Comprehensive analysis of the prevalence of hepatitis B virus escape mutations in the major hydrophilic region of surface antigen

Escape mutations in the major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg) are reported widely worldwide; these mutations lead to diagnostic problems, emergence of vaccine-escape mutants, and hepatitis B immunoglobulin (HBIG) therapy failure. However, the prevalence of these mutations in different genotypes remains to be studied systematically. In the current study, 11,221 non-redundant hepatitis B virus (HBV) sequences of 8 genotypes (from A to H), obtained from the National Center for Biotechnology Information (NCBI), were analyzed to determine the prevalence of HBsAg escape mutations that were previously described. Eight important mutations associated with diagnostic failure, P120T, T126S, Q129H, G130N, S143L, D144A, and G145A/R, were prevalent in one or more genotypes, with the frequency of no less than 1%. With regard to escape variants that evade vaccine or immunoglobulin therapy, mutations were located mainly at positions 120, 126, 129, 130, 133, 134, 137, 140, 143, 144, and 145. The majority of such mutations showed genotypic heterogeneity, indicating the different distribution of the escape mutations. Most of the escape mutations clustered in the "a" determinant, indicating that this region was more likely to be affected by immune selection or antiviral therapy than other regions. Understanding the prevalence and heterogeneity of escape mutations could provide useful guidance for the improvement of diagnostic assays, design of new vaccines, and prevention of failure of HBIG therapy.     

Naturally occurring MHR variants in Turkish patients infected with hepatitis B virus

Major B-cell epitopes are located at the major hydrophilic region (MHR) of hepatitis B virus (HBV) surface antigen (HBsAg). The genotypes, subtypes, and naturally occurring amino acid (aa) substitutions of MHR were analyzed in 81 Turkish adult patients (41 inactive HBsAg carriers and 40 patients with chronic hepatitis B) by direct sequencing of the S gene fragment. All the isolates were genotype D according to the phylogenetic analysis. The most common HBsAg subtype was ayw2, followed by ayw3 while one isolate specified ayw4 by encoding Leu127. MHR variants were detected in 22 of the 81 (27.2%) isolates. The prevalence was significantly higher in the chronic hepatitis B group (42.5%) compared to inactive HBsAg carriers (12.2%). Twenty-two samples had a total of 26 amino acid substitutions involving 14 positions. The majority of the patients had a single variation. Most of the amino acid substitutions were located at the HBs1 region of the MHR, while 9 of the 26 were in the classic "a" determinant (aa 124-147). When samples with "a" variants were evaluated by two different commercial HBsAg tests, only the isolate with Ser143Leu variation had a decreased reactivity in the assay using monoclonal antibodies for capture and detection. In conclusion, the findings of the study was in accordance with previous studies showing HBV genotype and subtype homogeneity (genotype D/ayw) in Turkey. Naturally occurring MHR and "a" determinant variants were common, especially among chronic hepatitis B patients. The influence of detected "a" variants on diagnostic assays was limited.     

Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing

The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.     

Characterization of hepatitis B virus genome variability in Iranian patients with chronic infection, a nationwide study

Hepatitis B virus (HBV) isolates from Iranian patients around the country were characterized. Eighty-one complete genomes from HBV isolates were sequenced and analyzed. The studied population was grouped into three categories including inactive carriers, patients with chronic hepatitis, and patients with liver cirrhosis. Molecular and phylogenetic analyses revealed that Iranian patients were infected with HBV genotype D and subgenotype D1. The most common subtype was ayw2, followed by ayw3 and ayw4. Several deletions and insertions that had no correlation with disease outcome were observed in the HBV genomes. The most frequent mutation in the major hydrophilic region (MHR) of HBV surface antigen (HBsAg) was sP120S. Almost half of the patients studied carried precore (PC) mutant variants and one-third of the studied population was infected with variants carrying basal core promoter (BCP) mutations. PC and BCP mutations were observed in older patients, especially in those with chronic liver disease. Sixty-seven patients (82.7%) were HBeAg negative, and the prevalence of precore mutant isolates (G1896A) was higher in this group than in HBeAg-positive patients. Lamivudine drug resistance mutations were detected after 1 year of treatment in about 30% of lamivudine-treated patients. In conclusion, these results demonstrate that HBV subgenotype D1 is the only subgenotype circulating in Iran, and there is no evidence of any exotic genotype in the region. The HBV PC (G1896A) mutation may play an important role in the clinical outcome of the disease by increasing the risk of progressive liver disease among Iranian patients infected with HBV.     

Gender disparity in distribution of the major hydrophilic region variants of hepatitis B virus genotype C according to hepatitis B e antigen serostatus

Hepatitis B virus (HBV) e antigen (HBeAg) seroconversion during chronic HBV infection is known to play an important role in disease progression and patient response to antiviral agents. The aim of the present study was to analyze gender disparity in distribution of major hydrophilic region (MHR) variants according to HBeAg serostatus. Prevalence of MHR variants from 68 Korean patients with chronic hepatitis (31 HBeAg-positive and 37 HBeAg-negative) was examined in terms of HBeAg serostatus and sex by direct sequencing analysis of the MHR. Gender disparity was observed in the distribution of MHR variants according to HBeAg serostatus. In male patients, the prevalence of MHR variants was significantly higher in HBeAg negative patients than in HBeAg positive patients [58.8% (10/17 patients) vs. 14.3% (3/21 patients), P=0.004]. However, the same was not true in female patients [55.0% (11/20 patients) vs. 60.0% (6/10 patients), P=1.000)]. In addition, 2 mutation types (L110I and G145A) related to HBeAg serostatus were found. In conclusion, HBeAg seroconversion in male chronic patients infected with genotype C could lead to mutations of MHR, major target to host immune response, which might in turn contribute to HBV persistence and immune evasion.     

Molecular epidemiology of chronic hepatitis B virus infection in Greece

Virological data on chronic hepatitis B virus (HBV) infection in Greece are limited. HBV genotypes, surface antigen (HBsAg) subtypes, and HBsAg “a” determinant mutations among patients infected chronically with HBV, were investigated. Serum samples from 135 HBsAg positive patients were tested. Serologic (HBsAg, anti‐HBs, HBeAg, and anti‐HBe), virologic (HBV‐DNA quantitation) and biochemical markers (serum alanine aminotransferase/ALT and aspartate aminotransferase/AST) were analyzed. HBV genotypes and HBsAg subtypes were determined by partial sequencing of the S gene. Genotyping was performed by using the National Center for Biotechnology Information online Genotyping tool and phylogenetic analysis. Nucleotide sequences were aligned pair wise with ClustalW and phylogenetic trees were constructed by the neighbor‐joining method. Sequences were also used to predict HBV HBsAg subtypes. In six patients (4%), simultaneous presence of HBsAg and anti‐HBs was determined, whereas 47 patients (35%) were HBeAg positive, 84 (62.5%) were anti‐HBe positive, and four patients (3%) were characterized by the simultaneous presence of HBeAg and anti‐HBe. Mean ALT was 238 IU/L (standard deviation = 576.84), and HBV‐DNA levels ranged from 1.02 × 10⁵ to 2.2 × 10⁷ IU/ml. Genotype D was predominant (98%), with viral groups D/ayw2 (73%) and D/ayw3 (27%). Group A/adw accounted for 1% of cases. Genotypes B and C were found exclusively in the Chinese immigrants (1%). Single or multiple point mutations were found in 35 cases (26%). Some of the most common mutations occurred at amino acid positions 129, 133, 134, 144, 145, including the “vaccine escape” mutation G145R. Mutations analysis revealed that amino acid substitutions did not affect detection by commercial immunoassays. J. Med. Virol. 83:245–252, 2011. © 2010 Wiley‐Liss, Inc.     

Mutations in the major hydrophilic region (MHR) of Hepatitis B virus genotype C in North China

Hepatitis B virus (HBV) can evolve by mutations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) to permit its escape from neutralization by antibodies such as HBV surface antibody (anti‐HBs) and from host immune responses. This study investigated the prevalence and pattern of MHR mutations in North China and the clinical correlations of these mutations. The MHRs of 161 HBsAg‐positive patients were amplified using nested PCR, and directly sequenced to identify MHR mutations. It was showed that among the 161 patients infected with HBV genotype C in North China, the overall frequency of MHR mutation was 46.6%. Furthermore, MHR mutations were associated with high white blood cell counts (P = 0.025), high bilirubin levels (P = 0.048), and cirrhosis (P = 0.010). The most frequent mutations in patients with both HBsAg‐positive and anti‐HBs‐positive were located in subregion 1 and 3 of MHR, specifically, residue Q101 (29.9%) and I126 (70.6%), which was different from the mutation pattern found in Western Europe and the United States. Taken together, these data indicated important virological and clinical aspects of HBV evolution in terms of the surface gene of genotype C, which might be important for its response to the currently available HBV vaccine. J. Med. Virol. 84:1901–1906, 2012. © 2012 Wiley Periodicals, Inc.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

Molecular epidemiological study of hepatitis B virus in blood donors from five Chinese blood centers

Although the genetic variability of hepatitis B virus (HBV) in HBV-infected patients has been extensively studied, reports on genotypes, subtypes and mutations in the S region of HBV strains from Chinese blood donors are limited. In this study, 245 blood samples from HBsAg-positive blood donors were collected from five geographically diverse blood centers in China. The S region of HBV was amplified, and the HBV genotype and subtype were determined. The amino acid sequences of the S region were aligned, and mutations related to the failure of immunization and HBsAg detection were determined. Of the 245 samples, 228 (93?%) were genotyped successfully. We found that genotypes B, C, D and A accounted for 58.8?%, 21.9?%, 6.6?% and 3.95?% of the isolates, respectively. The distribution of HBV antigen subtypes was as follows: adw (67.6?%), adr (23.3?%) and ayw (8.7?%). Mutations were present in 39 (17.1?%) of 228 samples in the major hydrophilic region (MHR) of the S region. This study demonstrated that HBV genotype/subtype B/adw was the most frequent strain circulating in HBV-infected Chinese blood donors, followed by C/adr. The occurrence of MHR mutants in HBV-infected blood donors and the potential failure to detect some of them in collected units poses a threat to transfusion safety.     

Molecular epidemiological study of hepatitis B virus among migrant workers from Cambodia,Laos, and Myanmar to Thailand

Although hepatitis B virus (HBV) infection is endemic in Southeast Asia, molecular epidemiological data on HBV circulating in some countries are limited. The aims of this study were to evaluate the seroprevalence of HBV and its genetic variability among migrant workers from Cambodia, Laos, and Myanmar in Thailand. Sera collected from 1,119 Cambodian, 787 Laotian, and 1,103 Myanmarese workers were tested for HBsAg. HBV DNA was amplified and the pre‐S/S region was sequenced for genotyping and genetic mutation analysis. HBsAg was detected in 282 (9.4%). The prevalence of HBsAg among migrant workers from Cambodia, Laos, and Myanmar was 10.8%, 6.9%, and 9.7%, respectively. Of 224 subjects positive for HBV DNA, 86% were classified as genotype C (99% were sub‐genotype C1) and 11.6% were genotype B (30.8%, 34.6%, and 30.8% were sub‐genotypes B2, B3, and B4, respectively). Various point mutations in the “a” determinant region were detected in approximately 18% of these samples, of which Ile126Ser/Asn was the most frequent variant. Sequencing analysis showed that 19.1% of samples had pre‐S mutations, with pre‐S2 deletion as the most common mutant (7.7%) followed by pre‐S2 start codon mutation (3.8%) and both pre‐S2 deletion and start codon mutation (3.3%). High prevalence of HBV infection (approximately 7–11%) was found among migrant workers from Cambodia, Laos, and Myanmar, which may reflect the current seroprevalence in their respective countries. The data also demonstrated that HBV sub‐genotype C1 was the predominant strain and various mutations of HBV occurring naturally were not uncommon among these populations. J. Med. Virol. 82:1341–1349, 2010. © 2010 Wiley‐Liss, Inc.     

Application strategies of serum HBV DNA detection in HBV infection patients: A retrospective study of 5611 specimens

The detection of hepatitis B virus (HBV) DNA plays a critical role in determining the level of viral replication in HBV-infected patients. However, how to select appropriate HBV DNA detection method, low-sensitivity (ls) and hypersensitivity (hs) remains unclear. In this study, hepatitis B surface antigen (HBsAg), hepatitis B e-antigen (HBeAg), alanine transaminase (ALT), aspartate transaminase (AST), and hs HBV DNA titers in serum of 5611 cases with suspected HBV infection were reviewed. Besides, the dynamic changes of HBV DNA and HBsAg in 85 chronic hepatitis B (CHB) patients receiving peginterferon α (PegIFNα) or entecavir (ETV) were observed. The results showed the positive rate of HBV DNA was 32.8%, of which low viral load (20 to 500 IU/mL) accounted for 51.8%. In the 5611 cases, when the HBsAg was less than 1000 IU/mL, the proportion of low viral load was 76.3%. Moreover, in patients receiving antiviral treatment, when HBsAg was less than 2000 IU/mL (PegIFNα) or HBsAg was less than 3500 IU/mL (ETV), the proportion of patients with low viral load was 79.5% or 78.0%, respectively. We developed a strategy of serum HBV DNA detection in HBV-infected patients. When HBsAg was negative, HBV DNA detection should be unnecessary. When HBsAg was 0.05 to 1000 IU/mL, hs HBV DNA should be detected in patients with abnormal level of ALT, AST, or HBeAg. While HBsAg was greater than or equal to 1000 IU/mL, ls HBV DNA was recommended. Moreover, the cutoff value of HBsAg increased during antiviral therapy of CHB patients. In conclusion, hs HBV DNA is of great value in HBV-infected patients with low viral load. HBV DNA detection methods should be selected reasonably according to the levels of HBsAg, HBeAg, ALT, and AST.     

Circulating microRNA‐122 levels are important predictor of hepatitis B virus surface antigen seroclearance

It is currently unclear what impact serum microRNA‐122 (miR‐122) levels have on clearance of hepatitis B virus (HBV) surface antigen (HBsAg) in HBV‐infected patients who had not received antiviral therapy. The current study evaluated the impact of serum miR‐122 levels on HBsAg seroclearance in 367 consecutive HBV‐infected patients who had not received antiviral therapy between their initial and last visit, and investigated the predictive factors of HBsAg seroclearance. Cumulative HBsAg seroclearance rates were 13.5%, 32.0%, and 37.4% after 10, 20, and 30 years, respectively. The yearly incidence of HBsAg seroclearance over the investigated 30‐year period was 1.25%. A significant and strong correlation was observed between serum miR‐122 and HBsAg levels. Moreover, there was a significant correlation between serum miR‐122 levels and the levels of HBV DNA, hepatitis B e‐antigen, and HBV core‐related antigen. The HBsAg seroclearance rate in patients with a <1.0‐fold change of serum miR‐122 levels was significantly higher than in those with a ≥1.0‐fold change. Multivariate analysis identified age (≥30 years), HBV DNA levels (<2.2 log U/mL), HBV genotype (non‐C), and serum miR‐122 levels (<1.0‐fold change) as significant predictors of HBsAg seroclearance. Our results indicated that serum miR‐122 level is an important predictor of HBsAg seroclearance in Japanese patients who do not receive antiviral therapy. Understanding the complexity of the interactions among various virus‐related and host‐related factors could potentially help in the design of new therapies that enhance HBsAg seroclearance.     

Relationship between serum hepatitis B virus DNA and surface antigen with covalently closed circular DNA in HBeAg‐negative patients

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is responsible for viral persistence. This study aimed to investigate the serum surrogate markers for cccDNA and to evaluate the intrahepatic viral events associated with disease activity in HBeAg‐negative chronic hepatitis B patients. Thirty‐three treatment‐naïve patients with a negative HBeAg who had a liver biopsy were studied. Active disease was defined as a serum alanine aminotransferase >40 IU/L and a serum HBV DNA >10,000 copies/ml. This study showed significant correlation between serum HBV DNA and both log cccDNA (r = 0.41, P = 0.018) and log total intrahepatic HBV DNA (r = 0.71, P < 0.0001). No significant correlation was observed between serum HBsAg and log cccDNA (P = 0.15) or log total intrahepatic HBV DNA (P = 0.97). Fourteen and 19 patients had inactive and active disease, respectively. The median log cccDNA and log total intrahepatic HBV DNA (copies/10⁶ cells) were significantly higher in patients with active disease compared with those with inactive disease (4.11 vs. 3.53, P = 0.03 and 5.46 vs. 4.64, P < 0.001, respectively). The HBV replicative efficiency, defined as the ratio of serum HBV DNA to cccDNA, was approximately 20% higher in patients with active disease. No significant difference was observed in the HBsAg levels and the ratio of serum HBsAg to cccDNA between the two groups. In conclusion, serum HBV DNA, but not HBsAg, reflects the amount of cccDNA and the replication efficiency of HBV in patients with HBeAg‐negative chronic hepatitis B. J. Med. Virol. 82:1494–1500, 2010. © 2010 Wiley‐Liss, Inc.     

Antigenic Diversity of Hepatitis B Virus Strains of Genotype F in Amerindians and Other Population Groups from Venezuela

The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.     

Prevalence of naturally occurring surface antigen variants of hepatitis B virus in Korean patients infected chronically

Although Korea is one of the endemic areas of hepatitis B virus (HBV) infection, the prevalence of naturally occurring variants in the major hydrophilic region (MHR) of the surface (S) gene of HBV has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical state, and HBeAg serostatus in a large series of Korean patients with chronic HBV infection by direct sequencing analysis of part of the S gene containing the MHR of HBV isolated from 101 chronic HBV patients (51 HBeAg-positive and 50 HBeAg-negative): 37 were asymptomatic carriers, 21 had chronic hepatitis, 20 had liver cirrhosis, and 23 had hepatocellular carcinoma (HCC). Forty-seven MHR variants (46.5%) of the 101 patients were detected, involving a total of 59 amino acid substitutions at 12 positions inside and 14 position outside the 'a' determinant, and 33 'a' determinant variants (32.7%). A total of 17 novel variants and 14 novel mutation patterns were detected. The prevalence of MHR variants in HBeAg-negative patients tended to be higher than in HBeAg-positive patients (54.0% vs.39.2%) and the prevalence of MHR variants in HCC and liver cirrhosis tended to be higher than in asymptomatic carriers (65.2% vs. 40.5% and 50.0% vs. 40.5%, respectively). In conclusion, three important findings were found in the present study. First, an unexpectedly high prevalence of naturally occurring MHR variants was found in Korean chronic patients. Second, several novel variants associated with mutations outside the 'a' determinant were detected. Finally, a higher prevalence of MHR variants was associated with HBeAg-negative serostatus and severe liver disease, particularly HCC.     

Hepatitis B virus genotypes and serotypes in western India: lack of clinical significance

To determine hepatitis B virus genotype and subtype distribution among HBV infected individuals with different clinical manifestations in western India, serum samples from 19 asymptomatic hepatitis B surface antigen carriers, 30 chronic hepatitis B patients, 8 acute hepatitis B patients, 5 fulminant hepatitis B patients, and with circulating HBV DNA were genotyped and subtyped on the basis of the nucleotide sequence analysis of S region of the HBV genome. Genotype D was the predominant genotype circulating in western India (57/62; 91.93%). All 19 asymptomatic hepatitis B surface antigen carriers, 8 acute hepatitis B patients, 5 fulminant hepatic failure patients and 25/30 chronic hepatitis B patients were circulating genotype D and ayw3/ayw2 subtypes. HBV genotype A was prevalent in 8% (5/62) of the total number of patients and all belonged to chronic hepatitis B category. Subtyping analysis showed that all genotype A isolates were of subtype adw2. As most of the patients from different clinical categories were infected with HBV genotype D, it is concluded that this genotype did not influence the outcome of HBV infection.     

Clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis B

To evaluate therapeutic immunostimulation nine chronic hepatitis B patients received six monthly intradermal vaccinations with HBsAg in combination with daily lamivudine. Another five patients received six doses of the vaccine and daily lamivudine together with daily Interleukin-2 (IL-2) s.c. within 3 months in an open-labeled trial. Clinical efficacy was assessed by alanine transaminase levels and HBV serology. The induction of specific T and B cell responses was analyzed serially by 3H-thymidine uptake, ELISA and ELISPOT assays. After the therapy was stopped, seven of nine vaccine/lamivudine and two of five vaccine/lamivudine/IL-2 recipients did not have detectable HBV DNA. Four complete responders cleared the virus and had normalized ALT levels, however, one of these patients showed transient disease reactivation followed by spontaneous viral clearance and normal ALT five months later. Low frequencies of anti-HBs producing B cells and HBV specific T helper cells secreting predominantly interferon-gamma were induced by i.d. vaccine therapy. The ELISPOT technique demonstrated transient induction of HBV peptide specific cytotoxic T cells in seven HLA-A2 positive chronic HBV carriers. The preliminary data from this study demonstrate that the HBV surface antigen vaccine in combination with antiviral or immunomodulating drugs induced antiviral immune responses and consequently viral elimination may be achieved in patients with unfavorable prognosis.     

Ultradeep Sequencing for Detection of Quasispecies Variants in the Major Hydrophilic Region of Hepatitis B Virus in Indonesian Patients

Quasispecies of hepatitis B virus (HBV) with variations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) can evolve during infection, allowing HBV to evade neutralizing antibodies. These escape variants may contribute to chronic infections. In this study, we looked for MHR variants in HBV quasispecies using ultradeep sequencing and evaluated the relationship between these variants and clinical manifestations in infected patients. We enrolled 30 Indonesian patients with hepatitis B infection (11 with chronic hepatitis and 19 with advanced liver disease). The most common subgenotype/subtype of HBV was B3/adw (97%). The HBsAg titer was lower in patients with advanced liver disease than that in patients with chronic hepatitis. The MHR variants were grouped based on the percentage of the viral population affected: major, ≥20% of the total population; intermediate, 5% to <20%; and minor, 1% to <5%. The rates of MHR variation that were present in the major and intermediate viral population were significantly greater in patients with advanced liver disease than those in chronic patients. The most frequent MHR variants related to immune evasion in the major and intermediate populations were P120Q/T, T123A, P127T, Q129H/R, M133L/T, and G145R. The major population of MHR variants causing impaired of HBsAg secretion (e.g., G119R, Q129R, T140I, and G145R) was detected only in advanced liver disease patients. This is the first study to use ultradeep sequencing for the detection of MHR variants of HBV quasispecies in Indonesian patients. We found that a greater number of MHR variations was related to disease severity and reduced likelihood of HBsAg titer.