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.

Significance of hepatitis B virus surface antigen, hepatitis C virus expression in hepatocellular carcinoma and pericarcinomatous tissues

AIM： To investigate the correlation between hepatitis B virus surface antigen （HBsAg）, hepatitis C virus （HCV） expression in hepatocellular carcinoma （HCC）, the HAI score of the noncancerous region of the liver and the serum Alpha fetoprotein （AFP） level. METHODS： The patterns of HBsAg and HCV in 100 cases of HCC and their surrounding liver tissues were studied on paraffin-embedded sections with immunohistochemistry, the histological status was determined by one pathologist and one surgeon simultaneously using the hepatitis activity index （HAIl score, and AFP was detected by radioimmunity. The study included 100 consecutive patients who underwent curative resection for HCC. Based on HBsAg and HCV expression, the patients were classified into 4 groups： patients positive for HBsAg （HBsAg group）, patients positive for HCV （HCV group）, patients negative for both HCV and HBsAg （NBNC group） and patients positive for both HBsAg and HCV （BC group）. RESULTS： The BC group had significantly higher HAI scores than the other three groups. （BC 〉 HCV 〉 HBsAg 〉 NBNC）. HBV and HCV virus infection was positively correlated with HAI （rs = 0.39, P = 0.00011. The positive rate of AFP （85.7%） and the value of AFP （541.2 ng/mL） in the group with HBV and HCV co-infection were the highest among the four groups. The positive rate （53.3%） of AFP and the value of AFP （ 53.3 ng/mL） in the group with none-infection of HBV and HCV were the lowest. HBV and HCV virus infection was positively correlated with AFP（rs = 0.38, P = 0.0001）. CONCLUSION： The AFP increase in patients with liver cancer was positively correlated with the infection of HBV and HCV. The-serum AFP elevation by the infection of HBV and HCV is one of mechanisms which lead to hepatocarcinogenesis, and the antivirus intervening treatment of hepatitis is significant for the prognosis of liver cancer. From our Spearman＇s rank correlation analysis, we can conclude that the severity of virally induced inflammation is correlated with HBsAg and HCV expression in HCC tissues and noncancerous tissues. Prior co-infection of HBV in HCV patients may be an adverse risk factor for intrahepatic inflammation.

Association of Cytokines with Alanine Aminotransferase, Hepatitis B Virus Surface Antigen and Hepatitis B Envelope Antigen Levels in Chronic Hepatitis B

Background： Cytokines play an important role in occorrence and recovery of hepatitis B virus （HBV） infection. The aim of this study was to investigate the changes ofcytokines concentration and its correlation to alanine aminotransferase （ALT）, HBV deoxyribonucleic acid （HBV-DNA）, hepatitis B envelope antigen （HBeAg）, and HBV surface antigen （HBsAg） in the development of chronic hepatitis B （CHB）. Methods： Thirteen healthy individuals （HI）, 30 chronic HBV-infected patients in immune tolerant （IT） phase, and 55 CHB patients were enrolled between August 2015 and May 2017. The peripheral blood samples were collected from all individuals. Tile levels of interferon （IFN）-α2, interleukin （IL）-10, transforming growth factor （TGF）-β1, HBV-DNA, HBsAg, and HBeAg and liver function were measured. The quantitative determinations of cytokines levels, including I FN-ct2, IL-10, and TGF-[31 were performed using Luminex multiplex technology. The correlation of cytokines to ALT, HBV-DNA, HBsAg, and HBeAg was analyzed by linear regression analysis. Results： IFN-ct2 levels were similar between HI and IT groups （15.35 [5.70, 67.65] pg/ml vs. 15.24 [4.07, 30.73] pg/ml, Z = -0.610, P - 0.542）, while it elevated significantly in CH B group （35.29 [ 15.94, 70.15] pg/ml vs. 15.24 [4.07, 30.73] pg/ml; Z = -2.522, P = 0.012）. Compared with HI group （3.73 [2.98, 11.92] pg/ml）, IL-10 concentrations in IT group （5.02 [2,98, 10.11] pg/ml）, and CHB group （7.48 [3. I 0, 18.00] pg/ml） slightly increased （X^2 = 2.015, P - 0.365）, and there was no significant difference between IT and CHB group （Z =- 1.419, P = 0.156）. The TGF-β1 levels among HI （3.59 ±0.20 pg/ml）, IT （3.62 ±0.55 pg/ml）, and CHB groups （3.64±0.30 pg/ml） were similar （X^2=2.739, P = 0.254）. In all chronic HBV-infected patients （including patients in IT and CHB groups）, the elevation of IFN-α2 level was significantly associated with ALT level （β = 0.389, t = 2.423, P = 0.018）, and was also negatively correlated to HBV-DNA load （β = -0.358, t=-2.308, P = 0.024）, HBsAg （β = -0.359, t = -2.288, P = 0.025）, and HBeAg contents （β = -0.355, t = -2.258, P = 0.027）. However, when both ALT level and cytokines were included as independent variable, HBV-DNA load, HBsAg, and HBeAg contents were only correlated to ALT level （[β= -0.459, t = -4.225, P = 0.000; β = -0.616, t = -6.334, P = 0.000; and β = -0.290, t = -2.433, P = 0.018; respectively）. Conclusions： IFN-α2 elevation was associated with ALT level in patients with chronic HBV infection. However, in CHB patients, only ALT level was correlated to HBV-DNA, HBsAg and HBeAg contents.

Occult hepatitis B virus and hepatocellular carcinoma

Occult hepatitis B virus(HBV)infection(OBI)is a challenging pathobiological and clinical issue that has been widely debated for several decades.By definition,OBI is characterized by the persistence of HBV DNA in the liver tissue(and in some cases also in the serum)in the absence of circulating HBV surface antigen(HBsAg).Many epidemiological and molecular studies have indicated that OBI is an important risk factor for hepatocellular carcinoma(HCC)development.OBI may exert direct pro-oncogenic effects through the activation of the same oncogenic mechanisms that are activated in the course of an HBsAg-positive infection.Indeed,in OBI as in HBV-positive infection,HBV DNA can persist in the hepatocytes both integrated into the host genome as well as free episome,and may maintain the capacity to produce proteins-mainly X protein and truncated preS-S protein-provided with potential transforming properties.Furthermore,OBI may indirectly favor HCC development.It has been shown that the persistence of very low viral replicative activity during OBI may induce mild liver necro-inflammation continuing for life,and substantial clinical evidence indicates that OBI canaccelerate the progression of liver disease towards cirrhosis that is considered the most important risk factor for HCC development.

Mutations in surface and polymerase gene of chronic hepatitis B patients with coexisting HBsAg and anti-HBs

AIM： To investigate the clinical significance and presence of mutations in the surface （S） and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs. METHODS： Twenty-three patients with chronic hepatitis B were studied. Of the 23 patients, i i were both positive for hepatitis B virus （HBV） surface antigen （HBsAg） and antibody to HBV surface antigen （anti-HBs）, 12 were negative for anti-HBs while positive for HBsAg. DNA was extracted from 200 μL serum of the patients. Nucleotide of the surface and overlapping polymerase gene from HBV-infected patients was amplified by PCR, and the PCR products were sequenced. RESULTS： Forty-one mutations were found within the surface gene protein of HBV in 15 patients （10 with coexisting HBsAg and anti-HBs）. Six （14.6%） out of 41 mutations were located at ＂α＂ determinant region in 5 patients （4 positive for HBsAg and anti-HBs）. Eleven mutations （26.8%） occurred in the downstream or upstream of ＂α＂ determinant region. Lamivudine （LMV）- selected mutations were found in three patients who developed anti-HBs, which occurred in amino acid positions （196, 198, 199） of the surface protein and in YMDD motif （M204I/V） of the polymerase protein simultaneously. Presence of these mutations did not relate to changes in ALT and HBV DNA levels.CONCLUSION： Besides mutations in the ＂α＂ determinant region, mutations at downstream or upstream of the ＂α＂ determinant region may contribute to the development of anti-HBs. These mutations do not block the replicating competency of HBV in the presence of high titer of anti-HBs.

Applicability and efficacy of a model for prevention of perinatal transmission of hepatitis B virus infection:Single center study in Egypt

AIM: To identify possible maternal risk factors for hepatitis B virus (HBV) acquisition and assess the efficacy of immunoprophylaxis given to infants born to hepatitis B virus surface antigen (HBsAg) positive mothers.

Basic Study Hepatitis B virus detected in paper currencies in a densely populated city of India: A plausible source of horizontal transmission?

BACKGROUND The recent rise in the incidence of hepatitis B virus(HBV)infections in a densely populated city of eastern India(“mixing vessel”of people of varied socioeconomic and immune status)prompted this study.Applying saliva on fingers for enumerating bank notes is a common practice in the Indian subcontinent.Paper notes may be a potential source of“horizontal”transmission of this virus,especially if there are cuts/bruises on the oral mucous membrane or skin.AIM To investigate whether paper currencies could be a plausible mode of horizontal transmission of HBV infection.METHODS Polymerase chain reactions(PCR)followed by nucleotide sequencing was done for the detection of HBV.Hepatitis B virus surface antigen enzyme-linked immunosorbent assay(HBsAg ELISA)was performed on all HBV deoxyribonucleic acid-positive samples to check the detectability of the virus.Atomic force microscopy(AFM)was carried out for visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings.RESULTS HBV-specific PCRs on pellets obtained after ultracentrifugation/immunoprecipitation of the currency paper washings detected potentially intact/viable HBV(genotype D2)in 7.14%of samples(n=70).AFM gave the visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings.However,HBV isolates from the currency notes could not be detected by HBsAg ELISA.CONCLUSION It is a common practice in the Indian subcontinent to count paper currencies by applying saliva on fingertips.Paper notes may be a potential source of“horizontal”transmission of this virus,especially if there are cuts/bruises on the oral mucous membrane or skin,but it was practically not possible to demonstrate experimentally such transmission.Detection of potentially intact/viable and“occult”HBV from currency poses potential risk of silent transmission of this virus among the general population.

Nationwide retrospective study of hepatitis B virological response and liver stiffness improvement in 465 patients on nucleos(t)ide analogue

BACKGROUND Hepatitis B virus(HBV)nucleos(t)ide analog(NA)therapy reduces liver disease but requires prolonged therapy to achieve hepatitis B surface antigen(HBsAg)loss.There is limited North American real-world data using non-invasive tools for fibrosis assessment and few have compared 1st generation NA or lamivudine(LAM)to tenofovir disoproxil fumarate(TDF).AIM To assess impact of NA on virological response and fibrosis regression using liver stiffness measurement(LSM)(i.e.,FibroScan®).METHODS Retrospective,observational cohort study from the Canadian HBV Network.Data collected included demographics,NA,HBV DNA,alanine aminotransferase(ALT),and LSM.Patients were HBV monoinfected patients,treatment naïve,and received 1 NA with minimum 1 year follow-up.RESULTS In 465(median 49 years,37%female,35%hepatitis B e antigen+at baseline,84%Asian,6%White,and 9%Black).Percentage of 64(n=299)received TDF and 166 were LAM-treated with similar median duration of 3.9 and 3.7 years,respectively.The mean baseline LSM was 11.2 kPa(TDF)vs 8.3 kPa(LAM)(P=0.003).At 5-year follow-up,the mean LSM was 7.0 kPa in TDF vs 6.7 kPa in LAM(P=0.83).There was a significant difference in fibrosis regression between groups(i.e.,mean-4.2 kPa change in TDF and-1.6 kPa in LAM,P<0.05).The last available data on treatment showed that all had normal ALT,but more TDF patients were virologically suppressed(<10 IU/mL)(n=170/190,89%)vs LAM-treated(n=35/58,60%)(P<0.05).None cleared HBsAg.CONCLUSION In this real-world North American study,approximately 5 years of NA achieves liver fibrosis regression rarely leads to HBsAg loss.

Risks of Viral Hepatitis B Transmission in Mother-to-Infant of Pregnant Women Carriers of Chronic Viral Hepatitis B in Cote d’Ivoire

The aim of this study was to identify the risk factors of mother-to-child transmission of HBV in positive Ag Hbs pregnant women in Cote d’Ivoire. Methods: This was a transversal prospective study that took place over a period of 7 months (from February 2016 to August 2016) in 2 university hospital and 2 private clinics. We consecutively recruited 91 pregnant women who were positive for HBs Ag in prenatal consultations. For each pregnant woman record included in the study, we provided Socio-demographic (Age, marital status, education level, social rank, gravidity, parity) and biological data (HBs Ag, Anti-HBc Total Ac, Hbe Ag, Ac anti-Hbe Ac, DNA-VHB, Ac anti-HCV Ac, retroviral serology, transaminases). All of these data were collected using a survey sheet developed for the study. Results: The age of our pregnant women HBs positive ranged from 18 years to 44 years with a mean age of 30.10 years. The age group from 20 to 39 years was the most represented with a frequency of 92.31%. Almost of all positive HBs Ag pregnant women was HBe Ag negative, only 3.3% was HBe Ag positive. The viral load above 2000 IU/ml was found in 21 (23.03%) patients. There were 4 co-infected patients, which 3 HBV-HIV and 1 HBV-HCV. Only 19 (20.88%) pregnant HBs Ag positive women were able to bring back the supplementary virological assessment within a period less than one month. Conclusion: According to our work the virologic profile of positive HBs Ag in pregnant women in Cote d’Ivoire is characterized by an important viral replication objectified by a high viral load in about 23% pregnant women, a negativity of HBe antigen in 96.6% of them.

Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice

AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system.METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 μL phosphate buffered saline (PBS)at an interval of 3 wk. Group 1: PBS only; group 2:gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminalfragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95 ℃ heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination.RESULTS: The average titer of serum anti-HBsAg antibodyin the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay.CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.