Physical interaction of human papillomavirus virus-like particles with immune cells
Diane M. Da Silva1,2,
Markwin P. Velders1,
John D. Nieland41,
John T. Schiller3,
Brian J. Nickoloff1,2 and
W. Martin Kast1,2
1 Cancer Immunology Program, Cardinal Bernardin Cancer Center and
2 Department of Microbiology & Immunology, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA
3 Laboratory of Cellular Oncology, National Institutes of Health, 36 Convent Drive, MSC 4040, Bethesda, MD 20892-4040, USA
Correspondence to:
W. M. Kast, Cardinal Bernardin Cancer Center, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA
 |
Abstract
|
---|
Human papillomavirus virus-like particles (HPV VLP) and chimeric VLP are immunogens that are able to elicit potent anti-viral/tumor B and T cell responses. To investigate the immunogenicity of VLP, we determined which cells of the immune system are able to bind HPV-16 VLP. VLP were found to bind very well to human and mouse immune cells that expressed markers of antigen-presenting cells (APC) such as MHC class II, CD80 and CD86, including dendritic cells, macrophages and B cells. mAb blocking studies identified Fc
RIII (CD16) as one of the molecules to which the VLP can bind both on immune cells and foreskin epithelium. However, transfection of a CD16 cell line with CD16 did not confer binding of VLP. Splenocytes from Fc
RIII knockout mice showed a 33% decrease in VLP binding overall and specifically to subsets of APC. These combined data support a role for CD16 as an accessory molecule in an HPV VLPreceptor complex, possibly contributing to the immunogenicity of HPV VLP.
Keywords: antigen presentation, CD16, Fc receptor, papillomavirus, vaccine, virus-like particles
 |
Introduction
|
---|
Human papillomavirus type-16 (HPV-16) infection in humans is associated with cervical intra-epithelial neoplasia and invasive cervical cancer (1). Papillomaviruses are members of the papovavirus family of viruses and consist of 55-nm, non-enveloped, icosahedral-shaped virions. Like most viruses, HPV is presumed to gain access to cells by attaching to cell membranes through an interaction between the viral capsid and cell-surface proteins. The 7.9-kb circular, double-stranded DNA viral genome contains open reading frames for early proteins that are responsible for viral DNA replication, transcription and cellular transformation, and late proteins that make up the virus capsid (2). In vitro, the HPV-16 L1 and L2 major and minor capsid proteins are able to self-assemble into virus-like particles (VLP) (3,4). VLP have been used to characterize the binding of papillomaviruses to a range of cell types from different tissues and animal species by various techniques, and the results indicate that at least one receptor is widely expressed and conserved, and that there is likely to be more than one receptor (511).
Papillomavirus VLP are also lead candidates as prophylactic and therapeutic vaccines against HPV infection and HPV-induced lesions. VLP mimic infectious virions in their ability to induce high titers of virus-neutralizing antibody, even in the absence of adjuvant (3,12). Additionally, VLP can be modified to carry non-viral plasmid DNA (i.e. pseudovirions) (13) or immunogens such as peptides recognized by cytotoxic T lymphocytes (CTL) or whole proteins (i.e. chimeric VLP), which upon vaccination elicit a CTL response against tumor cells expressing the appropriate tumor rejection antigen (1417). Since the ability of VLP to stimulate cell-mediated immune responses presumably resides in their ability to efficiently deliver antigen to professional antigen-presenting cells (APC), we were interested in determining if VLP are specifically targeted to professional APC through a virusreceptor type of interaction.
In this study, we determined which cells of the immune system of both mice and humans are most efficient at binding HPV-16 VLP. We report here that VLP strongly bind MHC class II+ APC and APC that express immune co-stimulatory molecules, including dendritic cells, macrophages, monocytes and B lymphocytes. mAb antibody blocking studies performed support the conclusion that the low-affinity IgG receptor, Fc
RIII (CD16), is involved in VLP binding to immune cells, although transfection experiments indicated that this is likely in the role of an accessory molecule. Splenocytes from mice lacking Fc
RIII were tested for VLP binding and were shown to have a reduced ability to bind VLP, indicating that Fc
RIII may play a role in the immune response against VLP-derived proteins.
 |
Methods
|
---|
VLP, cell lines, mice and antibodies
HPV-16 L1L2 VLP were produced in Trichoplusia ni (High Five) cells by infection with HPV-16L1L2 recombinant baculovirus. VLP were purified from cells by sucrose and CsCl gradient centrifugation, quantitated, and examined by electron microscopy and ELISA as previously described (14,18). DG75, an EpsteinBarr virus-negative, Burkitt's lymphoma B cell line, was kindly provided by Dr Magnus Evander (Umeå University, Umeå, Sweden). DG75 was cultured in IMDM (Biowhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 µg/ml kanamycin and 50 mM 2-mercaptoethanol. C57Bl/6 female mice were purchased from Taconic (Germantown, NY). Fc
RIII-deficient mice were a gift from Dr J. Sjef Verbeek (Leiden University Medical Center, Leiden, The Netherlands). Mouse thymocytes were isolated from 4- to 6-week-old C57Bl/6 mice. Human peripheral blood mononuclear cells (PBMC) from healthy donors were collected by apheresis and mononuclear cells were isolated over a Ficoll gradient. The following antibodies were purchased from PharMingen (San Diego, CA): anti-I-Ab (AF6-120.1)phycoerythrin (PE), anti-mouse B220 (RA3-6B2)FITC, anti-mouse CD4 (GK1.5)FITC, anti-mouse CD8a (53-6.7)PE, anti-mouse CD80 (16-10A1)FITC, anti-mouse CD11b (M1/70)FITC, anti-mouse CD49f (
6 integrin, GoH3), anti-mouse CD104 (ß4 integrin, 346-11A), anti-mouse CD16/CD32 (2.4G2), anti-NK cell (Ly49C, 5E6), goat anti-rat IgGFITC, goat anti-mouse IgGFITC, streptavidinallophycocyanin, streptavidinPE, streptavidinFITC, mouse IgG1 (A112-2)FITC, mouse IgG1, mouse IgG2a (G155-178), mouse IgG2b (49.2)FITC, mouse IgG2b, mouse IgG3 (A112-3), mouse IgM (G155-228), rat IgG2a (B39-4)PE, rat IgG2a, rat IgG2b (A95-1)FITC, rat IgG2b, anti-human CD1a (HI149)FITC, anti-human CD4 (RPA-T4)FITC, anti-human CD8 (RPA-T8)FITC, anti-human CD16 (3G8), anti-human CD19 (HIB19), anti-human CD23 (M-L233), anti-human CD32 (FLI8.26), anti-human CD49f (
6 integrin, GoH3), anti-human CD56 (B159), anti-human CD64 (10.1), anti-human CD80 (BB1), anti-human CD86 (2331), anti-human CD104 (ß4 integrin, 4399B) and anti-HLA-DR,DP,DQ (TU39)FITC. Anti-NLDC145 (dendritic cell marker) hybridoma was purchased from ATCC (Rockville, MD). Anti-human CD16 (Leu-11b, G022) was purchased from Becton Dickinson (San Jose, CA). Anti-human CD16 antibodies (VIFcRIII and DJ130c) were purchased from Dako (Carpinteria, CA). Anti-human CD16 (GRM-1) was purchased from Southern Biotechnology Associates (Birmingham, AL). Anti-HPV-16 VLP antibody H16.V5 was a generous gift from Dr Neil Christensen (Penn State University, Hershey, PA).
Biotinylation of VLP
VLP were biotinylated with sulfo-NHS-biotin (Pierce, Rockford, IL). One milligram of HPV-16 L1L2 VLP was incubated with biotin at a final concentration of 0.5 µg/ml at 4°C for 30 min. Biotinylation was quenched by the addition of serum containing medium. Excess biotin was removed by dialysis against two changes of 0.5 M NaCl/PBS overnight, and particles were aliquoted and stored at 80°C for future use. Antigen-capture ELISA (18) and electron microscopy analysis were performed to ensure that biotinylation did not interfere with the display of antibody-neutralizing epitopes and there was no disruption of capsid morphology.
VLP binding assay and FACS analysis
Splenocytes or thymocytes were isolated from C57Bl/6 mice or CD16 knockout mice and single-cell suspensions were made by passage of organs through a mesh filter. Cells (106) were incubated with 1µg of biotinylated HPV-16 L1L2 VLP for 1h on ice. After extensive washing, streptavidinallophycocyanin was added in combination with the indicated cell-specific marker antibody, followed by the appropriate secondary antibody if necessary. Fluorescence was analyzed by two- or three-color flow cytometry on a Becton Dickinson FACSCalibur using CellQuest software (Becton Dickinson, San Jose, CA). The FACSCalibur cytometer was calibrated with CaliBRITE beads using FACSComp software (Becton Dickinson). In blocking experiments, cells were preincubated with the indicated antibody at a concentration of 2 µg/106 cells or with a 50 times excess of unlabeled VLP.
Transfection of DG75 cells
Human Fc
RIII
chain (CD16) cDNA (kindly provided by Dr J. Ravetch, Rockefeller University, NY) was amplified in a standard PCR reaction with PFU polymerase (Stratagene, La Jolla, CA) using the forward primer: 5'-AATGGATCCATCATGTGGCAGCTGCTCCTC-3' introducing a BamHI restriction site and the reverse primer: 5'-ATTTCTA GATCAAATGTTTGTCTTCACAGAGAAATATAGTCC-3' introducing an XbaI restriction site. The PCR product was cloned into the BamHIXbaI sites of pCDNA3 (Invitrogen, Carlsbad, CA) and sequenced. The human universal
chain cDNA (kindly provided by Dr J.-P Kinet, Beth Israel Deaconess Medical Center, Boston, MA) was subcloned into the EcoRI site of pCDNA3. DG75 cells (107) were transfected by electroporation using 5 µg DNA of each plasmid containing CD16 and the
chain in 0.35 ml serum-free IMDM with the Genepulser (BioRad) at 960 µF and 0.25 kV in a 0.4 cm cuvette. Stable transfectants were selected in culture medium containing 1 mg/ml G418. CD16 expressing transfectants were identified and sorted by FACS analysis.
Immunohistochemistry
Fresh human foreskin was obtained from circumcisions performed at Loyola University Medical Center (Maywood, IL). Tissue was obtained from four tissue donors and suspended in 1xEarle's balanced salt solution (Sigma, St Louis MO). Four-millimeter punch biopsies were taken and embedded in Gum tragacanth (Sigma). Embedded biopsies were quick frozen in isopentane and stored at 20°C prior to sectioning. Non-specific avidinbiotin binding was blocked by pretreating sections with an avidinbiotin blocking kit as per the manufacturer's instructions (Vector, Burlingame, CA). Endogenous peroxidase activity was blocked by preincubating slides in 0.3% hydrogen peroxide for 5 min. For VLP staining, acetone-fixed human foreskin sections were incubated with biotinylated VLP at a concentration of 10 µg/ml for 30 min at 22°C and washed 3 times in PBS. Peroxidase-labeled avidin (Sigma) was then added to slides at a concentration of 2 µg/ml for 1 h at 22°C. Detection of binding was performed by adding 3-amino 9-ethyl carbazole (AEC) substrate. For antibody blocking studies, anti-CD16 antibodies (3G8 and DJ130c) were preincubated with sections at a concentration of 10 µg/ml for 30 min at 22°C. Slides were washed in PBS and VLP binding was performed as described above. For CD16 immunostaining, anti-CD16 antibody (3G8) or an isotype control antibody was added to sections at a concentration of 10 µg/ml for 1 h at 22°C. After washing 3 times in PBS, peroxidase-labeled goat anti-mouse IgG was added at a concentration of 5 µg/ml for 1 h at 22°C. Detection of antibodies was performed by addition of AEC substrate. Sections were counterstained with hematoxylin, washed thoroughly and analyzed by light microscopy. Digital images were captured using a SPOT Cooled color digital camera and included software (Diagnostic Instruments, Sterling Heights, MI).
 |
Results
|
---|
Binding of VLP to cells of the immune system
Because VLP and chimeric VLP are capable of stimulating B and T cell responses when injected into animals, we wanted to determine the distribution of VLP binding to freshly isolated cells of the immune system. If VLP are specifically targeted to professional APC, this finding would begin to explain why VLP are so effective in priming both MHC class I and class II immune responses at low antigen doses in the absence of adjuvant. Single-cell suspensions of freshly isolated murine splenocytes and thymocytes or human PBMC were tested for VLP binding by indirect immunofluorescence using biotinylated VLP and flow cytometry. The data are summarized in Table 1
. Cell populations within the splenocytes and PBMC were positive for VLP binding by varying degrees. The cells with the highest capability of binding were typically those that expressed markers of APC, e.g. MHC class II, CD80 and CD86 (Table 1
). Among the cell types that were highly positive for binding were dendritic cells, B cells, macrophages and monocytes. The majority of cells within the mouse thymus are immature double-positive cells (CD4+CD8+) that are in the process of going through thymic selection. Immature thymocytes were surprisingly negative for VLP binding even though they highly express the reported candidate receptor for papillomaviruses,
6ß4 (9,19,20,21). The same binding patterns were seen when non-biotinylated VLP and a monoclonal conformational-specific antibody for HPV-16 (clone H16.V5) were used for the VLP binding assay, indicating that binding was not an artifact of biotinylation (data not shown).
Antibodies to CD16 block VLP binding to cells
To test the hypothesis that the interaction between VLP and the immune cells could be mediated through the
6 integrin, we tested whether VLP binding to cells could be inhibited by pretreatment of the cells with integrin chain-specific mAb. Because a mAb to
6 but not ß4 has previously been shown to partially inhibit binding of VLP to an
6ß4-expressing cell line (9), we expected to see similar results if VLP bind to immune cells using the same molecular complex. Unexpectedly, pretreatment of human PBMC with anti-
6 antibody only partially blocked binding of VLP to cells, and no more than an isotype control antibody, suggesting that the specificity of the antibody did not determine blocking (Fig. 1
). We therefore hypothesized that the blocking of VLP to immune cells could be due to binding of any antibody to Fc receptors via the Fc tail. We tested this hypothesis by preincubating cells with antibodies against Fc
RI (CD64), Fc
RII (CD32), Fc
RIII (CD16), Fc
RI (CD23) and appropriate isotype control antibodies before testing the cells for VLP binding. While low-level inhibition by all antibodies was detected, VLP binding to human MHC class II+ PBMC was specifically blocked only by the antibody against Fc
RIII (clone GO22) (Fig. 1
). Inhibition of binding was 80% of the blocking seen when cells were preincubated with an excess of unlabeled VLP compared to 30% blocking with the isotype control antibody (Fig. 1
). Other anti-human CD16 antibodies (clones 3G8 and DJ130c) recognizing presumably different epitopes on CD16 also had the ability to partially inhibit VLP binding to class II+ PBMC. However, not all antibodies directed against CD16 (clones VIFcRIII and GRM-1) could inhibit binding, indicating the epitope specificity of the antibody has a profound effect on the ability to block (data not shown). The antibody clones that block VLP binding are not reported to cross-react with any other molecules, therefore the data suggest that CD16 is indeed the molecule with which the VLP are interacting, either directly or indirectly.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1. Blocking of HPV-16 L1L2 VLP binding to MHC class II+ PBMC. PBMC were preincubated with no antibody, 50 times unlabeled VLP (cold VLP), mouse IgM, anti-human CD16 (GO22, mouse IgM), mouse IgG1, anti-human CD64 (10.1, mouse IgG1), anti-human CD23 (M-L233, mouse IgG1), mouse IgG2b, anti-human CD32 (FLI8.26, mouse IgG2b), rat IgG2a or anti-human 6 integrin (GoH3, rat IgG2a) prior to adding biotinylated VLP. VLP binding was detected with the addition of streptavidinallophycocyanin followed by flow cytometry. Cells were counterstained with anti-HLA-DP,DQ,DRFITC. Shown is the percentage of VLP blocking on gated MHC class II+ cells with each of the blocking treatments. Values are the average of four independent experiments using different donor cells. Percentage of blocking was calculated using the following equation: [(MFI of VLP binding) (MFI of VLP binding with blocking treatment)]/[(MFI of VLP binding) (MFI of VLP binding with unlabeled VLP block)]x100.
|
|
Expression of Fc
RIII (CD16) in a receptor-negative B cell line
In order to determine whether Fc
RIII is capable of mediating VLP attachment by itself, a cell line which has been characterized as a receptor-negative B cell line (9), DG75, was stably transfected with the cDNA for the Fc
RIII complex and VLP binding analyzed. Fc
RIII consists of a hetero-oligomeric receptor complex consisting of a ligand-binding
chain associated with disulfide-linked CD3
, FcR
or FcR ß chains (2224). The associated
,
or ß chains are required for cell-surface expression of the CD16
chain, and are involved in signal transduction and receptor-mediated internalization (2527). Therefore the cDNAs for the Fc
RIII
chain and the FcR
signaling subunit were co-transfected into DG75 cells and stable transfectants expressing high levels of surface CD16 were selected and cloned. The parental cell line, DG75, did not express surface CD16 as determined by flow cytometry and VLP bound minimally, however evident, to these cells (Fig. 2A and B
). DG75 cells transfected with the vectors alone without the coding sequences for CD16 and the
chain (DG75-mock) showed the same flow cytometric profiles as DG75 (Fig. 2C and D
). Although the CD16-transfected DG75 cells expressed high levels of surface CD16 (Fig. 2E
), VLP did not bind to these cells any more than to the parental DG75 (Fig. 2F
). In contrast, VLP bound significantly to HeLa, a cervical cancer cell line that is CD16 (Fig. 2G and H
). The combined antibody blocking and transfection results suggest that the Fc
RIII
/
chain complex can contribute to VLP binding but is neither sufficient nor absolutely necessary by itself to confer binding of VLP to a non-binding cell line.
VLP binding to cells in CD16 knockout mice is diminished
Given that VLP bind equally well to mouse splenocytes as human PBMC, we were interested in determining if Fc
RIII might also function as an accessory molecule or co-receptor for VLP in the mouse. Similar antibody blocking experiments with mouse splenocytes as were done with human PBMC were complicated by the fact that the only available mouse FcR antibody 2.4G2 cannot distinguish between Fc
RII and Fc
RIII. Therefore, to test whether Fc
RIII specifically could be involved in VLP binding in the mouse system, we obtained splenocytes from Fc
RIII
-chain-deficient mice (28). VLP binding to splenocytes from Fc
RIII-deficient mice was reduced overall by 33% compared to binding to splenocytes of wild-type C57Bl/6 mice (Fig. 3
). Both a decrease in the percentage of cells binding VLP and in the intensity of staining was evident in the APC subsets of cells resident in the spleen but not in CD4+ cells, indicating the effect is cell-type specific. (Table 2
). These results indicate that either Fc
RIII (CD16) or a complex containing this molecule is involved in VLP binding to splenocytes and may contribute to antigen uptake during immunization of mice with VLP.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3. Analysis of VLP binding to splenocytes of C57Bl/6 or Fc RIII-deficient mice by flow cytometry. Shaded histogram corresponds to the fluorescence intensity for cells incubated with biotinylated VLP. Unshaded histogram corresponds to cells exposed only to the secondary reagent, streptavidinallophycocyanin, alone. In each case, 20,000 viable total splenocytes are shown after gating on forward and side scatter and exclusion of propidium iodide. Marker is set at 20 on the log scale in each panel. Representative of three experiments.
|
|
CD16 antibodies block VLP binding to human foreskin sections
Because of our identification of CD16 as an accessory molecule for VLP binding, we determined if the expression of CD16 was coincident with that of VLP binding in a tissue relevant for high-risk HPV infection. Tissue sections of human foreskin epithelium were analyzed immunohistochemically for CD16 expression and for VLP binding. CD16 in foreskin was expressed in the two upper layers of the skin, the stratum corneum and stratum granulosum on the skin side of foreskin (Fig. 4A
), and on the superficial portion of the mucosal side of foreskin (Fig. 4D
). No significant staining was observed with control antibodies or secondary antibodies alone and staining patterns were identical in all four donor foreskins analyzed (data not shown). HPV-16 L1L2 VLP were used to stain foreskin sections to determine where VLP binding occurs. The heaviest VLP binding was in the stratum corneum of the skin (Fig. 4B
) and on the superficial mucosa (Fig. 4E
). The distribution of CD16 staining overlapped that of VLP binding (Fig. 4A and D
versus B and E). When foreskin sections were preincubated with anti-CD16 antibodies prior to the VLP binding assay, VLP binding was reduced significantly in both skin and mucosa sections (Fig. 4C and F
). Preincubation with a control antibody did not reduce binding to either skin or mucosa (data not shown). The combined results indicate that CD16 may play a role as an accessory molecule for binding of HPV-16 not only in immune cells, but also in tissues relevant for virus infection.

View larger version (81K):
[in this window]
[in a new window]
|
Fig. 4. Immunohistochemical analysis of CD16 expression and VLP binding in human foreskin epithelium. Frozen section of foreskin were incubated with anti-human CD16 (clone 3G8) antibody (A and D) or biotinylated HPV-16 L1L2 VLP (B, C, E and F). Sections in (C) and (F) were preincubated with anti-CD16 (3G8 and DJ130c) antibodies prior to incubation with biotinylated VLP. Binding of anti-CD16 was detected with peroxidase-labeled goat anti-mouse IgG antibody. Binding of VLP at the skin (B and C) and mucosa (E and F) was detected with peroxidase-labeled avidin. Reactivity was visualized by the addition of AEC substrate. Sections were counterstained with hematoxylin and analyzed at a magnification ~40x.
|
|
 |
Discussion
|
---|
Productive HPV infections (i.e. infections of cells that are permissive for the virus life cycle) only occur in keratinocytes of stratified epithelium undergoing terminal differentiation. Since the papillomavirus receptor is reported to be widely expressed and evolutionarily conserved, infection appears to be limited by downstream cellular events following uptake (5). The study of HPV and its candidate receptors is complicated by the fact that native infectious HPV virions cannot be isolated in large enough quantities either in vitro or in vivo. Therefore most HPVreceptor interactions are studied with VLP that morphologically mimic native virions. It has previously been shown that VLP are capable of binding to cell lines of different tissue and cell lineages and different species (58). In this study, we investigated primarily the distribution of HPV VLP binding to cells of the immune system in order to determine what cells would potentially be targeted by VLP after immunization.
HPV, bovine papillomavirus and cottontail rabbit papillomavirus VLP all bind a proteinaceous molecule that is trypsin sensitive (6,7,29). The only reported candidate receptor for papillomaviruses until recently was the
6 integrin complexed with either the ß4 integrin or ß1 integrin (9). Evander et al. proposed the
6ß4 as a candidate receptor based on the ability of HPV-6b VLP to immunoprecipitate proteins with a mol. wt corresponding to the integrin subunits, and the ability of laminin and an antibody against
6 to block VLP binding. However, only basal epithelial cells, immature thymocytes, T cells and several types of tumor cells express the
6 integrin (68,19,20,30). The cellular distribution of
6 integrin therefore cannot account for specific VLP interactions with professional APC. HPV-11 VLP have recently been shown to interact with low affinity to cell-surface glycosaminoglycans (10). Glycosaminoglycans have been established as a receptor of relatively low specificity for a number of viruses (3136). Low-affinity interaction of the viruses with glycosaminoglycans would be followed by specific binding to and internalization by a higher-affinity specific protein component(s) of the host cell. VLP binding to a very wide range of cell types and tissues and to
6 cell lines (11) suggests that, like Herpes simplex virus (37), more than one distinct receptor exists for papillomaviruses.
The antibody-blocking and knockout mouse data suggest that Fc
RIII (CD16) is involved in binding of HPV-16 VLP on immunocytes. The 33% reduction in VLP binding to Fc
RIII-deficient mouse splenocytes was not as pronounced as the mAb blocking to human PBMC most likely because Fc
RII (CD32) is still expressed in these mice. Fc
RII and Fc
RIII are 95% homologous in mice and the extracellular domains of the two molecules are not easily distinguishable (38). Therefore, Fc
RII might contribute to VLP binding in the absence of or in addition to Fc
RIII in mice. Although immune cells are not likely to be relevant to the infectious life cycle of HPV, they are highly relevant for induction of immune responses after immunization with VLP and chimeric VLP.
Fc
RIII is a trypsin-sensitive, low-affinity IgG receptor that is present on human and mouse NK cells, macrophages, monocytes, subsets of T and B cells, granulocytes, and immature dendritic cells. These are also the cells that were found to bind HPV-16 VLP. An unexpected finding was that human NK cells positive for CD16 and CD56 were not cells highly targeted by VLP. Low VLP binding to CD16+ cells could be explained by the anti-CD16 antibody blocking the receptor; however, a non-blocking anti-CD16 antibody also yielded the same results indicating this explanation is not likely. The binding results could also be explained if the specific isoform of Fc
RIII found on NK cells is different from that found on APC, as there are many different forms and functions of CD16 (39). NK cells may lack the receptor needed for high-affinity binding of VLP, since CD16 alone does not confer binding to non-binding cells. As evident from Table 1
, mouse CD16+ splenocytes had a higher bias for VLP binding than human CD16+ PBMC. Expression of CD16 in human is much more restricted to certain cells types than in the murine system, possibly owing to the differential results observed between these species.
In contrast to
6 integrin transfection (9), expression of Fc
RIII in the DG75 B cell line did not lead to increased VLP binding. This result can be explained if the presence of Fc
RIII requires a specific modification or another molecule to form a complex capable of being a receptor for papillomavirus VLP. Fc
RIII may not bind VLP directly, but may function primarily as an accessory molecule, perhaps interacting with other molecules that bind VLP directly. As a result CD16 does not confer binding by itself; however, as observed here, blocking of CD16 results in (partial) loss of VLP binding. Additionally, although surface expression of Fc
RIII was detected by antibodies, it is not clear whether the complex could function to capture Fc immune complexes. As seen for many other viruses, high-affinity binding and penetration by HPV may require more than one cellular receptor for the initial high-affinity binding (4043). Fc
RIII is unlikely to function as an entry receptor for basal epithelial cells since this molecule is not detectably expressed in this layer or on epithelial cells that have been transformed by the virus (44). However, on immunocytes Fc
RIII may be able to function both for VLP binding and internalization under the proper conditions.
Fc
RIII normally functions to capture antigenantibody immune complexes and internalize them for cellular processing. Binding of VLP to Fc
RIII (CD16) on immune cells also raises the possibility that binding of a multivalent antigen such as a VLP would be able to cross-link and signal through the molecule. The Fc
RIII complex does contain a signaling component that normally results in tyrosine kinase activation and internalization of the molecule with its bound ligand (26,27). Cell signaling through the Fc receptor could lead to enhanced antigen uptake and presentation by professional APC. It has been shown that dendritic cell maturation and MHC class I antigen presentation can occur after FcR-mediated internalization of immune complexes (45). There is no source of any exogenous antibodies in the VLP preparations that could lead to the formation of VLPimmune complexes. However, VLP by themselves can induce maturation of immature dendritic cells leading to up-regulation of co-stimulatory molecules and secretion of Th1-promoting cytokines (M. P. Rudolf et al., submitted). Whether this VLP-induced maturation and up-regulation of co-stimulatory molecules on dendritic cells is Fc receptor mediated is currently under investigation.
High Fc
RIII expression is found on glandular epithelium in the endocervix and transformation zone, foreskin and rectal epithelium, and in the oral mucosa (44,4648). These sites are also the target tissues of mucosotropic papillomaviruses and our finding that HPV-16 VLP specifically bind to at least one of these sites indicates that Fc
RIII may be involved in the initial virus attachment at the site of infection. Our study shows that VLP binding co-localizes with Fc
RIII expression in foreskin and again that blocking CD16 with antibodies blocks VLP binding to this region. We show that Fc
RIII is expressed in the upper layers of the differentiating keratinocyte layer and in the superficial layer of the mucosa. Attachment to the superficial mucosa would allow the virus to migrate towards the cell type that leads to virus infection by way of microwounds or dermal dendritic cells that catch antigens and traffic with them to draining lymph nodes. Alternatively, binding of VLP to the outer portions of the mucosa may serve as a barrier to gaining entry to the deeper layers of the skin. Our data differ from that of Evander et al. in that their study demonstrated HPV-6b VLP binding to the basal and suprabasal epithelial cells in monkey esophageal sections (9). It is unlikely that the differences seen between our data and the previous data could be due to the nature of the particles used in the studies (HPV-6 VLP versus HPV-16 VLP used in our study) since VLP of different types have had similar cell binding profiles in previous studies (58). The nature of the tissues used to examine VLP binding may be the more critical difference.
Elucidation of the role Fc
RIII plays in binding and uptake of chimeric VLP during immunization will begin to unravel the mechanism of VLP as antigen delivery vehicles. The findings in this study provide useful information for understanding how VLP preferentially interact with immune cells, particularly APC. Determination of the molecule(s) responsible for VLP binding and entry into APC can lead to strategies to enhance immune responses against viral proteins.
 |
Acknowledgments
|
---|
This work was supported by grants P01 CA74182, R01 CA74397 and R01 CA78399 from the National Institutes of Health (to W. M. K.). D. M. Da S. is a fellow of the Arthur J. Schmitt Foundation. M. P. V. is a fellow of the Cancer Research Institute. The authors thank Ms Patricia Simms for technical assistance in the flow cytometry facility.
 |
Abbreviations
|
---|
AEC 3-amino 9-ethyl carbazole |
APC antigen-presenting cells |
CTL cytotoxic T lymphocyte |
HPV-16 human papillomavirus type 16 |
PE phycoerythrin |
PBMC peripheral blood mononuclear cells |
VLP virus-like particle |
 |
Notes
|
---|
5Present address: MediGene AG, Lochhamer Strasse 11, 82152 Martinsried/Munich, Germany.
The first two authors contributed equally to this work
Transmitting editor: J. Banchereau
Received 1 October 2000,
accepted 24 January 2001.
 |
References
|
---|
-
Walboomers, J. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J., Peto, J., Meijer, C. J. and Munoz, N. 1999. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189:12.[ISI][Medline]
-
zur Hausen, H. 1996. Papillomavirus infectionsa major cause of human cancers. Biochim. Biophys. Acta 1288:F55.[ISI][Medline]
-
Kirnbauer, R., Booy, F., Cheng, N., Lowy, D. R. and Schiller, J. T. 1992. Papillomavirus L1 major capsid protein self-assembles into virus- like particles that are highly immunogenic. Proc. Natl Acad. Sci. USA 89:12180.[Abstract]
-
Kirnbauer, R., Taub, J., Greenstone, H., Roden, R., Durst, M., Gissmann, L., Lowy, D. R. and Schiller, J. T. 1993. Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles. J. Virol. 67:6929.[Abstract]
-
Roden, R. B., Kirnbauer, R., Jenson, A. B., Lowy, D. R. and Schiller, J. T. 1994. Interaction of papillomaviruses with the cell surface. J. Virol. 68:7260.[Abstract]
-
Müller, M., Gissmann, L., Cristiano, R. J., Sun, X. Y., Frazer, I. H., Jenson, A. B., Alonso, A., Zentgraf, H. and Zhou, J. 1995. Papillomavirus capsid binding and uptake by cells from different tissues and species. J. Virol. 69:948.[Abstract]
-
Volpers, C., Unckell, F., Schirmacher, P., Streeck, R. E. and Sapp, M. 1995. Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells. J. Virol. 69:3258.[Abstract]
-
Qi, Y. M., Peng, S. W., Hengst, K., Evander, M., Park, D. S., Zhou, J. and Frazer, I. H. 1996. Epithelial cells display separate receptors for papillomavirus VLP and for soluble L1 capsid protein. Virology 216:35.[ISI][Medline]
-
Evander, M., Frazer, I. H., Payne, E., Qi, Y. M., Hengst, K. and McMillan, N. A. 1997. Identification of the alpha6 integrin as a candidate receptor for papillomaviruses. J. Virol. 71:2449.[Abstract]
-
Joyce, J. G., Tung, J. S., Przysiecki, C. T., Cook, J. C., Lehman, E. D., Sands, J. A., Jansen, K. U. and Keller, P. M. 1999. The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell- surface glycosaminoglycans on human keratinocytes. J. Biol. Chem. 274:5810.[Abstract/Free Full Text]
-
Sibbet, G., Romero-Graillet, C., Meneguzzi, G. and Campo, M.S. 2000.
6 integrin is not the obligatory cell receptor for bovine papillomavirus type 4. J. Gen. Virol. 81:327.[Abstract/Free Full Text]
-
Christensen, N. D., Hopfl, R., DiAngelo, S. L., Cladel, N. M., Patrick, S. D., Welsh, P. A., Budgeon, L. R., Reed, C. A. and Kreider, J. W. 1994. Assembled baculovirus-expressed human papillomavirus type 11 L1 capsid protein virus-like particles are recognized by neutralizing monoclonal antibodies and induce high titres of neutralizing antibodies. J. Gen. Virol. 75:2271.[Abstract]
-
Touze, A. and Coursaget, P. 1998. In vitro gene transfer using human papillomavirus-like particles. Nucleic Acids Res. 26:1317.[Abstract/Free Full Text]
-
Greenstone, H. L., Nieland, J. D., de Visser, K. E., De Bruijn, M. L., Kirnbauer, R., Roden, R. B. S., Lowy, D. R., Kast, W. M. and Schiller, J. T. 1998. Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model. Proc. Natl Acad. Sci. USA 95:1800.[Abstract/Free Full Text]
-
Peng, S., Frazer, I. H., Fernando, G. J. and Zhou, J. 1998. Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway. Virology 240:147.[ISI][Medline]
-
Schafer, K., Müller, M., Faath, S., Henn, A., Osen, W., Zentgraf, H., Benner, A., Gissmann, L. and Jochmus, I. 1999. Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection. Int. J. Cancer 81:881.[ISI][Medline]
-
Nieland, J. D., Da Silva, D. M., Velders, M. P., de Visser, K. E., Schiller, J. T., Müller, M. and Kast, W. M. 1999. Chimeric papillomavirus virus-like particles induce a murine self-antigen-specific protective and therapeutic antitumor immune response. J. Cell. Biochem. 73:145.[ISI][Medline]
-
Müller, M., Zhou, J., Reed, T. D., Rittmuller, C., Burger, A., Gabelsberger, J., Braspenning, J. and Gissmann, L. 1997. Chimeric papillomavirus-like particles. Virology 234:93.[ISI][Medline]
-
Sonnenberg, A., Linders, C. J., Daams, J. H. and Kennel, S. J. 1990. The alpha 6 beta 1 (VLA-6) and alpha 6 beta 4 protein complexes: tissue distribution and biochemical properties. J. Cell Sci. 96:207.[Abstract]
-
Wadsworth, S., Halvorson, M. J. and Coligan, J. E. 1992. Developmentally regulated expression of the beta 4 integrin on immature mouse thymocytes. J. Immunol. 149:421.[Abstract/Free Full Text]
-
McMillan, N. A., Payne, E., Frazer, I. H. and Evandert, M. 1999. Expression of the alpha 6 integrin confers papillomavirus binding upon receptor-negative B-cells. Virology 261:271.[ISI][Medline]
-
Hibbs, M. L., Selvaraj, P., Carpen, O., Springer, T. A., Kuster, H., Jouvin, M. H. and Kinet, J. P. 1989. Mechanisms for regulating expression of membrane isoforms of Fc gamma RIII (CD16). Science 246:1608.[ISI][Medline]
-
Lanier, L. L., Yu, G. and Phillips, J. H. 1989. Co-association of CD3 zeta with a receptor (CD16) for IgG Fc on human natural killer cells. Nature 342:803.[ISI][Medline]
-
Kurosaki, T., Gander, I., Wirthmueller, U. and Ravetch, J. V. 1992. The beta subunit of the Fc epsilon RI is associated with the Fc gamma RIII on mast cells. J. Exp. Med. 175:447.[Abstract]
-
Wirthmueller, U., Kurosaki, T., Murakami, M. S. and Ravetch, J. V. 1992. Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain. J. Exp. Med. 175:1381.[Abstract]
-
Bonnerot, C., Amigorena, S., Choquet, D., Pavlovich, R., Choukroun, V. and Fridman, W. H. 1992. Role of associated gamma-chain in tyrosine kinase activation via murine Fc gamma RIII. EMBO J. 11:2747.[Abstract]
-
Amigorena, S., Salamero, J., Davoust, J., Fridman, W. H. and Bonnerot, C. 1992. Tyrosine-containing motif that transduces cell activation signals also determines internalization and antigen presentation via type III receptors for IgG. Nature 358:337.[ISI][Medline]
-
Hazenbos, W. L., Gessner, J. E., Hofhuis, F. M., Kuipers, H., Meyer, D., Heijnen, I. A., Schmidt, R. E., Sandor, M., Capel, P. J., Daeron, M., van de Winkel, J. G. and Verbeek, J. S. 1996. Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice. Immunity 5:181.[ISI][Medline]
-
Roden, R. B., Hubbert, N. L., Kirnbauer, R., Breitburd, F., Lowy, D. R. and Schiller, J. T. 1995. Papillomavirus L1 capsids agglutinate mouse erythrocytes through a proteinaceous receptor. J. Virol. 69:5147.[Abstract]
-
Sonnenberg, A., Calafat, J., Janssen, H., Daams, H., Raaij-Helmer, L. M., Falcioni, R., Kennel, S. J., Aplin, J. D., Baker, J., Loizidou, M. and Garrod, D. 1991. Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J. Cell Biol. 113:907.[Abstract]
-
Zhu, Z., Gershon, M. D., Gabel, C., Sherman, D., Ambron, R. and Gershon, A. 1995. Entry and egress of varicella-zoster virus: role of mannose 6-phosphate, heparan sulfate proteoglycan, and signal sequences in targeting virions and viral glycoproteins. Neurology 45:S15.[ISI][Medline]
-
WuDunn, D. and Spear, P. G. 1989. Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J. Virol. 63:52.[ISI][Medline]
-
Secchiero, P., Sun, D., De Vico, A. L., Crowley, R. W., Reitz, M. S., Jr, Zauli, G., Lusso, P. and Gallo, R. C. 1997. Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans. J. Virol. 71:4571.[Abstract]
-
Chung, C. S., Hsiao, J. C., Chang, Y. S. and Chang, W. 1998. A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate. J. Virol. 72:1577.[Abstract/Free Full Text]
-
Klimstra, W. B., Ryman, K. D. and Johnston, R. E. 1998. Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor. J. Virol. 72:7357.[Abstract/Free Full Text]
-
Trkola, A., Gordon, C., Matthews, J., Maxwell, E., Ketas, T., Czaplewski, L., Proudfoot, A. E. and Moore, J. P. 1999. The CC chemokine RANTES increases the attachment of human immunodeficiency virus type 1 to target cells via glycos- aminoglycans and also activates a signal transduction pathway that enhances viral infectivity. J. Virol. 73:6370.[Abstract/Free Full Text]
-
Krummenacher, C., Nicola, A. V., Whitbeck, J. C., Lou, H., Hou, W., Lambris, J. D., Geraghty, R. J., Spear, P. G., Cohen, G. H. and Eisenberg, R. J. 1998. Herpes simplex virus glycoprotein D can bind to poliovirus receptor-related protein 1 or herpesvirus entry mediator, two structurally unrelated mediators of virus entry. J. Virol. 72:7064.[Abstract/Free Full Text]
-
Ravetch, J. V. and Kinet, J. P. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[ISI][Medline]
-
Van de Winkel, J. G. J. and Capel, P. J. A. 1993. Human IgG Fc receptor heterogeneity: Molecular aspects and clinical implications. Immunol. Today 14:215.[Medline]
-
Wu, L., Gerard, N. P., Wyatt, R., Choe, H., Parolin, C., Ruffing, N., Borsetti, A., Cardoso, A. A., Desjardin, E., Newman, W., Gerard, C. and Sodroski, J. 1996. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature 384:179.[ISI][Medline]
-
Fantini, J., Cook, D. G., Nathanson, N., Spitalnik, S. L. and Gonzalez-Scarano, F. 1993. Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor. Proc. Natl Acad. Sci. USA 90:2700.[Abstract]
-
Wickham, T. J., Mathias, P., Cheresh, D. A. and Nemerow, G. R. 1993. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 73:309.[ISI][Medline]
-
Stevenson, S. C., Rollence, M., White, B., Weaver, L. and McClelland, A. 1995. Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain. J. Virol. 69:2850.[Abstract]
-
Hussain, L. A., Kelly, C. G., Fellowes, R., Hecht, E. M., Wilson, J., Chapman, M. and Lehner, T. 1992. Expression and gene transcript of Fc receptors for IgG, HLA class II antigens and Langerhans cells in human cervico-vaginal epithelium. Clin. Exp. Immunol. 90:530.[ISI][Medline]
-
Regnault, A., Lankar, D., Lacabanne, V., Rodriguez, A., Thery, C., Rescigno, M., Saito, T., Verbeek, S., Bonnerot, C., Ricciardi-Castagnoli, P. and Smigorena, S. 1999. Fc
receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med. 189:371.[Abstract/Free Full Text]
-
Hussain, L. A., Kelly, C. G., Hecht, E. M., Fellowes, R., Jourdan, M. and Lehner, T. 1991. The expression of Fc receptors for immunoglobulin G in human rectal epithelium. AIDS 5:1089.[ISI][Medline]
-
Hussain, L. A. and Lehner, T. 1995. Comparative investigation of Langerhans' cells and potential receptors for HIV in oral, genitourinary and rectal epithelia. Immunology 85:475.[ISI][Medline]
-
Ulstein, M., Jensen, T. S. and Matre, R. 1995. Cyclic variation in the expression of Fc gamma receptors in human endometrium. Immunol. Lett. 46:21.[ISI][Medline]