Note: Descriptions are shown in the official language in which they were submitted.
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HOMOGENEOUS HUMAN PAPILLOMA VIRUS CAPSOMERE CONTAINING COMPOSITIONS, METHODS
FOR
MANUFACTURE, AND THE USE THEREOF AS DIAGNOSTIC, PROPHYLACTIC OR THERAPY
FIELD OF THE INVENTION
The present invention relates to stable human
papillomavirus (HPV) capsomeres and compositions contain-
ing produced by enzymatic, chemical and/or recombinant
methods. Such HPV capsomeres and compositions containing
are useful, e.g., as vaccines for conferring protection
against human papillomavirus infection and as diagnostic
agents for detection of antibodies specific to HPV L1
proteins. The present invention further relates to modi-
fied HPV L1 nucleic acid sequences which have been modi-
fied by site specific mutagenesis and/or deletion in order
to favor the formation of stable capsomeres which are
substantially incapable of assembly into virus-like parti-
cles.
Background of the Invention
Papillomaviruses infect a wide variety of different
species of animals including humans. Infection is typi-
iR
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cally characterized by the induction of benign epithelial
and fibro-epithelial tumors, or warts at the site of
infection. Each species of vertebrate is infected by a
distinct group of papillomavirus, each papillomavirus
group comprising several different papillomavirus types.
For example, more than sixty different human papilloma-
virus (HPV) genotypes have been isolated. Papillomavir-
uses are highly species-specific infective agents. For
example, canine and rabbit papillomaviruses cannot induce
papillomas in heterologous species such as humans. Neu-
tralizing immunity to infection against one papillomavirus
type generally does not confer immunity against another
type, even when the types infect a homologous species.
In humans, papillomaviruses cause genital warts, a
prevalent sexually transmitted disease. HPV types 6 and
11 are most commonly associated with benign genital warts
condyloma acuminata. Genital warts are very common and
subclinical or inapparent HPV infection is even more
common than clinical infection. While most HPV induced
lesions are benign, lesions arising from certain papillo-
mavirus types, e.g., HPV-16 and HPV-18, can undergo malig-
nant progression. Moreover, infection by one of the
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malignancy associated papillomavirus types is considered
to be a significant risk factor in the development of.
cervical cancer, the second most common cancer in women
worldwide. Of the HPV genotypes involved in cervical
cancer, HPV-16 is the most common, being found in about
50% of cervical cancers.
In view of the significant health risks posed by
papillomavirus infection generally, and human papilloma-
virus infection in particular, various groups have re-
ported the development of recombinant papillomavirus
antigens and their use as diagnostic agents and as.pro-
phylactic vaccines. In general, such research has been
focused toward producing prophylactic vaccines containing
the major capsid protein (L1) alone or in combination with
the minor capsid protein (L2). For example, Ghim et al,
Virology, 190:548-552 (1992) reported the expression of
HPV-1.Ll protein using a vaccinia expression in Cos cells
which displayed conformational epitopes and the use there-
of as a vaccine or for serological typing or detection.
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Also, Suzich et al, Proc. Natl. Acad. Sci.,
U.S.A., 92:11553-11557 (1995) report that the immunization
of canines with a recombinant COPV expressed in a baculo-
virus/insect cell system complet.ely prevented the develop-
ment of viral mucosal papillomas. These results are
important given the significant similarities between many
HPVs and COPV. For example, COPV, similar to HPVs associ-
ated with anogenital and genital papillomas cancer, in-
fects and induces lesions at a mucosal site. Also, the,Li
sequences of COPV shares structural similarities to HPV Li
sequences both at the level of DNA and protein. Given
these similarities, the COPV/beagle model is useful for
investigation of L1 protein containing vaccines, e.g.,
investigation of the protective immune response, protec-
tion from natural infection and optimization of vaccina-
tion protocols. (id.)
Also, a research group from the University of Roch-
ester reported the production of human papillomavirus
major capsid protein (L1) and virus-like particles. using a
baculovirus/insect cell expression system. (Rose et al,
University of Rochester, WO 94/20137, published on Septem-
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ber 15, 1994). In particular, they reported the expres-
sion of the L1 major capsid protein of HPV-6 and HPV-11
and the production of HPV-6, HPV-i1, HPV-16 and HPV-18
virus-like particles.
Further, a University of Queensland research group
also purportedly disclosed the recombinant manufacture of
papillomavirus L1 and/or L2 proteins and virus-like parti-
cles as well as their potential use as vaccines. (Frazer
et al, WO 93/02189, published on February 4, 1993).
Still further, a United States government research
group reported recombinant papillomavirus capsid proteins
purportedly capable of self-assembly into capsomere struc-
tures and viral capsids that comprise conformational
antigenic epitopes. (U.S. Patent No. 5,437,951, Lowy et
al, issued on August 1, 1995.) The claims of this patent
are directed to a specific HPV-16 DNA sequence which
encodes an Ll protein capable of self-assembly and use
thereof to express recombinant HPV-16 capsids containing
said HPV-16 L1 protein.
With respect to HPV capsid protein containing vac-
cines, it is widely accepted by those skilled in the art
that a necessary prerequisite of an efficacious HPV L1
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major capsid protein-based vaccine is that the L1 protein
present conformational epitopes expressed by native human
papillomavirus major capsid proteins (See, e.g., Hines et
al, Gynecologic Oncology, 53:13-20 (1994); Suzich et al,
Proc. Natl. Acad. Sci., U.S.A., 92:11553-11557 (1995)).
Both non-particle and particle recombinant HPV Li
proteins that present native conformational HPV Li epi-
topes have been reported in the literature. It is known
that Li is stable in several oligomeric configurations,
e.g., (i) capsomeres which comprise pentamers of the Li
protein and (ii) capsids which are constituted of seventy-
two capsomeres in a T=7 icosahedron structure. Also, it
is known that the Li protein, when expressed in eukaryotic
cells by itself, or in combination with L2, is capable of
efficient self-assembly into capsid-like structures gener-
ally referred to as virus-like particles (VLPs).
VLPs have been reported to be morphologically and
antigenically equivalent to authentic virions. Moreover,
immunization with VLPs has been reported to elicit the
production of virus-neutralizing antibodies. More specif-
ically, results with a variety of animal papillomaviruses
(canine oral papillotmavirus and bovine papillocnavirus-4)
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have suggested that immunization with VLPs results in
protection against subsequent papillomavirus infection.
Consequently, VLPs composed of HPV Li proteins have been
proposed as vaccines for preventing diseases associated
with human papillomavirus infections.
Additional studies have examined the effects of L1
deletions on capsid assembly. For example, Paintsil et al
recently reported that certain carboxy-terminal residues
of the BPV-1 L1 protein are not required for capsid forma-
tion. (Paintsil et al, Virol., 223:238-244 (1996)).
Moreover, at page 239, a schematic summary of the results
obtained upon expression of various BPV L1 deletions
generated by PCR mutagenesis is provided. In particular,
this summary indicates whether such fragments result in
proper (icosahedon) capsids, aberrant capsids, unstruc-
tured Ll aggregates or capsomeres. (Id.) Also, Paintsil
et al teach a specific carboxy-deletion wherein residues
451-495 (AC1) of the BPV-1 L1 protein were deleted.
However, there is no indication that the resultant capso-
meres present conformational epitopes of native BPV-1 or
whether they elicited neutralizing antisera. Also,
Paintsil et al (Id.), further notes at page 21, that
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"these truncation results suggested that deletion of a
more conserved region upstream of [amino acid] 471 (Fig.
4) completely perturbs the proper folding of the L1 pro-
tein". Therefore, these results would teach against
making modifications involving significant carboxy-termi-
nal deletions. Also, their experiments are limited to
BPV-1 Li deletions and mutations.
Therefore, notwithstanding what has been previously
reported, there still exists a need in the art for novel
HPV major capsid protein containing compositions that
present conformational epitopes associated with native
(wild-type) HPVs, and methods for their manufacture.
OBJECTS OF THE INVENTION
It is an object of the invention to provide stable
HPV capsomeres which present conformational epitopes
expressed by authentic (wild-type, infectious) HPV
virions.
It is a more specific object of the invention to
provide stable HPV capsomeres which present at least one
virus-neutralizing conformational epitope(s) expressed by
T =
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authentic HPV virions by enzymatic (trypsin) digestion of
purified HPV capsomere and/or virus-like particles.
It is another more specific object of the invention
to produce stable HPV capsomeres by chemical methods,
i.e., by "capping-off" one or more cysteine residues,
e.g., by reacting reduced cysteine residues with compounds
that prevent oxidation of sulfhydryls, e.g., alkylating
agents, thereby preventing disulfide bond formation and
VLP assembly.
It is another specific object of the invention to
provide stable HPV capsomeres by expression of a DNA
encoding a modified HPV L1 sequence wherein such modifi-
cation comprises a carboxy deletion and/or at least one
site specific mutation wherein such modification(s), upon
expression, results in capsomeres that are substantially
incapable of VLP assembly.
It is another specific object of the invention to
provide modified HPV L1 nucleic acid sequences which upon
expression result in stable HPV capsomeres that are sub-
stantially incapable of VLP assembly.
It is a more specific object of the invention to
provide modified HPV L1 nucleic acid sequences which
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comprise a carboxy-terminal deletion and/or substitution
or deletion modification involving at least one cysteine
residue which upon expression results in capsomeres sub-
stantially incapable of VLP assembly.
It is another object of the invention to provide
vaccines or diagnostic compositions containing stable HPV
capsomeres produced by enzymatic, chemical, and/or recom-
binant methods.
It is still another object of the invention to use
the stable HPV capsomeres of the present invention for the
manufacture of neutralizing polyclonal and monoclonal
antibodies.
It is another object of the invention to use the
stable HPV capsomeres of the present invention as vaccines
for conferring protection against HPV infection.
It is another object of the invention to use the
stable HPV capsomeres of the present invention as diag-
nostic agents for detection of anti-HPV antibodies.
BRIEF DESCRIPTION OF THE INVENTION
Therefore, the invention generally relates to stable
HPV capsomeres which present at least one virus-neutraliz-
r u
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ing conformational epitope expressed by an Ll protein
expressed by a native (infectious) HPV, methods for their
manufacture, and use thereof as diagnostic, prophylactic,
and therapeutic agents.
According to one aspect of the present invention,
there is provided stable human papillomavirus (HPV)
capsomeres which (i) have a reduced capacity to assemble
into virus-like particles (VLPs) relative to a corresponding
non-modified HPV Ll protein, wherein reduced capacity means
that said capsomeres assemble into VLPs less than 50%
relative to a corresponding non-modified HPV L1 protein;
(ii) present at least one virus-neutralizing conformational
epitope of the major capsid protein (L1) expressed by a
native (wild-type infectious) HPV virus; and (iii) induce
the production of HPV neutralizing antibodies.
According to another aspect of the present
invention, there is provided a composition for eliciting
neutralizing antibodies against a particular HPV type which
comprises the stable HPV capsomeres as described herein in
an amount sufficient to elicit neutralizing antibodies to
said HPV upon challenge, and a pharmaceutically acceptable
carrier.
According to still another aspect of the present
invention, there is provided use of a prophylactically
effective amount of the stable HPV capsomeres as described
herein for conferring protection to HPV infection in a
susceptible host.
According to yet another aspect of the present
invention, there is provided a diagnostic composition
comprising a diagnostically effective amount of the stable
HPV capsomeres as described herein, and a pharmaceutically
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acceptable carrier.
According to a further aspect of the present
invention, there is provided a human papillomavirus (HPV) Ll
DNA that contains a modification which upon expression
results in the production of stable HPV capsomeres which
have a reduced capacity to assemble into virus-like
particles relative to a corresponding non-modified HPV Ll
protein, wherein reduced capacity means that said capsomeres
assemble into VLPs less than 50% relative to a corresponding
non-modified HPV Ll protein, which present at least one
virus-neutralizing conformational epitope expressed by a
native HPV virion, and which induce the production of
HPV neutralizing antibodies.
As discussed in greater detail infra, it has been
surprisingly discovered that HPV Li proteins, which have
been modified to favor capsomere formation, and
substantially prevent the assembly of such capsomeres into
VLPs, express conformational epitopes of native, infectious
HPVs. In particular, they react with conformational
antibodies produced against native, infectious HPVs and
elicit the production of neutralizing antibodies.
These results are surprising given the significant
adverse effects that even minor modifications to L1 proteins
may incur. For example, it has been reported that a point
mutation in the HPV-16 Ll protein disrupts assembly of the
protein into virus-like particles and also impairs
conformation, (Roden et al, J. Virol., 68(11):1-5 (1994)).
Also, these capsomeres afford further advantages
because they should result in highly homogeneous vaccine
compositions. As the subject capsomeres, unlike non-
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modified L1 proteins, do not give rise to virus-like
particles or other aggregates to any significant extent,
compositions according to the invention should be sub-
stantially free of higher molecular weight forms of the Ll
proteins. This is advantageous from a clinical standpoint
wherein supplying a product of high consistency may be
very important, indeed essential. Also, because capso-
meres are significantly smaller than VLPs, they may be
easier to purify on conventional chromatographic media,
thereby increasing the ease of manufacture.
Definitions-
Ma-ior capsid protein or L1 protein
This refers to the structural protein of papillom-
avirus (PV) which constitutes the major portion of the PV
capsid structure. This protein has reported application
in the preparation of HPV vaccines and as a diagnostic
agent.
Major capsid protein or L2 protein
This refers to the structural protein of papilloma-
virus which constitutes a minor portion of the PV viral
capsid structure.
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Virus-like particles or VLPs
This refers to the capsid-like structures which
result upon expression and assembly of a papillomavirus Ll
DNA sequence alone or in combination with an L2 DNA se-
quence. VLPs are morphologically and antigenically simi-
lar to authentic virions. VLPs may be produced in vivo,
in suitable host cells, e.g., mammalian and insect host
cells, or may form spontaneously upon purification of
recombinant Ll proteins.
Capsomeres
This refers to an oligomeric configuration of the L1
protein which is constituted of L1 pentamers. Therefore,
capsomeres comprise the "monomer" units which constitute
the viral capsid structure.
Stable Car)someres
This refers to capsomeres which are substantially
incapable of assembly into virus-like particles. "Stable"
in this context refers to the fact that these capsomeres
substantially retain their capsomere morphology instead of
assembling into VLPs. This is preferably accomplished by
(i) removing enough of the carboxy-terminal portion of the
L1 protein to prevent or substantially inhibit VLP assem-
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bly and/or (ii) preventing or substantially inhibiting
disulfide bond formation. Disulfide bond formation may be
prevented by elimination of one or more cysteine residues,
e.g., by a substitution or deletion modification or by
chemical means, e.g., by incubation and storage in sulf-
hydryl reducing agents, e.g., by reaction with (3-mercap-
toethanol, dithiothreitol, cysteine or other compounds
which prevent oxidation of sulfhydryl groups. The free
sulfhydryls on capsomeres generated by thiol reduction of
VLPs are then "capped off", e.g., by reaction with alkyl-
ating agents, e.g., iodoacetamide or N-ethylmaleimide.
The subject stable capsomeres will express at least one
conformational neutralizing epitope expressed by the Li
protein of a corresponding native HPV virion.
Capsids
This refers to the structural portion of the papillo-
mavirus which is comprised of capsomeres. More specifi-
cally, it is constituted of seventy-two capsomeres in a
T=7 icosahedron structure.
Conformational Ll HPV Epitope
This refers to an epitope e-xpressed on the surface of
the subject stable papillomaviras capsomeres which is also
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expressed by an L1 protein of a corresponding native
(wild-type), infectious HPV. it is well accepted by those
skilled in the art that the presentation of conformational
epitopes is essential to the efficacy (both as prophylac-
tic and diagnostic agents) of HPV Li protein immunogens.
Conformational Neutralizing L1 HPV Epitope
This refers to an epitope expressed on the surface of
the subject stable papillomavirus capsomeres which is also
expressed by an Li protein of a corresponding native
(wild-type), infectious HPV, and which elicits neutraliz-
ing antibodies. It is well accepted by those skilled in
the art that the presentation of conformational neutraliz-
ing epitopes is essential to the efficacy (both as prophy-
lactic and diagnostic agents) of HPV Ll protein immuno-
gens.
Conformational Antibody
This refers to an antibody that specifically binds an
epitope expressed by a naturally occurring (wild-type) HPV
Li protein, e.g., major capsid protein expressed on the
surface of a native HPV.
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Modified HPV Ll DNA
This refers to an HPV Li DNA modified such that upon
expression it results in an HPV L1 protein that presents
at least one virus-neutralizing HPV L1 conformational
epitope, and forms stable capsomeres that are substan-
tially incapable of assembly into virus-like particles.
"Substantially incapable" is relative to non-modified HPV
Ll proteins which are known to spontaneously assembly into
VLPs when expressed by suitable host cells, e.g., mammali-
an or insect cells, or in vitro during protein purifica-
tion. Preferably, the modification will prevent VLP
assembly altogether. However, the invention embraces
modifications which substantially inhibit VLP formation,
e.g., by at least 50%, more preferably by at least 90%,
and most preferably by at least 95%. This can be deter-
mined by known methods, e.g., by visual detection of VLPs,
e.g., by electron microscopy. The HPV L1 DNA may further
comprise additional modification(s), e.g., additions,
substitutions, deletions, provided that they do not ad-
versely affect presentation of conformational neutralizing
epitope(s). Preferably, modifications which prevent VLP
assembly will comprise carboxy-terminal deletions and/or
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removal of at least one cysteine residue that inhibits or
prevents VLP assembly.
Modification which inhibits disulfide bond formation
This refers to a modification of an HPV Li DNA or
corresponding protein which inhibits disulfide bond forma-
tion. Preferably, this is effected by deletion and/or
substitution of at least one cysteine residue. However,
such modifications also include the replacement of one or
more amino acids, which are sufficiently proximate to a
cysteine residue, e.g., to sterically hinder the cysteine
residue and thereby prevent disulfide bond formation or
interfere with capsomere-capsomere interactions. Alterna-
tively, cysteine residues involved in VLP assembly can be
chemically "capped-off", e.g., reaction with ~-mercapto-
ethanol or dithiothreitol, followed by 0-alkylation with
iodoacetamide or N-ethylmaleimide. As discussed in great-
er detail infra, such cysteine residue(s) are comprised,
in particular, in the carboxy-terminal portion of the pro-
tein, more specifically in the region of the L1 protein
spanning amino acids 30 to 86, inclusive, relative to the
carboxy terminal end of the Ll protein. In the specific
case of HPV-11, a conserved cysteine is found at position
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424 of the L1 protein, which apparently affects VLP assem-
bly.
DETAILED DESCRIPTION OF FIGURES
Figure 1: SDS/PAGE analysis of purified HPV-11 Li
protein. The protein was mixed with sample preparation
buffer in the absence (lane 1) or presence (lane 2) of 2
mM DTT and boiled for 2 minutes prior to gel electrophore-
sis. Shown on the left are the positions at which molecu-
lar weight standards (in Da x 10-3) migrated.
Figure 2: 30% sucrose cushion analysis of HPV-11 VLP
disassembly. HPV-11 preparations were treated at 4 C as
described in the text, and samples were taken at the top
(T) or bottom (B) of the sucrose cushion prior to gel
electrophoresis. Group 1, untreated, purified HPV-11 VLP
starting material in PBS. Group 2, VLPs incubated with 5%
i3ME for 16 hours. Group 3, VLPs incubated with 5% l3ME for
1 hour. Group 4, VLPs incubated with 2% SME for 16 hours.
Group 5, VLPs incubated with 0.5% f3ME for 16 hours. Group
6, VLPs incubated with 10 mM DTT, 5mM EDTA for 16 hours.
Figure 3: 5-20% linear sucrose gradient analysis of
disassembled HPV-11 VLPs. VLPs in PBS were incubated with
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5% f3ME (a), or 200 mM NaHCO3, pH 9.6(b) for 16 hours at 4 C
and then centrifuged on a 5-20% linear sucrose gradient as
described in the text. The gradient was collected in 25
fractions (0.5 ml), and the pellet (P) was resuspended in
0.5 ml PBS. Shown is an immunoblot demonstrating the
position of the L1 protein across the gradient. Also
indicated are the peak positions at which sedimentation
standards migrated when run on separate gradients.
Figure 4: 10-65% linear sucrose gradient analysis of
HPV-11 VLPs in various states of assembly. An aliquot of
purified VLP starting material (a) was incubated with 5%
l3ME for 16 hours at 4 C (b) . A portion of f3ME-treated VLPs
were then reassembled by dialysis into PBS-0.5 NaCl to
remove reducing agent (c) . The samples are then centri-
fuged on 10-65% linear sucrose gradients as described in
the text. Each gradient was collected in 12 fractions (1
ml), and the pellet (P) was resuspended in 1 ml PBS.
Shown are immunoblots demonstrating the positions at which
the Li protein migrated on the different gradients. Also
indicated are the peak positions at which sedimentation
standards migrated, as in Fig. 3.
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Figure 5: Electron micrographs of HPV-11 VLPs in
various states of assembly. VLPs, treated as described,
were stained with 2% phosphotungstic acid, applied to
grids, and photographed at magnifications of 15-25,000
times. a, purified VLP starting material, b, VLPs disas-
sembled to the level of capsomeres by incubation with 5%
i3ME for 16 hours at 4 C. c, VLPs reassembled from disas-
sembled VLPs by dialysis into PBS-0.5 NaCl, d, the central
region of image c at greater magnification. Scale bar:
a,c = 200 nm; b,d,= 100 nm.
Figure 6: Reaction of intact and disassembled VLPs
with HPV-11 structure-specific monoclonal antibodies.
HPV-11 L1 VLP starting material (A), VLPs disassembled by
treatment with 5% i3ME either without (B) or with (C)
subsequent dialysis into PBS-0.5 M NaCl to remove reducing
agent, and VLPs disassembled in the presence of 200 mM
carbonate, pH 9.6 and then dialyzed into PBS-0.5 M NaCl
(D) were attached to the wells of microtiter plates. HPV-
11 structure-specific monoclonal antibodies H11.F1 (HPV-11
neutralizing; V) and H11.A3 (HPV-11 non-neutralizing; =)
were tested for immunoreactivity to the bound antigens in
an ELISA as described in the Materials and Methods.
i ~
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Reactivity with monoclonal antibody AU1 (~), which recog-
nizes a linear epitope found on HPV-11 L1, was used as a
control to demonstrate antigen attachment to the micro-
titer wells.
Figure 7: Anti-HPV-11 capsid and anti-HPV-11 capso-
mere sera were incubated with HPV-11 virions for 75 min at
37 C before addition to HaCaT cells. Alternatively,
virions were added to cells without pre-incubation with
serum, or virions were pre-incubated with pre-immune serum
prior to infection of HaCaT cells. Six day post-infec-
tion, the cells were harvested and total RNA was extract-
ed. Ten percent of the total RNA was used for cDNA syn-
thesis with an oligo d-T primer. Ten percent of the cDNA
was then used as template for nested PCR using primers
specific for the HPV-11 E1AE4 spliced message. PCR prod-
ucts were separated on 2% agarose gels. Gels were stained
with ethidium bromide and examined under UV light for the
presence of the 0.6 kb El'E4 band (top). PCR amplifica-
tion of 9-actin was performed on all cDNA samples as an
internal control (bottom). The expected size of the 9-
actin band is 0.6 kb. Lane A contains molecular size
markers. Lanes B and C contain PCR products obtained with
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RNA isolated from cells infected with HPV-11 pre-incubated
with a 10-2 dilution of normal rabbit serum. The ability
to detect the ElAE4 PCR product indicates that virus is
not neutralized by this control serum. Lanes D and E
represent reactions carried out with RNA from cells incu-
bated with virus that had not been pre-incubated with
serum, and lane F and G are from cells incubated without
virus. As expected, the ElAE4 band is detected in virus-
infected but not in uninfected cells. Lanes H-L contain
PCR products from cells infected with virus that had been
pre-incubated with serial loglo dilutions of anti-HPV-11
capsid antiserum (10-3-10-7) . This antiserum neutralizes
the virus at all dilutions tested. Lanes M-Q show PCR
products obtained when cells were infected with virus pre-
incubated with serial loglo dilutions of anti-HPV-11 capso-
mere serum (10-3-10-'). This antiserum effectively neutral-
izes the virus out to a 10-6 dilution.
DETAILED DESCRIPTION OF THE INVENTION
As discussed, the present invention generally relates
to stable HPV capsomeres which present at least one virus-
neutralizing conformational epitope of a native fiafec-
t . . . . .. . . 0
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tious) HPV, which are substantially incapable of assembly
into VLPs (when subjected to conditions that normally
result in VLP assembly).
The present invention was based, in part, on obser-
vations made by the inventors during quantitative disas-
sembly and reassembly of HPV-11 VLPs in vitro. Specifi-
cally, it was observed that maximal VLP disassembly re-
quired prolonged exposure to very high concentrations of
reducing agent. This suggested that stabilizing disulfide
bonds are buried and inaccessible, and that exposure of
these bonds to solvent by local fluctuations is very
infrequent.
However, this observation conflicted to some extent
with other reported experiments which suggested that
residues in the C-terminal end of the Ll protein may be
important to capsomere formation which is a portion of the
L1 molecule which would have been predicted to be rela-
tively accessible to solvent. Specifically, it was re-
ported that the BPV Li protein which contained a relative-
ly small carboxy-terminal deletion (24 carbon-terminal
amino acids removed) still assembled into VLPs. By con-
trast, a BPV-1 Li protein crontaining alarger carboxy
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deletion (44 amino acids) exhibited significantly inhibit-
ed ability to form capsids (Paintsil et al, Virology,
223:238-244 (1996)). Moreover, it was recently reported
that an HPV-1 protein containing an even larger C-terminal
deletion (86 amino acids) yielded capsomeres apparently
incapable of forming capsid-like structures. (Li et al,
J. Virol., 71:2988-2995 (1997)). These results suggested
the importance of C-terminal residues in VLP formation.
In this regard, it has been reported that in the 3.8
A structure of the SV40 virus, that the C-terminal domain
of the VP1 protein is involved in the formation of intra-
capsomeric bonds which stabilize the capsid, by extending
across the space between capsomeres and forming part of
the extended 0-sheet of the neighboring capsomeric Li pro-
tein. However, the requirement of disulfide bonds for
this interaction was not resolved in the crystal structure
because of disorder in this portion of the molecule
(Liddington et al, Nature, 354:278-284 (1991)). Also, it
was previously reported that 15 A strands connecting
capsomeres can be seen, at low resolution, in the cryo-
electron microscopic reconstruction of the BPV structure
(Baker et al, Biophys J., 60:1445-1456 (1991); Belnap et
r a
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al, J. Mol. Biol., 259:249-263 (1996)); and also in nega-
tively-stained HPV virions (Yabe et al, Virology, 227:13-
23 (1997)). These results suggested that linker arms may
stabilize papillomavirudae capsids. [However, it is noted
that these references did not provide any information
concerning what specific residues or role of other factors
which potentially could have affected PV capsid formation
and stability.]
The present invention was further based on experi-
ments which demonstrated that capsomeres generated from
HPV-11 VLPs, even after long exposure to high concentra-
tion of reducing agent, still retain structural epitopes
found on native VLPs (disclosed infra in the Examples).
This was demonstrated in part based on their reactivity
with a panel of antibodies that specifically react with
HPV-il Ll VLPs but do not with "denatured" Li proteins.
These results were surprising as it had previously been
reported that the binding of two of the tested antibodies
was VLP-dependent. (Ludmeyer et al, J. Virol., 71:3834-
3837 (1997)). Also, it was demonstrated (also disclosed
infra) that HPV-11 capsomeres are capable of eliciting the
production of virus-neutralizirn3 anttibodies (also dis-
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closed infra in Example 6). Therefore, these results and
observations in combination suggested to the inventors the
suitability of stable HPV capsomeres as immunogens for
conferring protection against HPV infection.
As discussed, given their resistance to VLP forma-
tion, the stable capsomeres potentially will give rise to
vaccines of enhanced homogeneity. Also, HPV capsomeres,
because of their smaller size and molecular weight rela-
tive to VLPs, should be easier to purify then VLPs, there-
by facilitating vaccine manufacture.
More specifically, these results, most especially the
fact that capsomere formation was favored by very high
reducing agent, suggested that one or more cysteine resi-
dues are likely involved in VLP assembly and disassembly.
Moreover, the fact that deletions in the carboxy terminal
portion of the L1 protein inhibits (or prevents) VLP
assembly further suggested that at least one cysteine
residue involved in VLP assembly is apparently comprised
in the carboxy-terminal portion of the L1 protein.
In general, stable capsomeres will be produced by one
of two general methods. The first method comprises ex-
pression of a modified HPV L1 DNA which contains at least
~
IF
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one modification(s) such that upon expression it results
in the production of stable capsomere. This first method
comprises the following steps:
(i) obtaining a desired HPV L1 DNA sequence;
(ii) introducing suitable modification(s) therein by
deletion and/or site-specific mutagenesis that prevent VLP
assembly and result in stable capsomere upon expression;
(iii) expressing said modified HPV Li DNA in suitable
host cells, and
(iv) recovering capsomere from said host cells.
The second method comprises expression of a non-
modified HPV L1 protein, and modifying the resultant
expression product to produce stable capsomere. Specif-
ically, the second method will comprise the following
steps:
(i) obtaining a desired HPV L1 DNA;
(ii) expressing said HPV Ll DNA in suitable host
cells;
(iii) purifying the resultant VLPs under conditions
(e.g. high reducing agent concentration) that provide for
the dissociation of said VLPs into capsomere and chemical-
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ly "capping off" to prevent VLP reassembly, e.g., by
alkylating free sulfhydryls; or
(iv) purifying the resultant VLPs or capsomere pro-
duced upon expression of the HPV L1 DNA during step (ii)
and digesting said VLPs or capsomeres with a protease
(e.g. trypsin) under conditions that result in stable
capsomere wherein a sufficient amount of the carboxy-
terminal portion of the Ll protein has been removed which
is required for VLP assembly.
Many HPV L1 DNAs have been reported in the literature
and are publicly available. (See, e.g., Baker, Sequence
Analysis of Papillomavirus, Genomes, pp. 321-384; Long et
al, U.S. Patent No. 5,437,931, Cole et al, J. Mol Biol.,
193:599-608 (1987); Danos et al, EMBO J., 1:231-236
(1982) ; Cole et al J. Virol., 38 (3) :991-995 (1986).)
Also, it is well known that HPV Li DNAs exhibit signifi-
cant homology. Therefore, a desired HPV Ll DNA can easily
be obtained, e.g., by the use of a previously reported HPV
L1 DNA or a fragment thereof as a hybridization probe or
as a primer during polymerization chain reaction (PCR) am-
plification. Indeed, numerous HPV Ll DNAs have been
cloned and expressed.
i =
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Preferably, the HPV Li DNA said in the subject inven-
tion will be derived from an HPV which is involved in
cancer or condyloma acuminata, e.g., HPV-16, HPV-18, HPV-
31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, and
HPV-56 are involved in cancer, and HPV-6, HPV-11, HPV-30,
HPV-42, HPV-43, HPV-44, HPV-54, HPV-55,and HPV-70, are
involved in warts. However, the subject stable capsomeres
may be produced from any desired HPV Li DNA.
According to the first embodiment of the invention,
the selected HPV Li DNA will be modified such that the
modified Li DNA upon expression results in stable cap-
someres which do not significantly assemble into larger
oligomers such as VLPs and which present at least one
virus-neutralizing conformational epitope of a native,
infectious HPV. This will be effected by introducing one
or both of the following modifications:
(i) deletion of up to 86 carboxy-terminal residues,
and more preferably deletion of from about 30 to 86 car-
boxy-terminal residues inclusive; and
(ii) introducing one or more site specific modifi-
cations which inhibit or prevent disulfide bond formation,
in particular disulfide bond(s) which are involved in VLP
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assembly. It is noted that if the L1 protein contains
modifications that prevent or inhibit disulfide bond
formation, it may not be necessary to delete carboxy-
terminal amino acids to generate stable capsomers.
Methods for introducing desired deletions in DNA
sequences are well known in the art. For example, car-
boxy-terminal deletions can be introduced by cleaving the
L1 DNA with appropriate restriction enzyme(s). Alterna-
tively, Li deletions can be constructed by PCR mutagenesis
of the L1 gene such as described by Paintsil et al (Virol-
ogy, 223:238-244 (1996)). Still alternatively, an HPV L1
DNA containing a deletion in the carboxy-terminal end of
the Li gene can be made by DNA synthesis.
Similarly, methods for introducing desired site
specific mutations in DNA sequences are well known.
Preferably, the selected HPV Li DNA will be mutagenized by
removal of at least one cysteine residue involved in VLP
assembly. As discussed, this can be accomplished by a
substitution modification, i.e., substituting a selected
cysteine codon with another codon, or by deletion. Sub-
stitution mutations are preferred as this may reduce the
potential of adversely affecting the three-dimensional
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structure of the resultant protein and the presentation of
desired conformational epitopes. Especially preferred
cysteine residues targeted for modification are those at
the carboxy-terminal portion of the protein, most espe-
cially those contained within carboxy-terminal 86 amino
acids of the L1 protein. However, other cysteine residues
may also potentially be modified provided that they do not
impair capsomere production and presentation of confor-
mational epitopes upon expression. Suitable methods for
introducing mutations in PV L1 DNAs are also disclosed in
Paintsil et al (Id.). In the specific case of HPV-11, a
cysteine residue targeted for modification is found at
position 424.
Alternatively, disulfide bond formation may be pre-
vented by modification of residues proximate to cysteine
residues, e.g., by introduction of amino acids which
sterically hinder disulfide bond formation or interfere
with capsomere-capsomere interactions.
While stable capsomeres should result from either of
these modifications alone, the introduction of both car-
boxy deletions and cysteine residue modifications into an
HPV-L1 sequence may itrrpart synergistic effects. Moreover,
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as noted in this embodiment of the invention, the deletion
of fewer than 86 carboxy-terminal amino acids may be
effected, thereby potentially retaining conformational
epitopes that may be present in this portion of the L1
protein. These modified or non-modified Ll DNA will then
be expressed in a selected expression system.
The selected host and expression vector will be
cultured under conditions that favor the production of the
subject stable HPV capsomeres. This will largely depend
upon the selected host system and regulatory sequences
contained in the vector, e.g., whether expression requires
induction. After expression, the HPV capsomeres will be
recovered from the host cells. The means of recovery will
also depend to some extent on the host/vector system.
For example, if an intracellular expression vector is
selected, the host cells will need to be lysed and the HPV
capsomeres recovered from the lysate. By contrast, if the
expression vector contains sequences that facilitate
secretion, HPV capsomeres can be recovered directly from
the culture medium. Methods for recovery of heterologous
proteins from recombinant host cells and culture medium
are well known in the art.
* _
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The subject modified HPV L1 sequences may be ex-
pressed in any host cell that provides for the expression
of recoverable yields of-HPV capsomeres. Suitable host
systems for expression of recombinant proteins are well
known and include, by way of example, bacteria, mammalian
cells, yeast, and insect cells. A preferred expression
system comprises the baculovirus/insect cell system used
in the examples as this system provides for high protein
yields. However, HPV L1 proteins can be produced in other
systems, in particular bacteria and yeast.
Suitable vectors for cloning of expression of the
subject HPV L1 encoding DNA sequences are well known in
the art and commercially available. Further, suitable
regulatory sequences for achieving cloning and expression,
e.g., promoters, polyadenylation sequences, enhancers,
selectable markers are also well known. The selection of
appropriate sequences for obtaining recoverable protein
yields is routine to one skilled in the art.
As discussed, when expressing the subject modified
HPV L1 DNAs, the host cells should only express HPV Ll
protein in the form of HPV capsomeres (as the L1 sequence
has been modified to prevent VLP assembly). However, as
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discussed, the present invention further embraces the
expression of non-modified HPV Ll DNAs, and the use of the
resultant expression product to produce stable HPV capso-
meres.
This aspect of the invention will preferably be
conducted by expression of non-modified HPV Li DNAs in a
selected host/vector system, recovery of resultant HPV
VLPs, and the conversion of said VLPs into stable capso-
meres. It is well known that non-modified HPV L1 DNAs
upon expression in eukaryotic cells may spontaneously
assemble into VLPs, or in the case of cells wherein assem-
bly does not occur in vivo (bacterial cells) this may
occur during purification.
While VLPs have reported application in HPV vaccines
and diagnostics, the object of the present invention is to
produce stable capsomeres, as it has been discovered that
they present conformational neutralizing epitopes and
induce neutralizing antisera.
This can be accomplished, e.g., by enzymatic treat-
ment of the HPV VLPs with an appropriate enzyme, e.g.,
trypsin, under conditions that remove the carboxy-terminal
portion of the HPV VLPs. In the specific case of HPV-11
w a
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VLPs, proteotype digestion with trypsin has been reported
to remove the 86 terminal amino acids. (Li et al, J.
Virol 71:2988-2995 (1997)). Moreover, said limited pro-
teolytic digestion of purified HPV-11 VLPs results in a
homogeneous population of stable HPV capsomeres, i.e.,
which are resistant to VLP assembly. (Li et al, J.
Virol., 71:2988-2995 (1997)).
Alternatively, the expressed HPV L1 proteins can be
disassembled during the course of HPV capsomere purifi-
cation. This can be effected by, e.g., conducting all the
purification steps in the presence of high concentration
of reducing agent, e.g., on the order of 1% to 5% by
weight of reducing agent, e.g., (3-mercaptoethanol. This
is discussed in greater detail in the Examples.
Reactive sulfhydryls on the purified capsomeres can
be chemically "capped-off", thereby preventing disulfide
bond formation (between cysteine residues on different
capsomeres) by reaction of cysteine residues with alkylat-
ing agents, such as iodoacetamide after reduction.
As discussed, the present invention should be broadly
applicable to any HPV Li sequence. There are a variety of
PV types known in the art. Further, particular types of
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PVs are associated with particular infections such as flat
warts, cutaneous warts, epidermodysplasia verruciformis,
lesions and cervical cancer. Over 60 different HPV types
have been identified in clinical lesions by viral nucleo-
tide sequence homology studies. See, for example, Jenson
et al, "Human papillomaviruses" In: Belshe, R. ed., Text-
book of human virology, Second Edition, MASS:PSG, 1989:951
and Kremsdorf et al, J. Virol., 52:1013-1018 (1984). The
HPV type determines, in part, the site of infection, the
pathological features and clinical appearance as well as
the clinical course of the respective lesion.
Because it is believed that there is little or no
cross-immunity for HPV types and immunity to infection is
HPV type-specific, it will be necessary to produce recom-
binant HPV capsomere for each specific HPV type upon which
protection or treatment is needed. However, due to the
homology between the Ll proteins and genes, hybridization
techniques can be utilized to isolate the particular Li
gene of interest. Nucleotide probes selected from regions
of the L1 protein which have been demonstrated to show
sequence homology, can be utilized to isolate other L1
genes. Methods for hybridization are known in the art.
T _
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See, for example, Nucleic Acid Hybridization, A Practical
Approach, IRL Press, Washington, D.C. (1985); Molecular
Cloning, A Laboratory Manual, Maniatis et al, eds., Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY (1982);
and Molecular Cloning, A Laboratory Manual, Sambrook et
al, eds., Cold Spring Harbor Laboratory, Second Edition,
Cold Spring Harbor, NY (1989). Alternatively, PCR methods
can be utilized to amplify Ll genes or gene fragments.
See, for example, U.S. Patent Nos. 4,683,195; 4,683,202;
and 4,800,159.
Virus particles can also be isolated for a particular
papillomavirus type, the DNA cloned, and the nucleic acid
sequences encoding Li proteins isolated. Methods for
isolation of viral particles and cloning of virus DNAs
have been reported. See, for example, Heilman et al. J.
Virology, 36:395-407 (1980); Beaudenon et al, Nature,
321:246-249 (1986); Georges et al, J. Virology, 51:530-538
(1984); Kremsdorf et al, J. Virology, 52:1013-1018 (1984);
Clad et al, Virology, 118:254-259 (1982); DeVilliers et
al, J. Virology, 40:932-935 (1981); and European Patent
Application 0133123.
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Alternatively, the Li protein for a particular human
papillomavirus can be isolated, the amino acid sequence
determined and nucleic acid probes constructed based on
the predicted DNA sequence. Such probes can be utilized
in isolating the L1 gene from a library of the papilloma-
virus DNA. See, for example, Suggs et al, PNAS,
78 (11) :6613-6617 (1981). See also, Young and Davis, PNAS,
80:1194 (1983).
Since the HPV capsomere must express at least one
neutralizing conformational epitope expressed by an in-
tact, infectious HPV, the particular expression system is
important to the invention. An expression system must be
utilized which provides for the production of capsomeres
having appropriate conformation. Such expression systems
should desirably also produce high levels of capsomere.
Generally, the expression system will comprise a vector
having the L1 protein of interest and the appropriate
regulatory regions as well as a suitable host cell.
As discussed, baculovirus vectors are preferably
utilized. A baculovirus system offers the advantage that
a large percentage of cells can be induced to express
protein due to the use of infection rather than transfec-
T iff
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tion techniques. While baculovirus is an insect virus and
grows in insect cells (SF9), these cells retain many of
the eucaryotic mechanisms for processing of proteins
including glycosylation and phosphorylation which may be
important for generating proteins of appropriate conforma-
tion. Baculovirus vector systems are known in the art.
See, for example, Summers and Smith, Texas Agricultural
Experimental Bulletin No. 1555 (1987); Smith et al, Mol.
Cell Biol., 3:2156-2165 (1985); Posse, Virus Research,
5:4359 (1986); and Matsuura, J. Gen. Virol., 68:1233-1250
(1987). Also, it has been reported that baculovirus/-
infected cells express HPV L1 proteins exhibiting appro-
priate conformation.
For expression in an appropriate expression system,
an L1 gene or modified L1 gene is operably linked into an
expression vector and introduced into a host cell to
enable the expression of the L1 protein by that cell. The
gene with the appropriate regulatory regions will be
provided in proper orientation and reading frame to allow
for expression. Methods for gene construction are known
in the art. See, in particular, Molecular Cloning, A
Laboratory Manual, Sambrook et al, eds., Cold Spring
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Harbor Laboratory, Second Edition, Cold Spring Harbor, NY
(1989) and the references cited therein.
A wide variety of transcriptional and regulatory
sequences may be employed. The signals may be derived
from viral sources, where the regulatory signals are
associated with a particular gene which has a high level
of expression. That is, strong promoters, for example, of
viral or mammalian sources, will be utilized. In this
manner, the optimum conditions for carrying our the inven-
tion include the cloning of the Li gene into an expression
vector that will overexpress conformationally-dependent
virus-neutralizing epitopes of the Li protein in trans-
fected or infected target cells.
The suitability of the particular stable HPV capso-
meres produced according to the invention as vaccines or
as diagnostic agents is confirmed by reaction with anti-
bodies or monoclonal antibodies which react or recognize
conformational epitopes present on the intact virion and
based on their ability to elicit the production of neu-
tralizing antiserum. Suitable assays determining whether
neutralizing antibodies are produced are known to those
skilled in the art. Further, this application teaches (in
w a
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Example 6) an in vitro assay suitable for determining
whether the HPV capsomere elicits neutralizing antibodies.
This is an essential characteristic of HPV capsomeres
which are to be used in HPV vaccines. In this manner, it
can be verified whether the HPV capsomeres will confer
protection against HPV infection. Thus, other expression
vectors and expression systems can be tested for use in
the invention.
As discussed, the capsomeres of the present invention
can be utilized to detect, diagnose, serotype, and treat
papillomavirus infection. When used for diagnosis or
serotyping, capsomeres according to the invention may be
labeled using any of a variety of labels and methods of
labeling. Examples of types of labels which can be used
in the present invention include, but are not limited to,
enzyme labels, radioisotopic labels, non-radioactive
isotopic labels, fluorescent labels, toxin labels, and
chemiluminescent labels.
Examples of suitable enzyme labels include malate
hydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast-alcohol dehydrogenase, alpha-glycerol
phosphate dehydrogenase, triose phosphate isomerase,
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peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, cata-
lase, glucose-6-phosphate dehydrogenase, glucoamylase,
acetylcholine esterase, etc.
Examples of suitable radioisotopic labels include 3H,
1251 , 131I, 32p, 35S, 14C, 51Cr, 57To, 56Co,59Fe, 75Se, 152Eu, 90Y
,
67Cu1 217C.i, 211At, 212Pb, 47SC, and 109Pd.
Examples of suitable fluorescent labels include a
152Eu label, a fluorescein label, an isothiocyanate label,
a rhodamine label, a phycoerythrin label, a phycocyanin
label, and allophycocyanin label, an o-phthaldehyde label,
an fluorescamine label, etc.
Examples of suitable toxin labels include diphtheria
toxin, ricin, and cholera toxin. Examples of chemilumi-
nescent labels include a luminal label, an isoluminal
label, an aromatic acridinium ester label, and imidazole
label, and acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, an aequorin label,
etc.
Those of ordinary skill in the art will know of other
suitable labels which may be employed in accordance with
the present invention. The binding of these labels to
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consomers can be accomplished using standard techniques
commonly known to those of ordinary skill in the art.
Typical techniques are described by Kennedy, J.H., et al,
Clin. Chim. Acta, 70:1-31 (1976), and Schurs, A.H.W.M., et
al, Clin. Chim. Acta, 81:1-40 (1977). Coupling techniques
mentioned in the latter are the glutaraldehyde method, the
periodate method, the dimaleimide method, the m-maleimido-
benzyl-N-hydroxy-succinimide ester method, all these
methods incorporated by reference herein.
The detection of the anti-HPV antibodies using the
subject capsomere can be improved through the use of
carriers. Well-known carriers include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases,
natural and modified celluloses, polyacrylamides, agaroses
and magnetite. The nature of the carrier can be either
soluble to some extent or insoluble for the purposes of
the present invention. Those skilled in the art will note
many other carriers suitable for binding proteins, or will
be able to ascertain the same by use of routine experimen-
tation.
The most important aspect of the present invention,
however, inwlves the develUgrnerrt af PV type-specific
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vaccines. The vaccines of the invention will contain an
amount of the subject stable HPV capsomeres sufficient to
induce formation of neutralizing antibodies in the host
contained in a pharmaceutically acceptable carrier.
Administration of the subject capsomere containing
vaccines may be effected by any pharmaceutically accept-
able means, e.g., parenterally, locally or systemically,
including by way of example, oral, intranasal, intrave-
nous, intramuscular, and topical administration. The
manner of administration depends on factors including the
natural route of infection. The dosage administered will
depend upon factors including the age, health, weight,
kind of concurrent treatment, if any, and nature and type
of the particular human papillomavirus. The vaccine may
be employed in dosage form such as capsules, liquid solu-
tions, suspensions, or elixirs, for oral administration,
or sterile liquid formulations such as solutions or sus-
pensions for parenteral or intranasal use. An inert,
immunologically acceptable carrier is preferably used,
such as saline or phosphate-buffered saline.
The vaccines will be administered in therapeutically
effective amounts. That is, in amounts sufficient to
I s
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produce a protective immunological response. Generally,
the vaccines will be administered in dosages ranging from
about 0.1 mg protein to about 20 mg protein, more general-
ly about 0.001 mg to about 100 mg protein. A single or
multiple dosages can be administered.
The method of the present invention makes possible
the preparation of HPV capsomere containing vaccines for
preventing papillomavirus infection. Further, by follow-
ing the methods of the invention, vaccines for any immuno-
genic type of human specific papillomavirus can be made.
As more than one PV type may be associated with PV
infections, the vaccines may comprise stable HPV capso-
meres derived from more than one type of PV. For example,
as HPV 16 and 18 are associated with cervical carcinomas,
therefore a vaccine for cervical neoplasia may comprise
capsomere of HPV 16; of HPV 18; or both HPV 16 and 18.
In fact, a variety of neoplasia are known to be
associated with PV infections. For example, HPVs 3a and
10 have been associated with flat warts. A number of HPV
types have been reported to be associated with epider-
modysplasia verruciformis (EV) including HPVs 3a, 5, 8, 9,
10, and 12. HPVs 1, 2, 4, and 7 have been reported to be
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associated with cutaneous warts and HPVs 6b, lla, 13, and
16 are associated with lesions of the mucus membranes.
See, for example, Kremsdorf et al, J. Virol., 52:1013-1018
(1984); Beaudenon et al, Nature, 321:246-249 (1986);
Heilman et al, J. Virol., 36:395-407 (1980); and
DeVilliers et al, J. Virol., 40:932-935 (1981). Thus, the
subject vaccine formulations may comprise a mixture of
capsomere from different HPV types depending upon the
desired protection.
As indicated, the HPV capsomeres of the invention can
also be utilized for serotyping and for incorporation in
serotyping kits.
For serological testing, the kits will comprise the
subject HPV capsomere and means for detection such as
enzyme substrates, labelled antibody, and the like.
Having now generally described the invention, the
following examples are offered by way of illustration and
not intended to be limiting unless otherwise specified.
EXAMPLES
The following materials and methods were used in the
Examples.
r a
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Chemicals
The anti-HPV mouse monoclonal antibody AU1 was pur-
chased from Berkely Antibody Co., monoclonal antibodies
H11.F1 and H11.A3 were purchased from Pennsylvania State
University (Christensen et al., 1990), horseradish peroxi-
dase (HRP)-labelled goat anti-mouse IgG was purchased
either from Southern Biotechnology Associates, Inc. or
Gibco/BRL, lOX phosphate-buffered saline (PBS) minus Ca2'
or Mg2+ was from Gibco/BRL, ECL reagents were purchased
from Amersham, protein molecular weight standards were
purchased from BioRad, and formvar/carbon-coated copper
grids and phosphotungstic acid were purchased from Elec-
tron Microscopy Sciences. All other reagents were pur-
chased from Sigma.
HPV-11 VLPs
HPV-11 Ll proteins were heterologously expressed in
Trichoplusia ni (High Five ) cells infected with recom-
binant baculovirus encoding the complete L1 open reading
frame downstream of the polyhedron promoter as described
(Ghim et al, Immunology of Human Papillomaviruses, Plenum,
NY, pp 147-152 (1994). Cells were harvested approximately
72 hours post-infection, pelleted by centrifugation, and
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frozen. For preparation of VLPs (all steps performed at 40
C), the cell paste was resuspended in homogenization
buffer (20 mM NaH2POõ 150 mM NaCl, pH 7.4, containing 10
mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin
A) and lysed at -3,000 PSI in a microfluidizer (Micro-
fluidics model HC8000/3A). The homogenized lysate was
then centrifuged at 100,000 x g for 90 minutes, and the
supernatant removed. The pellet, containing HPV-11 VLPs,
was resuspended in PBS containing CsCl (405 g/L) and
centrifuged at 66,000 x g for 90 minutes to remove a
contaminating buoyant layer. The clarified lysate was
then centrifuged overnight at 83,000 x g in a vertical
rotor, and the VLP band was collected (at a density of
1.28 g/cm3). The VLPs were diluted > 2-fold in PBS-0.5M
NaCl, to reduce the density of the solution, and layered
over a two component step gradient composed of 30% and 63%
sucrose (w/w in PBS -0.5M NaCl). The gradients were
centrifuged at 167,000 x g for 3 hours in a vertical
rotor, and the purified VLP band was collected at the
interface between the 30% and 63% sucrose solutions. The
VLPs were then dialyzed into selected buffers (either PBS,
or PBS with NaCl added to a final concentration of 0.3 M
I in
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or 0.5 M), and stored at 4 C. Protein concentration was
determined by the Bradford assay (Bradford, 1976) using
bovine serum albumin as the reference protein, and L1
content was determined as described by Suzich et al.,
Proc. Nat1. Acad. Sci. USA, 92:11553-11557 (1995). Start-
ing with 25-30 g of wet cell paste, the above protocol
yielded 15-25 mg of HPV-11 VLPs.
Sucrose gradient centrifugation
Three types of sucrose gradients were used in these
experiments. First, centrifugation on 30% sucrose cush-
ions was used to identify conditions which favored the
disassembly of VLPs into smaller, soluble components.
100-200 l reaction mixtures containing VLPs (50-100 g
total protein) plus or minus potential disrupting agents
were layered atop 5 ml centrifuge tubes filled with 4.8 ml
of 30% sucrose (w/w in PBS-0.5M NaCl) and centrifuged at
197,000 x g for 2 hours at 4 C in a swinging bucket rotor.
A 50 l aliquot was taken from the very top of the tube,
and mixed with 2X Laemmli sample preparation buffer
(Laemmli, Nature, 227:680-685 (1970). The remainder of
the 30% sucrose cushion was removed by pipet, and the
"pellet" (typically none was visible) avas resaspended in
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100 l of 1X Laemmli sample preparation buffer. The
presence of HPV-11 L1 protein at the top or bottom of the
30% sucrose cushion was then determined by SDS/PAGE, and
the relative amount of Li quantified by analysis of digi-
tized gels. Second, the state of disassembled VLPs was
determined by rate-zonal centrifugation through 5-20%
linear sucrose gradients. Disassembled VLPs (100-200 g
total protein in 400 ml) were layered atop preformed 11.6
ml gradients composed of 5-20% sucrose (w/v in PBS-0.5M
NaCl), and centrifuged at 111,000 x g for 24 hours at 4 C
in a swinging bucket rotor. Fractions (0.5 ml) were col-
lected across the gradient, and the "pellet" (typically
none was visible) was resuspended in 0.5 ml of PBS by
dounce homogenization. The position of HPV-11 Li protein
across the gradient was determined by immunoblotting. The
gradients were calibrated using standard proteins with
established sedimentation coefficients (E. coli R-galac-
tosidase, 19 S; catalase, 11.3 S; bovine serum albumin,
4.3 S), and the percentage of sucrose in the fractions was
determined by refractometry.
Third, the state of initial, disassembled, and reas-
sembled VLPs was determined by rate-zonal centrifugation
11 a
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through 10-65% linear sucrose gradients. HPV-11 Ll protein
(100-200 g total protein in 400 l) in various states of
assembly was layered atop preformed 11.6 ml gradients
composed of 10-65% sucrose (w/v in PBS-0.5M NaCl), and
centrifuged at 188,000 x g for 2.5 hours at 4 C in a
swinging bucket rotor. The gradients were collected (in
1.0 ml fractions), analyzed, and calibrated as above, with
parvovirus B19 (70 S) and HPV-18 L1 VLPs (160 S) used as
additional calibration standards.
Gel Electrophoresis
SDS/PAGE SDS/PAGE was performed largely according to
the method of Laemmli, Nature, 227:680-685 (1970). Sam-
ples were mixed with sample preparation buffer, boiled for
2 minutes, briefly spun in a minifuge, and loaded onto
7.5% (Fig. 1) or 10% (Figs. 2-4) minigels with a 4% stack-
ing gel. Gels were run for approximately 1 hour at 20 mA
constant current at room temperature, and protein was
visualized by staining with Coomassie brilliant blue R250.
InmLunoblotting
Electroblots of HPV-11 L1 from SDS/PAGE gels were
prepared largely according to the method of Towbin et al.,
Proc. Natl. Acad. Sci. USA, 76:4350-4354 (1979). The blots
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were blocked with 1% nonfat milk protein in PBS overnight
at 4 C. The blots were probed with AU1, a mouse mono-
clonal directed against a linear epitope on papillomavirus
L1 proteins (Lim et al., J. Infect. Dis., 162:1263-1269
(1990) for 90 minutes, washed with PBS, 0.1% Tween-80, and
then reblocked for 30 minutes. The blots were then incu-
bated with HRP-labeled goat anti-mouse IgG (Southern
Biotechnology Associates, Inc.) for 40 minutes, and washed
as above. The blots were then developed with ECL Western
blotting reagent, and exposed to X-ray film.
Analysis of gels
The Mr of monomeric and oligomeric L1 were determined
from their Rf values on 7.5$ SDS/PAGE, in comparison to
standard proteins (See and Jackowski, In T. E. Creighton
(ed) Protein structure: a practical approach, IRL Press,
NY, pp 1-21, (1989). When indicated, gels and immunoblots
were digitized on a Hewlett Packard Scanjet Plus flatbed
densitometer, and the relative intensity of bands was
determined using Scan Analysis software (Version 2.2;
Specom Research).
Electron microscopy
~
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Protein samples were allowed to settle on formvar and
carbon-coated copper grids, blotted dry, and stained with
freshly-filtered 2% phosphotungstic acid (pH 6.8). Grids
were examined in a JEOL model 1005 transmission electron
microscope at an accelerating voltage of 100 KV and photo-
graphed at nominal magnifications of 15-25,000x.
Enzyme-linked imanunosorbent assay (ELISA)
HPV-11 Li VLPs (0.5 - 1.0 mg/ml L1) in PBS-0.3 M
NaCl were either stored without treatment at 4 C, or
incubated overnight at 4 C following addition of (3ME (to a
final concentration of 5%) or 2.0 M carbonate buffer, pH
9.6 (to a final concentration of 200 mM carbonate). A
portion of the treated samples were then dialyzed against
4 x 1L PBS-0.5 M NaCl at 4 C for about 24 hrs. All sam-
ples were diluted to a concentration of 0.8 g Li/ml and
distributed into the wells of microtiter plates (80 ng Li
per well). Untreated VLPs and dialyzed material were
diluted into PBS. Sample treated with (3ME without subse-
quent dialysis was diluted into PBS containing 5% (3ME, and
undialyzed sample incubated in 200 mM carbonate was dilut-
ed into 200 mM carbonate, pH 9.6. Following incubation at
37 C for 1 hr, the plates were washed with PBS containing
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0.1% Tween 20 (PBS-Tw) and blocked with 5% nonfat milk
protein in PBS. Monoclonal antibodies were diluted in 1%
nonfat milk in PBS and added to the wells. Following a 1
hr incubation at room temperature, the plates were washed
with PBS-Tw and HRP-labeled goat anti-mouse IgG
(Gibco/BRL) was added. After 1 hr at room temperature,
the plates were washed as above and developed with HRP
substrate. Optical density measurements were made at 405
nm at the 15 min end-point. Averages of duplicate wells
were calculated as the final optical density values.
EXAMPLE 1
Quantitative disassembly of HPV-11 VLPs
Relatively large quantities of HPV-11 L1 VLPs were
prepared as starting material for the study of VLP dis-
assembly and reassembly. HPV-11 Ll VLPs were isolated
from recombinant baculovirus-infected High Five, cells by
CsCl and sucrose gradient centrifugation. The calculated
purity of these Li preparations, based on densitometric
analysis of SDS/PAGE, ranged between 70-90% (see Fig. 1,
lane 2) . In addition, in linear sucrose gradients most of
the protein migrated as expected for a mixture of individ-
r T
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ual and clumped VLPs (Fig. 4a), and in the electron micro-
scope a mixture of intermediate and full-size (50-55 nm)
particles were apparent (Fig. 5a).
The covalent and non-covalent interactions which
stabilize the assembled L1 VLPs are not entirely known,
but earlier work on papillomavirus VLPs and related poly-
omavirus virions and VLPs suggested the importance of
ionic strength, divalent cations (Brady et al, J. Virol.,
23:717-724 (1977); Salunke et al, Biophys. J., 56:887-900
(1987), and disulfide bonds (Sapp et al, J. Gen. Virol.,
76:2407-2512 (1995); Volpers et al, Virology, 200:504-512
(1994). In particular, Sapp and co-workers had demon-
strated by immunoblotting that -50 percent of the L1
protein of HPV-33 VLPs was disulfide-bonded into a range
of larger oligomers with an apparent Mr consistent with
trimers of Ll, and that mild reducing conditions partially
broke down HPV-33 VLPs to the level of capsomeres (Sapp et
al, J. Gen. Virol., 76:2407-2412 (1995); Volpers et al,
Virol., 200:504-512 (1994). In our studies, in the ab-
sence of reducing agents only a portion of the HPV-11 L1
protein migrated on SDS/PAGE with an apparent Mr of 55,000
Da (Fig. 1, Lane 1) = Approxicrrately 40%r (the percentage
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varied between different VLP preparations) of the L1
protein of HPV-11 VLPs was disulfide-bonded into larger
oligomers (Fig. 1, Lane 1), with predicted Mr values of
approximately 144,000 Da (possibly L1 trimer) and
210,000 Da (possibly Li tetramer). The L1 oligomers did
not migrate as a single band, and appeared to be heteroge-
neous in size. The -200,000 Da oligomer was also observed
on immunoblots by Sapp and coworkers (Sapp et al, J. Gen.
Viro1., 76:2407-2412 (1995); Volpers et al, Virol.,
200:504-512 (1994), as part of a broad higher molecular
weight band. These results indicate that a portion of the
L1 proteins in HPV-11 VLPs are disulfide-linked into
higher oligomers. To study the role of disulfide linkages
and other interactions in VLP stability, a rapid screening
assay for VLP disassembly was developed. Purified HPV-11
Li VLPs, both before and after various treatments, were
layered atop 30% sucrose cushions, centrifuged, and the
distribution of L1 protein at the top and bottom of the
30% cushion was visualized by SDS/PAGE. Intact VLPs were
expected to pellet through the 30% sucrose cushion;
non-aggregated capsomeres and Ll monomer were expected to
remain on the top of the cushion. An example of this
T 11
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assay is shown in Fig. 2. To quantitate the relative
disposition of L1 protein, the gels were digitized, the
total intensity of the Li bands at the top and the bottom
of the cushion was determined, and then the percentage of
the Li staining intensity found at either position was
calculated. The results of a number of such determina-
tions are tabulated in Tables 1 and 2. As demonstrated in
Fig. 2, the purified VLP starting material sedimented
through the 30% sucrose, as predicted, with no Ll apparent
at the top. However, upon incubation with a high concen-
tration of the reducing agent R-mercaptoethanol (~ME), Li
protein was found largely at the top of the 30% sucrose
cushion, indicating that the reducing agent had disassem-
bled the HPV-11 VLPs to smaller, non-aggregated compo-
nents. Interestingly, maximal disassembly of the VLPs
typically required exposure to a very high concentration
of reducing agent (in this instance 5%, or 713 mM, RME)
for a relatively long duration (-16 hours at 40 C). Lower
concentrations of reducing agent or shorter durations of
reduction were not as reliably effective at VLP disassem-
bly. Addition of a low concentration of a chelating agent
did riot enhance disassembly (Fig. 2 and Table 2)
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In addition to reductants, the other important vari-
ables for quantitative disassembly of VLPs were found to
be the ionic strength during the disassembly reaction and
the solubility of the VLP starting material. As observed
earlier for polyomavirus virions, lower ionic strength
conditions destabilize VLPs (Brady et al, J. Virol.,
23:717-724 (1977), although Sapp et al, J. Gen. Virol.,
76:2407-2412 (1996) reported that generation of HPV-33
capsomeres from VLPs was insensitive to salt concentration
between 0.15M and 0.6 M NaCl. For HPV-11 VLPs, maximum
disassembly (-90%) of VLPs exposed to 5% RME for 16 hours
was observed at "physiological" ionic strength (i.e., 0.15
M NaCl), but became correspondingly less effective as the
ionic strength was increased (Table 1). The stabilizing
effect of increased ionic strength could be partially
overcome by incubating the VLPs with reducing agents for
longer durations or at elevated temperatures. However,
while incubating the VLPs with 5% (3ME for 120 hours at 4
C, or for 24 hours at 24 C increased the extent of disas-
sembly to 60-70% at 0.5 M NaCl, disassembly was still far
from complete (data not shown). Furthermore, for quanti-
tative disassembly, the degree af aggregation of the VLP
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starting material was also important. In the experiments
reported here, the VLP solutions were dialyzed into dif-
ferent ionic strength buffers and stored at 4 C until use
in disassembly trials. After several days, particularly
at 0.15 M NaCl, the solutions became slightly cloudy,
indicating some degree of aggregation (although little or
no precipitate was observed). Treatment of the clouded
VLP solutions with reducing agents did not yield the same
degree of disassembly as was observed with the initial
soluble VLP solution, indicating that the aggregated VLPs
were resistant to disassembly. However, upon removal of
the aggregated material (which ranged from 10-50% of the
total VLPs depending on the age of the preparation) by
filtration, the remaining soluble VLPs again could be
disassembled to the same extent as the initial soluble VLP
starting material.
Interestingly, even at high concentrations of
chelators, chelation of cations did not significantly
influence VLP disassembly. Dialysis of VLPs into 200 mM
EDTA or EGTA buffers (PBS-0.3 M NaCl, pH 7.4) led to no
apparent disassembly, and the addition of 10 mM dithio-
threitol (DTT) to the dialysis buffers had little effect
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(Table 2). The inability of high concentrations of chela-
tors to disassemble VLPs was confirmed by electron micro-
scopic analysis, although EDTA (but not EGTA) appeared to
swell the VLPs slightly (data not shown). Either these
concentrations of chelator are insufficient to extract
tightly bound, structurally-important ions, or cations are
not essential to maintaining VLP structural integrity.
Conversely, addition of a concentrated aliquot of NaHCO3
buffer (pH 9.6) to a solution of VLPs, to a final concen-
tration of 200 mM carbonate (in PBS-0.3 M NaCl), caused
significant breakdown of the VLPs (Table 2). Addition of
DTT (to a final concentration of 10 mM), did not further
enhance carbonate-induced breakdown. Incubation of VLPs
with 200 mM carbonate/10 mM DTT is commonly used to dena-
ture HPV virions or VLPs in ELISAs (Favre et al, J.
Virol., 15:1239-1237 (1975); Christensen and Kreider, J.
Viro1., 64:3151-3156 (1990); Christensen et al, J. Gen.
Virol., 75:2271-2276 (1994). The effect of carbonate
appears to be buffer specific, and not merely a function
of pH, as incubation of HPV-11 VLPs with pH 9.6 glycine
buffer (200 mM final concentration) caused very little VLP
breakdown, as measured by the 30% sucrose cushion assay
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(Table 2). Similarly, Brady et al, J. Virol., 23:717-724
(1977), observed that carbonate buffer at alkaline pH, but
not alkaline pH alone, dissociated polyomavirus virions.
However, the specific effect of carbonate at pH 9.6 does
not appear to be due to carbonate's potential chelating
ability, as suggested by Brady et al, J. Virol., 23:717-
724 (1977), as 200 mM EDTA at pH 9.6 (+/- 10 mM DTT) was
completely ineffective at VLP disassembly (data not
shown).
EXAMPLE 2
Characterization of disassembled VLPs
Following long-term exposure to high concentrations
of reducing agent, the purified VLPs appear to be broken
down to the level of capsomeres. As shown in Fig. 3a, the
disassembled VLPs generated by incubation with 5% (3ME for
16 hours at 40 C migrated on 5-20% linear sucrose gradients
with an average sedimentation coefficient of 11.3 1.5 S
(n = 5), determined relative to sedimentation standards.
Larger species, with a calculated sedimentation coeffi-
cient of 16-18 S (perhaps dimeric capsomeres), and even
pelleted materials were occasionally observed. However,
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less than 10% of the L1 was detected at the top of the
gradient (expected position for Li monomer) or in the
pellet (expected position for intact VLPs or aggregated
capsomeres), suggesting that the purified VLP starting
material was largely disassembled to the level of individ-
ual capsomeres upon prolonged reduction. This conclusion
is supported by electron microscopic analysis of VLPs
following prolonged incubation with 5% ~ME, which depicted
a field of homogeneous capsomeres (Fig. 5b) averaging
9.7 1.2 nm (n = 15) in diameter, with occasionally a few
larger aggregated structures apparent (monomeric Li would
not be detected with this technique). The estimated capso-
mere diameter is slightly smaller than that observed by
cryoelectronmicroscopy (11-12 nm) (Baker et al, Biophys.
J., 1991; Hagensee et al, J. Virol., 68:4503-4505, (1994);
Belnap et al, J. Mol. Biol., 259:249-263 (1996), perhaps
due to shrinkage during electron microscope grid prepara-
tion. The data demonstrated in Figs. 3a and 5b indicate
that prolonged exposure to high concentrations of reduct-
ants quantitatively disassembles purified, soluble VLPs to
a homogenous population of capsomeres.
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Capsomeres generated from HPV-11 VLPs upon long term
exposure to high concentrations of reducing agent contain
structural epitopes found on intact VLPs. A panel of
HPV-ii-specific monoclonal antibodies has been described
which react with intact HPV-11 Ll VLPs but not with "dena-
tured" Li. These monoclonals include H11.F1, which has
been demonstrated to recognize a dominant neutralizing
epitope on HPV-11 virions, and H11.A3, a distinct
non-neutralizing structure-dependent antibody (Christensen
and Kreider, J. Virol., 64:3151-3156 (1990); Christensen
et al, J. Virol., 64:5678-5681 (1990). As anticipated,
H11.F1 and H11.A3 reacted strongly with the purified
HPV-11 VLP starting material when analyzed by ELISA (Fig.
6a). However, these antibodies also reacted with capso-
meres generated from the VLP starting material by exposure
to reducing agent (Fig. 6b). Thus, capsomeres possess at
least some of the structure-dependent epitopes found on
the surface of intact VLPs and authentic virions, in
agreement with studies performed by Li et al, J. Virol.,
71:2988-2995 (1997) on HPV-11 capsomeres expressed in E.
coli. These results further demonstrate that monoclonal
antibodies H11.F1 and H11.A3, while requiring a "native-
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like" conformation for binding, are not VLP-dependent as
has been previously described (Ludmerer et al, J. Virol.,
71:3834-3839 (1997)).
In contrast, monoclonal antibodies H11.F1 and H11.A3
fail to recognize HPV 11 VLPs dissociated by treatment
with carbonate buffer at pH 9.6 (data not shown;
Christensen et al, J. Gen. Virol., 75:2271-2275 (1994).
Carbonate treatment did not lead to a homogeneous solution
of capsomeres, but instead appeared as an indistinct
mixture of small objects, partially aggregated, when
examined by electron microscopy (data not shown). This
view was partially confirmed by analysis of carbonate-
treated VLPs on 5-20% linear sucrose gradients, in which
the Ll protein largely migrated at -4 S, although a small
population at 9-11 S was observed (Fig. 3b), in agreement
with the effects of carbonate buffer (at pH 10.6, with 10
mM DTT) upon BPV virions (Favre et al, J. Viro1., 15:1239-
1247 (1975)). Finally, while treatment with glycine
buffer at pH 9.6 did not dissociate VLPs to smaller,
individual particles (Table 2), it did have some effect.
VLPs treated with pH 9.6 glycine appeared in the electron
microscope as a poorly-defined mixture of intact, and
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partially-broken down and aggregated VLPs (data not
shown).
EXAMPLE 3
Use of Isolated HPV-11 L1 Capsomeres to Produce induce
virus-Neutralizing Antibodies
Purified L1 capsomeres were used to produce poly-
clonal antisera in rabbits, and three-fold serial dilu-
tions of these antisera along with antisera raised previ-
ously against recombinant HPV-il, 16 and 18 capsids, where
tested against intact capsids of HPV types 11, 16 and 18.
HPV-11 capsomere-specific and capsid-specific polyclonal
antisera were each equally immunoreactive with intact HPV-
11 capsids at all dilutions tested but did not react with
HPV-16 or HPV-18 capsids. These results are consistent
with previous observations made using HPV-11, 16 and 18
capsid-specific polyclonal antisera (Rose et al, J. Gen.
Virol., 75:2445-2449 (1994)), indicating that HPV-11
isolated Li capsomeres are highly immunogenic and retain
genotype-restricted antigenicity exhibited by intact Li
capsids and native HPV virions. When tested in an in
vitro neutralization assay involving infectious HPV-11
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virions, post-immune HPV-11 Ll capsomere-specific poly-
clonal antisera exhibited a neutralization titer of 10-5 -
10-6 which is comparable to that obtained with an antiserum
raised against intact HPV-11 virions in vivo (Rose et al,
J. Gen. Virol., ( Id .)). Pre - immune sera from rabbits
immunized with isolated L1 capsomeres exhibited no virus-
neutralizing activity, nor did a polyclonal antiserum
raised previously against HPV-16 capsids. Thus, isolated
HPV-il capsomeres display a highly immunogenic, genotype-
restricted capsid neutralizing antigenic domain of HPV-11
virions and intact capsids, and should be a useful immuno-
gen for the prevention of genital HPV disease.
TABLE 1
Disassembly of HPV-11 L1 VLPs'; Effects of reducing agentsa
Disassembly Condition 0.15 M NaCI 0.3 m NaCI 0.5 M NaCI
Top Bottom Top Bottom Top Bottom
Starting Material 3.8 0.7 96.3 f 0.8 3.2 f 1.4 96.8 f 1.4 4.2 0.3.4 95.9 f 0.6
5% J3ME, 16 hr 87.7t3.2 12.4t3.1 70.9f 12 29.1 f 12 53.2f6.8 46.8 6.8
5 BME, 1 hr 68.1 t 11 31.9 f 11 68.0 f 10 32 f 10 - -
2% l3ME, 16 hr 72.1 f 2.7 27.9 t 2.7 67.6 t 21 32.3 t 612 - -
0.5%l3ME, 16hr 45.8f18 54.2f16 28.8t16 71.2t16 - -
10 mM DTT, 16hr 44 .5 t 11 55 .5 t 11 43. 8 t 20 56.2 t 20 - -
10 mM DTT, 1 hr 9.5 f 6.4 90.5 t 6.4 - - - -
9
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lOmM DTT, 5mM 55.9 t 6.2 44.1 t 6.2 - - -
EDTA, 16 hr
aVLPs (0.5-1.0 mg/ml protein) were treated as indicat-
ed at 4 C, and the distribution of L1 across a 30% sucrose
cushion was determined as described in the Methods sec-
tion. Shown are the means of multiple determinations
(n=3-7) the standard deviation.
TABLE 2
Disassembly of HPV-11 L1 VLPs; Effects of chelators and
buffersa
Disassembly Condition Top Bottom
200 mM EDTA, pH 7.4 4 t 3 96 f 3
200 mM EDTA, 10 mM DTT 10 f 6 90 t 6
200 mM EGTA, pH 7.4 13 t 11 87 t 11
200 mM EGTA , 10,M DTT 11 f 6 89t6
200 mM NaHCO3, pH 9.6 81 2 19t2
200 mM NaHC03 , 10 mM DTT 74 f 11 26 t 11
200 mIvl glycine, pH 9.6 11 t 1 89 t 1
200 mM glycine, 10 mM DTT 41 t 12 59 t 11
aVLPs (0.5-1.0 mg/ml protein) were treated as indicat-
ed for 16 hours at 4 C, and the distribution of Li across
of 30% sucrose cushion was determined as described in the
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Methods section. Shown are the averages of duplicate
determinations the range.
The invention may be embodied in other specific forms
without departing from its spirit or essential character-
istics. The described embodiments are to be considered in
all respects only as illustrative and not restrictive, and
the scope of the invention is, therefore, indicated by the
appended claims rather than by the foregoing description.
All modifications which come within the meaning and range
of the lawful equivalency of the claims are to be embraced
within that scope.
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