Characterization Of Molecular And Cellular Immune Responses In Occult Hepatitis B Virus Infection From Blood Donor Population

Background and AimsThe occurrence and development mechanism of chronic hepatitis B virus is closely related to hepatitis B virus(HBV)mutation and immune status,gender,age,genetics,and liver inflammatory activity in the patient.However,occult HBV infection(OBI)has not been clearly described in terms of virological characteristics,co-infection,host immune response,and epigenetics.In particular,studies on molecular and cellular immune function of OBI carriers are rarely reported.The purpose of this study was to investigate the characterization of HBV-specific molecular and cellular immune responses and the mechanism of OBI occurrence in OBI carriers.SubjectsA total of 193 blood samples were collected from voluntary blood donors at Guangzhou Blood Center,China,during the period from August 2016 to December 2017.Six groups of blood donor cohorts were established in the study,including 37 OBI carriers,53 CHB patients(including 42 CHB-HBeAg-and 11 CHB-HBeAg+),47 blood donors with spontaneously resolved HBV infection,and 56 blood donors without HBV infection markers(including 33 HBV vaccinated donors and 23 no markers of healthy control).MethodsThe hepatitis B surface antigen(HBsAg)was detected by chemiluminescence in study population.HBV DNA was detected by nucleic acid detection(NAT).Viral load was quantified by qPCR,viral gene amplification was performed by nested PCR and the nucleotide sequences were determined by sequencing commercially.Human peripheral blood mononuclear cells(PBMCs)from blood donors were stimulated with HBV core and polymerase peptides in vitro.T cell proliferation assays(CFSE)was used for detecting T cell proliferation,enzymelinked immunospot assay(ELISPOT)for detecting the frequency of HBV-specific IFN-y secreted T cells,intracellular cytokine staining assay(ICS)for detecting the frequency and cell origin of CD4+and CD8+T Cells of IFN-γ,TNF-α,IL-2,IL-17A,IL-21,IL-10 and TGF-β,and cytometric bead array(CBA)test for detecting the level of individual cytokine secretion,respectively.SPSS 20.0 statistical analysis software was used for statistical analysis.The normal distribution of measurement data was used for two independent sample t test;analysis among multiple groups was used One-Way ANOVA;for sets comparison between groups the Mann-Whitney U test was used in non-normal data;the correlation test analysis was used by Spearman’s method.P<0.05 was considered statistically significant.Results1.The characteristics of OBI in blood donor population.The study found that the rate of OBI in HBcAb-positive donors was higher,especially in those with positive HBcAb alone.In addition,the rate of OBI increased with age and was higher in males than females.The genotype B was predominant in OBI in Guangzhou blood donors.2.The characteristics of T cell proliferation.The proliferation of CD8+T lymphocytes was dominated by CFSE with nonspecific stimuli PHA stimulation.There was no difference in overall comparison of the proliferation of T lymphocytes among six groups of cohorts(P=0.403),but the proliferation rates in the OBI and CHB groups were lower than those in the healthy controls,respectively(74.0%,78.1%vs.82.1%),while the proliferation of CD4+T lymphocytes was higher with the stimulation of HBV core and polymerase peptides.The overall comparison of the T-lymphocyte proliferations among six groups of cohorts was statistically significant(P<0.001).The proliferation rates of OBI and CHB groups were higher than those of healthy controls,respectively(3.0%vs.1.7%,P=0.016;3.3%vs.1.7%,P<0.001).3.The frequency of HBV specific IFN-y secretion T cells was tested by ELISPOT.The immune responses of lymphocytes from the OBI group and the CHB group were significantly higher than those from the other three groups(P=0.004).The overall comparison of the response intensities of the specific T-cells among six groups was significantly different by the stimulation of three recombinant proteins(HBcAg,HBsAg-ayw,HBsAg-adw)and peptides pool(P<0.05).The specific T cell response intensity in OBI group(160 SFC/106 PBMCs)was higher than that of the healthy control group(95 SFC/106 PBMCs)stimulated by HBcAg recombinant proteins,but was lower than CHB-HBeAg-group(208 SFC/106 PBMCs).Both OBI(25 SFC/106 PBMCs)and CHB-HBeAg-groups(25 SFC/106 PBMCs)showed higher response intensities than the healthy control group with HBV core and polymerase peptides pool(5 SFC/106 PBMCs).Additionally,the overall positive rate of HBV Core peptides pool stimulation was significantly higher than that of HBV Pol peptides pool(44.6%vs.16.1%).The ELISPOT positive rate of T cells stimulated by HBV core peptide pool was the highest in the OBI group(64.0%),and followed by the HBV spontaneously resolved group(53.2%).The positive rate of these two groups was significantly higher than that of the normal control group(21.7%).4.Intracellular cytokine T cell frequencies and extracellular secretory cytokine levels.The ICS data showed that the frequencies of CD4+and CD8+T cells secreting IFN-y,TNF-α,IL-2,IL-10,and TGF-β in OBI and CHB groups were both higher than that in healthy control group by the PMA stimuli.The frequencies of T cells secreting IL-17A and IL-21 in OBI were lower than that of CHB group.Moreover,the frequencies of CD4+and CD8+T cells secreting IFN-γ,TNF-α,IL-17A,IL-21,and TGF-β with stimulation of HBV core peptides were higher in the resolver group than those in both OBI and CHB groups(P<0.05),while the frequencies of T cells secreting IL-2 and IL-10 were higher in both OBI and CHB groups(P<0.05).The frequencies of CD4+and CD8+T cells secreting IFN-γ,TNF-α,IL-17A,and IL-21 were the lowest in CHB group with HBV polymerase peptide pool stimuli(P<0.05),while the frequencies of T cells secreting IL-10 and TGF-β were the lowest in the OBI group(P<0.05).The secretion cytokine levels tested by CBA showed that the levels of IFN-γ,IL-2,IL-10 and IL-17A were higher in both OBI and the CHB groups than those in the healthy control group with HBV core peptides stimuli(P<0.05).The levels of IFN-γ,IL-2 and IL-17A were also higher in both OBI and CHB groups than those in the healthy control group with HBV core peptides stimuli(P<0.05).The data of intracellular and extracellular cytokines obtained by ICS and CBA were basically consistent.5.The frequencies of MDSCs.According to the cell subpopulation counts,the level of M-MDSCs in PBMCs of OBI carriers was significantly lower than that of CHB patients(P<0.001),but not significantly different from that of normal controls(P=0.860).There was no difference in the levels of G-MDSCs among the five groups of cohorts(P=0.914).There was no correlation between M-MDSCs and G-MDSCs in peripheral blood with liver function parameters such as ALT,AST,TBIL,DBIL,TBA,ALB,ADA,CHE,γ-GT and TP in OBI carriers and CHB patients.ConclusionsThis study showed that the HBV-specific T-lymphocyte proliferative responses were higher in both OBI carriers and CHB patients than those in HBV resolver group and non-infected patients.OBI carriers,HBV resolvers and CHB patients showed high frequency of specific IFN-y secreting T cells.The IFN-γ response rate was the highest in OBI group,followed by the resolver group and CHB-HBeAg-group,but was lowest in CHB-HBeAg+group.The frequencies of CD4+and CD8+effector T cells specifically secreting IFN-γ,TNF-α,IL-17A,and IL-21 cytokines in the resolver group were significantly higher than the other groups,whereas the frequencies of suppressor T cells secreting IL-10 cytokines were high in both OBI and CHB group.In addition,the lower frequency of effector T cells in the CHB group and relatively higher frequency of T cells secreting IL-2 and IL-17A in the OBI group were observed.Consequently,the level of HBV-specific T-effector response in OBI carriers is between the levels of HBV resolvers and CHB patients,while the CHB patients have higher T-suppressor cell response,which make three different outcomes from HBV infected individuals.Innovation1.The blood donors with different outcomes of HBV infection who were not treated with antiviral drugs were enrolled in this study.The immune function changes of lymphocytes due to cryopreservation and reconstitution were avoided by using freshly isolated PBMCs in the experiments.2.Unlike previous studies by using HBV overlapping peptides,in this study the HBV core and Pol peptides were synthesized according to HLA-I and HLA-II restrictions that produced specific cellular immune responses.It could reflect the immune response status of T lymphocytes from the different outcome statuses of HBV infected individuals.3.The frequencies of 7 specific cytokines secreted by T-cell subsets and the level of 7 secreted cytokines were analyzed on the different outcomes of HBV-infected blood donors.Furthermore,the molecular and cellular immune mechanism was elucidated according to three statuses of outcomes from OBI carriers,CHB patients and HBV resolvers.