U.S. patent application number 10/446060 was filed with the patent office on 2003-11-06 for infectious papillomavirus pseudoviral particles.
Invention is credited to Lowy, Douglas R., Roden, Richard B., Schiller, John T..
Application Number | 20030207446 10/446060 |
Document ID | / |
Family ID | 21807834 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207446 |
Kind Code |
A1 |
Lowy, Douglas R. ; et
al. |
November 6, 2003 |
Infectious papillomavirus pseudoviral particles
Abstract
The invention provides an infectious papillomavirus pseudoviral
particle useful in gene transfer comprising: (a) a papillomavirus
vector DNA which comprises an E2 binding site and an expression
cassette comprising a gene and a sequence controlling expression of
said gene; and (b) a papillomavirus capsid which comprises L1 and
L2 structural proteins, such that said capsid encapsidates said
vector DNA, wherein said gene is derived from a first biological
species and said L1 structural protein is derived from a second
biological species and said first biological species is different
from said second biological species.
Inventors: |
Lowy, Douglas R.; (Bethesda,
MD) ; Schiller, John T.; (Silver Spring, MD) ;
Roden, Richard B.; (Washington, DC) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
21807834 |
Appl. No.: |
10/446060 |
Filed: |
May 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10446060 |
May 27, 2003 |
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09509748 |
Mar 30, 2000 |
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6599739 |
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09509748 |
Mar 30, 2000 |
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PCT/US97/12115 |
Jul 14, 1997 |
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60022104 |
Jul 17, 1996 |
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Current U.S.
Class: |
435/325 ;
424/93.2; 435/235.1; 435/456 |
Current CPC
Class: |
A61K 2039/5256 20130101;
A61K 48/00 20130101; A61K 39/00 20130101; C12N 7/00 20130101; A61P
35/00 20180101; C12N 2710/20023 20130101; A61P 17/00 20180101; C07K
16/084 20130101; A61K 2039/5258 20130101; C12N 2710/20022 20130101;
C07K 14/005 20130101; C12N 2810/6009 20130101; A61P 7/04 20180101;
C12N 15/86 20130101; Y10S 977/916 20130101 |
Class at
Publication: |
435/325 ;
435/456; 435/235.1; 424/93.2 |
International
Class: |
A61K 048/00; C12N
007/00; C12N 015/86 |
Claims
What is claimed is:
1. An infectious papillomavirus pseudoviral particle comprising:
(a) a papillomavirus vector DNA which comprises an E2 binding site
and an expression cassette comprising a gene and a sequence
controlling expression of said gene; and (b) a papillomavirus
capsid which comprises L1 and L2 structural proteins, such that
said capsid encapsidates said vector DNA.
2. The infectious papillomavirus pseudoviral particle of claim 1,
wherein each of said L1 and L2 structural proteins is derived from
a human papillomavirus.
3. The infectious papillomavirus pseudoviral particle of claim 1,
wherein said gene is a human gene.
4. A method of making infectious papillomavirus pseudoviral
particles comprising: (a) providing a cell line which expresses
papillomavirus E2 DNA binding protein and L1 and L2 structural
proteins; (b) transforming said cell line with a papillomavirus
vector DNA which comprises an E2 binding site and an expression
cassette comprising a gene and a sequence controlling expression of
said gene, wherein said papillomavirus E2 binding site is a cognate
binding site of said E2 DNA binding protein; (c) providing
conditions for the encapsidation of said vector DNA by a capsid
which comprises said L1 and L2 structural proteins to generate said
particles; and (d) harvesting said particles.
5. The method of claim 4, wherein said cell line is a mammalian
cell line, an insect cell line, or a yeast cell line.
6. A cell line comprising the infectious papillomavirus pseudoviral
particle of claim 1.
7. Infectious papillomavirus pseudoviral particles made by the
method of claim 4.
8. A method of transferring a gene into a cultured mammalian cell
comprising: (a) providing the infectious papillomavirus pseudoviral
particle of claim 1; and (b) infecting a cultured mammalian cell
with said particle such that said cultured mammalian cell is
transformed with said gene.
9. A method of screening for infectious papillomavirus pseudoviral
particles comprising administering the infectious papillomavirus
pseudoviral particles of claim 7 as test particles to cultured
non-infected mammalian cells and scoring for infectivity.
10. A composition comprising the infectious papillomavirus
pseudoviral particle of claim 1, wherein said gene encodes a
product capable of having a therapeutic effect when administered in
a therapeutically effective amount to a host subject in need
thereof.
11. A composition comprising the infectious papillomavirus
pseudoviral particle of claim 1, wherein said gene encodes a
product capable of having an immunogenic effect when administered
in an immunogenically effective amount to a host subject in need
thereof.
12. An infectious papillomavirus pseudoviral particle for use as a
medicament upon infecting cells of a human in vivo, wherein said
particle comprises: (a) a papillomavirus vector DNA which comprises
an E2 binding site and an expression cassette comprising a gene and
a sequence controlling expression of said gene, wherein said gene
encodes a therapeutic protein and said cells express a
therapeutically effective amount thereof; and (b) a papillomavirus
capsid which comprises L1 and L2 structural proteins, such that
said capsid encapsidates said vector DNA.
13. The method of claim 12, wherein said cells are epithelial cells
and said therapeutic protein has a systemic effect.
14. The method of claim 12, wherein said cells are epithelial cells
and said therapeutic protein has a local effect on said epithelial
cells.
15. The method of claim 13, wherein said therapeutic protein is
Factor IX and the expression of said therapeutic protein results in
treatment of hemophilia.
16. The method of claim 14, wherein said therapeutic protein is
herpes simplex virus thymidine kinase and the expression of said
therapeutic protein results in treatment of skin cancer.
17. An infectious papillomavirus pseudoviral particle for use as a
vaccine upon infecting cells of a human in vivo, wherein said
particle comprises: (a) a papillomavirus vector DNA which comprises
an E2 binding site and an expression cassette comprising a gene and
a sequence controlling expression of said gene, wherein said gene
encodes an immunogenic protein and said cells express an
immunogenically effective amount thereof; and (b) a papillomavirus
capsid which comprises L1 and L2 structural proteins, such that
said capsid encapsidates said vector DNA.
18. A second infectious papillomavirus pseudoviral particle, which
differs from a first infectious papillomavirus pseudoviral
particle, each particle for use as a vaccine upon infecting cells
of a human in vivo, wherein each particle comprises: (a) a
papillomavirus vector DNA which comprises an E2 binding site and an
expression cassette comprising a gene and a sequence controlling
expression of said gene, wherein said gene encodes an immunogenic
protein and said cells express an immunogenically effective amount
thereof; and (b) a papillomavirus capsid which comprises L1 and L2
structural proteins, such that said capsid encapsidate said vector
DNA; such that said second infectious papillomavirus pseudoviral
particle differs from said first infectious papillomavirus
pseudoviral particle in that said second is a different serotype
from said first.
Description
FIELD OF THE INVENTION
[0001] The field of the invention is related to infectious
papillomavirus pseudoviral particles useful in gene transfer.
BACKGROUND OF THE INVENTION
[0002] Gene transfer is a laboratory strategy in which the genetic
repertoire of eukaryotic cells is modified. Essentially, gene
transfer involves the delivery, to target cells, of an expression
cassette made up of one or more genes and the sequences controlling
their expression. The transfer process is accomplished by delivery
of the cassette to the cell where it can function
appropriately.
[0003] Considerable effort has been made to develop delivery
systems to express foreign proteins in eukaryotic cells. These
systems can be divided into two types: transfection and
infection.
[0004] The first type of delivery system for introducing cloned
DNAs into eukaryotic cells involves transfection. Calcium
phosphate- or DEAE-dextran-mediated transfection is the most widely
used method. The polycation Polybrene allows the efficient and
stable introduction of plasmid DNAs into cultured cells that are
relatively resistant to transfection by other methods (Kawai, S.,
and Nishizawa, M., 1984, Mol. Cell. Biol. 4, 1172; Chaney, W. G.,
et al., 1986, Somatic Cell Mol. Genet. 12, 237). In protoplast
fusion, protoplasts derived from bacteria carrying high numbers of
copies of a plasmid of interest are mixed directly with cultured
mammalian cells, and fusion of the cell membranes is accomplished
with polyethylene glycol, with the result that the contents of the
bacteria are delivered into the mammalian cells (Schaffner, W.,
1980, Proc. Natl. Acad. Sci. USA 77, 2163; Rassoulzadegan, M., et
al. 1982, Nature 295, 257). Electroporation features the
application of electric pulses to mammalian and plant cells so that
DNA is taken directly into the cell cytoplasm (Neumann, E., et al.,
1982, EMBO J. 1, 841; Zimmermann, U., 1982, Biochim. Biophys. Acta
694, 227). Artificial membrane vesicles, such as liposomes and
cationic lipids, are useful as delivery vehicles in vitro and in
vivo (Mannino, R. J., and Gould-Fogerite, S., 1988, BioTechniques
6, 682; Felgner, P. L., and Holm, M., 1989, Bethesda Res. Lab.
Focus 11, 21; Maurer, R. A., 1989, Bethesda Res. Lab. Focus 11,
25). Direct microinjection into nuclei is effective, but it cannot
be used to introduce DNA on a large scale (Capecchi, M. R., 1980,
Cell 22, 479). Finally, naked DNA can, by itself, be placed into
cells by particle bombardment (Yang, N. S., et al., 1990, Proc.
Natl. Acad. Sci. USA 87, 9568), or taken up by cells, particularly
when injected into muscle (Wolff, J. A., et al., 1990, Science 247,
1465).
[0005] The other type of delivery system is mediated by infection
and involves the use of viral expression vectors derived from
simian virus 40 (SV40) (Elder, J. T., et al., 1981, Annu. Rev.
Genet. 15, 295; Gething, M.-J., and Sambrook, J., 1981, Nature 293,
620; Rigby, P. W. J., 1982, Genetic engineering, R. Williamson,
ed., Academic Press, London, vol. 3, p. 83; Rigby, P. W. J., 1983,
J. Gen. Viral. 64, 255; Doyle, C., et al., 1985, J. Cell. Biol.
100, 704; Sambrook, J., et al., 1986, Mol. Biol. Med. 3, 459),
vaccinia virus (Mackett, M., et al., 1985, DNA cloning: A practical
approach, D. M. Glover, ed., IRL Press, Oxford, vol. 2, p. 191;
Moss, B., 1985, Virology, B. N. Fields, et al., eds., Raven Press,
New York, p. 685; Fuerst, T. R., et al., 1986, Proc. Natl. Acad.
Sci. USA, 83, 8122; Fuerst, T. R., et al., 1987, Mol. Cell. Biol.
7, 2538), adenovirus (Solnick, D., 1981, Cell 24, 135; Thummel, C.,
et al., 1981, Cell 23, 825; Thummel, C., et al., 1982, J. Mol.
Appl. Genet. 1, 435; Thummel, C., et al., 1983, Cell 33, 455;
Mansour, S. L., et al., 1985, Proc. Natl. Acad. Sci. USA 82, 1359;
Karlsson, S., et al., 1986, EMBO J. 5, 2377; Berkner, K. L., 1988,
BioTechniques 6, 616), retroviruses (Dick, J. E., et al., 1986,
Trends Genet. 2, 165; Gilboa, E., et al., 1986, BioTechniques 4,
504; Eglitis, M. A., and Anderson, W. F., 1988, BioTechniques 6,
608), and baculoviruses (Luckow, V. A., and Summers, M. D., 1988,
Bio/Technology 6, 47).
[0006] Expression of proteins from cloned genes in eukaryotic cells
has been used for a number of different purposes: to confirm the
identity of a cloned gene by using immunological or functional
assays to detect the encoded protein, to express genes encoding
proteins that require posttranslational modifications such as
glycosylation or proteolytic processing, to produce large amounts
of proteins of biological interest that are normally available in
only limited quantity from natural sources, to study the
biosynthesis and intracellular transport of proteins following
their expression in various cell types, to elucidate
structure-function relationships by analyzing the properties of
normal and mutant proteins, to express intron-containing genomic
sequences that cannot be transcribed correctly into mRNA in
prokaryotes, and to identify DNA sequence elements involved in gene
expression. Because expression of proteins can serve so many
different purposes, there is a need for new delivery systems to
meet the challenge of getting foreign DNA into eukaryotic cells.
The invention satisfies this need.
[0007] These and other objects of the invention will be apparent to
one of ordinary skill in the art upon consideration of the
specification as a whole.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention provides an infectious
papillomavirus pseudoviral particle.
[0009] In another aspect, the invention provides a HPV16{BPV1}
virion.
[0010] In still another aspect, the invention provides an
infectious papillomavirus pseudoviral particle comprising: a
papillomavirus vector DNA which comprises an E2 binding site and an
expression cassette comprising a gene and a sequence controlling
expression of the gene; and a papillomavirus capsid which comprises
L1 and L2 structural proteins, such that the capsid encapsidates
the vector DNA, where the gene is derived from a first biological
species and the L1 structural protein is derived from a second
biological species and the first biological species is different
from the second biological species.
[0011] In yet another aspect, the invention provides the
here-described infectious papillomavirus pseudoviral particle,
where the first biological species is BPV1 and the second
biological species is HPV16.
[0012] In a different embodiment, the invention relates to a method
of making infectious papillomavirus pseudoviral particles
comprising: providing a cell line which expresses papillomavirus E2
DNA binding protein and L1 and L2 structural proteins; transforming
the cell line with a papillomavirus vector DNA which comprises an
E2 binding site and an expression cassette comprising a gene and a
sequence controlling expression of the gene, where the
papillomavirus E2 binding site is a cognate binding site of the E2
DNA binding protein, and where the gene is derived from a first
biological species and the L1 structural protein is derived from a
second biological species and the first biological species is
different from the second biological species; providing conditions
for the encapsidation of the vector DNA by a capsid which comprises
the L1 and L2 structural proteins to generate the particles; and
harvesting the particles.
[0013] In the above method, the cell line may be a mammalian cell
line, an insect cell line, or a yeast cell line.
[0014] In yet a different embodiment, the invention relates to a
cell line comprising the here-described infectious papillomavirus
pseudoviral particle.
[0015] In still a different embodiment, the invention relates to a
method of transferring a gone into a cultured mammalian cell
comprising: providing the here-described infectious papillomavirus
pseudoviral particle; and infecting a cultured mammalian cell with
the particle such that the cultured mammalian cell is transformed
with the gene.
[0016] In another manifestation, the invention provides a method of
screening for infectious papillomavirus pseudoviral particles
comprising administering test particles to cultured mammalian cells
capable of being infected thereby and scoring for infectivity
thereof.
[0017] In a further manifestation, the invention provides a
composition comprising the here-described infectious papillomavirus
pseudoviral particle, where the gene in the expression cassette
encodes a product capable of having a therapeutic effect when
administered in a therapeutically effective amount to a host
subject in need thereof.
[0018] In an additional manifestation, the invention provides a
composition comprising the here-described infectious papillomavirus
pseudoviral particle, where the gene in the expression cassette
encodes a product capable of having an immunogenic effect when
administered in an immunogenically effective amount to a host
subject in need thereof.
[0019] The invention also relates to a method of providing a human
with an immunogenic protein comprising: infecting cells of the
human in vivo with the here-described infectious papillomavirus
pseudoviral particle, where the gene in the expression cassette
encodes the immunogenic protein, the cells expressing an
immunogenically effective amount of the immunogenic protein.
[0020] The invention further relates to a method of providing a
human with a therapeutic protein comprising: infecting cells of the
human in viva with the here-described infectious papillomavirus
pseudoviral particle, where the gene in the expression cassette
encodes the therapeutic protein, the cells expressing a
therapeutically effective amount of the therapeutic protein.
[0021] In this method, the cells may be epithelial cells, and the
therapeutic protein may have a systemic effect. Or the therapeutic
protein may have a local effect on the epithelial cells. Or the
therapeutic protein may be Factor IX and the expression of the
therapeutic protein may result in treatment of hemophilia. Or the
therapeutic protein may be herpes simplex virus thymidine kinase
and the expression of the therapeutic protein may result in
treatment of skin cancer.
[0022] This method may involve serial administration of different
serotypes and thus comprise infecting cells of the human in viva
with a second infectious papillomavirus pseudoviral particle where
the second infectious papillomavirus pseudoviral particle differs
from the first infectious papillomavirus pseudoviral particle in
that the second is a different serotype from the first.
[0023] The invention additionally relates to an infectious
papillomavirus pseudoviral particle comprising a papillomavirus
genome, which comprises an E2 binding site and an expression
cassette comprising a gene and a sequence controlling expression of
the gene, and a papillomavirus capsid, which comprises L1 and L2
structural proteins, such that the capsid encapsidates the genome,
where the E2 binding site is derived from a first papillomavirus
serotype and the L1 structural protein is derived from a second
papillomavirus serotype and the first papillomavirus serotype is
different from the second papillomavirus serotype.
[0024] The invention moreover relates to a method-of making
infectious virus pseudoviral virions in nonmammalian cells
comprising: providing a nonmammalian cell line which expresses the
nonstructural protein(s) of the virus for packaging the viral
genome of the virus in the empty capsid of the virus, and which
expresses the structural proteins of the virus capsid; transforming
the cell line with the viral genome which comprises the packaging
signal, and which further comprises an expression cassette
comprising a gene and a sequence controlling expression of the
gene, and where the gene is derived from a first biological species
and the viral capsid is derived from a second biological species
and the first biological species is different from the second
biological species; providing conditions for the encapsidation of
the viral genome by the viral capsid to generate the virions; and
harvesting the virions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1. A model for L2-mediated assembly of papillomavirus
virions. This model is discussed at length in the text. Briefly, it
is proposed that L2 acts to mediate papillomavirus assembly by
causing the concentration of the virion components within the PODs.
L2 will localize in the PODs independently of other viral proteins.
The L2 localization will cause the subsequent recruitment of E2
with the bound genome and L1. These events are independent of each
other. This L2-L1-E2-genome association within the PODs confers an
appropriate environment and/or concentration for virion
assembly.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0026] The invention satisfies the need for new delivery systems to
meet the challenge of getting cloned DNA into eukaryotic cells by
providing infectious papillomavirus pseudoviral particles. Section
I describes in vitro generation of infectious BPV virions and
infectious HPV16{BPV1} pseudoviral particles in mammalian cells.
Section II elaborates the requirements for the papillomavirus
capsid proteins, the viral transcription/replication protein, E2,
and POD nuclear structures for encapsidation. Section III details
the in vitro generation of infectious BPV virions in nonmammalian
cells. Section IV describes the use of infectious papillomavirus
pseudoviral particles in a specialized case of gene transfer, that
of gene therapy and gene immunization.
[0027] I. In Vitro Generation of a Human Papillomavirus Type 16
Virion Pseudotype
[0028] Using the protocol described in Example 1, a system was
developed for generating infectious papillomaviruses in vitro that
facilitates the analysis of papillomavirus assembly and
infectivity. Cultured hamster BPHE-1 cells harboring autonomously
replicating bovine papillomavirus type 1 (BPV1) genomes were
infected with defective Semliki Forest Viruses (SFVs) that express
the structural proteins of BPV1. When plated on C127 cells,
extracts from cells expressing L1 and L2 together induced numerous
transformed foci that could be specifically prevented by BPV
neutralizing antibodies, demonstrating that BPV infection was
responsible for the focal transformation. Extracts from BPHE-1
cells expressing L1 or L2 separately were not infectious. Although
SFV-expressed L1 self-assembled into virus-like particles, viral
DNA was detected in particles only when L2 was co-expressed with
L1, indicating that genome encapsidation requires L2. Expression of
human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1
cells also yielded infectious virus. These pseudotyped virions were
neutralized by antiserum to HPV16 virus-like particles (VLPs)
derived from European (114/K) or African (Z-1194) HPV16 variants,
but not by antisera to BPV VLPs, to a poorly assembling mutant
HPV16 L1 protein, or to VLPs of closely related genital HPV
types.
[0029] BPV L1 expressed from recombinant SFV in mammalian cells
binds L2 and assembles into VLPs. SFV is a simple positive strand
RNA virus. The pSFV-1 expression vector contains the gene for the
SFV RNA replicase, the inserted gene and a cis acting virion
packaging signal. In vitro synthesized RNA from this vector is
co-transfected with a helper vector (pHelper-2) RNA that encodes
the SFV structural genes. Upon transfection, the replicase is
translated and initiates successive rounds of RNA replication and
translation, thereby amplifying the viral RNAs. Translation of the
helper RNA leads to production of the SFV virion proteins and
encapsidation of the expression vector RNA, but not that of the
helper, which lacks the packaging signal. Therefore, the high titer
virus generated is defective because it does not encode the SFV
virion proteins. Upon infection of susceptible cells (e.g., BHK-21
or BPHE-1), the replicase again amplifies the infecting RNA.
Amplification of subgenomic RNAs encoding the cloned gene leads to
high level expression of the encoded protein.
[0030] Defective BPV1 L1 and BPV1 L2 recombinant Semliki Forest
Viruses (SFV-BL1 and SFV-BL2) were generated by co-transfecting
BHK-21 cells with in vitro transcribed Helper-2 RNA (Life
Technologies) (Berglund, P., et al., 1993, BioTechnology 11,
916-920) and a recombinant pSFV-1 RNA encoding the BPV1 L1 or BPV1
L2 gene. BHK-21 cells were infected with the recombinant SFVs and
harvested 72 h later. Expression of BPV1 L1 and L2 was demonstrated
by Western blot analysis with monoclonal antibody 1HB (Chemicon)
(Cowsert, L. M., et al., 1988, Virology 165, 613-15)) far L1 and
rabbit antiserum to a bacterially-produced
glutathione-S-transferase-- BPV1 L2 fusion protein for L2
(Kirnbauer, R., et al., 1992, Proc. Natl. Acad. Sci. USA 89,
12180-84). Cell fractionation studies demonstrated that at least
80% of both L1 and L2 resided in the nuclear fraction at the time
of harvest.
[0031] BHK-21 cells were infected for 3 days with either SFV-BL1
alone or SFV-BL2 alone or were co-infected with the two defective
viruses. The cells were harvested and VLPs were prepared by
centrifugation through a 40% (w/v) sucrose cushion and cesium
chloride isopycnic density gradient centrifugation (Kirnbauer, R.,
et al., 1993, J. Virol. 67, 6929-36). A visible band with a density
of approximately 1.28 g/cm.sup.3 was extracted from cesium chloride
density gradients of the SFV-BL1 alone and SFV-BL1 plus SFV-BL2
infected cell extract and dialysed into PBS containing 0.5 M NaCl.
A corresponding band was not detected in the gradient containing
the extract from the cells infected with only the SFV-BL2.
Transmission electron microscopy of the BPV1 L1 alone and the L1
plus L2 preparations demonstrated large numbers of 55 nm diameter
particles with a morphology similar to BPV virions that were absent
from the L2 alone preparation. Analysis of the L1 and L1 plus L2
preparations on a 10% Coomassie stained SDS-PAGE gel revealed a
single 55 kDa protein band corresponding to L1. Full length
(.about.70 kDa) L2 was detected by Western blot analysis with
rabbit antiserum to bacterially expressed
glutathione-S-transferase-BPV1 L2 fusion protein in the L1 plus L2,
but not the L1 alone, preparation. Co-immunoprocipitation and
co-purification of L1 and L2 showed that L2 co-assembled with L1
into VLPs.
[0032] Infectious BPV1 virions are generated by co-expression of
both BPV1 L1 and L2 in BPHE-1 cells. Since expression of the
recombinant SFVs led to efficient assembly of VLPs in mammalian
cells, generation of infectious BPV in vitro and determination of
which capsid proteins were required for virion formation was
attempted. To this end, the SFV recombinants were used to infect a
hamster cell line, BPHE-1, that maintains 50-200 copies of episomal
BPV1 genomes per cell (Zhang, Y.-L., et al., 1987, J. Virol. 61,
2924-2928). The BPHE-1 cells were infected with either SFV-BL1
alone or SFV-BL2 alone or co-infected with the two recombinant
viruses. The cells were maintained for 30 h, harvested and lysed by
sonication, and the extracts were incubated in the medium of
monolayers of mouse C127 fibroblasts for 1 h at 37.degree. C. The
cells were washed and maintained for 3 weeks in complete medium and
stained, and the foci were counted (Dvoretzky, I., et al., 1980,
Virology 103, 369-375). Approximately 50 foci occurred in plates of
C127 cells treated with BPHE-1 extracts expressing both BPV L1 and
L2, but no foci were produced by extracts expressing only BPV L1 or
only BPV L2 in multiple experiments.
[0033] To determine if focal transformation was due to transfer of
BPV1 DNA to the mouse C127 cells, six of the foci were ring cloned
and expanded for further analysis (Law, M. F., et al., 1981, Proc.
Nat. Acad. Sci. USA 78, 2727-2731). A Hirt extract (Hirt, B., 1967,
J. Mol. Biol. 26, 365-369) from each of the six clones was
separated on a 0.8% agarose gel, Southern blotted and probed with a
[.sup.32P]-labeled fragment of the BPV genome. High copy number
episomal BPV genomic DNA was detected in the extracts of all six
clones.
[0034] It is possible that the BPV DNA was transferred to the C127
cells by transfection rather than infection by in vitro generated
virions. Since neutralizing antibodies should not inhibit
transfection, extracts from the L1 and L2 co-expressing BPHE-1
cells were incubated for 1 h at 4.degree. C. in the presence of a
1:100 dilution (10 .mu.l) of rabbit antiserum to either BPV1 or
HPV16 L1 VLPs (purified from insect cells) or denatured BPV virions
(DAK0) prior to addition to the C127 cells. The L1 plus L2 extract
treated with antiserum to BPV VLPs did not produce any foci,
whereas extracts treated with antiserum to HPV16 VLPs or denatured
BPV virions (which do not neutralize BPV) produced similar numbers
of foci as the untreated extract. Treatment of the same extract
with monoclonal antibody 5B6 that neutralizes BPV (Roden, R. B. S.,
et al., 1994, J. Virol. 68, 7570-74), but not a control monoclonal
antibody (PAb 101) of the same IgG subtype, also inhibited focus
formation. The conformationally-dependent and type-specific
neutralization of focal transforming activity demonstrates that
infection by BPV virions and not transfection of BPV DNA was
responsible for the transformation of the C127 cells.
[0035] L2 is required for efficient encapsidation of the BPV
genome. L1 assembles into VLPs when expressed in eukaryotic cells,
but the function of L2 in generating infectious virus is less clear
(Kirnbauer, R., et al., 1992, Proc. Natl. Acad. Sci. USA 89,
12180-84). L2 may be necessary for some step during the infectious
process and/or is necessary for encapsidation of the genome (Zhou,
J., et al., 1993, J. Gen. Virol. 74, 763-68). To explore the latter
possibility further, ten 500 cm.sup.2 plates of BPHE-1 cells were
infected with SFV-L1 alone or SFV-L2 alone or were co-infected with
SFV-L1 and SFV-L2. The cells were harvested 30 h post infection,
sonicated and treated with DNAsel (2000 U) for 1 h at 37.degree.
C., and particles were purified. The cesium chloride gradients were
fractionated, and the density of each fraction measured. Nucleic
acid was purified from 200 .mu.l of each fraction, and BPV DNA was
detected by Southern blot analysis. 0.1 ng of BPV-pML plasmid DNA
was run as a size standard for uncut, DNAsel-resistant BPV genomes
(Sarver, N., et al., 1982, Proc. Natl. Acad. Sci. USA 79,
7147-7151). Only that fraction from the BPHE-1 extracts expressing
both L1 and L2 demonstrated significant accumulation of
DNAsel-resistant BPV DNA. This fraction had a density (1.31 g/ml)
consistent with that of infectious BPV virions obtained from warts
under the same conditions (1.32 g/ml).
[0036] This fraction was examined by cryo-electron microscopy.
Unlike transmission electron microscopy of negatively stained
particles, cryo-electron microscopy allows the DNA inside the full
capsids to be visualized directly as an electron dense core as
opposed to the lower density core of empty particles. Many well
formed particles were observed with electron dense cores, as well
as a smaller fraction that had a lower density core or were damaged
or rod shaped. It was not possible to estimate the number or
percentage of full versus empty particles, as the L1 was spread
over a large number of fractions as determined by Western blot
analysis. However, comparative Southern blot analysis using the
cloned BPV genome as a standard indicated that approximately 1 ng
of full length DNAsel-resistant DNA was observed in these extracts,
which corresponds to approximately 10.sup.8 DNA molecules. In
contrast, only 10.sup.4 infectious units were isolated from this
preparation, indicating that the particle to infectivity ratio is
high, approximately 10.sup.4. Using the same procedures, the number
of infectious units and the amount of DNasel-resistant BPV genomic
DNA present in a BPV virion preparation purified from bovine
papillomas were measured. The values for the particle to
infectivity (as measured by in vitro transformation of C127 cells)
ratio obtained were very similar for BPV virions isolated from
warts (2.times.10.sup.4) or generated in BPHE-1 cells
(1.times.10.sup.4).
[0037] Generation and neutralization of infectious HPV16{BPV1}
pseudotyped virions. Having demonstrated that co-expression of BPV1
L1 and L2 can result in encapsidation of BPV genomes, the question
was asked whether genome encapsidation was type specific. L1 and L2
derived from HPV16 were therefore tested for the ability to
encapsidate the BPV genome and thereby generate infectious
pseudotyped virions. L1 and L2 derived from a wild type HPV16
isolate (114K) were cloned into SFV vectors and expressed in BPHE-1
cells (Heino, P., et al., 1996, Virology 214, 349-359; Kirnbauer,
R., et al., 1993, J. Virol. 67, 6929-36). Expression was confirmed
by Western blot analysis using monoclonal antibody CamVir-1
(Pharmingen) for L1 and rabbit antiserum to bacterially expressed
glutathione-S-transferase-HPV16 L2 fusion protein for L2.
Production of infectious virions was assessed using the C127 focus
forming assay, as described above. Expression of the L1 and L2
derived from HPV16 in BPHE-1 cells consistently produced infectious
virions, referred to as HPV16{BPV1} virus, although approximately 5
to 10-fold less efficiently than BPV L1 and L2. No foci were
observed when BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were
coexpressed, but low-efficiency encapsidation by heterologous pairs
of capsid proteins cannot be discounted. Expression in BPHE-1 cells
of L1 and L2 derived from a capsid-assembly deficient mutant of
HPV16 did not produce any foci (Kirnbauer, R., et al., 1993, J.
Virol. 67, 6929-36; Seedorf, K., et al., 1985, Virology 145,
181-185).
[0038] Type-specific neutralization of pseudotyped virions.
Treatment of the HPV16{BPV1} extracts with 5 or 50 .mu.l of rabbit
antiserum to 114K HPV16 VLPs prevented focus formation, whereas
addition of antiserum to BPV1 VLPs, denatured BPV virions, or
assembly deficient HPV16 L1 of the prototype strain did not prevent
focus formation. Both antiserum to HPV16 L1 alone and antiserum to
L1/L2 VLPs were neutralizing. Antiserum generated to the L1 VLPs of
a divergent Zairian isolate of HPV16 also neutralized the
HPV16{BPV1} virions (Cheng, G., et al., 1995, J. Infect. Dis. 172,
1584-1587). This finding demonstrates that infectious virus with
HPV16 capsids, not BPV capsids, were produced and that infection of
the C127 cells and not transfection by the BPV DNA had
occurred.
[0039] The ability of antisera raised against VLPs derived from low
risk HPV 6b or 11 and high risk HPV18, 31, 33, or 45 to prevent
infection by HPV16{BPV1} virions was also tested. All of these sera
contain high titers of antibodies (.gtoreq.10.sup.4, described in
Radon, R. B. S., et al., 1996, J. Virol. 70, 3298-3301) that
recognize their corresponding VLPs in ELISA and hemagglutination
inhibition assays. However, none of the sera were able to prevent
infection of the HPV16{BPV1} virions when 50 .mu.l (or 5 .mu.l) was
added to the pseudovirion extract.
[0040] Discussion. Despite some progress, difficulties in
generating infectious papillomavirus virions in vitro and
manipulating them genetically continue to limit studies of this
tumor virus (Hagensee, M., and Galloway, D., 1993, Papillomavirus
Report 4, 121-124). Use of a mouse xenograft system has led to the
limited production of HPV11 and an in vivo infectivity assay
(Christensen, N. D., and Kreider, J. W., 1990, J. Virol. 64,
3151-3156; Kreider, J. W., et al., 1987, J. Virol. 61, 590-93). As
an alternative approach, raft cultures of human keratinocytes can
undergo relatively normal terminal differentiation, thereby
permitting expression of the late proteins and virion biosynthesis
(Dollard, S. C., et al., 1992, Genes Dev. 6, 1131-42; Meyers, C.,
et al., 1992, Science 257, 971-73). Small quantities of
morphologically correct HPV31b virions have been produced by this
method, but no quantitative infectivity assay has been developed
using this system (Meyers, C., et al., 1992, Science 257, 971-73).
Furthermore, neither the xenografts nor raft cultures are readily
amenable to genetic manipulation.
[0041] Using recombinant vaccinia virus as a vector for BPV1 L1 and
L2, Zhou and colleagues have previously concluded that both L1 and
L2 were necessary to encapsidate viral DNA and to generate
infectious BPV virions (Zhou, J., et al., 1993, J. Gen. Virol.
763-68). Because their BPV preparations contained infectious
vaccinia virus, which is cytotoxic for many cell types, including
C127, they used transient expression of viral RNA as their marker
for infectivity. One notable difference between the results
reported in that study and those obtained here was that their
infectivity marker was neutralized by antiserum to denatured BPV1
virions (DAK0). In contrast, the present SFV-derived or cattle
papilloma-derived virions induced focal transformation that was not
inhibited by any of the several lots of this sera that were tested,
in agreement with previous reports that DAK0 sera or other sera to
denatured virions are non-neutralizing. The results of the Zhou et
al. study are therefore ambiguous.
[0042] As described here, infectious papillomavirus have been
produced by expressing the virion capsid proteins in trans, via
defective SFV vectors, in cells that contain an intact viral
genome. Production of infectious BPV was monitored by a standard,
quantitative, in vitro BPV infectivity assay (Dvoretzky, I., et
al., 1980, Virology 103, 369-375). BPV induced focal transformation
of C127 cells was specifically inhibited by incubating infectious
preparations with neutralizing BPV-antisera, which confirmed that
the transformation resulted from BPV infection and not from
transfection of viral DNA.
[0043] This method of virus production provides the opportunity to
determine the functions of the virion proteins in virion formation
and to generate virions with specific modifications.
[0044] The presence of DNAsel-resistant full length BPV DNA in the
extracts expressing L1 plus L2, but not either L1 alone or L2
alone, demonstrates that L2 is required for encapsidation of the
BPV genome.
[0045] The ability of L1 and L2 derived from HPV16 to encapsidate
the BPV genome establishes that any viral DNA packaging signal that
exists for papillomavirus genomes is conserved between BPV1 and
HPV16, and that, because BPV1 and HPV16 are so highly
evolutionarily divergent, such a signal is moreover conserved among
papillomaviruses.
[0046] The apparent inability to generate infectious virus with BPV
L1 and HPV16 L2 or HPV16 L1 and BPV L2 implies L2 from widely
divergent papillomaviruses prevented the generation of infectious
virus, but this result would not be expected to occur for closely
related papillomaviruses.
[0047] Expression of HPV16 L1 and L2 in cells containing the BPV
genome produced infectious pseudotyped virions with HPV16 capsids.
They induced typical BPV type foci, and their infectivity was
neutralized by HPV16 antisera and not by BPV antisera. Since HPV16
is not more closely related to BPV1 than are other high risk HPV
types, it is expected that a strategy similar to the one reported
here for HPV16 can be used to generate infectious pseudotypes for
other high risk HPVs, and presumably for any papillomavirus. See
Example 4.
[0048] Although the focal transformation assay requires 2 to
3-weeks, this problem should in principle be circumvented by
incorporation of a rapid and easily detectable marker in the BPV
genome.
[0049] Results from a number of laboratories have indicated that
despite their strict host range, papillomaviruses bind to a variety
of cell types derived from diverse species (Muller, M., et al.,
1995, J. Virol. 69, 948-54; Roden, R. B. S., et al., 1994, J.
Virol. 68, 7260-66; Volpers, C., et al., 1995, J. Virol. 69,
3258-3264.). The ability of HVP16{BPV1} virions to induce focal
transformation of C127 cells establishes that C127 cells express
the cell surface receptor for HPV16 virions and are competent to
perform the subsequent steps of internalization and uncoating that
are required for initiating viral infection. The simplest
interpretation of these observations is that BPV and HPV16 share a
common intracellular pathway of infection as well as a common cell
surface receptor.
[0050] The in vitro generation of HPV16{BPV1} pseudotyped virus has
allowed, for the first time, the development of an antibody
neutralization assay for HPV16 and other high risk HPVs, since
there is neither a source of infectious HPV16 or other high risk
HPVs, nor an easily scored quantitative assay for the genome of
HPV16 or other high risk HPVs. Titers of neutralizing antibodies
induced by vaccination are the best correlate of protection for
most previously developed prophylactic vaccines (Robbins, J. B., et
al., 1995, J. Infect. Dis. 171, 1387-98), as also seems true for
the animal papillomavirus protection studies (Breitburd, F., et
al., 1995, J Virol. 69, 3959-63, Suzich, J. A., et al., 1995, Proc.
Natl. Acad. Sci. USA 92, 11553-11557). It is therefore important to
investigate whether the HPV16 VLPs induce high titers of
neutralizing antibodies and to determine the degree of
cross-protection between various genital HPV types. Until now, it
has been necessary to rely on surrogate assays for neutralization,
such as ELISA and hemagglutination inhibition (Roden, R. B. S., et
al., 1995, J. Virol. 69, 5147-51, Roden, R. B. S., et al., 1996, J.
Virol. 70, 3298-3301, Rose, R. C., et al., 1994, J. Gen. Viral. 75,
2445-49). Compared with neutralization, the VLP ELISA is relatively
non-stringent because it may recognize non-neutralizing antibodies,
while hemagglutination may be overly stringent because a class of
neutralizing antibodies (defined for BPV, CRPV and HPV11) does not
score in that assay (Roden, R. B. S., et al., 1996, J. Virol. 70,
3298-3301). It is no longer necessary to rely on these surrogate
assays for neutralization since presented with the described
quantitative in vitro neutralization assay. See Example 5.
[0051] The assembly-deficient mutant L1 of the reference HPV16
strain did not induce detectable neutralizing antibodies,
reinforcing the concept that most neutralizing epitopes are
displayed only on intact particles. The observation that antibodies
to a divergent assembly-competent variant (Zaire 1194 (Cheng, G.,
et al., 1995, J. of Infect. Dis. 172, 1584-1587)), which differs
from the 1141K HPV16 isolate at seven L1 amino acids, can
efficiently neutralize the HPV16{BPV1} virions made with the 1141K
isolate further suggests that VLPs of a single HPV16 variant will
induce protection against divergent HPV16 variants (Cheng, G., et
al., 1995, J. of Infect. Dis. 172, 1584-1587). However, the
pseudotyped virions were not neutralized by antiserum to VLPs
derived from six genital HPV types or BPV1. This was true even
though two of the VLP types tested, HPV31 and HPV33, are among
those most closely related to HPV16, with 84% and 81% L1 amino acid
sequence identity, respectively. These anti-VLP sera had titers in
ELISA and hemagglutination assays based on the homologous VLP type
of at least 10,000 (Roden, R. B. S., et al., 1996, J. Virol. 70,
3298-3301); therefore the negative results in the HPV16{BPV1}
neutralization assay were not due to a poor antibody response to
these VLPs. The data support the concept that HPV16 is a single
serotype, distinct from other genotypes.
[0052] The finding that antibodies elicited by assembled HPV16 VLPs
can efficiently inhibit infection by the HPV16{BPV1} virions
supports the potential utility of these VLPs as prophylactic
vaccine candidates. To make an informed decision for the components
of a multivalent VLP-based vaccine to prevent genital HPV
infection, it will be necessary to evaluate to what extent
antibodies generated against one type of HPV VLP will neutralize
infection by other types. The data that rabbit antibodies raised
against VLPs derived from other genital HPV types did not
neutralize HPV16{BPV1} infection suggest that protection obtained
by neutralizing antibodies in humans against these genital HPVs
will be type specific. The development of pseudotyped virions of
other HPV types, along with HPV16{BPV1}, could be used to more
broadly examine the question of cross-neutralization in animal
studies and in early phases of human vaccine trials.
[0053] II. The Papillomavirus Minor Capsid Protein, L2, Induces
Localization of the Virion Components and the Viral
Transcription/Replication Protein, E2, to POD Nuclear
Structures
[0054] Using the protocol described in Example 2, the subcellular
localization of structural and nonstructural bovine papillomavirus
(BPV) proteins in cultured cells has been examined by
immunofluorescent staining and confocal microscopy. When expressed
separately, L1, the major capsid protein, showed a diffuse nuclear
distribution, while the minor capsid protein, L2, was found to
localize to punctate nuclear regions identified as PML oncogenic
domains (PODs). Coexpression of L1 and L2 induced a relocation of
L1 into the PODs, leading to the colocalization of L1 and L2.
[0055] The effect of L2 expression on the distribution of the viral
DNA genome and the nonstructural viral proteins E1 and E2, which
are required for maintenance of the genome and viral DNA synthesis,
was examined. The localization of the E1 protein was unaffected by
L2 expression. However, the pattern of anti-E2 staining was
dramatically altered in L2-expressing cells. Similar to L1, E2 was
shifted from a dispersed nuclear locality into the PODs and
colocalized with L2. The recruitment of full-length E2 by L2
occurred in the absence of other viral components. Additionally, in
BPV-transformed fibroblasts the autonomously replicating BPV genome
was found to be coalesced in an adjoining nuclear region in an
L2-dependent manner.
[0056] L2 has been shown here to be essential for the generation of
infectious BPV. The current results provide evidence for a role for
L2 in the organization of virion components by recruiting them to a
distinct nuclear domain. This L2-dependent colocalization probably
serves as a mechanism to promote assembly of papillomaviruses
either by increasing the local concentration of virion constituents
or by providing the physical architecture necessary for efficient
packaging and assembly. The data also establish a role for a
nonstructural viral protein, E2, which binds a conserved sequence
motif in papillomavirus genomes, in the localization of the viral
genome to the PODs.
[0057] Subnuclear localization of BPV capsid proteins. BPHE-1 is a
hamster fibroblast cell line that is latently infected with
multiple copies of autonomously replicating BPV genomes and
expresses the nonstructural viral proteins (Zhang, Y.-L., et al.,
1987, J. Virol. 61, 2924-2928). The SFV expression system was used
to introduce the L2 minor capsid protein into BPHE-1 cells and
localize the L2 protein by immunofluorescent staining and laser
scanning confocal microscopy. The typical distribution of L2 6
hours after SFV infection indicates the protein was displayed in a
distinct intranuclear punctate pattern.
[0058] To rule out the possibility that this distribution depended
upon the BPV components in the BPHE-1 cells, L2 was expressed, via
the SFV vector, in cells that did not harbor papillomavirus
sequences. A similar punctate nuclear pattern of L2 staining was
also observed in these other cells types, including COS-7, BHK-21
and the human fibroblast cell line 1634. Therefore, this distinct
L2 localization is dependent only upon cellular factors and appears
to be independent of cell lineage. To determine if this
localization was a common feature of papillomavirus L2, the
distribution of the human papillomavirus 16 (HPV16) L2 protein,
expressed via an SFV vector, was also examined in these cell lines.
The pattern with HPV16 L2 protein was similar to that seen with BPV
L2, establishing that this localization is characteristic of
papillomavirus L2.
[0059] L2-containing punctate structures are PODs. To identify the
nuclear domains in which the BPV L2 protein localized, double
staining experiments against a number of described nuclear proteins
and L2 were performed. No colocalization of the L2 protein was
found with coiled bodies, the retinoblastoma protein, p53 or the
splicing factor SC35. Although the staining pattern seen with the
anti-SC35 antibody was similar to that seen with the anti-L2
antibody, it was evident from the merged image that these regions
were exclusive. However, when the distribution of the L2 protein
was compared with that of anti-promyelocytic leukemia (PML) protein
staining, a nearly complete overlap in protein distribution was
observed.
[0060] The PML protein is a putative growth suppressor gene product
that localizes in subnuclear organelles termed PODs (Chang, K. S.,
et al., 1995, Blood 85, 3646-3653; Dyck, J. A., et al., 1994, Cell
76, 333-43). The PML distribution appeared to be unaffected by the
expression of the L2 protein, and the localization of L2 in the
PODs was unrelated to the level of L2 in the cell. This was
observed no matter whether the cells were expressing high,
intermediate or low levels of L2. All the cells expressing L2
showed a similar punctate distribution, in which L2 colocalized
with PML in every cell. Therefore, it is unlikely that this
colocalization is an artifact of overexpression.
[0061] L2 redirects L1 to PODs. As L1 and L2 coassemble into
capsids, the question was asked whether L1 might display a nuclear
staining pattern similar to L2. However, when L1 was expressed in
BPHE-1 cells, the distribution of L1 protein differed markedly. L1
was present in a nuclear pattern that varied from a diffuse to
slightly speckled arrangement with nucleolar exclusion.
[0062] This result led to the exploration of the possibility that
the subcellular distribution of L1 protein might be affected by
coexpression of L2. Therefore, BPHE-1 cells were coinfected with
recombinant L1 SFV and recombinant L2 SFV, which are the conditions
that lead to the formation of infectious BPV in BPHE-1 cells. The
L1 staining pattern was dramatically altered from the diffuse
nuclear pattern seen after L1 SFV infection alone. The L2 staining
pattern in the coinfected cells was consistent with the
distribution of L2 observed in the absence of L1. The distributions
of L1 and L2 overlapped substantially in the merge of the two
images. In general, L1 did not appear as tightly coalesced as L2.
In some cells L1 was observed mostly surrounding, rather than
overlapping, the L2 domain. This variability may be due to
differences in the kinetics of the infection of individual cells or
may reflect intermediate stages in L1 relocation. The conclusion
can be drawn that L2 induced the redirection of a substantial
proportion of L1 to PODs.
[0063] L2 induces colocalization of E2. Next examined was the
effect of the expression of the BPV capsid proteins on the
distribution of the nonstructural viral protein E2, which is
involved in viral genome replication and viral transcription
(Chiang, C.-M., et al., 1992, Proc. Natl. Acad. Sci. USA. 89,
5799-5803; Spalholz, B. A., et al., 1985, Cell 42, 183-191; Ustav,
M., and Stenlund, A., 1991, EMBO J. 10, 449-457). In BPHE-1 cells,
E2 was detected as a nuclear protein with a diffuse distribution.
There was no apparent effect on the localization of this protein
when the L1 capsid protein was expressed in these cells. In
contrast, L2 expression shifted E2 into punctate regions similar to
those observed with the anti-L2 staining pattern. Although it did
not interfere with determining the localization of E2, the levels
of E2 often decreased substantially during recombinant SFV
infection, presumably due to the well documented inhibition of host
protein synthesis by SFV (Strauss, J. H., and Strauss, E. G., 1994,
Micro. Rev. 58, 491-562). This effect is partially due to
interference with the Na+K+transporter by SFV (Carrasco, L., 1977,
FEBS Lett. 76, 11-15; Garry, R. F., et al., 1979, Virology 96,
108-120). A decrease in E2 was also observed in control infections
with unrelated SFV recombinants. Infection in the presence of 100
mM KCl helped counteract this problem.
[0064] To determine if L2 induced the redistribution of the E2
protein into the L2-staining PODs, double staining of the BPHE-1
cells after infection with the L2-SFV was performed. The majority
of the cells showed a diffusely distributed nuclear pattern of E2
staining. However, many cells demonstrated a relocation of E2 into
a punctate pattern. All of the L2-expressing cells showed solely a
punctate pattern of L2 staining. The coincidence of the E2 and L2
staining was striking in the infected cells that maintained
detectable levels of E2.
[0065] L2 is sufficient to redistribute full-length E2.
BPV-transformed cells with autonomously replicating genomes express
three forms of the E2 protein: a full-length 48 kD form that
functions in genome replication and transcriptional transactivation
and two smaller forms which act as repressors of viral
transcription (Hubbert, N. L., et al., 1988, Proc. Natl. Acad. Sci.
USA. 85, 5864-5868; Lambert, P. F., J. Virol. 63, 3151-3154;
McBride, A. A., et al., 1991, J. Biol. Chem. 266, 18411-18414). The
antibody used in the immunofluoroscent studies recognizes an
epitope in the C-terminal DNA binding domain common to all three
proteins and would not distinguish among them. Another feature of
the BPHE-1 cells is that an unknown proportion of E2 molecules are
bound to the viral genome. Therefore, it was unclear whether the
L2-dependent redistribution of E2 in the BPHE-1 might depend on the
presence of the viral genome in the cells.
[0066] To determine if the L2-dependent redistribution of E2
observed in the BPHE-1 cells could occur between L2 and the full
length E2 protein, independently of the viral genome, BHK-21 cells
(which do not contain the papillomavirus genome) were infected with
both the L2-SFV recombinant and a SFV recombinant expressing the
full-length E2. Since the RNA for E2 was produced entirely by the
SFV RNA-dependent polymerase in the cytoplasm, production of the
alternative E2 mRNAs was precluded. As expected, only the 48 kD
form was detected on Western blots of SFV-E2 infected cell
extracts. As noted earlier, the L2 distribution in BHK-21 cells was
similar to that observed with the BPHE-1 cells. When E2 was
expressed in BHK-21 cells, in the absence of L2, the majority of
the protein was present in a diffuse nuclear distribution. When the
cells were coinfected with L2 and E2, the L2 pattern was unaltered,
but the E2 assumed the punctate staining pattern of L2 in the cells
that coexpressed the two proteins. These results indicate that
L2-dependent localization of the full-length E2 to PODs is
independent of the viral genome and viral gene products other than
L2.
[0067] L2 does not induce the redistribution of E1. The
localization of E1 was examined, which participates in viral DNA
replication and so is presumably expressed in BPHE-1 cells. The
immunostaining with an anti-E1 antibody in BPHE-1 cells was weak.
This result was expected, as only low levels of E1 expression from
steady state autonomously replicating BPV genomes have been
reported (Sun, S., et al., 1990, J. Virol. 64, 5093-5105). No
change in the speckled staining pattern was observed after
SFV-mediated expression of either capsid protein. Because the
intensity of the staining was so low, and the parental line of
BPHE-1 was not available as a control, no firm conclusions could be
drawn from the E1 analysis in these cells.
[0068] To overcome these problems, BHK-21 cells were infected with
an E1 recombinant SFV, which resulted in clear immunostaining in a
speckled nuclear pattern, while uninfected cells were negative.
Coinfection with the L2 and E1 recombinant SFVs resulted in the
typical punctate L2 staining pattern, but this expression did not
alter the E1 pattern in the coinfected cells. Therefore, the L2
protein does not directly induce a redistribution of E1. However,
these results do not preclude the possibility that E1 may localize
to PODs indirectly through its well documented interaction with E2
and the viral genome (see below) (Mohr, I. J., 1990, Science 250,
1694-99, Wilson, V. G., and Ludes-Meyer, J., 1991, J. Virol. 65,
5314-5322).
[0069] Distribution of the viral genome is altered by L2
expression. It has been estimated that each BPHE-1 nucleus contains
50-200 autonomously replicating copies of the BPV genome (Zhang,
Y.-L., et al., 1987, J. Virol. 61, 2924-2928). To determine if
expression of the BPV virion proteins might influence the
distribution of the BPV genome, FISH analysis was performed on
BPHE-1 cells that were infected with L1 or L2 recombinant SFV. A
fluorescein-labeled PCR probe was generated to the upstream
regulatory region of the genome and hybridized in situ after DNA
denaturation. Only faint diffuse fluorescent speckles were detected
when the genome distribution was examined in cells that were
uninfected or infected with L1-SFV. However, in some cells that
expressed the L2 protein, the fluorescent probe bound more
discrete, coalesced areas. The fluorescent signal could be removed
by pretreatment of the cells with DNase, but was unaffected by
RNase treatment.
[0070] When the location of the genome was compared to that of the
L2-POD structures, the DNA was found to be situated adjacent to
these domains. Anti-L2 staining performed after FISH revealed that
the hybridization and washing procedures resulted in less intense
protein detection than seen previously. Nevertheless, the
characteristic punctate pattern of L2 was still seen. The cells
that were uninfected showed diffuse, barely detectable
fluorescence. However, in the cells that expressed high levels of
L2, the fluorescent probe bound strongly in about 10-12 spots
within the nucleus. In the merged images, it was apparent that the
BPV DNA and the L2 protein were located in adjoining domains. This
hybridization pattern was not detected in cells that did not
express L2. However, this distribution was apparent in only 20-25%
of the L2-expressing cells. This variation may be due to
differences in the copy number of the infected cells, timing of the
particular infection or cell cycle variability, but does not
detract from the conclusion about L2 inducing localization of
virion components and viral proteins to PODs.
[0071] Discussion. As described here, the minor capsid protein 12
has been found to possess the intrinsic capacity to localize to
PODs in the absence of other viral components. Further, the
presence of L2 in PODs is associated with the recruitment of the
major capsid protein L1, the nonstructural protein E2, as well as
the viral genome. It is therefore attractive to speculate that PODs
are the main structure in which papillomaviruses assemble.
[0072] PODs are interchromatinic matrix-bound nuclear bodies with
average diameters of 0.3 mm to 0.5 mm in most cells. The cellular
function(s) of PODs is largely unknown (Ascoli, C., and Maul, G.
J., 1991, J. Cell. Biol. 112, 785-795; Grande, M. A., et al., 1996,
J. Cell. Biochem. 63, 280-91). They have also been designated Kr
bodies or nuclear domain 10 (ND10) based on the average number of
bodies per cell, although their number actually varies and may be
higher in transformed cells (Ascoli, C., and Maul, G. J., 1991, J.
Cell. Biol. 112, 785-795; Lamond, A. I., and Carmo-Fonseca, M.,
1993, Trends in Cell. Biol. 3, 198-204). PODs may be required for
normal maturation of myeloid cells, as their fragmentation is often
seen in acute promyelocytic leukemia (Dyck, J. A., et al., 1994,
Cell 76, 333-43). Disruption of PODs in this leukemia is associated
with heterodimer formation between the normal PML protein and a
PML-retinoic acid receptor a (PML-RARa) fusion protein that results
from a characteristic t(15;17) chromosomal translocation (de The,
H., et al., 1990, Nature 347, 558-561; de The, H., et al., 1991,
Nature 347, 558-561, Kakizuka, A., et al., 1991, Cell 66, 663-674).
In addition to PML, PODs contain at least 6 other proteins. These
include the SP100 protein, which was originally identified as an
autoantigen in patients with primary biliary cirrhosis, Int-6, the
PIC-1 protein, as well as 52 kD (NP52), 55 kD (NDP55), and 65 kD
proteins (Ascoli, C., and Maul, G. J., 1991, J. Cell. Biol. 112,
785-795; Boddy, M. N., et al., 1996, Oncogene 13, 971-982; Desbois,
C., et al., 1996, Science 273, 951-53; Epstein, A. L., 1984, J.
Virol. 50, 372-379; Szostecki, D., et al., 1990, J. Immunol. 145,
4338-4347).
[0073] Some associations have been reported between PODs and the
replication of other DNA viruses. Productive viral replication
appears to commence in association with PODs for herpes simplex
virus 1 (HSV-1), adenovirus 5 (Ad-5), and simian virus 40 (SV40)
(Carvalho, T., et al., 1995, J. Cell. Biol. 131, 45-56; Doucas, V.,
et al., 1996, Genes Dev. 10, 196-207; Everett, R. D., and Maul, G.
G., 1994, EMBO J. 13, 5062-69; Jiang, W. Q., et al., 1996, Exp.
Cell. Res. 229, 289-300; Maul, G. G., et al., 1996, Virology 217,
67-75; Puvion-Dutilleul, F., 1995, Exp. Cell. Res. 218, 9-16).
Despite the remarkable convergence to this structure for these
three genetically unrelated viruses, the role that this
localization plays in the virus-cell interaction has remained
unclear.
[0074] A number of potential roles in viral replication have been
suggested for the association of viral components with PODs. It has
been proposed that POD association may be a cellular mechanism that
has evolved to limit initial virus replication (Ishov, A. M., and
Maul, G. G., 1996, J. Cell. Biol. 134:815-826). The fact that Ad-5
E4-ORF3 and HSV-1 ICPO encode proteins that disrupt PODs as
infection proceeds has been taken as evidence supporting this
possibility (Doucas, V., et al., 1996, Genes Dev. 10, 196-207;
Everett, R. D., and Maul, G. G., 1994, EMBO J. 13, 5062-69; Maul,
G. G., et al., 1996, Virology 217, 67-75; Puvion-Dutilleul, F.,
1995, Exp. Cell. Res. 218, 9-16). Also, the observation that
interferon upregulates the expression of POD proteins is consistent
with PODs acting as an antiviral defense mechanism (Chelbi-Alix, M.
K., et al., 1995, Leukemia 9, 2027-2033; Grotzinger, T., et al.,
1996, Mol. Cell. Biology 16, 1150-56; Lavau, C., et al., 1995,
Oncogene 11, 871-876).
[0075] Alternatively, POD association may possibly play a positive
role in viral replication. This localization might: 1) increase
local concentration of viral products and so promote assembly, 2)
interfere with normal differentiation and/or apoptotic responses to
the viruses in the epithelial cells that are their usual sites of
initial replication, 3) facilitate access to cellular transcription
and/or replication factors (although there is little evidence that
PODs possess these functions), 4) promote essential processing of
viral products. In the latter regard, it is interesting that a
ubiquitin-dependent protease has recently been shown to be POD
associated (Boddy, M. N., et al., 1996, Oncogene 13, 971-982). The
findings reported here that the conversion from latent to
productive papillomavirus infection in the in vitro system is
associated with a redistribution of the relevant viral products to
PODs lend strong support to the view that PODs play a positive role
in the replication of papillomaviruses.
[0076] While studies of Ad-5, HSV, SV40 and Epstein-Barr virus
(EBV) have identified products of early genes that interact with,
and in some cases disassemble, PODs, they have not determined which
gene(s) is responsible for POD localization of the virion
components (Doucas, V., et al., 1996, Genes Dev. 10, 196-207;
Everett, R. D., and Maul, G. G., 1994, EMBO J. 13, 5062-69; Jiang,
W. U., et al., 1996, Exp. Cell. Res. 229, 289-300; Maul, G. G., et
al., 1996, Virology 217, 67-75; Puvion-Dutilleul, F., 1995, Exp.
Cell. Res. 218, 9-16). In this study it was demonstrated that the
association of the various papillomavirus components with PODs
during productive infection depends upon the L2 minor capsid
protein. In the absence of L2, which is essential for the
generation of infectious virus, the other viral components display
indistinct, heterogeneous distributions. The results indicate that
L2 may function to facilitate virion production by inducing the
colocalization of the other components required for virion
assembly. The recruitment to PODs is likely to represent an
important feature that distinguishes productive from latent
papillomavirus infection. It is possible that the POD-binding
proteins HSV-1 ICPO and EBV EBNA-5, which have been implicated in
the escape from latency, may serve an analogous function for their
respective viruses.
[0077] The results of this study suggest the following model for
the productive phase of the papillomavirus life cycle (FIG. 1). The
productive cycle begins when L1 and L2 expression is induced by
differentiation specific signals in the infected epithelial cells
(Dollard, S. C., et al., 1992, Genes Dev. 6, 1131-42; Meyers, C.,
et al., 1992, Science 257, 971-73). SFV-mediated expression of
these two genes substitutes for this induced expression in the
present system and demonstrates that differentiation per se is not
required for virus production. Virus assembly appears to be
triggered by the association of L2 with PODs and the colocalization
of L1. It is likely that L1 association with the PODs is the result
of a direct interaction of L1 with L2, as stable L1/L2 complexes
form in both fully assembled VLPs in vivo and also in partially
assembled viral capsid structures, including L1 pentamers, in
vitro. Although L1 can self-assemble into VLPs in the absence of L2
(Kirnbauer, R., et al., 1992, Proc. Natl. Aced. Sci. USA. 89,
12180-84; Kirnbauer, R., et al., 1993, J. Virol. 67, 6929-36), L2
increases VLP production 4-fold in insect cells and 100 fold in
mammalian cells (Hagensee, M. E., et al., 1993, J. Virol. 67,
315-22; Kirnbauer, R., et al., 1993, J. Virol. 67, 6929-36; Zhou,
J., et al., 1993, J. Gen. Virol. 74, 763-68). This greater
efficiency could be the result of an increased rate of capsid
assembly as a consequence of the L2-mediated concentration of L1 at
the PODs.
[0078] In some cells containing L2, it appeared that the L1 protein
was predominately located around the perimeter of the L2 domains
rather than overlapping them. These variations may reflect temporal
differences in the SFV infection of individual cells, since all
infections appeared to show a mixture of the two patterns. It is
likely that a variety of L1 assembly states was detected with the
anti-L1 antibody employed here. In vitro, the antibody recognizes
pentameric L1 as well as intact virions. It is possible that the L1
detected around the POD perimeter is due to mature virions that
have been released from the sites of assembly and show a diminished
reactivity with the anti-L2 antibody. Alternatively, the peripheral
anti-L1 staining could be due to L1 pentamers in the process of
assembling with L2.
[0079] L2 also induced the redistribution of E2. The experiments in
the BHK-21 cells clearly demonstrated that E2 association with the
PODs is L2 dependent, but is independent of L1, other early
papillomavirus gene products, or the viral genome. However, there
is no evidence that E2 interacts directly with L2. Despite
considerable efforts, including coimmunoprecipitation experiments
and cosedimentation in sucrose gradients, soluble E2-L2 complexes
have not been detected in vivo or in vitro. At present, it has not
been feasible to distinguish between the possibilities that the L2
and E2 bind with relatively low affinity, that E2 binds to a
component of the PODs that has been altered by L2, or that E2, L2
and a POD component form a trimolecular complex.
[0080] It has been unclear how papillomaviruses preferentially
package their genomes over cellular DNA, as neither the individual
capsid proteins nor the assembled VLPs bind the genome in a
sequence specific manner (Mallon, R. G., et al., 1987, J. Virol.
61, 1655-1660; Zhou, J., et al., 1994, J. Virol. 68, 619-25). The
present findings on the distribution of the viral genome may have
important implications for understanding this process. In latently
infected cells, the viral DNA displayed a dispersed distribution.
In contrast, the present analysis localized the viral DNA to the
PODs in at least some of the cells in the cultures capable of
producing infectious virions, suggesting the preferential packaging
of the viral genome into virions may result, at least in part, from
this directed localization.
[0081] Although the mechanism for this relocalization of the viral
genome has not been experimentally tested, one can speculate that
it is dependent upon the translocation of E2 to the PODs, as E2
avidly binds multiple sites on the viral genome (Androphy, A. J.,
et al., 1987, Nature 325, 70-73; Li, R., et al., 1989, Genes Dev.
3, 510-526). Since it has not been possible to detect E2 in
infectious BPV virions extracted from cattle warts, E2 is
visualized as acting catalytically in the process of virion
assembly. SV40 T antigen, which is a nonstructural protein of that
virus, may be functionally analogous to E2 in this regard. T
antigen is a viral genome binding transcription/replication factor
that associates with PODs, but does not cause their disruption
(Jiang, W. Q., et al., 1996, Exp. Cell. Res. 229, 289-300). A
signal on the SV40 viral genome that is required for its packaging
into virions has been mapped to a segment on the viral DNA that
includes the T antigen binding sites (Oppenheim, A., 1992, J.
Virol. 66,5320-5328).
[0082] III. In Vitro Generation of Infectious BPV Virions in Insect
Cells
[0083] Using the protocol described in Example 3, infectious BPV
has been obtained in Sf9 insect cells by transfecting the cells
with full-length circular BPV DNA and infecting them with
baculoviruses expressing various combinations of BPV proteins.
[0084] Production of infectious BPV requires E2. When Sf9 cells
transfected with the BPV genome were infected with baculoviruses
expressing L1 alone, L2 alone, or L1 and L2 together and the
extracts were tested in C127 cells, no focal transformation was
obtained. However, typical BPV foci were obtained on C127 cells
when extracts from BPV DNA transfected Sf9 cells infected with
baculoviruses expressing L1+L2 and E2 were examined. The addition
of a further baculovirus, which expressed E1, in addition to L1+L2
and E2 baculoviruses, actually resulted in extracts that induced
fewer foci on C127 cells, and infection with baculoviruses
expressing L1+L2 and E1 did not yield focus forming activity on
C127 cells. In addition, focal transformation on C127 cells was not
obtained if cells infected with L1+L2 and E2 baculoviruses had been
transfected with the BPVpML plasmid (which contains the bacterial
pML-2d plasmid inserted within the BPV genome), rather than the
isolated religated BPV genome. As with infectious BPV, focal
transformation of C127 cells could be prevented if the infectious
extract from the L1, L2, and E2 baculovirus infected Sf9 cells was
incubated with a neutralizing anti-BPV serum.
[0085] Requirement for exogenous E2. The requirement for the E2
baculovirus, in addition to L1 and L2, to obtain infectious virus
was different from that of the mammalian cell system. One possible
explanation for the difference from the mammalian cell system,
which does not require exogenous E2, is that the BPV genes encoding
nonstructural viral proteins might not be expressed in the BPV DNA
transfected Sf9 insect cells, in contrast to mammalian cells that
harbor the BPV genome. To test for this possibility, Sf9 cells
transfected with BPV DNA were examined by Western blotting for
expression of the BPV E6 protein by probing with a 1:500 dilution
of rabbit antiserum to ACII BPV E6 fusion protein (Androphy, E. J.,
et al., 1985, Science 230, 442-445). No E6 expression was detected
under these conditions. These results demonstrate that, in contrast
to mammalian cells, the BPV genes were not expressed from the
transfected BPV genome.
[0086] E2 is a viral transcription/replication regulator. The BPV
full-length E2 gene encodes a protein that functions as a dimer
that binds to sequences present in multiple copies in the upstream
regulatory region (URR) of all papillomaviruses (Turek, L., 1994,
Adv. Virus Res. 44, 305-356). The E2 protein has been shown to
stimulate viral RNA synthesis and viral DNA synthesis. Both of
these activities depend upon the binding of E2 to its cognate
binding sites (E2BS) in the URR, while viral DNA replication
requires the viral E1 gene in addition to E2 protein (Chiang,
C.-M., et al., 1992, Proc. Natl. Acad. Sci. USA 89, 5799-5803).
[0087] Exogenous E2 does not stimulate BPV transcription. To
determine if E2 expression in BPV DNA transfected Sf9 cells might
activate expression of other papillomavirus genes required for
virion assembly, the presence of the BPV E6 protein (whose
expression in mammalian cells is regulated by E2) was sought
following infection with the E2 baculovirus. By Western blot
analysis, no E6 expression was detected in BPV DNA transfected Sf9
cells expressing E2, whether they were singly infected with the E2
baculovirus or infected with the E2 and L1+L2 baculoviruses. These
findings establish that expression of nonstructural viral genes was
not activated under these conditions.
[0088] Exogenous E2 does not stimulate BPV DNA synthesis. To
examine the possibility that the E2 baculovirus might be promoting
BPV DNA replication of the input BPV DNA in the transfected Sf9
cells, BPV E1, E2, in L1 and L2 were expressed from recombinant
baculoviruses separately and in combination in BPV DNA transfected
Sf9 cells. After 3 days the cells were harvested, and Hirt extracts
prepared (Hirt, B., 1967, J. Mol. Biol. 26, 365-369). The
development of resistance to digestion by Dpn I was used to assay
for BPV DNA replication in the Sf9 cells. The extrachromosomal DNA
was digested with excess Dpn I, separated on a 1% agarose gel, and
Southern blotted. The presence of BPV DNA was detected with a
[.sup.32P]-labeled probe generated by random priming from the Spe
I-Kpn I fragment of BPV DNA. No evidence of Dpn I resistant BPV DNA
was observed (to a sensitivity of 1 ng of DNA per sample). These
results indicate that viral replication had not occurred in the
insect cells in the presence of E1, or E2, or both proteins.
Therefore E2 is not required in BPV DNA transfected Sf9 cells to
replicate the BPV genome for packaging.
[0089] Exogenous EZ does not increase the amount of BPV L1 or L2.
Having ruled out that E2 was stimulating expression of a
nonstructural viral gene or fostering the replication of BPV DNA in
the Sf9 cells, the question was asked whether E2 expression might
be increasing the amount of L1 or L2 to a level critical for virion
assembly. To address this possibility, Western blot analysis was
used to assess the levels of L1 and L2 in BPV DNA transfected Sf9
cells that had been infected with recombinant baculoviruses
expressing L1, L2 and E2 in all combinations and maintained for 3
days. E2 did not increase the level of capsid gene expression;
rather, increasing the number of different baculoviruses used for
each infection of a plate of BPV-transfected Sf9 cells tended to
decrease gene expression from each.
[0090] Discussion. In mammalian cells that stably harbor multiple
copies of the BPV DNA genome, expression of BPV L1 and L2 via
semliki forest virus vectors leads to infectious BPV (neutralizable
by BPV antisera). In the same system, expression of HPV16 L1 and L2
via semliki forest virus vectors leads to an infectious pseudotype
composed of the BPV genome surrounded by HPV16 L1/L2 capsids
(neutralizable by HPV16 antisera). Encapsidation of viral DNA and
generation of infectious virus both require expression of L1 and
L2, although L1 by itself makes empty capsids.
[0091] In insect cells, infectious BPV can be obtained if the BPV
DNA genome is introduced into cells, and the cells are infected
with baculoviruses expressing an appropriate combination of BPV
proteins. It is expected that a similar approach for other
papillomaviruses, including HPV, will also produce infectious virus
of that serotype. It is also expected that this approach will be
successful for other nonmammalian systems, including yeast, in
which expression of structural viral proteins leads to
self-assembly of properly folded viral capsids (Sasagawa, T., et
al., 1995, Virology 206, 126-35).
[0092] The operative finding from the studies carried out in insect
cells is that the production of infectious BPV in these cells
requires the exogenous production of E2, in addition to the viral
genome and the viral structural proteins, L1 and L2. The lack of
the requirement for exogenous E2 in mammalian cells is concluded to
arise from the fact that E2 is expressed from the episomal BPV
genome in mammalian cells, while this is not the case in insect
cells. Since BPV is a mammalian virus, it is not surprising that
mammalian cells are permissive for expression of nonstructural
genes from the viral genome (Turek, L., 1994, Adv. Virus Res. 44,
305-356), while evolutionarily divergent insect cells are
nonpermissive for expression of these genes from the viral
genome.
[0093] The results of experiments conducted in insect cells negate
the possibility that exogenous E2, when expressed from recombinant
baculovirus, is stimulating BPV DNA synthesis, BPV transcription,
or increasing the amount of BPV L1 or L2 protein. It is envisioned
that E2 has a different role, which is that of mediating
encapsidation of the BPV genome by structural viral proteins.
Although the mechanism by which E2 functions to obtain this result
is unknown, a model can be postulated. In this model, E2 dimers
when bound to their cognate E2 binding sites in the viral genome
form a complex that associates with the viral capsid as it is being
formed into fully assembled particles. Presumably some aspect of
the E2-DNA complex recognizes the assembling viral capsid in a
manner that fosters encapsidation of the viral DNA. In this model,
E2 might be incorporated into the viral capsid along with the viral
genome. Alternatively, encapsidation of the viral DNA might lead to
the release of E2, in which case it might be re-used to assist in
the encapsidation of other viral DNA genomes. This latter
possibility is favored, as the examination of infectious BPV from
bovine warts has thus far failed to detect E2 (at a sensitivity of
1 E2 molecule per 10 virions). The key feature of this model is
that it provides specificity for genome encapsidation through the
well documented high affinity binding of E2 to viral DNA sequences.
It is expected that all papillomaviruses encapsidate their genomes
via this mechanism. The results showing that L2 can recruit E2 and
the viral genome to PODs are consistent with this model. The
ability to pseudotype the BPV genome with HPV16 L1 and L2 in
mammalian cells establishes that the mechanism of encapsidation is
conserved among papillomaviruses. Furthermore, the model, in which
a nonstructural DNA binding protein brings viral DNA to the
developing virion, may apply to other viruses as well. In most
instances, the DNA binding protein (or RNA binding protein for RNA
viruses) will be virally encoded. However, there could be viruses
in which the DNA binding protein is cell encoded. See Example
6.
[0094] Based on the general features of this model, encapsidation
of a DNA by papillomavirus machinery requires that the DNA contain
the E2BS. This means that any DNA sequence, such as a gene and a
preferred regulatory element, can be encapsidated as a
papillomavirus pseudotype so long as it contains the E2BS.
Determination of the rules regarding the number and location of
E2BS, and whether the E2BS-containing DNA must also be episomal,
circular, and approximately 8 kb, is empirical. It is also expected
to be possible to eliminate virtually all the viral genes from the
DNA and still have the E2BS-containing DNA efficiently pseudotyped
by papillomavirus capsid proteins. The finding that HPV16 L1 and L2
can pseudotype the BPV genome shows that E2 can function with
heterologous capsids. This means that making a new viral pseudotype
does not require using the homologous E2. Instead, the requirements
are for the E2 DNA binding protein and its cognate DNA binding site
incorporated into a papillomavirus vector DNA, with papillomavirus
capsids that can be heterologous in relation to E2. To the extent
that the mechanism may apply to other viruses, it can also be
extended to them.
[0095] Papillomavirus pseudotypes can be made in mammalian cells
(Section I). Given the success in making infectious BPV in insect
cells (Section III), it is expected additionally to be possible to
make papillomavirus pseudotypes in insect cells, providing the
cells express E2, L1 and L2. Since L1 and L1/L2 virus-like
particles have been made in yeast (Sasagawa, T., et al., 1995,
Virology 206, 126-35), and E2 has been shown to function as an
E2BS-dependent transcription factor here (Lambert, P., et al.,
1989, Genes Dev. 3, 38-48; Morrissey, L., et al., 1989, J. Virol.
63, 4422-4425), it is expected therefore to be possible to make
papillomavirus pseudotypes in yeast, with the requirements in yeast
being similar to those in insect and, presumably, mammalian cells.
Addition of a DNA replication origin that functions in yeast or
insect cells (papillomaviruses already have a replication origin
that functions in mammalian cells) may increase infectious virus
production in the homologous system. Moreover, virus-like particles
have now been expressed in bacteria (Nardelli-Haefliger et al.,
Dec. 1-6, 1996, 15 Intl. Papillomavirus Workshop, 290), which
finding raises the expectation that papillomavirus pseutotypes can
be made in bacteria that are engineered to operably encode E2, L1
and L2 genes. Taken together, these properties may make it
relatively inexpensive to produce papillomavirus pseudotypes.
[0096] Additionally, it should be possible to make viral
pseudotypes in the test tube, which would avoid using cells. The
interaction of E2 with the other viral components, L1, L2, and
E2BS-containing DNA, provides the start of an assay to determine
the requirements for in vitro packaging of viral DNA. Efficient
packaging may also require cellular components or structures, such
as those present in PODs, a fact that would be established
empirically.
[0097] Because papillomavirus pseudotypes should be infectious for
a wide variety of cells, they are expected to have broad
application in gene transfer.
[0098] IV. Use of Infectious Papillomavirus Pseudoviral Particles
in Gene Therapy and Gene Immunization
[0099] Gene transfer in the context of gene therapy and gene
immunization is a clinical strategy in which the genetic repertoire
of somatic cells is modified for therapeutic or immunogenic
purposes. (Crystal, R. G., 1995, Science 270, 404-410; Mulligan, R.
C., 1993, Science 260, 926-932). Essentially, gene transfer, in
this context, also involves the delivery, to target cells, of an
expression cassette made up of one or more genes and the sequences
controlling their expression. This can be carried out ex vivo in a
procedure in which the cassette is transferred to cells in the
laboratory and the modified cells are then administered to the
recipient. Alternatively, gene transfer can be done in vivo, in a
procedure in which the expression cassette is transferred directly
to cells within an individual. In both strategies, the transfer
process is usually aided by a vector that delivers the cassette to
the cell where it can function appropriately.
[0100] The choice of an ex vivo or in vivo strategy and of the
vector used to carry the expression cassette is dictated by the
clinical target. The vector systems for which data are available
from clinical trials (retroviruses, adenoviruses, and
plasmid-liposome complexes) transfer expression cassettes through
different mechanisms and thus have distinct advantages and
disadvantages for different applications.
[0101] Replication-deficient, recombinant retrovirus vectors can
accommodate up to 9 kb of exogenous information. Retroviruses
transfer their genetic information into the genome of the target
cell. This is an advantage when treating hereditary and chronic
disorders, but it has risks, including the potential for toxicity
associated with chronic overexpression or insertional mutagenesis.
The use of retrovirus vectors is limited by the sensitivity of the
vector to inactivation, by the fact that target cells must
proliferate in order to integrate the proviral DNA into the genome,
and by production problems associated with recombination,
rearrangements, and low titers.
[0102] Adenovirus vectors in current use accommodate expression
cassettes up to 7.5 kb. Adenovirus vectors are well suited for
transfer applications because they can be produced in high titers
and they efficiently transfer genes to nonreplicating and
replicating cells. The transferred genetic information remains
extrachromosomal, thus avoiding the risks of permanently altering
the cellular genotype or of insertional mutagenesis. However,
adenovirus vectors in current use evoke nonspecific inflammation
and antivector immunity. These responses, together with the
extrachromosomal position of the expression cassette, limit the
duration of expression to periods ranging from weeks to months.
Thus adenovirus vectors will have to be readministered periodically
to maintain their persistent expression. Although it is unlikely
that repeat administration will be risky, it is not known whether
antibodies directed against vector capsid proteins will limit the
efficacy of repetitive administration of these vectors.
[0103] In theory, plasmid-liposome complexes have many advantages
as gene transfer vectors, in that they can be used to transfer
expression cassettes of essentially unlimited size, cannot
replicate or recombine to form an infectious agent, and may evoke
fewer inflammatory or immune responses because they lack proteins.
The disadvantage of these vectors is that they are inefficient,
requiring that thousands of plasmids be presented to the target
cell in order to achieve successful gene transfer.
[0104] One of the obstacles to successful gene transfer is
obtaining the perfect vector. The ideal vector will overcome the
hurdles presented by current vectors, including reduction of the
risk for insertional mutagenesis in retrovirus vectors,
minimization of the amount of immunity and inflammation evoked by
the adenovirus vectors, and enhancement of delivery of the gene to
the cell for the plasmid-liposome complexes.
[0105] The advantage of using a papillomavirus vector in gene
therapy and gene immunization is that it has many desireable
qualities as a vector, for it reduces the risk for insertional
mutagenesis characteristic of retrovirus vectors (by virtue of its
distinct life cycle), it minimizes the amount of immunity and
inflammation attributable to adenovirus vectors (see below), and it
enhances delivery in contrast to the problem intrinsic to
plasmid-liposome complexes (based on its being an animal
virus).
[0106] An attractive feature of papillomavirus vectors is that
there are many different serotypes whose neutralizing antibodies
cross-react poorly or not at all. Since neutralizing antibodies can
interfere with viral infection, the existence of multiple serotypes
means that patients who have developed neutralizing antibodies to
one papillomavirus serotype would remain susceptible to infection
by other viral serotypes. Encapsidation of the same DNA in
different capsid types would allow for multiple "boosts" in a gene
therapy or gene immunization protocol without progressive loss in
effectiveness of delivery. It would be reasonable to change
serotypes by switching L1 molecules, but it may also be necessary
to switch L2 molecules as well as L1. Although L1 contains the
major neutralizing epitopes, L2 also contains minor neutralizing
epitopes. This point may be important in a sequential
administration protocol.
[0107] The benefit of using animal viruses to obtain increased
delivery is offset by the limitation that these viruses must be
propagated in mammalian cells. This kind of propagation is
expensive and potentially dangerous due to the possibility of
contamination with occult viruses that might be infectious and
pathogenic for humans. A strength of the papillomavirus vector is
that, even though it is an animal virus, it can be propagated in
nonmammalian cells, such as insect and yeast cells, thus enjoying
the advantages of being an animal virus while avoiding the
pitfalls.
[0108] The pseudoviral particles of the invention are useful in the
context of gene therapy and gene immunization. The papillomavirus
vector uses a E2BS-containing DNA as a base, with the viral genes
possibly being deleted from the virus. The expression cassette is
inserted, and the infectious papillomavirus is produced in a
packaging cell line that contains the E2, L1 and L2 sequences that
provide the proteins necessary to package the virus. The vector
with its expression cassette enters the target cell via a specific
receptor, gets internalized into the cytoplasm, and is uncoated to
deliver its DNA genome with the expression cassette into the
nucleus, where it functions in an epichromosomal fashion to direct
the expression of its product. See Example 7.
[0109] In some cases, it might be desireable to provide in the
papillomavirus vector a gene encoding E1, which is known to be
required for stable maintenance of the viral genome as an episome.
This addition would tend to prevent integration of the DNA into the
host genome. Of course, the size of the expression cassette would
have to be correspondingly adjusted.
[0110] Examples of genes carried by the expression cassettes are
genes that encode expression products, such as proteins,
polypeptides, and peptides (that may be modified by glycosylation,
phosphorylation, or amidation, etc.) that are useful in gene
therapy or gene immunization (see below). Sequences controlling
their expression include promoters (for example, RSV or CMV),
enhancers, leader peptides, termination and palyadenylation
signals, splicing signals, viral replicons, and genes encoding
selectable markers.
[0111] Gene therapy is understood to be applicable to the treatment
of inherited diseases and, also, acquired diseases, ranging from
cardiovascular disorders to cancer to AIDS. Examples of cancer are
melanoma, renal cell, ovarian, cervical, neuroblastoma, brain, head
and neck, lung, liver, breast, colon, prostate, mesothelioma,
leukemia, lymphoma, multiple myeloma, and skin. Examples of other
diseases amenable to gene therapy include hemoglobinopathies,
severe combined immunodeficiency, hemophilias, familial
hypercholesterolemia, inherited emphysema, cystic fibrosis,
muscular dystrophy, lysosomal storage diseases, Gaucher's disease,
purine nucleoside phosphorylase deficiency, alpha-1 antitrypsin
deficiency, Fanconi's anemia, Hunter's syndrome, chronic
granulomatous disease, rheumatoid arthritis, peripheral vascular
disease, Parkinson's disease, diabetes, osteoporosis, chronic
wounds, psoriasis, and atopic dermatitis.
[0112] As for gene immunization, it is understood to be applicable
to raising a desired immune reaction, generating desired
antibodies, or eliciting a desired CTL response. It typically
results in protection against disease. These diseases include
infectious disease, like viral disease (for example, viral
influenza), bacterial disease, and parasitic disease.
[0113] Examples of genes carried by the pseudoviral particles of
the invention are those that encode, without limitation,
constituents of hemoglobin, adenosine deaminase, blood clotting
factors (e.g., Factor VIII, Factor IX), receptors (for example, LDL
receptor, ACh receptor, hormone receptors), purine nucleoside
phosphorylase, alpha-1 antitrypsin, ion channels (for example,
CFTR), dystrophin, lysosomal enzymes, insulin, calcitonin,
hormones, growth factors, cytokines; growth hormone,
erythropoietin, parathyroid hormone, TNF, CSF, IGF, MDR, IL-1,
IL-2, IL-4, interferons, p53; suicide gene products (for example,
herpes simplex virus thymidine kinase, cytosine deaminase,
vericella thymidine kinase); antibodies and fragments thereof,
components of MHC complexes (for example, HLA-B7), and minor
histocompatibility antigens; antisense and triple helix agents;
oncogenes; tumor suppressor genes; viral antigens, bacterial
antigens, parasitic antigens; connective-tissue proteins (e.g.,
collagen, elastin, and fibronectin) and foreign proteins (for
example, lysozyme and BSA).
[0114] It is to be understood that the pseudoviral particles of the
invention are used in gene transfer by infecting cells. While ex
vivo approaches are plausible, in vivo protocols are preferred.
Using either scenario, any cells that express the appropriate cell
surface receptors by which the particles gain entry are amenable to
infection. Papillomavirus pseudoviral particle-mediated gene
transfer into epithelial cells is particularly useful. Use of a
tissue is contemplated as a bioreactor (to produce proteins for
systemic release to treat disease), or to treat the tissue itself.
Use of the epithelium, particularly the epidermis, is thus
envisioned as a bioreactor, or to treat the epithelium, or the
epidermis, itself.
[0115] The pharmacologically or biologically active compounds of
this invention are generally administered to animals, particularly
humans.
[0116] These active compounds can be processed in accordance with
conventional methods of galenic pharmacy to produce medicinal
agents for administration to patients, e.g., mammals, including
humans.
[0117] The compounds of this invention can be employed in admixture
with conventional excipients, i.e., pharmaceutically acceptable
organic or inorganic carrier substances suitable for parenteral,
enteral (e.g., oral) or topical application which do not
deleteriously react with the active compounds. Suitable
pharmaceutically acceptable carriers include water, salt solutions,
alcohols, vegetable oils, synthetic fatty vehicles, etc. The
pharmaceutical preparations can be mixed with auxiliary agents,
e.g., lubricants, preservatives, stabilizers, and the like which do
not deleteriously react with the active compounds. They can also be
combined where desired with other active agents, e.g.,
vitamins.
[0118] For parenteral application, particularly suitable
formulations are injectable, sterile solutions, preferably oily or
aqueous solutions, as well as suspensions, emulsions, or implants.
These formulations are, if desired, mixed with auxiliary agents,
e.g., preservatives, stabilizers, buffers or salts for influencing
osmotic pressure, etc.
[0119] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules. A syrup,
elixir, or the like can be used where a sweetened vehicle is
employed.
[0120] For topical application, these are employed as nonsprayable
forms, viscous to semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, aerosols, etc. For topical application, also
suitable are sprayable aerosol preparations where the active
ingredient, preferably in combination with a solid or liquid inert
carrier material, is packaged in a bottle or in admixture with a
pressurized volatile, normally gaseous propellant, e.g., a
freon.
[0121] These compositions can be administered intravenously,
orally, or through the nose or lung. They can also be administered
parenterally or subcutaneously. Administration to the epithelium,
or the epidermis, can be by bombardment (for example, with a gene
gun) or topical application (for example, with a gene cream) which
may or may not require exposure of underlying cells by tape
stripping or penetration enhancers.
[0122] It will be appreciated that the actual preferred amounts of
active compound in a specific case will vary according to the
specific compound being utilized, the particular compositions
formulated, the mode of application, the particular situs, and the
organism being treated. Dosages for a given host can be determined
using conventional considerations, e.g., by customary comparison of
the differential activities of the subject compounds and of a known
agent, and, e.g., by means of an appropriate, conventional
pharmacological protocol.
[0123] The logic underlying the usefulness of papillomavirus
vectors in gene transfer is compelling, and put in the context of
gene therapy and gene immunization, the impact of this technology
for innovative therapies and prophylactics is enormous.
EXAMPLES
[0124] Particular aspects of the invention may be more readily
understood by reference to the following examples, which are
intended to exemplify the invention, without limiting its scope to
the particular exemplified embodiments.
Example 1
[0125] Reagents. BPHE-1 cells were obtained from A. Lewis (NIH,
Bethesda) (Zhang, Y.-L., et al., 1987, J. Virol. 61, 2924-2928).
C127 Clone C cells were obtained from W. Vass (NIH, Bethesda), and
BHK-21 cells were obtained from the ATCC. All antisera to VLPs
(Roden, R. B. S., et al., 1996, J. Virol. 70, 3298-3301) and
monoclonal antibodies have been described previously (Cowsert, L.
M., et al., 1988, Virology 165, 613-15; Roden, R. B. S., et al.,
1994, J. Virol. 68, 7570-74). Unless otherwise stated, all other
reagents, including the SFV expression vectors, were from Life
Technologies Inc., Gaithersburg.
[0126] Generation of recombinant pSFV-1 plasmids. In order to
remove an internal Spe I site, BPV L1 was amplified by PCR in two
separate reactions from Bam HI-cut and religated BPVpML DNA using
oligonucleotides
CCGCTGGATCCCACTATTATATAGCACCATGGCGTTGTGGCAACAAGGCCAG (SEQ ID NO:1)
and CAGTTGAGACTAGAGAGCCAC (SEQ ID NO:2) for one reaction, and
GTGGCTCTCTAGTCTCAACTG (SEQ ID NO:3) and
GCGGTGGATCCTTATTTTTTTTTTTTTTTTGCA- GGCTTACTGGAAGTTTTTTGGC (SEQ ID
NO:4) for the second. The products were gel purified and mixed, and
the full length L1 gene reamplified by using the outside primers.
The product (.about.1.5 kB) was gel purified, digested with Bam HI
and cloned into the Bam HI site of pSFV-1 (Liljestrom, P., and
Garoff, H., 1991, BioTechnology 9, 1356-1361). The clone was
sequenced to confirm the orientation and absence of the Spe I site
and amplification errors. BPV L2 was amplified by PCR from Bam
HI-cut and religated BPVpML DNA using
GCGGTAGATCTAATATGAGTGCACGAAAAAGAGTAAAACGTGCCAG- T (SEQ ID NO:5) and
CCGCTAGATCTAGGGAGATACAGCTTCTGGCCTTGTTGCCACAACGC (SEQ ID NO:6) for
primers. The product (.about.1.5 kB) was gel purified, digested
with Bgl II, cloned into the Bam HI site of pSFV-1 and sequenced.
Wild type (114/K) and capsid assembly deficient mutant (pAT) HPV16
L1 were excised from pEVmod using Bgl II and subcloned into pSFV-1.
HPV16 L2 was subcloned from a pEVmod vector into the Bam HI site of
pSFV-1.Nrul (which is linearized using Nru I rather than Spe I).
All plasmids were purified from E. coli HB101 by alkaline lysis and
cesium chloride isopynic density centrifugation.
[0127] Generation of recombinant SFV stocks. The recombinant pSFV-1
clones and pHelper-2 (Berglund, P., et al., 1993, BioTechnology 11,
916-920) plasmid were linearized using Spe I (or Nru I for
pSFV-1.Nrul based clones). The DNAs were phenol/chloroform
extracted and ethanol precipitated. To generate SFV RNA, 1 .mu.g of
each linearized pSFV-1 clone and 1 .mu.g of pHelper-2 were
resuspended in 100 .mu.l reactions containing 1 mM ATP, 1 mM CTP, 1
mM UTP, 0.5 mM GTP, 1 mM RNA capping analog m7G(5')ppp(5')G, 5 mM
DTT, 100 .mu.l human placental RNase inhibitor, 75U SP6 RNA
polymerase in 1.times. SP6 reaction buffer. The reaction mixtures
were incubated for 1 h at 37.degree. C. and 2.5 .mu.l was analyzed
on a 0.7% agarose gel to assess the integrity of the SFV RNAs. The
remaining RNA was diluted in 1 ml OptiMEM medium, mixed with 100
.mu.l of Lipofectin in 1 ml of OptiMEM and incubated for 15 min at
ambient temperature. BHK-21 cells in a T-75 tissue culture flask
were washed and covered with 2 ml of OptiMEM. The RNA/Lipofectin
mix was added, and the cells were incubated for 4 h at 37.degree.
C. The cells were washed once and maintained for 24 h in 13 ml of
complete medium (5% fetal calf serum, 10% tryptose phosphate broth,
10 mM Hepes pH 7.4, 1.times. nonessential amino acids, 100 U/ml
penicillin and 100 .mu.g/ml streptomycin in Glasgow's MEM). The
medium was harvested, clarified by centrifugation (1000.times.g, 10
min), aliquoted and stored at -80.degree. C.
[0128] Generation of papillomavirus in BPHE-1 cells. The
recombinant SFV stock was rendered infectious by incubation with
0.5 mg/ml chymotrypsin A4 (Boehringer Mannheim) for 30 min on ice
and treatment with 0.5 mg/ml aprotinin (Sigma). 4.times.10.sup.6
BPHE-1 cells maintained for 12-20 h in DMEM containing 10% fetal
calf serum, 100 U/ml penicillin and 100 .mu.g/ml streptomycin in a
100 mm tissue culture plate were washed in D-PBS (containing 0.9 mM
calcium and 0.5 mM magnesium). The cells were incubated for 2 h at
37.degree. C. with activated recombinant SFV (titrated to give
maximum expression levels, but generally 0.5 ml of each high titre
stock) diluted to 25 ml in D-PBS. The virus was aspirated, replaced
with complete medium and maintained for 30 h. The cells were
scraped from the dish into the medium, which was collected and
centrifuged (1000.times.g, 10 min), and the cell pellet was
resuspended in 1 ml of D-PBS. The cells were lysed by sonication
(10 s, 60% power, Fischer model 150 sonic dismembranator with a
microtip).
[0129] In vitro focal transformation assay. Cell lysates were added
to the medium (OMEM containing 10% fetal calf serum and 100 U/ml
penicillin and 100 .mu.g/ml streptomycin) of monolayers of C127
Clone C cells in 60 mm tissue culture plates. The cells were
incubated at 37.degree. C. for 1 h, washed and maintained in DMEM
containing 10% fetal calf serum for 3 weeks. The cells were stained
with 0.5% (w/v) methylene blue, 0.25% (w/v) carbol fuschin in
methanol, and the number of foci scored (Dvoretzky, I., et al.,
1980, Virology 103, 369-375).
[0130] Purification of particles from mammalian cells. For
preparation of VLPs, BHK-21 cells were maintained for 3 days post
infection with recombinant SFV. To generate full virions, BPHE-1
cells were maintained for only 30 h after infection with
recombinant SFV. Ten 500 cm.sup.2 culture dishes of cells were
scraped from the plates into the medium which was centrifuged
(1000.times.g, 10 min, 4.degree. C.) and the cell pellet
resuspended in 5 ml ice cold PBS. The cells were lysed by
sonication (1 min, 60% power) and treatment with 0.5% NP-40.
Extracts were layered over 30 ml 40% (w/w) sucrose in PBS cushion
and centrifuged for 150 min at 80,000.times.g at 4.degree. C. The
pellets were resuspended in 12 ml of 27% (w/w) cesium chloride in
PBS and centrifuged for 20 h at 275,000.times.g. The isopynic
density gradient was fractionated and the density of each fraction
determined using an Abbe3L refractometer (Milton Roy, Rochester,
N.Y.) (Kirnbauer, R., et al., 1993, J. Virol. 67, 6929-36).
[0131] Southern blot analysis. Cesium chloride gradient samples
were mixed with 2.5 volumes of ethanol and stored overnight at
-20.degree. C. The samples were centrifuged (16,000.times.g, 10
min, 4.degree. C.); the pellets were washed with 70% ethanol and
resuspended in 10 mM Tris, 1 mM EDTA, pH 8 (TE). Each sample was
treated with proteinase K, phenol/chloroform extracted, ethanol
precipitated and resuspended in TE. Samples were separated on a
0.8% agarose gel, transferred to nylon membrane (Hybond N,
Amersham) and UV cross-linked (12 .mu.J, UV Autocrosslink 1800,
Stratagene). BPV DNA was detected using [.sup.32P]-labeled random
primed SpeI-KpnI fragment of BPVpML under high stringency
conditions (Sambrook, J., Fritsch, E. F., and Maniatis, T., 1989,
Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0132] Electron microscopy. Transmission electron microscopy was
performed by binding 5 .mu.l samples to carbon-coated copper grids,
staining with 1% (w/v) uranyl acetate and examination using a
Philips EM400RT electron microscope at 100 KV. Samples for
cryo-electron microscopy were spun for 15 min in an airfuge onto
carbon-coated copper grids, frozen in liquid ethane and also
examined in a Philips EM400RT electron microscope at 100 KV (Booy,
F. P., et al., 1991, Cell 64, 1007-1015).
Example 2
[0133] Antibodies. The monoclonal antibody, B201, directed against
the BPV E2 protein, and the polyclonal antiserum, 150-1, which
recognizes the BPV E1 protein, were provided by Dr. Elliot Androphy
(New England Medical Center, Tufts University School of Medicine).
The monoclonal antibody, 5B6, which recognizes BPV L1 capsid
protein, and the rabbit polyclonal antiserum, 17128, raised against
the full length BPV L2 capsid protein, have been previously
described (Roden, R. B. S., et al., 1994, J. Virol. 68, 7570-74).
The monoclonal antibody, 6A8, directed against the BPV L2 protein
was provided by A. Bennett Jenson (Georgetown University) (Jin, X.
W., et al., 1989, J. Gen. Virol. 70, 1133-40). The antibody against
SC35 was purchased from Sigma Immunochemicals (St. Louis, Mo.). The
anti-PML antibody, 5E10, was generated by R. van Driel (University
of Amsterdam) and was a kind gift of Dr. Louis Staudt (NCI, NIH)
(Stuurman, N., 1992, J. Cell. Science 101, 773-84). FITC-conjugated
goat anti-mouse immunoglobulin G (IgG) and Texas-red conjugated
goat anti-rabbit IgG were purchased from Jackson Immunoresearch
(West Grove, Pa.).
[0134] Cell lines. BPHE-1 cells, obtained from A. Lewis (NIH,
Bethesda), were grown in DMEM supplemented with antibiotics and 10%
FCS (Zhang, Y.-L., et al., 1987, J. Virol. 61, 2924-2928). BHK-21
cells, obtained from the ATCC, were grown in Glasgow's medium
supplemented with 10% tryptose phosphate broth, antibiotics,
nonessential amino acids, HEPES and 5% FCS. For microscopic
analyses, cells were seeded onto acid-washed #01 coverslips in 24
well plates at a density of 1.times.10.sup.5 cells/well and
cultured overnight.
[0135] Recombinant Semliki Forest virus expression system. The
production of recombinant SFV RNAs and replication defective virus
expressing the BPV L1 or L2 capsid protein and the SFV infection
protocols are as described here. BPV E1 and E2 were cloned into the
BamHI site of pSFV-1 as PCR products amplified from the BPV genome,
the primers for E1 being: 5'
CCGCTGGATCCGCACCATGGCAAACGATAAAGGTAGC(SEQ ID NO:7) and 3'
GCGGTGGATCCGATCTTGCAACTTATCACTAC (SEQ ID NO:8), and the primers for
E2 being: 5' CCGCTGGATCCGCACCATGGAGACAGCATGCGAACG(SEQ ID NO:9) and
3' GCGGTGGATCCGAAGAAAAGGCAATGGCAGTG(SEQ ID NO:10). Recombinant
viruses expressing each gene were generated as described for L1 and
L2. For infection of cells, high titer recombinant SFV stock was
treated with 500 .mu.g/ml of chymotrypsin A4 on ice for 30 minutes
and then aprotinin was added to 500 .mu.g/ml for an additional 10
minutes. The activated virus was diluted in Dulbecco's PBS with
calcium and magnesium to {fraction (1/100)} and added to cells in
24 well plates. After 60 minutes at 37.degree. C., virus-containing
medium was removed and replaced with the normal growth medium
supplemented with 100 mM KCl for the remainder of the infection to
maintain cellular protein expression. Infections were allowed to
continue for 5-6 hours prior to cell fixation and
immunolocalization. Although SFV infection will induce cell death
in 48 hr., the morphology of the infected cells was not visibly
altered at this early time point.
[0136] Immunofluorescent staining. Cells were washed three times
with cold PBS pH 7.4, fixed by 10 min. incubation at room
temperature with 1.0% paraformaldehyde diluted in PBS, and washed
three times with PBS/200 mM glycine. Cells were then incubated with
primary antibody diluted in PBS/0.1% polyoxyethylene 20 cetyl ether
(Brij)(Sigma Chemicals, St. Louis, Mo.) and incubated at 4.degree.
C. Polyclonal antisera were used at a dilution of {fraction
(1/1000)}. Monoclonal antibodies used as hybridoma supernatants
were diluted {fraction (1/100)}. Purified antibodies were used at a
concentration of 5 .mu.g/ml. For double immunofluorescent staining
the primary antibodies were incubated in unison. After incubation,
coverslips were washed three times with PBS/0.1% Brij. Secondary
antibodies were diluted to 5 .mu.g/ml in PBS/0.1% Brij and
incubation was performed at 4.degree. C. After this incubation,
cells were washed thoroughly in PBS/0.1% Brij and inverted onto
Fluoromount-G mounting solution (Southern Biotechnology Associates,
Birmingham, Ala.) on a glass slide. Fluorescence was examined using
a BioRad MRC 1024 laser scanning confocal system attached to a
Zeiss Axioplan microscope. All images were acquired with a Zeiss
63.times. N.A. 1.4 planapo objective using the photon counting
mode. Control coverslips established that fluorescence in green and
red channels was not overlapping and that antibody binding was
specific for the intended antigen. Images were collaged and
scale-adjusted using the Adobe Photoshop program.
[0137] Fluorescent in situ hybridization (FISH). A probe to the
upstream regulatory region of the BPV genome (7173-28) was PCR
amplified using the 5' oligo, CGGCAAGCTTGCAATGTGCTGTGTCAGTTG (SEQ
ID NO:11), and the 3' oligo, CGCGAAGCTTAACGGTGATGGTGTGATTAT (SEQ ID
NO:12). The HindIII cloning site is in bold and the BPV sequence
overlap is underlined. The PCR reactions were performed using a
fluorescein labeling mix (Boehringer Mannheim Indianapolis, Ind.)
in which fluorescein-labeled dUTP is incorporated into the PCR
product. Cells were fixed in 2% paraformaldehyde/PBS with 5 mM
MgCl2 for 10 min. at room temperature. After 3 washes in PBS/200 mM
glycine, the cells were permeabilized with 0.2% TritonX-100 in PBS
(v/v) for 5 min. and rewashed with PBS. Immediately prior to
hybridization cells were washed with 2.times.SSC (1.times.SSC is
150 mM NaCl and 15 mM sodium citrate) at room temperature.
[0138] Labeled probe (150 ng/coverslip) was brought to a final
volume of 10 .mu.l with 1.times.SSC and dried under vacuum. The
probe was resuspended in 7 .mu.l of 100% deionized formamide and
heated to 90.degree. C. for 5 min. 7 .mu.l of hybridization buffer
was added to the probe to give a final concentration of 50%
formamide, 2.times.SSC, 1.times. Denhardt's solution, 10% dextran
sulfate and 50 mM Tris pH 7.5. In rapid succession, this mixture
was applied to the coverslip, inverted onto a glass slide, covered
with a second glass slide spaced with a 1 mm spacer, sealed with
Parafilm (American National Can, Greenwich, Conn.) and incubated
for 10 minutes at 90.degree. C. The slides were then transferred to
a humid 37.degree. C. chamber overnight. After overnight
incubation, the coverslips were washed in several changes of 50%
formamide/2.times.SSC at 37.degree. C. for 60 min., 2.times.SSC at
37.degree. C. for 30 min. and 2.times.SSC at room temperature for
30 min.
[0139] For experiments in which protein localization was also
desired, after the final posthybridization wash, antibody staining
was performed as described above except that detergent was not
included in the incubations or washes.
Example 3
[0140] Generation of BPV genome. To produce the circular BPV DNA,
cesium chloride purified BPVpML plasmid DNA, which contains the BPV
genome cloned via its unique Bam HI site, was digested with Bam HI,
phenol/chloroform extracted, and precipitated. The DNA was
resuspended at 500 .mu.g/ml in ligation buffer (50 mM TrisHCl pH
7.6, 5 mM MgCl.sub.2, 1 mM ATP, 1 mM DTT) containing 0.05 Weiss
units/.mu.g of T4 DNA ligase and incubated overnight at 16.degree.
C. to promote self-ligation of the BPV genome. The religated DNA
was precipitated with ethanol, washed and resuspended overnight in
TE (10 mM TrisHCl pH 8.0, 1 mM EDTA) at 1 .mu.g/.mu.l.
[0141] Generation of baculoviruses expressing BPV late and/or early
genes. Baculoviruses (Summers, M. D., and Smith, G. E., 1987, A
manual of methods for baculovirus vectors and insect cell culture
procedures. Bulletin No. 1555, Texas Agricultural Experiment
Station, College Station, Texas) that expressed BPV L1 alone, L2
alone, or L1 plus L2 (L1+L2) together have been described
(Kirnbauer, R., et al., 1993, J. Virol. 67, 6929-36). Similar
baculoviruses expressing BPV E1 (Blitz, I. L. and Laimins, L. A.,
1991, J. Virol. 65, 649-656) or E2 (Monini, P., et al., 1993, J.
Virol. 67, 5668-5676) were obtained from Elliot Androphy, New
England Medical Center, Boston, Mass.
[0142] Generation of papillomaviruses in insect cells. Prior to
infection, the Sf9 cells were maintained in spinner flasks at
27.degree. C. with Grace's medium containing 10% fetal calf serum
and 0.01% (v/v) pluronic F-68. Cells were harvested by
centrifugation (300.times.g, for 5 min) and resuspended at
10.sup.6,ml in serum-free Grace's medium. 3.times.10.sup.6 Sf9
cells were plated per 60 mm tissue culture dish and allowed to
adhere for 30 min. For each plate, 15 .mu.g of ligated BPV DNA in 1
ml of serum-free Grace's medium was mixed gently with 35 pi of
Lipofectin (Life Technologies) in 1 ml of serum-free Grace's medium
in a polystyrene tube and incubated for 30 min at room temperature.
The medium in the culture dishes was aspirated and replaced with
the DNA/Lipofectin complex in 2 ml of serum free Grace's medium.
The cells were incubated for 4 h at 27.degree. C., the medium
aspirated, and replaced with 2 ml of serum-free Grace's medium
containing 33 .mu.l of the various combinations of L1, L2, E1, and
E2 expressing recombinant baculoviruses (MOI .about.10). After a
one hour infection, the medium was removed and replaced with 5 ml
of Grace's medium containing 10% fetal calf serum, 100 U/ml
penicillin G and 100 .mu.g/ml streptomycin. The cells were
maintained at 27.degree. C. for 72 h in a humidified atmosphere and
harvested by scraping from the plate. The plates were washed once
with PBS to remove all remaining cells, and the cells were then
collected by centrifugation (300.times.g, 5 min). The medium was
aspirated and the cell pellet stored at -80.degree. C.
[0143] In vitro focal transformation assay. To test for the
production of infectious BPV, a standard focal transformation assay
on C127 cells was carried out (Dvoretzky, I., et al., 1980,
Virology 103, 369-375). One milliliter of D-PBS (containing 0.9 mM
calcium and 0.5 mM magnesium) was added to each cell pellet and the
cells were lysed by 15 sec of sonication on ice (microtip, 60%
power, Fisher sonic dismembranater model 150). For neutralization
studies, antibody was added at this stage and incubated for 1 h on
ice. The cell lysates were mixed into 5 ml of medium (DMEM
containing 10% fetal calf serum, 100 U/ml penicillin G, and 100
.mu.g/ml streptomycin maintained at 37.degree. C. in a humidified
5% CO.sub.2/95% air atmosphere) over confluent monolayers of low
passage number mouse C127 clone C cells in 60 mm culture dishes.
After a 1 h incubation at 37.degree. C., the cells were washed once
and then 5 ml of fresh medium was added. The following day the
medium was replaced with DMEM containing 10% fetal calf serum that
was used to maintain the cells for 2-3 weeks, replenishing twice
weekly. The foci were stained with methylene blue (Sigma, M9140)
and carbol fuchsin (Sigma, C-4165) in methanol and scored.
Example 4
[0144] A strategy similar to the one reported here for HPV16 is
used to generate infectious pseudotypes for other papillomaviruses.
The HPV11 genome is produced by isolating a HPV11 DNA clone.
Baculoviruses expressing the E2, L1 and L2 genes of HPV18 are
prepared. Sf9 insect cells are transfected with the HPV11 genome
and infected with the E2, L1 and L2 expressing baculoviruses.
Conditions are provided for generation of HPV18{HPV11} pseudotypes,
and the particles are harvested. To test for the production of
infectious pseudotypes, cultured mammalian cells are identified
that can be infected by HPV18 virions. An in vitro infectivity
assay is then carried out using these cells. Although focal
transformation may not be the endpoint, infection can be identified
by incorporation of a rapidly and easily detectable marker into the
papillomavirus genome. To determine whether transformation is due
to transfection or infection, neutralization studies are conducted.
Neutralizing antibodies are generated against HPV18 virions. The
HPV18{HPV11} pseudotypes are preincubated with these antibodies and
then administered to cells. If the neutralization step blocks
infectivity, then infection and not transfection by the
HPV18{HPV11} virions will have occurred.
Example 5
[0145] Papillomavirus VLPs are attractive candidates for vaccines
against papillomavirus infections because they present
conformational virion surface epitopes but lack the potentially
oncogenic viral genome. Supporting the vaccine potential of VLPs
are the findings that they induce high titers of apparently
type-specific neutralizing antibodies against infectious
papillomaviruses. In addition, vaccination with VLPs stimulated
type-specific, antibody-mediated in vivo protection against
high-dose experimental infection by papillomaviruses. As described
here, in vitro assays have now been developed that directly measure
neutralizing antibodies to high-risk HPVs, e.g., HPV16. This
required the in vitro generation of HPV16 virions and the
development of a quantitative in vitro assay for infectivity. This
assay is used in this protocol, for example, to monitor the
generation of neutralizing antibodies in the development of an
immunoprophylactic vaccine against papillomavirus infection using
papillomavirus VLPs, or in the monitoring of protection for
previously developed immunoprophylactic vaccines since titer of
neutralizing antibodies are the best correlate of protection.
Although the focal transformation assay requires 2 to 3 weeks, this
problem is circumvented by incorporation of a rapid and easily
detectable marker into the papillomavirus genome. Accordingly, a
.beta.-galatosidase expression cassette is substituted for most of
a viral genome, such as the BPV genome, leaving only those
cis-elements, such as the E2 binding sites, required for efficient
encapsidation. In this example, the BPV genomes are encapsidated
with HPV16 L1 and L2 structural proteins to produce
HPV16{BPV1/.beta.-galactosidase} pseudotyped virions. Protection
for immunoprophylactic vaccines against HPV16 infection using HPV16
VLPs is monitored by testing for neutralizing antibodies against
HPV16. A sample is obtained from a vaccinee and mixed with
HPV16{BPV1/.beta.galact- osidase} pseudotypedvirions. Infection of
C127 cells with HPV16{BPV1/.beta.-galactosidase}, where infection
is indicated by a color change, represents a quantitative in vitro
neutralization assay that can be conducted in 3-4 days.
Neutralizing antibodies will block infectivity. The presence of
neutralizing antibodies can be determined by relating the amount of
infectivity measured with the amount of infectivity measured for a
control sample known to be free of neutralizing antibodies. The
concentration of neutralizing antibodies can be established by
relating the amount of infectivity measured with the amount of
infectivity measured for samples containing known amounts of
neutralizing antibodies. The waning of neutralizing antibodies in a
vacinee is used as an indication that a booster inoculation with
the HVP16 VLP vaccine is warranted.
Example 6
[0146] The following is a laboratory protocol for preparing
infectious herpesvirus pseudoviral virions in non-mammalian cells.
The herpesvirus genome is produced by isolating a herpesvirus DNA
clone. An expression cassette is prepared operably encoding a
cloned DNA. The expression cassette is substituted for the viral
genes, while maintaining the packaging signal, to obtain a
herpesvirus vector DNA. Baculoviruses expressing the nonstructural
protein(s) for the packaging the viral genome in the empty capsid,
and expressing the structural proteins of the herpesvirus capsid
are prepared. Sf9 insect cells are transfected with the herpesvirus
vector DNA and infected with the nonstructural and structural
protein-expressing baculoviruses. Conditions are provided for
generation of herpesvirus pseudotypes, and the virions are
harvested. To test for the production of infectious virions,
cultured mammalian cells are identified that can be infected by
wild type virions. An in vitro infectivity assay is then carried
out using these cells. Infection is identified by testing for the
production of the protein encoded by the cloned DNA. To determine
whether transformation is due to transfection or infection,
neutralization studies are conducted. Neutralizing antibodies are
generated against wild type virions. The herpesvirus pseudotypes
are preincubated with these antibodies and then administered to
cells. If the neutralization step blocks infectivity, then
infection and not transfection by the pseudotyped virions will have
occurred. The infectious herpesvirus pseudoviral virions are used
to transfer the cloned DNA into mammalian cells of the central
nervous system.
Example 7
[0147] The following is a clinical protocol for treatment of
wrinkles of the face. The HPV1 genome is produced by isolating a
HPV1 DNA clone. An expression cassette is prepared operably
encoding proelastin, the precursor for elastin, a molecule found in
the connective tissue of the skin. The expression cassette is
substituted for the viral genes, while maintaining the E2BS, to
obtain a HPV1 vector DNA. Baculoviruses expressing the E2, L1 and
L2 genes of HPV1 are prepared. Sf9 insect cells are transfected
with the HPV1 vector DNA and infected with the E2, L1 and L2
expressing baculoviruses. Conditions are provided for generation of
HPV1{HPV1/elastin} pseudotypes, and the particles are harvested. To
test for the production of infectious particles, cultured mammalian
cells are identified that can be infected by HPV1 virions. An in
vitro infectivity assay is then carried out using these cells.
Infection is identified by testing for the production of elastin.
To determine whether transformation is due to transfection or
infection, neutralization studies are conducted. Neutralizing
antibodies are generated against HPV1 virions. The HPV1 pseudotypes
are preincubated with these antibodies and then administered to
cells. If the neutralization step blocks infectivity, then
infection and not transfection by the HPV1{HPV1/elastin} virions
will have occurred. Infectious HVP1{HPV1/elastin} particles are
formulated in a cream. The cream is applied topically to the face
of a patient. The particular dose of virus is selected based on
clinical trials in which increasing the concentration does not
appreciably increase the efficiency of gene transfer and decreasing
the concentration results in the efficiency of gene transfer being
significantly decreased. About 10 days following the administration
of virus, the patient is evaluated for reduction of wrinkles to the
face, and the cream is reapplied for another treatment on an
as-needed basis.
[0148] While particular embodiments of the invention have been
described in detail, it will be apparent to those skilled in the
art that these embodiments are exemplary, rather than limiting. The
true scope of the invention is that defined within the attached
claims and equivalents thereof. All references cited herein are
hereby expressly incorporated by reference.
Sequence CWU 1
1
12 1 52 DNA Artificial Sequence PCR Primer 1 ccgctggatc ccactattat
atagcaccat ggcgttgtgg caacaaggcc ag 52 2 21 DNA Artificial Sequence
PCR Primer 2 cagttgagac tagagagcca c 21 3 21 DNA Artificial
Sequence PCR Primer 3 gtggctctct agtctcaact g 21 4 55 DNA
Artificial Sequence PCR Primer 4 gcggtggatc cttatttttt tttttttttt
gcaggcttac tggaagtttt ttggc 55 5 47 DNA Artificial Sequence PCR
Primer 5 gcggtagatc taatatgagt gcacgaaaaa gagtaaaacg tgccagt 47 6
47 DNA Artificial Sequence PCR Primer 6 ccgctagatc tagggagata
cagcttctgg ccttgttgcc acaacgc 47 7 37 DNA Artificial Sequence PCR
Primer 7 ccgctggatc cgcaccatgg caaacgataa aggtagc 37 8 32 DNA
Artificial Sequence PCR Primer 8 gcggtggatc cgatcttgca acttatcact
ac 32 9 36 DNA Artificial Sequence PCR Primer 9 ccgctggatc
cgcaccatgg agacagcatg cgaacg 36 10 32 DNA Artificial Sequence PCR
Primer 10 gcggtggatc cgaagaaaag gcaatggcag tg 32 11 30 DNA
Artificial Sequence PCR Primer 11 cggcaagctt gcaatgtgct gtgtcagttg
30 12 30 DNA Artificial Sequence PCR Primer 12 cgcgaagctt
aacggtgatg gtgtgattat 30
* * * * *