Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium1
Institute of Immunology and Transfusion Medicine, Ernst Moritz Arndt University, Greifswald, Germany2
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, VA, USA3
Departamento de Bioquimica y Biologia Molecular, Universidad Complutense, Madrid, Spain4
Author for correspondence: Peter Vanlandschoot. Fax +32 9 240 63 11. e-mail Peter.Vanlandschoot{at}rug.ac.be
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Abstract |
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Introduction |
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One of the most remarkable features of HBV is its production of three different virus particles: infectious virions (Dane particles) and two types of non-infectious sub-virus particles, termed hepatitis B surface antigen (HBsAg). HBsAg consists mainly of spherical particles and a small amount of filamentous particles. These spheres and filaments can accumulate to several hundred µg/ml in blood of HBV-infected patients. The spherical particles contain virally encoded membrane proteins and approximately 30% (by weight) of host cell-derived lipids. The S protein accounts for more then 90% of the protein contained in HBsAg. The L and M protein forms the remainder. S, M and L share 226 C-terminal amino acids. The M protein contains an N-terminal extension of 55 amino acids, termed the pre-S2 sequence. The L protein is similar to M but is elongated at its N-terminal end with another 120128 amino acids (pre-S1 sequence). Both glycosylated and non-glycosylated forms of these viral membrane proteins are present in the particles (Ganem, 1996 ; Seeger & Mason, 2000
). Several cellular membrane and serum proteins have been shown to interact with the viral membrane proteins or HBsAg. The pre-S2 region is considered to be involved in the attachment to polymerized human serum albumin, an unusual glycan structure, fibronectin and the transferrin receptor (Imai et al., 1979
; Franco et al., 1992
; Gerlich et al., 1993
; Budkowska et al., 1995
). In recent years, it has been suggested that the pre-S1 domain contains an attachment site for glyceraldehyde-3-phosphate-dehydrogenase, an IgA receptor, IL-6, asialoglycoprotein, an 80 kDa protein and an SCCA1 homologue (Petit et al., 1992
; Pontisso et al., 1992
; Neurath et al., 1992
; Treichel et al., 1994
; Ryu et al., 2000
; De Falco et al., 2001
). Attachment of the S protein to annexin V and apolipoprotein H has been reported (Hertogs et al., 1993
; Mehdi et al., 1994
).
The lipid composition of HBsAg has been determined as well. The main lipid components are phospholipids (67%), cholesteryl ester (
15%), cholesterol (
14%) and triglycerides (
3%). Phosphatidylcholine (PC) accounts for approximately 90% of the phospholipids, while phosphatidylethanolamine accounts for 24%. Trace amounts of phosphatidylserine (PS), sphingomyelin, lysophosphatidylcholine and lysophosphatidylethanolamine are present (Kim & Bisell, 1971
; Gavilanes et al., 1982
). Phospholipid components of HBsAg have been suggested to be involved in the binding of particles to two proteins, apolipoprotein H and annexin V (Neurath & Strick, 1994
; De Meyer et al., 1999
; Stefas et al., 2001
). Removal of the lipids reduces the helical content of the HBsAg proteins and recognition by monoclonal antibodies (mAbs) (Gavilanes et al., 1990
). Reconstitution of these particles stripped of their lipids with both neutral and negatively charged phospholipids restores the original morphology of the particles and topology of the proteins. However, the helical content and antigenic activity is restored only with acidic phospholipids (Gomez-Gutierrez et al., 1994
, 1995
). Several observations suggest that the lipid content affects the immunogenicity of HBsAg. Triton X-100-extracted particles were reported to be more immunogenic than the native particles (Skelly et al., 1981
), while incorporation of HBsAg into liposomes composed of PC and cholesterol induced higher levels of antibodies (Manesis et al., 1979
). Furthermore, treatment of HBsAg with phospholipase C, which removes the phosphoalcohol head groups of the phospholipids, enhances its immunogenicity (Baijot, 1991
; Diminsky et al., 2000
).
Recently, we observed the preferential binding of biotinylated recombinant HBsAg (b-rHBsAg) to the CD14+ cell population of PBMCs. Binding to monocytes was enhanced by a heat-labile serum protein. It was shown further that rHBsAg suppressed the LPS- and IL-2-induced production of cytokines (Vanlandschoot et al., 2002 ). Here, it is reported that rHBsAg particles bind to monocytes through interaction with the LPS-binding protein (LBP) and the well known LPS receptor CD14. It is demonstrated further that attachment of rHBsAg occurs through a domain on CD14 that is identical to or largely overlaps with the LPS-binding pocket. We demonstrate also that plasma-derived HBsAg (pHBsAg) is not endowed with the property to bind in an LBP-dependent manner to the surface of CD14-expressing cells. Experimental evidence is presented that charged phospholipids not present in pHBsAg determine the interaction with (cellular) receptors.
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Methods |
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rHBsAg was biotinylated using an ECL protein biotinylation module (RPN 2202, Amersham Pharmacia), as described before (Vanlandschoot et al., 2002). Biotinylated rHBsAg (b-rHBsAg) was purified by gel filtration on a Sephadex G25 column using PBS. Fractions of 1 ml were collected and the two b-rHBsAg peak fractions were pooled.
pHBsAg.
pHBsAg (subtype adw or ayw) was purified by precipitation with PEG-8000, potassium bromide floatation, caesium chloride-gradient ultracentrifugation, agarose 4B gel filtration and matrix cellufine sulfate-affinity chromatography. Three different pHBsAg preparations were used. The first contained 280 µg/ml of protein and was stored at 4 °C for several months after purification. The second was stored in lyophilized form and contained 500 µg/ml of protein upon reconstitution. The third was stored at -70 °C immediately after purification from plasma and contained 500 µg/ml of protein.
pHBsAg was stripped of its phospholipids and reconstituted with different phospholipids, as described previously (Gavilanes et al., 1990 ; Gomez-Gutierrez et al., 1994, 1995). The phospholipids used were PC (from egg), PS (from bovine brain) and 1,2-dioleoyl-phosphatidylglycerol (DOPG). pHBsAg, pHBsAg stripped of its lipids (Del-pHBsAg) and pHBsAg reconstituted with PC (PC-pHBsAg), PS (PS-pHBsAg) and DOPG (DOPG-pHBsAg) were in 10 mM TrisHCl, pH 7·0, 50 mM NaCl buffer containing 280, 300, 160, 300 and 290 µg/ml of protein, respectively.
Soluble CD14 (sCD14) and LBP.
sCD14 and LBP were expressed in CHO cells and purified, as described elsewhere (Stelter et al., 1999 ), or were purchased from Biometec.
Antibodies.
Mouse anti-human CD14 and anti-CD14FITC (clone P9) and streptavidinephycoerythrin (StrepPE) were from Becton Dickinson. Mouse anti-human CD14 and anti-CD14FITC (clone My4) were obtained from Immunotech. Mouse anti-human CD18FITC (clone 6.7) was purchased from Pharmingen. Ascites fluid of mouse anti-human CD14 clones biG4 (IgG1) and biG11 (IgG1), purified mouse anti-human CD14 clone biG2 (IgG2a) and rabbit anti-human CD14 antiserum were from Biometec. Mouse anti-d and anti-y were a kind gift from DiaSorin. Human anti-HBsAg clones F47B and F9H9 were a kind gift from L. Sillekens (Centraal Laboratorium van de Bloedbank, Amsterdam). Human anti-a was developed in the laboratory. The following isotypic controls were used: mouse IgG1 (Becton Dickinson) or ascites fluid of LMBH6 (Vanlandschoot et al., 1998 ) and IgG1FITC (Becton Dickinson), mouse IgG2a (Pharmingen), mouse IgG2aFITC (Caltag), mouse IgG2b and IgG2bFITC (Coulter) and rabbit IgG (Sigma). For FACS analysis, non-labelled antibodies were detected with goat anti-rabbit IgGFITC (Pharmingen) and rabbit anti-mouse F(ab')2FITC (DAKO). Human antibodies were detected with rabbit anti-human F(ab')2FITC. Goat anti-humanHRP (Sigma) serum was used for ELISA.
Cells.
Human PBMCs were isolated from buffy coats using Ficollhypaque (density=1·077 g/ml, Nycomed Pharma) centrifugation. Cells were stored in liquid nitrogen. CHO cells expressing human CD14, CD14(3941,43,44)A and CHO cells transfected with the vector only (Stelter et al., 1997 ; Jack et al., 1995
) were grown in MEM
without nucleosides and ribonucleosides (Gibco BRL) supplemented with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin and 100 nM (plasmid-transfected CHO cells) or 500 nM methotrexate (CD14-expressing CHO cells). Cultured cells were detached mechanically or by using non-enzymatic cell-dissociation buffer (Sigma), washed twice with 2% non-heat-inactivated human AB serum (HS, BioWhittaker) in Hanks' balanced salt solution (Gibco BRL) (2% HSHBSS) and stained as described below.
Staining of cells.
Cells were incubated with (b)-rHBsAg in 200 µl 12% HSHBSS (or 0·5 µg/ml LBP in HBSS or 0·5 µg/ml LBP in 1% HSHBSS, as indicated) for 1 h on ice. After two washes with the same solution, cells were incubated with StrepPE- and/or FITC-labelled antibodies in 2% HSHBSS for 1 h on ice. After two washes, cells were resuspended in 1 ml 2% HSHBSS or PBS containing propidium iodide (PI) and analysed on a FACScan flow cytometer (Becton Dickinson). Dead cells that incorporated PI were gated out of analysis. At least 5000 cells were counted per analysis. Fluorescence (530 nm for FITC and 580 nm for PE) was measured; median fluorescence was determined in each case. The signals were acquired in a logarithmic mode for Fl1 (FITC) and Fl2 (PE). Threshold levels were set according to negative (StrepPE only) and isotypic controls. Non-biotinylated HBsAg particles were detected using S-specific mAbs followed by rabbit anti-humanFITC or rabbit anti-mouse F(ab')2FITC.
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Results |
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CD14-specific antibodies can inhibit attachment of b-rHBsAg to monocytes
PBMCs were incubated with increasing amounts of anti-CD14FITC clone My4 or P9. After washing, the cells were incubated with b-rHBsAg. As shown in Fig. 1, a partial inhibition of attachment of b-rHBsAg was obtained when cells were pre-incubated with increasing concentrations of antibody P9. My4 almost completely inhibited the binding of b-rHBsAg to the monocytes, even at the lowest concentration used. (Fig. 1b
). A mAb directed against CD18, another LPS-binding molecule, had no effect on b-rHBsAg attachment (data not shown). These results strongly suggest that CD14 is indeed involved in the attachment of rHBsAg to monocytes.
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Effect of phospholipids on the attachment of b-rHBsAg to PBMCs
Cells were incubated with PC, PS and DOPG in 2% HSHBSS. After washing, PBMCs were incubated with b-rHBsAg. Of the three lipids examined, PC did not inhibit attachment of b-rHBsAg. Both PS and DOPG reduced strongly the binding of b-rHBsAg to monocytes (Fig. 6). Because the lipids were removed before b-rHBsAg (in fresh 2% HSHBSS) was added to the PBMCs, the lipids attached to the cells caused the inhibition.
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The LBP- and CD14-dependent attachment of rHBsAg, pHBsAg, Del-pHBsAg, PC-pHBsAg, PS-pHBsAg and DOPG-pHBsAg to the membrane of CHO cells was examined. As shown in Fig. 7, LBP already induced attachment of rHBsAg to CHO-DHFR control cells. Of the different pHBsAg preparations, only DOPG-pHBsAg bound to CHO-DHFR cells. Stronger binding of rHBsAg, PS-pHBsAg and DOPG-pHBsAg to CHO-CD14 was demonstrated, while even in the presence of LBP, no attachment was seen for pHBsAg, Del-pHBsAg and PC-pHBsAg to these cells. However, Del-pHBsAg and PC-pHBsAg are hardly recognized by mAb F47B, which was used to detect the particles on the cell surface. Therefore, the capacity of the different HBsAg preparations to inhibit binding of b-rHBsAg to PBMCs was examined (Fig. 8
). Of the different preparation, 10 and 50 µg/ml, together with 0·5 µg/ml LBP in 1% HSHBSS, were used to saturate all possible binding sites on the monocytes. After washing, PBMCs were incubated with b-rHBsAg. The use of 10 µg/ml of rHBsAg clearly inhibited (80%) the attachment of b-rHBsAg. Hardly any inhibition, even with 50 µg/ml, was obtained with pHBsAg, Del-pHBsAg and PC-pHBsAg. Both PS- and DOPG-pHBsAg inhibited binding of b-rHBsAg very efficiently. Taken together, these experiments show that plasma-purified HBsAg does not bind to the surface of cells that express CD14. pHBsAg can gain this characteristic when certain phospholipids are incorporated into the particles.
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Using CHO-CD14 cells, it is possible to detect non-biotinylated rHBsAg on the cell membrane and, therefore, to study the effect of the other mAbs (anti-d, anti-a, F9H9 and F47B) on the attachment of rHBsAg to the cell surface. All these mAbs detected rHBsAg bound to the cell surface of CHO-CD14 cells (data not shown). Binding of rHBsAg pre-incubated with the different mAbs was compared to the binding of rHBsAg only. Particles were detected with the mouse mAb anti-d. As a control, the effect of the mAbs on the recognition of rHBsAg already bound to the cell surface by mAb anti-d was determined. As shown in Fig. 9, identical signals were observed if rHBsAg was pre-incubated or not with mAb anti-d, which suggests that this mAb does not inhibit binding. Human mAbs anti-a and F9H9 did not interfere with recognition by anti-d of rHBsAg already bound to the cells. Pre-incubation of rHBsAg with these mAbs did not result in reduced detection by mAb anti-d, which suggests that these two human mAbs do not inhibit binding. Surprisingly, mAb F47B interfered with the detection by mAb anti-d of rHBsAg on the cell surface. Nevertheless, after pre-incubation of rHBsAg with F47B, a further reduced detection by mAb anti-d was obtained; this suggests that mAb F47B can (partially) inhibit the attachment of rHBsAg to the cell surface, an observation made before. Taken together, these results indicate that the C-terminal region of the S protein might also play a role in the attachment of rHBsAg to CD14-expressing cells.
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Discussion |
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In contrast to rHBsAg, which binds to the membrane of CD14+ cells through interaction with LBP, pHBsAg does not. The results from the reconstitution experiments demonstrate clearly that this capacity to bind to the cell surface is determined by the phospholipid content of the particles. Attachment to CD14 cells does not occur upon reconstitution of pHBsAg with PC. Upon reconstitution of Del-pHBsAg with PS or DOPG, highly efficient attachment is observed. The lipid content of plasma-purified and S. cerevisiae-derived HBsAg particles has been determined (Gavilanes et al., 1982 ; Van der Meeren et al., 1994
). PC and neutral lipids are the major components, phosphatidylethanolamine being a minor one. The most striking difference is the abundance (2732%) of PI in the yeast-derived particles. This phospholipid is not present in natural HBsAg. Like PS, PI has been shown to bind to LBP and CD14. Based on these observations, it is proposed here that PI and PS are the phospholipids that determine the LBP-dependent attachment of rHBsAg to the membrane of CD14-expressing cells. The question remains whether attachment of rHBsAg to the cells results solely from a phospholipidreceptor interaction. PC does not bind to CD14 or LBP, while PS is known to bind to LBP and CD14. DOPG is known to bind LBP (Schromm et al., 1996
; Wurfel & Wright, 1997
; Yu et al., 1997
). Because DOPG blocks binding of rHBsAg, DOPG most probably binds to the cell surface through interaction with LBP and CD14. Therefore, it is attractive to conclude that binding of rHBsAg to the cells results solely from a phospholipidreceptor interaction. However, a precise conformational structure of the S protein, which depends on the presence of PS and PI, might be required for HBsAg to interact with CD14 and LBP. Indeed, the possible involvement of the C-terminal end of the S protein cannot be excluded, as demonstrated by the antibody-inhibition experiments. This region, amino acids 160207, has been predicted to form two membrane-spanning domains (Stirk et al., 1992
). However, several mAbs have been reported to bind to this region, which was suggested recently to lie on the surface of the membrane with the hydrophilic face in contact with an aqueous environment (Paulij et al., 1999
; Jolivet-Reynaud et al., 2001
). Mutations in the region of amino acids 112145 can affect the recognition of the 160207 amino acid region by mAbs. It is thought that the C-terminal region is close to a region around residue 120 of the same monomer or the adjacent monomer (Chen et al., 1996
). The observation that mAb F47B interferes with binding of mAb anti-d supports further this new structural concept.
The total lack of PI and the presence of only trace amounts of PS in pHBsAg is remarkable, if one considers that HBsAg, when expressed in mammalian cells (like CHO, human hepatoma cells and mouse fibroblast cells) or yeast cells (S. cerevisiae and Hansenula polymorpha) contains always 47% of PS and/or phosphatidylinositol at least (Gavilanes et al., 1982 ; Van der Meeren et al., 1994
; Satoh et al., 1990
, 2000
; Diminsky et al., 1997
). Furthermore, reconstitution experiments have demonstrated that good recovery of antigenicity is obtained only when negatively charged phospholipids, like PI and PS, are used (Gomez-Gutierrez et al., 1994
, 1995
). Although the results of such reconstitution experiments do not prove that these lipids are required for correct folding in vivo, the fact that HBsAg particles assemble in the membrane of the endoplasmic reticulum, which contains 1020% molar PI, makes the lack of PI in pHBsAg even more puzzling. However, LBP and sCD14 are known to catalyse the transfer of charged phospholipids (Schromm et al., 1996
; Wurfel & Wright, 1997
; Yu et al., 1997
). This raises the possibility that during HBV infection, PI and PS are initially present in particles produced by infected hepatocytes and that these two lipids are removed by LBP, CD14 or other lipid transfer molecules.
Whatever the in vivo relevance of our observations, the discovery of the rHBsAgCD14 interaction is probably very important for future HBV vaccine development. Recombinant vaccines replaced the plasma-derived vaccines in the course of the 1990s. These former vaccines have proven to induce a similar rate of seroconversion and protection against HBV as the latter (Leroux-Roels et al., 2001 ). However, considering the effects of the phospholipid composition on the antigenic and immunogenic structure (Skelly et al., 1981
; Manesis et al., 1979
; Baijot, 1991
; Gomez-Gutierrez et al., 1994
, 1995
; Diminsky et al., 2000
), it seems likely that qualitative and quantitative differences can exist in the antibodies obtained upon immunization with different HBsAg preparations. Indeed, reduced anti-HBsAg levels after immunization with the yeast-derived vaccine compared to the plasma-derived vaccine has long been recognized (Heijtink et al., 1985
; Jilg et al., 1984
). It was proposed recently that this is due to differences in the antigenic structure and incompatibility of HBsAg used in the quantification assays (Heijtink et al., 2000
). Our observations indicate that the anti-inflammatory and immunosuppressive potential of yeast-expressed HBsAg is another factor that might affect the induction of HBsAg-specific antibodies.
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Acknowledgments |
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Received 2 April 2002;
accepted 14 May 2002.