Hepatol Int (2012) 6:707–717 DOI 10.1007/s12072-011-9316-5
ORIGINAL ARTICLE
Naturally occurring core immune-escape and carboxy-terminal mutations\truncations in patients with e antigen negative chronic hepatitis B Ranjit Chauhan • Shiv K. Sarin • Manoj Kumar Jayashree Bhattacharjee
•
Received: 16 May 2011 / Accepted: 1 October 2011 / Published online: 2 December 2011 Ó Asian Pacific Association for the Study of the Liver 2011
Abstract Introduction Hepatocellular injury is often progressive in patients with hepatitis B e antigen negative chronic hepatitis B (HBeAg -ve CHB). There is scant data on association of core mutations occurring in patients with HBeAg -ve CHB with severity of liver disease. Materials and methods Hundred and eighteen patients with chronic infection who were HBeAg negative, antiHBe, and HBV DNA positive were enrolled. Precore and core regions were amplified, sequenced, and analyzed for precore, T helper, cytotoxic T lymphocytes (CTLs), B-cell epitope, and core carboxy-terminal region mutations. Results Majority of patients were infected with HBV genotype D: 96 (81%) [D1: 16, D2: 55 and D5: 25] followed by genotype A1: 15 (13%) and genotype C: 7 (6%) [C1: 5 and unidentified subgenotype C: 2]. Classical (A1896) as well as nonclassical precore region mutations were detected in 30 (25%) and in 9 (7.6%) patients, respectively. Core immune escape, core carboxy-terminal mutations and truncations were detected in 61 (52%), 11 (9.3%), and 14 (12%) patients, respectively. Three core immune escape mutations were significantly higher in patients with coexisting precore stop codon compared with patients without precore stop codon mutation, cT12S (43 vs. 8%, p \ 0.001), cS21T (16 vs. 3.4%, p \ 0.026), and cE77D (30 vs. 4.5%, p \ 0.002). When frequency of core R. Chauhan S. K. Sarin (&) M. Kumar Department of Gastroenterology and Advanced Centre for Liver Diseases, G.B. Pant Hospital, Room No. 201, New Delhi 110002, India e-mail: [email protected] R. Chauhan J. Bhattacharjee Department of Biochemistry, Lady Hardinge Medical College, New Delhi 110002, India
immune escape mutations was compared among CHB and decompensated patients, and cT12S: (27 vs. 10%, p \ 0.05), cS21T (16 vs. 1.35%, p \ 0.01), cT67P/N: (20 vs. 4%, p \ 0.001), cE113D (11.37 vs. 1.35%, p \ 0.05), and cP130T/Q (7 vs. 0%, p \ 0.001) mutations were found to be significantly higher in decompensated patients. Conclusion Core immune-escape mutations cT12S, cS21T, cT67P, cE113D, and cP130T/Q are significantly higher in decompensated liver disease patients and could influence the severity of liver disease in HBeAg -ve CHB patients. Keywords HBV Core Mutation Genotype HBeAg Immune-escape Carboxy-terminal
Introduction Hepatitis B virus (HBV) infection affects 400 million people globally. It is estimated that between 235,000 and 328,000 people die annually due to liver cirrhosis and hepatocellular carcinoma, respectively [1]. There are four phases in the natural history of chronic HBV infection. First phase is the immune-tolerant phase defined by hepatitis B e antigen (HBeAg) positive, with normal liver enzyme levels, and high serum HBV DNA concentrations. The second phase is the immune-clearance phase defined by HBeAg positive, abnormal liver enzymes, and lower HBV DNA concentrations. The third phase is defined by seroconversion from HBeAg to anti-HBe and has normal hepatic enzyme levels and low HBV DNA levels. In the fourth phase, hepatic enzymes are fluctuating and HBV DNA is high; this phase is also termed as HBeAg negative (-ve) chronic hepatitis B (CHB) or reactivation phase [2]. Particularly in areas where HBV transmission is
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predominantly perinatal, despite seroconversion, HBV continues to proliferate and could cause cirrhosis and liver cancer [3, 4]. There is an alarming rise in HBeAg -ve CHB worldwide [5], such patients have cirrhosis-related complications and 15–20% of patients develop liver decompensation within 5 years if left untreated [6, 7]. The core gene encodes two polypeptides, precore and core. Initiation of translation at the first start codon (AUG) results in a 25-kDa precore protein that is secreted as HBeAg. Initiation of translation at the second AUG leads to the synthesis of core protein that assembles to form 27 nm particles that comprise virion nucleocapsid (HBcAg). Although HBeAg and HBcAg are serologically distinct, these antigens are cross-reactive at the level of T-cell recognition because they are colinear throughout most of their primary sequence. The HBcAg possesses unique immunologic features: (1) HBcAg can function as both a T-cell-independent and a T-cell-dependent antigen; (2) it is *1,000-fold more immunogenic than HBeAg; (3) it preferentially primes Th1 cells, whereas, HBeAg preferentially primes Th2 cells; (4) HBcAg-specific Th cells mediate anti-envelope as well as anti-HBc antibody production; and (5) HBcAg is an effective carrier platform for heterologous epitopes [8]. Core carboxy terminal region varies and is heterogeneous [9]. In one of our recent studies, we identified impaired expression of TAP and LMP1 leading to defective antigen processing and presentation [10]. However, it is vital to know the profile of core mutations predominating during reactivation phase, which would add to the understanding of the interplay between virus and the host immune system. Viruses have developed a number of mechanisms to combat the host immune system and one of them is by escaping the immune system through mutations. Recent studies have identified hot-spots of mutations and deletions in the core gene of HBV in patients with severe liver disease [11, 12]. Core gene variations have also been related to ethnic background and HBV genotypes [13]. Lack of such a study from Indian subcontinent prompted us to profile complete core gene mutations in HBeAg -ve CHB patients and to investigate their association with severity of associated liver disease and HBV genotype.
Patients and methods Patients who fulfilled the following criteria were enrolled: (1) HBsAg positive, anti-HBe positive, and HBeAg negative for [6 months; (2) serum HBV DNA [104 copies ml-1. A total of 118 patients were enrolled from January 2002 to August 2008 based on the above criteria. The enrolled patients were also negative for anti-HCV, HIV 1 and 2 antibodies, anti-HDV, ANA, and ASMA. None of them consumed significant alcohol or was exposed
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to antiviral or immunosuppressive therapy. All liver biopsies were evaluated by a liver histopathologist and histological lesions were graded according to the classification proposed by Ishak et al. [14]. A total of 83 patients underwent liver biopsy to determine the grading and staging of the liver disease. Patients were further categorized into three groups based on the clinical, radiological, and/or histological examination. Group I included CHB (n = 52), with no evidence of cirrhosis. Group II included patients with compensated cirrhosis (n = 22). Group III included patients with decompensated cirrhosis (n = 44). Decompensated liver disease was defined by a serum bilirubin level [2.5 times the upper limit of normal, a prothrombin time prolonged by [3 s, a serum albumin level \3 g dl-1, or a history of ascites, variceal hemorrhage, or hepatic encephalopathy. Informed consent was obtained from all patients and the study was approved by ethical committee of GB Pant Hospital, New Delhi. HBV DNA isolation and amplification of precore/core and surface gene HBV DNA was isolated from 100 ll of plasma with the help of standard phenol–chloroform method. Briefly, 100 ll of serum samples were treated with 10 ll of serum lysis buffer (20 mM Tris pH 7.5, 10 mM EDTA, 150 mM NaCl), 5 ll proteinase K (15 mg ml-1) and 5 ll of 20% SDS, and incubated at 37°C for 3 h. Subsequently the supernatant obtained after treatment with tris–saturated phenol (pH 7.9), chloroform, and chloroform–isoamyl alcohol (24:1) was left overnight for DNA precipitation in the presence of 3 M sodium acetate (pH 5.2) and absolute ethanol. After centrifugation, the obtained pellet was washed with 70% ethanol, dried, and dissolved in 30 ll of 19 TE buffer (10 mM Tris 1 mM EDTA). The region encoding complete precore and core was amplified by nested polymerase chain reaction (PCR) using a proofreading Taq DNA polymerase (MBI Fermentas, USA), primers used for amplification of precore and core region were TTT GTA GGA GGC TGT AGGC (1,767–1,788) and AAA GAC AGG AAC AGT AGA AGA ATA (2,493–2,516). The amplification profile used for PCR was 94°C for 3 min followed by 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min, and a final extension for 72°C for 7 min that generated a 749-bp product. Samples detected negative in the first round of amplification were subjected to the second round PCR using internal primers GTA GGC ATA AAT TGG TCT GCG C (1,783–1,804) and CCC ACC TTA TGA GTC CAA G (2,465–2,483). Cycling conditions were same as given in the first round PCR and a positive amplification yielded a 700-bp product. In randomly selected samples, a part of the HBV surface region (nucleotide position from
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425 to 840) was also amplified by nested PCR using the following sets of primers SUR-F1: (401–419) TTCCTC TTCATCCTGCTGCT and SUR-R1: CAAGGTACCC CAACTTCCAA (890–909). The first set of primers amplified a 509-bp fragment. A second nested PCR amplification was performed using internal primers SUR-F2: GTATG TTGCCCGTTTGTCCT (459–478) and SUR-R2: GCCCCA ACGTTTGGTTTTAT (842–861). The PCR reaction conditions for both the rounds were: initial denaturation at 94°C for 5 min followed by 37 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, and final extension at 72°C for 7 min. Ten microliters of nested PCR product (403 bp) were analyzed on 1.5% agarose gel in 19 TBE buffer stained with ethidium bromide. Purified HBV DNA from a recombinant vector was used as the positive control. DNA extracted from serum samples of healthy individuals and commercially available molecular biology grade water served as negative and reagent controls, respectively. Each set of PCR amplifications included HBV positive and negative controls, and all negative controls from the first round amplification were included in the second round PCR amplification. Direct DNA sequencing of PCR products Amplicons were purified using the Qiaquick PCR Purification kit (QIAGEN, Valencia, CA). Purified 700 bp fragments of core and 403 bp of surface region were sequenced with internal primers in an automated DNA sequencer, ABI prism 310. Briefly, 200–250 ng of purified template in a 5-ll volume were mixed with 4-ll sequencing reaction mixture supplied by Perkin Elmer and 1 ll each of forward and reverse primer (3 pmol ll-1) in two different sets of reactions. Sequencing reaction was performed under cyclic conditions of 96°C for 30 s, 55°C for 30 s, and 60°C for 4 min. After completion of 30 cycles, the sequencing reaction product was precipitated by adding 90 ll of H2O, 10 ll of 3 M NaOAc (pH 4.6), and 250 ll of absolute alcohol at room temperature for 10 min, and centrifuged at 12,000 rpm for 15 min. Subsequently, 70% alcohol was added to the pellet and centrifuged at the same speed for 10 min and at 37°C, the dried pellet was dissolved in 10 ll of gel-loading dye (formamide and dextran blue). Samples were denatured for 5 min at 95°C and then quenched on ice for 5 min and loaded in an automated DNA sequencer 310 (ABI Prism Perkin Elmer, Foster City, USA). Determination of HBV genotypes HBV genotypes were determined based on the multiplex genotyping on 118 HBV DNA isolates as described previously [15], genotypes were confirmed based on the precore and core region sequences. All sequences which
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encoded genotype A had two codon insertions corresponding to amino acid (aa) 153 and 154 in the core gene. In genotype C, isolates at nucleotide position 1858C and 1858T were checked. Amino acid at position 27 of core gene was also checked for the presence of isoleucine [16, 17]. In addition, genotypes were reconfirmed on the basis of 403 bp nucleotide region of the surface gene from 25 randomly selected isolates, 13 from genotype D, 8 from genotype A, and 4 from genotype C. Phylogenetic trees were constructed by Neighbor Joining (NJ) method. To confirm the reliability of the pairwise comparison and phylogenetic analysis of the trees, bootstrap resampling and reconstruction was carried out 1,000 times. Phylogenetic analysis was done using MEGA software version 7. Analysis of sequences for detection of mutations Obtained sequences were translated into aa sequences using expasy translational software. Precore and core regions were analyzed at aa level for the occurrence of mutations. Phenylalanine residue at position 17 in precore region, which is characteristic of genotype A1, was not considered as a mutation. All aa residues of core region were analyzed, that is, total aa 183 residues in genotypes D and C and aa 185 in genotype A were grouped based on the immunodominant epitopes of T helper, CTL, and B cell. Regions considered for analysis of mutations in B-cell epitopes were aa 74–89, aa 130–138, and aa 107–118; T helper cell epitopes: aa 1–20, aa 50–69, and aa 117–131; HLA-restricted cytotoxic T-cell epitopes: aa 18–27 and aa 141–151. Cysteine residues important for core protein assembly: aa 48, 61, 107, and 183; and arginine-rich C-terminal region important for pregenomic RNA encapsidation (aa 150–157) and HBV RNA– DNA binding domain (aa 150–177) were analyzed. Mutations were determined based on the respective genotypes obtained from the genebank. Following accession numbers representative of each genotype were used. Genotype A: AY233274, AY934772, AF297621, M57663, and AY233290; genotype C: M38636, D23680, D50519, AB033556, and X01587; and genotype D: AY945307, DQ315780, M32138, Z35716, X02496, and X72702. HBV DNA quantitation HBV DNA was quantified by a commercially available signal amplification hybrid capture assay (Ultra sensitive kit, Digene Hybrid Capture assay, Digene Diagnostics, Beltsville, MD, USA) with a lower limit of detection of 4,700 copies ml-1. Statistical analysis Continuous variables were presented as mean ± SD or median (range) and categorical variables as frequency and
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percentage. The unpaired Student’s t test was applied for comparisons of normally distributed variables and Chisquare test was used for nominal categorical variables. The statistical significance of inter-group differences, for nonnormal distributed data, was evaluated by means of Mann– Whitney tests. A significance level of 0.05 was used.
Results Demographic profile of the patients The mean age of the patients was 34.1 ± 14 years, majority of them were male (M: 89; F: 29). The distribution of age, sex, serum alanine amino transferase levels, serum viral load, histological activity index (HAI), and fibrosis score of each group of patients is shown in Table 1. When demographic data were analyzed based on the presence and absence of precore stop codon mutation, we found patients harboring precore stop codon mutation (A1896) to have higher HAI compared with patients without precore stop codon (Table 2).
position of nonclassical precore region mutations. In core region 80 (44%) aa sites were mutated, of which 31/80 (38.75%) were spanning outside the immunodominant region. Forty-nine (61.25%) sites were mutated in immunodominant regions, distribution of mutations based on each epitopic regions was as follows: 20/49 (40.8%) of sites were mutated in T helper region, 12/49 (24.4%) in CTL region, and 17/49 (34.7%) in B epitopic region. In T helper region, frequently occurring mutations were detected at positions cT12S 20/118 (17.9%), cE64D 11/118 (9.3%), cT67P/N 12/118 (10.1%), cS69G 8/118 (6.7%), and cM66I 7/118 (5.9%). Common mutations detected in CTL region were cS21T 8/118 (6.7%), cF140S/P 6/118 (5%), cT142P 7/118 (5.9%), and cT147C/P 6/118 (5%), whereas in the B-cell region common mutated sites detected were at positions cV74G/A 13/118 (11%), cE77D 14/118 (11.8%), cA80I/T/V 11/118 (9.3%), cD83E 7/118 (5.4%), and cE113D 6/118 (5%). All mutations detected in core T helper, CTL, and B-cell immunodominant region are given in Fig. 2. Coexistence of precore stop codon (A1896) and core mutations
Prevalence of HBV genotypes Three genotypes were prevalent in the study population, the most prevalent genotype was genotype D: 96/118 (81.3%) (D1: 16, D2: 55, and D5: 25) followed by genotype A1: 15/118 (12.7%) and genotype C: 7/118 (5.9%) (C1: 5 and unidentified subgenotype C: 2). Frequency and profile of precore and core region mutations One-fourth of HBeAg -ve CHB patients were infected with classical precore stop codon mutation 30/118 (25%), whereas nonclassical precore region mutations were detected in 9/118 (7.6%) patients. Figure 1 describes the
Based on the stop codon mutation in the precore region (A1896), patients were categorized into two categories: category I included patients with wild-type aa at position G1896 (n = 88) and category II with precore stop codon at A1896 (n = 30). Mutations in the T helper, CTL, B cell epitope and core terminal region were compared between both the groups. Majority of the patients with precore stop codon mutation (A1896) were found to harbor coexisting core mutations compared with the wild type (G1896) [45/ 88 (51%) vs. 24/30 (80%), p \ 0.05]. Significant differences were noted in the T helper and CTL region, that is, at position cT12S [13/30 (43%) vs. 7/88 (8%), p \ 0.001], cS21T [5/30 (16%) vs. 3/88 (3.4%) p \ 0.026], and cE77D [10/30 (33.3%) vs. 4/88 (4.5%) p \ 0.002]. However,
Table 1 Demographic profile of patients Group I (n = 52)
Group II (n = 22)
Group III (n = 44)
p value
Age (years)
32.9 ± 14.5
36.1 ± 16.2
36.6 ± 15.1
NS
Gender (M:F)
33:19
19:3
37:7
NS
-1
Bilirubin (mg dl ) mean (range)
1 (0.2–1.6)
0.8 (0.2–1.8)
2 (1.2–16.8)
\0.028
Albumin (g dl-1) mean ± SD
3.9 ± 0.28
3.5 ± 0.53
2.7 ± 0.59
\0.05
ALT (IU l-1) median (range)
51.5 (25–81)
41.5 (22–70)
83.5 (22–345)
\0.05
HAI mean ± SD
4.4 ± 2.1
5.4 ± 2.1
5.9 ± 2.2
\0.05
Fibrosis mean ± SD
2.1 ± 0.7
3.2 ± 0.42
3.6 ± 0.5
\0.02
DNA log copies mean ± SD Biopsy available
6.4 ± 1.3 33
6.6 ± 1.4 22
6.2 ± 1.0 28
NS
Significant values reported if p \ 0.05, statistical analysis was performed with Mann–Whitney or Fisher’s exact test
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Table 2 Demographic and clinical profile of patients with and without precore mutant forms Precore mutant (A1896) (n = 30)
Without precore mutant (G1896) (n = 88)
p value
Mean age (years)
37.0 ± 13
34.2 ± 15.5
NS
Gender (M:F)
24:6
65:23
NS
S bilirubin (mg dl-1) mean ± SD
1.4 ± 1.0
1.2 (0.6–16.8)
NS
-1
ALT (IU l ) median (range)
56 (22–345)
55 (22–345)
NS
Albumin (g dl-1) mean ± SD
3.8 ± 0.42
3.9 ± 0.5
NS
DNA log copies mean ± SD
6.3 ± 1.2
6.4 ± 1.2
NS
HAI mean ± SD
8.0 ± 2.1
5.9 ± 1.2
\0.05
Fibrosis mean ± SD
3.2 ± 1.0
2.7 ± 1.1
NS
Significant values reported if p \ 0.05, Statistical analysis was performed with Mann–Whitney test or Fisher’s exact test Profile of non-classical precore region mutations amino acid Category Genotype Pt ID ID-2 CHB D ID-8 CHB D ID-47 CHB D ID-48 CHB D ID-50 CHB D Compen ID-67 D Compen ID-72 D ID-80# Decomp A Decomp ID-82 D
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 M Q L F H L C L I I S C S/T C P T V/F Q A S K L C L G W L W F Y V P L F F T F St St L
29 G D
D
Fig. 1 Profile of non-classical precore region mutations. CHB Chronic hepatitis B, Compen compensated cirrhosis, and Decomp decompensated patients. Precore region mutations are given in black box, whereas one patient (#ID80) with Genotype A 1 was harboring a
stop codon mutation at precore position 21. Amino acid characteristics of genotype A are given in white box. Pt ID Patient identification number
when statistical analysis was carried out by comparing nonclassical precore region mutations and core immunodominant region mutations, no significance was found.
Genotype D
Commonly occurring core mutations with respect to genotype Genotype A Several core mutations were detected in HBV genotype A infected patients. The mutational profile of T helper region in genotype A patients was cV13G/A [4/15 (27%)], cE14D [1/15 (7%)], and cP50H/S [4/15 (27%)]. In B-cell region a single mutation cL84Q [1/15 (7%)] was present. Two mutations outside the immunodominant region were also detected, cY38F 1/15 (7%) and cP45S 2/15 (13%). Genotype C In genotype C infected patients, none of the T helper and CTL region mutations was found. However, two mutations were detected in B-cell epitope region at positions cV85I 2/7 (28.5%) and cA137G 1/7 (14%) and were present in two and one patient, respectively. Similar to the above finding, a single mutation outside the immunodominant was detected at position cW71G 1/7 (14%) in one patient.
In genotype D infected patients T helper region mutations were detected at position cT12S [21/96 (21.8%)] and A69G [8/96 (8.3%)]. In B-cell region, mutations were detected at positions cE77D [11/96 (11.4%)] and cE113D [6/96 (6.2%)]. The CTL region mutation cS21T [8/96 (8.3%)] was exclusively detected in genotype D infected patients. Association of immune escape mutations with severity of liver disease Mutations clustering regions (MCRs) as well as point mutations occurring in CTL, T helper, and B-cell regions were compared for their association with severity of liver disease. Mutations clustering in CTL region 18–27 were significantly more common in decompensated cirrhotics compared with CHB patients [9/44 (20%) vs. 4/74 (9%), p = 0.016] as well as mutations clustering in T helper regions 1–20 and 50–69 were also significantly more common in decompensated patients as compared with compensated patients [13/44 (29%) vs. 6/74 (8%), p \ 0.05] and [13/44 (29%) vs. 9/74 (12%), p \ 0.02]. Table 3 describes all three regions, however, no significant association between B-cell clustering region with severe liver disease patients was found. When comparison was
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Fig. 2 Prevalence and profile core immunodominant mutation. The figure describes the frequency and profile of core immune epitope mutations. White horizontal bar with black dots represents the number
of mutations detected in each immunodominant region; size of the dot depicts the frequency. More than one type of substitution detected is given by / sign. CTL Cytotoxic T lymphocytes
done by considering single point mutations with severe liver disease patients, mutations at five positions were found to be significantly associated with decompensated liver disease patients, T helper point mutations were cT12S [12/44 (27%) vs. 8/74 (10%), p \ 0.05] and cT67P/N [9/44 (20%) vs. 3/74 (4%), p \ 0.001]. B-cell region mutation was cE113D [5/44 (11.3%) vs. 1/74 (1.35%), p \ 0.05] and CTL region mutation was cS21T [7/44 (16%) vs. 1/74 (1.35%), p \ 0.05]. cP130T/Q mutation spanning in both B-cell epitope as well as CTL region was exclusively detected in four decompensated cirrhotic patients (p \ 0.001). Table 4 describes the
association of various individual mutations with severity of liver disease patients.
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Profile of naturally occurring core carboxy-terminal mutations Core carboxy-terminal region mutations were detected in 11/118 (9.3%) patients. The distribution of core carboxyterminal region aa substitutions was comparable among the disease category group [p = nonsignificant (NS)]. Two patients were infected with genotype A1 strain, and were harboring cR151Stop and cT162E mutation each.
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Table 3 Association of mutated immunodominant region with severity of liver disease Immunodominant region (amino acid)
Group I ? II (n = 74) (%)
Group III (n = 44) (%)
p value
CTL (18–27)
4 (9)
9 (20)
\0.017
T helper (1–20)
6 (8)
13 (29)
\0.05
T helper (50–69)
9 (12)
13 (29)
\0.02
14 (19)
12 (27)
NS
B cell (74–89)
Association of mutated immunodominant region with severe decompensated patients is described in this table. Statistical analysis was performed with Chi-square test Table 4 Association of individual core immunodominant mutations with severity of liver disease Codon
Total n (118) (%)
Group I (52) (%)
Core 12
20 (16.9)
3 (5.7)
Core 21
8 (6.7)
Core 64
11 (9.3)
Core 67
12 (10)
Group II (22) (%)
Group III (44) (%)
Mutation
p value
4 (18)
13 (29.5)
T ?S
\0.05*
1 (4.5)
7 (16)
S? T
\0.05*
3 (5.7)
4 (9)
6 (13.6)
E ?D
NS
2 (3.8)
1 (4.5)
9 (20)
T ?N/P
\0.01*
–
Core 69
8 (6.7)
6 (11.5)
2 (4.5)
A? S
NS
Core 77
14 (11.8)
7 (13.4)
3 (13.6)
4 (9)
E ?D/Q
NS
Core 80
11 (9.3)
4 (7.6)
3 (13.6)
4 (9)
A? I/V/T
NS
Core 113
6 (5)
1 (3.8)
–
5 (11.3)
E ?D
\0.05*
Core 130
4 (2.5)
–
4 (9)
P? Q/T
\0.05*
–
–
Data were analyzed using Chi-square test or Fisher’s exact test –, represents no mutation detected * Comparisons were made between Group I ? II versus Group III
Interestingly, three patients were harboring important SPRRR motif mutations. One patient ID45 with precore stop codon mutation was harboring triple mutation in core carboxy-terminal region; these were at position cG153R, cR154Q, and cS155V. Two additional patients, patient ID56 and ID63 were harboring a mutation at position 169 substituting glutamine to lysine and other patient was harboring mutation at c176 substituting serine to threonine. Mutational profile of genotype D patients harboring core carboxy-terminal mutations is given in Fig. 3. Of the nine patients with genotype D, three (33.33%) were harboring precore stop codon mutation (A1896). In none of the patients core internal deletion was detected. Profile of naturally occurring of core carboxy-terminal truncations Core carboxy-terminal truncations were present in 14/118 (12%) patients. Of the 14, 3 (21%) and 11/14 (78.5%) patients were harboring genotype A and genotype D, respectively. Two patients with genotype D infection were harboring coexisting precore stop codon mutation (A1896). Two types of truncations, smaller (3–7 bp) and longer (12–29 bp) truncations were detected in seven patients each. SPRRR-III motif was disrupted/truncated in two patients (ID29, 117), SPRRR-II and SPRRR-III motifs
were disrupted/truncated in three patients (Pt ID20, 31, 51), and SPRRRI-II and III motifs were disrupted/truncated in two patients (ID73 and ID25). Interestingly, one patient with SPRRRI-III disrupted motif (ID73) was harboring coexisting precore stop codon mutation (A1896). Whereas smaller truncations were spanning downstream to the SPRRR-III motif, they were detected in seven (50%) patients. Core carboxy-terminal truncations were comparable among the disease category group (p = NS). Profile of all 14 truncations is detailed in Fig. 4. Association of core mutations with HBV DNA levels HBV DNA levels were significantly high in patients infected with mutations at positions cP130 and cS21T compared with the wild type (7.1 9 1011 ± 1.3 9 102 vs. 5.7 9 106 ± 1.1 9 102; p \ 0.002) and (3.7 9 109 ± 1.2 9 102 vs. 4.9 9 106 ± 1.1 9 102; p \ 0.005). However, none of the mutations was significantly associated with ALT levels.
Discussion The salient features of the present study in HBeAg -ve CHB are: detection of genotype C1, identification of
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714 Fig. 3 Profile of core carboxyterminal mutations. CHB Chronic hepatitis B, Compen compensated cirrhosis and Decomp decompensated patients. This figure describes core carboxy-terminal region mutations identified in Genotype D (upper row) and A (lower row) infected patients. Core carboxy-terminal mutations were considered downstream to amino acid 150 of core region
Hepatol Int (2012) 6:707–717 Profile of Core Carboxy-terminal mutations amino Patient ID ID4 ID22 ID45 ID49 ID56 ID63 ID70 ID74 ID99
acid Category CHB CHB CHB CHB Compen Compen Compen Compen Decomp
amino Patient ID ID71 ID136
151 153 154 155 160 162 169 176 178 179 180 182 acid Category nt 1896 R G R S T T Q S S R E Q Compen Wild • • • • • E • • • • • • Decomp Wild St • • • • • • • • • • •
nonclassical precore region mutations in *8% of patients and association of immune escape mutations with severe liver disease patients. In addition, the data shows coexistence of precore stop codon mutation and immune escape mutations, and the presence of core terminal truncations and mutations in such patients. Several reports suggest that occurrence of core mutations is high in HBeAg seroconvertors with immune clearance phase compared with HBeAg positive patients [18, 19]. Very few studies focussed on core immune escape mutations in reactivating severe liver disease patients, and secondly, none of the studies simultaneously examined mutations of precore region in such patients [20]. This is one of the comprehensive studies reporting the prevalence of classical precore stop codon mutation as well as nonclassical precore mutations coexisting with core immune escape mutation in HBeAg -ve CHB patients. In this study, we found that the overall rate of core gene substitutions was significantly higher in decompensated patients compared with CHB patients; the substitution hotspots identified were at positions cT12S, cS21T, cT67P/ N, cE113D, and cP130T. Identification of cT12S, cS21T, and cE113D mutations in the previous reports in 65% of occult hepatocellular carcinoma as well as progressing hepatocellular carcinoma patients [21, 22] suggests their pathogenetic role. The aa position cT12S is in close proximity with the HLA A2 CTL restricted epitope and selection of cT12S mutation could lead to immune escape of the virus. Contrary to our findings, an in vitro study suggested that deletion at similar position blocks the virion formation [23]. We speculate that this could retain the viral machinery within the hepatocytes, possibly by disruption of the polyadenylation signal, generating longer transcripts and leading to severe liver disease. Interestingly, mutations at position cT12S and cS21T were coexisting with precore stop (A1896) codon mutation, the interplay between precore stop codon mutation and cT12S/cS21T mutations needs to be further studied. A second mutation, mutation at
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nt 1896 Wild Mutated Mutated Wild Wild Wild Wild Wild Mutated
151 153 154 155 160 162 169 176 178 179 180 182 R G R S T T Q S S R E Q • • K • • • • • • • • • C R • • • • • • • L M • • R Q V • • • • • • • • • • • • S • • • • • • • • • • • • • K • • • • • • • • • • • • T • • • • • • • • • • • • • • I M • • A • • • • • • • • • • C • T • • • • • • • •
position cT67 in T helper region, have been detected in patients with fulminant and hepatocellular carcinoma [21, 24]. A recent study by Homs et al. [25] also reported the presence of cE64 and cT67 mutations in patients treated with interferon. It is quite possible that mutation in this region could activate dendritic cells and modulate the immune response. Coexisting mutations at these two sites also suggest their mutual cooperativity and strict conservation in one single protein. Identification of cP130T mutation in four decompensated patients was surprising, the DNA levels were found to be significantly high in these patients. Similar to our findings a previous study reported the occurrence of P130T mutation in patients experiencing ALT exacerbations [26]. Codon 130 spans in both T- and B-cell epitope and it is exposed on the surface of mature HBeAg and HBcAg, one of the reasons for such nature could be enrichment of fulllength transcripts. Two mutations cS49T and cE77D detected in 3 and 14 patients, respectively, in the present study were significantly associated with precore stop codon mutation. Similar mutations have earlier been identified in three cirrhotic and portal hypertension patients from India [27]. One of the mutation ‘‘cE77D’’ have been detected in HBV genotype A patients from late HBeAg-positive phase, displaying extremely low HBe antigenicity [28]. Glutamine residues at positions 77 and 113 of the core gene are responsible for peptide anchoring to the B pocket of HLA B-4001 [29]. A recent study from New Zealand suggested that immune escape mutations occurring at positions cE77 and cE113 could decrease peptide binding and prevent presentation to CD8 T cells. However, their role in causing severe liver disease is yet to be investigated [30]. There were four genotype D infected patients in whom important SPRRR motif core terminal region mutations were found. In the first patient (ID45), a coexisting triple mutation at positions cG153R, cR154Q, and cS155V was
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Profile of Core Carboxy tuncations 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 Pt ID
Group
ID20
CHB
ID21
CHB
ID29
CHB
ID31
CHB
ID51
CHB
ID73*
Compen
ID94
Decomp
H
ID115*
Decomp
H
ID116
Decomp
ID117
Decomp
ID118
Decomp
Pt ID
Group
R
ID25
CHB
T
ID76
Decomp
ID79
Decomp
R
S
P
R
R
R
T
P
S
P
R
R
R
R
S
P
S
P
R
R
R
R
S
Q
S
R
E
S
Q
C
L S
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I
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159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 R
R
T
P
S
P
R
R
R
R
S
P
S
P
R
R
R
R
S
Q
S
R
E
S
Q
C
H F
Fig. 4 Profile of core carboxy-truncations. CHB Chronic hepatitis B, Compen compensated cirrhosis, and Decomp decompensated patients. The above row shows patients infected with Genotype D strain and lower row shows patients infected with Genotype A strain. Patient ID
with esterik sign were mutated at precore position A1896. Truncated region is represented by sign approximately. Pt ID Patient identification number
found; in the second patient (ID56) cQ169K was detected, the third patient (ID63) was harboring cS176T mutant, and in fourth patient (ID99) double mutants cG153C and cS155T were detected. Interestingly, two of these patients (ID45, ID99) were harboring coexisting precore stop codon mutation (A1896) and patient (ID99) had decompensated liver. In addition, one patient with genotype A1 was harboring unique mutation, leading to stop codon mutation at position cR152Stop. Changes in core SPRRR motif at positions 155 and 176 could alter the phosphorylation status of core protein and may modulate the severity of liver disease. Two of the studies suggested that mutations in core carboxy terminal could change the distribution of HBcAg in hepatocytes, leading to variable packaging of pregenomic RNA within the nuleocapsids [31, 32]. In an earlier study, we had shown that nearly one-third of the HBeAg -ve CHB patients were infected with exclusive basal core promoter regulatory TA1-3 region mutations. However, no association of these mutations and genotypes as well with severity of liver disease was found [33]. In the present study, we further found that 7.6% HBeAg -ve reactivating patients do harbor nonclassical precore region mutations. However, we could not detect any significant association of nonclassical precore region mutations with decompensated severe liver disease patients. This is the first study reporting the presence of nonclassical precore region mutations, including precore gene translation initiation codon mutation in HBeAg -ve CHB patients in India. Interestingly, two earlier studies reported selection of precore gene translation initiation codon mutations in HBeAg -ve reactivating phase and
support our findings [34, 35]. It is quite possible that selection of this variant is one of the strategies of the virus to cope with the vigorous host immune system. Recently, genotype C has been reported from occult HBV-infected patients and blood donors from Eastern and Southern parts of India [36, 37]. Similarly, we reported the prevalence of genotype C in North India in asymptomatic patients with raised ALT [15]. However, this is the first study from Northern India reporting the prevalence of HBV subgenotype C1 in 4.2% of HBeAg -ve CHB patients with severe liver disease. Phylogenetic analysis of core and surface gene revealed that five of genotype C sequences clustered with South East Asia (C1) and not with East Asian origin (C2) [38]. Of the seven patients with genotype C infection, two were of Tibetan origin and remaining five were from urban population and had international travel history. Based on the partial gene sequence, we identified C/D recombinant in two of these Tibetan patients (data not shown). However, we classified them as unidentified subgenotype C. These two strains could not firmly be classified as C/D recombinant until whole genome sequencing and analysis are done. Such an approach is underway for characterizing them. A recent report from Afghanistan revealed the presence of genotype C HBV infection in [30% of their population. It is quite possible that this virus would have transmitted via human trafficking across and within the borders [39]. The spontaneous coevolution of the virus with rapidly migrating humans could be one of the reasons of the changing molecular epidemiology. In conclusion, the present study reports the characterization of immune escape mutations in HBeAg -ve CHB patients with and without precore stop codon mutation. The
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results indicate that patients with severe liver disease should be tested for HBV genotypes and mutational profile, as more diverse viral population may be selected in severe liver disease patients in future and there is an urgent need for individualized immunomodulatory therapies. Acknowledgements Authors are thankful to Indian Council of Medical Research for providing funds through Advanced Center for Liver Diseases project. Authors thank Dr Scott Bowden (VIDRL) for critically reviewing the manuscript.
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ORIGINAL ARTICLE
Naturally occurring core immune-escape and carboxy-terminal mutations\truncations in patients with e antigen negative chronic hepatitis B Ranjit Chauhan • Shiv K. Sarin • Manoj Kumar Jayashree Bhattacharjee
•
Received: 16 May 2011 / Accepted: 1 October 2011 / Published online: 2 December 2011 Ó Asian Pacific Association for the Study of the Liver 2011
Abstract Introduction Hepatocellular injury is often progressive in patients with hepatitis B e antigen negative chronic hepatitis B (HBeAg -ve CHB). There is scant data on association of core mutations occurring in patients with HBeAg -ve CHB with severity of liver disease. Materials and methods Hundred and eighteen patients with chronic infection who were HBeAg negative, antiHBe, and HBV DNA positive were enrolled. Precore and core regions were amplified, sequenced, and analyzed for precore, T helper, cytotoxic T lymphocytes (CTLs), B-cell epitope, and core carboxy-terminal region mutations. Results Majority of patients were infected with HBV genotype D: 96 (81%) [D1: 16, D2: 55 and D5: 25] followed by genotype A1: 15 (13%) and genotype C: 7 (6%) [C1: 5 and unidentified subgenotype C: 2]. Classical (A1896) as well as nonclassical precore region mutations were detected in 30 (25%) and in 9 (7.6%) patients, respectively. Core immune escape, core carboxy-terminal mutations and truncations were detected in 61 (52%), 11 (9.3%), and 14 (12%) patients, respectively. Three core immune escape mutations were significantly higher in patients with coexisting precore stop codon compared with patients without precore stop codon mutation, cT12S (43 vs. 8%, p \ 0.001), cS21T (16 vs. 3.4%, p \ 0.026), and cE77D (30 vs. 4.5%, p \ 0.002). When frequency of core R. Chauhan S. K. Sarin (&) M. Kumar Department of Gastroenterology and Advanced Centre for Liver Diseases, G.B. Pant Hospital, Room No. 201, New Delhi 110002, India e-mail: [email protected] R. Chauhan J. Bhattacharjee Department of Biochemistry, Lady Hardinge Medical College, New Delhi 110002, India
immune escape mutations was compared among CHB and decompensated patients, and cT12S: (27 vs. 10%, p \ 0.05), cS21T (16 vs. 1.35%, p \ 0.01), cT67P/N: (20 vs. 4%, p \ 0.001), cE113D (11.37 vs. 1.35%, p \ 0.05), and cP130T/Q (7 vs. 0%, p \ 0.001) mutations were found to be significantly higher in decompensated patients. Conclusion Core immune-escape mutations cT12S, cS21T, cT67P, cE113D, and cP130T/Q are significantly higher in decompensated liver disease patients and could influence the severity of liver disease in HBeAg -ve CHB patients. Keywords HBV Core Mutation Genotype HBeAg Immune-escape Carboxy-terminal
Introduction Hepatitis B virus (HBV) infection affects 400 million people globally. It is estimated that between 235,000 and 328,000 people die annually due to liver cirrhosis and hepatocellular carcinoma, respectively [1]. There are four phases in the natural history of chronic HBV infection. First phase is the immune-tolerant phase defined by hepatitis B e antigen (HBeAg) positive, with normal liver enzyme levels, and high serum HBV DNA concentrations. The second phase is the immune-clearance phase defined by HBeAg positive, abnormal liver enzymes, and lower HBV DNA concentrations. The third phase is defined by seroconversion from HBeAg to anti-HBe and has normal hepatic enzyme levels and low HBV DNA levels. In the fourth phase, hepatic enzymes are fluctuating and HBV DNA is high; this phase is also termed as HBeAg negative (-ve) chronic hepatitis B (CHB) or reactivation phase [2]. Particularly in areas where HBV transmission is
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predominantly perinatal, despite seroconversion, HBV continues to proliferate and could cause cirrhosis and liver cancer [3, 4]. There is an alarming rise in HBeAg -ve CHB worldwide [5], such patients have cirrhosis-related complications and 15–20% of patients develop liver decompensation within 5 years if left untreated [6, 7]. The core gene encodes two polypeptides, precore and core. Initiation of translation at the first start codon (AUG) results in a 25-kDa precore protein that is secreted as HBeAg. Initiation of translation at the second AUG leads to the synthesis of core protein that assembles to form 27 nm particles that comprise virion nucleocapsid (HBcAg). Although HBeAg and HBcAg are serologically distinct, these antigens are cross-reactive at the level of T-cell recognition because they are colinear throughout most of their primary sequence. The HBcAg possesses unique immunologic features: (1) HBcAg can function as both a T-cell-independent and a T-cell-dependent antigen; (2) it is *1,000-fold more immunogenic than HBeAg; (3) it preferentially primes Th1 cells, whereas, HBeAg preferentially primes Th2 cells; (4) HBcAg-specific Th cells mediate anti-envelope as well as anti-HBc antibody production; and (5) HBcAg is an effective carrier platform for heterologous epitopes [8]. Core carboxy terminal region varies and is heterogeneous [9]. In one of our recent studies, we identified impaired expression of TAP and LMP1 leading to defective antigen processing and presentation [10]. However, it is vital to know the profile of core mutations predominating during reactivation phase, which would add to the understanding of the interplay between virus and the host immune system. Viruses have developed a number of mechanisms to combat the host immune system and one of them is by escaping the immune system through mutations. Recent studies have identified hot-spots of mutations and deletions in the core gene of HBV in patients with severe liver disease [11, 12]. Core gene variations have also been related to ethnic background and HBV genotypes [13]. Lack of such a study from Indian subcontinent prompted us to profile complete core gene mutations in HBeAg -ve CHB patients and to investigate their association with severity of associated liver disease and HBV genotype.
Patients and methods Patients who fulfilled the following criteria were enrolled: (1) HBsAg positive, anti-HBe positive, and HBeAg negative for [6 months; (2) serum HBV DNA [104 copies ml-1. A total of 118 patients were enrolled from January 2002 to August 2008 based on the above criteria. The enrolled patients were also negative for anti-HCV, HIV 1 and 2 antibodies, anti-HDV, ANA, and ASMA. None of them consumed significant alcohol or was exposed
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to antiviral or immunosuppressive therapy. All liver biopsies were evaluated by a liver histopathologist and histological lesions were graded according to the classification proposed by Ishak et al. [14]. A total of 83 patients underwent liver biopsy to determine the grading and staging of the liver disease. Patients were further categorized into three groups based on the clinical, radiological, and/or histological examination. Group I included CHB (n = 52), with no evidence of cirrhosis. Group II included patients with compensated cirrhosis (n = 22). Group III included patients with decompensated cirrhosis (n = 44). Decompensated liver disease was defined by a serum bilirubin level [2.5 times the upper limit of normal, a prothrombin time prolonged by [3 s, a serum albumin level \3 g dl-1, or a history of ascites, variceal hemorrhage, or hepatic encephalopathy. Informed consent was obtained from all patients and the study was approved by ethical committee of GB Pant Hospital, New Delhi. HBV DNA isolation and amplification of precore/core and surface gene HBV DNA was isolated from 100 ll of plasma with the help of standard phenol–chloroform method. Briefly, 100 ll of serum samples were treated with 10 ll of serum lysis buffer (20 mM Tris pH 7.5, 10 mM EDTA, 150 mM NaCl), 5 ll proteinase K (15 mg ml-1) and 5 ll of 20% SDS, and incubated at 37°C for 3 h. Subsequently the supernatant obtained after treatment with tris–saturated phenol (pH 7.9), chloroform, and chloroform–isoamyl alcohol (24:1) was left overnight for DNA precipitation in the presence of 3 M sodium acetate (pH 5.2) and absolute ethanol. After centrifugation, the obtained pellet was washed with 70% ethanol, dried, and dissolved in 30 ll of 19 TE buffer (10 mM Tris 1 mM EDTA). The region encoding complete precore and core was amplified by nested polymerase chain reaction (PCR) using a proofreading Taq DNA polymerase (MBI Fermentas, USA), primers used for amplification of precore and core region were TTT GTA GGA GGC TGT AGGC (1,767–1,788) and AAA GAC AGG AAC AGT AGA AGA ATA (2,493–2,516). The amplification profile used for PCR was 94°C for 3 min followed by 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min, and a final extension for 72°C for 7 min that generated a 749-bp product. Samples detected negative in the first round of amplification were subjected to the second round PCR using internal primers GTA GGC ATA AAT TGG TCT GCG C (1,783–1,804) and CCC ACC TTA TGA GTC CAA G (2,465–2,483). Cycling conditions were same as given in the first round PCR and a positive amplification yielded a 700-bp product. In randomly selected samples, a part of the HBV surface region (nucleotide position from
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425 to 840) was also amplified by nested PCR using the following sets of primers SUR-F1: (401–419) TTCCTC TTCATCCTGCTGCT and SUR-R1: CAAGGTACCC CAACTTCCAA (890–909). The first set of primers amplified a 509-bp fragment. A second nested PCR amplification was performed using internal primers SUR-F2: GTATG TTGCCCGTTTGTCCT (459–478) and SUR-R2: GCCCCA ACGTTTGGTTTTAT (842–861). The PCR reaction conditions for both the rounds were: initial denaturation at 94°C for 5 min followed by 37 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2 min, and final extension at 72°C for 7 min. Ten microliters of nested PCR product (403 bp) were analyzed on 1.5% agarose gel in 19 TBE buffer stained with ethidium bromide. Purified HBV DNA from a recombinant vector was used as the positive control. DNA extracted from serum samples of healthy individuals and commercially available molecular biology grade water served as negative and reagent controls, respectively. Each set of PCR amplifications included HBV positive and negative controls, and all negative controls from the first round amplification were included in the second round PCR amplification. Direct DNA sequencing of PCR products Amplicons were purified using the Qiaquick PCR Purification kit (QIAGEN, Valencia, CA). Purified 700 bp fragments of core and 403 bp of surface region were sequenced with internal primers in an automated DNA sequencer, ABI prism 310. Briefly, 200–250 ng of purified template in a 5-ll volume were mixed with 4-ll sequencing reaction mixture supplied by Perkin Elmer and 1 ll each of forward and reverse primer (3 pmol ll-1) in two different sets of reactions. Sequencing reaction was performed under cyclic conditions of 96°C for 30 s, 55°C for 30 s, and 60°C for 4 min. After completion of 30 cycles, the sequencing reaction product was precipitated by adding 90 ll of H2O, 10 ll of 3 M NaOAc (pH 4.6), and 250 ll of absolute alcohol at room temperature for 10 min, and centrifuged at 12,000 rpm for 15 min. Subsequently, 70% alcohol was added to the pellet and centrifuged at the same speed for 10 min and at 37°C, the dried pellet was dissolved in 10 ll of gel-loading dye (formamide and dextran blue). Samples were denatured for 5 min at 95°C and then quenched on ice for 5 min and loaded in an automated DNA sequencer 310 (ABI Prism Perkin Elmer, Foster City, USA). Determination of HBV genotypes HBV genotypes were determined based on the multiplex genotyping on 118 HBV DNA isolates as described previously [15], genotypes were confirmed based on the precore and core region sequences. All sequences which
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encoded genotype A had two codon insertions corresponding to amino acid (aa) 153 and 154 in the core gene. In genotype C, isolates at nucleotide position 1858C and 1858T were checked. Amino acid at position 27 of core gene was also checked for the presence of isoleucine [16, 17]. In addition, genotypes were reconfirmed on the basis of 403 bp nucleotide region of the surface gene from 25 randomly selected isolates, 13 from genotype D, 8 from genotype A, and 4 from genotype C. Phylogenetic trees were constructed by Neighbor Joining (NJ) method. To confirm the reliability of the pairwise comparison and phylogenetic analysis of the trees, bootstrap resampling and reconstruction was carried out 1,000 times. Phylogenetic analysis was done using MEGA software version 7. Analysis of sequences for detection of mutations Obtained sequences were translated into aa sequences using expasy translational software. Precore and core regions were analyzed at aa level for the occurrence of mutations. Phenylalanine residue at position 17 in precore region, which is characteristic of genotype A1, was not considered as a mutation. All aa residues of core region were analyzed, that is, total aa 183 residues in genotypes D and C and aa 185 in genotype A were grouped based on the immunodominant epitopes of T helper, CTL, and B cell. Regions considered for analysis of mutations in B-cell epitopes were aa 74–89, aa 130–138, and aa 107–118; T helper cell epitopes: aa 1–20, aa 50–69, and aa 117–131; HLA-restricted cytotoxic T-cell epitopes: aa 18–27 and aa 141–151. Cysteine residues important for core protein assembly: aa 48, 61, 107, and 183; and arginine-rich C-terminal region important for pregenomic RNA encapsidation (aa 150–157) and HBV RNA– DNA binding domain (aa 150–177) were analyzed. Mutations were determined based on the respective genotypes obtained from the genebank. Following accession numbers representative of each genotype were used. Genotype A: AY233274, AY934772, AF297621, M57663, and AY233290; genotype C: M38636, D23680, D50519, AB033556, and X01587; and genotype D: AY945307, DQ315780, M32138, Z35716, X02496, and X72702. HBV DNA quantitation HBV DNA was quantified by a commercially available signal amplification hybrid capture assay (Ultra sensitive kit, Digene Hybrid Capture assay, Digene Diagnostics, Beltsville, MD, USA) with a lower limit of detection of 4,700 copies ml-1. Statistical analysis Continuous variables were presented as mean ± SD or median (range) and categorical variables as frequency and
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percentage. The unpaired Student’s t test was applied for comparisons of normally distributed variables and Chisquare test was used for nominal categorical variables. The statistical significance of inter-group differences, for nonnormal distributed data, was evaluated by means of Mann– Whitney tests. A significance level of 0.05 was used.
Results Demographic profile of the patients The mean age of the patients was 34.1 ± 14 years, majority of them were male (M: 89; F: 29). The distribution of age, sex, serum alanine amino transferase levels, serum viral load, histological activity index (HAI), and fibrosis score of each group of patients is shown in Table 1. When demographic data were analyzed based on the presence and absence of precore stop codon mutation, we found patients harboring precore stop codon mutation (A1896) to have higher HAI compared with patients without precore stop codon (Table 2).
position of nonclassical precore region mutations. In core region 80 (44%) aa sites were mutated, of which 31/80 (38.75%) were spanning outside the immunodominant region. Forty-nine (61.25%) sites were mutated in immunodominant regions, distribution of mutations based on each epitopic regions was as follows: 20/49 (40.8%) of sites were mutated in T helper region, 12/49 (24.4%) in CTL region, and 17/49 (34.7%) in B epitopic region. In T helper region, frequently occurring mutations were detected at positions cT12S 20/118 (17.9%), cE64D 11/118 (9.3%), cT67P/N 12/118 (10.1%), cS69G 8/118 (6.7%), and cM66I 7/118 (5.9%). Common mutations detected in CTL region were cS21T 8/118 (6.7%), cF140S/P 6/118 (5%), cT142P 7/118 (5.9%), and cT147C/P 6/118 (5%), whereas in the B-cell region common mutated sites detected were at positions cV74G/A 13/118 (11%), cE77D 14/118 (11.8%), cA80I/T/V 11/118 (9.3%), cD83E 7/118 (5.4%), and cE113D 6/118 (5%). All mutations detected in core T helper, CTL, and B-cell immunodominant region are given in Fig. 2. Coexistence of precore stop codon (A1896) and core mutations
Prevalence of HBV genotypes Three genotypes were prevalent in the study population, the most prevalent genotype was genotype D: 96/118 (81.3%) (D1: 16, D2: 55, and D5: 25) followed by genotype A1: 15/118 (12.7%) and genotype C: 7/118 (5.9%) (C1: 5 and unidentified subgenotype C: 2). Frequency and profile of precore and core region mutations One-fourth of HBeAg -ve CHB patients were infected with classical precore stop codon mutation 30/118 (25%), whereas nonclassical precore region mutations were detected in 9/118 (7.6%) patients. Figure 1 describes the
Based on the stop codon mutation in the precore region (A1896), patients were categorized into two categories: category I included patients with wild-type aa at position G1896 (n = 88) and category II with precore stop codon at A1896 (n = 30). Mutations in the T helper, CTL, B cell epitope and core terminal region were compared between both the groups. Majority of the patients with precore stop codon mutation (A1896) were found to harbor coexisting core mutations compared with the wild type (G1896) [45/ 88 (51%) vs. 24/30 (80%), p \ 0.05]. Significant differences were noted in the T helper and CTL region, that is, at position cT12S [13/30 (43%) vs. 7/88 (8%), p \ 0.001], cS21T [5/30 (16%) vs. 3/88 (3.4%) p \ 0.026], and cE77D [10/30 (33.3%) vs. 4/88 (4.5%) p \ 0.002]. However,
Table 1 Demographic profile of patients Group I (n = 52)
Group II (n = 22)
Group III (n = 44)
p value
Age (years)
32.9 ± 14.5
36.1 ± 16.2
36.6 ± 15.1
NS
Gender (M:F)
33:19
19:3
37:7
NS
-1
Bilirubin (mg dl ) mean (range)
1 (0.2–1.6)
0.8 (0.2–1.8)
2 (1.2–16.8)
\0.028
Albumin (g dl-1) mean ± SD
3.9 ± 0.28
3.5 ± 0.53
2.7 ± 0.59
\0.05
ALT (IU l-1) median (range)
51.5 (25–81)
41.5 (22–70)
83.5 (22–345)
\0.05
HAI mean ± SD
4.4 ± 2.1
5.4 ± 2.1
5.9 ± 2.2
\0.05
Fibrosis mean ± SD
2.1 ± 0.7
3.2 ± 0.42
3.6 ± 0.5
\0.02
DNA log copies mean ± SD Biopsy available
6.4 ± 1.3 33
6.6 ± 1.4 22
6.2 ± 1.0 28
NS
Significant values reported if p \ 0.05, statistical analysis was performed with Mann–Whitney or Fisher’s exact test
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Table 2 Demographic and clinical profile of patients with and without precore mutant forms Precore mutant (A1896) (n = 30)
Without precore mutant (G1896) (n = 88)
p value
Mean age (years)
37.0 ± 13
34.2 ± 15.5
NS
Gender (M:F)
24:6
65:23
NS
S bilirubin (mg dl-1) mean ± SD
1.4 ± 1.0
1.2 (0.6–16.8)
NS
-1
ALT (IU l ) median (range)
56 (22–345)
55 (22–345)
NS
Albumin (g dl-1) mean ± SD
3.8 ± 0.42
3.9 ± 0.5
NS
DNA log copies mean ± SD
6.3 ± 1.2
6.4 ± 1.2
NS
HAI mean ± SD
8.0 ± 2.1
5.9 ± 1.2
\0.05
Fibrosis mean ± SD
3.2 ± 1.0
2.7 ± 1.1
NS
Significant values reported if p \ 0.05, Statistical analysis was performed with Mann–Whitney test or Fisher’s exact test Profile of non-classical precore region mutations amino acid Category Genotype Pt ID ID-2 CHB D ID-8 CHB D ID-47 CHB D ID-48 CHB D ID-50 CHB D Compen ID-67 D Compen ID-72 D ID-80# Decomp A Decomp ID-82 D
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 M Q L F H L C L I I S C S/T C P T V/F Q A S K L C L G W L W F Y V P L F F T F St St L
29 G D
D
Fig. 1 Profile of non-classical precore region mutations. CHB Chronic hepatitis B, Compen compensated cirrhosis, and Decomp decompensated patients. Precore region mutations are given in black box, whereas one patient (#ID80) with Genotype A 1 was harboring a
stop codon mutation at precore position 21. Amino acid characteristics of genotype A are given in white box. Pt ID Patient identification number
when statistical analysis was carried out by comparing nonclassical precore region mutations and core immunodominant region mutations, no significance was found.
Genotype D
Commonly occurring core mutations with respect to genotype Genotype A Several core mutations were detected in HBV genotype A infected patients. The mutational profile of T helper region in genotype A patients was cV13G/A [4/15 (27%)], cE14D [1/15 (7%)], and cP50H/S [4/15 (27%)]. In B-cell region a single mutation cL84Q [1/15 (7%)] was present. Two mutations outside the immunodominant region were also detected, cY38F 1/15 (7%) and cP45S 2/15 (13%). Genotype C In genotype C infected patients, none of the T helper and CTL region mutations was found. However, two mutations were detected in B-cell epitope region at positions cV85I 2/7 (28.5%) and cA137G 1/7 (14%) and were present in two and one patient, respectively. Similar to the above finding, a single mutation outside the immunodominant was detected at position cW71G 1/7 (14%) in one patient.
In genotype D infected patients T helper region mutations were detected at position cT12S [21/96 (21.8%)] and A69G [8/96 (8.3%)]. In B-cell region, mutations were detected at positions cE77D [11/96 (11.4%)] and cE113D [6/96 (6.2%)]. The CTL region mutation cS21T [8/96 (8.3%)] was exclusively detected in genotype D infected patients. Association of immune escape mutations with severity of liver disease Mutations clustering regions (MCRs) as well as point mutations occurring in CTL, T helper, and B-cell regions were compared for their association with severity of liver disease. Mutations clustering in CTL region 18–27 were significantly more common in decompensated cirrhotics compared with CHB patients [9/44 (20%) vs. 4/74 (9%), p = 0.016] as well as mutations clustering in T helper regions 1–20 and 50–69 were also significantly more common in decompensated patients as compared with compensated patients [13/44 (29%) vs. 6/74 (8%), p \ 0.05] and [13/44 (29%) vs. 9/74 (12%), p \ 0.02]. Table 3 describes all three regions, however, no significant association between B-cell clustering region with severe liver disease patients was found. When comparison was
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Fig. 2 Prevalence and profile core immunodominant mutation. The figure describes the frequency and profile of core immune epitope mutations. White horizontal bar with black dots represents the number
of mutations detected in each immunodominant region; size of the dot depicts the frequency. More than one type of substitution detected is given by / sign. CTL Cytotoxic T lymphocytes
done by considering single point mutations with severe liver disease patients, mutations at five positions were found to be significantly associated with decompensated liver disease patients, T helper point mutations were cT12S [12/44 (27%) vs. 8/74 (10%), p \ 0.05] and cT67P/N [9/44 (20%) vs. 3/74 (4%), p \ 0.001]. B-cell region mutation was cE113D [5/44 (11.3%) vs. 1/74 (1.35%), p \ 0.05] and CTL region mutation was cS21T [7/44 (16%) vs. 1/74 (1.35%), p \ 0.05]. cP130T/Q mutation spanning in both B-cell epitope as well as CTL region was exclusively detected in four decompensated cirrhotic patients (p \ 0.001). Table 4 describes the
association of various individual mutations with severity of liver disease patients.
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Profile of naturally occurring core carboxy-terminal mutations Core carboxy-terminal region mutations were detected in 11/118 (9.3%) patients. The distribution of core carboxyterminal region aa substitutions was comparable among the disease category group [p = nonsignificant (NS)]. Two patients were infected with genotype A1 strain, and were harboring cR151Stop and cT162E mutation each.
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Table 3 Association of mutated immunodominant region with severity of liver disease Immunodominant region (amino acid)
Group I ? II (n = 74) (%)
Group III (n = 44) (%)
p value
CTL (18–27)
4 (9)
9 (20)
\0.017
T helper (1–20)
6 (8)
13 (29)
\0.05
T helper (50–69)
9 (12)
13 (29)
\0.02
14 (19)
12 (27)
NS
B cell (74–89)
Association of mutated immunodominant region with severe decompensated patients is described in this table. Statistical analysis was performed with Chi-square test Table 4 Association of individual core immunodominant mutations with severity of liver disease Codon
Total n (118) (%)
Group I (52) (%)
Core 12
20 (16.9)
3 (5.7)
Core 21
8 (6.7)
Core 64
11 (9.3)
Core 67
12 (10)
Group II (22) (%)
Group III (44) (%)
Mutation
p value
4 (18)
13 (29.5)
T ?S
\0.05*
1 (4.5)
7 (16)
S? T
\0.05*
3 (5.7)
4 (9)
6 (13.6)
E ?D
NS
2 (3.8)
1 (4.5)
9 (20)
T ?N/P
\0.01*
–
Core 69
8 (6.7)
6 (11.5)
2 (4.5)
A? S
NS
Core 77
14 (11.8)
7 (13.4)
3 (13.6)
4 (9)
E ?D/Q
NS
Core 80
11 (9.3)
4 (7.6)
3 (13.6)
4 (9)
A? I/V/T
NS
Core 113
6 (5)
1 (3.8)
–
5 (11.3)
E ?D
\0.05*
Core 130
4 (2.5)
–
4 (9)
P? Q/T
\0.05*
–
–
Data were analyzed using Chi-square test or Fisher’s exact test –, represents no mutation detected * Comparisons were made between Group I ? II versus Group III
Interestingly, three patients were harboring important SPRRR motif mutations. One patient ID45 with precore stop codon mutation was harboring triple mutation in core carboxy-terminal region; these were at position cG153R, cR154Q, and cS155V. Two additional patients, patient ID56 and ID63 were harboring a mutation at position 169 substituting glutamine to lysine and other patient was harboring mutation at c176 substituting serine to threonine. Mutational profile of genotype D patients harboring core carboxy-terminal mutations is given in Fig. 3. Of the nine patients with genotype D, three (33.33%) were harboring precore stop codon mutation (A1896). In none of the patients core internal deletion was detected. Profile of naturally occurring of core carboxy-terminal truncations Core carboxy-terminal truncations were present in 14/118 (12%) patients. Of the 14, 3 (21%) and 11/14 (78.5%) patients were harboring genotype A and genotype D, respectively. Two patients with genotype D infection were harboring coexisting precore stop codon mutation (A1896). Two types of truncations, smaller (3–7 bp) and longer (12–29 bp) truncations were detected in seven patients each. SPRRR-III motif was disrupted/truncated in two patients (ID29, 117), SPRRR-II and SPRRR-III motifs
were disrupted/truncated in three patients (Pt ID20, 31, 51), and SPRRRI-II and III motifs were disrupted/truncated in two patients (ID73 and ID25). Interestingly, one patient with SPRRRI-III disrupted motif (ID73) was harboring coexisting precore stop codon mutation (A1896). Whereas smaller truncations were spanning downstream to the SPRRR-III motif, they were detected in seven (50%) patients. Core carboxy-terminal truncations were comparable among the disease category group (p = NS). Profile of all 14 truncations is detailed in Fig. 4. Association of core mutations with HBV DNA levels HBV DNA levels were significantly high in patients infected with mutations at positions cP130 and cS21T compared with the wild type (7.1 9 1011 ± 1.3 9 102 vs. 5.7 9 106 ± 1.1 9 102; p \ 0.002) and (3.7 9 109 ± 1.2 9 102 vs. 4.9 9 106 ± 1.1 9 102; p \ 0.005). However, none of the mutations was significantly associated with ALT levels.
Discussion The salient features of the present study in HBeAg -ve CHB are: detection of genotype C1, identification of
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714 Fig. 3 Profile of core carboxyterminal mutations. CHB Chronic hepatitis B, Compen compensated cirrhosis and Decomp decompensated patients. This figure describes core carboxy-terminal region mutations identified in Genotype D (upper row) and A (lower row) infected patients. Core carboxy-terminal mutations were considered downstream to amino acid 150 of core region
Hepatol Int (2012) 6:707–717 Profile of Core Carboxy-terminal mutations amino Patient ID ID4 ID22 ID45 ID49 ID56 ID63 ID70 ID74 ID99
acid Category CHB CHB CHB CHB Compen Compen Compen Compen Decomp
amino Patient ID ID71 ID136
151 153 154 155 160 162 169 176 178 179 180 182 acid Category nt 1896 R G R S T T Q S S R E Q Compen Wild • • • • • E • • • • • • Decomp Wild St • • • • • • • • • • •
nonclassical precore region mutations in *8% of patients and association of immune escape mutations with severe liver disease patients. In addition, the data shows coexistence of precore stop codon mutation and immune escape mutations, and the presence of core terminal truncations and mutations in such patients. Several reports suggest that occurrence of core mutations is high in HBeAg seroconvertors with immune clearance phase compared with HBeAg positive patients [18, 19]. Very few studies focussed on core immune escape mutations in reactivating severe liver disease patients, and secondly, none of the studies simultaneously examined mutations of precore region in such patients [20]. This is one of the comprehensive studies reporting the prevalence of classical precore stop codon mutation as well as nonclassical precore mutations coexisting with core immune escape mutation in HBeAg -ve CHB patients. In this study, we found that the overall rate of core gene substitutions was significantly higher in decompensated patients compared with CHB patients; the substitution hotspots identified were at positions cT12S, cS21T, cT67P/ N, cE113D, and cP130T. Identification of cT12S, cS21T, and cE113D mutations in the previous reports in 65% of occult hepatocellular carcinoma as well as progressing hepatocellular carcinoma patients [21, 22] suggests their pathogenetic role. The aa position cT12S is in close proximity with the HLA A2 CTL restricted epitope and selection of cT12S mutation could lead to immune escape of the virus. Contrary to our findings, an in vitro study suggested that deletion at similar position blocks the virion formation [23]. We speculate that this could retain the viral machinery within the hepatocytes, possibly by disruption of the polyadenylation signal, generating longer transcripts and leading to severe liver disease. Interestingly, mutations at position cT12S and cS21T were coexisting with precore stop (A1896) codon mutation, the interplay between precore stop codon mutation and cT12S/cS21T mutations needs to be further studied. A second mutation, mutation at
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nt 1896 Wild Mutated Mutated Wild Wild Wild Wild Wild Mutated
151 153 154 155 160 162 169 176 178 179 180 182 R G R S T T Q S S R E Q • • K • • • • • • • • • C R • • • • • • • L M • • R Q V • • • • • • • • • • • • S • • • • • • • • • • • • • K • • • • • • • • • • • • T • • • • • • • • • • • • • • I M • • A • • • • • • • • • • C • T • • • • • • • •
position cT67 in T helper region, have been detected in patients with fulminant and hepatocellular carcinoma [21, 24]. A recent study by Homs et al. [25] also reported the presence of cE64 and cT67 mutations in patients treated with interferon. It is quite possible that mutation in this region could activate dendritic cells and modulate the immune response. Coexisting mutations at these two sites also suggest their mutual cooperativity and strict conservation in one single protein. Identification of cP130T mutation in four decompensated patients was surprising, the DNA levels were found to be significantly high in these patients. Similar to our findings a previous study reported the occurrence of P130T mutation in patients experiencing ALT exacerbations [26]. Codon 130 spans in both T- and B-cell epitope and it is exposed on the surface of mature HBeAg and HBcAg, one of the reasons for such nature could be enrichment of fulllength transcripts. Two mutations cS49T and cE77D detected in 3 and 14 patients, respectively, in the present study were significantly associated with precore stop codon mutation. Similar mutations have earlier been identified in three cirrhotic and portal hypertension patients from India [27]. One of the mutation ‘‘cE77D’’ have been detected in HBV genotype A patients from late HBeAg-positive phase, displaying extremely low HBe antigenicity [28]. Glutamine residues at positions 77 and 113 of the core gene are responsible for peptide anchoring to the B pocket of HLA B-4001 [29]. A recent study from New Zealand suggested that immune escape mutations occurring at positions cE77 and cE113 could decrease peptide binding and prevent presentation to CD8 T cells. However, their role in causing severe liver disease is yet to be investigated [30]. There were four genotype D infected patients in whom important SPRRR motif core terminal region mutations were found. In the first patient (ID45), a coexisting triple mutation at positions cG153R, cR154Q, and cS155V was
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715
Profile of Core Carboxy tuncations 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 Pt ID
Group
ID20
CHB
ID21
CHB
ID29
CHB
ID31
CHB
ID51
CHB
ID73*
Compen
ID94
Decomp
H
ID115*
Decomp
H
ID116
Decomp
ID117
Decomp
ID118
Decomp
Pt ID
Group
R
ID25
CHB
T
ID76
Decomp
ID79
Decomp
R
S
P
R
R
R
T
P
S
P
R
R
R
R
S
P
S
P
R
R
R
R
S
Q
S
R
E
S
Q
C
L S
H I L A
I
N A I
K
159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 R
R
T
P
S
P
R
R
R
R
S
P
S
P
R
R
R
R
S
Q
S
R
E
S
Q
C
H F
Fig. 4 Profile of core carboxy-truncations. CHB Chronic hepatitis B, Compen compensated cirrhosis, and Decomp decompensated patients. The above row shows patients infected with Genotype D strain and lower row shows patients infected with Genotype A strain. Patient ID
with esterik sign were mutated at precore position A1896. Truncated region is represented by sign approximately. Pt ID Patient identification number
found; in the second patient (ID56) cQ169K was detected, the third patient (ID63) was harboring cS176T mutant, and in fourth patient (ID99) double mutants cG153C and cS155T were detected. Interestingly, two of these patients (ID45, ID99) were harboring coexisting precore stop codon mutation (A1896) and patient (ID99) had decompensated liver. In addition, one patient with genotype A1 was harboring unique mutation, leading to stop codon mutation at position cR152Stop. Changes in core SPRRR motif at positions 155 and 176 could alter the phosphorylation status of core protein and may modulate the severity of liver disease. Two of the studies suggested that mutations in core carboxy terminal could change the distribution of HBcAg in hepatocytes, leading to variable packaging of pregenomic RNA within the nuleocapsids [31, 32]. In an earlier study, we had shown that nearly one-third of the HBeAg -ve CHB patients were infected with exclusive basal core promoter regulatory TA1-3 region mutations. However, no association of these mutations and genotypes as well with severity of liver disease was found [33]. In the present study, we further found that 7.6% HBeAg -ve reactivating patients do harbor nonclassical precore region mutations. However, we could not detect any significant association of nonclassical precore region mutations with decompensated severe liver disease patients. This is the first study reporting the presence of nonclassical precore region mutations, including precore gene translation initiation codon mutation in HBeAg -ve CHB patients in India. Interestingly, two earlier studies reported selection of precore gene translation initiation codon mutations in HBeAg -ve reactivating phase and
support our findings [34, 35]. It is quite possible that selection of this variant is one of the strategies of the virus to cope with the vigorous host immune system. Recently, genotype C has been reported from occult HBV-infected patients and blood donors from Eastern and Southern parts of India [36, 37]. Similarly, we reported the prevalence of genotype C in North India in asymptomatic patients with raised ALT [15]. However, this is the first study from Northern India reporting the prevalence of HBV subgenotype C1 in 4.2% of HBeAg -ve CHB patients with severe liver disease. Phylogenetic analysis of core and surface gene revealed that five of genotype C sequences clustered with South East Asia (C1) and not with East Asian origin (C2) [38]. Of the seven patients with genotype C infection, two were of Tibetan origin and remaining five were from urban population and had international travel history. Based on the partial gene sequence, we identified C/D recombinant in two of these Tibetan patients (data not shown). However, we classified them as unidentified subgenotype C. These two strains could not firmly be classified as C/D recombinant until whole genome sequencing and analysis are done. Such an approach is underway for characterizing them. A recent report from Afghanistan revealed the presence of genotype C HBV infection in [30% of their population. It is quite possible that this virus would have transmitted via human trafficking across and within the borders [39]. The spontaneous coevolution of the virus with rapidly migrating humans could be one of the reasons of the changing molecular epidemiology. In conclusion, the present study reports the characterization of immune escape mutations in HBeAg -ve CHB patients with and without precore stop codon mutation. The
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results indicate that patients with severe liver disease should be tested for HBV genotypes and mutational profile, as more diverse viral population may be selected in severe liver disease patients in future and there is an urgent need for individualized immunomodulatory therapies. Acknowledgements Authors are thankful to Indian Council of Medical Research for providing funds through Advanced Center for Liver Diseases project. Authors thank Dr Scott Bowden (VIDRL) for critically reviewing the manuscript.
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