Hepatitis B Surface Antigen Mutants Research Articles

Genetic diversity, haplotype analysis, and prevalence of Hepatitis B virus MHR mutations among isolates from Kenyan blood donors.

The rapid spread of HBV has resulted in the emergence of new variants. These viral genotypes and variants, in addition to carcinogenic risk, can be key predictors of therapy response and outcomes. As a result, a better knowledge of these emerging HBV traits will aid in the development of a treatment for HBV infection. However, many Sub-Saharan African nations, including Kenya, have insufficient molecular data on HBV strains circulating locally. This study conducted a population-genetics analysis to evaluate the genetic diversity of HBV among Kenyan blood donors. In addition, within the same cohort, the incidence and features of immune-associated escape mutations and stop-codons in Hepatitis B surface antigen (HBsAg) were determined. In September 2015 to October 2016, 194 serum samples were obtained from HBsAg-positive blood donors residing in eleven different Kenyan counties: Kisumu, Machakos, Uasin Gishu, Nairobi, Nakuru, Embu, Garissa, Kisii, Mombasa, Nyeri, and Turkana. For the HBV surface (S) gene, HBV DNA was isolated, amplified, and sequenced. The sequences obtained were utilized to investigate the genetic and haplotype diversity within the S genes. Among the blood donors, 74.74% were male, and the overall mean age was 25.36 years. HBV genotype A1 (88.14%) was the most common, followed by genotype D (10.82%), genotype C (0.52%), and HBV genotype E (0.52%). The phylogenetic analysis revealed twelve major clades, with cluster III comprising solely of 68 blood donor isolates (68/194-35.05%). A high haplotype diversity (Hd = 0.94) and low nucleotide diversity (π = 0.02) were observed. Kisumu county had high number of haplotypes (22), but low haplotype (gene) diversity (Hd = 0.90). Generally, a total of 90 haplotypes with some consisting of more than one sequence were observed. The gene exhibited negative values for Tajima's D (-2.04, p<0.05) and Fu's Fs (-88.84). Several mutations were found in 139 isolates, either within or outside the Major Hydrophilic Area (MHR). There were 29 mutations found, with 37.9% of them situated inside the "a" determinant. The most common mutations in this research were T143M and K122R. Escape mutations linked to diagnostic failure, vaccination and immunoglobulin treatment evasion were also discovered. Also, one stop-codon, W163STP, inside the MHR, was found in one sample from genotype A. In Kenya, HBV/A1 is still the most common genotype. Despite limited genetic and nucleotide diversity, haplotype network analysis revealed haplotype variance among HBV genotypes from Kenyan blood donors. The virological properties of immune escape, which may be the source of viral replication endurance, were discovered in the viral strains studied and included immune-escape mutations and stop-codon. The discovery of HBsAg mutations in MHR in all isolates highlighted the need of monitoring MHR mutations in Kenya.

Prevalence of occult hepatitis B virus infection and characterisation of hepatitis B surface antigen mutants among adults in western Croatia

Introduction and ObjectivesOccult hepatitis B virus (HBV) infection (OBI) is characterised by low levels of hepatitis B virus (HBV) DNA in the blood/liver of patients with negative hepatitis B surface antigen (HBsAg). This study aimed to determine the OBI prevalence and virological characteristics (viral genotypes and HBsAg mutants) in patients with an “anti-HBc only” serological profile. Materials and MethodsA total of 24 900 serum samples were routinely screened for hepatitis B markers over a five-year period. All anti-HBc-positive/HBsAg-negative/anti-HBs-negative sera were selected and analysed for the presence of HBV DNA. Mutational analyses of the HBs gene and polymerase gene sequences were performed. Results1749 (7.02%) sera were anti-HBc positive, and 113 (0.45%) sera had an “anti-HBc only” serological profile (HBsAg/anti-HBs negative). HBV DNA was detected in 12/113 (10.61%) “anti-HBc only” positive sera, representing 0.048% of all routinely tested samples. Due to extremely low viremia, HBV genome was successfully sequenced in only two sera where subgenotype D3 was confirmed. Mutational analyses of the S gene revealed multiple missense mutations. In addition to the M133I, Y134F, and G145R mutations, already associated with diagnostic escape, we also found nine novel OBI-related S-gene mutations - S136Y, F158L, K160N, E164G, S167L, A168V, L175S, S210I and F212C. ConclusionsWe detected multiple known and novel S gene mutations in 2/12 (16.6%) OBI cases, nevertheless, further studies are required to determine their role in the pathogenesis of OBI. Understanding the frequencies of clinically relevant HBV mutations may contribute to improvement of diagnostic protocols.

A small molecule targeting hepatitis B surface antigen inhibits clinically relevant drug-resistant hepatitis B virus.

Currently approved oral antivirals for chronic HBV infection target the reverse transcriptase (RT) domain of the HBV polymerase. Emergence of drug resistance has been reported in a small proportion of chronic HBV patients on prolonged treatment with antivirals. We recently reported ZINC20451377, a small molecule targeting hepatitis B surface antigen (HBsAg) that effectively inhibits both WT HBV and tenofovir-resistant HBV. Due to the partial overlap between the RT domain and HBsAg, drug-resistant mutants are associated with corresponding mutations in HBsAg. To evaluate the efficacy of ZINC20451377 against nine clinically relevant drug-resistant HBV mutants that lead to simultaneous mutations in the overlapping HBsAg gene. Huh7 cells were transfected with 1.2× HBV replicons corresponding to WT HBV or drug-resistant HBV mutants and treated with different concentrations of ZINC20451377. We assessed the IC50 values of ZINC20451377 for HBsAg levels in the culture supernatants using ELISAs. HBV secretion was measured by immunocapture of secreted virions followed by real-time PCR quantitation of virion-associated DNA. ZINC20451377 led to a dose-dependent inhibition of secreted HBsAg encoded by WT HBV and all nine drug-resistant mutants tested and the IC50 values were in the low micromolar range. ZINC20451377 inhibited HBV secretion from drug-resistant mutants except for mutants harbouring the rtL180M + rtM204V (MV) mutation. The small molecule ZINC20451377 inhibits HBsAg and virion secretion in some of the clinically relevant drug-resistant HBV mutants. ZINC20451377 has a modest overall effect, and it was not effective against the MV mutants (lamivudine- and entecavir-resistant mutants).

1066. Immune Escape Mutant Detection Using Commercially Available Methods for Hepatitis B Surface Antigen Serological Testing

BackgroundAlthough overall infection rates of Hepatitis B virus (HBV) in the United States (US) remain stable, as many as 2.2 million persons are still chronically infected with Hepatitis B Virus (HBV)1. Persons who inject drugs (PWID) are at a higher risk of HBV infection and since 2009 three states (KY, TN, WV) have reported up to a 114% increase in cases of acute HBV infection due to higher infection rates among a non-Hispanic white populations (30–39 years), and injection drug users2. Hepatitis B vaccination is recommended as primary prevention for adults who are at increased risk for HBV infection, including PWID. However, data from the National Health Interview Survey indicate that hepatitis B vaccination coverage is low among adults in the general population3, and it is likely to be lower among injection drug users.Hepatitis B Surface Antigen (HBsAg) is the first serological marker to appear after HBV exposure and infection; this marker is included in the recommended panel for acute hepatitis diagnosis and accurate detection is necessary for early and accurate diagnosis. Serological testing challenges exist for HBsAg due to the high degree of genetic variability which can further be exacerbated by endogenous and exogenous pressures. The immuno-dominant region may have one or more mutations described as immune escape mutations which can decrease or abrogate HBsAg binding to antibodies used in immunoassays. Although the prevalence of these mutations is not well documented in the United States, international studies have shown that up to 79% of HBV-reactivated patients (vs 3.1% of control patients; p< 0.001) carry HBsAg mutations localized in immune-active HBsAg regions4.MethodsA study was conducted using a panel of 10 unique recombinant HBsAg immune escape mutants. Panel members were tested by commercially available HBsAg serological immunoassays.ResultsIt was found that although commercially available HBsAg immunoassays are the primary diagnostic tool for HBV diagnosis, not all HBsAg immune escape mutants are detected, with some method detecting as few as 5 out of 10 of these mutant samples.Figure 1 ConclusionImprovement is needed in commercially available methods for the accurate detection of HBsAg.Disclosures Robert Gish, MD, Abbott (Consultant)AbbVie (Consultant, Advisor or Review Panel member, Speaker’s Bureau)Access Biologicals (Consultant)Antios (Consultant)Arrowhead (Consultant)Bayer (Consultant, Speaker’s Bureau)Bristol Myers (Consultant, Speaker’s Bureau)Dova (Consultant, Speaker’s Bureau)Dynavax (Consultant)Eiger (Consultant, Advisor or Review Panel member)Eisai (Consultant, Speaker’s Bureau)Enyo (Consultant)eStudySite (Consultant, Advisor or Review Panel member)Exelixis (Consultant)Fujifilm/Wako (Consultant)Genentech (Consultant)Genlantis (Consultant)Gilead (Consultant, Advisor or Review Panel member, Speaker’s Bureau)GLG (Consultant)HepaTX (Consultant, Advisor or Review Panel member)HepQuant (Consultant, Advisor or Review Panel member)Intercept (Consultant, Speaker’s Bureau)Ionis (Consultant)Janssen (Consultant)Laboratory for Advanced Medicine (Consultant)Lilly (Consultant)Merck (Consultant)Salix (Consultant, Speaker’s Bureau)Shionogi (Consultant, Speaker’s Bureau)Viking (Consultant)

Hepatitis B Virus Mutant Infections in Hemodialysis Patients: A Case Series

Rationale & ObjectiveHepatitis B virus (HBV) transmission in hemodialysis units has become a rare event since implementation of hemodialysis-specific infection control guidelines: performing hemodialysis for hepatitis B surface antigen (HBsAg)-positive patients in an HBV isolation room, vaccinating HBV-susceptible (HBV surface antibody and HBsAg negative) patients, and monthly HBsAg testing in HBV-susceptible patients. Mutations in HBsAg can result in false-negative HBsAg results, leading to failure to identify HBsAg seroconversion from negative to positive. We describe 4 unique cases of HBsAg seroconversion caused by mutant HBV infection or reactivation in hemodialysis patients.Study DesignFollowing identification of a possible HBsAg seroconversion and mutant HBV infection, public health investigations were launched to conduct further HBV testing of case patients and potentially exposed patients. A case patient was defined as a hemodialysis patient with suspected mutant HBV infection because of false-negative HBsAg testing results. Confirmed case patients had HBV DNA sequences demonstrating S-gene mutations.Setting & ParticipantsCase patients and patients potentially exposed to the case patient in the respective hemodialysis units in multiple US states.Results4 cases of mutant HBV infection in hemodialysis patients were identified; 3 cases were confirmed using molecular sequencing. Failure of some HBsAg testing platforms to detect HBV mutations led to delays in applying HBV isolation procedures. Testing of potentially exposed patients did not identify secondary transmissions.LimitationsLack of access to information on past HBsAg testing platforms and results led to challenges in ascertaining when HBsAg seroconversion occurred and identifying and testing all potentially exposed patients.ConclusionsMutant HBV infections should be suspected in patients who test HBsAg negative and concurrently test positive for HBV DNA at high levels. Dialysis providers should consider using HBsAg assays that can also detect mutant HBV strains for routine HBV testing.

Frequency of hepatitis B surface antigen variants (HBsAg) in hepatitis B virus genotype B and C infected East- and Southeast Asian patients: Detection by the Elecsys® HBsAg II assay

BackgroundTo avoid false negative results, hepatitis B surface antigen (HBsAg) assays need to detect samples with mutations in the immunodominant ‘a’ determinant region, which vary by ethnographic region. ObjectiveWe evaluated the prevalence and type of HBsAg mutations in a hepatitis B virus (HBV)-infected East- and Southeast Asian population, and the diagnostic performance of the Elecsys® HBsAg II Qualitative assay. Study designWe analyzed 898 samples from patients with HBV infection from four sites (China [Beijing and Guangzhou], Korea and Vietnam). HBsAg mutations were detected and sequenced using highly sensitive ultra-deep sequencing and compared between the first (amino acids 124–137) and second (amino acids 139–147) loops of the ‘a’ determinant region using the Elecsys® HBsAg II Qualitative assay. ResultsOverall, 237 distinct amino acid mutations in the major hydrophilic region were identified; mutations were present in 660 of 898 HBV-infected patient samples (73.5%). Within the pool of 237 distinct mutations, the majority of the amino acid mutations were found in HBV genotype C (64.8%). We identified 25 previously unknown distinct mutations, mostly prevalent in genotype C-infected Korean patients (n = 18) followed by Chinese (n = 12) patients. All 898 samples were correctly identified by the Elecsys® HBsAg II Qualitative assay. ConclusionsWe observed 237 distinct (including 25 novel) mutations, demonstrating the complexity of HBsAg variants in HBV-infected East- and Southeast Asian patients. The Elecsys® HBsAg II Qualitative assay can reliably detect HBV-positive samples and is suitable for routine diagnostic use in East and Southeast Asia.

Clade homogeneity and low rate of delta virus despite hyperendemicity of hepatitis B virus in Ethiopia

BackgroundAlthough hepatitis B virus (HBV) is hyperendemic and heterogeneous in its genetic diversity in Ethiopia, little is known about hepatitis D virus (HDV) circulating genotypes and molecular diversity.MethodsA total of 321 hepatitis B surface antigen (HBsAg) positives (125 HIV co-infected, 102 liver disease patients and 94 blood donors) were screened for anti-HDV antibody. The anti-HDV positive sera were subjected to Real time PCR for HDV-RNA confirmation. The non coding genome region (spanning from 467 to 834 nucleotides) commonly used for HDV genotyping as well as complete HDV genome were sequenced for genotyping and molecular analysis.ResultsThe anti-HDV antibody was found to be 3.2% (3) in blood donors, 8.0% (10) in HIV co-infected individuals and 12.7% (13) in liver disease patients. None of the HIV co-infected patients who revealed HBV lamivudine (3TC) resistance at tyrosine-methionine/isoleucine-aspartate-aspartate (YM(I)DD) reverse transcriptase (RT) motif with concomitant vaccine escape gene mutants was positive for anti-HDV antibody. The HDV viremia rate was 33.3%, 30.0% and 23.1% in respect to the above study groups. All the six isolates sequenced were phylogenetically classified as HDV genotype 1 (HDV-1) and grouped into two monophyletic clusters. Amino acid (aa) residues analysis of clathrin heavy chain (CHC) domain and the isoprenylation signal site (Py) at 19 carboxyl (C)-terminal amino acids (aa 196–214) and the HDV RNA binding domain (aa 79–107) were highly conserved and showed a very little nucleotide variations. All the sequenced isolates showed serine at amino acid position 202. The RNA editing targets of the anti-genomic HDV RNA (nt1012) and its corresponding genomic RNA (nt 580) showed nucleotides A and C, respectively.ConclusionsThe low seroprevalence and viraemic rates of HDV in particular during HIV-confection might be highly affected by HBV drug resistance selected HBsAg mutant variants in this setting, although HDV-1 sequences analysis revealed clade homogeneity and highly conserved structural and functional domains. Thus, the potential role of HBV drug resistance associated polymerase mutations and concomitant HBsAg protein variability on HDV viral assembly, secretion and infectivity needs further investigation.

Analysis of Antigenic and Conformational Changes in Hepatitis B Surface Antigen (HBsAg) Identified in Iranian Patients with Chronic Hepatitis B

Background: The ‘a’ determinant domain of hepatitis B surface antigen (HBsAg) (positions 124 to 147) is recognized by antibodies raised either naturally or induced by vaccine. Failure to protect against hepatitis B virus (HBV) infection may occur due to the conformational changes of ‘a’ determinant induced by mutations. Objectives: The present study analyzed the molecular and three-dimensional (3D) characteristics of the HBsAg ‘a’ determinant mutations among Iranian chronic hepatitis B (CHB) patients, who were vaccine and drug naive. Methods: Eighty patients with HBsAg positive test results were selected according to the data extracted from questionnaires. Serologic and molecular assays were performed using real-time Polymerase Chain Reaction (PCR) and subsequently surface nested PCR on CHB patients. Next, an extensive mutational analysis was applied following direct sequencing on HBsAg amplified genes. The potential impacts of altered antigenic and 3D properties of amino acid substitutions were carried out using bioinformatics approaches. Results: All patients were negative for HBeAg and positive for anti-HBe. Mutational analysis showed that 60 (75%) of 80 patients had at least one amino acid substitution. Several mutations were found in ‘a’ determinant (P127L, P127T, G130N, and S136Y). Bioinformatics investigations indicated that all mutations induced a conformational change in ‘a’ determinant region. P127L substitution led to a considerable decreased HBsAg antigenicity compared to other mutants. Conclusions: The current analyses revealed that the studied mutations induced a local change in the ‘a’ determinant conformation. These findings could be useful for the design of HBsAg detection assays, which may significantly improve the ability to detect particular HBsAg mutants.

Detection and molecular characterisation of a diagnosis escape variant associated with occult hepatitis B virus in Brazil.

BACKGROUND Many studies have identified mutations in the hepatitis B surface antigen (HBsAg) as important factors limiting the ability of commercial serological assays to detect this viral antigen. However, an association between mutations in the HBsAg gene and the occurrence of occult HBV infection (OBI) in patients has not been established.OBJECTIVES To detect hepatitis B virus (HBV) DNA in patients with anti-HBc as a unique serological marker, a previously published, cost-effective TaqMan-based real-time polymerase chain reaction (PCR) test with minor groove binding probes was adapted for use in this study. The current study also aimed to investigate HBsAg mutations and genotypes of HBV in OBI at the Viral Hepatitis Ambulatory Clinic in Rio de Janeiro to determine any possible association.METHODS Intra-assay and inter-assay reproducibility were determined, and the mean coefficient of variation values obtained were 2.07 and 3.5, respectively. Probit analysis indicated that the 95% detection level was 25 IU/mL. The prevalence of OBI was investigated in 35 serum samples with an ‘anti-HBc alone’ profile from individuals who attended our clinic between 2011 and 2013.FINDINGS HBV DNA was detected in only one sample, resulting in an OBI rate of 2.9%. Nucleotide sequencing of the pre-S/S region was performed to genotype and analyse mutations within the HBsAg gene of this HBV DNA. The HBV in the OBI case was classified as sub-genotype A1, and a sequence analysis of the small S gene revealed 12 mutations in the major hydrophilic region compared to the consensus A1 sequence. Most of these mutations occurred in amino acid residues that have been reported as clinically relevant because they have been implicated in vaccine escape and/or inability to detect HBsAg by commercial serological assays.MAIN CONCLUSIONS Our study suggests the importance of specific HBsAg mutations, different from those in D, B, and C genotypes, in sub-genotype A1 HBV associated with OBI.

The Thr to Met substitution of amino acid 118 in hepatitis B virus surface antigen escapes from immune-assay-based screening of blood donors.

Hepatitis B surface antigen (HBsAg) is the main diagnosis marker for hepatitis B virus (HBV) infection. In this study, a novel HBV mutant from an HBV-positive blood donor with false-negative results during HBsAg screening was identified. DNA sequencing discovered two mutations at nt 353 (A to T) and nt 349 (T to A), leading to Thr to Met and Ser to Thr substitutions at aa 118 and 117 of HBsAg, respectively. Further analysis showed that eight of ten HBsAg ELISA kits failed to detect this HBsAg mutant. A mutagenesis assay indicated that the Thr to Met substitution at aa 118 was the determinant for escape from HBsAg ELISA detection. A small-scale screening of blood donors identified two individuals infected by this unique HBV mutant, suggesting a certain level of prevalence among the general population. In conclusion, our study identified the aa 118 mutation in HBV surface antigen and provided information for improvement of HBV diagnosis products.

HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia.

In Ethiopia, HBV and HIV are co-circulating. Since patients are not routinely tested for HBV, the use of antiretroviral drugs could contribute to unintended HBV drug resistance and surface gene variability during HIV coinfection. A total of 161 hepatitis B surface antigen (HBsAg)-positive sera from 58 HIV-coinfected and 103 drug-naive HBV-monoinfected individuals were characterized for HBV drug resistance and immune escape HBsAg variants. HBV polymerase/surface gene fragment of 716 bp was analysed by direct sequencing. In 34 out of 161 study subjects (21.1%) HBV drug resistance mutations (DRMs) were detected with a frequency of 3.1% rtL80F/I, 0.6% rtA181V, 1.2% rtT184S, 6.2% rtV173L, 10.6% rtL180M, 10.6% rtM204V/I and 8.1% rtI233V. The prevalence of the major DRMs in HBV-HIV-coinfected individuals was significantly higher than monoinfected individuals (41.4% versus 10.7%). Lamivudine selected DRMs, that is, rtL180M (29.3%) and rtM204V/I (29.3%) and rtV173L (15.5%) were more prevalent in HBV-HIV-coinfected individuals but absent in HBV-monoinfected individuals. Despite the finding that rtL180M and rtM204V/I were higher among ART-experienced individuals, the overall prevalence of DRMs (48.0% versus 36.4%) showed no significance difference among antiretroviral therapy (ART) status. The study also revealed higher frequency and heterogeneity of putative and known immune escape HBsAg mutations both in the major hydrophilic region (MHR; 68.3%) and outside the MHR (82.5%) of the surface gene. In particular, the 'a' determinant surface gene mutations (sT125S, sA128V, sQ129H/R, sT131I, sC137S, sT143M, sD144D/E, sG145R, sT148P) and the majority of clustered/multiple as well as drug selected immune escape HBsAg mutations were more prevalent in HBV-HIV-coinfected individuals. HIV therapy without HBV co-management in Ethiopia fosters emergence and circulation of HBV variants of public health importance. It is highly recommended to include HBV testing and co-management as part of routine HIV care programmes for a better ART selection.

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples

BackgroundHepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays. ObjectivesTo evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations. Study designA selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia®, bioMérieux, Marcy-L’Étoile, France. Alere Determine HBsAg™, Iverness Biomedical Innovations, Köln, Germany. Quick Profile™, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative® assay (Abbott Laboratories, Sligo, Ireland). ResultsThe sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile™ assay with a virus harboring a D144A mutation. ConclusionsThe evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.

Hepatitis B surface antigen genetic elements critical for immune escape correlate with hepatitis B virus reactivation upon immunosuppression.

Hepatitis B virus (HBV) reactivation during immunosuppression can lead to severe acute hepatitis, fulminant liver failure, and death. Here, we investigated hepatitis B surface antigen (HBsAg) genetic features underlying this phenomenon by analyzing 93 patients: 29 developing HBV reactivation and 64 consecutive patients with chronic HBV infection (as control). HBsAg genetic diversity was analyzed by population-based and ultradeep sequencing (UDS). Before HBV reactivation, 51.7% of patients were isolated hepatitis B core antibody (anti-HBc) positive, 31.0% inactive carriers, 6.9% anti-HBc/anti-HBs (hepatitis B surface antibody) positive, 6.9% isolated anti-HBs positive, and 3.4% had an overt HBV infection. Of HBV-reactivated patients, 51.7% were treated with rituximab, 34.5% with different chemotherapeutics, and 13.8% with corticosteroids only for inflammatory diseases. In total, 75.9% of HBV-reactivated patients (vs. 3.1% of control patients; P<0.001) carried HBsAg mutations localized in immune-active HBsAg regions. Of the 13 HBsAg mutations found in these patients, 8 of 13 (M103I-L109I-T118K-P120A-Y134H-S143L-D144E-S171F) reside in a major hydrophilic loop (target of neutralizing antibodies [Abs]); some of them are already known to hamper HBsAg recognition by humoral response. The remaining five (C48G-V96A-L175S-G185E-V190A) are localized in class I/II-restricted T-cell epitopes, suggesting a role in HBV escape from T-cell-mediated responses. By UDS, these mutations occurred in HBV-reactivated patients with a median intrapatient prevalence of 73.3% (range, 27.6%-100%) supporting their fixation in the viral population as a predominant species. In control patients carrying such mutations, their median intrapatient prevalence was 4.6% (range, 2.5%-11.3%; P<0.001). Finally, additional N-linked glycosylation (NLG) sites within the major hydrophilic loop were found in 24.1% of HBV-reactivated patients (vs. 0% of chronic patients; P<0.001); 5 of 7 patients carrying these sites remained HBsAg negative despite HBV reactivation. NLG can mask immunogenic epitopes, abrogating HBsAg recognition by Abs. HBV reactivation occurs in a wide variety of clinical settings requiring immune-suppressive therapy, and correlates with HBsAg mutations endowed with enhanced capability to evade immune response. This highlights the need for careful patient monitoring in all immunosuppressive settings at reactivation risk and of establishing a prompt therapy to prevent HBV-related clinical complications.

Clinical significance of hepatitis B surface antigen mutants

Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the "a" determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications.

Hepatitis B Virus (HBV) Infection in Liver Disease Patients in Mumbai, India with Special Reference to Hepatitis B Surface Antigen (HBsAg) Mutant Detection.

To determine the prevalence of Hepatitis B Surface antigen (HBsAg) in patients attending the Hepatology Out Patient Department (OPD) of a tertiary care hospital and to compare the routinely used HBsAg detection kit with the mutant detection kit to find out the presence of mutants in a given setting. A cross-sectional study was carried out in adult patients with liver disease attending the Hepatology OPD, of a tertiary care hospital in Mumbai, India. Age, gender and clinical history of the patient were recorded. Blood specimen was tested for HBsAg (Microscreen(TM) ELISA, Span diagnostics, India) and HBsAg mutants (Hepanostika HBsAg Ultra(TM) ELISA, Biomerieux, France). The samples with discordant results between these two ELISAs were confirmed by Hepatitis B Virus (HBV) Deoxyribonucleic Acid (DNA) Polymerase Chain Reaction (PCR) (Cobas Taqman(TM), Roche Molecular Systems, USA). Seven hundred and eighteen patients were enrolled in the study. The mean age of patients in the study group was 41 years (range 17 to 69 years). Four hundred and ninety seven (69.22%) were males and remaining were females. The prevalence of HBsAg was found to be 17.4%. The positivity amongst the male population was 18.1% which was higher than the female population (15.8%). Of the 718 samples tested, 120 were positive for HBsAg by Microscreen(TM) ELISA and 132 were positive by Hepanostika HBsAg ultra(TM). Of the 12 discordant samples, HBV DNA was detected in five samples indicating 0.7% prevalence of mutants. Hepatitis B is prevalent in liver disease patients. The mutant detecting assay is recommended in set-ups where missing HBsAg in patients would have tremendous impact on the outcome such as in blood donors, organ or tissue donors and antenatal screening of mothers. It is also helpful in chronic liver disease patients where the routine HBsAg detection test is negative and the other causes of chronic liver disease have been ruled out. However, it is not recommended for use in routine diagnostic set-ups where high false positivity would lead to over-diagnosis of the condition.

Decreased antigenicity profiles of immune-escaped and drug-resistant hepatitis B surface antigen (HBsAg) double mutants

BackgroundSelective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. Because of the overlap of the open reading frame (ORF) S for the HBsAg and ORF P for viral polymerase, rtM204I and rtM204V mutations in the polymerase would produce sI195M and sW196S in the HBsAg. The combined effects of immune-escaped mutations (sT118M, sG145K, sG145R) and drug-resistant mutations (rtM204I, rtM204V) on the antigenicity profiles of HBsAg has not been widely explored.MethodsTo determine the combined effects of immune-escaped and drug-resistant mutants on the antigenicity profiles of HBsAg, recombinant plasmids encoding HBsAg double mutants were constructed using site-directed mutagenesis. The supernatant from each plasmid transfection was analyzed for HBsAg in the western-blotting and five of the most commonly used commercial ELISA kits in China.ResultsWestern-blotting assay showed the successful expression of each HBsAg mutant. All five ELISA kits manifested similar avidity, which were demonstrated by the slope of the curves, for the sT118M mutant, and sT118M-rtM204I (sT118M-sI195M) and sT118M-rtM204V (sT118M-sW196S) double mutants, suggesting that drug-resistant YMDD mutants caused negligible losses in the antigenicity of immune-escaped sT118M HBsAg. In contrast, the presence of the rtM204I (sI195M) mutation, but not rtM204V (sW196S) in combination with the sG145K mutation significantly reduced the avidity of sG145K HBsAg. The rtM204I (sI195M) mutation also decreased the antigenicity profiles for sG145R HBsAg.ConclusionsDrug-resistant mutations rtM204I (sI195M) and rtM204V (sW196S) caused significant reduction in antigenicity for the immune-escaped HBsAg mutants sG145K and sG145R, which may hamper HBV diagnosis and disease control from HBV blood-transfusion transmissions in China. The development of ELISA kits with a greater sensitivity for drug-resistant and immune-escaped HBsAg warrants further consideration.

An Overview of Hepatitis B Virus Surface Antigen Mutant in the Asia Pacific

Hepatitis B virus infection is a serious health problem worldwide, and more than 350 million people are chronic carriers, constituting a major global threat. Southeast Asia and the Western Pacific have the highest levels of endemicity in the world, with an estimated seroprevalence ranging between 2% and 31%. Mutations in the hepatitis B surface antigen (HBsAg) have been reported in many parts of the world but are most common in Asian infants; such mutants have several clinical effects, such as the development of hepatocellular carcinoma. Diagnostic failures by commercial assays have reduced the diagnostic effectiveness of HBsAg detection. For example the substitution of an amino acid in the major hydrophilic region of the S gene reduces the binding of hepatitis B surface antibodies leading to immune escape. The safety of blood transfusion may be compromised by current screening tests due to escape from being neutralised by antibodies induced by HBsAg mutants, and undetectable levels of viral surface protein. Data on the epidemiology of HBsAg mutation in Asia Pacific are scant; however, this manuscript has reviewed the available information on the epidemiology of HBsAg mutation in Asia Pacific.

Hepatitis B virus subgenotype A1, occurrence of subgenotype D4, and S gene mutations among voluntary blood donors in Kenya

Kenya is one of the high endemic zones for hepatitis B virus (HBV) infection. The consensuses on prevalence of the HBV genotypes and the existence of their variants have not been fully established in Kenya. Hence, there is a need to further monitor the diversity of HBV. This study aimed to extend the current molecular and epidemiological information about the geographical distribution of HBV genotypes and subgenotypes, as well as to describe the hepatitis B surface antigen (HBsAg) variants circulating in different Regional Blood Transfusion Centres of Kenya. A total of 32 HBsAg positive blood units from five different blood transfusion centers in Kenya were used in the study. The HBV DNA preS/S-gene was amplified and sequenced. Alignments of S gene were applied using reference sequence from GeneBank. Phylogenetic analysis was performed using the MEGAv4.0 software with the neighbor-joining and maximum composite likelihood methods. Twenty-one plasma samples (65.6%) were DNA positive and were successfully sequenced. Eighteen out of the twenty-one isolates (85.7%) belonged to subgenotype A1 Afro-Asian: six were from Nairobi, four from Kisumu, two from Embu, and three each from Eldoret and Mombasa. The other three strains (14.3%, 3/21) belonged to subgenotype D4 from Mombasa. The HBsAg mutations were detected in nine isolates (42.9%, 9/21). The HBV/A1 and HBV/D4 are dominant among blood donors in Kenya. This demonstrates that continuous monitoring of the HBV diversity would help reveal circulating genotypes and subgenotypes as well as mutants of clinical significance in Kenya.

Construction and expression of hepatitis B surface antigen escape variants within the "a" determinant by site directed mutagenesis.

The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ''a'' determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

Functional analysis of hepatitis B virus immune escape mutants with insertion mutations in the surface antigen

To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen (HBsAg) on viral antigenicity and replication. Hepatitis B virus (HBV) DNA was obtained from patients with HBsAg-positive chronic hepatitis B (CHB) infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B (AB073826) and genotype C (AF286594). Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site-directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants' effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay (CMIA) and Southern blot hybridization, respectively. The viral antigenicity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp-Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein's surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone. Two CHB patients harbored HBV with insertion mutations in HBsAg: one with two (NT) and one with three (NTT) inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA (P = 0.02). The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites (with an average increase of 0.13). The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability (range: -0.55 to 2.97; affecting eight proximal amino acids) and the flexibility (range: -0.01 to 1.1; affecting five proximal amino acids). The NTT three amino acids insertion in the HBsAg open reading frame, between 114 and 115 of the wild-type sequence, detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.