| BackgroundHIV and HBV have similar routes of transmission,so HIV/HBV co-infection is common.Overall,5 to 20 percent of HIV infected patients can be infected with HBV in worldwide.HIV/HBV co-infection can promote the progress of the disease,that the progress of end-stage liver disease in HIV/HBV co-infection is significantly accelerated compared with HBV mono-infection;that immune reconstruction in HIV/HBV co-infection is delayed compared with HIV mono-infection.Highly active antiretroviral therapy(HAART)can simultaneously inhibit HIV and HBV replication,but cannot completely eliminate HIV reservoirs and HBV reservoirs(HBV cccDNA in liver cells).End-stage liver disease has become the leading cause of death in HIV infected patients.The transmission mode of HBV is mainly mother-to-child transmission in china,and the virus strain is mainly B type and C type.In view of the different epidemiological characteristics and worse prognosis of HBV in China,the uniqueness of HBV infection in China should be fully considered in the treatment of HIV/HBV co-infected patients.HBV genomic sequence variations contain many forms,including deletions in the PreS region.The PreS/S region encodes a translated polypeptide comprising plenty of important epitopes of HBV,and the deletion of the PreS region causes an immunogenic change of the epitope,which allows the virus to evade the host's immune surveillance and cause a sustained immune response.HBV quasispecies are populations in which HBV replication produces a large number of mismatches but does not constitute genotypic variation.The gradual development of dominant quasispecies with the ability to evade host immune attack is an important reason for the persistent and chronic HBV infection.There is no comprehensive research report on the characteristics of PreS region quasispecies variation of HIV/HBV co-infection before treatment and whether it affects the HBV virologic response and inflammatory response during treatment.Previous studies of our research group found that the deletion of HBV PreS quasispecies before treatment was related to the immune reconstruction of HIV/HBV co-infected patients after treatment(the number of CD4+T lymphocytes and the recovery of CD4/CD8 ratio),suggesting that HBV genome sequence variation in HIV/HBV co-infected cohort before treatment may have an impact on the treatment effect and even the prognosis.Previous studies have found that HBV genomic variants related to HCC,such as PreS,A1762T/G1764 A,are more common in HIV/HBV co-infection,and HBV genomic specific sequence variants(mainly including point mutations and deletion of PreS region)are significantly correlated with the increased incidence of end-stage liver diseases in HBV mono-infection.So,are HBV sequence variants in the HIV/HBV co-infected cohort associated with accelerated disease progression? What mechanisms might lead to accelerated disease progression? To answer this question,we need to rely on the longitudinal treatment cohort of HIV/HBV coinfection to study and analyze the variation characteristics of HBV sequence before treatment and its influence on HBV virologic response and inflammatory response during long-term treatment.The purpose of this study was to study the variation characteristics of HIV/HBV coinfection cohort and its effect on long-term HBV virologic response and host inflammatory response,so as to explore the possible mechanism of accelerated disease progression of HIV/HBV co-infection.The research of this subject is divided into three parts: The first part is based on the cross-sectional HBV whole genome sequence,analyzes the variation characteristics related to liver disease progression in HIV/HBV co-infected patients and the correlation between HBV variation and host immunity.The second part of the study is to analyze the characteristics and heterogeneity of HBV PreS quasispecies deletion before HIV/HBV co-infected cohort treatment on the basis of the genome-wide analysis in the first part.The third part is based on the study of the second part.Based on the longitudinal treatment cohort of HIV/HBV co-infection followed up for 6 years,we studied and analyzed the long-term effects on virologic response and inflammatory response of pre-treatment HBV quasispecies deletion from the perspectives of HBV virologic response(using relevant indicators: HBV DNA,HBsAg,HBV PgRNA)and inflammatory response activation(using relevant indicators: sCD14,sCD163,IP-10,IL-18).Part ? The Characterization of HBV full-length variation in HIV/HBV co-infection before treatmentObjectives The variation characteristics of the whole HBV genome sequence before treatment of HIV/HBV co-infection were analyzed in depth,so as to provide a basis for further study on the possible influence of the HBV sequence variation before treatment on the longitudinal treatment cohort of HIV/HBV co-infection.Methods HIV/HBV co-infection and HBV mono-infection were taken as the research objects.After collect the patient's peripheral blood before antiviral,DNA was extracted from serum and was used as template for HBV genome-wide segmented nested PCR amplification.The PCR products were confirmed by electrophoresis and sent to Invitrogen Company for sequencing.Contig Express sequences were spliced and the splicing sequences were compared with the corresponding HBV wild-type strains by Bio Edit7.0 software.The incidence of variation related to liver disease progression(A1762T,G1764 A,G1896A,G1613 A,C1653T,T1753 V,A1846T,G1899 A,Pre S1 deletion,Pre S2 deletion)and HBV Pre S/S epitopes variation in the two groups were compared;The incidence of progression-related variation of liver disease between the two groups was compared taking the number of CD4+T lymphocytes in HIV/HBV co-infection as the critical value;The proportion of point mutations between Pre S deletion and non-Pre S deletion in HIV/HBV co-infection.The phylogenetic tree analysis was completed out by MEGA 6.0 software.Office2010 and SPSS19.0 software were used for statistical analysis,P<0.05 indicate that the difference was statistically significant.Univariate and multivariate Logistic regression analysis was used to analyze the related factors in multivariate model.Results 1.82 cases of HIV/HBV co-infection and 50 cases of HBV mono-infection were successfully amplified,and there was no significant difference in sex composition,age,HBe Ag status,genotype,ALT,AST and HBV DNA.2.Comparison of variants related to liver disease progression between the two groups,most of variation related to the progress of liver disease in HIV/HBV co-infected patients were higher and T1753 V had statistical significance.3.The point mutation rate of Pre S deletion in HIV/HBV co-infection group is higher than that non-Pre S deletion except for C1653 T.The variation rates of A1762 T,G1764A,G1613 A and T1753 V are statistically significant.4.Univariate and multivariate Logistic regression analysis,HIV infection is a related factor for T1753 V,HBV gene C is a related factor for A1762 T and G1764 A,HBV gene B is a related factor for G1896 A,and HBe Ag negative is a related factor for G1896 A,A1846T and G1899 A.5.Except for C1653 T and G1899 A,the variation rate of group of CD4+T cell number?100/ul is higher than CD4+T cell number>100/ul 6.Genetic evolution analysis show that the evolutionary relationship of patients with Pre S deletion was further.The analysis of HBV Pre S/S epitopes variation show that the incidence of cytotoxic T cell(CTL)epitope variation in HIV/HBV co-infected group is higher than HBV mono-infected group,and the comparison of Pre S2 aa1-15 epitope is statistically significant.The deletion rate of Pre S2 aa1-15 epitopes is also significantly higher.There is no significant difference in the incidence of B cell epitopes and all Th cell epitopes except for Pre S2 aa1-26.Conclusions The variation related to liver disease progression and the HBV Pre S/S epitopes variation in HIV/HBV co-infected patients is higher than HBV mono-infected patients.There was more variation related to liver disease progression in HIV/HBV co-infection with low immune function,suggesting that HBV variation was related to host immune status,and HIV/HBV co-infected patients had a higher risk of liver disease progression.Part ? The Characteristics of quasispecies variation of Pre S region in HIV/HBV co-infectionObjectives To analyze the variation characteristics and heterogeneity of quasispecies in Pre S region of HIV/HBV co-infected patients and their correlation with host immune status from the view of quasispecies.Methods HIV/HBV co-infection and HBV mono-infection were taken as the research objects.The peripheral blood of the patients before treatment was collected,and the DNA in serum was extracted and amplified by nested PCR.The HBV Pre S amplification positive product was selected for TA cloning.After the monoclonal PCR,the positive cloning bacteria solution was selected and sent to Invitrogen company for sequencing.The Chromas 2.4 software and Bio Edit7.0 software were used to compare with the corresponding HBV wild-type strains.Comparison of the rate of patients with Pre S deletion,Pre S quasispecies deletion frequency,heterogeneity in the two groups.The heterogeneity was calculated by Golang language package and MEGA 6.0.Office2010 and SPSS19.0 software were used for statistical analysis,P<0.05 indicate that the difference was statistically significant.Results 1.71 cases of HIV/HBV co-infection and 76 cases of HBV mono-infection were successfully amplified,and there was no significant difference in sex composition,age,HBe Ag status,genotype,ALT,AST and HBV DNA.2.Comparison of the rate with Pre S deletion in HIV/HBV co-infected patients and HBV mono-infected patients(one HBV Pre S deletion in the quasispecies sequence was classified as Pre S deletion)was completed.The rate of Pre S deletion in HBV mono-infected patients is higher than HIV/HBV co-infected patients,and the rate is not statistically significant.3.The quasispecies frequency of Pre S deletion in HIV/HBV co-infected patients is higher than HBV mono-infected patients.The quasispecies frequency of Pre S1 deletion in HIV/HBV co-infected patients is significantly higher than HBV mono-infected patients,which was statistically significant.The quasispecies frequency of Pre S2 deletion in HIV/HBV co-infection was higher than HBV mono-infection.The frequency of quasispecies deletion in HBV Pre S functional region was higher in HIV/HBV co-infection.the comparison of S protein promoter region and heat shock protein 70 binding site was statistically significant.4.The HIV/HBV co-infected patients(55 patients)had lower HBV quasispecies heterogeneity than HBV mono-infected patients(32 patients),had a narrow evolutionary width.Conclusions HIV/HBV co-infected patients were more likely to have higher frequency Pre S1 and Pre S2 quasispecies deletion,and the Pre S1 deletion was the main one.The frequency of quasispecies deletion in functional regions of HIV/HBV co-infection was generally high,and the deletion frequency of s-protein promoter region and heat shock protein 70 binding site was the highest.The heterogeneity of HBV quasispecies in HIV/HBV co-infection was low,and the evolutionary width of quasispecies in HIV/HBV co-infection was narrow,suggesting that the combination of HIV infection had significant influence on the quasispecies variation in HBV Pre S region.Part ? The effect of HBV Pre S quasispecies deletion before treatment in HIV/HBV co-infected patients on HBV viral response and inflammatory response after treatmentObjectives According to the results of the second part,based on the longitudinal treatment cohort of HIV/HBV co-infection followed up for 6 years.The influence of HBV Pre S quasispecies deletion on HBV virologic response and inflammatory response in the long-term antiviral treatment was analyzed.From the perspective of virology and immunology,the possible pathogenesis mechanism of HBV Pre S deletion in HIV/HBV co-infection was studied.Methods Plasma was collected from patients with HIV/HBV co-infection before treatment and at 2,4,and 6 years after treatment.The ELISA method was used to quantitatively detect s CD14,s CD163,IP-10 and IL-18.The electrochemiluminescence method was used to quantitatively detect HBs Ag and PCR-fluorescent probe method was used to quantitatively detected HBV DNA and HBV Pg RNA.According to the quasispecies deletion of HBV Pre S,Pre S1,Pre S2 before treatment,the corresponding experimental group and control group were settled up.Longitudinal changes of HBV virology response indexes(HBV DNA,HBs Ag,HBV Pg RNA)and inflammatory factors(s CD14,s CD163,IP-10,IL-18)were analyzed.Office2010 and SPSS19.0 software were used for statistical analysis,and the Graph Pad Prism 6.0 software was performed for drawing.P < 0.05 indicates that the difference was statistically significant.Results 1.Comparison of HIV/HBV co-infected group with Pre S quasispecies deletion(42 cases)and non-Pre S deletion(29 cases).(1)there was no statistically significant difference in plasma HBV DNA at all time points.(2)the HBs Ag detection value of plasma in the group with Pre S deletion was significantly lower than non-Pre S deletion before treatment(0 years),which is statistical significance,and the time points of 2,4 and 6 years after treatment were continuously lower than that non-Pre S deletion.(3)the plasma HBV Pg RNA detection values in the group with Pre S deletion were significantly higher than non-Pre S deletion before treatment(0 years)and 2 years after treatment,which is statistical significance.At the time points of 4 years and 6 years after treatment,which is no statistical significance.2.Comparison of HIV/HBV co-infected group with Pre S1 quasispecies deletion(34 cases)and non-Pre S1 deletion(37 cases).(1)there was no statistically significant difference in plasma HBV DNA,HBs Ag at all time points.(2)the plasma HBV Pg RNA detected value was significantly higher(0 years)and 2 years after treatment,which is statistical significance,while there was no statistical significance in the comparison between 4 years and 6 years after treatment.3.Comparison of HIV/HBV co-infected group with Pre S2 quasispecies deletion(22 cases)and non-Pre S2 deletion(49 cases).(1)there was no statistically significant difference in plasma HBV DNA,HBV Pg RNA at all time points.(2)the HBs Ag detection value of plasma in the group with Pre S2 deletion was significantly lower than non-Pre S2 deletion before treatment(0 years),which is statistical significance,and the time points of 2,4 and 6 years after treatment were continuously lower than that non-Pre S2 deletion.4 There were 59 cases of longitudinal detection of serial plasma inflammatory factors(s CD14,s CD163,IP-10,IL-18)in the HIV/HBV co-infected cohort.(1)Compared with the group without Pre S quasispecies deletion(27 cases),the group with Pre S quasispecies deletion(32 cases)found that the detection values of s CD14,s CD163 and IL-18 in plasma were significantly higher before treatment(0 years),which is statistical significance;there was no statistical significance at the time points of 2,4 and 6 years after treatment;and there was no statistical significance of the detection of IP-10 at all-time points.(2)Compared with the group without Pre S1 quasispecies deletion(33 cases),the group with Pre S1 quasispecies deletion(26 cases)found that the detection values of s CD14,s CD163 and IL-18 in plasma were higher before treatment(0 years),that detection values of s CD14 and s CD163 were statistically significant,and the detection values of s CD163 tended to be higher in 2,4 and 6 years after treatment;the detection values of s CD14 and IL-18 in 2,4,6 years after treatment and the detection values of IP-10 at all-time points had no statistical significance.(3)Compared with the group without Pre S2 quasispecies deletion(41 cases),the group with Pre S2 quasispecies deletion(18 cases)found that the detection values of s CD14 and s CD163 in plasma were higher before treatment(0 years)and had statistical significance,and there was no significant difference in 2,4,6 years after treatment.The detection values of IL-18 at all-time points were higher,with statistical significance after treatment in 4 and 6 years.There was no statistical significance of the detection of IP-10 at all-time points.Conclusions HBV Pre S quasispecies deletion before treatment in HIV/HBV co-infected cohort affected HBV virologic response and host inflammatory response during treatment,especially Pre S2 deletion,which was associated with low levels of plasmatic HBs Ag and high levels of plasmatic IL-18 in long-term HAART.It is suggested that HBV Pre S deletion may lead to abnormal activation of inflammatory response through abnormal HBs Ag secretion,thus accelerating the progression of HIV/HBV co-infection. |
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