Characterization of occult hepatitis B virus from blood donors carrying genotype A2 or genotype D strains q

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1 Journal of Hepatology 49 (2008) Characterization of occult hepatitis B virus from blood donors carrying genotype A2 or genotype D strains q Daniel Candotti 1, Piotr Grabarczyk 2, Paola Ghiazza 3, Roberto Roig 4, Natalia Casamitjana 5, Paola Iudicone 6, Michael Schmidt 7, Arthur Bird 8, Robert Crookes 9, Ewa Brojer 2, Michelina Miceli 6, Azin Amiri 10, Chengyao Li 1,11, Jean-Pierre Allain 10, * 1 National Health Service Blood and Transplant, Cambridge Blood Centre, Cambridge, United Kingdom 2 Institute of Haematology and Transfusion Medicine, Warsaw, Poland 3 Struttura di Qualificazione Biologica del Sangue, Ospedale Sant Anna, Torino, Italy 4 Centro Transfusion Comunidad Valencia, Valencia, Spain 5 Centre de Transfusió i Banc de Teixits, Barcelona, Spain 6 Azienda Ospedaleria S Camillo-Forlanini, Rome, Italy 7 Institute of Transfusion Medicine and Immunohematology, Johann Wolfgang Goethe University, Frankfurt, Germany 8 Western Province Blood Transfusion Service, Cape Town, South Africa 9 South African National Blood Service, Johannesburg, South Africa 10 Department of Haematology, University of Cambridge, Cambridge Blood Centre, Long Road, Cambridge CB2 2PT, United Kingdom 11 Southern Medical University, Guangzhou, China Background/Aims: Nucleic acid testing (NAT) for hepatitis B virus (HBV) DNA in blood donations identified occult HBV infection (OBI) as a potential threat to blood safety. Methods: A collaborative study was undertaken to explore the molecular basis of OBIs prevalent in Europe in relation to clinical and serological data. Results: Ninety-one percent of 77 donor samples of European origin HBV DNA positive but HBV surface antigen (HBsAg) negative were confirmed. Viral load ranged between unquantifiable and 5640 IU/mL (median 25 IU/mL). Fifty-two strains were genotyped (14 HBV A2 and38hbv D ). Compared to HBsAg+ samples, genotype D was significantly more frequent than genotype A2 in OBIs from Poland or Italy (P < 0.04). Amino acid substitutions were concentrated in the immunologically active parts of the Pre-S/S proteins (P < ) affecting both cellular CD8 T-cell epitopes and B-cell neutralizing Major Hydrophilic Region epitopes. Substitutions were more frequent in OBIs than in HBsAg+ strains of both genotype D (P < 0.001) and A2 (P < 0.01), in OBIs of genotype D than A2 in the a region (P < 0.001) but not cellular epitopes, and in anti-hbs+ than anti-hbs OBIs (P < 0.001). Conclusions: Results support the hypothesis that humoral and cellular immune pressure on the HBV envelope proteins are major mechanisms generating OBI. Ó 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Occult HBV; HBV; Blood donors; Europe Received 6 February 2008; received in revised form 8 April 2008; accepted 19 April 2008; available online 2 June 2008 Associate Editor: F. Zoulim q None of the co-authors has any conflict of interest to declare concerning the material presented in this manuscript. Dr. Allain is an occasional lecturer for the Chiron/Novartis Corporation. * Corresponding author. Tel.: ; fax: address: jpa1000@cam.ac.uk (J.-P. Allain) /$34.00 Ó 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi: /j.jhep

2 538 D. Candotti et al. / Journal of Hepatology 49 (2008) Introduction HBV DNA screening revealed the presence of cases of occult HBV infections (OBI) defined as HBV DNA positive but hepatitis B surface antigen (HBsAg) negative among apparently healthy blood donors [1,2]. Preliminary data previously published indicated that, in addition to anti-hbc being the most frequent serological marker of HBV infection in OBI, anti-hbs was present in approximately half of the cases, raising the issue of possible multiple mechanisms of OBIs [2]. OBIs were thought to originate from individuals who had recovered from infection and kept virus or viral nucleic acid persisting in sanctuaries such as the liver, unrecognized by the host immune system and occasionally detectable at low level in the peripheral blood [3,4]. It may also reflect the progressive decline of HBsAg in long-term chronic infection resulting in levels of surface antigen undetectable by standard immunoassays [1]. From a transfusion point of view, distinguishing between these two potential origins of OBI is critical as the presence of anti-hbs is generally considered to prevent infection by transfusion and blood units containing >100 IU/L of anti-hbs were considered safe for transfusion [5]. The donors deferred on the basis of diagnosis of OBI carriage need to be informed of the potential clinical consequences of their condition. A study group called HBV blood safety was constituted within the Transfusion Transmitted Infection working party from the International Society of Blood Transfusion (ISBT) [6]. The data presented strongly suggests that a majority of OBIs of genotype A2 or D (dominant in Europe) are the result of strong immune pressure reflected by a remarkable frequency of amino acid substitutions in the large, middle and small surface proteins concentrated on immunoactive sequences. 2. Materials and methods 2.1. Collaborative centers and sample identification Blood centers participating in the study were from Poland (coordinated by the Warsaw Institute of Hematology and Transfusion Medicine), from Italy the Italian Red Cross Blood Centre, Rome and the Blood Biological Qualification Department, Sant Anna Hospital, Turin, from Spain the Centre de Transfusió i Banc de Teixits, Barcelona and the Community Blood Centre, Valencia and from Germany, the German Red Cross Blood Center in Frankfurt. In addition, three genotype D samples came from donors of European origin in the Western Province Blood Transfusion Service in Cape Town, and two in the South African National Blood Service in Johannesburg. All samples were from HBsAg negative/hbv DNA positive blood donors. The algorithms used for HBV DNA screening differed to a considerable extent between centers. The two centers in Italy and in Valencia, Spain screened directly for HBV DNA in individual donation samples using a triplex transcription-mediated amplification assay (PROCLEIX Ò ULTRIO TM assay, Chiron/Gen-Probe, Emeryville/San Diego, CA). Reactive donations were retested with a discriminatory assay for HBV DNA. The 95% detection limit for HBV DNA was 11 IU/mL [2,7]. The screening scheme in Poland has been previously reported [2]. The blood centers in Frankfurt and Barcelona tested for anti-hbc and HBV DNA as described [8,9]. Blood centers in South Africa used the fully automated PROCLEIX Ò TIGRIS Ò System with the PROCLEIX Ò ULTRIO TM assay [10]. Confirmation was performed either with an alternative commercial assay (Cobas Ampliscreen HBV; Roche Diagnostics, Manheim, Germany for Poland) or in-house PCR or real time PCR (other centers). All quantification data come from the Cambridge coordinating center by real time PCR (QPCR) as described [12] Serological testing Each collaborating center provided results of serological HBV markers. Assays used for HBsAg detection were Ortho Antibody to HBsAg ELISA Test System 3 and chemoluminescence-based HBsAg assay (Ortho-Clinical Diagnostics, Raritan, NJ, USA) in Poland, Bioelisa HBsAg colour (BIOKIT S.A., Barcelona, Spain) in Rome and HBsAg chemoluminescence assay on the PRISM instrument (Abbott Laboratories, Abbott Park, IL, USA) in Barcelona, Frankfurt, Turin and Valencia as well as South Africa. The sensitivity of the different HBsAg assays ranged between 0.1 and 0.3 IU/mL. Anti-HBc was detected with Monolisa anti-hbc Plus (Bio-Rad, Marnes-la-Coquette, France), or Wellcozyme anti-hbc (Abbott/Murex, Dartford, United Kingdom) or microparticle enzyme immunoassay core (Abbott Labs, USA). Anti-HBs were detected with Hepanostika anti-hbs (Organon Teknika/bioMerieux, Boxtel, The Netherlands), Murex Anti-HBs EIA (Abbott/Murex) or microparticle enzyme immunoassay Ausab IMx (Abbott Labs, USA) HBV DNA analyses Viral DNA was extracted from 500 ll of plasma [11,12]. After ultracentrifugation of ml of plasma according to the volume available, full-length HBV genome minus 50 bp in the pre-core region (3150 bp), Pre-S/S region (1190 bp), 450 bp in the S region including the antigenic loop, and 300 bp in the basic core promoter/pre-core region (BCP/PC) were amplified using nested-pcrs [12]. PCR products were directly sequenced and phylogenetic analyses were performed [11,12]. Deduced amino acid sequences were compared to sequences of HBV strains of similar genotype obtained from HBsAg-positive individuals available in the GenBank database (genotype A2: AB116076, AB116077, AB116078, AB116079, AB116081, AB126580, AB246337, AB246338, AF537372, AJ012207, AJ309369, AM282986, AY233280, AY233286, X51970, X70185, Z35717, and Z72478; genotype D: AB090269, AB090270, AB104709, AB104711, AB109475, AB109476, AB120308, AB126581, AB246348, AF043593, AF121240, AJ344117, AY233291, AY233292, AY233293, AY233294, AY233295, AY233296, AY661793, AY721606, AY721607, AY721608, AY721611, AY721612, AY741797). Whenever possible, HBsAg+ sequences selected for comparison fulfilled the following criteria: (i) being isolated from HBsAg+ chronic carriers over age 30, HBeAg positive and without mutations preventing the expression of pre-core; (ii) having a geographical origin comparable to the OBI strains. Diversity was defined as the mean value for pair-wise difference between sequences within the same group, calculated as the number of amino acid differences between two individual sequences [11] Statistical analysis Analyses were performed using the SPSS software (version 15.0). Categorical variables were compared using Fisher s exact test and, for continuous variables, the non-parametric Mann Whitney test. 3. Results 3.1. Detection of occult HBV infection in European or of European origin blood donors Seventy-seven samples initially classified as occult HBV infection by six collaborating European blood

3 D. Candotti et al. / Journal of Hepatology 49 (2008) centers and five samples from Europeans who donated blood to South African blood programs were investigated. Eight samples (9.4%) were repeatedly negative by QPCR and nested PCR assays after ultracentrifugation. Although these samples may contain very low levels of HBV DNA, they were considered not confirmed and excluded from further analysis. The 74 samples confirmed HBV DNA positive by QPCR and/or nested PCR were collected from 13 females and 61 males. Donor age ranged between 19 and 73 (median: 51 years). All individuals were clinically asymptomatic and had levels of ALT below 50 IU/L (Table 1). All but two samples contained anti-hbc (P19 not tested, P6 remained negative with a follow-up sample), and 40/74 samples (54%) contained anti-hbs. Levels of anti-hbs in 17 quantified samples ranged between 5 and >1000 IU/L (median 22.5 IU/L) Prevalence of occult HBV infection in European blood donor populations The screening data obtained from four European countries are summarized in Table 2. Only samples from Poland were representative of the whole country, the others were from five large cities. The yield of OBI in blood donors was low in the German center since the prevalence of anti-hbc was the lowest. In Poland, Italy and Valencia, only donations screened in individual samples were taken into account. In three European countries with anti-hbc prevalence ranging between 4 and 7% (Italy, Poland and Spain), the prevalence of occult HBV carriage ranged between 1:1943 and 1:14,717. This frequency appears high compared to HBsAg but the population of mostly repeat donors was unscreened for HBV DNA but most HBsAg+ donors had already been excluded. To estimate the respective frequency of OBI and HBsAg carriage, only first time donors (unscreened for both HBV DNA and HBsAg) were considered. The ratio between HBsAg and OBI carriers ranged between 7.6 and 71 (Table 2) Molecular characterization of OBI The BCP/PC region, the Pre-S/S region, and the whole HBV genome were successfully amplified and sequenced from 53 (72%), 49 (66%), and 16 (22%) samples, respectively. Phylogenetic analysis indicated that 10 and 39 sequences clustered with HBV genotype A2 and genotype D, respectively (bootstrap values of 97% and 100% over 1000 replicates, respectively) (data not shown). Analysis of eight additional S sequences showed two groups of four sequences clustering with genotype A and D (bootstrap values of 82% and 97%, respectively) (data not shown). Overall 14 OBIs of European origin were genotype A( 2 ) (24.6%) and 43 (75.4%) were genotype D (Table 1). In Poland, Italy and Spain, the distribution of occult HBV infections was stratified according to genotype and compared to the distribution observed in HBsAg+ strains from either blood donors or patients [13 19]. A consistent excess of genotype D occult HBV was highly significant in Poland (P < 0.001), significant in Italy (P = 0.04) and just below significance in Spain (P = 0.06) (Table 3). The HBV DNA load ranged between unquantifiable (13 cases) and 5640 IU/mL (median 25 IU/mL, 39 IU/ ml when including only quantifiable samples). Similar viral load distribution was observed in genotype A2 (median: 19.5 IU/mL; range: < IU/mL) or genotype D (median: 34 IU/mL; range: <1 372 IU/mL) samples. Viral load was significantly higher in anti-hbs negative samples (median: 39 IU/mL; range: < IU/mL) than in anti-hbs positive samples (median: 16 IU/mL; range: <1 270 IU/mL) (P = 0.007). The difference remained significant in genotype D samples (38 IU/mL [<1 372 IU/mL] and 13 IU/mL [<1 47 IU/mL] (P = 0.028) but not in genotype A2 samples. HBV DNA quantification was not obtained in 2 and 10 genotype A2 and genotype D samples, respectively (12/ 57). In the 17 samples not genotyped (data not shown) the median HBV DNA load was 11 IU/mL, ranging between <5 and 237 IU/mL. All samples contained anti-hbc and anti-hbs was detected in 10/17 samples (58.8%), at a titer >100 IU/L in six. When compared for age, gender, anti-hbc and anti-hbs reactivity, the 57 donors with and 17 without identified genotype were not significantly different, except for viral load (P = 0.01). The genotyped group was considered representative of occult HBV in blood donors PreS/S region analysis The genomic and deduced amino acid (aa) sequences of the genotyped strains described here were examined at three different levels. First, genotype A2 and D OBI sequences were compared to their respective HBsAg positive counterparts. Secondly, within OBIs, features of genotype A2 (OBI A2 ) and D (OBI D ) sequences were compared. Finally within a given genotype, sequences from samples anti-hbs positive or negative were compared. The pre-s1/pre-s2, and S aa sequences were obtained from 49 (10 OBI A2 and 39 OBI D ) and 57 (14 OBI A2 and 43 OBI D ) OBI strains, respectively. Diversity was significantly higher in OBIs than in HBsAg+ strains and in OBI D than OBI A2 (Table 4). Substitutions appeared concentrated in immunodominant regions including the antigenic loops Major Hydrophilic Region (MHR) of the S antigen and the CD8+ cytotoxic T-lymphocyte (CTL) epitopes (Table 4, Figs. 1 and 2). When aa envelope protein sequences were divided between immunore-

4 540 D. Candotti et al. / Journal of Hepatology 49 (2008) Table 1 General information on 57 genotyped cases of occult HBV infection identified in blood donors Samples a Gender Age Viral load (IU/ml) Anti-HBc Anti-HBs IU/L b ALT level IU/L Genotype Full genome HBV vaccine Poland P1 M Pos Pos 32 D Yes No P2 M 50 <1 c Pos D No P3 M Pos 8 20 A2 No P4 M NA 18 Pos A2 Yes No P5 M 42 <1 Pos Pos 16 D No P6 M Neg A2 No P18 M 55 <1 Pos Pos ND A2 NK P20 M Pos Pos 21 A2 No P7 M Pos Neg 22 A2 Yes P8 F 40 9 Pos Neg 10 D No P9 M Pos Neg 38 A2 Yes No P10 M Pos Neg 27 D Yes No P11 F Pos Neg 18 D No P12 M Pos Neg 29 D No P13 M Pos Neg 10 D Yes P14 M Pos Neg 19 D No P15 M Pos Neg 20 D Yes No P16 M Pos Neg 23 A2 No P17 M 42 <1 Pos Neg 39 D NK P19 M ND Neg 33 A2 NK P21 M Pos Neg 38 D NK Median Italy R1 M 51 <1 Pos D No R2 M Pos D No R3 M Pos Neg 35 D Yes No R4 M 49 <1 Pos Neg 27 D Yes No T1 M 56 4 Pos D No T2 M Pos Pos 15 D No T3 M Pos Neg 14 D No T4 M Pos Pos 29 D Yes No T5 M Pos Neg 15 D Yes No T6 M 56 2 Pos Neg 23 D No T7 M Pos Neg 29 D No T8 M Pos Neg 17 D Yes No Median Spain B1 M Pos 5 25 A2 No B2 M Pos 6 20 D No B3 M 64 <1 Pos A2 No B4 M Pos D No B5 F 54 <1 Pos D No B6 M Pos 73 ND D No B7 M Pos 89 ND D No B8 M Pos Neg 18 D No V1 M Pos D Yes No V2 M Pos Neg ND A d No V3 M Pos Neg ND D Yes No V4 M 44 <1 Pos D No V5 M 47 <1 Pos D No V6 F Pos D Yes No V7 M Pos Neg 14 D No V8 M 59 <1 Pos D No V9 M 30 6 Pos Neg 6 A d No Median Germany F1 M 55 <1 Pos Pos 22 D No F2 M 68 8 Pos Pos 26 A2 No Line missing

5 D. Candotti et al. / Journal of Hepatology 49 (2008) Table 1 (continued) Samples a Gender Age Viral load (IU/ml) Anti-HBc Anti-HBs IU/L b ALT level IU/L Genotype Full genome HBV vaccine South Africa CT1 M Pos Neg D No CT2 M Pos Neg ND D Yes No CT3 M Pos Neg ND D No J1 F 57 2 Pos D Yes No J2 M 32 4 Pos Neg <40 D Yes No Total median Samples from which the pre-ss sequences were not available a shorter S region sequences was obtained. Genotyping was obtained on the basis of an S sequence only, which does not allow differentiating between A1 and A2 genotypes. a Code for sample identification stand P, Poland; R, Rome; T, Turin; B, Barcelona; V, Valencia; F, Frankfurt; CT, Cape Town; J, Johannesburg; ND, not done; NK, not known. b Result given as positive or negative unless quantification was performed in miu/ml. c Viral load <1 indicates no C t with the real time PCR quantitative assay. Despite this non-quantifiable viral load in plasma, sequences were obtained after ultracentrifugation. d Samples providing only S sequences that did not allow differentiating between geneotype A1 and A2. active (180 aa including the 63 aa of the MHR reactive for humoral immunity and the 117 aa of 12 cellular epitopes) and non immunoreactive 229 aa for genotype A2 and 220 aa for genotype D, a significant difference in frequency of aa substitutions compared to the consensus wild-type sequence was observed (5.7% vs 2.6% for OBIs genotype A2; P < and 6.9% vs 1.5% for OBIs genotype D; P < ). In contrast, the frequency of substitutions in the HBsAg positive sequences of both genotype A2 was 0.8% ( %) irrespective of immune reactivity and 0.9% and 0.5% in immunoreactive and inactive regions, respectively (P = 0.046); (P < compared to OBIs). The average aa diversity was significantly higher in the OBI D strains than in the OBI A2 strains (P < ) in the MHR loops, excluding aa known responsible for serotypes (Table 4). No genotype D but 5/24 genotype A2 OBI sequences were wild-type. In addition, the anti-hbs+ OBI A2 and OBI D subgroups presented significantly higher average aa intra-subgroup diversity than the corresponding anti-hbs subgroups within the MHR (8.9 aa [range: 0 17] vs 4.0 aa [range: 0 8] in OBI A2 [P < ] and 11.9 aa [range: 0 24] vs 9.8 aa [range: 0 23] in OBI D [P < ]). However, when aa sequences were compared to the HBsAg+ consensus sequence, the number of aa substitution between genotype D anti-hbs+ and anti-hbs sequences was not significantly different (Fig. 1). Substitutions previously associated with immune escape phenotype, and particularly in a number of cysteines occurred more fre- Table 2 Prevalence of occult HBV infection in different areas of Europe Country City Population of donors Number of donation screened Number of OBI (frequency) Number of donations HBsAg+ (frequency) Poland NA a Total 250, (1:14,717) 1st time (1:7630) 570 (1:107) Italy Rome Total 35,016 b 8 (1:4502) 16/28,401 (1:1755) Turin Total 236, (1:13,111) 1st time 8781 c 5 (1:1756) 38 (1:230) Spain Barcelona d Total 15,545 8 (1:1943) 6/15,545 (1:2591) Valencia Total 117, (1:9819) 29/84,226 (1:2904) 1st time 23,563 1 (1:23,563) 47 (1:501) Germany Frankfurt e Total 1,348,759 7 (1:192,680) 4/1,240,859 (1:310,214) 1st time 107, (1:674) a Not applicable: samples were collected from several regional Polish blood centers. b Reported in Vox Sanguinis, 2006; Suppl. 3; p. 58. c Data are from published first time donors [32] during the period of the OBI study although with a different denominator. d Identified from anti-hbc positive donations [9]. This study being a subset of the total donor population, the respective frequency of HBsAg and OBI in first time donors could not be obtained. e Identified from pools of 96 according to a previously published method [8]. HBsAg+ column, top line corresponds to repeat donors; second line corresponds to first time donors.

6 542 D. Candotti et al. / Journal of Hepatology 49 (2008) Table 3 Distribution of HBV genotypes in HBsAg positive blood donors and patients and occult HBV in three European countries Country HBsAg DNA Genotype A2 Genotype D Other genotype(s) Poland + a + 50 (79.4) 13 (20.6) 0 + b + 99 (80.5) 18 (14.6) 6 (5.2) + 6 (37.5) 10 (62.5) * 0 Italy + c + 59 (26.1) 165 (73.0) 2 (0.9) (100) ** 0 Spain + d (39.5) 234 (48.1) 60 (12.3) + e (64.2) 62 (23.1) 34 (12.7) + 4 (28.6) 10 (71.4) *** 0 a Blood donors, unpublished. b References [13 15]. c References [16,17]. d Reference [18]. e Reference [19]. * P < between lines 1 and 3. ** P = *** P = 0.06 between lines 1 and 3. quently in anti-hbs+ subgroups irrespective of genotype (Fig. 1). In the region s119g to s124c, substitutions were described as possibly interfering with infectivity of HDV-containing particles [20]. Although s121c and s124c were essentially conserved (1 and 3 substitutions, respectively), aa at other positions were substituted in 27/44 and 6/14 genotype D and genotype A2 sequences, respectively. One, two and nine CD8+ CTL epitopes (numbered 1 12 in Fig. 2) have been previously identified within the pre-s1, pre-s2, and S regions of HBV genotype D strains, respectively [21,22]. In genotype A2, the consensus sequences of epitopes 1, 4, 5, 7 9 and 12 are identical to genotype D. Genotype A2 epitope 3, epitopes 2 and epitopes 6, 10 and 11 differ by three, two and one aa from genotype D, respectively. The OBI strains showed greater aa variability within CTL epitopes than seen in the non-obi strains, irrespective of genotype (Fig. 2). Two OBI A2 (20%) and six OBI D (15%) showed six or more mutated CTL epitopes, and only four OBI D (10%) showed no mutation. In both genotypes A2 and D, epitopes 1, 3, 4 and 5 were highly conserved. In contrast, epitopes 8 and 9 had mutations, often multiple. Epitope 2 was more mutated in genotype D than in genotype A2 (5.5% vs 4% of aa positions mutated, respectively). However, the overall frequency of substitutions was similar between the two genotypes. The average diversity of the S promoter was 3.8 nt (range: 1 8) and 8.8 nt (range: 1 16) in the OBI A2 and OBI D groups, respectively. Only OBI D sequences showed a significantly higher variability than HBsAg+ sequences (8.8 nucleotides [range: 1 16] vs 5.2 [range: 0 11]; P < ) Analysis of HBV pol, Core, and X genes The characteristic low viral load associated with OBI might reflect low level of viral DNA replication, inefficient viral morphogenesis, or alteration of viral gene expression affected by mutations in the pol, core or X genes. Aa sequences of the full-length pol gene product were obtained for two OBI A2 and 14 OBI D strains. The diversity was significantly higher in the OBI D sequences than in the HBsAg+ counterpart (Table 4). One to three aa substitutions were found in three identified RT domain CTL epitopes (rt : rtl121f, rtr123g, rtv125i, rta126s/t and rtl128v; rt : V220L/M); rt : rtl242m/s, rts243a/y, rti246l/v, rth247q, and rtl248i) in 15, 6, and 11 OBI samples, respectively. Table 4 Average intra- and inter-group amino acid diversity of occult HBV of genotype A2 and D HBV proteins Genotype A2 Genotype D OBI strains HBsAg+ strains P value OBI/HBsAg+ OBI vs HBsAg+ strains OBI strains HBsAg+ strains P value OBI/HBsAg+ OBI vs HBsAg+ strains PreS1 4.3 (1 11) 2.4 (0 9) < (0 11) 4.6 (0 13) 1.6 (0 6) < (0 12) PreS2 4.3 (0 11) 1.2 (0 4) (0 8) 4.1 (0 13) 1.7 (0 7) < (0 11) S 15.5 (2 30) 4.6 (0 14) < (1 30) 21.4 (0 42) 3.4 (0 10) < (0 29) MHR 7.1 (0 17) 1.7 (0 5) < (0 16) 10.3 (0 24) 1.7 (0 7) < (0 17) Polymerase NA NA NA 46.5 (16 63) 24.8 (1 50) < (13 66) Terminal protein NA NA NA 5.3 (2 8) 4.1(0 9) 5.3 (2 9) Spacer 10.9 (2 24) 4.6 (0 15) 7.8 (0 22) 10.9 (0 23) 6.5 (0 21) 9.7 (0 25) Polymerase/RT b domain 9.1 (1 17) 5.3 (0 13) 7.3 (0 17) 12.9 (5 21) 9.4 (0 20) 15.5 (4 27) RNaseH NA NA NA 5.0 (0 10) 3.4 (0 8) 4.3 (0 10) Core NA NA NA 5.1 (0 12) 3.2 (0 10) 4.4 (0 11) HBx NA NA NA 7.7 (2 14) 5.7 (0 15) 6.8 (2 16) a Different numbers of sequences were analyzed for each region according to data availability: OBI genotype D sequences: PreS1 and PreS2, n = 39; S, n = 43; polymerase including terminal protein and RnaseH domains, n = 11; spacer domain, n = 38; polymerase/rt domain, n = 11; Core and HBx, n = 14; OBI genotype A2 sequences: PreS1 and PreS2, n = 10; S, n = 14; polymerase, n = 11; genotype A2 and genotype D control sequences, n = 18 and n = 25, respectively. b Putative thumb domain was not included in the genotype A2 OBI sequences analyzed. NA, not available.

7 D. Candotti et al. / Journal of Hepatology 49 (2008) Fig. 1. Amino acid substitutions in the MHR of the S protein of OBIs compared to wild-type consensus sequences. Deduced amino acid sequences of the a region of 57 OBI strains. Twenty genotype D strains from anti-hbs+ samples (A), 23 genotype D strains anti-hbs negative (B) and 14 genotype A2 strains (8 anti-hbs positive in bold and 6 anti-hbs negative) (C) are shown. The samples code is as in Table 1. The amino acid identification is with the one letter code spanning amino acid of the S protein. Cysteines and the corresponding substituted amino acids are in italic. Other substitutions known to be involved in lower HBsAg assay reactivity are in bold [23 26]. Dots indicate identity with the consensus sequence for the genotype given on top of each group of sequences. Donors P13 and T5 donors were vaccinated against HBV. The two OBI A2 and 14 OBI D core aa sequences available did not have significant mutations compared to the HBsAg+ groups. A limited number of substitutions was found within the CD4+ T-cell epitopes and CD8+ T- cell epitopes were essentially conserved. The average aa variability within the X protein was not significantly different between OBI D and HBsAg+ strains (Table 4). There were no deletions or insertions in the X gene Pre-core and regulatory elements analysis The precore region showed four different mutational mechanisms preventing HBeAg synthesis in OBI A2 and OBI D strains. The G1896A mutation was found in 10 OBI D strains (22%). In two OBI A2 (18%) and 13 OBI D (29%) strains, the precore ATG start codon was abolished by a point mutation at any of the three nucleotide positions. A C1817T nucleotide mutation introduced a

8 544 D. Candotti et al. / Journal of Hepatology 49 (2008) Fig. 2. Variability of CTL epitopes within the HBV envelope proteins of OBI and HBsAg-positive strains. CTL epitopes are numbered 1 12 on top of each consensus sequence. The top two panels correspond to genotype A2 10 OBI and 10 HBsAg+ reference sequences. The bottom two panels correspond to genotype D epitopes from 39 OBI and 25 HBsAg+ reference sequences. Amino acid positions are indicated at each end of the individual epitopes according to the large S protein numbering. Only amino acid changes are indicated with the one letter code and deletions are noted with dashes. For each epitope, the number of strains carrying the mutation(s) is indicated on the left hand side. stop signal at codon 2 in two OBI D strains (4%), and the insertion of an additional thymidine at position 1895 was responsible for a frame-shift and a stop signal at codon 33. The analysis of regulatory elements controlling viral replication and gene expression scattered over the entire OBI genomes did not reveal major changes. Altogether, 55% and 14% of OBI D and OBI A2, respectively, had mutations preventing HBeAg synthesis. 4. Discussion The data presented from a large number of blood donation samples negative for HBsAg but positive with commercial nucleic acid testing (NAT) assays for HBV DNA provide a solid basis to characterize occult HBV carriage in asymptomatic blood donors. The vast majority (91%) of the samples identified by screening were confirmed except when sample volume prevented ultracentrifugation or viral load was below confirmatory assay sensitivity or were NAT screening false-positive. Donors with OBI were generally older by approximately one decade (median 51 years) than either total donor or HBsAg carrier populations (Table 1). OBI carriers had normal ALT levels, and detectable anti-hbc (Table 1). Approximately half of these individuals also carried detectable anti-hbs. The frequency of OBI varied geographically (Table 2). In Eastern and Southern

9 D. Candotti et al. / Journal of Hepatology 49 (2008) Europe blood donor anti-hbc prevalence is >4% and OBI prevalence ranged between 1:2000 and 1:15,000. These differences might be related to epidemiology (genotype), to the intensity and duration of human intervention on HBV such as vaccination or to differences in sensitivity of the testing methods. Different HBsAg screening assays were used in this study that might have different ability to detect HBsAg variants introducing a potential bias into the estimation of OBI frequency [23 25]. Among anti-hbc carrying blood donors, anti-hbs ranges between 88 and 91% (1). OBIs presented an excess of anti-hbc-only positive donations. This may simply be related to a longer period of carriage in older donors who may start having a relatively less efficient immune control of the infection but may also indicate that low level HBV DNA replication might be more frequent in chronic HBV carriers having lost detectable HBsAg. The overall distribution between genotypes D and A2 in the European countries represented in this study was 73 and 27%, respectively. Compared to HBsAg+ genotype distribution, OBI D frequency was significantly higher in Italy and Poland and approaching significance in Spain (Table 3). Short of extensive details regarding the natural history of HBV in these patients and donors, no explanation can be offered at this time. Further characterization of OBIs relied essentially on analyzing viral genome sequences. The low viral load limited the success of this approach. The data summarized in Table 4 and Figs. 1 and 2 strongly suggest a predominant role played by the host immune system in determining the OBI condition. Several lines of evidence support such a role. First, the diversity of aa in the Pre-S/S proteins of OBIs was significantly higher than in the HBsAg+ groups, irrespective of genotype (Table 4). Importantly, this frequency of aa substitutions was significantly higher in the immunoreactive regions of these proteins whether related to humoral or cellular immunity (P < ). In nearly half of OBIs, the presence of anti-hbs clearly indicated humoral immune pressure against the MHR of the surface proteins that was not present in the HBsAg+ strains. In addition, the frequency of substitutions was significantly higher in the subgroup of samples containing anti-hbs than in those without this marker (Table 4). Of note, most substitutions previously reported as escape mutants interfering with HBsAg detection were found (see Fig. 1), many others individually or in combination may also interfere with surface antigen detection [24 26]. It is also possible that, anti- HBs evolving in parallel with HBsAg, some mutated antibodies may not be detected by the available commercial assays. Such diversity was not observed in other viral structural proteins coded by the pol or the core genes. The considerably lower level of aa substitutions affecting the pol gene reading-frame indirectly supports the hypothesis. Second, the mutations leading to aa substitutions in the major S protein of OBI strains were concentrated in the antigenic loops ( a region of MHR) previously described as affected in escape mutants elicited by passive or active HBsAg immunity [27,28] (Table 4 and Fig. 1). If the potential role of s in infectivity of Delta virus is confirmed in HBV, the high frequency of mutations in these six aa (61 and 43% for genotype D and A2, respectively) might participate in the mechanism of OBI occurrence. The pattern of highly mutated genotype D but also genotype A2 strikingly contrasts with a virtual absence of such mutations in the recently described genotype E OBIs [11]. However, the overall frequency of these mutations might be affected by the variable ability of the HBsAg screening assays to detect MHR mutants as already mentioned. Thirdly, high levels of aa substitutions were observed in putative CD8 T-cell epitopes located within pre-s1, pre-s2 and S regions (Fig. 2). It has been suggested that a strong polyclonal CTL response to HBV plays an essential role in viral clearance and clinical recovery during acute hepatitis and that an ineffective CTL response is the primary determinant of viral persistence [29]. A diminished CTL response that allows viral persistence might select cellular immunity escape mutants that would be even less well recognized by an already ineffective immune system and thereby contribute further to persistent infection [29]. CTL epitopes located in the core and pol regions were essentially conserved suggesting that different HBV genes evolved independently according to differences in the immune pressure. Interestingly, the two donors from Poland (P7 and P13) who reported having been vaccinated against HBV but whose plasmas did not contain detectable anti-hbs had the most heavily substituted sequences in the a region of genotype A2 and D, respectively. This observation is compatible with the possibility that the heavily mutated anti-hbc-only OBIs such as P10, P17, V7 or CT2 might correspond to donors who had recovered from HBV infection and mounted an effective humoral response but whose anti-hbs was either no longer detectable or directed against mutants escaping detection with the current assays (Table 1, Fig. 1). Little mutated or wild-type strains such as P9, P11, T6, V2, V9, CT1 and CT3 may correspond to chronic infections relatively well controlled by the immune system and having lost detectable HBsAg. From these converging observations, it appears clear that a mechanism of immune escape, humoral, cellular or both, plays an important role in the genesis of occult HBV infections. The molecular data obtained from 16 whole genome sequences and a larger number of BCP/PC and Pre-S/S sequences provided some insight into potential viral mechanisms involved in occult HBV carriage. Some degree of diversity was found in domains important for gene expression and virus replication as previously

10 546 D. Candotti et al. / Journal of Hepatology 49 (2008) reported [11,30,31], but there is no evidence that the mutations observed play a critical role in occult HBV carriage. However, mutations in the pre-core region preventing the production of the HBe antigen are known to play a role in or to be associated with lowering the viral load but may also allow the production of anti-hbe. The main feature in the BCP/PC region in genotype D strains was the relatively high frequency (24%) of mutated pre-core start codon ( ) and low frequency of 1896 stop codon (22%) in individuals with very low viral load. From eight genotype A2 sequences, only one pre-core start codon mutation was observed. Consequently, 7/8 A2 strains but only 53% of genotype D strains showed the genetic ability to express HBe antigen. However, the difference in the ability to produce HBe antigen was not reflected in viral load, which was similar, irrespective of genotype. In conclusion, occult HBV strains showed a higher genetic heterogeneity in the S gene compared to strains from HBsAg+ individuals. The clustering of substitutions in critical immunoactive sites suggest that the host immune system, whether humoral or cellular, likely plays a critical role in selecting or allowing the establishment and/or maintenance of occult HBV carriage. Acknowledgement The authors recognize the role of the International Blood Transfusion Society (ISBT) Transfusion Transmitted infection (TTI) working party for initiating the establishment of the HBV safety subgroup and encouraging its activities. The companies supporting the working party are thanked for making this possible. Dr. Nico Lelie is thanked for his role in establishing the subgroup network and shipment of samples. The molecular work involved in this study was supported in part by a grant from the National Health Service Blood and Transplant, England (BS 2003/2), the ISBT Foundation and Novartis/Chiron Corporation. The HBV DNA positive blood donor study in Poland was supported by a grant from the Ministry of Science and Education (No. N /3601). References [1] Allain J-P. Occult hepatitis B virus infection: implications in transfusion. Vox Sang 2004;86: [2] Brojer E, Grabarczyk P, Liszewski G, Mikulska M, Allain J-P, Letowska M, et al. Characterization of HBV DNA+/HBsAg blood donors in Poland identified by triplex NAT. Hepatology 2006;44: [3] Rehermann B, Ferrari C, Pasquinelli C, Chisari FV. The hepatitis B virus persists for decades after patients recovery from acute viral hepatitis despite active maintenance of a cytotoxic T- lymphocyte response. Nat Med 1996;2: [4] Raimondo G, Pollicino T, Cacciola I, Squadrito G. Occult hepatitis B virus infection. J Hepatol 2007;46: [5] Allain J-P, Reesink HW, Lucey C. A European perspective on the management of donors and units testing positive for hepatitis B virus DNA. Transfusion 2006;46: [6] Allain J-P. International collaborative study proposal for the characterization of occult hepatitis B virus infection identified by nucleic acid or anti-hbc screening. Vox Sang 2007;92: [7] Koppelman MH, Sjerps MC, Reesink HW, Cuypers HT. Evaluation of COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen HBV DNA, HCV RNA and HIV-1 RNA amplification and detection. Vox Sang 2005;89: [8] Schmidt MC, Nubling M, Scheiblauer H, Chudy M, Walch LA, Seifried E, et al. Anti-HBc screening of blood donors: a comparison of nine anti-hbc tests. Vox Sang 2006;91: [9] Sauleda S, Maymo RM, Piron M, Candotti D, Allain J-P, Hernandez JM, et al. Serological and molecular markers of hepatitis B virus in Spanish blood donors: evidence of occult hepatitis B. Vox Sang 2006;93:63. [10] Heyns ADP, Swanevelder JP, Lelie PN, Crookes RL, Busch MP. The impact of individual donation NAT screening on blood safety. ISBT Science Series. Vox Sang 2006: [11] Zahn A, Li C, Danso K, Candotti D, Owusu-Ofori S, Temple J, et al. Molecular characterization of occult hepatitis B in genotype E-infected subjects. J Gen Virol 2008;89: [12] Candotti D, Opare-Sem O, Rezvan H, Sarkodie F, Allain J-P. Molecular and serological characterization of hepatitis B virus in deferred Ghanaian blood donors with and without elevated alanine amino transferase. J Viral Hepat 2006;13: [13] Abou-Jaoudé G, Sureau C. Role of the antigenic loop of the hepatitis B virus envelope proteins in infectivity of Delta virus. J Virol 2005;79: [14] Dzierzanowska-Fangrat K, Woynarowski M, Szczygielska I, Jozwiak P, Cielecka-Kuszyk J, Dzierzanowska D, et al. Hepatitis B virus genotypes in children with chronic hepatitis B in Poland. Eur J Gastroenterol Hepatol 2006;18: [15] Bielawski KP, Charmuszko U, Dybikowska A, Stalke P, Podhajska AJ. Genetic variability of hepatitis B virus isolates in Poland. Virus Genes 2006;33: [16] Bielawski KP, Dybikowska A, Lisowska-Charmuszko U, Stalke P, Gregorowicz K, Trocha H, et al. Distribution of HBV genotypes and mutants among hepatitis B infected patients from northern Poland. Int J Mol Med 2004;14: [17] Dal Molin G, Poli A, Croce LS, D Agaro P, Biagi C, Comar M, et al. Hepatitis B virus genotypes, core promoter variants, and precore stop codon variants in patients infected chronically in North-Eastern Italy. J Med Virol 2006;78: [18] Medici MC, Aloisi A, Martinelli M, Abelli LA, Casula F, Valcavi P, et al. HBV genotypes and antiviral-resistant variants in HBV infected subjects in Northern Italy. New Microbiol 2006;29: [19] Rodriguez-Frias F, Jardi R, Buti M, Schaper M, Hermosilla E, Valdes A, et al. 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11 D. Candotti et al. / Journal of Hepatology 49 (2008) after universal vaccination in Taiwan. Hepatology 1999;30: [25] Coleman PF, Chen YCJ, Mushahwar IK. Immunoassay detection of hepatitis B surface antigen mutants. J Med Virol 1999;24: [26] Jolivet-Reynaud C, Lesénéchal M, O Donne B, Becquart L, Foussadier A, Forge F, et al. Localization of hepatitis B surface antigen epitopes present on variants and specifically recognized by anti-hepatitis B surface antigen monoclonal antibodies. J Med Virol 2001;65: [27] Carman WF, Zanetti AR, Karayiannis P, Waters J, Manzillo G, Tanzi E, et al. Vaccine-induced escape mutant of hepatitis B virus. Lancet 1990;336: [28] Carman WF, Owsianka A, Wallace LA, Dow BC, Mutimer DJ. Antigenic characterization of pre- and post-liver transplant hepatitis B surface antigen sequences from patients treated with hepatitis B immune globulin. J Hepatol 1999;31: [29] Bertoletti A, Costanzo A, Chisari FV, Levrero M, Artini M, Sette A, et al. Cytotoxic T lymphocyte response to a wild-type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. J Exp Med 1994;180: [30] Pollicino T, Raffa G, Costantino L, Lisa A, Campello C, Squadrito G, et al. Molecular and functional analysis of occult hepatitis B virus isolates from patients with hepatocellular carcinoma. Hepatology 2007;45: [31] Chaudhuri V, Tayal R, Nayak B, Kumar Acharya S, Kumar Panda S. Occult hepatitis B virus infection in chronic liver disease: full-length genome and analysis of mutant surface promoter. Gastroenterology 2004;127: [32] Ghiazza GP, Cesale Ros E, Cornagliotto G, Demarin G, Demarchi G, Gariglio V, et al. HBV-DNA and HBcAb IgG in blood donor population. Vox Sanguinis 2006;91:51.

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