Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CHIMERIC PAPILLOMAVIRUS-LIKE PARTICLES
FIELD OF THE INVENTION
The present invention relates to chimeric papillomavirus-like particles and
related synthetic DNA molecules,
host cells, methods and vaccines.
BACKGROUND OF THE INVENTION
Papillomaviruses inflect the epithelia of humans and a wide variety of
animals, where they generally induce
benign proliferation at the site of infection. However, in some cases the
lesions induced by certain papillomaviruses
undergo malignant progression. There is a strong association between malignant
progression of human genital lesions
and certain human papillomavirus (HPV) types, such as HPV16. Infection by one
of these types is considered the
most significant risk factor in the development of cervical cancer, one of the
most common cancers of women
worldwide (zur Hausen, H., Science 254:1167 (1991); Schiffman, M.H., J. Natl.
Cancer Inst. 84:394 (1992)). The
majority of cervical carcinomas contain and express HPV early genes, such as
E6 and E7, and these genes have been
shown to have potent transforming and immortalizing activity in cultured cells
(Werness, B.A., Munger, K. & Howley,
P.I~. (1991) Advances in Oncology, eds. Dellita V-T., Hellman, S. & Rosenberg,
S.A. (Lipponcott, Philadelphia) pp.3-
18).
Papillomaviruses are non-enveloped double-stranded DNA viruses about 55 nm in
diameter with an
approximately 8 kb genome in the nucleohistone core (faker, et al., Biophys J
60:1445 (19911). The capsids are
composed of two virally-encoded proteins, L1 and L2, that migrate on SDS-PAGE
gels at approximately 55 kDa and
75 kDa, respectively (Mose Larson et al., J. Virol. 61:3596 (1987)). L1, which
is the major capsid protein, is
arranged in 72 pentameters which associate with T-7 icosahedral symmetry. The
function and position within the
virion of L2 are unclear (Baker, et al., Biophys J 60:1445 (1991)).
The L1 protein has the capacity to self-assemble so that large amounts of
virus-like particles (VLPs) may
be generated by expression of the L1 protein from a number of species of
papillomavirus in a variety of recombinant
expression systems (Hagensee et al., J Virol 67:315 (1993); Kirnbauer et al.,
Proc Natl Acad Sci USA 89:12180
(1992); Kirnbauer et al., J Virol 67:6929 (1993); Rose et al., J Viroi 67:1936
(1993)). Although not required for
assembly, L2 is incorporated into VLPs when co-expressed with L1 (L1/L2 VLPs)
in cells.
Immunization of rabbits with native virions or L1 VLPs, but not with non-
assembled L1 expressed in E. cvli,
induces high titers of neutralizing serum antibodies (Christensen, N.D. and
Kreider, J.W., J Virol 64:3151 (1990);
Kirnbauer et al., Proc Natl Acad Sci USA 89:12180 (19921; Pilacinski et al.,
Bioffechnology 2:356 (1984); Segre
et al., Am. J. Vet. Res. 16:517 (19551). The polyclonal and monoclonal
antibodies generated against native particles
recognize conformationally dependent epitopes (Christensen, N.D. and Kreider,
J.W., Virus Res 28:195 (1993);
Christensen et al., J Virol 64:5678 (1990); Christensen et al., Virology
181:572 (1991)).
Neutralizing antibodies generated against VLPs also recognize conformationally
dependent epitopes. Using
infectious BPV1, which can be readily obtained from bovine lesions, and a
quantitative in vitro BPV1 infectivity assay
(Ovoretzky et al., Virology 103:369 (1980)), workers showed VLPs from bovine
papillomavirus induced high levels
of neutralizing antibodies (Kirnbauer et al., Proc Natl Acad Sci USA 89:12180
(1992)). The neutralizing antibodies
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were directed against conformationally dependent epitopes, in that
denaturation of the
particles prior to immunization abolished the ability of the preparation to
induce
neutralizing activity (id. ).
When the L1 gene of a HPV16 isolate derived from a nonprogressed lesion was
used to express the L1 major capsid protein in insect cells via recombinant
baculoviruses,
L1 self assembled into VLPs at a yield 3 orders of magnitude higher than what
had been
obtained using L1 derived from the prototype HPV16 (originally isolated from a
cancerous
lesion), and formed VLPs that were morphologically similar to native virions
(Kirnbauer et al., J Virol 67:6929 (1993)). DNA sequence comparison identified
a single
nonconserved amino acid change to be responsible for the inefficient self
assembly of the
prototype L1 (id.). The L1 of the assembly-competent clone is thus considered
to be the
wild-type gene, and the prototype L1 of the assembly-defective clone a mutant.
Using HPV16 VLPs of the wild-type L1 protein as antigens, an ELISA was
developed that detected serum antibodies in patients infected with HPV 16
(Kirnbauer et al. ,
J. Natl. Cancer Inst. 86:494 (1994)). In contrast, neither denatured HPV16
particles nor
preparations of the prototype Ll protein could detect these antibodies (id.).
These results
demonstrate that the prototype L1 protein does not present conformational
epitopes.
2o Rabbit serum raised against self-assembled wild-type HPV16 L1/L2 virus-like
particles was discovered to prevent HPV 16 VLP binding to cell surface
molecules
(Roden et al., J. Virol., 68:7260-7266 (1994)). In contrast, serum raised
against the
prototype strain of HPV16 L1/L2 did not prevent such binding (id.). The data
show that
the prototype HPV16 strain lacks conformational epitopes.
2 5 Rabbits immunized with intact cottontail rabbit papillomavirus (CRPV)
virus-like
particles composed of L1 or L1/L2 were protected from subsequent experimental
challenge
by infectious CRPV (Breitburd et al., J. Virol. 69:3959-3963 (June, 1995). In
contrast,
those immunized with denatured particles were not protected (id. ). These
findings are
consistent with the conclusion that VLPs presenting conformational epitopes
are able to
3 o induce protective immunity.
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VLPs composed of capsid proteins are attractive candidates for prophylactic
vaccines to prevent papillomavirus infection. However, it is unlikely that
these VLP
vaccines would have therapeutic effects against established papillomavirus
infections. The
capsid proteins, unlike E6 and E7, are not detectably expressed in progressed
lesions or in
infected basal epithelial cells, which are the presumed targets in immune
regression of
papillomas.
There is evidence from experimental models that immunity against
papillomavirus
1 o proteins other than L1 and L2 might help control papillomavirus infection.
Since E6 and
E7 are selectively maintained during oncogenic progression, there is the
possibility that
peptides derived from these oncoproteins could serve as targets for cell-
mediated immune
responses to HPV-containing tumor cells. Studies in animal models suggest the
E7 protein
of HPV16 acts as a tumor rejection antigen (Chen et al., Proc. Natl. Acad.
Sci. USA
88:110 (1991); Feltkemp et al., Eur. J. Immunol. 23:2242 (1993)). Moreover,
the
frequency of HPV infection, persistence of HPV infection, and risk of
developing cervical
cancer and other HPV-related cancers is increased in patients with depressed
cellular
immunity (Allout et al., Br. Med. J. 298:153 (1989); Laga et al., Int. J.
Cancer 50:45
(1992)). These observations suggest
WO 96!11274 PCT/US95/12914
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cell-mediated immunity is important in the defense against HPV infection and
its associated tumor development. The
induction of such immunity might be therapeutic, as well as prophylactic.
It has been demonstrated that foreign peptides can be incorporated into viral
capsid-like structures and these
chimeric particles can be used to present foreign antigens to the immune
system. Published examples include
hepatitis B core antigen particle presentation of human rhinovirus type 2
epitopes (Francis et al., Proc. Natl. Acad.
Sci. USA 87:2545 (1990)), gp41 of HIV (Borisova et al., FEBS Lett. 259:121
(1989)), and B19 parvovirus particle
presentation of peptides from herpes simplex virus 1 and murine hepatitis
virus (Brown et al., Virology 198:477
(1994)). The parvovirus chimeras protected mice from experimental challenge
with the corresponding virus. In all
of the above systems, foreign sequences have been inserted in proteins
integral to the capsid structure and have been
limited to less than 20 amino acids. However a recent study (Miyamura et al.,
Proc. Natl. Acad. Sci. USA 91:8507
(1994)) has demonstrated that the entire 147 as hen egg white lysozyme protein
can be incorporated into B19
parvovirus particles when fused to the parvovirus L1 minor capsid protein. The
lysozyme remained biologically active
and elicited an immune response when injected into rabbits. In perhaps less
relevant studies, hepatitis B virus
surface antigen particles (which are lipid membrane structures) containing 84
as of HIV-1 envelope glycoprotein
(Michel et al., J. Virol. 64:2452 (1990)) and yeast Ty virus-like particles
containing a portion of HIV-1 V3 loop
(Griffiths et al., J. Virol. 65:450 (1991)) have also been shown to produce an
immune response to the inserted
peptides when inoculated into animals. With respect to papillomaviruses, it
was recently reported that hepatitis B
core antigen particles containing HPV16 E7 peptides (all less than 20 aa)
induced peptide specific antibodies and T-
helper responses in mice (Tindle et al., Virology 200:547 (19941).
Chimeric particles based on self-assembled papillomavirus L1 have not been
reported, nor has the use of
L2 as a viral fusion partner for purposes of generating chimeric VLPs been
described. The chimeric particle studies
cited above involve viruses that are unrelated to papillomaviruses and thus
cannot predict the results of chimeric
particle studies involving papillomaviruses. Indeed, in the papillomavirus
study by Kirnbauer et al., J Virol 67:6929
(1993), supra, it was demonstrated that a single nonconserved amino acid
change in L1 is responsible for efficient
self-assembly of L1 into VLPs and the presentation of conformational epitopes,
which seem to be required for
induction and detection of clinically relevant immune reactivity. Thus, the
studies using viruses unrelated to
papillomavirus cannot predict whether a papillomavirus L2 containing a foreign
peptide or protein can co-assemble
with papillomavirus L1 into particles, given that a single amino acid
substitution in L1 can abolish efficient self-
assembly. Neither can these studies predict whether any resulting chimeric
particles will retain the ability to induce
or detect neutrafaing antibodies or other immune related responses, given that
a single amino acid substitution in
L1 can bar the presentation of conformational ep-ttopes.
It is an object of the present invention to provide chimeric papillomavirus-
like particles. These chimeric
particles may function as platforms for multivalent antigen presentation. Or
they may serve for delivery into cells
of proteins for processing into peptides and subsequent presentation of these
peptides within the context of MHC
molecules to elicit a cell-mediated immune response. The chimeric particles
represent a cost effective way to
generate an effective papillomavirus vaccine with a broad spectrum of utility.
Alternatively, the chimeric
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papillomavirus-like particles may be applied to VLP and/or
fusion partner purification. Or, the particles may operate
to deliver into cells intact and active proteins, for
example, enzymes, or toxins or drugs.
SUMMARY OF INVENTION
This invention provides a papillomavirus-like particle
having conformational epitopes comprising a papillomavirus
L1 fusion product. This invention also provides a
composition comprising the aforementioned
papillomavirus-like particle.
This invention also provides a composition comprising
a first synthetic DNA molecule characterized as encoding a
papillomavirus L1 product and a second synthetic DNA
molecule characterized as encoding a papillomavirus L2
fusion product, wherein said molecules are capable of
directing expression in a transformed cell of
papillomavirus-like particles characterized as having
conformational epitopes, wherein said particles comprise
said papillomavirus L1 product and papillomavirus L2 fusion
product.
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According to one aspect of the invention, there is provided a papillomavirus-
like
particle, characterized as having conformational epitopes, comprising a
papillomavirus L1
product and a papillomavirus L2 fusion product.
The papillomavirus L2 fusion product may be characterized as being a human
papillomavirus L2 fusion product or a bovine papillomavirus L2 fusion product.
The human papillomavirus L2 fusion product may be characterized as being a
HPV 16 L2 fusion product and the bovine papillomavirus L2 fusion product may
be
1 o characterized as being a BPV 1 L2 fusion product.
The papillomavirus L2 fusion product may comprise a fusion partner
characterized
as being a peptide or a full-length protein, or may comprise fusion partners
that include
peptides or full-length proteins or combinations thereof linked in tandem.
The fusion partner may be a papillomavirus E6 or E7 product.
The papillomavirus L2 fusion product may be characterized as being fused at
its 5'
or 3' end to a fusion partner.
The papillomavirus L2 fusion product may be characterized as having a fusion
partner inserted between L2 amino acids.
The papillomavirus L1 product may be characterized as being a human
2 o papillomavirus L1 product or a bovine papillomavirus L1 product.
The human papillomavirus L1 product may be characterized as being a HPV16 L1
product and the bovine papillomavirus L1 product may be characterized as being
a
BPV1 L1 product.
The papillomavirus-like particle may consist essentially of HPV16L1 and
2 5 HPV 16L2-HPV 16E7 (full-length), where HPV 16E7 is fused to the 3' end of
HPV 16L2 ,
BPVL1 and BPVL2-HPV 16E7 (full-length), where HPV 16E7 is fused to the 3' end
of
BPVL2, or BPVLl and BPVL2-HPV16E7 (amino acids 1-30), where HPV16E7 is fused
to
BPVL2 between L2 amino acids 274 and 275.
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According to another aspect of the invention, there is provided one or more
synthetic DNA molecule or molecules characterized as singly or doubly encoding
a
papillomavirus L1 product and a papillomavirus L2 fusion product where the
molecule or
molecules direct expression in a transformed host cell of a papillomavirus-
like particle,
characterized as having conformational epitopes, comprising the papillomavirus
L1 product
and the papillomavirus L2 fusion product.
The transformed host cell may be an insect host cell (such as Sf9 insect host
cell)
1 o and the DNA molecule or molecules may further comprise an insect cell
vector (such as a
baculovirus vector), or the transformed host cell may be a mammalian host cell
and the
DNA molecule or molecules may further comprise a mammalian cell vector (such
as a
vaccinia virus vector), or the transformed host cell may be a yeast host cell
and the DNA
molecule or molecules may further comprise a yeast cell vector.
WO 96/11274 -5- ~~ ~ ~ ~ ~ ~ PCTIUS95/12914
According to another aspect of the invention, there is provided a host cell
transformed with the DNA
molecule or molecules of above.
According to yet another aspect of the invention, there is provided a method
for using the DNA molecule
or molecules of above comprising the steps of: providing conditions for the
molecule or molecules of above to direct
the above expression; and recovering the papillomavirus-like particle from the
above transformed host cell.
According to still another aspect of the invention, there is provided a method
for producing a papillomavirus-
like particle, characteraed as having conformational epitopes, comprising a
papillomavirus L1 product and a
papillomavirus L2 fusion product, which method comprises the step of providing
conditions for the DNA molecule or
molecules of above to direct the above expression in the above transformed
host cell of the papillomavirus-like
particle.
The invention also provides a method of purification of the papillomavirus-
like particle of above comprising
the step of exposing the papillomavirus-like particle to an affinity
chromatography column, comprising antibodies that
bind to a fusion partner of the papillomavirus L2 fusion product of the
papillomavirus-like particle, resulting in the
purification of the particle.
The invention further provides a method of purification of a fusion partner of
the papillomavirus L2 fusion
product of the papillomavirus-like particle of above comprising the step of
isolating the papillomavirus-like particle
of above resulting in the purification of the fusion partner.
The invention additionally provides a method of delivery into a cell of a
fusion partner of the papillomavirus
L2 fusion product of the papillomavirus-like particle of above comprising the
step of administering the papillomavirus-
like particle of above to the cell resulting in the delivery into the cell of
the fusion partner.
The invention moreover provides a vaccine comprising the papillomavirus-like
particle of above.
DETAILED DESCRIPTION OF THE INVENTION
This invention arises from the result that a papillomavirus L2 fusion product
can become incorporated into
a papillomavirus L1 product-based papillomavirus-like particle that presents
conformational epitopes.
This result was unexpected.
In the papillomavirus study by Kirnbauer et al., J Virol 67:6929 (1993),
supra, it was demonstrated that
a single nonconserved amino acid change in L1 is responsible for efficient
self-assembly of L1 into VLPs and the
presentation of conformational epitopes, which seem to be required for
induction and detection of clinically relevant
immune reactivity. Thus, it could not be predicted whether a papillomavirus L2
containing a foreign peptide or protein
could co-assemble with papillomavirus L1 into particles, given that a single
amino acid substitution in L1 can abolish
efficient self-assembly. NeE~her could it be predicted whether any resulting
chimeric particles would retain the ability
to induce or detect neutralaing antibodies or other immune related responses,
given that a single amino acid
substitution in L1 can bar the presentation of conformational epitopes.
A papillomavirus L2 fusion product is meant to include a chain of amino acids
in which part of the chain
comes from a L2 protein sequence and part of the chain comes from another
protein sequence (or other protein
sequences).
2J,6r
u,
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L2 fusion products are produced by splicing (in frame) an open reading frame
for one protein (or a number
of proteins) next to or into an open reading frame for L2.
Protein engineering is used to determine the structure of the L2 fusion
product. In a routine exercise for
protein engineers, they generate variants of the natural protein L2. The
changes they make can be educated guesses
based on detailed knowledge of the structure of L2; alternatively, changes can
be made on a purely random basis.
Or a combination of structural information with random mutagenesis and
selection can have dramatic results.
For example, the selection of L2 amino acids between which to insert a fusion
peptide or protein can be
made on this basis. The existence of a region where amino acid sequence and
length vary between papillomaviruses
suggests that this region represents a structure that is nonessential for the
integrity of L2 andlor its incorporation
into particles. The observed ability to insert a fusion peptide or protein
within L2 implies that such a region exists.
Determination of a structure for the l2 fusion product is performed on the
basis of the ability of the L2
fusion product to become incorporated into a papillomavirus L1 product-based
papillomavirus-like particle that presents
conformational epitopes.
Preferred means of determining a structure include assays that measure the
efficiency andlor authenticity
of incorporation of an L2 fusion product into a papillomavirus L1 product-
based papillomavirus-like particle.
The efficiency andlor authenticity of formation of L1lL2 VLPs is related to
the presentation of
conformational epitopes (supral.
The chimeric L2 will thus become incorporated into L1-based VLPs with an
efficiency andlor authenticity
similar to that of incorporation of wild-type L2.
Other preferred means of determining a structure far the L2 fusion product
include assays that measure
the induction andlor detection of neutralizing antibodies.
The induction andlor detection of neutralizing antibodies is related to the
presentation of conformational
epitopes, because neutralizing antibodies are directed against
conformationally dependent epitopes (supra).
The incorporation of the chimeric L2 will thus not inhibit the induction
andlor detection of neutralizing
antibodies by chimeric papillomavirus-like particles as compared to L1 or
L11L2 VLPs.
For purposes of the invention, a chimeric L2 fusion product is capable of
becoming incorporated into a
papillomavirus L1 product-based papillomavirus-like particle that presents
conformational epitopes, whether identified
by the above means or any other means known in the art.
The structure of l2 and -'tts fusion partner (and L1) is meant to include a
full-length L2 protein (and L1
protein) and a full-length fusion partner protein, and their peptide
fragments, whether 5' fragments, 3' fragments,
or internal fragments, having at least about 20 amino acid residues,
advantageously at least about 10 amino acid
residues, and preferably at least about 5 amino acid residues. Type, subgroup
and strain variations of L2 (and L1),
and human allelic and species variations of the fusion partner protein, are
expressly contemplated as falling within
the scope of the invention. The invention also includes conservative variants
of the full-length L2 protein (and L1
proteiN and the full-length fusion partner protein, and their peptide
fragments, where conservative amino acids are
substituted for amino acid residues of wild-type L2 (and L1) and the fusion
partner protein. To account for
WO 96!11274 , ~j ~~ ~ PCT/US95l12914
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degeneracy of the genetic code, the invention also includes DNA coding for the
same amino acid residues as does
the DNA of the L2 (and L1) and fusion partner gene.
The chimeric papillomavirus-like particle itself is envisioned as
incorporating any L2 fusion product with any
L1 product from any papillomavirus, whether the genomes are closely related,
or are distantly related, so long as
incorporation into particles occurs. Thus, a L2 fusion product, for example,
related to any of BPV-1, BPV-2, BPV-4,
' CRPV, DPV, EEPV, HPV-1, HPV-5, HPV-6, HPV-8, HPV-11, HPV-16, HPV-18, HPV-31
or HPV-33, can be incorporated
into particles with any L1 product, for example, from any of the above virus,
or any type, subgroup or strain
variation of papillomavirus.
Because VLPs present conformationally dependent epitopes required for the
induction of high titer
neutralizing serum antibodies, VLP chimeras containing L2 fusion products can
operate prophylactically as optimized
subunit vaccines for the stimulation of humoral immunity to prevent
papillomavirus infection and thereby preclude
the development of papillomavirus associated cancers and other papillomavirus
associated pathologies.
Because it is unlikely VLPs will prove effective as therapeutic vaccines to
induce regression of existing
papillomavirus proliferative lesions, as discussed above, chimeric VLPs can
function to address this long-felt and
heretofore unsatisfied need to develop a therapeutic vaccine.
Chimeric VLPs are expected to bind specific cell surface receptors, get
internalized and be released into the
cytoplasm, and thus be more likely to promote the presentation of peptides in
conjunction with class I MHC
molecules for display to cytotoxic T cells for the generation of cell-mediated
immunity. This is in contrast to
uncomplexed proteins that would not be expected to specifically enter cells,
or to promote the presentation of
peptides in the context of Class I MHC molecules to elicit a cytotoxic T cell
response (being more likely, if at all,
to promote the presentation of peptides to be linked to Class II MHC molecules
and displayed to helper T cells).
Inclusion of one or more fusion partners as L2 fusion products would clearly
be a cost effective way to
generate an effective papillomavirus vaccine with a broad spectrum of utility,
including therapy and improved
prevention of clinical lesions.
The fusion partner may be selected from the list consisting of those fusion
partners that would provide a
method for expanding the potential targets of a VLP-based vaccine, for
example, E6 or E7 peptide or full-length E6
or E7, other papillomavirus peptides or proteins, or peptides or proteins of
other STD or infectious agents, eg.,
Herpes simplex, HIV, Chlamydia trachomatis, Neisseria gonorrhoeae, and
Treponema pallidum.
The L2 fusion product is not limited to a single fusion partner per L2
molecule, and may include additional
fusion partners, for example, additional peptides or full-length proteins or
combinations thereof derived from the same
or different proteins linked in tandem. Also, more than one L2 fusion product
may be co-assembled into a single VLP.
For example, a L2 fusion product or chimeric VLP containing a viral target
epitope may also be engineered to contain
a binding domain of a co-stimulatory protein or an accessory receptor or
ligand involved in immune reactivity, eg.,
B7 (which interacts w'tth CD28 on T cellsl, an intercellular adhesion molecule
(ICAM), a lymphocyte functional antigen
(LFA), a vascular cell adhesion molecule (VCAM-1), and a heat stable antigen
(HSA). (To target the co-stimulators
or ligands to the surface of VLPs, see Example 14.)
~)
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It will be appreciated that the actual preferred amounts of chimeric
papillomavirus-like particles as vaccines
in the prevention andlor treatment of disease will vary according to the
specific 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, eg., by means of an appropriate, conventional
vaccination protocol. ,
Alternatively, chimeric VLPs can used in a method of purification of VLPs.
VLPs are useful pei se as
prophylactic vaccines and in immunodiagnostics (supra). Briefly, antibodies to
the fusion partner are generated using o
standard immunological techniques, an affinity chromatography column is
constructed using the antibodies, and the
VLPs are subsequently purified in an affinity chromatography step.
Or, chimeric VLPs can be used in a method of purification of a fusion partner.
For example, VLPs containing
a L2-E7 fusion product may be useful as a means of obtaining correctly folded
E7. It has been reported that a
greater percentage of cervical cancer patients are seropositive for
conformationally correct E7 than for denatured
E7, as isolated from bacteria (Viscidi et al., Int. J. Cancer 55:780 (1993)).
The in vitro transcription-translation
system used to generate E7 in this report is laborious and expensive, and uses
radio-labeled E7. It would be
advantageous to purify chimeric E7 VLPs and use the material in an E7 ELISA.
To avoid complications of
seroreactivity with human L1 or L2, BPV-based particles could be used.
Monitoring E7 seroreactivity, which
correlates with cancer as opposed to premalignant disease, has been proposed
to be useful to follow the course of
disease in cervical cancer patients and to screen for reoccurrences pdl.
Optionally, chimeric VLPs can be used in a method of delivery of an intact and
active protein into cells.
This protein may be, for example, an enryme, or a toxin or a drug. The
chimeric VLPs are administered to the cells,
either in vitro, in vivo, in situ, or ex vivo, and the protein is subsequently
delivered into the cell where it functions
for its intended purpose, for example, as an enzyme, or a toxin or a drug.
We have succeeded at generating chimeric papillomavirus-like paPticles
incorporating E7 protein or peptide
fused to L2.
A papillomavirus E7 gene when fused in frame to the 3' end of the L2 gene or
within part of the L2 open
reading frame expressed an L2-E7 fusion protein that, in the presence of Lt
protein, was incorporated into virus-like
particles. Efficiency and authenticity of incorporation of LZ-E7 into VLPs was
similar to that of the wild-type L2
protein. BPV L11L2-E7 virus-like particles were found to induce neutralizing
antibodies as effectively as BPV L11L2
particles. Chimeric particles were observed to elicit humoral immunity
specific for fusion partner epitopes in that
rabbits immunized with L2-E7 chimeric particles generated antibodies directed
against E7.
Having succeeded at generating chimeric papillomavirus-like particles opens
the door to fusing virtually any
protein or peptide to an L2 product for incorporation into chimeric
papillomavirus-like particles. '
We accomplished the production of chimeric VLPs by generating L2-E7 fusion
proteins and expressing these
proteins along with L1 via recombinant baculoviruses in insect cells. We
generated three chimeric VLPs: HPV16L1
+ HPV16L2-HPV16E7 (full-length), BPVL1 + BPVL2-HPV16E7 (full-length) and BPVL1
+ BPVL2-HPV16E7 (aa 1-30).
~2~ 160 ~
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BPV L2 was fused at its 3' end to HPY16 E7. HPY16 L2 was fused at its 3' end
to HPV16 E7.
Additionally, the first 30 codons of HPV16 E7 were inserted between codons 274
and 275 of BPV L2.
Sucrose gradient co-sedimentation and co-immunoprecipitation of L1 and the L2-
E7 chimeras demonstrated
the incorporation of the chimeric protein into the YLPs.
Transmission electron microscopy revealed that particles containing the l2-E7
fusion protein assembled with
- the same efficiency as L1 or L11L2 VLPs, and were morphologically
indistinguishable.
In vitro BPV1 neutralization assays demonstrated that the BPY L1-containing
chimeric VLPs were capable
of inducing neutralizing antisera. Titers were comparable to those obtained
using BPV L11L2 VLPs. Equivalent
neutralizing titers of 30,000 were obtained for both BPYL11L2 YLPs and
BPYL11L2-HPV16E7 (full-length) chimeric
VLPs.
Rabbits immunized with L2-E7 chimeric particles generated antibodies directed
against E7, demonstrating
the induction by chimeric VLPs of humoral immunity specific for fusion partner
epitopes, and also establishing the
location of E7 epitopes as external to VLPs.
As set forth in Example 1, chimeric recombinant baculoviruses can be
generated. Genes for the chimeras
may be obtained from genomic sources or cDNAs, by direct synthesis, or by any
combination thereof. L2 and its
fusion partner genes can be amplified by recombinant PCR techniques, for
example, with oligos containing restriction
enzyme sites and complementarities that facilitate fusion and cloning into
expression, transfer andlor cloning vectors,
eg., plasmids.
Fused genes can be cloned into a baculovirus expression vector. Another
baculovirus expression vector
containing L1 can be generated. Or the fused genes can be cloned into a
baculovirus double expression vector that
already contains L1.
Example 2 sets forth a typical procedure for selection of recombinant
baculoviruses. CsCI-purified (or
equivalent) recombinant plasmid can be cotransfected with baculovirus DNA into
Sf9 insect cells by using Lipofectin
(or equivalent). The recombinant baculoviruses can then be plaque-purified
(for example) using conventional
baculovirus vector and insect cell culture procedures.
It may be advantageous to use two single expression baculovirus vectors
instead of a double expression
baculovirus vector. In this case, chimeric VLPs can be produced by infecting
Sf9 cells with two recombinant
baculoviruses, one encoding a L1 product and the other encoding the L2 fusion
product. Locating the genes on
different vectors facilitates manipulation of the amount of L1 product and L2
fusion product produced. This approach
permits one to change the ratio of L2 fusion product to Lt product in a VLP.
Although in native virions, L2 is the
minor component as compared to L1, we can achieve greater incorporation of L2
fusion product into VLPs using two
single expression vectors.
As set forth in Example 3, chimeric particles can be purified by banding in
cesium chloride (or equivalent).
Sf9 insect cells can be infected, for example, at a multiplicity of infection
of 10 w-'tth recombinant baculoviruses.
After 72 hours (or so), cells can be harvested and sonicated in a phosphate-
buffered saline (or equivalent) for 60 sec
(or sol. After low speed clarification (or equivalent), the lysates can be
subjected to centrifugation, for exa~r:f7ie, at
WO 96/11274 ~ ~ ~ PCT/US95/12914
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110,000 x g for 2.5 h through a 40% (wtlvol) sucroseIPBS cushion (SW-28
rotor). The resuspended pellets can be
centrifuged to equilibrium, for example, at 141,000 x g for 20 h at room
temperature in a 10-40% (wtlwt) CsCIIPBS
gradient. The visible band can be harvested, centrifuged to equilibrium again
by using the identical conditions,
dialyzed extensively against PBS, and stored at 4°C (for example).
As set forth in Example 4, co-sedimentation of chimeric complexes can be
established, for example, by
analytical gradient centrifugation. E.g., a 12 to 45% sucrose step gradient
can be allowed to linearize overnight at
4°C, dialyzed samples can be layered on top, and the gradient can be
centrifuged at 41,000 rpm (288,000 x g) for
2 h in a SW-41 rotor. Fractions can be harvested and analyzed for co-
sedimentation, for example, by Western
blotting or co-immunoprecipitation.
As set forth in Example 5, co-sedimentation can be established, for example,
by co-immunoprecipitation.
As set forth in Example 6, antisera can be produced. This can be done to
conduct a BPV1 neutralization
assay (or equivalent) as is described in Dvoretsky et al., Virology 103:369
(1980). E.g., antisera can be produced
as follows. Rabbits can be immunized by subcutaneous injection of 330,u1 of
CsCI gradient-purified particles in PBS
at a concentration of 1 mglml. Rabbits can then be boosted with the same
amount of particles two weeks and four
weeks after the primary injection.
As set forth in Example 7, a BPV1 neutralization assay (or equivalent) can be
performed to test whether
BPV chimeric particles present conformational epitopes. E.g., serial dilutions
of sera obtained 3 weeks after the
second booster injection can be incubated with w 500 focus-forming units of
BPV1 virus for 30 min, the virus can
be adsorbed to C127 cells for 1 h, and the cells can be cultured for 3 weeks.
Foci can then be stained with 0.5%
methylene blue10.25% carbol fuchsin in methanol. Neutralizing titers can be
obtained for chimeric VLPs and control
BPVL1-L2 VLPs. Equivalent neutralizing titers will indicate proper folding of
the chimeric particles effective to present
conformational epitopes.
As set forth in Example 8, chimeric particles can be assayed, for example, by
transmission electron
microscopy. E.g., purified particles can be adsorbed to carbon-coated grids,
stained with 1 % uranyl acetate, and
examined with a Philips electron microscope model EM 400T at x36,000
magnification. Efficiency of incorporation
and morphology of chimeric particles and Lt or LltL2 VLPs can be compared and
contrasted. Indistinguishable
efficiency of incorporation and morphology will indicate proper self-assembly
of the chimeric particles.
As set forth in Example 9, chimeric particles can be assayed, for example, for
induction of humoral immunity
specific for fusion partner epitopes. E.g., rabbits can be inoculated with
chimeric VLPs. The sera can be analyzed
for antibodies, for example, by subjecting a sample of the fusion partner
antigen to SDS-PAGE and then Western
blotting. Immune and preimmune (control) sera can be applied at an appropriate
dilution. Detection of the fusion
partner band in the Western blot by serum from the immune rabbit indicates the
induction of antibodies specific for
fusion partner epitopes.
As set forth in Example i0, chimeric particles can be assayed, for example,
for induction of cell-mediated
immunity specific for fusion peptides, for instance, by injecting chimeric
VLPs into mice, and measuring antigen-
specific T cell proliferation.
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As set forth in Example 11, chimeric particles can be assayed, for example,
for induction of prophylactic
immunity against challenge infection, for instance, by immunizing rabbits with
chimeric CRPV VLPs and nonchimeric
CRPV VLPs (control), and subsequently challenging with infectious CRPV, to
demonstrate that L2 fusions do not
abrogate prophylactic immunity; or by immunizing experimental animals with
chimeric VLPs containing an STD agent,
subsequently challenging with the STD agent, and measuring increased survival
against lethal challenge or decreased
infection following sub-lethal challenge.
As set forth in Example 12, chimeric particles can be assayed, for example,
for induction of therapeutic
immunity against pre-existing papillomas, for instance, by immunizing rabbits
(that have pre-existing papillomas) using
chimeric CRPV VLPs and nonchimeric CRPV VLPs (control), and measuring
regression of the pre-existing papillomas.
As set forth in Example 13, chimeric particles can be assayed, for example,
for immunotherapy and
immunoprevention of tumors.
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
Generation of chimeric recombinant baculoviruses
Three chimeras were generated: HPV16L2-HPV16E7, BPVL2-HPV16E7, and BPVL2-
HPV16E7 (aa 1-30).
HPV16L2-HPV16E7 contained the full-length HPV16E7 fused to the C-terminus (aa
473) of HPV16L2. BPVL2-
HPVi6E7 contained the full-length HPV16E7 fused to the C-terminus of BPVL2 (aa
469). BPVL2-HPV16E7 (aa 1-30)
contained the first 30 amino acids of HPV16E7 fused to the middle of BPVL2
between amino acids 274 and 275.
L2-E7 chimeric genes were generated via recombinant PCR techniques (Higuchi,
R. (1990) in PCR Protocols;
A Guide to Methods and Applications, ed. Innis, MA, Gelfand, DH, Sninsky, JJ,
and White, TJ. (Academic, New
York), pp. 177-1801. For the chimeras containing full-length HPV16E7, L2 was
amplified with a 5' oligo containing
a restriction enzyme site, and a 3' oligo which was complementary to the E7 5'
oligo. In an independent reaction,
E7 was amplified with a 5' oligo complementary to the L2 3' oligo and a 3'
oligo containing a restriction enzyme
site. The L2 and E7 genes were then fused in a second primer extension
reaction using only the outside (L2 5', and
E7 3') oligos. For the chimera containing only amino acids 1-30 of HPV16E7,
the "internal" primers encoded the
first 30 as of HPV16E7.
The fused genes were then cloned into the baculovirus double expression vector
pSynwtVl-, (which already
contained L1 cloned under the polyhedrin promoter (Kirnbauer et al., Virol.
67:6929 (1993)) immediately downstream
of the pSyn promoter.
The BPVL2-HPV16E7 chimeras were cloned as 5' Bglll to 3'Bglll fragments. The
HPV16L2-HPV16E7
chimera was cloned as a 5' Sstll to 3' Sstll fragment. The primers used for
BPVL2-HPV16E7 (full-length) were:
V
BPVL2 sense,
5'GCGGTAGATCTACCTATAAATATGAGTGCACGAAAAAGAGTAAAACGT3'(SEQ ID N0:1),
and antisense,
5'GCAATGTAGGTGTATCTCCATGCATGGCATGTTTCCGTTTTTTTCGTTTCCTCAACAAGGAGGG3'(SEO ID
N0:2),
~20160j
WO 96/11274 PCT/US95J12914
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HPV16E7, sense,
5'CCCTCCTTGTTGAGGAAACGAAAAAAACGGAAACATGCCATGCATGGAGATACACCTACATTGC3'(SEO ID
N0:3),
and antisense,
5'CCGCTAGATCTGGTACCTGCAGGATCAGCCATGG3' (SEO ID N0:4).
The primers used for BPVL2-HPV16E7 (aa 1-30) were as follows: BPVL2, sense:
same as above.
Internal primer, antisense,
5'CAGTTGTCTCTGGTTGCAAATCTAACATATATTCATGCAATGTAGGTGTATCTCCATGCATGGATGAAAACACTTCA
GG
ATCTTCCGTGGGC3' (SEO ID N0:5).
Internal primer, sense,
5'GCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACCAAACATTTG
C
AAACCCACTGTATGAAGCAGAACC3' (SEO ID N0:6).
BPVL2, antisense,
5'CCGCTAGATCTAGGGAGATACAGCTTCTGGCCTTGTTGCCACAACGC3'(SEO ID N0:7).
The primers used for HPV16L2-HPV16E7 chimeras were as follows: HPV16L2, sense,
5'GCGGTCCGCGGAATATGCGACACAAACGTTCTGCAAAACGCACAAAACGT3'(SEQ ID N0:8),
and antisense,
5'ATCTCCATGCATGGCAGCCAAAGAGAC3' (SEO ID N0:9),
HPV16E7, sense,
5'GTCTCTTTGGCTGCCATGCATGGAGAT3' (SEO ID N0:101.
and antisense,
5'GCTCC GGGGTACCTGCAGGATCAGCC3' (SEQ ID N0:11).
EXAMPLE 2
Selection of recombinant baculovirusea
CsCI-purified recombinant plasmid was cotransfected with baculovirus DNA
(Baculo-Gold; PharMingen, San
Diego, CA) into Sf9 insect cells (ATCC, CRL 1711) by using Lipofectin
(GIBCOIBRL, Gaithersburg, MD) (Hartig, P.C.
Biotechniques 11:310 (1991)). The recombinant baculoviruses were plaque-
purified as described (Summers, M.D. &
Smith, G.E. (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures, Texas
Agricultural Experiment Station Bulletin (Tex. Agric. Exp. Stn., College
Station, TX), Vol 1555).
EXAMPLE 3
Purification of chimeric particles
Sf9 insect cells were infected at a multiplicity of infection of 10 with
recombinant baculoviruses. After
72 h, the harvested cells were sonicated in phosphate-buffered saline (PBS)
for 60 sec. After low-speed clarification,
Y
the lysates were subjected to centrifugation at 110,000 x g for 2.5 h through
a 40% (wtlvol) sucroseIPBS cushion
(SW-28 rotor). The resuspended pellets were centrifuged to equilibrium at
141,000 x g for 20 h at room temperature
in a 10-40% (wtlwt) CsCIIPBS gradient. The visible band was harvested,
centrifuged to equilibrium again by using
the identical conditions, dialyzed extensively against PBS, and stored at
4°C.
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EXAMPLE 4
Co-sedimentation of Ll/L2-E7 complexes
To determine whether the L2-E7 fusion protein was incorporated into particles,
analytical gradient centrifugation was performed as previously described for
determining
the co-assembly of L1 and L2 (Kirnbauer et al., Virol. 67:6929 (1993)).
Briefly, a 12 to 45 % sucrose step gradient was allowed to linearize overnight
at
4°C, dialyzed samples were layered on top, and the gradient was
centrifuged at 41,000 rpm
(288,000 x g) for 2 h in a SW-41 rotor. Fractions were harvested. The
fractions were
analyzed by Coomassie stained SDS-PAGE (L1) or Western blotting with an anti-
BPVL2
polyclonal Ab, or anti-HPV 16E7 polyclonal Ab, and l2sl_labeled anti-rabbit
lgG.
Association of the chimeric L2-E7 with the virus-like particles was
established by
co-sedimentation.
EXAMPLE 5
Co-immunoprecipitation of L1/L2-E7 complexes
2 o To obtain evidence that the L2-E7 fusion proteins formed stable complexes
with Ll,
co-immunoprecipitation experiments were performed.
Briefly, BPVL1/L2-HPV16E7 (full-length) and BPVL1/L2-HPV16E7 (aa 1-30)
VLP preparations were immunoprecipitated in PBS, 1 % Triton~ X-100 with anti-
L1 Mab
SB6 (Roden et al., J. Virol., [supra] (November, 1994)), an anti-L1 polyclonal
Ab,
pre-immune serum or an irrelevant Ab (anti-ElA Mab) and protein A-Sepharose
and
subjected to SDS-PAGE. Proteins were immunoblotted and probed with anti-BPVL2,
or
anti-HPV 16E7 sera, or an anti-HPV 16E7 Mab (Triton Diagnostics, Alameda, CA).
Association of the chimeric L2-E7 with the virus-like particles was
established by
co-immunoprecipitation.
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EXAMPLE 6
Production of antisera
Rabbits were immunized by subcutaneous injection of 330 ul of CsCI
gradient-purified particles in PBS at a concentration of 1 mg/ml. Rabbits were
boosted
with the same amount of particles two weeks and four weeks after the primary
injection.
EXAMPLE 7
BPVl neutralization assay
A focus-forming assay was performed as described (Kirnbauer et al., Proc.
Natl.
Acad. Sci. USA 89:12180 (1992)).
Briefly, serial dilutions of rabbit sera obtained 3 weeks after the second
booster
injection were incubated with X500 focus-forming units of BPV 1 virus for 30
min, the virus
was adsorbed to C 127 cells for 1 h, and the cells were cultured for 3 weeks
(Dvoretzky et al., Virology 103:369 (1980)). The foci were stained with 0.5%
methylene
2 o blue/0.25 % carbol fuchsin in methanol.
Equivalent neutralizing titers of 30,000 were obtained for the BPVLI/L2 VLPs
and
BPVL1/L2-HPV16E7 (full-length) chimeric VLPs.
EXAMPLE 8
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Electron microscopy
Transmission electron microscopy was performed as described (Kirnbauer et al.,
Proc. Natl. Aced. Sci. USA
89:12180 (1992))
Briefly, purified particles were adsorbed to carbon-coated grids, stained with
196 uranyl acetate, and
examined with a Philips electron microscope model EM 400T at x36,000
magnification.
Particles containing the L2-E7 fusion protein were found to be
indistinguishable from L1 or L11L2 VLPs in '
terms of morphology and efficiency of incorporation.
EXAMPLE 9
Induction of antibodies
Rabbits inoculated with BPVL11L2-HPV16E7 papillomavirus-like particles
produced antisera that recognized
E7.
To analyze the sera for E7 specificity, 2.5 ,ug of HPV16E7 and 2.5 Ng of BSA
(control) were subjected
to SDS-PAGE and then Western blotted. Immune and preimmune (control) sera were
applied at a dilution of 1:10.
. Serum from the immune rabbit specifically detected the HPV16E7 protein band
in the Western blot,
indicating the induction of antibodies specific for E7 epitopes.
Example 10
Induction of cell-mediated immunity
Chimeric particles are assayed for induction of cell-mediated immunity
specific for E7 peptides, for example,
by injecting chimeric VLPs into mice, and measuring antigen-specific T cell
proliferation.
Example 11
Prophylactic immunity
Chimeric particles are assayed for induction of prophylactic immunity against
challenge infection, for
example, by immunizing rabbits with E7 chimeric CRPV VLPs and nonchimeric CRPV
VLPs (control), and subsequently
challenging with infectious CRPV, to demonstrate that L2-E7 fusions do not
abrogate prophylactic immunity; or by
immunizing experimental animals with chimeric VLPs containing an STD agent,
subsequently challenging with the STD
agent, and measuring increased survival against lethal challenge or decreased
infection following sub-lethal challenge.
Example 12
Therapeutic immunity
Chimeric particles are assayed for induction of therapeutic immunity against
pre-existing papillomas, for
example, by immunizing rabbits (that have pre-existing papillomas) using E7
chimeric CRPV VLPs and nonchimeric
CRPV VLPs (control), and measuring regression of the pre-existing papillomas.
Example 13
Immunotherapy and immunoprevention of tumors
Chimeric particles are assayed for the ability to prevent tumor development or
to treat existing tumors, for
example, in experimental animals such as mice using tumor cells expressing,
eg., E7. Animals are immunized with,
CA 02201601 2000-OS-10
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e. g. , L2-E7 chimeric VLPs and tested for growth of inoculated tumorigenic
cells that
express, e.g., E7. This approach has been shown to work for animals immunized
with
noncomplexed E7 and a co-stimulatory protein (Chen et al., Proc. Natl. Acad.
Sci. USA
88:110 (1991)).
EXAMPLE 14
Targeting the surface of chimeric VLPs
It may be desirable for certain molecules, such as co-stimulators or ligands
that bind
to cell-surfaces, to be present on the surface of chimeric VLPs. Roden et al.,
J. Virol.,
[supra) (November, 1994) determined that regions of L2 are located on the
surface of
VLPs. A peptide composed of BPV L2 amino acids 44-173, when inoculated into
rabbits,
induced neutralizing antibodies that were active at a 1:1000 serum dilution.
These results
indicate that regions of L2 specify neutralizing epitopes, and that these
regions are
therefore accessible to the surface of native virions. Consequently, fusing a
non-L2
polypeptide to one of these regions would likely result in the targeting of
this polypeptide
to the surface of a chimeric VLP.
2 o 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, and the true scope of the invention is that defined within the
appended claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: The United States of America
(ii) TITLE OF THE INVENTION: CHIMERIC PAPILLOMA VIRUS-LIKE
PARTICLES
(iii) NUMBER OF SEQUENCES: 11
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GCGGTAGATC TACCTATAAA TATGAGTGCA CGAAAAAGAG TAAAACGT 48
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AGGG 64
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AAATGACCAA ACATTTGCAA ACCCACTGTA TGAAGCAGAA CC 102
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GCTCCGCGGG GTACCTGCAG GATCAGCC 28
