| It has been reported that the neonatal hepatitis B virus(HBV)vaccine is an effective strategy to protect children from chronic HBV infection and to reduce the positive rate of hepatitis B surface antigen(HBsAg)among those vaccinees.The vaccine can also decrease the incidence of hepatocellular carcinoma(HCC)and mortality among adolescents.Antibodies against HBsAg(anti-HBs)conferred by HBV vaccination at infancy waned or reached undetectable levels in many individuals 10-15 years later.Many investigators have conducted multiple booster tests in Chinese or other populations and found that booster could improve the production of anti-HBs in those vaccinees who were anti-HBs(-)before booster.The result indicated that the revaccination could trigger an anamnestic response to HBsAg in those previous vaccines.However,it is not a direct evidence of the presence of antiviral immunity.Like many other tumors,HCC usually occurs in adulthood.Many potential factors can promote the progression of HCC in HBV infected patients.It has been reported that the risk of HCC in patients with occult HBV infection(OBI)is also significantly increased.OBI has been reported among those vaccinated individuals.Adults who were born to HBsAg(+)mothers and had been protected at childhood by neonatal vaccination series are still at high risk of HBV infection,including OBI occurred.So,we needed to explore the long-term protection of HBV vaccination against OBI and possible mechanisms by which anti-HBs response is attenuated.This study consists of two parts:Part Ⅰ:Long-term effect on occult HBV infection after neonatal hepatitis B vaccination(Neonatal hepatitis B vaccination protects mature adults from occult virus infection,Hepatology International,2021.Published online)Background&Aim:Antibody against hepatitis B core antigen(anti-HBc)is one of the most classic markers for“the past HBV infection”.HBV-DNA fragment could be detected in sera of anti-HBc positive individuals,indicating the occurrence of OBI.Studies reported that HBV-DNA was frequently detected in the blood of the vaccinated individuals with serological markers of HBsAg(-)/anti-HBc(+),indicating that the OBI might occur in some adults who received vaccination.OBI can increase the risk of many chronic liver disease,like HCC.It is necessary to define the protection of neonatal vaccination against OBI in their mature adulthood Qidong County,China,was once a high prevalence of HBV infection compared with China as a whole.To study the efficacy of HBV vaccination against other liver diseases and HCC,the Qidong Hepatitis B Intervention Study(QHBIS)was conducted during 1983-1990,a window period when the vaccine was not available in any rural area of China.A total of 41 182 newborns were randomly assigned to the neonatal vaccination group,and 40 211(97.64%)of them were immunized with 5 μg of plasma-derived HBV vaccine each dose at day 0,month 1 and month 6 after birth.A total of 41 730 newborns were randomly assigned to the control group,and all the participants received neither vaccine nor placebo.In 2000,the children in both groups who were born during 1986-1990 received catch-up vaccination at age 10-14 years.23 368 participants in the control group received a three-dose catch-up vaccination.Each dose contained 10μg of recombinant vaccine.In 2008-2012,we followed the participants(19-28 years of age)of the QHBIS regarding serological survey and disease outcomes.In 2018,we conducted the serological survey among adults(28-35 years of age)who remained in the cohort by 2008-2012 to detected the long-term protection of neonatal vaccination against OBI.Methods&Results:1.Participants.We sampled approximately 10%of the adults who remained in the QHBIS by 2008-2012 to conduct the serological survey.To analyze the protection against OBI in vaccinated adults(28-35 years of age),each participant was tested for HBV serological markers,HBV-DNA,and relaxed circular DNA(rcDNA).A total of 6 715 individuals were enrolled.Among them,3 615 individuals from the neonatal vaccination group and 3 100 individuals from the control group donated blood samples.2.The neonatal vaccination kept the effective protection against HBV infection.Among the mature adults(28-35 years of age)in neonatal vaccination group,the prevalence of HBsAg was 1.58%(57/3 615),HBsAg(-)/anti-HBc(+)was 4.70%(170/3615),significantly lower than in the control group(7.45%,231/3 100,19.48%,604/3 100,respectively)(all p<0.001).3.The age is an uncertain factor affecting HBsAg(-)/anti-HBc(+)prevalence among adults who received neonatal vaccination.The prevalence of HBsAg(-)/anti-HBc(+)in the neonatal vaccination group was 4.31%(55/1 276)among the sampled participants at 28-29 years,4.66%(59/1 266)among the sampled participants at 30-31 years,5.74%(49/853)among the sampled participants at 32-33 years,but it was 4.29%(7/163)among the sampled participants at 34-35 years.The increase of HBsAg(-)/anti-HBc(+)with aging was uncertain among the neonatal vaccination group(P for trend=0.261).Nevertheless,HBsAg(-)/anti-HBc(+)prevalence in the control group increased significantly with aging(P for trend<0.001),from 17.16%of 28-29 years to 25.67%of 34-35 years old.4.Maternal HBsAg-positive status increased the HBV infection in adults who received neonatal vaccination.Among the vaccinated adults who were born to HBsAg(+)mothers,HBsAg prevalence was 8.5%(32/378),and HBsAg(-)/anti-HBc(+)was 10%(38/378).But among the vaccinated adults who were born to HBsAg(-)mothers,the prevalence was 0.8%(25/3 237)and 4.1%(132/3 237),respectively.5.OBI had happened in some adults who received neonatal vaccination.From blood samples of HBsAg(-)/anti-HBc(+)adults,29 blood samples were positive for HBV-DNA.In 130 blood samples from vaccination group,17(13.08%)were positive;in 287 samples from control group,12(4.18%)were positive for HBV-DNA.To confirm the presence of infectious virion in the HBsAg(-)/anti-HBc(+)bloods,we examined the presence of HBV rcDNA in the 29 sera tested positive for HBV-DNA.Of them,26(90%)of the samples had the positive signals of HBV rcDNA,indicating the true OBI happened among those individuals.6.No mutation of the S gene increased in HBV isolates from adults who received neonatal vaccination.We amplified and sequenced the HBV PreS-S region.Analysis showed no significant increase of variation in the S gene and the ’a’ epitope between the neonatal vaccination group and control group.However,we detected a high R48K mutation of PreS2 in isolates that were derived from the neonatal vaccination group.The same mutation was also detected in occult HBV infected children in Qidong.Summary:The protective effect of neonatal vaccination has lasted for more than 3 decades.But we found that the OBI had occurred in adults who received neonatal vaccination.The individuals who were born to HBsAg(+)mothers had significantly increased risk of developing HBV infections in adulthood.Part Ⅱ:The impacts of prenatal HBsAg exposure on the generation of T follicular helper cells and anti-HBs specific to HBsAg.Background&Aim:HBV vaccine consists of HBsAg protein which is a T-cel 1-dependent antigen,and the production of anti-HBs needs the help of CD4+T cells.T follicular helper cells(Tfh cells)are a subset of CD4+T cells that expressed high levels of CXCR5.Interleukin(IL)21 is an autocrine growth factor for Tfh cells and is crucial for the generation of Tfh cells.IL-21 has been also shown to be produced by natural killer T(NKT)cells.The Tth cells can promote the development of B cells,and the generation of plasma cells and memory B cells by IL21/IL21R,ICOS-ICOSL,and CD40/CD40L signals.Maternal HBsAg can be detected in the amniotic fluid and cord blood.It was reported that the immune responses in neonates born to HBV-positive mothers were different from those of neonates born to HBV-negative mothers in responses to microbial products.So,we speculated that prenatal HBsAg exposure impaired the differentiation of HBsAg-specific B cells into antibody-producing plasma cells by reducing Tfh cells generation,which might be a risk factor of vaccination failure.Methods&Results:1.T-cell responses to HBsAg in HBV-positive mothers were weak and generate less numbers of HBsAg-specific IFNγ-producing,IL4-producing and IL21-producing T cells.We obtained 11 human umbilical cord blood samples from HBsAg(+)and HBsAg(-)mothers to determine their T cell response to HBsAg.We determined maternal HBV-DNA and HBV serological markers two weeks before delivery,and then divided the cord blood samples into HBsAg(+)/HBV-DNA>105 IU/mL(n=2),HBsAg(+)/HBV-DNA ≤105 IU/mL(n=5),and HBsAg(-)/HBV-DNA(-)group(n=4).Lymphocytes were isolated from cord blood and were cultured in the presence of 5 g/ml HBsAg for 5 days.Compared with the cord blood T cells from HBsAg(-)mothers,the numbers of HBsAg-specific T cells that produced IFNy,IL4,and IL21 were lower of HBsAg(+)mothers.Notably,no HBsAg-specific IL21-producing T cells were detected when maternal HBV-DNA>105 IU/mL.2.A murine model mimicking human prenatal HBsAg exposure.We used female Albl-HBV mice(HBV-mother,HBV-M,C57BL/6J background)that constitutively express HBsAg within hepatocytes and secrete HBsAg persistently into the serum to mate with male wild-type C57BL/6J mice.On embryonic days 19-20(E19-E20),we collected their amniotic fluid,plasma,and liver tissues respectively,and analyzed HBsAg expression.All those first filial generation(HBV-M/F1)mice who were born to HBsAg(+)female mice had detectable HBsAg in the amniotic fluid,indicating that HB V-M/F1 mice might be used to mimic human prenatal HBsAg exposure status.HB V-M/F1+mice had detectable HBsAg in the liver tissues,blood,and amniotic fluid.And inserted HBsAg in liver genome was detected in HBV-M/F1+ mice.In the HB V-M/F1-mice,HBsAg was only detected in the amniotic fluid.Those HBV-M/F1-mice only exposed to HBsAg in amniotic fluid and had no HBV fragment in the hepatocytes.3.Prenatal HBsAg exposure impaired HBsAg-specific Tfh cell generation in response to HBsAg.Each HBV-M/F1 mouse or wild-type C57BL/6J mouse was immunized at 4 weeks old with hepatitis B vaccine contained 5μg HBsAg.Compared with wild-type C57BL/6J mice,the percentage of splenic CD4+CXCR5+PD1+Tfh was lower in HBV-M/F1+and HBV-M/F1-,particular in the HBV-M/F1+mice.All HBV-M/F1 mice HBsAg-specific CD4+T cells had significantly reduced the generation of IL-21(p<0.05).Then naive CD4+ T cells and dendritic cells were isolated from different mice and cocultured.Addition of exogenous IL-21 to the coculture system that contained naive CD4+T cells isolated from HBV-M/F 1-mice.The numbers of CD4+CXCR5+PD1+Tfh cells increased.Nevertheless,no improvement for the CD4+CXCR5+PD1+Tfh cells generation was observed after inclusion of IL-21 to the culture system that contained HBV-M/F1+naive CD4+ T cells.4.Prenatal HBsAg exposure impaired the differentiation of HBsAg-specific B cells into antibody-producing plasma cells.Each HBV-M/F1 mouse or wild-type C57BL/6J mouse was immunized at 4 weeks old with hepatitis B vaccine contained 5μg HBsAg.The stimulation with CD4+T from HBV-M/F 1 mice generated significantly fewer numbers of CD138+lgD-plasma cells and HBsAg-specific antibody secreting cells(ASCs)compared with the CD4+T cells in wild-type C57BL/6J mice(p<0.01).Then CD4+CXCR5+Tfh cells and B cells were isolated from different strains of mice and cocultured.Addition of exogenous IL-2 1 to the coculture system that contained CD4+CXCR5+ Tfh isolated from HBV-M/F1-mice.The numbers of CD1 38+IgD-plasma cells increased approximately two-fold.Nevertheless,the generation of CD138+IgD-plasma cells did not change with CD4+CXCR5+Tfh cells isolated from HBV-M/F1+mice after IL-21 addition.5.Booster vaccination of HBsAg improved anti-HBs generation in HBV-M/F1-mice.Each HBV-M/F1 mouse or wild-type C57BL/6J mouse was immunized at 4 weeks old with hepatitis B vaccine contained 5μg HBsAg for the first dose.The second and third dose of vaccines were given every two weeks.And we examined the HBsAg booster effect on the generation of anti-HBs.In the group of HBV-M/F1+ mice(n=12),anti-HBs remained undetectable after the third immunization.However,In the group of HBV-M/F1-mice(n=12),the numbers of anti-HBs generating mice increased from 58%(7/12)after the second dose to 92%(11/12)after the third dose of HBsAg immunization.In the group of wild-type C57BL/6J mice(n=12),the numbers of anti-HBs generating mice increased from 58%(7/12)after the second dose to 100%(12/12)after the third dose of HBsAg immunization.And no statistics significance of anti-HBs positive rate was observed between the HBV-M/F1-group and wild-type C57BL/6J group.The IgG2a/IgG1 ratio of immunized HBV-M/F1-mice was still lower than that of immunized wild-type C57BL/6J mice(p<0.01).Summary:Prenatal HBV exposure might reduce HBsAg-specific Tfh cells generation,and impair Tfh-dependent anti-HBs response by reducing IL21 production.However,HBV vaccine booster might increase the IL21 production,and then improve the differentiation of HBsAg-specific B cells into antibody-producing plasma cells,which could improve HBsAg-specific responses for uninfected individuals who were prenatally exposed to maternal HBsAg. |
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