107 IU/ml,P = 0.587).Additionally,the rates of anti-HBs ?10 mIU/ml or the anti-HBs levels were not significantly different between the children born by caesarean section and those born by vaginal delivery.Conclusions:With the recommended immunoprophylaxis against hepatitis B,caesarean section does not reduce the risk of mother-to-child transmission of HBV.Therefore,elective caesarean section should not be used in HBsAg-positive pregnant women to prevent the mother-to-child transmission of HBV.Part III:Influence of maternal hepatitis B carrier status on perinatal outcomes and child's growthObjective:To clarify whether maternal hepatitis B surface antigen(HBsAg)carrier status may add risk for adverse neonatal outcomes and even affect the child's growth.Methods:Totally 380 HBsAg-positive and 428 HBsAg-negative singleton pregnant women across Jiangsu province,who delivered their babies during 2002-2004,were retrospectively investigated.Birth weight,height,and adverse neonatal outcomes including preterm birth,stillbirth,neonatal death and congenital malformations,were compared between the two groups.During October 2009 and March 2010,we followed the children above and evaluated each child's growth including weight,height,and health conditions at follow-up.Blood sample was collected from each child to test for HBV serologic markers.Results:The birth weight and height in the infants of HBsAg-positive mothers was 3436.1 ± 388.1 g and 49.8 ± 2.4 cm respectively,each comparable to that in the infants of HBsAg-negative women(weight 3466.4 ± 435.2 g,P = 0.308 and height 49.9 ± 2.4 cm,P = 0.769).The overall prevalence of preterm birth was 2.1%(17/808).The rate of preterm birth in HBsAg-positive group was relatively higher than that in HBsAg-negative mothers(2.9%,11/380 vs.1.4%,6/428),however,it failed to reach statistical significance(P = 0.140).Additionally,there was no significant difference in other adverse neonatal outcomes including stillbirth(0.5%vs.0.2%),neonatal death(0.5%vs.0.5%)and congenital malformation(0.8%vs.1.4%).Logistic regression analyses further demonstrated that maternal HBsAg carrier status was not associated with neonatal outcomes(all P>0.05).Moreover,271(71.3%)children of HBsAg-positive mothers and 297(69.4%)of HBsAg-negative mothers were followed at the age of 5-7 years.The children's weights(22.7 ± 3.8 kg vs.22.7 ± 4.4 kg,P=0.960)or heights(116.8 ± 7.1 cm vs.117.0 ± 9.8 cm,P=0.860)were also comparable between the two groups.There were one(0.26%)child with cerebral palsy in HBsAg-positive group,and four(0.93%)children with abnormal health conditions in HBsAg-negative group,including one with low intelligence,two with congenital deaf and dumb and one infant death due to infantile pneumonia.No maternal death occurred in HBsAg-positive or-negative group.Conclusions:Our results suggest that maternal HBsAg carrier status does not pose any risk for adverse neonatal outcomes and even affect the child's growth.Chapter Two:Preliminary evaluation of cytomegalovirus(CMV)recombinant polypeptides for detection of CMV-specific IgG antibodies and IgG avidityObjective:To evaluate whether cytomegalovirus(CMV)recombinant polypeptides expressed in prokaryotic cells were suitable for detecting CMV-specific IgG antibodies and the IgG avidity index(AI).Methods:The fragment of gene coding for several CMV polypeptides with good antigenicity was cloned and expressed in E.coli,respectively,and the recombinant protein was purified,including phosphoprotein 150(pp150),pp28,non-structural protein pp52,and glycoproteins B(gB).We further established indirect ELISA based on purified recombinant protein above for detecting CMV IgG antibodies in the samples of 200 sera,and compared the results with those of CMV IgG ELISA kit(DIA.PRO Diagnostic Bioprobes,Italy).Moreover,a urea denaturation test included in the indirect ELISA based on selected recombinant polypeptides,which showed good consistence with the IgG ELISA kit(DIA.PRO,Italy),was performed to measure the CMV IgG AI of serum samples from primarily-infected or latently infected patients,respectively.Results:We constructed eight recombinant plasmids,and further acquired the recombinant proteins purified from prokaryotic cells,including pp150/1(aa 1-555),pp150/2(aa 555-705),pp150/3(aa 862-1048),pp150/4(aa 555-1048),pp28(aa 1-191),pp52/1(aa 297-434),pp52/2(aa 1-434)and gB(aa 552-635).Compared with the results of CMV IgG ELISA kit,recombinant polypeptide pp 150/4 showed the highest positive coincident rate(90.9%),followed by pp150/3(89.7%),pp150/2(88.9%)and pp150/1(80.2%),indicating that the antigen epitopes were predominantly distributed in carboxyl terminus region.The positive result of polypeptide gB accorded with the ELISA kit was relatively low(38.9%,49/126),but the negative coincident rate was highest(98.6%,73/74),followed by polypeptide pp150/4(97.0%),pp150/3(95.9%),pp150/2(94.6%)and pp150/1(89.2%).We further determined CMV IgG AI based on recombinant polypeptide pp 150/2,pp 150/3 or pp 150/4,which previously showed good consistence with CMV IgG ELISA kit.Of eight serum samples form primarly-infected patients with AI of 15.3-29.8(DIA.PRO,Italy),four showed AI>30%(39.0-60.4)based on polypeptide pp150/4.In addition,of seven serum samples from latently-infected patients with AI of 59.6-92.3 determined by the IgG ELISA kit,two showed AI<30%(22.9 and 25.1 respectively),indicating primary infection,which was not in accordance with actual infection status.Similarly,the results of CMV IgG AI established based on recombinant polypeptide pp 150/2 or pp150/3 were not in good consistence with the IgG ELISA kit.Conclusions:Recombinant pp150 protein showed good antigencity,and can be used to establish indirect ELISA for detecting CMV IgG antibodies.However,we could not correctly distinguish primary form non-primary infection by the IgG AI established based on recombinant pp150 protein,and therefore the recombinant polypeptides were not suitable for measuring CMV IgG AI.