Immunostimulatory potential of hepatitis B nucleocapsid preparations: lipopolysaccharide contamination should not be overlooked

Peter Vanlandschoot1, Freya Van Houtte1, Peter Ulrichts2, Jan Tavernier2 and Geert Leroux-Roels1


1 Virus Host Interactions Unit, Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium
2 The Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research (VIB9), Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium

Correspondence
Peter Vanlandschoot
Peter.Vanlandschoot{at}UGent.be


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The nucleocapsid of hepatitis B virus (HBV) allows insertions of heterologous peptides and even complete proteins. Because of its outstanding capacity to induce B-cell, T-helper and cytotoxic T-cell responses, this structure is considered to be an important instrument for future vaccine development. Most of the evidence for the unique immunogenic qualities of nucleocapsids has been generated in mice, which are not natural hosts of HBV. Moreover, most nucleocapsid preparations used in these studies were produced in a recombinant manner in Escherichia coli. Such preparations have been shown to contain lipopolysaccharide (LPS). Not unexpectedly, it is shown here that contaminating LPS, rather than the nucleocapsid structure itself, is responsible for the activation of human antigen-presenting cells. Careful examination of the literature dealing with the immunogenicity of HBV nucleocapsids suggests that the possible presence of LPS has been largely ignored or underestimated in several studies. This raises doubts on some of the underlying mechanisms that have been proposed to explain the unique immunogenicity of the HBV nucleocapsid.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The initial step in the activation of a B cell is the cross-linking of the antigen receptor. A second signal is required to enhance the response and to induce a switch from IgM to IgG (Bachmann & Zinkernagel, 1997; Baumgarth, 2000). This signal is normally delivered by specific T-helper (Th) cells. Multivalent repetitive structures are often capable of triggering strong, T cell-independent IgM responses. Many viruses exhibit such repetitive structures and, indeed, have been shown to induce IgM responses that are completely T cell-independent (T-independent type I response) (Bachmann & Zinkernagel, 1996). If help is not provided, the switch to IgG does not occur unless very high antigen doses are used (Szomolanyi-Tsuda & Welsh, 1998). As always, there are exceptions to this paradigm: the nucleocapsid of the hepatitis B virus (HBV) was shown to induce the switch from IgM to IgG in nude mice or CD4-depleted xid mice (Milich & McLachlan, 1986; Milich et al., 1997; Fehr et al., 1998). In immune-competent mice, a single injection of capsids stimulates IgM and IgG antibodies and Th-cell function. Immunizations with as little as 6 ng capsid in saline resulted in detectable anti-HBV core antigen (HBcAg) production 2 weeks after a single injection. Furthermore, capsids elicited primarily IgG2a and IgG2b antibodies, with a low level of IgG3 and no IgG1 antibodies. Cytokine production by capsid-primed Th cells confirmed this Th1 phenotype: capsid-primed T cells efficiently produced interleukin 2 (IL2) and gamma interferon (IFN-{gamma}), but low levels of IL4 (Milich & McLachlan, 1986; Milich et al., 1997). It was reported by Riedl et al. (2002) that encapsidated RNA facilitates the priming of this Th1 immunity in mice. Recent work has indeed demonstrated that single-stranded RNA (ssRNA) has strong immunostimulatory potential, and Toll-like receptor (TLR) 7 and 8 have been shown to mediate recognition of GU-rich ssRNA (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004; Scheel et al., 2004).

Because experiments with humans are limited, it is not known whether these observations also hold for man. HBV capsids induced the production of capsid-binding IgM molecules when peripheral blood lymphocytes (PBLs) of unprimed individuals together with capsids were transferred into the spleen of NOD/SCID recipient mice. This was observed with adult human and neonatal (cord blood) donors. In addition, capsids activated purified human B cells to produce anti-capsid IgM when injected into NOD/SCID mice, thus providing evidence that capsids behave as a T cell-independent antigen in humans. However, a switch from IgM to IgG production was not observed, even after a booster injection with capsids in vivo (Cao et al., 2001). This observation suggested that other signals needed to induce this switch were not or inefficiently triggered. Such signals might come from linked Th cells [not present in the experiments reported by Cao et al. (2001)], dendritic cells (DCs) or innate receptor ligands (Wykes et al., 1998; Poeck et al., 2004; Zinkernagel & Hengartner, 2004). Peripheral blood mononuclear cells (PBMCs) contain several types of antigen-presenting cells, such as monocytes, plasmacytoid DCs and myeloid DCs. Apparently, none of these cells were present in sufficient numbers or provided the signals necessary for the switch to IgG. Because unfractionated spleen cells and DCs of mice secreted IL12 when stimulated with nucleocapsids (Riedl et al., 2002), we investigated whether human antigen-presenting cells would be activated by nucleocapsids. We demonstrate here a strong activation of monocytes and DCs by recombinant nucleocapsid preparations. However, this activating potential of nucleocapsids is mediated by lipopolysaccharide (LPS) contaminants. LPS-free recombinant nucleocapsid preparations are difficult to obtain, especially when produced in Escherichia coli. Nevertheless, the presence of LPS in nucleocapsid preparations was and still is largely neglected in many studies. These observations cast doubt on some of the mechanisms that have been proposed to explain the extraordinary immunogenicity of the HBV nucleocapsid.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HBcAg.
Full-length HBcAg produced in E. coli (HBcAg-c) was obtained from Diasorin. Two full-length HBcAg nucleocapsid preparations, obtained from GlaxoSmithKline, were produced in Saccharomyces cerevisiae (HBcAg-y). To demonstrate the presence of encapsidated RNA, nucleocapsids were separated on a 1 % Tris/EDTA/acetic acid agarose gel, first stained with ethidium bromide, to visualize encapsidated RNA. Protein was visualized by staining with GelCode Blue Stain reagent (Pierce). The LPS content of the HBcAg-y preparations was determined by using the Coatest Chromo-LAL assay (Chromogenix).

Reagents.
Mouse anti-human CD14 (clone My4, IgG2b) was obtained from Immunotech. Purified mouse anti-human CD14 clone biG2 (IgG2a) was from Biometec. Mouse anti-human CD14 clone M5E2 (IgG2a), mouse IgG1 and IgG2a were from BD Biosciences Pharmingen. Mouse IgG2b was from Immunotech. Mouse anti-human CD14–fluorescein isothiocyanate (FITC), CD18–FITC, CD40–FITC, CD40–phycoerythrin (PE), CD80–FITC, CD80–PE, CD83–FITC, CD86–FITC, HLA-DR–FITC and streptavidin–PE (SAPE) were from BD Biosciences Pharmingen. Mouse IgG1–FITC, IgG2a–FITC and IgG2b–FITC were from BD Biosciences Pharmingen, Caltag and Immunotech, respectively. LPS (E. coli O111 : B4) and poly(I : C) were from Sigma.

Cytokine determinations.
The concentrations of human tumour necrosis factor alpha (TNF-{alpha}) and IL12p40 in cell supernatant were determined by using commercially available kits (Bioscource) according to the manufacturer's instructions.

Cells.
THP-1 cells were grown in cRPMI (RPMI 1640/10 % fetal calf serum/2 mM L-glutamine/1 mM sodium pyruvate/50 U penicillin ml–1/50 µg streptomycin ml–1/20 µM {beta}-mercaptoethanol). Human PBMCs were isolated from buffy coats by using Ficoll-Hypaque centrifugation (density, 1·077 g ml–1; Nycomed Pharma). Cells were stored in liquid nitrogen. Monocytes were enriched by plastic adherence for 2 h at 37 °C. To generate immature monocyte-derived DCs, enriched monocytes were cultured for 6 days in cRPMI in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF) (1600 U ml–1) and IL4 (100 ng ml–1). To induce maturation, cells were washed and cultured for 48 h in cRPMI with 100 ng LPS ml–1, 10 µg poly(I : C) ml–1 and 10 µg HBcAg ml–1.

Staining of cells.
Cells were incubated with PE- and/or FITC-labelled antibodies in PBS/0·8 % BSA for 1 h on ice. After 2 washes, cells were resuspended in the same buffer containing propidium iodide (PI) and analysed on a FACScan flow cytometer (Becton Dickinson). Dead cells that incorporated PI were gated out of the analysis. At least 5000 cells were counted per analysis. Fluorescence was measured at 530 nm for FITC and 580 nm for PE. 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 (SAPE only) and isotypic controls.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To study the activating potential of HBV nucleocapsids for human antigen-presenting cells, pre-monocytic THP-1 cells and PBMCs, monocytes and DCs from different donors were used. Three nucleocapsid preparations were used, one produced in E. coli (HBcAg-c) and two produced in yeast (HBcAg-y). All three preparations contained RNA that co-migrated with capsid proteins. HBcAg-c contained more RNA than the yeast-derived nucleocapsids (Fig. 1). The mobility of HBcAg-c was higher, probably because of the higher RNA content, which reduces the positive charge of the capsids. Where indicated, LPS and poly(I : C) were included as positive stimulation controls. As markers for cellular activation, the secretion of cytokines (TNF-{alpha} and IL12p40) and changes in the expression of indicated surface markers (CD14, CD18, CD40, CD80, CD83, CD86 and HLA-DR) were monitored. CD14 is an LPS co-receptor and can be up- or downregulated upon activation of monocytes and macrophages by LPS. Within the first few hours after stimulation, the expression of CD14 is upregulated, followed by a downregulation. After 24–48 h, expression is again upregulated (Antal-Szalmás, 2000). CD14 is not expressed on THP-1 cells. CD18 or integrin beta chain 2 is an integral cell-surface protein that participates in cell adhesion, as well as cell surface-mediated signalling. Prolonged LPS stimulation (more than 48 h) leads to its upregulation on THP-1 cells. CD40, CD80 and CD86 are costimulatory molecules that play a key role in immune activation, tolerance regulation and skewing of Th responses. Human monocytes express low levels of CD40 and CD80, which are upregulated when cells are activated by LPS. CD86 is expressed constitutively on monocytes and has been reported to be downregulated by LPS (Wolk et al., 2000). LPS stimulation of THP-1 cells for more than 48 h leads to upregulation of CD86 (Lim et al., 2002). CD40 and CD80 are both expressed on immature DCs, whereas CD86 is not. CD83 is one of the best-known maturation markers for human DCs. The fact that CD83 is strongly upregulated together with costimulatory molecules such as CD40, CD80 and CD86 during DC maturation suggests that it plays an important role in the induction of immune responses (Lechmann et al., 2002). HLA-DR is also upregulated during maturation of DCs.



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Fig. 1. HBcAg-c and HBcAg-y contain different amounts of encapsidated RNA. Five micrograms of HBcAg-c (lane 3) and HBcAg-y (lanes 1 and 2) preparations were separated on a 1 % agarose gel that contained ethidium bromide to visualize encapsidated RNA (a). The same gel was stained with GelCode Blue Stain reagent to visualize nucleocapsids (b).

 
Stimulation of THP-1 cells
Incubation of THP-1 cells for 48 h with 1 µg LPS ml–1 upregulated expression of CD40 and CD80. Expression of CD18 and CD86 was not affected (Fig. 2). In agreement with previous reports, expression of both markers was increased after 72 h stimulation (data not shown). A completely identical result was observed when THP-1 cells were incubated with 5 µg HBcAg-c ml–1. The addition of 5 µg HBcAg-y ml–1 did not result in upregulation of CD40 or CD80. When the secretion of TNF-{alpha} was determined, this cytokine was clearly present in cell supernatant of LPS- and HBcAg-c-stimulated THP-1 cells. The concentration of TNF-{alpha} in HBcAg-y-stimulated cells was below the detection limit of 15·6 pg ml–1 (Table 1a).



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Fig. 2. Activation of THP-1 cells by HBcAg-c, but not HBcAg-y. THP-1 cells (5x105 ml–1) were stimulated for 48 h with LPS (1 µg ml–1), HBcAg-c (5 µg ml–1) or HBcAg-y (5 µg ml–1) and analysed for cell-surface expression of the indicated molecules by flow cytometry. Black lines represent stimulated cells, grey lines represent non-stimulated cells and dashed lines represent isotypic controls.

 

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Table 1. Production of TNF-{alpha} and IL12p40 by THP-1 cells, PBMCs, monocytes and immature DCs

(a) THP-1 cells were stimulated for 48 h with LPS (1 µg ml–1) and HBcAg-c/y (5 µg ml–1). (b) PBMCs and monocytes were stimulated for 24 h with 5 µg HBcAg-c/y ml–1, 100 ng LPS ml–1 or 10 µg poly(I : C) ml–1. (c) PBMCs were stimulated for 24 h with 10 µg HBcAg-c/y ml–1, 100 ng LPS ml–1 or 10 µg poly(I : C) ml–1. (d) Immature DCs were stimulated for 48 h with 10 µg HBcAg-c/y ml–1, 100 ng LPS ml–1 or 10 µg poly(I : C) ml–1. ND, Not determined.

 
Stimulation of PBMCs and a fraction enriched in monocytes
As the THP-1 cell line did not respond to HBcAg-y, we investigated whether HBcAg-y would instead be able to activate primary monocytes. Incubation of PBMCs with 5 µg HBcAg-y ml–1 for 48 h upregulated expression of CD40, CD80 and CD14 on monocytes. Expression of CD86 did not change. Identical results were obtained when a cell fraction that was enriched for monocytes was used (Fig. 3). Determination of TNF-{alpha} and IL12p40 in supernatant of cultured PBMCs and monocytes demonstrated good induction of both cytokines by HBcAg-y (Table 1b). As HBcAg-y activated monocytes, we compared the concentration of TNF-{alpha} that was induced by HBcAg-y with the concentration induced by HBcAg-c. PBMCs were stimulated with 10 µg HBcAg-c/y ml–1, 100 ng LPS ml–1 or 10 µg poly(I : C) ml–1. As shown in Table 1(c), TNF-{alpha} levels induced by HBcAg-y were only threefold lower than those induced by HBcAg-c and LPS. Monocytes upregulated expression of CD40 and also secreted IL12p40 when stimulated with LPS and poly(I : C) (data not shown).



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Fig. 3. HBcAg-y activates PBMCs. PBMCs or partially purified monocytes from two donors were stimulated for 48 h with HBcAg-y (5 µg ml–1). Cell-surface expression of the indicated molecules was determined by flow cytometry. Black lines represent stimulated cells, grey lines represent non-stimulated cells and dashed lines represent isotypic controls.

 
Stimulation of immature DCs
Immature DCs were generated from enriched monocytes by incubation with IL4 and GM-CSF. Cells were stimulated for 48 h with 10 µg HBcAg-c/y ml–1. Incubations with 100 ng LPS ml–1 and 10 µg poly(I : C) ml–1 were performed to demonstrate the maturation potential of the immature DCs generated thus. Indeed, stimulation with LPS and poly(I : C) resulted in the maturation of the cells, as demonstrated by the upregulation of CD40, CD80, CD83, CD86 and HLA-DR (Fig. 4). Both ligands induced the secretion of TNF-{alpha} and IL12p40 (Table 1d). When immature DCs were incubated with HBcAg-c, full maturation was observed (Fig. 4) and cells produced TNF-{alpha} and IL12p40. However, this was not observed when cells were incubated with HBcAg-y.



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Fig. 4. Maturation of immature DCs by HBcAg-c, but not HBcAg-y. IL4- and GM-CSF-induced immature DCs were stimulated for 48 h with LPS (100 ng ml–1), poly(I : C) (10 µg ml–1), HBcAg-c (10 µg ml–1) or HBcAg-y (10 µg ml–1) and analysed for cell-surface expression of the indicated molecules by flow cytometry. Black lines represent stimulated cells, grey lines represent non-stimulated cells and dashed lines represent isotypic controls.

 
HBcAg-c and HBcAg-y contain different concentrations of LPS
The experiments described above revealed an unexpected difference in the activation capacity of the E. coli- and yeast-derived nucleocapsid preparations. HBcAg-c was capable of activating THP-1 cells, monocytes and immature DCs, whereas HBcAg-y only stimulated monocytes. In a recent paper, it was reported that recombinant nucleocapsid preparations produced in E. coli reproducibly contain 3–10 EU LPS (µg nucleocapsid)–1 (Geldmacher et al., 2004). Nucleocapsids produced in yeast are more likely to be free of LPS. Nevertheless, we determined the LPS content of these nucleocapsid preparations. The two HBcAg-y preparations contained 0·422 and 0·462 EU LPS (µg nucleocapsid)–1. This would correspond to ~50 pg LPS (µg HBcAg-y)–1. The LPS content of the HBcAg-c preparation was extremely high, 136 EU LPS (µg nucleocapsid)–1 or 16 ng LPS (µg HBcAg)–1 (as reported by Cao et al., 2001). It is well-known that monocytes, immature DCs and THP-1 cells are all susceptible to stimulation with LPS, but monocytes are considered to be the most sensitive, due to the expression of the LPS co-receptor CD14. The levels of LPS in the preparations could thus easily explain their different capacities to activate THP-1 cells, monocytes and immature DCs. Therefore, we investigated whether LPS, not nucleocapsids, was responsible for the activation of the cells.

LPS in the nucleocapsid preparations activates THP-1 cells and monocytes
LPS and HBcAg-c were boiled for 30 min and added to THP-1 cells. Boiling destroys the encapsidated RNA and the capsids, but not LPS. As shown in Fig. 5(a), this treatment did not inhibit the cytokine-inducing activity of LPS and HBcAg-c. This result suggests strongly that the high level of LPS in HBcAg-c is at least partly responsible for the activation of THP-1 cells, monocytes and immature DCs.



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Fig. 5. (a) The stimulatory capacity of HBcAg-c is not destroyed upon treatment at 100 °C for 30 min. THP-1 cells (5x105 ml–1) were stimulated with LPS (1 µg ml–1) or HBcAg-c (5 µg ml–1) that was boiled for 30 min (open bars) or kept on ice for 30 min (filled bars). The concentration of TNF-{alpha} in cell supernatant was determined after 24 h. (b) HBcAg-y-induced secretion of TNF-{alpha} by PBMCs is blocked by CD14-specific antibody My4. PBMCs (106 ml–1) were incubated with different antibodies (10 µg ml–1) for 30 min before LPS (0·2 ng ml–1, filled bars) or HBcAg-y (10 µg ml–1, open and grey bars) was added. The concentration of TNF-{alpha} in cell supernatant was determined after 24 h.

 
As HBcAg-y only stimulated monocytes, another assay was used to demonstrate that the low levels of LPS were responsible for the activation of monocytes. PBMCs were stimulated for 24 h with 10 µg yeast-derived nucleocapsids ml–1 or 0·2 ng LPS ml–1, together with three different CD14-specific antibodies. Only one of these (My4) is known to block the LPS-induced activation of monocytes efficiently. Indeed, the CD14-specific antibody My4 strongly inhibited LPS-induced production of TNF-{alpha}, whereas the other antibodies, M5E2 and Big2, had little or no effect (Fig. 5b). More importantly, only My4 inhibited the induction of TNF-{alpha} by the HBcAg-y preparation, which indicates strongly that even this very low concentration of LPS in HBcAg-y was responsible for the activation of monocytes.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In HBV-infected patients, the HBV nucleocapsid (HBcAg) seems to be significantly more immunogenic than the other viral proteins. High titres of anti-HBcAg are produced by all HBV-infected patients. Anti-HBcAg IgM appears early in acute hepatitis B and anti-HBcAg IgG can persist with slowly decreasing titres for many years and even for life. HBcAg and HBeAg share most of their sequence (149 aa) and are highly cross-reactive at the T cell. Nevertheless, HBcAg and HBeAg are recognized differently by B cells and the immune response toward these antigens appears to be regulated independently (reviewed by Vanlandschoot et al., 2003). To try to understand the unique immunogenicity of HBcAg in vivo, many researchers have used recombinant HBcAg, which was most often expressed in E. coli. Obviously, when proteins are purified from E. coli, the danger for contamination with LPS is very high and its presence should be monitored carefully. Recently, it was reported that recombinant nucleocapsid preparations, when produced in E. coli, reproducibly contain 3–10 EU LPS (µg nucleocapsid)–1 (Geldmacher et al., 2004). Nevertheless, nucleocapsids purified from E. coli by Neirynck et al. (1999) contained only 50–180 pg LPS (µg nucleocapsid)–1. Obviously, the level of contamination most probably depends on the care that is taken to prevent the introduction of LPS or the stringency with which LPS molecules are removed.

All three nucleocapsid preparations used here contained LPS and this was shown to be involved in the stimulation of antigen-presenting cells. The level of contamination was very high in the nucleocapsids produced in E. coli: 136 EU LPS (µg HBcAg-c)–1 (Cao et al., 2001). Extremely low levels of LPS were present in the capsids produced in yeast: 0·422 and 0·462 EU LPS (µg HBcAg-y)–1. Because S. cerevisiae does not synthesize LPS, we believe that a minor contamination of HBcAg-y with LPS may have occurred during the purification of the nucleocapsids. Because of the high LPS levels in HBcAg-c, it is not surprising that THP-1 cells, monocytes and immature DCs were activated when they were exposed to 5–10 µg HBcAg-c. Only primary monocytes, but not THP-1 cells or immature DCs, were activated when stimulated with 5–10 µg HBcAg-y. This difference can be explained by the expression of CD14 by primary monocytes and not by the other cell types. It is well-known that the presence of CD14 on cells makes these cells much more sensitive to activation by LPS (Fenton & Golenbock, 1998). The involvement of CD14 in the stimulation of monocytes by HBcAg-y was shown experimentally. Indeed, activation by HBcAg-y was inhibited by a CD14-specific antibody that is known to block LPS activation (Stelter et al., 1997). Immature DCs did not mature and did not secrete TNF-{alpha} or IL12p40 when stimulated with HBcAg-y, which suggests that the level of LPS was too low to trigger these responses. Although HBcAg-c and HBcAg-y contained vastly different (300-fold) amounts of LPS, TNF-{alpha} levels induced by HBcAg-y in PBMCs were only threefold lower. This is not surprising, as it is well-known that the LPS-binding protein present in serum strongly enhances the stimulation by LPS (Heumann et al., 1992; Dentener et al., 1993; Tada et al., 2002).

Based on our results, we examined whether other researchers checked for the possible contamination with LPS of their nucleocapsid preparations. Surprisingly, the LPS content has been reported in only a few papers (e.g. Neirynck et al., 1999; Cao et al., 2001; Riedl et al., 2002). A few other papers mention that LPS levels were measured, but quantities observed were regarded as ‘not significant’ (e.g. Manigold et al., 2003). Because the possible presence of LPS was and still is largely ignored, we wondered whether some of the proposed immunogenic qualities of nucleocapsids might be attributed to LPS. Two reports wherein the role of LPS may have been ignored are discussed below.

In 1986, it was reported that the nucleocapsid is both a T cell-dependent and a T cell-independent antigen (Milich & McLachlan, 1986). When capsids were injected into athymic (nude) mice, which are devoid of T cells, capsids induced IgM- and IgG-class antibodies. These experiments were performed by using Freund's complete or incomplete adjuvant, but even a single injection of 10 µg nucleocapsid dissolved in saline efficiently induced IgG2b and IgG3 isotype production and virtually no IgG1 or IgG2a (Milich et al., 1997). This induction of HBcAg-specific IgG without T-cell help is rather unique and was not observed in our hu-PBL-NOD/SCID model (Cao et al., 2001). However, mouse B cells are easily activated by LPS through interaction with two LPS receptors, TLR4 and RP105 (Miyake et al., 2000). On the contrary, human B cells do not respond to stimulation with LPS (Bernasconi et al., 2003; Wagner et al., 2004). This difference in susceptibility of B cells to LPS might perhaps explain why only naive B cells from mice produced HBcAg-specific IgG antibodies. Following nucleocapsid-induced cross-linking of the B-cell receptor, LPS might have delivered the signals required for the immunoglobulin switch to occur.

Nucleocapsids expressed in bacteria, yeast or mammalian cells and made of full-length core proteins always contain RNA of approximately 5–20 ng µg–1, varying in length from 30 to 3000 nt (Birnbaum & Nassal, 1990; Riedl et al., 2002). Deletion of the C-terminal, arginine-rich end generates capsids in which >98 % of RNA binding is lost. Immunization of mice with such truncated capsids no longer primes a Th1 immune response, but a clear Th2 response. Unprimed spleen cells and bone marrow-derived DCs stimulated with full-length capsids, but not truncated capsids, produced IL12p70. Based on these and additional observations, it was suggested that trace amounts of prokaryotic or eukaryotic RNA have a potent enhancing and modulating effect on the immune response towards the capsids. It was proposed that the RNA is protected from degradation by nucleases during its extracellular phase. Following uptake of capsids by endocytosis or macropinocytosis, particles would be disrupted in an acidic, late endosomal or early lysosomal compartment. The encapsidated RNA would probably be released, allowing it to interact with immunostimulatory receptors (Riedl et al., 2002). Recent papers have indeed demonstrated that ssRNA has immunostimulating capacities (Diebold et al., 2004; Heil et al., 2004; Lund et al., 2004; Scheel et al., 2004). However, the full-length nucleocapsid preparation used also contained very high amounts of LPS [1–4 ng LPS (µg protein)–1], whereas the truncated capsid preparations contained only 10–30 pg LPS (µg protein)–1 (Riedl et al., 2002). This very high concentration of LPS in the full-length nucleocapsid preparation has, without any doubt, strongly stimulated cells both in vivo and in vitro. This activation was probably much stronger than the activation by the ~100-fold lower amounts of LPS in the truncated capsid preparation. We suggest that perhaps it was not only the difference in RNA content that caused the different outcome of the immune response.

Finally, we observed that HBcAg-c and HBcAg-y preparations differed not only in their LPS content, but also in the presence of molecules that trigger cells via TLR2 agonists. Already, HBcAg-c at 62 ng ml–1 efficiently activated an NF-{kappa}B-dependent reporter gene in TLR2-transfected HEK293T cells, whereas up to 5 µg HBcAg-y ml–1 did not. The LPS used in our studies also triggered such TLR2-transfected HEK293T reporter cells (data not shown). It has indeed been demonstrated that standard, purified, E. coli-derived LPS preparations might contain a variety of extremely bioactive molecules, some of which signal through TLR2 (Skidmore et al., 1975; Morrison et al., 1976; Sultzer & Goodman, 1976; Hirschfeld et al., 2000). Similar molecules were probably not removed during the purification of HBcAg-c and may also have contributed to the strong stimulatory potential of HBcAg-c. We hope that, based on this paper, researchers who study the immunogenicity of HBV nucleocapsids or use this structure as a tool to study immunity or to develop new vaccines will bear in mind that the variable presence of TLR4 and TLR2 agonists might lead to incorrect conclusions.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Dr Martine Wettendorf (GlaxoSmithKline, Rixensart, Belgium) for providing yeast-expressed HBcAg. Research was supported by the Concerted Research Initiative of Ghent University (GOA project no. 12050203).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 September 2004; accepted 14 October 2004.