1 Division of Transfusion Medicine, Department of Haematology, University of Cambridge, Cambridge, UK
2 National Blood Service, Long Road, Cambridge CB2 2PT, UK
Correspondence
Jean-Pierre Allain
jpa1000{at}cam.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Heparin is a glycosaminoglycan (GAG) found in the secretory granules of mast cells (Rabenstein, 2002) and basophils (Hileman et al., 1998
). GAGs are negatively charged molecules that interact with positively charged amino acids by electrostatic forces, but, as they are structurally diverse, they can also interact specifically with proteins (Kjellen & Lindahl, 1991
; Shukla et al., 1999
). Recombinant HCV E2 envelope protein (Barth et al., 2003
) and hepatitis B surface antigen (HBsAg) (Einarsson et al., 1978
) bind to heparin. A wide variety of pathogens, including enveloped and non-enveloped viruses, such as flaviviruses (Chen et al., 1997
; Germi et al., 2002b
; Hilgard & Stockert, 2000
; Hulst et al., 2001
; Su et al., 2001
), human immunodeficiency virus type 1 (HIV-1) (Patel et al., 1993
), herpes simplex virus (Shieh et al., 1992
), Sindbis virus (Byrnes & Griffin, 1998
) and adenovirus type 2 (Summerford & Samulski, 1998
), use GAGs expressed on target cells as receptors or attachment factors.
Here, we examined the binding of HCV and hepatitis B virus (HBV) from patient plasma samples to heparin. IgG-free HCV and HBV interacted with immobilized heparin in preference to immunocomplexed virions. Based on the differential binding properties of viruses and plasma proteins to heparin, we have developed an efficient one-step method for purification of undamaged, largely IgG-free HCV and HBV from plasma that avoids ultracentrifugation.
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METHODS |
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Viral RNA/DNA extraction and quantification by RT-Q-PCR.
Viral DNA was extracted from plasma or column fractions by using a High Pure Viral Nucleic Acid kit (Roche Diagnostics). Viral RNA was extracted from plasma, column fractions, immunoprecipitated material and last washes after HCV binding to cells by using a QIAamp viral RNA mini kit (Qiagen).
Quantification was performed by Q-PCR using a PE Applied Biosystems Prism Model 7700 sequence detector for HCV RNA, as described previously (Hamaia et al., 2001), and the Mx4000 Multiplex quantitative PCR system (Stratagene) for HBV DNA, as described previously (Candotti et al., 2004
). Each sample was tested in duplicate and the mean result was determined. Variation between duplicates did not exceed 1 %. In each quantitative run, 10-fold serial dilutions of WHO international standards (HCV 96/790 I and HBV 97/746; National Institute for Biological Standards and Controls, Potters Bar, UK) or in-house reference plasmas validated against the international standards were included.
Analytical heparin-affinity chromatography.
Plasma samples were loaded onto 1 ml bed volume Hi-Trap heparin columns (Amersham Biosciences) equilibrated with 0·02 M Tris/HCl (pH 7·4), 0·15 M NaCl. The volume of plasma was adjusted to contain 1x106 IU virus, but never exceeded 1 ml. In cases of higher viral loads, samples were diluted in normal plasma to 0·5 ml final volume. In a few cases, a lower viral load was used, but not more than 1 ml of sample was used, in order to avoid saturating the column. After recirculation of the sample for 1 h at room temperature using a peristaltic pump, the column was washed with eight column volumes of equilibrating buffer (EB) and eluted with a 0·32 M NaCl step gradient in 0·02 M Tris/HCl over 20 bed volumes at a flow rate of 1 ml min1. Fractions of 35 ml were collected and viral nucleic acids were extracted and assessed by Q-PCR.
Estimation of the amount of IgG-free virus in plasma.
Plasma samples (volumes used as explained above) were loaded onto 1 ml bed volume Hi-Trap protein G columns (Amersham Biosciences) previously equilibrated with 0·05 M Tris/HCl (pH 7·5), 0·15 M NaCl (TBS). The column was washed with 10 ml TBS (IgG-free virus fraction), eluted with 5 ml IgG elution buffer (Pierce) and neutralized immediately with 1 M Tris/HCl, pH 9 (IgG-complexed virus fraction). Viral genomes were extracted from the flow-through (FT) plus washing and elution fractions and assessed by Q-PCR.
EH HCV purification by heparin-affinity chromatography.
HCV EH plasma in EB was applied to previously equilibrated Hi-Trap heparin columns and recirculated for 1 h at room temperature. Unbound plasma proteins were removed by washing with 8 ml 0·3 M NaCl EB (washing fraction) and bound material was eluted with 6 ml 0·4 M NaCl EB (elution fraction). The elution fraction was then concentrated approximately eightfold by using Centricon centrifugal filter devices with a 100 kDa cut-off (Amicon; Millipore) by centrifugation at 1000 r.p.m. at 4 °C. To adjust the concentration of NaCl to physiological levels, the devices were refilled with 0·05 M Tris/HCl (pH 7·4) and the sample was further concentrated. The virus quantity was monitored by RT-Q-PCR and the presence of the E2 envelope protein was checked by Western blotting for each fraction. Loss of virus associated with the concentration step was typically <30 %. Protein concentration was determined by using the BCA Protein assay (Pierce) with BSA as the standard. Purified virus was kept at 4 °C for not more than 2 days until used. The degree of purification was calculated as the ratio of IU HCV (mg total protein in eluted fraction)1 to IU HCV (mg total protein in original plasma)1.
Polyclonal antibody to EH.
Two EH plasmapheresis units were collected in 1998 and 2002 and a fragment (E1/E2) of EH HCV genome spanning aa 326502, including the hypervariable region (HVR-1) of E2 (aa 384411), was amplified, cloned and sequenced from both units. A consensus 15-mer peptide (EH1) was derived from 20 clones of the HVR-1 region sequences. The peptide (C)SGLAGLFTPGAKQNI was synthesized by Severn Biotech Ltd and an N-terminal cysteine residue was added for conjugation to maleimide-activated keyhole limpet haemocyanin (KLH) according to the manufacturer's guidelines (Pierce).
A polyclonal antibody against the conjugated KLHEH1 peptide was produced in rabbits (Harlan Sera-Lab). The antiserum was tested for antigen specificity and sensitivity, and total IgG (designated anti-EH1 antibody) was purified by using a Hi-Trap protein G column according to the manufacturer's recommendations. The antibody to EH1 recognized EH E2 envelope protein by Western blotting and was able to immunoprecipitate the virus from plasma (see below).
Electrophoresis and immunoblotting.
Volumes equivalent to 250 IU EH viral RNA from the FT plus 0·3, 0·4 and 2 M NaCl heparin fractions, and to 1000 IU viral RNA from the heparin-purified EH and concentrated fraction, were analysed by 10 % SDS-PAGE. Proteins were visualized by gel staining with Coomassie R-250 brilliant blue. For Western immunoblot analysis of the EH envelope E2 protein, volumes equivalent to 800 IU viral RNA from the heparin-purified viral preparation or concentrated fractions from the FT plus washing and the 0·3 M NaCl fractions were separated by 10 % SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell). Blots were probed with the anti-EH1 antibody (5 µg ml1) and revealed by using a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (diluted 1 : 10 000; Sigma) and the Supersignal West Pico chemiluminescent substrate for HRP (Pierce).
EH immunoprecipitation with anti-EH1 antibody.
IgG-depleted EH plasma (FT from HiTrap protein G column) or heparin-purified and concentrated EH plasma were incubated with 100 µl anti-EH1 antibody (40 µg ml1) for 2 h at 37 °C with gentle mixing, followed by 16 h incubation at 4 °C. A 50 % slurry of protein ASepharose (Sigma) was added to the mixture and incubated for 3 h at room temperature. The mixture was then centrifuged at 12 000 r.p.m. for 10 s and the resin was washed three times with TBS. After the last wash (which was kept to verify the absence of HCV RNA), viral RNA was extracted directly from the pelleted beads and quantified by RT-Q-PCR. Parallel experiments were carried out by using rabbit pre-bleed IgG (negative control) or IgG-depleted EH plasma, incubated directly with the protein ASepharose to determine the presence of any IgG-complexed virus that might be directly precipitated by protein ASepharose.
HCV binding to lymphocytes.
For binding experiments, Molt-4 T cells or U937 pro-monocyte cells (2x105) in the exponential phase of growth were used. Cells were washed with PBS and the pellets were resuspended in increasing concentrations of heparin-purified and concentrated EH plasma (300 µl final volume) and incubated at 37 °C for 90 min in 48-well plates within a humidified chamber. Cells were washed with ice-cold PBS, three times with 12 ml and once with 500 µl, and this final wash was saved to verify the absence of viral RNA. Total RNA was extracted from the cellular pellet and associated viral particles by using an RNeasy mini kit (Qiagen) following the manufacturer's instructions. To create a standard curve, 10 µl of 10-fold dilutions of HCV+ plasma ranging from 15 to 30 000 IU were mixed with 2x105 U937 cells and total RNA from these dilutions was extracted with an RNeasy mini kit.
Electron microscopy (EM) of HBV.
Glow-discharged, Formvar/carbon-coated electron microscope grids were floated for 30 s on 10 µl drops of the heparin-purified HBV fraction. After three washes on 10 µl drops of distilled water, samples were negatively stained with 2 % aqueous sodium phosphotungstate (pH 6·5) for 45 s, drained and examined by using a Philips CM 100 transmission electron microscope.
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RESULTS |
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Based on the differential binding to heparin of IgG-free HCV, IgG-complexed HCV and plasma proteins, we further analysed this interaction as a possible method for purification of Ig-free HCV from human plasma. From eight independent chromatography results of EH plasma, total protein concentration was reduced approximately 1000-fold in the 0·4 M elution fraction when compared with the input or the 0·3 M washing fraction (Fig. 1a). The degree of HCV purification reached in this fraction was calculated as approximately 1000-fold (930±76). The purity of the fractions was confirmed by SDS-PAGE. Only a few very faint bands were observed in the eluted fractions (Fig. 2a
). The number of bands did not increase significantly after silver staining (data not shown) or when the fraction was concentrated eight- to tenfold (see Methods) and stained with Coomassie blue (lane conc. in Fig. 2a
). Two of the most conspicuous bands in the heparin-purified and concentrated fraction (denoted with an asterisk in Fig. 2a
) matched the expected molecular mass of the HCV E2 (
68 kDa) and E1 (
36 kDa) envelope proteins. The presence of E2 protein in the concentrated fraction was confirmed by the detection of a specific 65 kDa band by Western blotting probed with an antibody directed against the HVR-1 region of EH (anti-EH1 antibody) (Fig. 2b, c
).
To confirm the presence of virions in the heparin-purified preparations, EH virus from plasma or from purified preparations was immunoprecipitated with the anti-EH1 antibody and assessed by Q-PCR. Similar amounts of virus (median, 20 %) were immunoprecipitated in both cases (Fig. 3a
), suggesting that, after heparin purification, antibody to the envelope E2 protein recognized the HVR-1 epitope and precipitated whole virions. Moreover, the treatment of heparin-purified HCV with non-ionic detergents (0·8 % Triton X-100 or 25 mM n-octyl-
-D-glucopyranoside) prevented the detection of viral RNA in the immunoprecipitated material (data not shown), further suggesting the presence of enveloped particles after the purification process.
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Overall, the results suggest that one-step heparin-affinity chromatography can be used to purify largely Ig-free, undamaged HCV particles from infected human plasma easily and efficiently.
HBV from infected human plasma binds to heparin and can be purified by heparin-affinity chromatography
Having developed a robust purification method for mostly Ig-free HCV, we extended our analysis to plasma samples from chronic HBV carriers. The stepwise elution used for HCV experiments yielded two different patterns of HBV binding to heparin (Fig. 4a and Table 1
). In four of eight samples, 5079 % of viral DNA was eluted at
0·4 M NaCl. In the other four samples, most viral DNA eluted at
0·3 M NaCl and only 1625 % eluted at higher NaCl concentrations. This difference was not related to viral load. The percentage of IgG-free HBV in circulation determined by viral retention on protein G columns ranged from <5 to 55 %, grossly correlating with retention on heparin columns (Table 1
).
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IgG-free HBV is the predominant fraction interacting with heparin
To determine whether, as for HCV, the heparin-bound viral fraction was composed preferentially of IgG-free virus, a series of experiments was designed, combining protein G and heparin chromatography (Table 2). The non-retained (enriched in IgG-free HBV) and the retained (IgG-complexed HBV) fractions of protein G chromatography (from sample HBV-3 in Table 1
) were loaded on heparin columns and bound virus was eluted by using stepwise concentrations of NaCl. Seventy per cent of the viral DNA, in the protein G non-retained fraction, was eluted from the heparin column with
0·4 M NaCl, whilst only 35 % of HBV DNA from the retained protein G fraction was eluted. Conversely, when the HBV sample was first applied to a heparin column and the dialysed 0·3 M (41 % of viral DNA) and
0·4 M NaCl (59 % of viral DNA)-eluted fractions were loaded on protein G columns, approximately 80 % of the virus present in the 0·3 M NaCl heparin fraction was retained on the protein G column, indicating the presence of IgG-associated virus. Only 12 % of the virus present in the
0·4 M NaCl heparin elution fraction was retained on the protein G column, most of it appearing as IgG-free virus. Comparable results were obtained with another HBV plasma sample (HBV-4 in Table 1
): when heparin fractions were applied to protein G columns (Table 2
), a clear enrichment in IgG-free virus was observed.
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DISCUSSION |
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The binding of HCV to heparin was used to purify the virus effectively from plasma, as only traces of contaminating plasma proteins co-eluted with HCV at 0·4 M NaCl (Figs 1 and 2a
). Our protocol is simple, gentle and does not require special technical skills or equipment, in particular caesium chloride- or sucrose-gradient ultracentrifugation, which are time-consuming and labour-intensive, may not remove contaminants with a density similar to the viral particles and may cause shedding of the envelope glycoproteins and disruption of viral particles (André et al., 2002
; Fujita et al., 2001
; Xiang et al., 1998
). In fact, following purification by heparin-affinity chromatography, HCV E2 was detected specifically (Fig. 2b and c
) and virions from plasma or heparin-purified fractions were similarly immunoprecipitated (Fig. 3a
), suggesting that the purification conditions did not disrupt the viral particles. Despite the high purity of the preparation after purification, detection of HCV by EM was unsuccessful, probably as a result of the low HCV particle concentration, as reported by Trestard et al. (1998)
. In contrast, the integrity of HBV particles after heparin chromatography was confirmed by transmission EM (Fig. 4b
). When purified HBV was concentrated by centrifugation as described in Methods for HCV purification and observed by using EM, complete particles were visible, suggesting that the purification and concentration procedures did not damage the HBV virions and thus were unlikely to have damaged the HCV particles. As a further criterion of the quality of the purified HCV, viral binding to target cells after heparin purification was investigated. Dose-responsive binding to both Molt-4 and U937 cells was observed reproducibly (Fig. 3b
), reaching saturation near to 1x105 IU, as reported previously (Hamaia et al., 2001
). This method is reproducible and rapid, as the virus purification can be completed within 1 day, and can readily be scaled up.
In contrast to the virus from an agammaglobulinaemic patient, the majority of HCV from immunocompetent chronic carriers was eluted at 0·3 M NaCl (Fig. 1
). This difference is best explained by the low percentage of IgG-free HCV in circulation (Table 1
; Hamaia et al., 2001
) compared with the agammaglobulinaemic patient, who cannot form HCV immune complexes. The increased percentage of HCV binding to heparin when the non-retained fraction from a protein G column (hence enriched in IgG-free virus) was submitted to heparin-affinity chromatography suggested that antibodies complexed to the virions interfere with the binding. For the HCV- and HBV-containing samples studied, we observed a higher percentage of heparin-bound than IgG-free (not retained on protein G columns) virus (Table 1
). This might indicate that a proportion of virions carrying fewer IgG molecules retain heparin-binding capacity. It would be easy to obtain IgG-free virus by a further protein G step, if required.
The recombinant HCV E2 protein binds to heparin with high affinity (dissociation constant of 5·2x109 M) (Barth et al., 2003), whilst the elution of virus in plasma at 0·4 M NaCl would suggest a weaker interaction. The ionic strength sufficient to elute HCV was similar to that used to elute other enveloped RNA viruses, such as Sindbis virus (Byrnes & Griffin, 1998
) and Japanese encephalitis virus (Su et al., 2001
). The apparent difference in affinity for heparin between recombinant E2 and whole viral particles might be explained by E2 forming part of a heterodimer with E1 at the viral surface. Alternatively, plasma proteins bound to virions may affect the binding to heparin. It is likely, however, that the system developed in this study is more representative of the interaction of HCV with GAGs. The GAG heparan sulphates are expressed at the surface of most, if not all, mammalian cells (Rabenstein, 2002
) and constitute an important cellular-attachment factor for many members of the family Flaviviridae, such as dengue virus (Chen et al., 1997
; Germi et al., 2002b
; Hilgard & Stockert, 2000
), classical swine fever virus (Hulst et al., 2001
), yellow fever virus (Germi et al., 2002b
) and Japanese encephalitis virus (Su et al., 2001
). Heparin is the closest homologue to liver heparan sulphate (Lyon et al., 1994
) and is often used experimentally to mimic the interaction with the latter. As IgG-free HCV eluted at higher-than-physiological salt concentrations (Fig. 1
; Table 1
), one could speculate that under these conditions, Ig-free, presumably infectious HCV (Hijikata et al., 1993
; Kanto et al., 1995
) may interact with heparan sulphate molecules expressed on target cells.
Heparin inhibits the binding of cultured HBV to HepG2 and primary hepatocytes (Ying et al., 2002). We studied the binding of HBV from chronic carriers to heparin and compared the results with those obtained with HCV. The data presented here indicated that HBV binding to and elution from heparin columns were similar to those for HCV (Fig. 4a
; Table 2
) and, as observed with HCV, there was also a positive correlation between the percentage of IgG-free HBV in circulation (not retained on protein G columns) and binding to heparin (Fig. 5
). HBsAg has previously been purified by heparin-affinity chromatography (Einarsson et al., 1978
; Tajima et al., 1992
) and we observed HBsAg in addition to intact virions in our heparin-purified HBV material (Fig. 4b
). During acute and chronic HBV infection, both complete virions and free, soluble HBsAg can be found in the serum of infected patients, the latter exceeding the concentration of virions by a factor of 104 or greater (Margolis et al., 1997
). It is possible that the differences in HBV binding to heparin between samples (Table 1
) could be explained by competition, depending on the ratio of whole virus to free surface antigen present. However, the chromatography volume input was in the order of 1050 µl (
1x106 IU virus) for the majority of samples and the heparin-binding sites were unlikely to be saturated. In fact, the same percentage of bound virus to heparin was obtained for two HBV samples when the volume input was up to 1 ml plasma. Thus, it appears that the virus-binding capacity to heparin is more probably related to the percentage of IgG-free virus in circulation. Supporting this, the HBV fraction not retained on protein G columns bound preferentially to heparin (Table 2
), even when the ratio of virus to free antigen should be lower for this fraction than for the original sample. Conversely, most of the purified HBV eluted from immobilized heparin did not bind to protein G (Table 2
). For some HBV samples, the percentage of virions retained on immobilized heparin was considerably higher than for HCV-containing samples, consistent with the absence of anti-HBsAg in the former and the presence of neutralizing antibodies in the latter.
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ACKNOWLEDGEMENTS |
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Received 17 September 2004;
accepted 30 November 2004.
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