U.S. patent application number 09/874506 was filed with the patent office on 2001-10-11 for papillomavirus cellular receptor.
Invention is credited to Kast, Wijbe M., Nieland, John D..
Application Number | 20010028883 09/874506 |
Document ID | / |
Family ID | 22234280 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010028883 |
Kind Code |
A1 |
Kast, Wijbe M. ; et
al. |
October 11, 2001 |
Papillomavirus cellular receptor
Abstract
The present invention provides a complex comprising a
biologically active substance and a ligand that recognizes
CD16.
Inventors: |
Kast, Wijbe M.;
(Willowbrook, IL) ; Nieland, John D.; (Munich,
DE) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Family ID: |
22234280 |
Appl. No.: |
09/874506 |
Filed: |
June 5, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09874506 |
Jun 5, 2001 |
|
|
|
09348573 |
Jul 7, 1999 |
|
|
|
6242176 |
|
|
|
|
60092637 |
Jul 13, 1998 |
|
|
|
Current U.S.
Class: |
424/178.1 ;
424/1.49; 424/134.1; 424/143.1; 424/183.1; 424/450; 435/343;
435/343.1; 435/343.2; 435/345; 435/5; 435/7.1; 435/7.24; 436/548;
514/44R; 530/351; 530/388.22; 536/23.1; 536/23.5; 536/23.52 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/283 20130101; A61K 38/00 20130101; A61K 39/00 20130101;
C07K 14/005 20130101; C12N 2710/20022 20130101 |
Class at
Publication: |
424/178.1 ;
424/1.49; 514/44; 435/5; 435/7.1; 435/7.24; 435/345; 435/343;
435/343.1; 435/343.2; 436/548; 530/351; 530/388.22; 424/134.1;
424/450; 424/143.1; 424/183.1; 536/23.1; 536/23.5; 536/23.52 |
International
Class: |
A61K 051/00; A61K
048/00; A61K 039/395; A61M 036/14; C12Q 001/70; G01N 033/53; C07H
021/02; C12N 005/06; C12P 021/08; C07K 001/00 |
Goverment Interests
[0002] This invention was made with Government support under Grant
Number RO1 CA74397 awarded by the National Institutes of Health.
The United States Government may have certain rights in this
invention.
Claims
What is claimed is:
1. A complex comprising a pharmacologically active substance and a
ligand that recognizes CD16.
2. The complex of claim 1, further comprising an agent for
tethering said pharmacologically active substance to said
ligand.
3. The complex of claim 1, further comprising a liposome.
4. The complex of claim 1, wherein said ligand is an antibody
recognizing an epitope on said CD16.
5. The complex of claim 1, wherein said ligand is a protein
comprising an external domain of a papillomavirus capsid
protein.
6. The complex of claim 1, wherein said pharmacologically active
substance comprises a cytotoxic protein, a chemotherapeutic agent,
or a radioactive agent.
7. The complex of claim 1, wherein said pharmacologically active
substance comprises a gene encoding a bioactive molecule.
8. The complex of claim 7, wherein said bioactive molecule is a
protein that promotes cell death.
9. The complex of claim 8, wherein said protein is an enzyme that
converts a prodrug into a cytotoxin.
10. The complex of claim 7, wherein said bioactive molecule is an
RNA having a sequence antisense to a portion of a papillomavirus
gene.
11. The complex of claim 1, comprising a chimeric protein having a
first domain comprising said ligand and a second domain comprising
said pharmacologically active substance.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of co-pending U.S. patent application
Ser. No. 09/348,573, filed on Jul. 7, 1999, claiming the benefit of
U.S. Provisional Application No. 60/092,637, filed Jul. 13,
1998.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates to methods and reagents for
treating or preventing papillomavirus infection.
BACKGROUND OF THE INVENTION
[0004] Papillomavirus are causative agents for several types of
epithelial and mucosal diseases. Of particular concern is that
certain strains of papillomavirus are associated with cervical and
penile cancers (see, e.g., Iwasawa et al., J. Urol., 149, 59-63
(1993); Koutsky et al., N. Engl. J. Med., 327, 1272-78 (1992)).
Considerable efforts, therefore, are underway to prevent the spread
of this virus by developing a prophylactic vaccine and novel
treatments for papillomavirus-induced lesions (see, e.g., Cason et
al., Vaccine, 11, 603-11 (1993); Crawford, Cancer Surv., 16, 215-29
(1993)). Such efforts, however, have been hampered by the
particular difficulties of working with papillomavirus in vitro. To
complete its life cycle, papillomavirus requires its host cell to
undergo a differentiation event. Currently, no in vitro culture
system duplicates this state adequately to permit efficient
papillomavirus growth in vitro.
[0005] Papillomaviruses are nonenveloped double-stranded DNA
viruses about 55 nm in diameter with an approximately 8-kb genome
in a nucleohistone core (Baker et al., Biophys J. 60, 1445-56
(1991)). The capsids include two viral proteins (L1 and L2) of
about 55 kDa and 75 kDa, respectively (Larson et al., J. Virol.,
61, 3596-3601 (1987)). L1 is the major capsid protein, and it is
arranged in 72 pentameres within the capsid. In fact, L1 has the
ability to self-assemble into virus-like particles (VLPs) upon
production of the L1 protein in eukaryotic cells (see, e.g.,
Hagensee et al., J. Virol, 67, 315-22 (1993); Kimbauer et al., J.
Virol., 67, 6929-36 (1993)). The function and position of L2 within
the virion are not clear, although the protein is assembled with L1
into VLPs when coexpressed in cells.
[0006] Because of the lack of suitable papillomavirus culture
conditions, VLPs typically are used for in vitro studies of
papillomavirus infection, as opposed to intact papillomavirus (see,
e.g., Roden et al., J. Virol., 68, 7260-66 (1994); Volpers et al.,
J. Virol., 69, 3258-64 (1995)). Using VLPs, it is now thought that
a putative cell receptor for papillomavirus is expressed across a
wide range of cell types and is highly conserved between a diverse
group of organisms (see, e.g., Muller et al., J. Virol., 69, 948-54
(1995); Volpers et al., supra; Roden et al., supra). Currently,
little is understood about papillomavirus infection, particularly
the identity of a cell surface receptor recognizing
papillomavirus.
[0007] In view of the foregoing problems, there exists a need for
methods and reagents for treating or preventing papillomavirus
infection.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention is predicated, at least in part, on
the discovery that papillomavirus selectively binds the CD16
protein present in a variety of cells. Using this discovery, the
present invention provides methods and reagents for treating or
preventing papillomavirus infection. In one aspect, the present
invention provides reagents and methods for attenuating the ability
of papillomavirus to bind to cells by blocking access of
papillomavirus to its cellular receptor. In another aspect, the
present invention provides a method of attenuating the ability of
papillomavirus to infect cells by reducing the free titer of
papillomavirus. In another aspect, the present invention provides a
complex comprising a biologically active substance and a ligand
that recognizes CD16 and a method of delivering a biologically
active substance to an HVP-infected cell using the complex. These
and other advantages of our invention, as well as additional
inventive features, will be apparent from the following detailed
description.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention provides methods and reagents for
treating or preventing papillomavirus infection. In one aspect, the
method attenuates the ability of papillomavirus to bind cells. One
method of attenuating the ability of papillomavirus to bind cells
is to expose the cells to a ligand that recognizes CD16 under
conditions sufficient for the ligand to bind CD16. The bound ligand
interferes with subsequent interaction between papillomavirus and
the CD16 molecules; thus, by such treatment, the ability of
papillomavirus to subsequently bind cells is attenuated.
[0010] In this context, the ligand is present on any molecule
suitable for blocking the interaction between papillomavirus and
the bound CD16 protein. While many types of molecules can provide
the ligand, generally the ligand is present as part of a protein.
For example, the ligand can be an antibody recognizing an epitope
on CD16. In other embodiments, the ligand is on a protein including
an external domain of an papillomavirus capsid protein (e.g., L1,
L2, or a soluble derivative of L1 or L2 retaining an extracellular
domain). The residues that comprise the ligand in a protein need
not necessarily be contiguous in the chain of amino acids that make
up the protein. In other words, the ligand can be generated by the
particular conformation of the protein, e.g., through folding of
the protein in such a way as to bring contiguous and/or
noncontiguous sequences into mutual proximity. Such proteinatious
species can be synthesized using standard direct peptide
synthesizing techniques (see, e.g., Bodanszky, Principles of
Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)), such as via
solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc.,
85, 2149-54 (1963); Barany et al., Int. J. Peptide Protein Res.,
30, 705-739 (1987); and U.S. Pat. No. 5,424,398). Alternatively,
such modified proteins can be chemically crosslinked, and a variety
of cross-linking agents are known in the art and widely available
(e.g., succinimidyl or maleimidyl cross-linkers). Methods for
conjugating peptides and polyamines are also well-known in the art
(see, e.g., Staros, Biochem., 21, 3990 (1982)). Alternatively, a
DNA fragment encoding the protein can be subcloned into an
appropriate vector using well known molecular genetic techniques.
The fragment is then transcribed and the peptide subsequently
translated in vitro within a host cell. Any appropriate expression
vector (see, e.g., Pouwels et al., Cloning Vectors: A Laboratory
Manual (Elsevior, NY: 1985)) and corresponding suitable host cells
can be employed for production of recombinant peptides. Expression
hosts include, but are not limited to, bacterial species, mammalian
or insect host cell systems including baculovirus systems (e.g., as
described by Luckow et al., Bio/Technology, 6, 47 (1988)), and
established cell lines such 293, COS-7, C127,3T3, CHO, HeLa, BHK,
etc. From such cells, the modified chimeric proteins can be
harvested by standard techniques.
[0011] The ligand is exposed to the cells, e.g., under conditions
suitable for the ligand to bind the CD16 protein. For example, for
cells in vitro, the ligand-bearing molecules can be added to the
cell culture for a time sufficient for them to bind the CD16
molecules on the cells. For cells in vivo, the ligand-bearing
molecules can be supplied in a pharmacologically acceptable carrier
(e.g., a solution, gel, magma, or salve). In this regard, the
ligand can be delivered topically to cells within a discrete organ
or tissue or systemically to attenuate papillomavirus infection on
a more wide-spread scale.
[0012] The ligand recognizing CD16 is exposed to the cells under
conditions sufficient for the ligand to bind the cells. The
concentration of ligand, as well as the conditions required for
efficient binding, will depend on the type of ligand employed and
location of delivery. However, it is within the routine skill in
the art to investigate the kinetic profile of such ligands in
advance of an application.
[0013] In accordance with the present invention, another method of
attenuating the ability of papillomavirus to infect cells is to
reduce the free titer of papillomavirus. In the context of the
invention, this can be achieved by exposing papillomavirus to a
ligand that recognizes the surface of an papillomavirus under
conditions sufficient for the ligand to bind the papillomavirus.
The resultant papillomavirus/ligand complex is less able to infect
cells than unbound papillomavirus (i.e., it forms a noninfective
complex).
[0014] In this context, the ligand can be present on any molecule
suitable for interacting with papillomavirus. While many types of
molecules can provide the ligand, generally the ligand is present
as part of a protein. For example, the ligand can be an antibody
recognizing the surface of the papillomavirus virion (or one of the
two capsid proteins). Many such antibodies are known in the art,
and some are commercially available. In other embodiments, the
ligand is present in a protein including an external domain from
the CD16 protein. For example, soluble derivatives of the CD16
protein are disclosed in published international application WO
89/11490. Where the ligand is proteinatious, the protein can be
manufactured by any suitable method, such as the methods discussed
above.
[0015] The ligand recognizing papillomavirus is exposed to the
virus under conditions suitable for the ligand to bind
papillomavirus. For example, to attenuate infection of cells in
vitro, the ligand can be added to the culture solution. To
attenuate in vivo infection, the papillomavirus-ligand can be
supplied in a pharmacologically acceptable carrier (e.g., a
solution, gel, magma, or salve). In this regard, for example, a
pharmacologically-acceptable carrier containing the ligand
recognizing papillomavirus can be injected into a patient (e.g.,
intravenously, subcutaneously, etc.) or applied to the skin or
mucosa to reduce the titer of papillomavirus. Alternatively, the
ligand recognizing papillomavirus can attenuate infection of cells
within a discrete organ or tissue (e.g., a tumor) by supplying the
carrier containing the ligand to the tissue.
[0016] This strategy can also be employed to reduce the
transmission of papillomavirus. For example, as papillomavirus is a
sexually-transmitted virus, the ligand recognizing papillomavirus
can be applied vaginally, for example in a creme, douche, sponge or
other suitable carrier. Alternatively, a carrier containing the
ligand can be used to coat a condom or similar device to attenuate
viral transmission between individuals.
[0017] To reduce viral titer, the ligand recognizing the
papillomavirus surface is exposed to the papillomavirus under
conditions sufficient for the ligand to bind the papillomavirus.
The concentration of ligand, as well as the conditions required for
efficient binding, will depend on the type of ligand employed and
location of delivery. However, it is within the routine skill in
the art to investigate the kinetic profile of such ligands in
advance of an application.
[0018] Regardless of the type of ligand employed (e.g., either a
ligand recognizing papillomavirus or a ligand recognizing CD16),
the present inventive method attenuates the ability of the virus to
infect cells. In many cases, the ability of papillomavirus to bind
a population of cells is reduced by at least an order of magnitude.
Typically, the method can substantially reduce, or even practically
eliminate, the ability of the virus to bind to, and therefore
infect, cells. However, even in protocols in which some residual
viral infection still occurs, the method is a useful prophylaxis or
therapy for papillomavirus-associated disorders. Indeed, any
reduction in the incidence of viral infection renders it less
likely that a given papillomavirus infection will spread or be
transmitted between individuals. Moreover, protocols, such as the
inventive method, that attenuate viral infection can be used in
combination with other regimens to combat papillomavirus infective
diseases.
[0019] Aside from attenuating the ability of papillomavirus to
infect cells, the discovery that papillomavirus binds CD16 affords
a method of delivering a pharmacologically active substance to a
papillomavirus-infected cell. This method uses a complex including
a pharmacologically active substance and a ligand recognizing CD16.
The complex is exposed to the papillomavirus-infected cells under
conditions sufficient for the ligand to bind CD16 on the cells. The
pharmacologically active substance is thus brought into proximity
to, and delivered to, the infected cell. The use of this method
permits the targeted delivery of the pharmacologically active
substance to the infected cells. This permits the employment of
relatively high concentrations of many pharmacologically active
agents to be delivered to the infected cells without many of the
concomitant side effects attributed to the activity of such agents
in noninfected cells.
[0020] For use in this method, the complex includes at least a
ligand recognizing CD16 and a pharmacologically active substance.
In this regard, the ligand is present on any molecule suitable for
interacting with the CD16 protein present on
papillomavirus-infected cells, such as the molecules described
above. The complex is delivered to the infected cells similarly to
the manner described above.
[0021] Within the complex, the pharmacologically active substance
can be any compound that exerts a biological effect on the infected
cells. For example, for treating tumors (e.g., cervical or penile
cancers), warts, or other papillomavirus-related lesions, the
pharmacologically active substance can be a medicament, cytotoxin,
chemotherapeutic agent, radioactive agent, etc.
[0022] In other embodiments, the pharmacologically active substance
is a gene encoding a bioactive molecule. In such circumstances, the
complex delivers the gene to the infected cells such that the cells
internalize the gene and express it to produce the bioactive
molecule. For combating tumors associated with papillomavirus
infection, the gene can encode a cytokine (e.g., tumor necrosis
factor (TNF), TGF-.alpha., TGF-.beta., interleukins (IL) such as
IL-1, IL-2, IL-3, etc., GM-CSF, G-CSF, M-CSF, co-stimulatory factor
B7, etc.), a protein that promotes cell death or an enzyme that
converts a prodrug into a cytotoxin (e.g., HSV-tk, cytosine
deaminase, xanthine/guanine phosphoribosyltransferase, cytochrome
p450 2B1, etc.). Still other bioactive molecules are RNA species
having sequences antisense to portions of papillomavirus genes
(e.g., the genes encoding L1, L2).
[0023] The complex can be formed in any suitable manner. In one
embodiment, the complex can be proteinatious (e.g., a chimeric
protein having the papillomavirus ligand as a first domain and the
pharmacologically active substance as a second domain). For
example, the complex can comprise all or a portion of the CD16
molecule fused to a toxin. A preferred chimeric complex includes a
soluble portion of the CD16 molecule fused to a portion of the
Pseudomonas endotoxin A (see, e.g., U.S. Pat. No. 5,587,455). Such
proteinatious agents can be manufactured by any appropriate method,
such as those discussed above.
[0024] Many such complexes include non-proteinatious components.
For example, such a complex can incorporate nucleic acids (e.g.,
encoding the genes described above). In other embodiments, the
complex can include an agent for tethering the pharmacologically
active substance to the ligand. For example incorporating lipids
into the complex (e.g., in the form of liposomes) enhances cellular
uptake of many types of pharmacologically active agents, especially
nucleic acids. The use of such lipids is especially preferred when
the ligand component of the complex is of viral origin (e.g., L1,
L2, a VLP, etc.) (see, e.g., Innes et al., J. Virol., 64, 957-61
(1990); Morishaita et al., Hypertension, 21, 894-99 (1993); U.S.
Pat. No. 5,635,380). Where a liposome is employed in the complex,
and where a nucleic acid is the pharmacologically active agent,
preferably the liposome contains cationic lipids, but can contain
neutral lipids as well. Preferred cationic lipids include
LIPOFECTIN (DOTMA) (Gibco BRL), LIPOFECTMINE (Gibco BRL), and DOTAP
(Boeringer-Mannheim), and others are known in the art (see, e.g.,
U.S. Pat. No. 5,736,392).
[0025] Such complexes including non-proteinatious components can be
created by mixing the ligand with the lipid and/or nucleic acid and
allowing the complexes to =form. The mixing can occur, for example,
in a serum-free culture medium, and can occur under any suitable
temperature.
[0026] While it is believed that one of skill in the art is fully
able to practice the present invention after reading the foregoing
detailed description, the following examples are set forth to
further illustrate some of its features. In particular, these
examples demonstrate that papillomavirus can selectively bind the
CD16 cell-surface molecule, that a ligand recognizing the CD16 cell
surface molecule can block papillomavirus attachment to cells, that
various papillomavirus-induced tumors express the CD16 molecule,
that CD16 expression is associated with HPV binding in vivo, and
that HPV binds cells poorly unless they express CD16.
[0027] The procedures employed in these examples, such as affinity
chromatography, Southern blots, PCR, DNA sequencing, vector
construction (including DNA extraction, isolation, restriction
digestion, ligation, etc.), cell culture (including antibiotic
selection), transfection of cells, protein assays (Western
blotting, immunoprecipitation, immunofluorescence), in situ
hybridization, etc., are techniques routinely performed by those of
skill in the art (see generally Sambrook et al., Molecular Cloning,
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989)). Accordingly, in the interest of brevity, the
basic experimental protocols are not discussed in detail. As these
examples are included for purely illustrative purposes, they should
not be construed to limit the scope of the invention in any
respect.
EXAMPLE 1
[0028] This example demonstrates that CD16 is a cellular receptor
for papillomavirus.
[0029] The ability of a ligand recognizing the CD16 protein to
block papillomavirus binding was assayed by FACS analysis using
cocktails of antibodies recognizing various cell surface proteins.
Single cell suspensions of human peripheral blood lymphocytes
obtained with the consent of a donor were created.
[0030] Papillomavirus binding was assessed using papillomavirus
L1/L2 VLPs (Greenstone et al., Proc. Nat. Acad. Sci. USA, 95,
1800-05 (1998)). To assess signal, these VLPs were biotinylated by
incubating them with N-hydroxysuccinimide-biotin for one hour at
room temperature. The VLPs were dialyzed against standard
phosphate-buffered saline for four hours and then stored at
-80.degree. C. for later use. Biotinylation was assessed using a
sandwich ELISA. At a 100-fold dilution, biotinylated VLPs
registered positive, while control VLPs exhibited little absorption
signal.
[0031] The cell populations were incubated on ice with the
biotinylated VLPs for two hours. Following the incubation,
populations of cells were incubated with streptavidin-APC and the
various antibody cocktails indicated in Table 1. Following this
second incubation, the cells were exposed to labeled secondary
antibody (rat or mouse FITC-conjugated Ig). In blocking
experiments, the cells were pre-incubated with antibodies for one
hour on ice. The antibodies employed recognized, respectively,
CD16, CD21, CD23, CD32, CD64, .alpha.6 integrin, and .beta.4
integrin. Fluorescence was assessed and quantified using a
FACS-Calibur with CELLQUEST software (Becton-Dickinson). The number
of cells gated for MCH class II and bound to biotinylated VLP were
counted.
[0032] The data revealed that only when the anti-CD16 antibody was
absent from the cocktail did the papillomavirus VLPs bind to the
cells with wild-type efficiency. Significantly, these results
demonstrate that exposing cells to a ligand recognizing CD16 (e.g.,
the anti-CD16 antibody used in these experiments) attenuates
papillomavirus binding. Importantly, the results indicate that
interfering with the interaction between the VLPs and the
.quadrature.6 integrin with the .alpha.6 antibody had no effect on
viral binding. This result is surprising because it is inconsistent
with a prior suggestion that the .alpha.6 integrin may be a
papillomavirus receptor (Evander et al., J. Virol., 71(3), 2449-56
(1997)).
EXAMPLE 2
[0033] This example demonstrates that cells associated with
papillomavirus infection express CD16 on the cell surface.
[0034] Various cell lines (i.e., RD cells (a monkey kidney cell
line), HeLa cells (a human papillomavirus strain 18-expressing
cervical cancer cell line), CV1 cells (a monkey kidney cell line),
BB49 cells (a human papillomavirus strain 6-expressing head and
neck cancer cell line), Caski cells (a human papillomavirus strain
16-expressing cervical cancer cell line), and Epstein-Barr
Virus-transformed cells)) were assayed for CD16 expression using an
antibody recognizing CD16. The total number of cells in each
population, and the number of such cells expressing CD16, were
measured by FACS analysis as described above.
[0035] The results revealed that the number of cells expressing
CD16 approximated the number of cells in each population,
indicating that papillomavirus-infected cells express CD16. These
results are consistent with published immunohistochemical data
demonstrating that CD16 is expressed in tissue associated with HPV
lesions, but not expressed in other tissues (compare Hussain et
al., AIDS, 5, 1089-94 (1991), Hussain et al., Clin Exp. Immunol.,
90, 530-38 (1992), and Hussain et al., Immunology, 85, 475-84
(1995)).
EXAMPLE 3
[0036] This example demonstrates that CD16 expression correlates
with HPV binding in situ.
[0037] Frozen sections of human foreskin epithelium were incubated
with either biotinylated HPV16 L1/L2 VLPs, an anti-human CD16
monoclonal antibody, an anti-human .alpha.6 integrin, or an
anti-human .beta.4 integrin. The sections exposed to VLPs were then
incubated with peroxidase-labeled streptavidin, and the sections
exposed to the antibodies were incubated with peroxidase-labeled
secondary antibodies. Subsequently, each section was developed with
peroxidase substrate.
[0038] Histological examination of each section revealed .alpha.6
and .beta.4 integrin staining in a well-defined deep stratum of
dermis, stratum basale, well away from the surface of the foreskin.
Conversely, CD16 staining was observed in a discrete layer of
tissue at the surface of the foreskin. HPV particles were detected
only in the region in which CD16 was detected, and not in the
regions in which .alpha.6 and .beta.4 integrin were detected. This
result is surprising because it is inconsistent with a prior
suggestion that .alpha.6 or .beta.4 integrins may be papillomavirus
receptors (Evander et al., supra).
EXAMPLE 4
[0039] This example demonstrates that HPV binding in vivo is
reduced in the absence of CD16.
[0040] Biotinylated HPV16 L1/L2 VLPs were incubated with
splenocytes from either C57 B1/6 or CD16 knockout mice.
Non-biotinylated HPV16 L1/L2 VLPs were used as a control. After
incubation, the cells were washed, and VLP binding was assayed
using allophycocyanin-labeled streptavidin. Fluorescence was
assessed using a Becton Dickinson FacsCalibur running CellQuest
software.
[0041] The data revealed that the VLPs bound the knockout cells
with only about half the efficiency to which they bound wild-type
cells expressing CD16. These results demonstrate that CD16 is
required for high-efficiency papillomavirus infection.
[0042] All of the references cited herein, including patents,
patent applications, and publications, are hereby incorporated in
their entireties by reference.
[0043] While the present invention has been described with an
emphasis upon preferred embodiments and illustrative examples, it
will be obvious to those of ordinary skill in the art that
variations of the preferred embodiments may be used and that the
present invention can be practiced otherwise than as specifically
described herein. Accordingly, the present invention includes all
modifications encompassed within the spirit and scope of the
invention as defined by the following claims.
* * * * *