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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 CD14fluorescein isothiocyanate (FITC), CD18FITC, CD40FITC, CD40phycoerythrin (PE), CD80FITC, CD80PE, CD83FITC, CD86FITC, HLA-DRFITC and streptavidinPE (SAPE) were from BD Biosciences Pharmingen. Mouse IgG1FITC, IgG2aFITC and IgG2bFITC 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-) 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 ml1/50 µg streptomycin ml1/20 µM -mercaptoethanol). Human PBMCs were isolated from buffy coats by using Ficoll-Hypaque centrifugation (density, 1·077 g ml1; 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 granulocytemacrophage colony-stimulating factor (GM-CSF) (1600 U ml1) and IL4 (100 ng ml1). To induce maturation, cells were washed and cultured for 48 h in cRPMI with 100 ng LPS ml1, 10 µg poly(I : C) ml1 and 10 µg HBcAg ml1.
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.
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RESULTS |
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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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DISCUSSION |
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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 510 µg HBcAg-c. Only primary monocytes, but not THP-1 cells or immature DCs, were activated when stimulated with 510 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-
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-
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 520 ng µg1, 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 [14 ng LPS (µg protein)1], whereas the truncated capsid preparations contained only 1030 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 ml1 efficiently activated an NF-B-dependent reporter gene in TLR2-transfected HEK293T cells, whereas up to 5 µg HBcAg-y ml1 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.
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ACKNOWLEDGEMENTS |
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Received 13 September 2004;
accepted 14 October 2004.