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*
Laboratory of Cellular Oncology, National Cancer Institute, and
Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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; however, the release of
these cytokines was delayed relative to LPS activation. Production of
IL-12p70 by VLP-exposed DCs required the addition of syngeneic T cells
or rIFN-
. Finally, BMDCs pulsed with HPV16 VLPs induced
Th1-dominated primary T cell responses in vitro. Our data provide
evidence that DCs respond to intact papillomavirus capsids and that
they play a central role in VLP-induced immunity. These results offer a
mechanistic explanation for the striking ability of papillomavirus
VLP-based vaccines to induce potent T and B cell responses even in the
absence of adjuvant. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs are a family of bone marrow-derived, professional APCs with a unique capacity to initiate and modulate cell-mediated immune responses (4, 5, 6). Immature DCs located in peripheral tissues (e.g., epidermal Langerhans cells) function as sentinels of the immune system in that they capture and process Ags (7, 8, 9). A wide range of stimuli, including infectious virus, bacterial Ags, or inflammatory cytokines, can induce maturation of DCs, which is associated with up-regulation of costimulatory molecules as well as those encoded by the MHC (10, 11, 12, 13). Following the acquisition of Ag, DCs migrate to regional lymph nodes, where the presentation of MHC-Ag complexes together with costimulatory molecules leads to T cell activation (14, 15). Thus, DCs function as outposts of immune surveillance in that they trigger primary immune reactions against infectious pathogens including viruses (13).
Most studies on the role of DCs in viral immunity have focused on infectious virus models, which cannot distinguish between the effects of direct interaction with virion structure and those induced by de novo production of viral gene products. It has been shown previously that DC maturation can be induced by viral products such as dsRNA (16), or following interaction between glycosylated viral envelope proteins and lectin receptors expressed on DCs (17). However, it remained unclear whether structural features of nonenveloped virion surfaces can deliver DC-activating signals that are sufficient for the induction of an immune response.
To examine the effects of viral structural components on DC activation, we used papillomavirus-like particles (VLPs) as a model system. Papillomavirus VLPs are nonenveloped icosahedral particles, composed of the L1 major capsid protein, which form via self-assembly in the cell nucleus after high level expression of L1. Papillomavirus VLPs were chosen, because they resemble capsids from infectious virions morphologically and antigenically, while lacking encapsidated viral genes (18, 19, 20, 21).
In several animal models, systemic vaccination with papillomavirus VLPs, engineered and expressed in insect cells, can induce type-specific, high-titer neutralizing Abs against conformationally dependent L1 epitopes, and confer Ab-dependent protection against experimental infection with wild-type virus (20, 22, 23, 24). Based on these characteristics, papillomavirus VLPs are attractive candidates for prophylactic subunit vaccines, with the long-term goal of inducing protection against human papillomavirus (HPV)-induced diseases, including cervical cancer (25, 26). HPV VLPs are currently being studied in early phase human clinical trials, which have confirmed that systemic vaccination even without adjuvant can induce high titers of neutralizing Abs (27, 70). In addition, chimeric VLPs, in which peptides derived from an HPV oncoprotein are fused to a viral capsid protein, can induce specific CTL and protect mice from tumor formation by a syngeneic cell line (28, 29, 30). VLPs can also serve as immunogenic vehicles for fused nonpapillomavirus Ags. For example, CTL can be induced by murine P815 tumor-associated Ag P1A (31), while a strong Ab response to a central self Ag, CCR5, occurred when a cell surface-exposed CCR5 peptide was presented as part of the regular array of assembled VLPs (32).
Parenteral immunization with papillomavirus VLP-based vaccines can induce potent humoral and cell-mediated immune responses even without adjuvant. However, the mechanism(s) that accounts for the high intrinsic immunogenicity of papillomavirus VLPs has not been determined. Therefore, we have begun to address this issue in this study by using immature bone marrow-derived DCs (BMDCs), to assess early events occurring after encounter with noninfectious papillomavirus VLPs and to investigate their ability to initiate primary T cell responses. As controls, we investigated the DC-activating effects of disordered papillomavirus capsomers and of VLPs from human polyomaviruses (BK and JC), which, like papillomavirus VLPs, resemble naked icosahedral virions. We found that only the fully assembled papillomavirus VLPs were capable of inducing acute phenotypic and functional maturation of DCs, resulting in the induction of Th1-dominated, primary immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following VLP preparations were generated using a
baculovirus expression system established in our laboratory
(20): 1) papillomavirus VLPs composed of L1 major capsid
proteins, HPV16(K)-L1 VLPs, the assembly-deficient mutant HPV16(P)-L1,
and bovine papillomavirus (BPV)-L1 VLPs; 2) papillomavirus VLPs
containing L1 and L2 minor capsid protein, HPV18-L1/L2 and BPV-L1/L2
VLPs; and 3) human polyomavirus VLPs composed of VP1 major capsid
proteins, JCV-VP1 VLPs and BKV-VP1 VLPs (Ref. 33 and
S. A. Frye, P. N. Jensen, M. Gravell, and E. O. Major.
Production of the major structural protein of the human polyomavirus
JCV and BKV: self-assembly into virus-like particles and use in
serological studies; manuscript in preparation). Briefly, recombinant
virus was propagated in Sf9 insect cells (ATCC, CRL 1711; American Type
Culture Collection, Manassas, VA) cultured in Graces’ insect medium
supplemented with 10% inactivated FBS, 0.5% calcium chloride, 2.8%
potassium chloride, 3.33% lactalbumin hydrolysate, 3.33% yeastolate,
and 0.1% pluronic F-68 (all Life Technologies, Grand Island, NY). Sf9
cells were adjusted to 3 x 106 cells/ml and
infected at a multiplicity of infection of 
10 with recombinant
baculovirus. After 72 h at 26.6°C, cells were harvested and
washed once in ice-cold PBS. The final pellet was either snap frozen in
liquid nitrogen and stored at -70°C or processed immediately. For
VLP preparation, the pelleted cells were lysed by short-pulse
sonification on ice (Fisher sonic dismembrator). The lysates were
loaded on top 40% (w/v) sucrose (ICN Biomedicals, Aurora, OH)/PBS
cushions and centrifuged at 25,000 rpm for 2.5 h in a SW-28 rotor
(Beckman, Fullerton, CA). The resulting pellet was resuspended in 2 ml
of 27% (w/w) cesium chloride (CsCl; Life Technologies)/PBS and
sonicated a second time. Cell lysates were subjected to two subsequent
centrifugations to equilibrium in 27% (w/w) CsCl (20 h/28,000
rpm/10°C). Fractions were harvested and analyzed by SDS/PAGE and
Coomassie blue staining or Western blotting using the cross-reactive
anti-L1 mAb Camvir-1 (PharMingen, San Diego, CA) for detection of
denatured L1. Immunoreactive fractions were dialyzed against 0.5 M NaCl
in PBS and quantified by Coomassie blue staining of 10% Tris-HCl gels
(Bio-Rad, Hercules, CA) by comparison with a BSA (Life Technologies)
standard. To investigate possible effects of contaminating Sf9 proteins
in the VLP preparations, a negative control preparation was generated
by pelleting uninfected Sf9 cells and processing as for VLP
purification.
Transmission electron microscopic analysis of VLP preparations
To determine the efficiency of VLP assembly, transmission electron microscopy was performed by absorbing particles onto carbon-coated grids, negative staining with 1% uranyl acetate, and examination with a Philips electron microscope (model EM 400T) at a x36,000 magnification.
Animals
C57BL/6 (H2b) inbred mice, ages 6–8 wk, were obtained from the National Cancer Institute Frederick Cancer Research Facility Animal Production Area (Frederick, MD) and housed under pathogen-free conditions. Mice were euthanized by CO2 asphyxiation for organ harvest.
Generation of BMDCs
BMDCs were obtained according to standard protocols with slight modifications (34). Briefly, hind extremities of C57BL/6 mice were collected, soft tissues removed, and bones rinsed in 70% ethanol. After cutting the ends of femurs and tibias, bone marrow was flushed out with RPMI 1640 (Life Technologies) and collected through a nylon mesh. Red cells were lysed with ACK lysing buffer (BioWhittaker, Walkersville, MD). DCs and precursors were enriched by negative selection of lineage marker-positive cells using magnetic beads (anti-CD4 (L3T4), anti-CD8a (Ly-2), anti-CD19, and anti-NK cell (DX5) MicroBeads; Miltenyi Biotec, Auburn, CA), according to the manufacturer’s protocol. The remaining cells were plated in 24-well plates at 1 x 106 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 5 x 10-5 M 2-ME, 25 mM HEPES, 50 µg/ml gentamicin, and 1x antibiotic/antimycotic solution (all from Life Technologies) (normal culture medium, NCM). Added to the cultures were murine rGM-CSF (1000 U/ml) and murine rIL-4 (1000 U/ml; both PharMingen). After 3 days at 37°C, cultures were fed by aspirating 75% of medium and adding back fresh NCM supplemented with GM-CSF and IL-4. At day 6, cells were harvested and preparations contained 67–88% DCs, as assessed by CD11c expression.
DC stimulation and pulsing
To investigate DC maturation, day 6 BMDCs were replated at 1 x 106 cells/ml in 24-well plates and exposed to various concentrations of VLPs (0.1–10 µg/ml). Control experiments were performed adding LPS or VLPs directly to the developing DCs without replating the cells. The results of these experiments were very similar to those obtained by replating the cells at day 6. By FACS analysis, the background staining of all the markers tested was lower on the cells that had not been replated as compared with background expressions on replated DCs. The phenotypic maturation of the DCs by LPS and VLPs was more pronounced if added directly to the cultures. Up-regulation of costimulatory molecules was observed at 24 h and did not increase after 48 h of exposure to LPS or VLPs. As a positive control, DCs were stimulated with LPS (Escherichia coli 026:b6; Sigma, St. Louis, MO) at 1 µg/ml. As a negative control, an uninfected Sf9 insect cell preparation, generated as described above, was used. At various time points, cells and cell supernatants were obtained and analyzed. For T cell assays, day 6 BMDCs were pulsed by adding HPV16(K)-L1 VLPs at 10 µg/ml. After overnight culture, excess VLPs were removed by thoroughly washing the cells.
Immunofluorescent staining and confocal microscopy
To detect cell-associated VLPs, day 6 BMDCs were adhered to
acid-washed number 01 cover slips in 24-well plates and cultured
overnight. VLPs were added to the cells at a concentration of 0.7
µg VLPs/105 cells. Dilutions were prepared in
cold PBS, and the VLPs were incubated with the cells for 15 min on ice.
Unbound VLPs were removed by washing four times with cold PBS.
Subsequently, the cells were either fixed directly to evaluate cell
surface binding of VLPs, or returned to 37°C in NCM supplemented with
GM-CSF and IL-4 to allow for VLP internalization. Cells were then fixed
at selected time points by a 15-min incubation in 1% paraformaldehyde
diluted in PBS. The method of immunofluorescent staining has been
previously described (35). Briefly, after fixation, the
cells were washed three times with 200 mM glycine/PBS. They were then
incubated with primary Ab diluted in 0.1% Brij 58/PBS and incubated at
4°C. Abs used were 5B6 for detection of BPV-L1 (36, 37),
and 5.12.2 and 3.1.1 to detect JC and JC/BK VLPs, respectively
(Novocastra Laboratories, Newcastle, U.K.). The secondary Ab,
FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West
Grove, PA), was diluted to 5 µg/ml in 0.1% Brij 58/PBS. To
distinguish VLP uptake by the BMDCs from that by other phagocytic
cells, double stainings using a PE-labeled anti-CD11c Ab
(PharMingen) were performed. Fluorescence was examined with a Bio-Rad
MRC 1024 laser-scanning confocal system attached to a Zeiss Axioplan
microscope. Images were acquired using the photon counting mode. Images
were collaged and subjected to identical scale adjustment with Adobe
Photoshop (Adobe Systems, Mountain View, CA) software.
FACS staining
Cells were collected into cold Hank’s BSS without Phenol Red
(Life Technologies) plus 0.1% BSA (Life Technologies) and 0.1% sodium
azide (Aldrich, Milwaukee, WI). To avoid nonspecific binding of labeled
Ab, FcBlock (purified rat anti-mouse CD16/CD32; PharMingen) was
added at 1 µg for 106 cells. Single or double
stainings were performed using mAbs against the following mouse surface
Ags (all PharMingen): CD3 (145-2C11, hamster IgG), CD4 (GK1.5, rat
IgG2b), CD8 (53-6.7, rat IgG2a), CD11c (HL3, hamster IgG), CD40 (3/23,
rat IgG2a), CD54 (3E2, hamster IgG), CD80 (16-10A1, hamster IgG), CD86
(GL1, rat IgG2a), H-2Kb (AF6-88.5, mouse IgG2a), I-Ab (A
b)
(AF6-120.1, mouse IgG2a), and appropriate control Abs. A total of
105 cells per assay was incubated with the
respective Abs at a concentration of 2.5 µg/sample for 30–45 min at
4°C. After two rounds of washings, cellular fluorescence was
monitored in a FACSCalibur and analyzed using CellQuest software (both
Becton Dickinson, Mansfield, MA). Propidium iodide (Aldrich) was added
to gate on viable cells.
Cytokine ELISAs
Cytokine levels in conditioned cell supernatants were assayed by
ELISAs for murine IL-4, IL-6, IL-10, IL-12p70, TNF-, and IFN-
(all R&D Systems, Minneapolis, MN), according to the manufacturer’s
instructions. Adsorbance was read at 450 nm with an automated
microplate ELISA reader. Cytokine levels were quantified from two to
three titrations using standard curves, and results were expressed in
pg/ml. The lower detection limits for the assays were as follows: IL-4,
7.8 pg/ml; IL-6, 15.6 pg/ml; IL-10, 15.6 pg/ml; IL-12p70, 7.8 pg/ml;
TNF-, 23.4 pg/ml; and IFN-, 9.4 pg/ml.
Syngeneic T cell assay
T cells were obtained from spleen cells of C57BL/6 mice by
positive selection using magnetic beads (anti-CD4 (L3T4) and
anti-CD8a (Ly-2) MicroBeads; Miltenyi Biotec) with a purity of
>95%. T cells were placed into 96-well round-bottom plates at 2
x 105 cells/well in NCM and stimulated with
either VLP-pulsed or untreated syngeneic DCs at a ratio of 10:1. After
7 days in culture, T cells were harvested, replated at 2 x
105 cells/well in NCM supplemented with murine
IL-2 (20 U/ml; PharMingen), and restimulated with DCs as before. Three
days after the second stimulation, T cell proliferation was assessed
using the CellTiter96 Aqueous assay (Promega, Madison, WI). Cell
proliferation was detected by
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(Owen’s reagent) bioreduction into formazan that can be determined by
absorbance at 490 nm using a microplate reader. To correct for the
presence of BMDCs in the cocultures, the absorbance obtained from DC
cultures was subtracted (corrected absorbance 490 nm). In addition,
culture supernatants were collected for cytokine detection (IFN-,
IL-4, and IL-10).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Papillomavirus VLPs have been shown to bind to a wide range of
cell types, including APCs from different sources (Refs.
38 and 39 ; W. M. Kast, unpublished data).
However, the ability of BMDCs to bind and internalize VLPs has
not been evaluated. To address this issue, we exposed BPV-L1/L2 VLPs to
immature mouse BMDCs and used confocal microscopy to monitor the
association of VLPs with the cell surface and their internalization
(Fig. 1
). We detected high levels of VLP
binding to the BMDCs, with a pattern similar to that of control mouse
fibroblasts, which are known to be infectable by BPV (40)
(Fig. 1 and data not shown). However, internalization of bound VLPs by
BMDCs was significantly more rapid than by the fibroblasts (data not
shown). Particles were internalized by the immature BMDCs as early as 5
min after incubation at 37°C (Fig. 1). Within 15 min, high levels of
VLPs were localized predominately in the dendrites and in cytoplasmic
vesicles located adjacent to the cell membrane. After 30 min, uptake
had reached saturation, and no further reduction of surface-bound VLPs
was observed at later time points, suggesting that the uptake capacity
of the cells was exhausted. Over the next 90 min, particles coalesced
into dense vesicles within the cells and relocalized to the perinuclear
region. Internalization of the VLPs by DCs was confirmed by costaining
for CD11c (data not shown). Thus, immature DCs effectively bind and
rapidly internalize papillomavirus VLPs.
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When DCs use internalized proteins to stimulate T cell responses,
the DCs must undergo maturation and migrate from the periphery to
regional lymph nodes. To determine whether VLPs induce phenotypic DC
maturation, BMDCs were exposed to serial dilutions of HPV16(K)-L1 VLPs,
and expression of cell surface markers was determined by flow
cytometry. We found a dose-dependent up-regulation of MHC class I and
II molecules as well as costimulatory molecules (CD40, CD80, CD86) and
the CD54 adhesion molecule (Fig. 2). At
VLP concentrations of 10 µg/ml, we observed maturation of DCs equal
to that after exposure to bacterial LPS (Table I). Phenotypic maturation occurred also
after exposure to VLPs at a concentration of 1 µg/ml (3 x
104 VLPs/DC) (Fig. 2), while no significant
response was detectable at 0.1 µg/ml (data not shown). The kinetics
of the induction of cell surface markers was similar for LPS and VLPs,
in that it required 24 h for the activated phenotype to be
present, and the activated state remained for 48 h with no further
up-regulation of any of the markers tested (Fig. 3).
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), indicating that DC activation is a
general characteristic of papillomavirus VLPs. To rule out the
possibility that components other than the VLPs in the preparations
might have induced the observed effects, a negative control sample,
prepared from uninfected Sf9 cells, was added to BMDCs, but it failed
to induce markers of activation (data not shown). Thus, papillomavirus
VLPs induce acute phenotypic maturation of BMDCs.
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To determine whether the assembly state of L1 influenced the
induction of BMDC maturation, we compared the stimulatory capacity of
fully assembled HPV16(K)-L1 VLPs with that of an assembly-deficient
mutant HPV16(P)-L1. This L1 mutant, from an HPV16 genome isolated from
cervical cancer, is defective in proper VLP assembly due to a single
amino acid substitution at residue 202 (21). The wild-type
and mutant L1s were purified concurrently using the same procedure. We
confirmed by electron microscopy that HPV16(K)-L1 had efficiently
assembled into complete spherical VLP structures of 55 nm in
diameter with a regular array of capsomers (Fig. 5). In contrast, preparations of the
mutant HPV16(P)-L1 contained predominately (>90%) unassembled
pentamers or irregular aggregates, with properly assembled VLPs
detected only rarely (Fig. 5). When added to immature BMDCs, the
HPV16(P)-L1 preparation was at least 10-fold less effective in inducing
phenotypic DC maturation than fully assembled HPV16(K)-L1 VLPs.
Although 10 µg/ml of wild-type virus induced the up-regulation of a
wide panel of activation markers, the mutant at this concentration
induced only one of them (CD80) (Fig. 6).
No up-regulation was seen with the mutant at 1 µg/ml (data not
shown), although at this concentration the wild type still induced
phenotypic maturation (Fig. 2
). Therefore, in the interaction of HPV16
capsid proteins with DCs, well-ordered particles are superior in their
capacity to activate DCs.
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To investigate the possibility that the DC-stimulatory capacity of
papillomavirus VLPs is a common characteristic of naked icosahedral
VLPs, we tested VLPs of two human polyomaviruses, BK and JC virus, for
their effect on BMDC maturation. These VLPs were chosen because
polyomaviruses resemble papillomaviruses morphologically in that they
all share an icosahedral structure composed of 72 pentamers of the
major capsid protein arranged in T = 7 symmetry, although
polyomavirus capsids are somewhat smaller than those of
papillomaviruses (Fig. 5). By confocal microscopy, using a panel of
monoclonal and polyclonal Abs, we found that immature BMDCs effectively
bound and internalized BKV-VP1 and JCV-VP1 VLPs (data not shown).
However, both polyomavirus VLPs failed to induce up-regulation of MHC
and costimulatory molecules, even at concentrations of VLPs that were
100 times higher than that sufficient for phenotypic activation by
papillomavirus VLPs (Fig. 6 and data not shown). Therefore, VLPs with
very similar structures can differ markedly in their capacity to
activate BMDCs.
Papillomavirus VLPs induce secretion of inflammatory cytokines by DCs
Given that HPV16 is the papillomavirus type most commonly associated with cervical cancer (26) and that HPV16-L1 VLPs are currently being evaluated in clinical trials, we focused on VLPs from this HPV type for subsequent experiments.
It is well established that DCs are a significant source of a wide
range of cytokines that are secreted in response to various stimuli,
e.g., viruses or bacteria and their products, such as LPS
(41, 42, 43). To investigate whether VLPs are capable of
inducing cytokine production in DCs, levels of IL-6, IL-12p70, and
TNF- were determined in supernatants of BMDCs cultured in the
presence or absence of different concentrations of HPV16(K)-L1 VLPs.
Bacterial LPS (1 µg/ml) was used as the positive control. Although
BMDCs secreted low to undetectable baseline levels of the above
cytokines, HPV16(K)-L1 VLPs induced the secretion of IL-6 and TNF-
in a dose-dependent manner (Fig. 7).
However, this treatment did not result in appreciable IL-12p70
production. When the kinetics of cytokine production was analyzed,
secretion of IL-6 and TNF- in response to HPV16 VLPs occurred more
slowly than that induced by LPS, which rapidly induced inflammatory
cytokines as early as 6 h after stimulation, with maximum levels
reached at 18 h (Fig. 7, A and B). However,
at 48 h, the VLPs (10 µg/ml) induced similar levels of TNF-
and higher levels of IL-6 compared with the levels induced by LPS at
this time point. In parallel experiments, BKV-VP1 and JCV-VP1 VLPs did
not induce DCs to secrete proinflammatory cytokines (IL-6, 31.6 ±
10 pg/ml; TNF-, 23.4 pg/ml (not above background)).
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(44, 45, 46). Therefore, we cocultured BMDCs with syngeneic T
cells in the presence or absence of VLPs. In this setting, HPV16(K)-L1
VLPs effectively induced IL-12p70 secretion by BMDCs. Moreover,
rIFN- was able to substitute for T cells in inducing significant
levels of IL-12 (Fig. 7C). Control experiments revealed that
HPV16(K)-L1 VLPs had no direct effect on T cell phenotype (the
expression of CD25 and CD154, as measures of T cell activation, was
unchanged over background) or IFN- production (data not shown).
These results suggest that exposing BMDCs to VLPs can
activate T cells, which in turn provide the signals, including IFN-,
sufficient for IL-12 secretion by the BMDCs. DCs pulsed with papillomavirus VLPs induce primary, Th1-dominated T cell responses
Because DCs are the only cell type capable of inducing primary T
cell responses, we investigated the effect of VLP-pulsed BMDCs on
activation of syngeneic, naive T cells. To exclude direct effects of
VLPs on T cells, BMDCs were pulsed overnight with VLPs to allow
processing and presentation of VLP proteins (39, 47) and
were then added to the T cells. BMDCs pulsed overnight with HPV16(K)-L1
VLPs induced significant T cell proliferation measured at day 3 after
the second stimulation (Fig. 8). In
addition, culture supernatants obtained from T cells conditioned with
VLP-pulsed DCs contained high levels of IFN- (Fig. 8), but lacked
detectable levels of the Th2 cytokines IL-4 or IL-10 (data not shown).
Thus, the VLP-pulsed BMDCs induce Th1-dominated immune responses in
naive, syngeneic T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); and 3) those that induce effector
functions (production of IL-12p70). Papillomavirus VLPs composed of L1
alone or L1 and minor capsid protein L2 induced maturation of BMDCs
(Fig. 4), correlating with in vivo data demonstrating that vaccination
with L1 or L1/L2 VLPs resulted in protective immune responses
(18, 22, 48). A threshold concentration of 10 µg/ml of
VLPs was required for optimal phenotypic and functional activation of
106 DC, although some response was seen at 1
µg/ml. Given that VLP concentrations applied during vaccination were
usually 1–100 µg VLPs per injection (18) and that as
few as 100 DCs are sufficient to induce an in vivo immune response in
mice (49), our results are likely to reflect the
physiologic situation after VLP vaccination. Therefore, these data
provide an experimental explanation for the pronounced immunogenicity
of papillomavirus VLP-based vaccines even in the absence of
adjuvant. Several observations support the conclusion that the DC activation seen in our assays resulted from specific recognition of VLP structural elements and not from copurified contaminants in the VLP preparations. First, neither similarly processed lysates of uninfected Sf9 insect cells nor similar preparations of polyomavirus VLPs, also derived from baculovirus-infected Sf9 cells, induced DC activation. Second, the assembly-deficient mutant L1 of the reference strain of HPV16 (21) inefficiently activated DCs. The low level residual activity of mutant L1 observed in our experiments may result from the low number of properly assembled VLPs in the preparation or from suboptimal activation of the DCs by disorganized aggregates of L1 capsomers.
Recently, so-called PRRs of the innate immune system have been
identified that mediate the immune recognition of microbial Ags by
means of certain conserved structural features (2). DCs
express several types of PRRs, including the mannose receptor DEC 205
and Toll-like receptors (3, 50), suggesting that they may
be important components of the innate immune system before initiation
of Ag-specific immune responses. However, pattern recognition of virion
structural elements by DCs is poorly understood. To our knowledge,
there is only one previous report that examined the effect of
noninfectious viral subunit vaccines on DC functions (51).
In that study, two different influenza virus subunit vaccine
preparations (one conventional viral subunit vaccine composed of viral
hemagglutinin and neuraminidase proteins alone, and one virosomal
subunit vaccine, in which influenza hemagglutinins were incorporated
into the surface of liposomes) were found to have no effect on the
phenotype or secretory functions of human DCs. Because an inactivated
whole virus influenza vaccine preparation used in that same study
induced DCs to up-regulate MHC and costimulatory molecules and to
secrete IL-12p70 and TNF-, the authors suggested that these positive
effects on DCs might have resulted from the presence of viral RNA
sequences analogous to bacterial DNA motifs containing unmethylated CpG
dinucleotides (52). In contrast to inactivated enveloped
viruses used in earlier studies (51, 53), our VLP
preparations do not contain the viral genome, other viral proteins, or
cellular membrane proteins of the heterologous cells used to produce
the virions. Therefore, the ability of papillomavirus VLPs to activate
immune functions in DCs is most likely related to the ordered
repetitive array of virion surface proteins and the interaction of
these proteins with yet to be identified DC cell surface receptors,
presumably leading to receptor oligomerization and subsequent
downstream signaling.
To characterize receptor-ligand interactions early after encounter of
immature DCs with papillomavirus VLPs, we performed binding and
internalization studies using confocal microscopy. The abundant
association of VLPs to the cell surface of our highly enriched murine
BMDC preparations and the rapid internalization observed support the
notion that BMDCs possess specific VLP receptors. These findings are
consistent with those of other investigators, who have shown, by FACS
analysis of bulk cell populations, that APCs from mouse spleen or human
PBMCs, as well as human Langerhans cells, bound HPV16 VLPs (W. M.
Kast, unpublished data). FcRIII (CD16) was proposed as a
candidate receptor for papillomaviruses in that study. Therefore, the
observed association of papillomavirus VLPs with the DC surface might
result, at least partially, from the binding to Fc receptors,
which are known to be highly expressed on DCs (54, 55, 56, 57).
Other possible receptors include cell surface proteoglycans
(58) and 6 integrin
(59). The latter is less likely to play a role in VLP-DC
interaction, as monocyte-derived DCs (60) and BMDCs do not
express this integrin (our own unpublished observation).
Secretion of cytokines such as IL-6, IL-12, or TNF- is a
critical component of immune regulation of B and T lymphocytes during
encounter with an infectious agent (61, 62, 63). As the
interaction of DCs with certain viruses (e.g., HIV) can induce mRNA for
a wide range of cytokines, but without cytokine production, cytokine
transcription and translation in DCs can in some instances be
differentially regulated (42). The effect of noninfectious
subunit vaccines on the cytokine secretory activity of DCs is less well
understood and seems to be critically dependent on the vaccine
preparation studied (51). We found that HPV16 VLPs were
potent stimulators of DC-derived IL-6 and TNF-, which were
detectable at the protein level as secreted proteins. Compared with
LPS, the overall cytokine response to VLPs was as pronounced, yet
relatively delayed. By contrast, the kinetics of phenotypic activation
of DCs after exposure to either stimulus was similar. If the time
course of maturation relative to cytokine release in VLP-activated DCs
is similar in vivo, these results suggest that most of the cytokines
would be released after the DCs reached the regional lymph nodes, which
appears to occur within 6–48 h after Ag exposure (64, 65). This may account, at least partially, for the observation
that immunization with HPV L1 VLPs did not induce substantial
inflammation at the side of injection in early phase clinical trials
(27 , 70). This observation might also, in
part, explain the characteristic persistence of localized productive
papillomavirus infection in the absence of inflammatory responses. The
rapid cytokine secretion induced by LPS is consistent with the strong
local inflammatory reaction after injection of LPS in vivo. It will be
interesting to determine whether viral and bacterial inducers of DC
activation generally exhibit differences in the kinetics of cytokine
release. An attractive hypothesis is that the acute response to
bacterial products has evolved to produce immediate inflammation to
focus the immune response to sites of infections, which tend to remain
localized. In contrast, infections by most viruses (with
papillomaviruses a notable exception) rapidly become disseminated, and
an unrestricted inflammatory response would likely be
counterproductive.
In previous studies, IL-12 production by DCs was tightly regulated and
required additional signals from T cells (44, 45, 46).
Similarly, we found that HPV16 VLPs failed to induce IL-12p70 directly,
but required help from syngeneic T cells. Given that papillomavirus
VLPs had no direct effect on IFN- production or on phenotypic
activation of T cells (data not shown), these findings indicate that
VLP-exposed DCs activate T cells, which in turn provide the signal(s),
including IFN , necessary for the induction of IL-12p70 by the DCs.
Consistent with this hypothesis, we found that rIFN- can substitute
for T cells in stimulating VLP-exposed DCs to secrete IL-12p70 and that
VLP-pulsed DCs stimulated T cells to secrete IFN- (Fig. 8).
Our data strongly suggest that DCs activated by papillomavirus
VLP may be highly effective inducers of Ag-specific cellular immunity.
We found that immature BMDCs pulsed with HPV16 VLPs induced
proliferation of syngeneic T cells, which also produced IFN- (but
not IL-4 or IL-10), thus initiating a strongly polarized Th1 immune
response. Using an intracellular cytokine assay, we have preliminary
data indicating that DCs armed with papillomavirus VLPs generate both
CD4+ and CD8+ T cell
responses (data not shown). These findings point to a central role of
DCs in the initiation and modulation of T cell responses to
papillomavirus VLPs and offer mechanistic explanations for the strong
adjuvant-independent antitumor immune response to chimeric
papillomavirus VLPs in mouse tumor models (28, 29, 30, 31).
Because DCs are critically involved in B cell differentiation and
isotype switching (66), VLP-activated DCs may also
facilitate the pronounced Ab response against conformational L1
epitopes observed after VLP vaccination in the absence of adjuvant.
There are several possible explanations for the striking difference in the response of DCs to the structurally similar papillomavirus and polyomavirus VLPs. Immune responses have evolved under considerable selective pressure imposed by pathogens, which may have resulted in the recognition of naked icosahedral virion structures through yet to be identified receptors. During natural infection with papillomaviruses, virions are shed to the exterior by the terminally differentiated epithelial cells and therefore are not readily exposed to the systemic immune system in large numbers. Given that DC activation appears to require interaction with multiple virus capsids (>1000 VLPs per cell in our in vitro assays), there may not be a strong selective pressure against the recognition of papillomaviruses by DCs. In contrast, some lytic viruses that induce persistent systemic infections as part of their normal life cycles (e.g., BK and JC viruses) may have counterevolved to escape acute pattern recognition by DCs. Other viruses, e.g., herpes simplex, measles, or vaccinia virus, are known to inhibit DC maturation (67, 68, 69), but this inhibition is thought to require DC infection. It is interesting that DCs retain the ability to bind and internalize BK and JC VLPs, suggesting that the absence of activation in the case of polyomaviruses may be due to differences in the specific receptors bound by the two groups of viruses. Alternatively, intracellular events may account for the differences in DC activation upon internalization of viral Ags.
Collectively, our results point to a central role of DCs in acute immune recognition of papillomavirus virions. Although papillomavirus structural elements were necessary and sufficient to activated DCs, it is clear that gross structural features of a virus capsid do not allow predictions about its effect on DC functions or provide insights into which VLPs will most likely serve as potent vaccine vehicles. These results contribute significantly to a better understanding of the mechanisms involved in the immune response to virion capsids and may provide knowledge for the rational design and development of future vaccines.
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Footnotes |
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2 Current address: Biotechnology Centre of Oslo-Bio, Forskningsparken, Gaustadalleen 21, 0137 Oslo, Norway.
3 Address correspondence and reprint requests to Dr. John T. Schiller, Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Building 36, Room 1D28, 36 Convent Drive MSC4040, Bethesda, MD 20892-4040.
4 Abbreviations used in this paper: PRR, pattern recognition receptor; DC, dendritic cell; BMDC, bone marrow-derived DC; BPV, bovine papillomavirus; HPV, human papillomavirus; NCM, normal culture medium; VLP, virus-like particle.
Received for publication November 8, 2000. Accepted for publication February 16, 2001.
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