Association between low molecular polypeptide 7 single nucleotide polymorphism and response to therapy in hepatitis C virus infection

AIM: To investigate the relationship between low molecular polypeptide-7 (LMP-7) gene polymorphism and response to interferon (IFN) therapy in chronic hepatitis C virus (HCV) patients. METHODS: LMP-7 polymorphism at codon 49 with nucleotide substitution from A to C was amplified in 104 chronic HCV patients of genotype 4. The amplicons were digested with restriction endonuclease Bsm I and the produced restriction fragment length polymorphism was analyzed. Patients received IFN + regional blood volume therapy for 48 wk and the frequency of thissingle nucleotide polymorphism (SNP) was statistically correlated with treatment response. The exclusion criteria for these patients were stated by the national health program for treating viral hepatitis. Main exclusion criteria included co-infection with hepatitis B virus or schistosomiasis, thyroid dysfunction, uncontrolled diabetes mellitus, history of long term drug or alcohol intake and autoimmune hepatitis. Multivariate analyses were done to correlate LMP-7 SNP plus several factors such as age, gender, weight, serum alpha-fetoprotein (AFP) and alanine aminotransferase levels, liver activity, fibrosis score and viral load with response to therapy. RESULTS: The data presented in this study clearly demonstrated statistically significant differences between sustained virological response (SVR) (defined as the absence of HCV RNA levels in the patient's sera at least 6 mo after discontinuation of treatment) and non-response (NR) (where HCV RNA levels in the patient's sera never become undetectable for 6 mo during or after treatment). Variables were described as odds ratio with 95%CI. The data were considered significant if P values were ≤ 0.05; highly significant if P < 0.01 and very highly significant if P < 0.001. Current data showed that 91.7% of patients carrying LMP-7 C/C allele were associated with SVR, while the other two genotypes C/A and A/A were associated with NR patients, 83.3% and 64.3% respectively, showing that genotype CC was strongly associated with response to interferon (95%CI: 12.0719-134.6572, P = 0.0001). The majority of parameters recorded in SVR and NR patients included higher values of mean age (P = 0.004), alanine aminotransferase (P = 0.001), AFP (P = 0.001), body weight (P = 0.025), viral load (P = 0.025), higher fibrosis and histological activity index indices among NR vs SVR patients. Also, the multivariate statistical analysis of the different factors of fibro-sis score, liver activity grade, genotypes and alleles of LMP-7 gene polymorphism in responders and NRs of HCV patients in this study showed that HCV patients with A allele had a very highly significant association with the NRs, high fibrosis and higher liver activity, while the C allele had a very highly significant association with the responders, low fibrosis and lower liver activity (95%CI: 3.5800-13.2519, P = 0.0001).CONCLUSION: LMP-7 SNP is a candidate gene that should be considered when designing a mathematical model for predicting response to therapy and disease progression in HCV patients.

Hepatitis B virus persistent infection-related single nucleotide polymorphisms in HLA regions are associated with viral load in hepatoma families

BACKGROUND Genome-wide association studies from Asia indicate that HLA-DP and HLA-DQ loci are important in persistent hepatitis B virus(HBV)infections.One of the key elements for HBV-related carcinogenesis is persistent viral replication and inflammation.AIM To examine genetic and nongenetic factors with persistent HBV infection and viral load in families with hepatocellular carcinoma(HCC).METHODS The HCC families included 301 hepatitis B surface antigen(HBsAg)carriers and 424 noncarriers born before the nationwide vaccination program was initiated in 1984.Five HBV-related single nucleotide polymorphisms(SNPs)—rs477515,rs9272105,rs9276370,rs7756516,and rs9277535—were genotyped.Factors associated with persistent HBV infection and viral load were analyzed by a generalized estimating equation.RESULTS In the first-stage persistent HBV study,all SNPs except rs9272105 were associated with persistent infection.A significantly higher area under the reciprocal operating characteristic curve for nongenetic factors vs genetic factors(P<0.001)suggests that the former play a major role in persistent HBV infection.In the second-stage viral load study,we added 8 HBsAg carriers born after 1984.The 309 HBsAg carriers were divided into low(n=162)and high viral load(n=147)groups with an HBV DNA cutoff of 105 cps/mL.Sex,relationship to the index case,rs477515,rs9272105,and rs7756516 were associated with viral load.Based on the receiver operating characteristic curve analysis,genetic and nongenetic factors affected viral load equally in the HCC family cohort(P=0.3117).CONCLUSION In these east Asian adults,the mechanism of persistent HBV infection-related SNPs was a prolonged viral replication phase.

No association between IRF3 polymorphism and susceptibility to hepatitis B virus infection in Chinese patients

AIM:To investigate the association between three tag single nucleotide polymorphisms (tagSNPs) in inter-feron regulatory factors (IRF3) and the genetic suscep-tibility to chronic hepatitis B virus (HBV) infection.METHODS:We performed a case-control study of 985 Chinese cases of chronic HBV infection and 294 self-limiting HBV-infected individuals as controls.Three tagSNPs in IRF3 (rs10415576,rs2304204,rs2304206) were genotyped with the Multiplex SNaPshot technique.The genotype and allele frequencies were calculatedand analyzed.RESULTS:The three SNPs showed no significant geno-type/allele associations with chronic HBV infection.Overall allele P values were:rs10415576,P=0.0908,odds ratio (OR) [95% confidence interval (CI)]=1.1798 (0.9740-1.4291);rs2304204,P=0.5959,OR (95% CI)=1.0597 (0.8552-1.3133);rs2304206,P=0.8372,OR (95% CI)=1.0250 (0.8097-1.2976).Overall genotype P values were:rs10415576,P=0.2106;rs2304204,P=0.8458;rs2304206,P=0.8315.There were no statisti-cally significant differences between patients with chron-ic HBV infection and controls.Haplotypes generated by these three SNPs were also not significantly different between the two groups.CONCLUSION:The three tagSNPs of IRF3 are not asso-ciated with HBV infection in the Han Chinese population.

Host nucleotide polymorphism in hepatitis B virusassociated hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is etiologically linked with hepatitis B virus(HBV) and is the leading cause of death amongst 80% of HBV patients. Among HBV affected patients, genetic factors are also involved in modifying the risk factors of HCC. However, the genetic factors that regulate progression to HCC still remain to be determined. In this review, we discuss several single nucleotide polymorphisms(SNPs) which were reportedly associated with increased or reduced risk of HCC occurrence in patients with chronic HBV infection such as cyclooxygenase(COX)-2 expression specifically at COX-2-1195G/A in Chinese, Turkish and Egyptian populations, tumor necrosis factor α and the three most commonly studied SNPs: PAT-/+, Lys939Gln(A33512C, rs2228001) and Ala499Val(C21151T, rs2228000). In genome-wide association studies, strong associations have also been found at loci 1p36.22, 11q22.3, 6p21(rs1419881, rs3997872, rs7453920 and rs7768538), 8p12(rs2275959 and rs37821974) and 22q11.21. The genes implicated in these studies include HLA-DQB2, HLA-DQA1, TCF19, HLA-C, UBE2L3, LTL, FDX1, MICA, UBE4 B and PG. The SNPs found to be associated with the above-mentioned genes still require validation in association studies in order to be considered good prognostic candidates for HCC. Screening of these polymorphisms is very beneficial in clinical experiments to stratify the higher or lower risk for HCC and may help in designing effective and efficient HCC surveillance programs for chronic HBV-infected patients if further genetic vulnerabilities are detected.

Association of-238G/A and -857C/T Polymorphisms of Tumor Necrosis Factor-Alpha Gene Promoter Region With Outcomes of Hepatitis B Virus Infection

Objectives To determine whether -238G/A and -857C/T polymorphisms of tumor necrosis factor-alpha （TNF-o0 gene promoter were associated with outcomes of hepatitis B virus infection. Methods A total of 246 HBV self-limited infected subjects and 443 chronic hepatitis B （HB） patients were recruited in this case-control study. TNF-α-238G/A and -857C/T gene promoter polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP）. Results The frequency of TNF-α-238 GG （90.7%） in chronic HB group was significantly lower than that （95.1%） in self-limited group （P=0.041）. The frequency of TNF-oc-857 CC （79.7%） in chronic HB patients was significantly higher than that （70.9%） in self-limited infected subjects （P=0.021）. Multiple logistic regression analysis revealed that both TNF-oc-238GA and -857CC were independently associated with chronic HB. Conclusions TNF-α promoter variants are likely to play a substantial role in influencing the outcomes of HBV infection.

Association of polymorphism of tumor necrosis factor-alpha gene promoter region with outcome of hepatitis B virus infection

AIM： To determine whether -238G/A and -857C/T polymorphisms of tumor necrosis factor-alpha （TNF-α）, gene promoter and hepatitis B （HB） viral genotypes were associated with outcomes of HBV infection. METHODS： A total of 244 HBV self-limited infected subjects, 208 asymptomatic carriers, and 443 chronic HB patients were recruited to conduct a case-control study. TNF-α -238G/A and -857C/T gene promoter polymorphisms were examined by polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP）, and HBV genotypes were examined by nested PCR. RESULTS： The positive rate of HBV DNA in asymptomatic carder group and chronic HB group was 46.6% and 49.9%, respectively. HBV genotype proportion among the asymptomatic carriers was 2.1% for genotype A, 25.8% for genotype B, 68.0% for genotype C, and 4.1% for genotype B＋C mixed infection, and 0.9% for genotype A, 21.7% for genotype B, 71.5% for genotype C, 5.9% for genotype B＋C mixed infection in chronic HB group. There was no significant difference in genotype distribution between the asymptomatic carrier group and chronic HB group （X^2 = 1.66, P = 0.647）. The frequency of -238GG genotype in self-limited group was 95.1%, significantly higher than 90.7% in chronic HB group and 89.0% in asymptomatic carrier group （P = 0.041 and P = 0.016, respectively）.The frequency of TNF-α-857 CC in chronic HB group was 79.7%, significantly higher than 64.4% in asymptomatic carrier group and 70.9% in self-limited group （P〈0.001 and P = 0.023, respectively）. A multiple logistic regression analysis revealed that TNF-α-238GA and -857CC were independently associated with chronic HB after gender and age were adjusted.CONCLUSION： TNF-α promoter variants are likely to play a substantial role in the outcome of HBV infection.

Single Nucleotide Polymorphisms rs2227284, rs2243283 and rs2243288 in the IL-4 Gene show no Association with Susceptibility to Chronic Hepatitis B in a Chinese Han Population

Objective To investigate the relationship between single nucleotide polymorphisms(SNPs) of the interleukin-4(IL-4) gene and outcome of hepatitis B virus(HBV) infection in a Chinese Han population.Methods Total of 501 patients with chronic hepatitis B virus(HBV) infection and 301 controls with selflimiting HBV infection were studied. Three tag SNPs in the IL-4 gene(rs2227284G/T, rs2243283C/G and rs2243288A/G) were genotyped by the Multiplex snapshot technique. The genotype and allele frequencies were calculated and analyzed.Results The three SNPs showed no significant genotype/allele associations with chronic HBV infection. Overall allele P values were: rs2227284, P = 0.655, odds ratio(OR) [95% confidence interval(CI)] = 1.070(0.793-1.445); rs2243283, P = 0.849, OR(95% CI) = 0.976(0.758-1.257); rs2243288, P = 0.659, OR(95% CI) = 1.060(0.818-1.375). Overall genotype P values were: rs2227284, P = 0.771; rs2243283, P = 0.571; rs2243288, P = 0.902. There were no statistically significant differences between patients with chronic HBV infection and controls. Haplotypes generated by these three SNPs also had no significant differences between the two groups. Conclusions The three tag SNPs of IL-4 were not associated with the outcome of HBV infection in the Han Chinese population.

Polymorphisms of microsomal triglyceride transfer protein in different hepatitis B virus-infected patients

AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and persistent infection. METHODS: A total of 316 subjects with self-limited HBV infection and 316 patients with persistent HBV infection (195 subjects without familial history), matched with age and sex, from the Chinese Han population were enrolled in this study. Polymorphisms of MTP at the promoter region -493 and at H297Q were determined by the allele specific polymerase chain reaction (PCR). RESULTS: The ratio of males to females was 2.13:1 for each group and the average age in the self-limited and chronic infection groups was 38.36 and 38.28 years, respectively. None of the allelic distributions deviated significantly from that predicted by the Hardy-Weinberg equilibrium. There was a linkagedisequilibrium between H297Q and -493G/T (D’ = 0.77). As the χ2 test was used, the genotype distribution of MTP -493G/T demonstrated a significant difference between the self-limited infection group and the entire chronic group or the chronic patients with no family history (χ2 = 8.543, P = 0.015 and χ2 = 7.199, P = 0.019). The allele distribution at the MTP-493 position also demonstrated a significant difference between the study groups without family history (χ2 = 6.212, P = 0.013). The T allele emerged as a possible protective factor which may influence the outcomes of HBV infection (OR: 0.59; 95% CI: 0.389-0.897). CONCLUSION: The polymorphism of the MTP gene, T allele at -493, may be involved in determining the HBV infection outcomes, of which the mechanism needs to be further investigated.

Associations of IFN-γ rs2430561 T/A,IL28B rs12979860 C/T and ERα rs2077647 T/C polymorphisms with outcomes of hepatitis B virus infection:a meta-analysis

Several studies investigated associations of IFN-γ rs2430561 T/A,IL28 B rs12979860 C/T and ERα rs2077647 T/C gene polymorphisms with outcomes of hepatitis B virus（HBV） infection,but the results were controversial.Therefore,we performed a meta-analysis of all published observational studies to address this inconsistency.Literature was searched in online database and a systematic review was conducted based on the search results.A total of 24 studies were included and dichotomous data were presented as odds ratio（OR） with a 95%confidence interval（CI）.The rs2430561 T allele was associated with reduced persistent HBV infection risk（T vs.A：OR,0.690;95%CI,[0.490,0.971]）,while the rs2077647 T allele significantly increased the risk of persistent HBV infection（T vs.C：OR.1.678;95%CI,[1.212,2.3231）.Rs 2077647 CC might play a role in protecting individuals against HBV persistence（TT vs.CC：OR,4.109;95%CI,[2.609,6.473]）.Furthermore,carriers of the rs2430561 TT genotype were more likely to clear HBV spontaneously compared with those of the AA genotype（TT vs.AA：OR,0.555;95%CI,[0.359,0.856]）.For rs12979860 C/T polymorphism,no significant correlation with HBV infection outcomes was found.In subgroup analyses,the results were similar to those of overall analysis.However,for rs2077647 TT vs.TC＋CC,significantly increased risks were observed in the Asian and hospital-based population,but not in the overall analysis.IFN-γrs2430561 T/A and ERα rs2077647 T/C genetic polymorphisms were associated with outcomes of HBV infection,but no association was found between IL28 B rs12979860 C/T and HBV infection.

HMGB1 gene polymorphisms in patients with chronic hepatitis B virus infection

AIM: To characterize high mobility group box chromosomal protein 1(HMGB1) polymorphisms in patients infected with hepatitis B virus(HBV) and determine the different patterns in patient subgroups.METHODS: A total of 1495 unrelated Han Chinese HBV carriers were recruited in this hospital-based casecontrol study.The HMGB1 1176 G/C polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism assay.RESULTS: A significant association was observed between HMGB1 1176 G/C polymorphism and outcome of HBV infection.The subjects bearing 1176G/G genotype had an increased risk of susceptibility to chronic hepatitis B,liver cirrhosis and severe hepatitis B when compared with those bearing at least one 1176C allele.CONCLUSION: Patients with 1176G/G genotype of HMGB1 gene are more likely to have a progressive status in HBV infection.

RANTES gene single nucleotide polymorphisms and expression in patients with chronic hepatitis B virus infection

Background Regulated on activation, normal T-cell expressed and secreted (RANTES) plays a critical role in T-lymphocyte activation and proliferation. The process is involved in both acute and chronic phases of inflammation. The present study was to ascertain the possible correlations between chronic hepatitis B virus (HBV) infection and the RANTES gene polymorphisms and their expression. Methods The study included 130 HBV negative healthy donors and 152 patients with chronic hepatitis B (CHB) virus infection. The polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLPs) were used to detect RANTES gene single nucleotide polymorphisms (SNPs). RANTES levels in the platelet depleted plasma were detected by enzyme linked immunosorbent assay (ELISA). Results RANTES alleles -403G, -28C and In1.1T were the predominant alleles in the subjects studied. No significant correlation was found between CHB infection and the RANTES alleles, while a significant correlation was found between CHB infection and increased RANTES expression in platelet depleted plasma (P<0.05). Conclusions SNPs in RANTES gene do not affect chronic HBV infection or the outcome of interferon-α treatment in patients positive for HBV “e” antigen (HBeAg+). However, patients with CHB infection express the higher levels of plasma RANTES, which is thus associated with CHB infection.

Relationship between cytokine gene polymorphisms and chronic hepatitis B virus infection

Hepatitis B virus (HBV) infection is still a public health problem worldwide, being endemic in some parts of the world. It can lead to serious liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular cancer. The differences in host immune response can be one of the reasons for the various clinical presentations of HBV infection. Polymorphisms of genes encoding the proinflammatory and antiinflammatory cytokines, which are responsible for regulation of the immune response, can affect the clinical presentation of the infection. Particularly, the polymorphisms of the genes encoding cytokines such as interleukin (IL)-1, IL-6, IL-8, IL-10, IL-18, IL-28B, interferon-&#x003b3;, tumor necrosis factor-&#x003b1;, tumor growth factor-&#x003b2;1, and regulatory molecules like vitamin D receptor and chemokine receptor 5 can be responsible for different clinical presentations of HBV infections. The genomic information about cytokines and other mediators can be important for determining high-risk people for developing chronic hepatitis or hepatocellular cancer and may be used to plan treatment and preventive approaches for these people. In this review, the current knowledge in the literature on the association between cytokine/regulatory molecule gene polymorphisms and clinical course of chronic HBV infection is summarized, and the clinical implementations and future prospects regarding this knowledge are discussed.

Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population

BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.

interleukin 28B genetic polymorphism and hepatitis B virus infection

Interleukin (IL) 28B genetic polymorphism is significantly associated with the sustained virological response rate in patients with chronic hepatitis C treated with pegylated interferon-&#x003b1; (PEG-IFN) plus ribavirin and with spontaneous hepatitis C virus clearance. However, a consensus on the relationship between IL28B genetic polymorphism and the favorable outcome of chronic hepatitis B virus infection defined by hepatitis B e antigen seroconversion, and/or hepatitis B surface antigen seroclearance in patients treated with interferon or PEG-IFN has not been reached. Several reports failed to show a positive association, while some studies demonstrated a positive association in certain subject settings. More prospective studies including large cohorts are needed to determine the possible association between IL28B genetic polymorphism and the outcome of interferon or PEG-IFN treatment for chronic hepatitis B.

IFIT1 polymorphisms predict interferon-α treatment efficiency for hepatitis B virus infection

AIM To investigate the association between interferoninduced protein with tetratricopeptide repeats 1(IFIT1) polymorphisms and interferon-α(IFNα) treatment efficiency among Chinese hepatitis B virus(HBV) infection patients.METHODS Two hundred and twenty five newly diagnosed chronichepatitis B(CHB) patients were enrolled in the study. All of these patients received IFNα treatment for a course of 48 wk, and were followed up for 24 wk after the treatment was end. Clinical information about virological response, hepatitis B e antigen(HBe Ag) seroconversion rate and combined response at the end of the treatment, as well as the sustained response by the time of following up 24 wk after the treatment, was collected. Four tag-single nucleotide polymorphisms(SNPs) of IFIT1 were selected and assessed for their association with these clinical outcomes.RESULTS At the end of the treatment, HBe Ag seroconversion was observed in 27.1% patients. Thirty-six point nine percent patients achieved virological response, and 15.6% patients exhibited combined response. Sustained response was obtained in 26.2% patients. The main HBV genotype of the study was genotype B. Patients who infected with HBV genotype B or C showed better treatment efficiency, no matter which clinical outcome was considered. Among the four SNPs assessed, rs303218(A > G) was found to be significantly associated with the end point virological response when assuming additive model [OR = 0.64(95%CI: 0.42-0.96), P = 0.032]. Patients who carried rs303218 GG genotype had a rather higher rate of achieving virological response(response rate: 52%, OR = 0.40, 95%CI: 0.18-0.91; P = 0.028) when compared to those had AA genotype(response rate: 27%). The most significant interaction was observed in patients who had relative lower baseline aspartate transaminase. No association between SNPs and HBe Ag seroconversion, combined response or sustained response was observed.CONCLUSION IFIT1 involves in the regulation of IFNα treatment for CHB and its polymorphism rs303218 can predict the end point virological response. The finding requires further validation.

Relationship between interleukin-6 polymorphism and susceptibility to chronic hepatitis B virus infection

AIM:To identify the relationship between tag single nucleotide polymorphisms(tag SNPs) of interleukin-6(IL-6) gene and susceptibility to chronic hepatitis B virus(HBV) infection in a Han Chinese population.METHODS:We performed a case-control study of501 Chinese patients with chronic HBV infection and301 self-limiting HBV-infected individuals as controls.Genomic DNA was isolated from the whole blood of all subjects using phenol/chloroform with MaXtract highdensity tubes. Tag SNPs were identified using genotype data from the panel(Han Chinese in Beijing) of the phase II HapMap Project. Four tag SNPs in IL-6(rs17147230A/T,rs2066992G/T,rs2069837A/G and rs2069852A/G) were genotyped by the Multiplex Snapshot technique. The genotype and allele frequencies were calculated and analyzed.RESULTS:Five haplotypes were involved in the analysis,with frequencies higher than 0.03. One of the haplotypes,TTAA,was significantly different between the two groups. Overall haplotype P values were:ATAA,P = 0.605,OR(95%CI) = 1.056(0.860-1.297); TGAG,P = 0.385,OR(95%CI) = 1.179(0.813-1.709); TGGG,P = 0.549,OR(95%CI) = 1.087(0.827-1.429); TTAA,P = 0.004,OR(95%CI) = 0.655(0.491-0.873); TTAG,P = 0.266,OR(95%CI) = 1.272(0.832-1.944). However,the four SNPs showed no significant genotype/allele associations with susceptibility to chronic HBV infection. Overall allele P values were:rs17147230,P = 0.696,OR(95%CI) = 1.041(0.850-1.276); rs2066992,P = 0.460,OR(95%CI)= 1.090(0.868-1.369); rs2069837,P = 0.898,OR(95%CI) = 0.983(0.759-1.274); rs2069852,P = 0.165,OR(95%CI) = 0.859(0.693-1.064). Overall genotype P values were:rs17147230,P = 0.625; rs2066992,P= 0.500; rs2069837,P = 0.853; and rs2069852,P =0.380.CONCLUSION:The four tag SNPs of IL-6 gene may be associated with susceptibility to chronic HBV infection in the Han Chinese population.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Th1/Th2 cytokines and their genotypes as predictors of hepatitis B virus related hepatocellular carcinoma

Hepatocellular carcinoma(HCC), the predominant type of primary liver cancer, is one of the most serious lifethreatening malignancies, worldwide. In majority of the cases, HCC develops after prolonged and persistent chronic liver disease. hepatitis B virus(HBV) or HCV infection is prominent etiological factors, attributing to this condition. It has been well documented that HBV, being the inducer of chronic inflammation, is the main causative agent in causing HCC, particularly in Asian countries. The HBV infection leads to a wide range of clinical symptoms from carrier state to malignancy. Cytokines being immune-modulatory molecules, are the key mediators in the defense mechanism against viral infection. In this regard, this review will detail the substantial role of key Th1: interleukin 1(IL-1), IL-2, IL-12, tumor necrosis factor-α, interferon-γ; Th2: IL-4, IL-10 and non Th1/Th2: IL-6, transforming growth factor-β1 cytokines genotypes in analyzing the variability in the clinical manifestations in an HBV-afflicted individual, which might finally, culminates into HCC. Since cytokine production is regulated genetically, the cytokine promoter region single-nucleotide polymorphisms induced changes, greatly affects the cytokine production, thus resulting into differential outcome of immune balance.