Mutations in the S gene and in the overlapping reverse transcriptase region in chronic hepatitis B Chinese patients with coexistence of HBsAg and anti-HBs

Abstract Background The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. Aims This research aimed to determine the clinical and virological features of the rare pattern. Methods A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. Results The amino acid variability within major hydrophilic region, especially the “a” determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the “a” determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. Conclusions In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence.

Analysis of Reverse Transcriptase Gene Mutations in the Hepatitis B Virus at a University Hospital in Korea

BACKGROUND: Most mutations in the reverse transcriptase (RT) gene of the hepatitis B virus (HBV) are related to resistance to antiviral agents. Cross-sectional studies on the mutations of this gene are rare. Thus, we analyzed the mutation patterns of RT genes and their biochemical parameters. METHODS: From 2009 to 2012, 301 blood specimens from patients with chronic hepatitis B at Daegu Catholic University Medical Center were retrospectively analyzed for the RT gene sequence of HBV, ALT, hepatitis B e antigen (HBeAg), and HBV DNA. The mutation patterns of the RT gene were compared with the biochemical parameters. RESULTS: Of the 301 patients, 100 (33.2%) had no RT gene mutations. The remaining showed the following mutation patterns: rtM204I/V (50.2%), rtL180M (39.2%), and rtA181T/V (19.6%). Combined mutations were found in 146 cases (48.5%). Of these, the combination of amino acid changes at rt180+rt204 (49.3%) was most frequently detected, followed by rt181+rt236 (11.0%) and rt173+rt180+rt204 (9.6%). In the mutated group, HBV DNA and HBeAg positive rates were significantly higher (P<0.05 for both). Phenotypic analysis showed that lamivudine resistance was most frequently detected (34.6%), followed by adefovir resistance (15.6%). Multidrug resistance was detected in 48 cases (15.9%). The adefovir-resistant group had a higher proportion of cases with HBV loads greater than 2,000 IU/mL. CONCLUSIONS: We found correlations between the mutation status of the RT domain and biochemical parameters such as HBV DNA and HBeAg positive rate. The presence of RT gene mutations could therefore be utilized to predict clinical status.

Genotypic resistance to entecavir in chronic hepatitis B patients / 대한간학회지

BACKGROUND/AIMS: The prevalence and clinical characteristics of entecavir (ETV) resistance is not well known. The aim of this study was to determine the frequency of genotypic resistance in nonresponders and virologic breakthrough (VBT) patients. METHODS: The medical records of 76 chronic hepatitis B patients treated for a least 6 months from October 2006 to October 2008 were reviewed retrospectively. We divided patients into two groups: nucleoside analogue (NA)-naive patients (n=38) and LAM experienced patients (n=38). NA-naive and LAM experienced patients received ETV at 0.5 and 1.0 mg/day, respectively. The virologic response and VBT were investigated in both groups. We used the multiplex restriction fragment mass polymorphism (RFMP) method to test genotypic resistance at the rtI169, rtT184, rtS202, rtM204, and rtM250 sites. RESULTS: Age, gender, serum ALT, and HBV DNA level before treatment did not differ between the groups. Neither VBT nor nonresponse was observed in the NA-naive group, whereas VBT and nonresponse were observed in three patients each in the lamivudine (LAM)-experienced group; all six patients had YMDD mutation at study enrollment, all three patients with VBT had genotypic resistance to ETV, but the three nonresponse patients did not have genotypic resistance to ETV. CONCLUSIONS: We suspect that VBT is mostly associated with genotypic resistance to ETV. However, nonresponse might be associated with the continuance or reselection of the YMDD mutant in LAM-experienced patients.

Long-term clevudine therapy in nucleos(t)ide-naive and lamivudine-experienced patients with hepatitis B virus-related chronic liver diseases / 대한간학회지

BACKGROUNDS/AIMS: Clevudine is an effective antiviral nucleoside analogue, but there are few data regarding its long-term effects, resistance, and safety. The aim of this study was to evaluate the long-term clinical efficacy of clevudine over a 1-year treatment period in nucleos(t)ide-naive and lamivudine-experienced chronic hepatitis B patients. METHODS: Nucleos(t)ide-naive (group A, n=196) and lamivudine-experienced (serum hepatitis B virus, HBV DNA >2,000 copies/mL without resistant mutants at the start of clevudine therapy, group B, n=75) patients were included in this study. Basic clinical characteristics including age, sex, the presence of cirrhosis, laboratory data, and hepatitis B surface antigen (HBeAg) positivity were similar between the two groups. Pretreatment serum levels of HBV DNA were 7.4 and 6.6 log10 copies/mL (P<0.001). The mean treatment duration was 8 months for both groups (range for group A: 3-21 months; range for group B: 3-20 months). Genotypic analysis for resistant mutations in the reverse transcriptase of HBV was performed after viral breakthrough. RESULTS: After 1 year of therapy, 75.0% and 51.9% of groups A and B, respectively, had HBV DNA levels of <2,000 copies/mL (P=0.032), and HBeAg seroconversion rates were 16.9% and 16.7%, respectively. The rates of viral breakthrough at 1 year were 10.0% (8/80) and 44.4% (12/27), respectively (P<0.001). Proven sites of mutation of HBV DNA polymerase in naive patients were, for example, L80I, L180M, A181V/T, M204I and V207I. Ten patients complained of prominent fatigue and revealed elevated serum levels of aspartate aminotransferase (AST) and creatine phosphokinase (CPK). Two of these patients presented with severe myopathy from which they recovered completely after quitting clevudine. CONCLUSIONS: Clevudine is one of the recommended first-line medicines for the treatment of chronic hepatitis B, but it is not free from resistance, particularly in patients with a history of previous lamivudine treatment, but also in naive patients. Clevudine should be avoided in previously lamivudine-exposed patients. In addition, reelevation of serum AST and CPK levels is not a rare occurrence, and close observation and follow-up tests are essential.

Human Monoclonal Antibody Inhibiting Reverse Transcriptase Activity of Hepatitis B Virus Polymerase Protein / 대한소화기학회지

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.

Prevalence of primary and secondary resistant mutations to antiretroviral drug in a population of Puerto Rican infected with HIV

INTRODUCTION: Several studies have reported increasing number of therapeutic failures with HAART in HIV-infected individuals. In order to assess the impact HIV antiretroviral resistance could have on treatment, we decided to determine the prevalence of primary and secondary antiretroviral resistant genotypes in a population of HIV-infected Puerto Ricans and compare the mutational distribution pattern with that reported in Europe and US. METHOD: In a total of 80 plasma samples from patients with detectable viral load of over 1,000 RNA copies/ml, the Trugene Visible Genetics HIV sequencing method was used to detect antiretroviral resistance mutations. RESULTS: We found 55 subjects (69 per cent) with high level of resistance to ZDV in the reverse transcriptase gene and 46 subjects (58 per cent) with high level of resistance to NFV in the protease gene. Mutation frequencies to the NRTI ranged in appearance from as high as 54 per cent (i.e., M184V) in the studied subjects to a low of less than 5 per cent (i.e., M184I and V75T). For the NNRTI the most common mutation was K103N in 40 per cent of the subjects and found to confer cross resistance to NVP, DLV and EFV. Another concerning finding is the increasing trend of the frequency of primary and secondary resistant mutations from year 2000 to 20001. Nine (23 per cent) of the total detected primary mutations, to either RTI or PI, showed an increase of at least 5 per cent from one year to the other. Similarly, there were 6 (11 per cent) secondary resistant mutations showing an increase of at least 5 per cent during the two years studied. CONCLUSIONS: In two year period we detected a tendency to increase in primary and secondary HIV-resistant mutation in a population of HIV-infected Puerto Ricans.

Delecion infrecuente en el codon 67 del gen de la transcriptasa reversa de HIV en falla al tratamiento antirretroviral / Infrequent deletion in 67 codon in HIV reverse transcriptase in antiretroviral treatment failure

Presentamos un caso clínico de falta al tratamiento antirretroviral, con aparición de residentes confirmada por genotipificación. También se muestra la evolución del patrón de mutaciones que confieren resistencia a inhibidores de proteasa y transcriptasa reversa conforme a la modificación del esquema terapéutico indicado a la paciente. Se encontró un raro patrón de resistencia a AZT, con una deleción del códon 667 en el gen de la transcriptasa reversa lo que enfatiza la importancia de la genotipificación de la resistencia a antivirales por métodos de sequenciación.