Medical Microbiology and Immunology Hepatitis B escape mutants in Scottish blood donors --Manuscript Draft-Manuscript Number: Full Title:
Hepatitis B escape mutants in Scottish blood donors
Article Type:
Original Investigation
Keywords:
Occult HBV infection; HBsAg screening; HBsAg mutation; Nucleic acid testing; Phylogenetic analysis; Immunoassay
Corresponding Author:
Osmany Larralde, Ph.D Scottish National Blood Transfusion Service Edinburgh, UNITED KINGDOM
Corresponding Author Secondary Information: Corresponding Author's Institution:
Scottish National Blood Transfusion Service
Corresponding Author's Secondary Institution: First Author:
Osmany Larralde, Ph.D
First Author Secondary Information: Order of Authors:
Osmany Larralde, Ph.D Brian Dow, PhD Lisa Jarvis, PhD Fiona Davidson, PhD Juraj Petrik, PhD, DSc
Order of Authors Secondary Information: Abstract:
Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralization site.
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Hepatitis B escape mutants in Scottish blood donors
Osmany Larralde, Brian Dow, Lisa Jarvis, Fiona Davidson and Juraj Petrik
Scottish National Blood Transfusion Service/NHS, Edinburgh, UK.
Corresponding author: Dr. Osmany Larralde, Microbiology and Components Group, RDI, Scottish National Blood Transfusion Service, 21 Ellen's Glen Road, Edinburgh EH17 7QT, United Kingdom Tel: +44-131-536 5870 Fax: +44-131-536 6012 Email:
[email protected]
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Abstract
Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralization site.
Keywords
Occult HBV infection; HBsAg screening; HBsAg mutation; Nucleic acid testing; Phylogenetic analysis; Immunoassay
Introduction Despite the continuous improvement of HBsAg serological assays since the 1970’s, HBV transmission is still considered the highest risk of viral transmission to blood components recipients in the UK (1.66 - 3.88 per million donations) [1, 2]. The implementation of HBV NAT in Scotland in March 2010 has reduced the residual risk of HBV transmission due to window period and has revealed cases of OBI; defined by the presence of HBV DNA without detectable HBsAg outside the acute phase window period, and most of the time accompanied by the presence of anti-HBc [3, 4].
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Since the current risk of HBV transfusion transmission seems to be mainly related to OBI and the emergence of escape mutant viruses that render blood donations false negative for HBsAg serological testing, this investigation was aimed to analyse the frequency of these rare events in Scottish blood donors and to use molecular techniques to study the effect and possible origin of these mutations.
Materials and methods
Donors and sample collection
This was a retrospective study looking at a selection of Scottish blood donors that have been found HBV positive either by serology or PCR (n=649). Samples from 1970 to 1988 were taken only from donors of west of Scotland, whereas samples from 1989 to 2011 included other areas of Scotland. All blood donors were voluntary and non-remunerated. Anonymised double-coded data was used in the study protocol in order to protect confidentiality of personal information. Plasma samples stored at -20oC were retrieved from archives and analysed for several HBV markers.
HBV NAT
Viral DNA was extracted from 200 µl of plasma sample using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Germany) according to the manufacturer’s instructions. For estimation of HBV load, a 10 µl aliquot of extracted HBV DNA was amplified using a nested PCR with secondary amplification and detection by real time PCR using the Roche Light Cycler V3.5. BVDV is used as an internal control, primers and probes are described elsewhere [5]. Reaction conditions were as follows: reverse transcription (required to amplify the internal control) at 48oC for 45 minutes, immediately followed by a single cycle of 94oC for 2 minutes then 30 rounds of amplification consisting of three stages: 30 seconds at 94oC, 21 seconds at 55oC and 1.5 minutes at 72oC. This is followed by a final cycle of 7 minutes at 72oC. Nested amplification and detection was performed on the Light Cycler using the following conditions, an initial denaturation for 2 minutes at 95C followed by 35 cycles, each consisting of 2 seconds at 94C, 20 seconds at 55C and 35 seconds at 72C. After this the samples are cooled to 40C. Primers (S1, S5, S3 and S6) and probes (F4 and P4) were designed to amplify a 157 base pair fragment of the surface gene (Table 1). An estimate of HBV DNA load was made by comparison with samples of known virus titre. Samples from 2010 were routinely tested for HBV DNA, HCV RNA and HIV RNA using Cobas TaqScreen multiplex nucleic acid test [6].
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HBV Serological Markers
Plasma samples were tested for HBsAg using the following commercial assays: BIOELISA HBsAg 3.0 (Biokit, SA, Spain), Monolisa HBsAg ultra (BIO-RAD, France), MUREX HBsAg (Abbott Murex Biotech, Ltd, UK) and PRISM HBsAg (Abbott Diagnostics, USA). Other serological markers of HBV infection investigated were HBeAg, anti-HBe, anti-HBc and IgM anti-HBc (Abbott Diagnostics, USA).
HBsAg Cloning and Expression
HBsAg gene was amplified by nested PCR using a high fidelity PFU DNA polymerase system (Promega, USA). All PCRs were done in 50 l reaction with 1 x PFU PCR buffer with MgSO4, 0.2 mM dNTPs, 1 M each primer, 1.25 U of PFU polymerase and 1 l template DNA. First round conditions consisted of initial denaturation for 2 minutes at 95C followed by 35 cycles, each consisting of 1 minute at 95C, 30 seconds at 57.7C (Ta) and 1 minute at 72C. Second round conditions were similar to first round, except Ta was 61.1 C. The final extension was 5 minutes at 72C for both rounds. Outer primers (O27 and O28) and inner primers (O12 and O13) were designed to incorporate BclI and SacI sites at each end of the amplified product for cloning HBsAg gene into BamHI/SacI of the mammalian expression vector pTriex-5 (Table 1). Positive colonies were identified by PCR and by KpnI/SacI digestion. Positive clones were sequenced in both directions using TriExUp and TriExDown primers (EMD/Merck Novagen, USA). This cloning strategy allows expression of the whole length HBsAg gene (681 nt) in frame with an Nterminal Strep-tag II and a C-terminal His-tag.
Constructs were purified using EndoFree Plasmid Maxi Kit (QIAGEN, Germany) and DNA quantified by NanoDrop spectrophotometer (Thermo Scientific) and gel electrophoresis. Equal amounts of maxiprep plasmid DNA were transiently transfected into a human hepatocarcinoma cell line (HepG2), purchased from ATCC-LGC Standards. HepG2 cells were grown in Advanced D-MEM with GlutaMAX (Life Technologies Invitrogen, USA) and 5% inactivated foetal calf serum. The number of passages from the master culture (passage 74) was minimised using a seed lot system. Briefly, HepG2 cells were seeded at a density of 1 x 10 6 cells in 60 mm cell culture dishes (FALCON) and transfected 24 hours later with 5 g of each construct and 15 μl of GeneJuice transfection reagent (EMD/Merck Novagen, USA). The transfection mixture was replaced by growth medium 6 h after. Cells were incubated for 3 days at 37°C (5% CO2) and the culture medium collected for analysis of secreted HBsAg.
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Sequence Analysis
HBV genotype was assessed by entering DNA sequences in the Genotyper tool of the HepSEQHepatitis B resource [7]. HBsAg serological subtype prediction was based on specific amino acid (aa) positions [8]. All data processing was performed using GENEIOUS PRO software [9]. Two hundred and forty three HBsAg nucleotide sequences were selected from NCBI and aligned to obtain consensus sequences of each HBV genotype. Analysis of HBsAg mutations was conducted by alignment with the consensus sequence of its genotype. The nucleotide sequence of escape mutants was blasted in GenBank and the closest related strains were used for construction of a dendrogram.
Results Eight of 649 HBV positive samples showed discordant results in four commercial HBsAg serological tests, while 5 samples were HBsAg negative and HBV DNA positive (table 2). Donors 2, 8, 10, 12 and 13 fulfil the criteria for OBI since in addition to being HBsAg negative in all assays, they had a very low viral load ( 125
0.48 Neg
0.97 Neg
1.37 Pos
1.36 Pos
Pos
Neg
Neg
ND
2
125
0.76 Neg
8.63 Pos
7.87 Pos
ND
Neg
Neg
Neg
ND
4
> 125
0.82 Neg
ND
1.04 Pos
Neg
Neg
Neg
Neg
Neg
5
> 125
0.55 Neg
0.28 Neg
10.21 Pos
ND
Neg
Pos
Neg
ND
6
> 125
0.60 Neg
3.98 Pos
2.62 Pos
3.74 Pos
Neg
Neg
Neg
Neg
7
> 125
0.45 Neg
1.57 Pos
2.28 Pos
1.65 Pos
Neg
Neg
Neg
Neg
8
