U.S. patent application number 09/822662 was filed with the patent office on 2002-12-26 for protecting against canine oral papillomavirus (copv).
This patent application is currently assigned to Georgetown University School of Medicine. Invention is credited to Ghim, Shin-Je, Jenson, A. Bennett, Schlegel, C. Richard.
Application Number | 20020197264 09/822662 |
Document ID | / |
Family ID | 26673341 |
Filed Date | 2002-12-26 |
United States Patent
Application |
20020197264 |
Kind Code |
A1 |
Schlegel, C. Richard ; et
al. |
December 26, 2002 |
Protecting against canine oral papillomavirus (COPV)
Abstract
Recombinantly produced LI major capsid proteins which mimic
conformational naturalizing epitopes on human and animal papilloma
virions including canine and equine papilloma virions are provided.
These recombinant proteins are useful as vaccines for conferring
protection against papillomavirus infection. Antibodies to the
recombinant protein are also provided. Such antibodies are useful
in the diagnosis and treatment of viral infection.
Inventors: |
Schlegel, C. Richard;
(Rockville, MD) ; Jenson, A. Bennett; (Rockville,
MD) ; Ghim, Shin-Je; (Washington, DC) |
Correspondence
Address: |
Pillsbury Winthrop LLP
Intellectual Property Group
Ninth Floor
1100 New York Avenue, NW
Washington
DC
20005-3918
US
|
Assignee: |
Georgetown University School of
Medicine
|
Family ID: |
26673341 |
Appl. No.: |
09/822662 |
Filed: |
April 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09822662 |
Apr 2, 2001 |
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09134377 |
Aug 14, 1998 |
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09134377 |
Aug 14, 1998 |
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08724281 |
Oct 1, 1996 |
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5874089 |
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60004691 |
Oct 2, 1995 |
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Current U.S.
Class: |
424/184.1 ;
424/186.1; 424/192.1; 424/199.1; 424/204.1; 435/235.1; 435/320.1;
435/69.1; 435/69.3; 536/23.72 |
Current CPC
Class: |
A61K 39/12 20130101;
Y10S 977/802 20130101; A61K 2039/552 20130101; Y10S 977/918
20130101; C07K 14/005 20130101; G01N 2333/025 20130101; A61K
2039/5252 20130101; A61P 31/12 20180101; C12N 2710/20022 20130101;
C12N 2710/14143 20130101; C12N 2710/20034 20130101; A61K 39/00
20130101 |
Class at
Publication: |
424/184.1 ;
424/204.1; 424/186.1; 424/192.1; 424/199.1; 435/235.1; 435/320.1;
435/69.1; 435/69.3; 536/23.72 |
International
Class: |
A61K 039/12; C07H
021/04; C12P 021/06; C12N 015/09; A61K 039/00; A61K 039/38; C12N
007/00; C12N 007/01; C12N 015/00; C12N 015/63; C12N 015/70; C12N
015/74 |
Claims
What is claimed is:
14. A vaccine for conferring protection against human
papillomavirus (HPV) infection in a human susceptible to human
papillomavirus infection which comprises (i) a prophylatically
effective amount of an extract comprising a formalin-treated human
papillomavirus L1 protein and (ii) a pharmaceutically acceptable
carrier.
16. The vaccine of claim 14 wherein said human papillomavirus is
selected from the group consisting of HPV 1, HPV 2, HPV 3, HPV 4,
HPV 6, HPV 7, HPV 10,HPV 11, HPV 12, HPV 16 and HPV 18.
17. A method of protecting a human against human PV infection
comprising administering a prophylatically effective amount of a
vaccine according to claim 14.
20. The method of claim 17, wherein said administered vaccine
comprises a formalin-inactivated human papillomavirus selected from
the group consisting of HPV1, HPV2, HPV3, HPV4, HPV6, HPV7, HPV10,
HPV11, HPV12, HPV16 and HPV18.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/134,377, filed Aug. 14, 1998, which is a divisional of U.S. Ser.
No. 08/724,281, filed Oct. 1, 1996, now U.S. Pat. No. 5,874,089,
and which claims benefit of priority from U.S. Provisional Serial
No. 60/004,691, filed Oct. 2, 1995.
FIELD OF THE INVENTION
[0002] The invention relates to the diagnosis, serotyping,
prevention and treatment of viral diseases, particularly
papillomavirus infections.
[0003] More particularly, the invention relates to the diagnosis,
serotyping, prevention and treatment of human papillomavirus
infections, equine papillomavirus infections and canine
papillomavirus infections.
BACKGROUND OF THE INVENTION
[0004] Papillomaviruses (PV) are members of the papovavirus family
and contain a double stranded circular DNA genome with a typical
size of about 7900 base pairs (bp). Human papillomaviruses (HPV)
are recognized as a cause of various epithelial lesions such as
warts, condylomas and dysplasias. See, Gissman, L., Cancer Survey,
3:161 (1984); Boshart et al, EMBO J., 3:1151 (1984); Romanczuk et
al, J. Virol., 65:2739-2744 (1991); Jenson et al, In
"Papillomaviruses and human cancer" (H. Pfister. Ed.), pp. 11-43,
CRC Press (1990); Schlegel, R., "Papillomaviruses and human cancer"
In: Viral pathogenesis (ed. Fujinami, R.), Seminars in Virology
1:297-306 (1990); and Jenson et al, "Human Papillomaviruses" In
Belshe, R. ed. Textbook of human virology, Second Edition:
MASS:PSG, 1989:951.
[0005] HPVs are grouped into types based on the similarity of their
DNA sequence. Two HPVs are taxonomically classified as being of the
same type if their DNAs cross-hybridize to greater than 50% as
measured by hybridization in solution under moderately stringent
hybridization conditions.
[0006] A number of distinct papillomaviruses have been shown to
infect humans. Papillomaviruses are highly species and
tissue-specific, and are characterized by a specific mode of
interaction with the squamous epithelia they infect. These small
DNA tumor viruses colonize various stratified epithelia like skin
and oral and genital mucosa, and induce the formation of
self-limiting benign tumors known as papillomas (warts) or
condylomas. These tumors are believed to arise from an initial
event in the infectious cycle where the virus enhances the division
rate of the infected stem cell in the epithelial basal layer,
before it is replicated in the differentiating keratinocyte.
[0007] The term papillomavirus covers a large number of viruses
which are considered responsible for several forms of viral
infection ranging from relatively benign warts of the skin or
mucous membranes to hyperplasias susceptible to progressing into
dysplasias or intra-epithelial neoplasms, and malignant conversion
to various forms of cancer, the most significant being that of the
female uterine cervix.
[0008] A number of HPVs types have been identified. Furthermore,
the preferential association of certain HPV types with anatomic
location and distinct types of lesions gives support to the
hypothesis that different HPV-induced lesions constitute distinct
diseases, and that the clinical patterns of lesions express
specific biological properties of distinct types of HPVs.
Distinctive histological features have been associated with the
infection of the skin or mucous membranes by different types of
HPVs.
[0009] The genomes of different HPV types have been cloned and
characterized. In particular, the genomes of two HPV types, HPV 16
and HPV 18, have been found to be associated with about 70% of
invasive carcinomas of the uterine cervix.
[0010] Human papillomaviruses which infect the genital tract mucosa
play a critical role in the development of cervical cancer. See,
Lorincz et al, Obstetrics & Gynecology, 79:328-337 (1992);
Beaudenon et al, Nature, 321:246-249 (1986); and Holloway et al,
Gynecol. Onc., 41:123-128 (1991). For example, the majority of
humans cervical carcinomas (95%) contain and express HPV DNA and it
is the expression of two viral oncoproteins, E6 and E7, which
appears to be critical for cellular transformation and maintenance
of the transformed state. Despite the detailed knowledge concerning
the molecular mechanism of action of these oncoproteins, there is
little information available on the biology of papillomavirus
infection, including the identity of viral receptors, the control
of viral replication and assembly, and the host immune response to
virus and virally-transformed cells. An effective vaccine against
HPV infection could potentially reduce the incidence of human
cervical dysplasia and carcinoma by 90-95%. However, there is no
tissue culture system which permits sufficient keratinocyte
differentiation to propagate the PV in-vitro. Because of the
widespread occurrence of HPV infection, methods for detecting,
preventing and treating viral infection are needed. Also, methods
for detecting, preventing and treating papillomavirus infection in
animals, e.g., equines and canines, are also needed.
[0011] Canine papillomas were one of the first animal systems
studied when McFaydean and Hobday transmitted the oral papilloma in
1898. Today, dogs are commonly used as models for a variety of
diseases and much is known about their physiology and immune
system. Papillomas affect many anatomic locations in dogs, similar
to the human diseases. Puppies may have marginal papillae on their
tongues which are normal anatomic structures resembling oral
papillomas. True papillomas can be found on the dorsal tongue and
buccal mucosa, ocular mucous membranes, mucous membranes of the
lower genital tracts of both males and females, and haired skin.
The lesions are characterized by epithelial proliferation on thin
fibrovascular stalks and there may be specific cytopathic effects
in the stratum granulosum in which the cells swell, develop large
keratohyalin-like granules, and may have intranuclear inclusions.
Group-specific papillomavirus antigens can be detected by the cells
exhibiting cytopathic effects.
[0012] The canine oral papillomavirus has been cloned and
characterized (Sundberg et al, Amer. J. Vet. Res., 47(5), 1142-1177
(1986)). The COPV viral genome was cloned into pBR322, a
restriction map constructed, with the completeness of the COPV
genome confirmed by comparison of restriction fragment sizes
derived from cloned and virion DNA. (Id.) It is known that COPV is
antigenically similar to other papillomaviruses. For example, it
has been reported that some of the antigenic and immunogenic
epitopes of HPV16 and bovine, canine and avian papillomaviruses are
shared. (Dillner et al, J. Virol., 65(12), 5862-6871, (1991)).
[0013] Strong evidence suggests that canine papillomaviruses play a
role in squamous cell carcinoma development. For example,
papillomavirus antigens are detected in penile and vulvar
carcinomas. Also, it has been reported that intramuscular injection
of canine oral papillomavirus results in the later development of
cutaneous squamous cell carcinoma.
[0014] Papillomas are also prevalent in equines. In fact,
papillomas are probably the most common equine tumor; however, few
are ever submitted to diagnostic laboratories for histologic
confirmation. Papillomas in equines generally affect the skin,
mouth, lower genital tract and eyes. Papillomavirus which causes
infection in equines is of the cutaneous type. Equine
papillomaviruses have also been isolated and cloned. It is also
known that equine papillomavirus infection causes millions of
dollars in losses annually to the equine industry. Thus, based on
the foregoing, it is clear that there exists a need for effective
vaccines against papillomaviruses including HPV's and animal
papillomaviruses such as COPV and equine papillomavirus.
SUMMARY OF THE INVENTION
[0015] Toward that end, a recombinantly produced L1 major capsid
protein which mimics conformational neutralizing epitopes on human
and animal papilloma virions is provided. The recombinant protein
reproduces the antigenicity of the intact, infectious viral
particle. The recombinant protein can be utilized to
immunoprecipitate antibodies from the serum of patents infected or
vaccinated with PV. Neutralizing antibodies to the recombinant
protein are also provided. The antibodies are useful for the
diagnosis and treatment of papilloma viral infection. The invention
additionally provides subviral vaccines for the prevention of human
and animal papillomavirus infection, e.g., for preventing equine
and canine papillomavirus infection.
[0016] More specifically, recombinantly provided L1 major capsid
proteins which mimic the conformational neutralizing epitopes on
human, equine and canine papilloma virions are provided. These
recombinant capsid proteins reproduce the antigenicity of the
intact infectious human, canine or equine virus particle. The
recombinant proteins can be utilized to immunoprecipitate
antibodies from the serum of humans, equines or canines infected or
vaccinated with the corresponding PV. Neutralizing antibodies to
the human, canine or equine papillomavirus capsid protein are also
provided. These antibodies are useful for the diagnosis and
treatment of human, canine or equine papilloma viral infections.
The invention further provides subviral vaccines for the prevention
of human, canine and equine papillomavirus infection.
[0017] The invention further provides a unique and relevant canine
animal model for the development of papillomavirus vaccines, in
particular canine and human papillomavirus vaccines; which unlike
the available rabbit and bovine papillomavirus models, utilizes the
canine oral papillomavirus (COPV) which is tropic for mucous
membranes and is assayable for infectivity under normal conditions
of exposure.
DESCRIPTION OF THE FIGURES
[0018] FIG. 1. Reactivity of rabbit polyclonal antisera and mouse
monoclonal antibodies with SDS-disrupted HPV-1 as determined by
immunoblot analysis.
[0019] Purified HPV-1 virions were denatured with SDS and their
constituent proteins separated by SDS polyacrylamide gel
electrophoresis. The HPV-1 proteins were then transferred
electrophoretically to nitrocellulose and reacted with 1:100
dilutions of the rabbit antisera or monoclonal antibodies (ascites
fluid). MAB45, which was produced as a hybridoma supernatant, was
only diluted 1:10. Primary antibody reactivity was detected using
alkaline phosphatase-labelled goat anti-rabbit or anti-mouse IgG
(Bio-Rad) at a dilution of 1:1000 in PBSA. Only rabbit antiserum #3
and MAB45, which both recognize denatured HPV-1 virions by ELISA,
were found to react significantly with denatured L1 protein (see
arrow).
[0020] FIG. 2. Construction of SV40 vector, pSJ-1, which expresses
the HPV-1 L1 gene.
[0021] The L1 gene of HPV-1 was amplified from cloned HPV-1 DNA
using 5' and 3' oligonucleotide primers which contained XhoI and
BamHI enzyme restriction sites, respectively. The plasmid,
designated pSJ-1, contained the HPV-1 L1 gene expressed by the SV40
late promoter. The plasmid also contained the SV40 origin of
replication (ori) as well as the SV40 VP1 intron and late
polyadenylation signals. The entire pSJ-1 L1 gene was sequenced in
its entirety and found to be identical to the genomic HPV-1 L1
sequence.
[0022] FIG. 3. immunoprecipitation of HPV-1 L1 protein from COS
cells transfected with pSJ-1.
[0023] COS cells, grown in 10 cm diameter plastic plates, were
transfected when 80% confluent with 10 .mu.g pSJ-1 plasmid DNA
using a calcium phosphate precipitation technique (Graham, F. L.,
and van der Eb., A. J., Virology 52:456-467 (1973). 48 hr later,
the cells were metabolically labelled with 500
.mu.Ci/ml.sup.35S-methionine for 4 hr in 2.5 ml cysteine and
methionine-free medium. The cells were then washed with PBS,
extracted with RIPA buffer, and immunoprecipitated with the
indicated rabbit antisera or mouse monoclonal antibodies. The
immunoprecipitated proteins were then analyzed by SDS-gel
electrophoresis and autoradiography. All immune polyclonal antisera
and monoclonal antibodies were able to immunoprecipitate L1 protein
(see arrow). Lanes 1 and 4 show the absence of L1 protein when
extracts were precipitated with either non-immune rabbit serum
(lane 1) or with non-immune murine serum (lane 4).
[0024] FIG. 4. Immunofluorescent staining of cos cells transfected
with pSJ-1.
[0025] COS cells grown on glass coverslips were transfected with 10
.mu.g pSJ-1 as described in FIG. 3. After 48 hr, the coverslips
were washed with PBS, fixed in cold acetone, and reacted with 1:250
dilutions of rabbit antisera or mouse monoclonal antibodies. The
reacted primary antibodies were stained with FITC-labeled goat
anti-IgG at the dilution of 1:10 in PBS (Cappel). Nuclei of
approximately 5-10% of transfected cos cells were positive by
immunofluorescence. The evaluated antibodies were R#3 (panel a),
R#7 (panel b), MAB45 (panel c), 334B6 (panel d), 339B6 (panel e),
D54G10 (panel f), and 405D5 (panel g). All antisera were
non-reactive with cos cells transfected with the parent pSVL vector
lacking the HPV-1 L1 gene, including R#3 (panel h).
[0026] FIG. 5 contains results of an experiment wherein beagle dogs
were administered serum obtained from beagle dogs vaccinated with a
formalin-inactivated canine oral papillomavirus (COPV) L1 protein;
or were administered serum from non-immune beagles, or lactate
Ringers solution. The results show that the dogs administered the
immune dog serum did not show any sign of papillomas after
challenge with live infectious COPV, whereas both the group
administered non-immune serum or lactate Ringers solution developed
papillomas.
[0027] FIG. 6 contains results of an experiment wherein a first
control group of dogs were mock vaccinated with PBS (Group I), a
second group vaccinated with formalin-fixed wart homogenates (Group
II), a third group vaccinated with 20 .mu.g L1 contained in PBS
(Group III), a fourth group with 20 .mu.g of L1 protein contained
in alum (Group IV), and a fifth group with 20 .mu.g L1 protein in
QS21 adjuvant (Group V) (wherein the COPV L1protein was produced in
recombinant baculovirus infected S9 cells).
[0028] FIGS. 7 and 8 contain the results of an experiment wherein
the antibody response against both linear and conformational COPV
L1 epitopes were compared after a first vaccination, after a second
vaccination, and after challenge with infectious COPV. In this
experiment, the animal groups were administered at 8 and 10 weeks
as follows: Group I mock vaccinated with 0.2 ml PBS; Group II
vaccinated with 0.2 ml formalin-fixed wart homogenate; Group III
vaccinated with 0.2 ml of a composition comprising 20 .mu.g of L1
protein in PBS; Group IV vaccinated with 20 .mu.g of a composition
comprising L1 protein in PBS containing alum, and Group V
vaccinated with 0.2 ml of a composition comprising 20 .mu.g L1
protein in QS21 adjuvant.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0029] Methods and compositions are provided for the prevention,
detection and treatment of papillomavirus (PV) infection. The
methods are based upon the production of a recombinant L1 major
capsid protein which is capable of reproducing the conformational
neutralizing epitopes on human and animal papillomavirus virions.
The invention is further drawn to antigenic fragments of such
recombinant L1 proteins.
[0030] Although papillomaviruses infect a wide variety of
vertebrate species, they exhibit a remarkable conservation of
genomic organization and capsid protein composition.
Papillomaviruses consist of small (about 55 nm), non-enveloped
virions which surround a genome of double-stranded, circular DNA.
The genome is approximately 8,000 bp in length and can be divided
into equal-length "early" and "late" regions. The "early" region
encodes 7-8 genes involved in such processes as viral DNA
replication (the E1 and E2 genes), RNA transcription (the E2 gene),
and cell transformation (the E5, E6 and E7 genes). The "late"
region encodes two structural proteins, L1 and L2, which represent
the major and minor capsid proteins, respectively. All of the
"early" and "late" genes are transcribed from the same strand of
viral DNA.
[0031] There are a variety of PV types known in the art. Further,
particular types of PVs are associated with particular infections
such as flat warts, cutaneous warts, epidermodysplasia
verruciformis, lesions and cervical cancer. Over 50 different HPV
types have been identified in clinical lesions by viral nucleotide
sequence homology studies. See, for example, Jenson et al, "Human
papillomaviruses" In: Belshe, R. ed., Textbook 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.
[0032] The L1 protein represents the most highly conserved protein
of all the papillomavirus proteins. The nucleotide sequence of the
L1 open reading frames of BPV-1, HPV-1A, and HPV-6B are given in
U.S. Pat. No. 5,057,411, which disclosure is incorporated herein by
reference. Furthermore, it is noted that L1 proteins and fusion
proteins have been produced recombinantly. However, prior to the
present invention, it was not known that L1 proteins with
sufficient fidelity to maintain a conformation equivalent to that
found in intact papillomavirus virions could be produced.
Previously, recombinant L1 protein was produced as linear molecules
which were incapable of protecting against papillomavirus
infection. The present invention, in contrast, provides
conformationally correct protein which is capable of inducing
neutralizing antibodies which protect against animal and human
papillomaviruses.
[0033] Because it is believed that there is little or no
cross-immunity for PV types and immunity to infection is PV
type-specific, it will be necessary to produce recombinant L1
protein for each specific PV type upon which protection or
treatment is needed. However, due to the homology between the L1
proteins and genes, hybridization techniques can be utilized to
isolate the particular L1 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.
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, N.Y. (1982); and Molecular Cloning, A Laboratory
Manual, Sambrook et al, eds., Cold Spring Harbor Laboratory, Second
Edition, Cold Spring Harbor, N.Y. (1989). Alternatively, PCR
methods can be utilized to amplify L1 genes or gene fragments. See,
for example, U.S. Pat. Nos. 4,683,195; 4,683,202; and
4,800,159.
[0034] Virus particles can also be isolated for a particular
papillomavirus type, the DNA cloned, and the nucleic acid sequences
encoding L1 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.
[0035] Alternatively, the L1 protein for a particular
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 papillomavirus DNA. See, for example, Suggs et al,
PNAS, 78(11):6613-6617 (1981). See also, Young and Davis, PNAS,
80:1194 (1983).
[0036] Since the recombinant L1 protein must be of suitable
conformation to mimic that of the intact virus particle, the
expression system is crucial to the invention. An expression system
must be utilized which produces L1 protein in the correct
conformation. That is, the recombinant L1 protein reproduces the
antigenicity of intact infectious virus particles. Such expression
systems should also produce high levels of capsid protein.
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. Typically a suitable host will be one
which provides eucaryotic mechanisms for processing of the
proteins.
[0037] Ideally, a strong promoter is utilized for high expression
of the recombinant protein. Of particular interest is the pSVL
vector. The pSVL vector contains an SV40 origin of replication and
when transfected in COS cells, which express Large T antigen,
replicates to a high copy number.
[0038] Alternatively, baculovirus vectors can be 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 transfection 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 conformation. 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).
[0039] In particular, this application exemplifies the expression
of the canine oral papillomavirus (COPV) L1 protein in Sf9 cells
using a baculovirus expression system and demonstrates that the
resultant L1 proteins comprise conformational epitopes and confer
protection when administered to naive dogs (beagles) upon challenge
with live infectious COPV.
[0040] COPV was selected for several reasons. First, because of the
high level of similarity between COPV and HPV's at the DNA and
amino acid sequence level, genetic organizational level, as well as
similar mucosal route of infection, COPV provides a highly suitable
in vivo model for study of HPV vaccines. More specifically, dogs
may be inoculated with COPV L1 proteins and challenged with live
COPV in order to provide relevant in vivo evidence regarding the
effectiveness of conformational PV L1 proteins to confer immunity
against the corresponding papillomavirus.
[0041] While this is tremendously advantageous by itself, the COPV
L1 protein is also important in its right. As discussed above, COPV
is a mucosal papillomavirus which results in papillomas in canines
that are found in the dorsal tongue and buccal mucosa; ocular
mucous membranes, mucous membranes of the lower genital tracts of
both males and females, and haired skin. Moreover, COPV is believed
to play a role in squamous cell carcinoma. Therefore, a vaccine
against COPV is highly desirable because it may be used to prevent
papillomas in canines, and also squamous carcinoma caused by
COPV.
[0042] Moreover, given the substantial similarities between COPV
and HPVs, in particular those which cause cancer in humans, the
COPV/beagle animal model has applicability in screening the
effectiveness of potential antiviral agents for treating human
papillomavirus infection. Essentially, this will involve
administering an antiviral agent predicted to be useful for
treating human papillomaviral infection to a beagle dog which has
been infected with COPV and ascertaining the effects of this
antiviral agent on the status of COPV infection. This may be
effected, e.g., by observing the size and number of papillomas in
the treated animal before and after treatment with the antiviral
agent. Antiviral agents which inhibit papilloma development or
result in their decrease in size and/or number in treated animals
should possess similar activity in humans for treating HPV
infection given the similarities between COPV and HPVs.
[0043] Another animal PV where L1 conformational proteins have
application in the design of vaccines is equine papillomavirus. As
noted, equine papillomavirus is probably the most common cause of
equine tumor. Squamous cell carcinomas, which are believed to be
caused by equine papillomaviral infection, are also common in
horses. This is substantiated by the fact that such carcinomas have
an anatomic distribution similar to papillomas. One of the most
common locations of such carcinomas is the lower genital tract.
Moreover, equine papillomavirus infection results in substantial
expense (many millions of dollars yearly) to the equine industry.
Therefore, an equine papillomavirus vaccine produced according to
the invention should possess tremendous potential for protecting
equines against equine papillomavirus infection and squamous cell
carcinoma caused thereby. As with the afore-described
papillomavirus vaccines, this vaccine will comprise a
prophylactically effective amount of recombinant equine
papillomavirus L1 proteins or fragments which exhibit the
conformation of L1 proteins expressed by native equine
papillomavirus virions. Based on the high level of sequence
similarities between the L1 sequences of different
papillomaviruses, the equine papillomavirus L1 sequence can readily
be cloned and expressed in a suitable expression system, e.g.,
baculovirus.
[0044] For expression in an appropriate expression system, the 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 Harbor Laboratory, Second
Edition, Cold Spring Harbor, N.Y. (1989) and the references cited
therein.
[0045] A wide variety of transcriptional and translational
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 out the invention include the cloning of the L1 gene into
an expression vector that will overexpress
conformationally-dependent epitopes of the L1 protein in
transfected or infected target cells.
[0046] The recombinant L1 protein is confirmed by reaction with
antibodies or monoclonal antibodies which react or recognize
conformational epitopes present on the intact virion. In this
manner, the L1 protein can be verified as having the suitable
conformation. Thus, other expression vectors and expression systems
can be tested for use in the invention.
[0047] As discussed, it is essential that the expressed L1 protein
be conformational, i.e., that it contain conformational epitopes
that are necessary for a protective immunogenic response. This will
typically be accomplished by expression of the entire L1 sequence
of the particular papillomavirus, e.g., COPV or a human
papillomavirus, e.g., HPV-6, HPV-11, HPV-16, HPV-18, among others.
However, the invention also embraces expression of L1 DNA
fragments, i.e., which do not comprise the entire L1 coding
sequence but which upon expression still produce conformational L1
proteins, i.e., L1 proteins which contain conformational
epitopes.
[0048] The specific L1 DNA fragments which results in the
expression of conformational L1 proteins may be identified, e.g.,
by expressing different fragments of a particular L1 DNA, and
ascertaining whether the resultant protein is conformational. This
may be effected, e.g., by determining whether the particular L1
fragment reacts with or elicits the production of antibodies
specific to conformational L1 epitopes.
[0049] To confirm that PV L1 DNA fragment encoding less than the
entire L1 protein may be obtained which upon expression result in
conformational L1 proteins, fragments of the COPV L1 open reading
frame were expressed. In particular, fragments of the COPV L1 open
reading frame were expressed which contained either a deletion in
the amino-terminal or carboxy-terminal portion of the L1 sequence.
It was found that the L1 protein containing the amino-terminal
deletion expressed in a SV40 vector in COS cells apparently did not
result in conformational L1 proteins (when tested with
conformationally-dependent antibodies). By contrast, the L1
sequence which contained a deletion in the carboxy-terminal region
when expressed in COS cells using the same SV40 vector system
resulted in conformational L1 proteins (as demonstrated by binding
to antibodies which recognize conformational epitopes). This
carboxy-deletion consisted of deletion of the 26 amino acid
fragment of the COPV L1 sequence, which was replaced by a 5 amino
acid nuclear sequence of a nonstructural viral protein (large T
protein) of SV40. The 26 amino acids of the COPV L1 sequence
deleted include the nuclear signal sequences necessary for
translocation of the native L1 protein into the cell nucleus. The
particular nuclear signal sequence is not critical and is only
necessary for transport into the nucleus. In this regard, many
nuclear signal sequences are well known in the art. Thus, these
results demonstrate that L1 fragments encompassing the carboxy
terminus of the L1 protein are not necessary for reproducing
conformational epitopes.
[0050] While only 26 amino acids of the carboxy-terminus were
deleted, it is reasonable to assume that larger deletions will also
result in conformational L1 proteins. As indicated, those deletions
which are operable, i.e., which result in conformational L1
proteins, may be identified based on the reactivity of the
resultant L1 fragments with conformational antibodies. This may be
determined, e.g., by immunofluorescence or immunoprecipitation
using conformational L1 antibodies.
[0051] Also, while only a carboxy-terminal deletion was
demonstrated to yield conformational L1 proteins upon expression,
it is believed that other deletions may also result in
conformational L1 proteins. For example, internal deletions may
also result in the formation of conformational L1 proteins. Also,
it is expected that substitution mutations may be identified which
result in conformational L1 proteins. Such substitutions may
potentially be made in both the conserved and hypervariable regions
of the L1 protein.
[0052] Moreover, while only COPV L1 fragments (containing deletion
of 26 carboxy-terminal amino acids) were demonstrated to yield
conformational L1 proteins, it is reasonable to expect that similar
results will be obtained with other PV L1 sequences, given their
high level of homology. It is especially reasonable to assume that
similar results will be observed with carboxy-terminal deletions of
HPV L1 sequences given the substantial similarities between HPVs
and COPV.
[0053] COPV and HPVs associated with human malignancy are highly
similar. They exhibit similar genetic organization, viral
structure, capsid protein sequences, and selectively infect a
mucosal site of infection. Based on these similarities,
carboxy-deletions of HPV L1 sequences should also result in
conformational L1 proteins when expressed according to the
invention. This can be confirmed by testing with conformational
antibodies specific to the particular HPV L1 fragment being
expressed.
[0054] Once the L1 protein of suitable conformation has been
expressed, antibodies can be raised against the recombinant protein
or antigenic fragments thereof. The antibodies of the present
invention may be prepared using known techniques. Monoclonal
antibodies are prepared using hybridoma technology as described by
Kohler et al, Nature, 256:495 (1975); Kohler et al, Eur. J.
Immunol., 6:511 (1976); Kohler et al, Eur. J. Immunol., 6:292
(1976); Hammerling et al, in: Monoclonal Antibodies and T-Cell
Hybridomas, Elsavier, N.Y., pages 563-681 (1981). Such antibodies
produced by the methods of the invention are capable of protecting
against PV infection.
[0055] The term "antibody" includes both polyclonal and monoclonal
antibodies, as well as fragments thereof, such as, for example, Fv,
Fab and F(ab).sub.2 fragments which are capable of binding antigen
or hapten. Such fragments are typically produced by proteolytic
cleavage, such as papain, to produce Fab fragments or pepsin to
produce F(ab).sub.2 fragments. Alternatively, hapten-binding
fragments can be produced through the application of recombinant
DNA technology or through synthetic chemistry.
[0056] As indicated, both polyclonal and monoclonal antibodies may
be employed in accordance with the present invention. Of special
interest to the present invention are antibodies which are produced
in humans or are "humanized" (i.e., non-immunogenic in a human) by
recombinant or other technology. Humanized antibodies may be
produced, for example, by placing an immunogenic portion of an
antibody with a corresponding, but non-immunogenic portion,
chimeric antibodies. See, for example, Robinson et al,
International Patent Publication PCT/US86/02269; Akira et al,
European Patent Application 184,187; Taniguchi, M. European Patent
Application 171,496; Morrison et al, European Patent Application
173,494; Neuberger et al, PCT Application WO86/01533; Cabilly et
al, European Patent Application 125,023; Better et al, Science,
240:1041-1043 (1988); Liu et al, PNAS, 84:3439-3443 (1987); Liu et
al, J. Immunol., 139:3521-3526 (1987); Sun et al, PNAS, 84:214-218
(1987); Nishimura et al, Cancer Research, 47:999-1005 (1987); Wood
et al, Nature, 314:446-449 (1985); and Shaw et al, J. National
Cancer Inst., 80:1553-1559 (1988). General reviews of "humanized"
chimeric antibodies are provided by Morrison, S. L., Science,
229:1202-1207 (1985) and by Oi et al, BioTechniques, 4:214
(1986).
[0057] The antibodies, or antibody fragments, of the present
invention can be utilized to detect, diagnose, serotype, and treat
papillomavirus infection. In this manner, the antibodies or
antibody fragments are particularly suited for use in
immunoassays.
[0058] Antibodies, or fragments thereof, 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.
[0059] Examples of suitable enzyme labels include malate
hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,
yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, acetylcholine esterase, etc.
[0060] Examples of suitable radioisotopic labels include .sup.3H,
.sup.125I, .sup.131I, .sup.32P, .sup.35S,.sup.14C, .sup.51Cr,
.sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu, .sup.90Y,
.sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47SC, and
.sup.109Pd.
[0061] Examples of suitable fluorescent labels include a .sup.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.
[0062] Examples of suitable toxin labels include diphtheria toxin,
ricin, and cholera toxin. Examples of chemiluminescent 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.
[0063] 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 antibodies or
fragments thereof can be accomplished using standard techniques
commonly known to those of ordinary skill in the art. Typical
techniques are described by Kennedy et al, Clin. Chim. Acta,
70:1-31 (1976), and Schurs 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-maleimidobenzyl-N-hydroxy-succinimide ester method, all these
methods incorporated by reference herein.
[0064] The detection of the antibodies (or fragments of antibodies)
of the present invention may 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 suitable carriers for binding
monoclonal antibody, or will be able to ascertain the same by use
of routine experimentation.
[0065] By raising antibodies against L1 proteins which mimic the
antigenicity of papillomavirus virions, the antibodies raised
against such recombinant proteins are neutralizing and protective
antibodies. The antibodies are able to prevent subsequent infection
of the same type of papillomaviruses from which the L1 protein was
derived. That is, if a recombinant L1 protein from papillomavirus
type 16 is utilized to raise antibodies, these antibodies will
protect against subsequent infection of papillomavirus type 16.
Thus, the method of the present invention provides for the
prevention, treatment or detection of any HPV type.
[0066] The antibodies of the invention can be utilized to determine
HPV types by serotyping as set forth in Jenson et al, J. Cutan.
Pathol., 16:54-59 (1989). Determining the HPV type may be
clinically important for determining the putative biological
potential of some productively infected HPV-associated lesions,
particularly benign and low-grade premalignant anogenital tract
lesions. Thus, the present invention makes it possible to treat and
prevent infection of any type of PV from which the L1 gene can be
obtained and neutralizing antibodies obtained.
[0067] The invention also provides for pharmaceutical compositions
as the antibodies can also be utilized to treat papillomavirus
infections in mammals. The antibodies or monoclonal antibodies can
be used in pharmaceutical compositions to target drug therapies to
sites of PV infection. In this manner, the drugs or compounds of
interest are linked to the antibody to allow for targeting of the
drugs or compounds. Methods are available for linking antibodies to
drugs or compounds. See, for example, EP 0,146,050; EP 0,187,658;
and U.S. Pat. Nos. 4,673,573; 4,368,149; 4,671,958 and
4,545,988.
[0068] Such drug therapies include antiviral agents, toxic agents
and photoactivatable compounds, such as coumarin, psoralen,
phthalocyanimes, methylene blue, eosin, tetracycline, chlorophylls,
porphyrins and the like. Such groups can be attached to the
antibodies by appropriate linking groups. Antibody conjugates
containing a photoactivatable compound are administered followed by
irradiation of the target cells.
[0069] The antibody or antibody conjugates of the present invention
can be formulated according to known methods to prepare
pharmaceutically useful compositions such as by admixture with a
pharmaceutically acceptable carrier vehicle. Suitable vehicles and
their formulation are described, for example, in Remington's
Pharmaceutical Sciences (16th Ed., Osol, A.Ed., Mack Easton Pa.
(1980)). To form a pharmaceutically acceptable composition suitable
for effective administration, such compositions will contain an
effective amount of antibody, either alone, or with a suitable
amount of carrier vehicle.
[0070] The therapeutic or diagnostic compositions of the invention
will be administered to an individual in therapeutically effective
amounts. That is, in an amount sufficient to diagnose or treat PV
infection. The effective amount will vary according to the weight,
sex, age and medical history of the individual. Other factors
include, the severity of the patient's condition, the type of PV,
mode of administration, and the like. Generally, the compositions
will be administered in dosages ranging from about 0.01 to about 2
picomoles/ml, more generally about 0.001 to about 20
picomoles/ml.
[0071] The pharmaceutically prepared compositions may be provided
to a patient by any means known in the art including oral,
intranasal, subcutaneous, intramuscular, intravenous,
intraarterial, parenteral, etc.
[0072] Another aspect of the present invention involves the
development of PV type-specific vaccines. The vaccines of the
invention are those that contain the necessary antigenic
determinants to induce formation of neutralizing antibodies in the
host;
[0073] possess high immunogenic potential; are safe enough to be
administered without danger of clinical infection; devoid of toxic
side-effects; suitable for administration by an effective route,
for example, oral, intranasal, topical or parenteral; mimics the
circumstances of natural infection; stable under conditions of
long-term storage; and, compatible with the usual inert vaccine
carriers.
[0074] The vaccines of the present invention include the
conformationally correct recombinant L1 proteins or fragments
thereof which provide the conformational epitopes present on the
intact virions. Such amino acid sequences of the L1 protein
comprise the antigenic component of the vaccine. It may be
necessary or preferable to covalently link the antigen to an
immunogenic carrier, i.e., bovine serum albumin or keyhole limpet
hemocyanin. The vaccines of the invention may be administered to
any mammal susceptible to infection with the papillomavirus. Human
and non-animal mammals may benefit as hosts.
[0075] Administration of the vaccines may be parenteral, but
preferably oral or intranasal, depending upon the natural route of
infection. The dosage administered may be dependent upon the age,
health, weight, kind of concurrent treatment, if any, and nature
and type of the papillomavirus. The vaccine may be employed in
dosage form such as capsules, liquid solutions, suspensions, or
elixirs, for oral administration, or sterile liquid formulations
such as solutions or suspensions for parenteral or intranasal use.
An inert, immunologically acceptable carrier is preferably used,
such as saline or phosphate-buffered saline.
[0076] The vaccines will be administered in therapeutically
effective amounts. That is, in amounts sufficient to 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 generally about 0.01 mg to about 100 mg
protein. A single or multiple dosages can be administered.
[0077] The method of the present invention makes possible the
preparation of subviral vaccines for preventing papillomavirus
infection. Further, by following the methods of the invention,
vaccines for any immunogenic type of specific papillomavirus can be
made.
[0078] As more than one PV type may be associated with PV
infections, the vaccines may comprise L1 antigenic amino acids from
more than one type of PV. For example, as HPV 16 and 18 are
associated with cervical carcinomas, a vaccine for cervical
neoplasias may comprise L1 protein of HPV 16; of HPV 18; or both
HPV 16 and 18.
[0079] In fact, a variety of neoplasias 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 epidermodysplasia verruciformis (EV)
including HPVs 3a, 5, 8, 9, 10, and 12. HPVs 1, 2, 4, and 7 have
been reported to be associated with cutaneous warts and HPVs 6b,
11a, 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, vaccine formulations may comprise a
mixture of L1 proteins from different PV types depending upon the
desired protection.
[0080] In the same manner, the pharmaceutical compositions may
contain a mixture of antibodies to different PV types.
[0081] As indicated, the L1 protein of the invention can be
utilized for serotyping.
[0082] That is, monoclonal antibodies capable of reacting with
conformationally correct L1 protein can be produced which can be
used to serotype PV. In this manner, tissue or serum can be
obtained from a patient and analyzed for the ability to
immunoprecipitate such antibodies
[0083] In a broader sense, the antibodies can be used for
serological screening. In this manner, populations of individuals
can be tested for the ability to immunoprecipitate conformationally
correct antibodies. Specific HPV type antibody responses can be
determined.
[0084] The invention lends itself to the formulation of kits,
particularly for the detection and serotyping of HPV. Such a kit
would comprise a carrier being compartmentalized to receive in
close confinement one or more containers, each container having
antibodies for a particular HPV type or a mixture of antibodies for
a variety of known HPV types. Other containers may contain means
for detection such as enzyme substrates, labelled
antigen/anti-antibody and the like.
[0085] For serological testing, the kits will comprise the
conformationally correct recombinant L1 protein. Such a kit could
also be utilized for vaccines.
[0086] While the present invention is generally directed to
producing by recombinant method conformationally correct
papillomavirus L1 proteins of any human or animal papillomavirus,
as well the use of such proteins as vaccines, and or diagnosis and
serotyping, in the preferred embodiments the recombinantly
produced, conformationally correct L1 proteins will comprise human
papillomavirus L1 proteins, canine oral papillomavirus (COPV) L1
proteins or equine papillomavirus L1 proteins.
[0087] As discussed supra, the canine oral papillomavirus (COPV)
animal model offers a unique and highly relevant animal model for
the development of human canine papillomavirus vaccines. Moreover,
unlike the available rabbit and bovine papillomavirus models, COPV
is tropic for mucous membranes and is assayable for infectivity
under natural conditions of exposure. Using a beagle colony which
exhibits a high, natural incidence of oral papillomas, the present
inventors have demonstrated that these tumors express viral capsid
proteins and contain intact viruses, which are preventable by
immunization with virus-containing tumor extracts. Moreover, as
described in greater detail infra, it has been demonstrated that
administration of formalin-inactivated COPV or recombinant COPV
conformational L1 proteins confers complete protection upon
challenge with the virus.
[0088] As discussed, infection of the oral mucous by COPV results
in the induction of well-differentiated, benign, squamous cell
tumors (warts). These lesions contain episomal DNAs which have been
cloned separately by two research groups (Sundberg et al, Amer. J.
Vet. Res., 47:1142-1144 (1986); Bregman et al, Vet Patriol.,
24:477-487, (1987). The COPV genome is slightly larger (8.2 Kb)
than most other papillomavirus genomes (8.0 Kb) but the two
isolates characterized to date exhibit identical restriction enzyme
cleavage patterns. Inoculation of beagles with wart extracts,
similar to the bovine and rabbit models, induces immunity to
subsequent reinfection [unpublished results]. Unfortunately, in a
small proportion of vaccinated animals, squamous cell carcinoma
develops at the site of injection (Bregman et al, Vet Pathol.,
24:477-487 (1987)). This presumably results from the neoplastic
transformation of cutaneous keratinocytes by COPV which become
entrapped in the needle during injection.
[0089] Sequencing results have demonstrated that the L1 gene of
COPV is highly homologous to the L1 gene of HPV-1. Moreover, this
virus possesses several critical characteristics which render it an
ideal animal model for the "malignancy-associated" human
papillomaviruses which distinguish it from the current rabbit and
bovine models.
[0090] In particular, COPV, in contrast to CRPV, BPV-1 and BPV-2,
infects and induces tumors at mucosal sites. This site mimics that
for the mucosotrophic HPV-16 and HPV-18 which infect genital mucosa
which are associated with cervical carcinoma. COPV has been
isolated from genital mucous but not from cutaneous sites. Thus,
COPV provides an ideal animal model for study of mucosotropic
papillomaviruses which infect genital mucosa, and for screening and
design of vaccines for providing immunity against such
mucosotrophic papillomaviruses. This is extremely beneficial
because of the fact that some mucosal HPVs, e.g., HPV-16 and HPV-18
are associated with cervical carcinoma.
[0091] Moreover, vaccines designed to prevent mucosal lesions may
have specific requirements for generating IgA responses and for
initiating an immune response in a specific subset of B
lymphocytes.
[0092] Additionally, unlike the currently available CRPV and BPV
models, COPV exhibits a high endogenous infection in a specific
beagle colony. Thus, it is possible to escalate the efficacy of
vaccines for preventing this naturally occurring infection. By
contrast, the bovine and rabbit models require artificial means of
infection (cutaneous abrasion) which may not necessarily reflect
the natural mechanism of mucosal infections. Therefore, the
beagle/COPV model should permit enhanced evaluation of the efficacy
of putative vaccines against mucosal papillomaviruses such as COPV,
HPV-16 and HPV-18 since it will better mimic in-vivo conditions
than the CRPV and BPV models.
[0093] Further, carcinomas can develop at the site of benign tumors
in a small percentage of animals as well as at the site of
injection of crude "live" wart extracts. The limited conversion of
benign lesions into carcinomas is also observed in human infected
by mucosal papillomaviruses (HPV-16 and HPV-18) and represent the
most serious consequence of HPV infection. Malignant conversion
does not occur with cutaneous BPV, but does occur with CRPV in
domestic rabbits.
[0094] This is highly significant because an effective vaccine
against human papillomaviruses cell potentially reduce the
incidence of human cervical dysplasia and carcinoma by 90-95%.
However, due to the species specificity of these viruses, there are
no animals into which HPV may be introduced to evaluate such
vaccines. Moreover, because there are currently no tissue culture
methods for propagating the virus, thereby eliminating the ability
to assay viral neutralization in vitro. The only viable mechanisms
for developing an HPV vaccine are to use prototype animal
papillomaviruses which closely mimic the human disease process.
[0095] Thus, in light of the above, COPV should afford significant
advantages over available rabbit and bovine papillomavirus animal
models. Further, because the capsid proteins of COPV are closely
related to HPV and since the biology of COPV closely mimics that of
certain human papillomaviruses, e.g., HPV-16 and HPV-18, the
identification of an effective COPV vaccine will yield direct
benefits both because of potential veterinary applications, and
more importantly to the development of vaccines against HPV's
associated with cervical carcinoma.
[0096] Therefore, the present invention provides for the production
of conformationally correct COPV L1 proteins, and the use of such
COPV L1 proteins as vaccines against COPV, as well as the use
thereof as an in vivo animal model for the development of human
papillomavirus vaccines.
[0097] The present inventors studied the ability of
conformationally correct COPV L1 proteins to afford immunity
against COPV challenge in a beagle colony which exhibits a natural
high incidence of oral warts formation as a consequence of viral
infection. However, it is expected that the present invention will
be applicable with any canine which is naturally susceptible to
COPV infection.
[0098] Additionally, the present COPV/canine animal model further
provides a means for delineating the role of antibodies against
conformational epitopes of the COPV L1 proteins, as well as the L2
protein, in providing for resistance against COPV infection upon
challenge with COPV.
[0099] This may be effected by injection of COPV wart extract
(known to contain COPV viral particles) into susceptible animals.
Also, the L1 and the L2 genes of COPV may be expressed in
expression vectors which provide for the production of
conformationally correct L1 and L2 proteins. As discussed supra,
this will preferably be effected using eukaryotic expression
vectors, e.g., mammalian, insect or yeast cells, e.g.,
Saccharomyces. Particularly preferred host cells for expression of
COPV L1 proteins include by way of example COS cells and
recombinant baculovirus infected Sf9 cells which produce L1
proteins which self-assemble into virus-like particles which
antigenically mimic the intact COPV virion.
[0100] The conformationally correct COPV L1 and/or L2 proteins will
be used to screen immune animal sera for the presence of L1 and L2
specific antibodies as well as to induce immunity in susceptible
animals. The present invention further facilitates the
identification of optimal conditions for inducing immunity in
susceptible animals against COPV infection. Additionally, given the
similarities between COPV and HPVs, the present invention further
enables the identification of optimal conditions for inducing
immunity against HPVs, in particular HPV-16 and HPV-18.
[0101] The ability of COPV L1 and L2 antibodies to inhibit
COPV-induced tumors can be evaluated using virions purified from
wart tissue or from other sources such as viral-producing tumors
grown in nude mice.
[0102] Also, the conformationally correct COPV L1 and L2 proteins
produced according to the invention can be used to generate
monoclonal antibodies which may be used as therapeutics for
providing passive immunization against COPV or as diagnostic
agents. Further, monoclonal antibodies generated against intact
virions may be used to define the molecular location of
conformational, neutralizing epitopes on the COPV L1 and L2
proteins. Moreover, due to the structural and immunogenic
similarity between COPV and HPVs, these antibodies may further have
potential applicability in the development of human papillomavirus
vaccines and diagnostic agents.
[0103] Immunization studies with COPV conformationally correct L1
and L2 proteins produced according to the present invention should
enable the precise identification of specific dosages, carriers,
adjuvants, frequency of administration and route of administration
which provide for optimal immunity against COPV infection and
possibly against HPV infection given the substantial similarities
of COPV and HPV5. Immunity will be determined by studying
vaccinated animals for the spontaneous appearance of oral
warts.
[0104] Additionally, at selected times pre- and post-vaccination,
animals will be evaluated for the presence of IgG, IgM and IgA
antibodies which react with intact virus, or with L1 or L2
proteins. The temporal production of antibodies, as well as the
ability of these antibodies to inhibit COPV infection will also be
tested. In this regard, the present inventors have determined that
injection of purified virus-like particles, with or without
adjuvant, by systemic intradermal route of administration protects
beagles from intraviral challenge with infectious COPV. Also, serum
produced against intact virions were found to develop rapidly and
to remain high in vaccinated beagles. By contrast, control, naive
beagles were highly susceptible to challenge administered by the
same route.
[0105] It has further been demonstrated by the present inventors
that immunoglobulin fractions from vaccinated beagles which are
passively transferred into weaning recipient beagles confer
complete protection against COPV challenge. This provides
additional evidence that the subject conformationally correct L1
and L2 proteins confer immunity against COPV by inducing a humoral
(antibody) response against conformational epitopes contained on
the L1 and L2 proteins.
[0106] Having now generally described the invention, the following
examples are offered by way of illustration and not intended to be
limiting unless otherwise specified.
EXPERIMENTAL
EXAMPLE 1
Materials and Methods (for Examples 1-3)
Animals
[0107] Female, athymic (nu/nu) mice were purchased from Harlan
Sprague Dawley, Madison Wis., and used for xenograft transplants
when 6 to 8 weeks old.
Virus
[0108] BPV-1 was purified from experimentally-induced bovine
cutaneous fibropapillomas as described by Lancaster, W. D. and
Olson, D., Demonstration of two distinct classes of bovine
papillomavirus, Virology, 89:372-279 (1978) (1978). The virus was
stored at -80.degree. C. until used.
Antibodies
[0109] All serum samples were heat-inactivated at 56.degree. C. for
30 min. Each antibody preparation was evaluated for reactivity with
intact and disrupted BPV-1 particles (Table I) before testing for
neutralization of BPV-1 induced transformation of C127 cells and
xenografts.
[0110] Bovine polyclonal antibodies. Bovine sera were obtained from
calves either vaccinated with BPV-1 L1 fusion proteins or
experimentally-infected with BPV-1.
[0111] Holstein X Angus calves were immunized with different
formulations of a recombinant BPV-1 L1::B-galactosidase vaccine
(Jin, X. W., Cowsert, L., Marshall D., Reed, D., Pilacinski, W.,
Lim, L. and Jenson, A. B., Bovine serological response to a
recombinant BPV-1 major capsid protein vaccine, Intervirology,
31:345-354 (1990)). The cloned L1 gene begins 76 bp down stream
from the start codon of the L1 open reading frame at nucleotide
5686 and is terminally fused to the E. coli B-galactosidase gene
(Pilacinski, W. P., Glassman, D. L., Richard, A. K. Sadowski, P. L.
and Alan, K. R., Cloning and expression in Escherichia coli of the
bovine papillomavirus L1 and L2 open reading frames, Bio/Technol.,
2:356-360 (1984)). Calves were vaccinated on days 0 to 21, and
challenged by intradermal inoculation of 2 sites with 10.sup.10
BPV-1 particles on day 56 (Jin, X. W., Cowsert, L., Marshall D.,
Reed, D., Pilacinski, W., Lim, L. and Jenson, A. B., Bovine
serological response to a recombinant BPV-1 major capsid protein
vaccine, Intervirology, 31:345-354 (1990)). The calves were bled on
days 3 (designated as pre-bleed), 55 (bleed 1) and 104 (bleed 2)
days of the trial and the sera tested for reactivity with intact
and disrupted BPV-1 particles by ELISA. Although 90% and 58% of
calves developed antibody responses to internal and external BPV-1
capsid epitopes respectively, all calves developed fibromas.
[0112] Two steer (926 and 921), acquired as calves from a
sequestered herd of cattle without prior exposure to BPV-1 or
BPV-2, were inoculated at multiple sites with finely ground
homogenates of BPV-1 induced fibropapillomas. Fibropapillomas
developed in the scarified sites and persisted for varying lengths
of time before undergoing spontaneous regression. The sera used in
this experiment were collected during the earliest signs of
fibropapilloma regression in both animals.
[0113] Rabbit polyclonal antibodies. Rabbit anti-sera were prepared
by inoculation with either intact BPV-1 or BPV-2 virions, or
denatured BPV-1 particles and then bled 2 weeks after the final
immunization (Jenson, A. B. Rosenthal, J. D., Olson, C., Pass, F.
W., Lancaster W. D. and Shah, K. I., Immunologic relatedness of
papillomaviruses from different species, J. Nat. Cancer Inst.,
64:495-500 (1980), Jenson, A. B., Kurman, R. J. and Lancaster, W.
D., Detection of papillomavirus common antigens in lesions of the
skin and mucosa, Clinics In Dermatol., 3:56-63 (1985); Cowsert, L.
M., Lake, P. and Jenson, A. B., Topographical and conformational
epitopes of bovine papillomavirus type 1 defined by monoclonal
antibodies, J. Nat. Cancer Inst., 79:1053-1057 (1987)).
[0114] Murine monoclonal antibodies. Two murine MAbs, 13D6 and JG,
were also used to test for neutralization. 13D6 recognizes
conformational epitopes on BPV-1, BPV-2 and deer papillomavirus
(DPV) intact particles (Cowsert, L. M., Lake, P. and Jenson, A. B.,
Topogaphical and conformational epitopes of bovine papillomavirus
type 1 defined by monoclonal antibodies, J. Nat. Cancer Inst.,
79:1053-1057 (1987)), whereas JG recognizes a BPV-1 type-specific
linear epitope internal to the capsid (data not shown).
1TABLE I ELISA REACTIVITY OF BOVINE, RABBIT AND HUMAN SERA AND
MURINE MAbs WITH INTACT AND DISRUPTED BPV-1 PARTICLES Serum.sup.1
or BPV-1 particles MAb samples Intact Disrupted Vaccinated
calves.sup.2 163 Pre-bleed 0.002 0.016 1 0.041 0.925 2 0.312 1.472
173 Pre-bleed 0.036 0.066 1 0.101 1.222 2 0.182 1.249 Rabbit.sup.3
NRS 0.065 0.073 BPV-1 1.454 0.095 BPV-2 1.621 0.085 BPV-1 (SDS)
0.319 1.358 MAbs 13D6 0.629 0.004 JG 0.004 0.423 Hyperimmune
steers.sup.4 926 0.296 0.033 921 0.397 0.202 Human 1 0.964 0.036 2
0.554 0.247 .sup.1RBPV-1 and RBPV-2 were diluted 1/2000; all other
samples were diluted 1/50. .sup.2Pre, pre-bleed sera from calves
163 and 173; 1, sera of calves 163 and 173 at the time of challenge
with BPV-1 virions; 2, sera of calves 163 and 173 at end of the
vaccine trial. .sup.3NRS, normal rabbit serum; BPV-1 (SDS) rabbit
serum prepared against SDS-disrupted BPV-1. .sup.4Steers (926 and
921), serum of steer inoculated at 24 different cutaneous sites
with BPV-1 homogenates.
Neutralization Assays
[0115] Two assays (xenografts in athymic mice and murine C127 cells
cultures) for detecting antibody-mediated neutralization of
infectious PV virions were compared for specificity.
[0116] Xenograft assay. To assay for neutralization of BPV-1
infectivity, a 1:10 dilution of polyclonal anti-sera in PBS was
added to aliquots of infectious BPV-1 in PBS and incubated for 1 hr
at 37.degree. C. BPV-1 in PBS alone was included as a positive
control for infectivity. Bovine fetal skin chips (5 to
10.times.2-.times.2-mm pieces) were added to each dilution and
incubated for 1 hr at 37.degree. C.
[0117] The chips were transplanted under the renal capsule of
athymic mice and cyst size (in mm) and morphology of its lining
epithelium was determined after 60 days. Cyst sizes were calculated
as geometric mean diameters (BMDs) by calculating the cubic root of
the length.times.width.times.height of cysts in mm.
[0118] Statistical analysis was accomplished by determining the
means of the GMDs of cysts and fibropapillomas for each anti-serum
and was compared with those for untreated controls by using the
Student's t-test.
[0119] C127 cells assay. Murine C127 cells were obtained from ATCC,
Rockville, MD, and grown as described by (Dvoretzky, I., Shober,
R., Chattopadhy, S. K. and Lowy, D. R., A quantitative in vitro
focus-forming assay for bovine papillomavirus, Virology,
103:369-375 (1980)). The neutralization assays were carried out in
Petri dishes (100 mm). C127 cells were seeded at approximately
10.sup.5 to 5.times.10.sup.5 cells, which were allowed to become 75
to 80% confluent, BPV-1 virions (10.sup.3 focus-forming units
[FFU]) were then incubated with either 0.5 ml DMEM as a positive
control for infectivity or an equal volume of the MAb or polyclonal
anti-serum (diluted 1:5) at 37.degree. C. for 1 hr prior to
inoculation of C127 cells. After 11/2 hrs adsorption. 10% FBS
supplemented MEM was added to each dish. The medium was replenished
the next day and then 3 times each week for 17 to 19 days, at which
time the dishes were fixed and stained 0.1% methylene blue in
methanol to count the number of FF per dish. Controls included
fetal calf sera and serum from a steer that had no history of
fibropapillomas.
Results
[0120] The specificity of 2 different assay systems, xenografts and
C127 cells, for measuring the neutralization of BPV-1 infection
were compared using selected animal sera and murine MAbs. The sera
and MAbs tested were: (1) sera from rabbits and cattle immunized
and/or infected with intact BPV-1 and BPV-2 virions (the immune
systems were exposed to both conformational and linear BP-1 capsid
surface epitopes); (2) sera from rabbits and cattle immunized with
denatured BPV-1 virions and L1 fusion proteins respectively (the
immune systems were exposed to denatured/linear BPV-1 capsid
epitopes); (3) selected sera from humans that reacted with intact
BPV-1; and (4) MAbs that define BPV-1 conformational surface
epitopes and epitopes that are internal to the BPV-1 capsid.
Epitope Topography
[0121] The sera evaluated in our study were tested initially for
reactivity with both intact and disrupted BPV-1 capsids, thus
defining the topographical location of the corresponding epitopes
as either external or internal to the BPV-1 capsid as previously
described (Cowsert, L. M., Lake, P. and Jenson, A. B.,
Topographical and conformational epitopes of bovine papillomavirus
type 1 defined by monoclonal antibodies, J. Nat. Cancer Inst.,
79:1053-1057 (1987)). (Table 1).
[0122] Rabbit sera produced against intact BPV-1 or BPV-2 virions
and sera from steers inoculated at multiple sites with infectious
homogenates of BPV-1 induced fibropapillomas as well as MAb 13D6
reacted primarily with intact virions. The two human sera selected
for this study reacted primarily with intact BPV-1 particles.
[0123] Rabbit serum prepared against SDS-disrupted BPV-1 viral
particles, and sera (bleed 2) from calves 163 and 173 at the end of
the vaccine trial, 48 days after challenge with BPV-1 virions,
reacted with both intact and disrupted viral particles. Calf 163
serum (bleed 1), immediately prior to challenge with infectious
BPV-1 virions, reacted only with disrupted BPV-1 particles. MAb JG
reacted only with disrupted BPV-1 virions.
[0124] Pre-bleed/normal rabbit and bovine sera (calves 163 and 173
did not react either with intact or with disrupted BPV-1 virions by
ELISA.
Neutralization Assays
[0125] Two different assays were compared for neutralization of
BPV-1 infectivity by the hyperimmune sera and MAbs: (i) xenografts
in athymic mice, and (ii) C127 cell cultures.
[0126] Xenograft neutralization assay. Polyclonal antisera
(non-absorbed) as well as negative control sera were tested for the
neutralization of BPV-1 infectivity of bovine fetal skin
transplanted beneath the renal capsule of athymic mice. Effective
neutralization was determined by comparing cyst size and
microscopic morphology (Table II).
[0127] Bovine fetal skin chips were incubated with BPV-1 which had
been preincubated for 1 hr with dilutions of the various polyclonal
antisera. The chips were grafted sub-renally in athymic mice, and
average geometric mean diameters of cyst sizes were determined 60
days later (Table II). A large and significant reduction in cyst
size was obtained for the sera from 2 rabbits inoculated with
intact BPV-1 or BPV-2 and both steer polyclonal anti-sera collected
from animals with regressing BPV-1-induced fibropapillomas. Neither
polyclonal anti-serum from the rabbit inoculated with denatured
BPV-1 particles nor pre-bleed, challenge or post-challenge bovine
sera from the recombinant vaccination study in calves and a
significant effect on cyst size at the dilution tested. Human sera
and MAbs reactive with intact BPV-1 particles or linear epitopes of
BPV-1 did not result in cyst-size reduction.
[0128] C127 cell neutralization assay. Pre-bleed rabbit and calf
163 and 173 sera, hyperimmune rabbit serum prepared against
SDS-disrupted BPV-1 virions, both human sera, and calf sera (163
and 173) following vaccination but immediately prior to challenge
with BPV-1, did not neutralize FF of C127 cells by BPV-1 virions
(Table III). However, rabbit sera produced by immunization with
intact BPV-1 and BPV-2 had neutralizing titers of 10.sup.6 and
10.sup.4 respectively, and the hyperimmune steer sera had a
neutralizing titer of 10.sup.6 (926) to 10.sup.3 (921). Calves 163
and 173 sera at the end of the vaccination trial had a neutralizing
titer of less than 10.sup.1, probably because of exposure to
infectious challenge virus, rather than a maturing immune response
against the vaccine.
[0129] Neither fetal calf sera nor selected adult steer serum from
non-immune animals inhibited FF in C127 cells.
2TABLE II CYST SIZE AND MORPHOLOGY OF BPV-1 INDUCED XENOGRAFTS
DEVELOPING AFTER VARIOUS SERUM PRETREATMENTS OF INFECTIOUS BPV-1
Cyst size.sup.2 Serum or (mean and MAb samples.sup.1 SEM in mm)
Morphology.sup.3 Vaccinated calves 163 Pre-bleed 5.8 (0.9).sup.4
5/6/6 1 4.0 (0.4) 4/4/4 2 4.6 (0.5).sup.4 5/6/6 173 Pre-bleed 6.7
(0.8).sup.4 6/6/6 1 5.2 (0.7).sup.4 6/6/6 2 5.3 (0.6).sup.4 6/6/6
Rabbit NRS 5.8 (0.6).sup.4 8/8/8 BPV-1 3.5 (0.2).sup.5,6 0/10/10
BPV-2 3.3 (0.5).sup.5,6 1/6/6 BPV-1 (SDS) 8.3 (0.5).sup.4,5 4/4/4
MAbs 13D6 4.4 (0.6).sup.4 6/6/6 JG 5.3 (0.7).sup.4 6/6/6
Hyperimmune steers 926 3.0 (0.6).sup.5 0/3/4 921 3.4 (0.4).sup.5
0/6/6 Human 1 6.5 (0.7).sup.4 6/6/6 2 5.0 (0.4).sup.4 6/6/6
.sup.1Serum samples from various sources described in Table I.
.sup.2Cyst sizes were determined from geometric mean diameters.
.sup.3Number of cysts morphologically transformed/number of
surviving cysts/number of grafts attempted. .sup.4Mean cyst size
significantly different (p.sub.5 < 0.05) from BPV-1 -infected
treatment group of (positive control for neutralization).
.sup.5Mean cyst size significantly different (p < 0.05) from
rabbit anti-intact BPV-1 (previously used as positive control for
BPV-1 xenograft neutralization studies). .sup.6Mean cyst size
significantly different (p < 0.05) from normal rabbit serum
(previously used as negative control for BPV-1 xenograft
neutralization studies).
[0130]
3TABLE III NEUTRALIZATION OF BPV-1 INFECTION OF C127 CELLS BY
BOVINE, RABBIT AND HUMAN SERA AND MURINE MAbs Serum or
Neutralization MAb samples.sup.1 titer.sup.2 Vaccinated calves 163
Pre-bleed 0 1 0 2 <10.sup.1 173 Pre-bleed 0 1 0 2 <10.sup.1
Rabbit NRS 0 BPV-1 10.sup.6 BPV-2 10.sup.2 BPV-1 (SDS) 0 MAbs 13D6
0 JG 0 Hyperimmune steers 926 >10.sup.6 921 >10.sup.3 Human 1
0 2 0 .sup.1Identification of different sera and MAbs as in Table
I. .sup.2The neutralization titer is expressed as the reciprocal of
the highest serum dilution required to neutralize focus formation
of murine C127 cells by BPV-1 virions.
Discussion
[0131] The xenograft system has provided an effective model for the
detection of antibody-mediated neutralization of productive PV
infections, including BPV-1 (Christensen, N. and Kreider, J. W.,
Antibody-mediated neutralization in vitro of infectious
papillomaviruses, J. Virol., 64:3151-3156 (1990)). However
neutralizing antibodies also prevent BPV-1 virions from inducing FF
in non-productively infected murine C127 cells in culture
(Dvoretzky, I., Shober, R., Chattopadhy, S. K. and Lowy, D. R., A
quantitative in vitro focus-forming assay for bovine
papillomavirus, Virology, 103:369-375 (1980)). To compare the
specificity of the 2 methods, and to determine the epitopes
responsible for neutralization, selected sera from cattle, rabbits
and humans and murine MAbs were tested for neutralizing
activity.
[0132] The papillomavirus genomes are encapsulated by L1 (major
capsid) and L2 (minor capsid) proteins (Banks, L. Matlashewski, G.
Pim, D., Churcher, M., Roberts, C. and Crawford, L., Expression of
human papillomavirus type-6 and type-15 capsid proteins in bacteria
and their antigenic characterization, J. Gen. Virol., 69:3081-3089
(1987), Christensen, N. Kreider, J. W., Cladel, N. M. and Galloway,
D. A., Immunological cross-reactivity to laboratory-produced HPV-11
virions of polysera raised against bacterially derived fusion
proteins and synthetic peptides of HPV-6b and HPV-16 capsid
proteins, Virology, 175:1-9(1990), Cowsert, L. M., Lake, P. and
Jenson, A. B., Topogaphical and conformational epitopes of bovine
papillomavirus type 1 defined by monoclonal antibodies, J. Nat.
Cancer Inst., 79:1053-1057 (1987), Cowsert, L. M., Pilacinski, W.
P. and Jenson, A. B., Identification of the bovine papillomavirus
L1 gene product using monoclonal antibodies, Virolog , 165:613-615
(1988), Doobar, J. and Gallimore, P. H., Identification of proteins
encoded by the L1 and L2 open reading frames of human
papillomavirus 1a, J. Virol., 61:2793-2799 (1987), Jin. X. W.,
Cowsert, L., Pilacinski, W. and Jenson, A. B., Identification of L2
open reading frame gene products of bovine papillomavirus type-1 by
monoclonal antibodies, J. Gen. Virol., 70:1133-1140 (1989), Komly,
C. A., Breitburd, F., Croissant, O. and Streeck, R. E., The L2 open
reading frame of human papillomavirus type 1a encodes a minor
structural protein carrying type-specific antigens, J. Virol.,
60:813,816 (1986), Kreider, J. W., Howett, M. K., Wolfe, S. A.,
Barlett, G. L., Zaino, R. J., Sedlacek, T. V. and Mortel, R.,
Morphological transformation in vitro of human uterine cervix with
papillomavirus from condylomata acuminata, Nature (Lond.),
317:639-640 (1985), Nakai, Y., Lancaster, W. D., Lim, L. Y. and
Jenson, A. B., Monoclonal antibodies to genus-and type-specific
papillomavirus structural antigens, Intervirology, 25:30-37 (1986),
Roseto, A., Pothier, P., Guillemin, M. C., Peries, J., Breitburd,
F., Bonneaud, N. and Orth, G., Monoclonal antibody to the major
capsid protein of human papillomavirus type 1, J. Gen. Virol.,
65:1319-1324 (1984)), to form virions in the nuclei of terminally
differentiating keratinocytes (Firzlaff, J. M., Kiviat, N. B.,
Beckmann, A. M., Jenison, A. and Galloway, D. A., Detection of
human papillomavirus capsid antigens in various squamous epithelial
lesions using antibodies directed against the L1 and L2 open
reading frames, Virology, 164:467-477 (1988), Jenson, A. B.,
Rosenthal, J. D., Olson, C., Pass, F. W., Lancaster, W. D. and
Shah, K., Immunologic relatedness of papillomaviruses from
different species, J. Nat. Cancer Inst., 64:495-500 (1980), Lim, P.
S., Jenson, A. B., Cowsert, L., Nakai, Y., Lim, L. Y. and Sundberg,
J., Distribution and specific identification of papillomavirus
major capsid protein epitopes by immunocytochemistry and epitope
scanning of synthetic peptides, J. Infect. Dis., 162:1263-1269
(1990), Sandberg, J. P., Junge, R. E. and Lancaster, W. D.,
immunoperoxidase localization of papillomaviruses in hyperplastic
and neoplastic epithelial lesions of animals, Amer. J. Vet. Res.,
45:1441-1446 (1984)). The PV L1 capsid protein in contrast to the
L2 protein, is highly conserved throughout the PV genus (Baker, C.
C., Sequence analysis of papillomavirus genomes, In: N. P. Salzman
and P. M. Howley (eds.), The papoviridae, Vol. 2, The
papillomaviruses, pp. 321-385, Plenum, New York (1987). However
only type-specific and minimally cross-reactive linear and
conformational epitopes of the L1 protein have been detected on the
virion surface by MAbs, Cowsert, L. M., Lake, P. and Jenson, A. B.,
Topographical and conformational epitopes of bovine papillomavirus
type 1 defined by monoclonal antibodies, J. Nat. Cancer Inst.,
79:1053-1057 (1987), Cowsert, L. M., Pilacinski, W. P. and Jenson,
A. B., Identification of the bovine papillomavirus L1 gene product
using monoclonal antibodies, Virology, 165:613-615 (1988) whereas
type-specific linear epitopes of the L2 protein appear to be
internal to the capsid (Jin. X. W., Cowsert, L., Pilacinski, W. and
Jenson, A. B., Identification of L2 open reading frame gene
products of bovine papillomavirus type-1by monoclonal antibodies,
J. Gen. Virol., 70:1133-1140 (1989), Komly, C. A., Breitburd, F.,
Croissant, O. and Streeck, R. E., The L2 open reading frame of
human papillomavirus type 1 a encodes a minor structural protein
carrying type-specific antigens, J. Virol., 60:813,816 (1986),
Tomita, Y., Shirasawa, H., Sekine, H. and Simizu, B., Expression of
human papillomavirus type 6b L2 open reading frame in Escherichia
coli::L2-.beta.-galactosidase fusion proteins and their antigenic
properties, Virology, 158:8-14 (1987)). In this study, only sera
from rabbits immunized with either intact BPV-1 or BPV-2 virions
and cattle infected with homogenates of productively infected
fibropapillomas were capable of neutralizing infectivity of BPV-1
in both murine C127 cells and in the xenografts.
[0133] Although the neutralization assay in murine C127 cells may
be more quantitative, primarily because the assay involves FF of
single cells in a monolayer, it is no more specific than the
xenograft system (Christensen, N. and Kreider, J. W.,
Antibody-mediated neutralization in vitro of infectious
papillomaviruses. J. Virol. 64:3151-3156 (1990)), which is more
analogous to neutralization of BPV-1 infection in the natural host
by prior vaccination with intact virions (Jarrett, W. F. H.,
O'Neill, B. W., Gaukroger, J. M., Laird, H. M., Smith, K. T. and
Campo, M. S., studies on vaccination against papillomaviruses: a
comparison of purified virus, tumor extract and transformed cells
in prophylactic vaccination. Vet. Rec. 126:449-452 (1990a),
Jarrett, W. F. H., O'Neill, B. W., Gaukroger, J. M., Laird, H. M.,
Smith, K. T. and Campo, M. S., Studies on vaccination against
papillomaviruses: the immunity after infection and vaccination with
bovine papillomaviruses of different types. Vet. Rec. 126:473-475
(1990b)). Bovine sera from vaccinated calves almost 2 months after
challenge with BPV-1 virions neutralized BPV-1 -induced FF of C127
cells, but did not prevent the development of fibropapillomas in
the xenografts. Although both assays were accomplished using
aliquots of the same sera, the differences in personnel, handling
of specimens, conditions of infection and neutralization, which
were performed at separate locations, could also explain the slight
difference in results.
[0134] Rabbit and bovine sera that were prepared against either
denatured BPV-1 capsids or recombinant BPV-1 L1 vaccine,
respectively, did not neutralize BPV-1 infectivity in either
neutralization assay. Since these sera only recognized continuous
BPV-1 L1 epitopes, it was concluded that linearized BPV-1 surface
epitopes were not capable of inducing neutralizing antibodies.
Neutralizing activity in this study appears to be largely dependent
upon conformational epitopes.
[0135] The 2-human sera that reacted with intact BPV-1 particles
did not prevent BPV-1-induced FF in C127 cells or transformation of
bovine fetal skin in the xenograft model. This suggests that human
sera either recognized a non-neutralizing mimeotope or defined
BPV-1 conformational epitopes that are not associated with
neutralization of BPV-1 infectivity. Nevertheless, these results
support the concept that significant exposure to intact BPV-1 viral
particles is necessary for the production of neutralizing
antibodies (Jarrett, W. F. H., O'Neill, B. W., Gaukroger, J. M.,
Laird, H. M., Smith, K. T. and Campo, M. S., studies on vaccination
against papillomaviruses: a comparison of purified virus tumor
extract and transformed cells in prophylactic vaccination. Vet.
Rec. 126:449-452 (1990a), Jarrett, W. F. H., O'Neill, B. W.,
Gaukroger, J. M., Laird, H. M., Smith, K. T. and Campo, M. S.,
Studies on vaccination against papillomaviruses: the immunity after
infection and vaccination with bovine papillomaviruses of different
types. Vet. Rec. 126:473-475 (1990b)).
[0136] Our study reveals that neutralization of BPV-1 infectivity
by serum antibodies can be measured by prevention of either FF in
C127 cells or transformation of bovine fetal skin in the xenograft
model. Since the results of the 2 assays were concordant, it is
concluded that (1) neutralization of FF of C127 cells and
transformation of bovine fetal skin in the xenografts both appear
to be true indicators of the capacity of antibodies to neutralize
BPV-1 infectivity, that is, the antibodies react with
conformationally correct L1 protein; and (2) neutralization of FF
by C127 cells can be used for studies of early BPV-1 virion-host
cell interaction to define functional epitopes.
EXAMPLE 2
Expression of a Prototype L1 Protein (HPV-1) by the pSVL Vector
Transfected into COS Cells
[0137] The L1 protein of HPV-1 was expressed because there exist
several monoclonal antibodies against HPV-1 which react with
conformational epitopes present on the intact virion. We reasoned
that if we were successful in generating HPV-1 L1 protein with
native conformation, these monoclonal antibodies might react with
the isolated, expressed L1 protein. This would confirm the ability
to produce L1 protein of suitable conformation to mimic that
present on the intact virus particle. It is critical to generate an
immune response against the conformational epitopes of the
papillomaviruses in order to produce a neutralizing antibody.
[0138] The choice of vector was based upon several criteria. We
desired to have expression vectors which produced high levels of
capsid protein which would not only facilitate their use for
vaccines but also potentially aid in achieving empty capsid
formation in the nucleus. The pSVL vector and the baculovirus
vectors both use very strong promoters and have been used
extensively for expressing proteins. In addition, the pSVL vector
contains an SV40 origin of replication and, when transfected in cos
cells which express Large T antigen, replicates to high copy
number. The replication of the input vector, combined with the
strong activity of the viral promoter, results in extremely high
levels of expressed protein. The cos cells are also permissive for
the assembly of SV40 virions and might potentially facilitate the
assembly of PV particles. The baculovirus system also offers the
advantage that a larger percentage of cells can be induced to
express protein (due to the use of infection rather than
transfection 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 (glycosylation and
phosphorylation) which might be important for generating proteins
of appropriate conformation.
[0139] The scheme for the cloning of the HPV-1 L1 protein into pSVL
is shown in FIG. 1.
[0140] The expression of the HPV-1 L1 protein by pSVL was first
assayed by immunofluorescence. COS cells were transfected with 1-10
.mu.g of the plasmid shown in FIG. 1. After 48 hrs, the cells were
fixed with cold methanol and then reacted with either non-immune
mouse ascites (a), rabbit antiserum generated against SDS-disrupted
BPV-1 (b), or mouse monoclonal antibody 405D5 which recognizes a
type-specific, conformational epitope on HPV-1. A positive nuclear
staining was seen with both antibodies and was absent from
non-transfected cells. In addition, the L1-expressing cells were
also reactive with several additional monoclonal antibodies which
specifically react with independent, conformational epitopes (data
not shown). After transfection cos cells were then fixed with
methanol and stained for reactivity with either control rabbit
serum, Dako antiserum generated against SDS-disrupted BPV-1
virions, or mouse monoclonal antibody 405D5 which reacts
specifically with HPV-1 virion conformational epitopes. Four
additional conformation-specific monoclonal antibodies gave an
identical immunofluorescence pattern and clearly indicate that the
L1 protein synthesized in cos cells retains conformational
epitopes. In addition, the L1 protein exhibits the anticipated
intranuclear localization, reflecting the appropriate processing
and translocation of this protein. This result demonstrates that
the conformational epitope identified by 405D antibody is present
entirely on the L1 protein (rather than L2 or a combination of
L1/L2). Most importantly, the reactivity of L1 with this monoclonal
antibody demonstrates the L1 protein has retained a conformational
epitope identical to that found in its virion-associated state.
Electron microscopy experiments are currently being performed to
evaluate whether the L1 protein is assembling into empty viral
particles. Thus, the pSVL vector is successful in producing HPV-1
L1 protein with a native conformation for generating antibody
responses which react with intact virus particles.
[0141] The synthesis of the L1 protein was also determined by
immunoprecipitation from transfected cos cells. At 48 hr
post-transfection, the cos cells were metabolically labelled with
S-35 methionine and cysteine for 4 hrs, extracted with RIPA buffer,
and immunoprecipitated with rabbit antiserum generated against
SDS-disrupted BPV-1 (Dako). We used this antibody for
immunoprecipitations since the solubilization of L1 protein with
denaturing detergents may abolish its recognition by the
conformation-dependent L1 antibody described above. An SDS-PAGE of
the immunoprecipitates indicates that the synthesized L1 protein is
full-length (55 kD). This series of immunofluorescence and
immunoprecipitation experiments demonstrates therefore that the
pSVL vector will be able to generate L1 protein which will be
suitable for inducing conformation-dependent antibodies.
EXAMPLE 3
[0142] Papillomavirus infections cause cutaneous warts and mucosal
condylomata in a wide variety of vertebrate animals (Olson, C., in
"The papovaviridae" (N. P. Salzman and P. M. Howley, Eds.), pp.
39-66, Plenum Press (1987)) and, in humans, are strongly associated
with the development of cervical dysplasia and carcinoma (Jenson,
A. B., and Lancaster, W. D., in "Papillomaviruses and human cancer"
(H. Pfister, Ed.) pp. 11-43, CRC Press (1990)). Each papillomavirus
type is highly species-specific and preferentially infects squamous
epithelium at a restricted number of anatomic locations. Vegetative
viral DNA replication occurs in the nucleus of terminally
differentiated keratinocytes where the viral genome becomes
encapsidated by the major (L1) and minor (L2) capsid proteins,
forming virions 55 nm in diameter. Unfortunately, there are no
tissue culture systems which permit sufficient keratinocyte
differentiation to propagate papillomaviruses in vitro and this
limitation has compromised the analysis of the late expression of
the L1 and L2 genes as well as the characterization of the host
immune response to their gene products.
[0143] Due to the etiologic role that human papillomaviruses
(HPV's) play in some human malignancies, recent attention has been
focused on the development of a recombinant capsid protein vaccine
to reduce the incidence of HPV infection and its neoplastic
sequelae. The first animal model for a potential vaccine utilized
bovine papillomavirus type 1 (BPV-1). The L1 protein of BPV-1 was
expressed in bacteria (Pilacinski, W. P., Glassmam, D. L., Krzyzek,
R. A., Sadowski, P. L., and Robbins, A. K., Biotechnology,
2:356-360 (1984)) and used to immunize cattle against subsequent
viral challenge (Pilacinski, W. P., Glassmam, D. L., Glassman, K.
L., Read, D. E., Lum, M. A., Marshall, R. F., and Muscoplat, C. C.,
In "Papillomaviruses: molecular and clinical aspects" (T. R. Broker
and P. M. Howley, Eds., pp. 257-271, Alan R. Liss, Inc., New York
(1985)). However, since the expressed L1 protein apparently lacked
native conformation (due to the insoluble, aggregate form of
over-expressed, fusion proteins in bacteria), it did not induce
antibodies which could either recognize or neutralize intact BPV-1
virions (Jin, X. W., Cowsert, L., Marshall, D., Reed, D.,
Pilacinski, W., Lim, L. Y., and Jenson, A. B., Intervirology,
31:345-354 (1990); and Ghim, S., Christensen, N. D., Kreider, J.
W., and Jenson, A. B., Int. J. Cancer 49:285-289 (1991)).
[0144] The ability of antibodies to neutralize papillomaviruses
appears to be related to their ability to react with type-specific,
conformational epitopes on the virion surface (Ghim, S.,
Christensen, N. D., Kreider, J. W., and Jenson, A. B., Int. J.
Cancer, 49:285-289 (1991); Christensen, N. D. and Kreider, J. W.,
J. Virol., 64:3151-3165 (1990); Christensen, N. D., Kreider, J. W.,
Cladel, N. M., Patrick, S. D., and Welsh, P. A., J. Virol.,
64:5678-5681 (1990); and Jarrett, W. F. H., O'neil, B. W.,
Gaukroger, J. M., Smith, K. T., Laird, H. M., and Campo, M. S.,
Vet. Rec., 126:437-475 (1990)) and, indeed, previous studies have
demonstrated that the predominant antibody response detected
against HPV-1 in humans is directed against such conformational
epitopes (Steele, J. C., and Gallimore, P. H., Virology,
174:388-398 (1990); and Anisimov, E., Bartk, P., Vlcek, D., Hirsch,
I., Brichacek, B., and Vonka, V., J. Gen. Virol., 71:419-422
(1990)). In the current study, we characterize a series of
antibodies for their reactivity with HPV-1 conformational epitopes
and demonstrate that HPV-1 L1 protein synthesized in cos cells
expresses these virion conformational epitopes. This expressed
protein can, therefore, be used for vaccine development as well as
serologic screening techniques.
[0145] The initial experiments were designed to characterize a
series of polyclonal and monoclonal antibodies for their reactivity
with HPV-1 virions which were either in an intact (native
conformation) or SDS-denatured (non-conformational) state. It was
essential to characterize these antibodies in detail so that they
could be used to evaluate the conformational state of expressed
HPV-1 L1 protein. A summary of the ELISA experiments and the
details for the isolation and purification of the HPV-1 virions are
given in Table IV. Briefly, microtiter plate wells were coated with
either intact or SDS-disrupted HPV-1 virions as described
previously (Cowsert, L. M., Lake, P., and Jenson, A. B., J. Natl.
Cancer Inst., 79:1053-1057 (1987)) and used to screen the indicated
antisera or monoclonal antibodies. The two hyperimmune rabbit sera
produced against HPV-1 have been described previously (Pass, F.,
and Maizel, J. V., J. Invest. Dermatol., 60:307-311 (1973)); rabbit
(R #3) antiserum was generated against disrupted HPV-1 particles
and rabbit (R #7) antiserum against intact particles. The four
monoclonal antibodies that recognize conformational epitopes on the
surface of HPV-1 particles were kindly provided by Dr. P. Pothier
(Bourgogn University, France). Monoclonal antibody (MAB45) defines
a linear epitope on the surface of the HPV-1 virion (Yaegashi, N.,
Jenison, S. A., Valentine, J. M., Dunn, M., Taichman, L. B., Baker,
D. A., and Galloway, D. A., J. Virol., 65:1578-1583 (1991)) and was
obtained through the generosity of Dr. D. A. Baker (State
University of New York, Stony Brook).
4TABLE IV Reactivity of rabbit polyclonal antisera and murine
monoclonal antibodies with intact and disrupted HPV1 virions.sup.a
as determined by ELISA. ELISA value Intact Disputed Antibody
Immunogen virions virions Rabbit Pass #7 intact HPV1 1.493 0.002
Pass #3 disrupted HPV1 0.918 0.616 Murine 334B6 intact HPV1 0.438
0.003 339B6 intact HPV1 0.520 0.000 405D5 intact HPV1 0.429 0.009
D5 4G10 intact HPV1 0.464 0.003 MAB45.sup.b L1 of HPV1 0.512 0.332
.sup.aHPV-1 virions were extracted from productively infected
plantar warts (Jenson, A.B., Lim, L.Y., and Singer, B., J. Cutan.
Pathol., 16:54-59 (1989)) and purified by equilibrium
centrifugation in a CsCl gradient (Cowsert, L.M., Lake, P., and
Jenson, A.B., J. Natl. Cancer Inst., 79:1053-1057 (1987)). Virions
(1.34 g/ml) and empty particles (1.29 g/ml) were collected
separately, dialysed against Tris buffer (20 mM Tris, 10 mM PMSF,
pH 7.5) and stored at -70.degree. C. Microtiter plate wells
(Immunolon II, Dynatech) were coated with either intact or
SDS-disrupted HPV-1 virions as described previously (Cowsert, L.M.,
Lake, P., and Jenson, A.B., J. Natl. Cancer Inst., 79:1053-1057
(1987)). The plates were then washed with PBS containing 0.05%
Tween 20 (PBST). The microtiter wells were further incubated with
PBS containing 1% bovine serum albumin (PBSA) for 1 hr at
37.degree. C. to prevent nonspecific protein binding. The plates
were washed again with PBST and incubated first with either rabbit
polyclonal antibodies or murine monoclonal antibodies as primary
antibody and subsequently with appropriate alkaline
phosphatase-conjugated goat anti-IgG diluated 1:1000 in PBSA
(Bio-Rad) for 1 hr at 37.degree. C. Following several washes, the
microtiter plates were developed with SIGMA 104 phosphatase
substrate (Sigma) in diethanolamine buffer (Voller, A., Bidwell,
D., and Bartlett, A., In "Manual of clinical immunology" (N. Rose
and H. Freedman, Eds.), pp. 359-371. American Society of
Microbiology, Washington, D.C. (1980) for 30 min at 37.degree. C.
Absorbance was measured at 410 nm using a Dynatech Micro-elisa
reader. .sup.bMAB45 is an abbreviated designation for MABDW45
(Baegashi, N., Jenison, S. A., Valentine, J. M., Dunn, M.,
Taichman, L. B., Baker, D. A., and Galloway, D. A., J. Virol.,
65:1578-1583 (1991)).
[0146] The ELISA data indicate that R#7 antiserum indeed is
specific for conformational epitopes on the surface of the HPV-1
virion since it reacts only with intact HPV-1 virions. This is also
true for monoclonal antibodies 334B6, 339B6, 405D5, and D54G10. On
the other hand, R#3 antiserum and monoclonal MAB45 also react well
with SDS-denatured virions, demonstrating their reactivity with
linear, non-conformational epitopes (Cowsert et al, J. Natl. Cancer
Inst., 79:1053-1057 (1987)).
[0147] To confirm the ELISA results shown in Table IV, we also
evaluated the same antibodies for reactivity with disrupted HPV-1
virions as determined by Western blotting (FIG. 1). This figure
demonstrates that only antibodies which recognized denatured HPV-1
virions by ELISA (R#3 and MAB45) showed significant reactivity with
SDS-denatured virion proteins by immunoblotting. However,
antibodies shown in Table IV to recognize only intact virions (R#7,
334B6, 339B6, D54G10, and 405D5) exhibited no or little reactivity
by immunoblotting analysis. Thus, two independent techniques verify
the specificity of the above antibodies for conformational and
non-conformational epitopes on the HPV-1 virion.
[0148] In an attempt to produce isolated L1 protein which retained
critical virion conformation epitopes, we expressed the HPV-1 L1
protein in mammalian cells. The HPV-L1 gene was amplified by PCR
and cloned into the pSVL vector as described in FIG. 2 using
standard molecular techniques (Maniatis, T., Fritsch, E. F., and
Sambrook, J., In "Molecular Cloning: A Laboratory Manual," Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). The
resulting plasmid, pSJ1-L1, expresses the HPV-1 L1 gene from a
strong SV40 late promoter. In addition, the plasmid also contains
the SV40 origin of replication and, when transfected into cos cells
by calcium phosphate precipitation (Graham et al, Virology,
52:456-467 (1973)), replicates to a high copy number.
[0149] COS cells were first evaluated for L1 protein synthesis by
immunoprecipitation techniques using the above antibodies. 48 hr
post-transfection, the cos cells were labelled with
.sup.35S-methionine (NEN, Express .sup.35S Protein labelling Mix)
for 4 hr, washed with buffer, and solubilized in RIPA buffer (which
contains a mixture of 1% NP-40, DOC, and 0.1% SDS detergents). The
cell extracts were then immunoprecipitated with the indicated
antibodies and analyzed by SDS-gel electrophoresis as previously
described (Goldstei et al, EMBO, 9:137-146 (1990)). The data in
FIG. 3 indicate that L1 protein could be efficiently precipitated
by conformation-dependent antibodies (such as R#7, 334B6, 339B6,
D54G10 and 405D5). In addition, the L1 protein could also be
immunoprecipitated with antibodies which recognize
non-conformational epitopes on the virion surface (R#3). These
findings indicate that the L1 protein expressed in COS cells
displayed conformational epitopes observed previously only on
intact virions. It is also obvious that the L1 extraction
conditions did not significantly denature the protein.
Characteristic of L1 protein isolated directly from virions, the
synthesized L1 protein was approximately 57 kD in size (Doorbar et
al, J. Virol., 61:2793-2799 (1987)). The retention of
conformational epitopes in RIPA buffer and the ability of
conformation-dependent antibodies to react with L1 indicates that
the affinity purification of L1 protein from transfected cells will
be possible.
[0150] COS cells were also evaluated for L1 protein synthesis by
immunofluorescence microscopy (FIG. 4). Cells, plated onto glass
coverslips in Dulbecco's modified Eagles medium (DMEM) supplemented
with 10% fetal calf serum, were transfected with 10 .mu.g plasmid
DNA, glycerol-shocked 48 hr later, washed with phosphate buffered
saline (PBS), and fixed for 5 min in cold acetone. The cells were
then reacted with appropriate dilutions of primary antibody
followed by fluorescein-conjugated goat anti-rabbit or goat
anti-mouse IgG. Incubations with primary and secondary antibodies
were performed at room temperature for 1 hr. Subsequent to a final
PBS wash, the coverslips were mounted in Elvanol and viewed with an
Olympus fluorescent microscope. The presence of L1 protein in cell
nuclei was clearly discernible in 5-10% of transfected cells 48
hours post-transfection, independent of whether the primary
antibody reacted with conformational and/or non-conformational
epitopes. All of the antibodies which were capable of
immunoprecipitating L1 were also successful by immunofluorescence.
As mentioned previously, antibodies produced against disrupted
virions recognize both internal and external virion linear epitopes
and therefore are capable of reacting with intact particles (e.g.,
R#3). However, such antibodies do not recognize conformational
epitopes and are not neutralizing (Ghim et al, Intl. J. Cancer,
49:285-289 (1991)). Thus, the staining pattern obtained with rabbit
antisera to native (R#7) or denatured (R#3) HPV-1 virions was
indistinguishable. These results, therefore, demonstrate
unequivocally that the L1 protein synthesized in the cos cells was
of a conformation similar to that found in intact virions. In
addition, the protein clearly translocated to the nucleus in a
normal fashion (Zhou et al, Virology, 185:625-632 (1991)).
[0151] The above findings suggest that the HPV-1 major capsid
protein, when expressed in the absence of other viral proteins, can
precisely reproduce/mimic the antigenicity of intact viral
particles. While we cannot be certain that no assembled viral
particles are present in the transfected cos cells, we have been
unsuccessful in visualizing such structures by electron microscopic
examination of either transfected cells or of immunoprecipitates
containing L1 protein (data not shown). Apparently it is not
essential to have viral particle formation in order to reproduce
the characteristic, viral conformational epitopes.
[0152] Since the neutralization sites present on papillomavirus
virions consist predominantly of conformational epitopes, it is
inferred in our studies that the L1 protein synthesized in cos
cells might serve successfully as a vaccine or for the serologic
detection and typing of papillomavirus infections. Due to the
similarities among the papillomaviruses with respect to genetic
organization, virion structure, and amino acid sequence of their
capsid proteins, it is also likely that our findings with HPV-1 L1
will have direct applicability to the study of other HPV's such as
HPV-16 and HPV-18 which have important contributory roles to the
development of cervical carcinoma.
5TABLE V ELISA VALUES AT 25 MIN IN SUBSTRATE Four rabbits were
inoculated with homogenates of COS cells containing intranuclear,
conformationally correct BPV-1 L1. Each of 4 rabbits received
homogenates of 1 .times. 10.sup.6 cells in Freund's incomplete
adjuvant on days 14 and 28, and were then exsanguinated on day 38.
Prebleed sera from the 4 rabbits were negative for reactivity with
intact and denatured BPV-1 virions by ELISA. At day 38, rabbit #1
2, 3 and 4 were tested for reactivity with intact and disrupted
BPV-1 particles after 25 min. incubation with substrate shown in
Table V. 1:50 1:100 1:500 1. .445 .266 .014 Intact BPV-1 (I) .042
.028 .001 Disrupted BPV-1 (D) .016 .001 -- .002 Phosphate buffer
saline (PBS) 2. .332 .210 .011 (I) .076 .047 .077 (D) .025 .018
.001 (PBS) 3. .157 .096 .003 (I) .027 .016 -- .001 (D) .022 .011 --
.001 (PBS) 4. .275 .159 .011 (I) .075 .044 .005 (D) .017 .011 .001
(PBS) *1H8 -- -- .022 (I) -- -- .880 (D) -- -- < .020 (PBS)
**Rabbit intact -- -- >2 .000 (I) BPV-1 Virions -- -- .058 (D)
-- -- < .020 (PBS) *MAb 1H8 recognizes only disrupted BPV-1
**Polyclonal Ab rabbit anti-intact BPV-1 recognized only intact
BPV-1
EXAMPLE 4
[0153] This experiment describes the successful use of a
formalin-inactivated canine oral wart homogenate as a vaccine to
prevent infection by COPV in Beagle dogs. In this experiment, 26
dogs received doses of phosphate buffered saline (PBS)
intradermally, and 99 dogs received two doses of a
formalin-inactivated vaccine containing 50 ng of COPV L1 capsid
protein. One month after the second dose, all 125 dogs were
challenged with infectious COPV by scarification of the oral
mucosa. All of the control group developed papillomavirus by 6-8
weeks after challenge. By contrast, none of the dogs immunized with
the formalin-inactivated vaccine containing COPV L1 conformational
capsid protein developed perceptible papillomas. The methodology of
this experiment is described in detail below.
Materials and Methods
[0154] Equal numbers of male and female outbred Beagle dogs were
obtained and maintained at Marshall Farms in North Rose, N.Y. The
animals were vaccinated for parvovirus (31, 38, 45, and 59 days of
age Northwest Tennessee Veterinary Services, Dresden, Tenn.);
canine parainfluenza-Bordatella bronchisepticum (26 days of age,
Intra-Trac-II, Schering-Plough Animal Health, Omaha, Nebr.); mink
distemper (69 days of age, Distem-R TC, Schering-Plough Animal
Health); canine distemper, adenovirus type-II, parainfluenza,
parvovirus, and Leptospira (105 days of age, Duramune, DA.sub.2
LP+PV, Fort Dodge Laboratories, Fort Dodge, Iowa); and rabies, (100
days of age, Imrab-1, Pitman-Moore, Mundelein, Ill.). Blood was
drawn routinely at 120 days of age for CBC, reticulocyte count, and
prothrombin times.
[0155] The dogs were nursed by their dams until 8 weeks and then
put on a commercial diet. After weaning, the dogs were housed in
open sheds in wire cages suspended above the ground. The dogs had
access to tap water ad libitum. The dogs were on a natural light
cycle.
Vaccine Preparation
[0156] Twenty-five (25) dogs were routinely inoculated as described
below with live COPV in order to induce papillomas that produced
infectious virions. After inoculation, the papillomas were removed
surgically eight weeks after induction by scarification. The
resultant papillomas were frozen in plastic vials and stored at
-70.degree. C. until used. For vaccine production or preparation of
the challenge inoculum, papillomas were thawed, placed on two
aluminum plates, and mascerated by hammering the plates together.
The material was then placed in a blender with chlorinated tap
water (2 grams of tissue into 100 ml total of water, 2% w/v) and
homogenized for 10 minutes at room temperature. The homogenate was
then passed twice through cheese cloth to remove large particulates
and then frozen at -70.degree. C. This preparation was thawed
slowly to room temperature and then used to challenge dogs to
induce productive oral papillomas or inactivated by the addition of
8 ml of neutral buffered 10% formalin to 240 mls (1:30 v/v) of the
filtered homogenate, stored at 4.degree. C. for 48 hours, and then
used as the vaccine. This crude vaccine contained COPV L1 protein
at concentrations ranging from 50-1000 ng/ml as determined by
quantitative immunoblotting and ELISA techniques.
Animal Vaccination
[0157] All dogs were injected intradermally twice, at 8 and 10
weeks of age. For each injection, 0.2 ml of vaccine formulation was
injected into the foot pad of the dew claw phalanx) using a TB
syringe with a 26 gauge needle. Twenty-six Beagle dogs received
phosphate buffered saline, pH 7.4, as a placebo. Ninety-nine Beagle
dogs received the formalin-inactivated vaccine in the same
manner.
Challenge of Vaccinated Does and Control Groups
[0158] All 125 dogs were then challenged with infectious live COPV
by scarification with a wire brush on the dorsal, buccal and
maxillary mucosa. Challenge with infectious virus was performed one
month after the second dose of vaccine or placebo solution. After
challenge, each dog was examined daily by a clinical veterinarian
or a trained veterinary technician for eight weeks.
Results
[0159] All of the control group dogs (26/26) which were injected
with PBS and challenged with infectious COPV developed oral
papillomas between six and eight weeks following exposure to the
virus. By contrast, none of the dogs which were injected with the
formalin-inactivated preparation (0/99) developed clinically
evident oral papillomas. These results are believed to provide
persuasive in vivo evidence that vaccination with wart extract
containing conformationally correct L1 proteins may be used to
protect Beagles against COPV infection. Additionally, given the
substantial genetic and structural similarities between COPV and
HPV, these results suggest that similar approaches may be applied
for the prevention of HPV infections.
Example 5
[0160] In this experiment, serum obtained from the above vaccinated
weanling dogs was passively transferred to naive dogs. The
recipient non-vaccinated dogs (which had received the immune serum
transfer) were then evaluated for protection against COPV
infection.
Materials and Methods
[0161] Serum samples were harvested by phlebotomy from either
non-immune 10 week old naive beagle weanlings or from immune 12
week old weanlings which had been vaccinated with a crude COPV wart
vaccine on weeks 8 and 10 following birth as described in Example
4. The serum immunoglobulin fraction was obtained from both groups
by ammonium sulfate differential precipitation and dialysis against
phosphate buffered saline.
[0162] After the immunoglobulin solutions were obtained from the
non-immune and the immune dogs, these solutions were then
administered intravenously to two groups of four dogs intravenously
over a 20 minute period at a dosage of 200 mg/kg. Additionally, a
control group of 4 dogs was administered lactate Ringers solution
intravenously over a 20 minute period at a dosage of 200 mg.kg.
These three groups of dogs were then challenged with infectious
live COPV by scarification as described in Example 4.
Results
[0163] The results, of this experiments are illustrated in FIG. 5.
As summarized therein, all the dogs which were administered lactate
Ringers solution as well as the dogs which were administered
non-immune dog serum developed papillomas after challenge with live
infectious COPV. By contrast, none of the dogs who received the
immune dog serum from the dogs which had been vaccinated with the
crude COPV wart extract showed any signs of papillomas after
challenge with live infectious COPV.
[0164] Therefore, these results provide evidence that immunity
induced by the wart vaccine is mediated by immunoglobulins and that
complete protection of animals can be achieved by these antibodies
without the need or cooperation of cellular immune responses. This
is a critical element in the design of a papillomavirus vaccine,
whether it be for COPV or HPV.
[0165] Also important is the observation that the dose of challenge
COPV used in this study was extremely high. Usually dogs require
6-8 weeks in order for tumors to become evident. However, in this
specific experiment, a concentrated preparation of wart extract was
used which generated tumors within 3 weeks. Thus, even when animals
are challenged with extraordinarily high titers of virus, they are
protected by passively transferred antibodies.
EXAMPLE 6
[0166] The previous two experiments described two critical elements
in the development of a COPV vaccine containing: (1) the success of
a formalin-inactivated wart extract containing COPV L1 proteins for
conferring immunity against COPV in Beagle dogs and (2) the use of
serum obtained from the above vaccinated dogs for the passive
transfer of immunity. The final critical element, which is the
object of this experiment, is to show that the isolated,
conformationally-correct form of COPV L1 protein is the essential
element in the vaccine which elicits immunity and that the COPV L1
protein is sufficient in itself in inducing protective
immunity.
Materials and Methods
[0167] The following experiment relates to the use of
conformationally correct recombinant COPV L1 proteins, in
particular COPV L1 proteins expressed in recombinant baculovirus
virus infected Sf9 cells, as a vaccine against COPV in Beagle
dogs.
[0168] The COPV DNA was originally isolated and cloned by Dr. John
Sundberg (Jackson Laboratories). Briefly, the COPV DNA was isolated
from virions which had been purified by cesium chloride gradients
from a canine oral papilloma. The COPV 8.2 kb DNA fragment has been
cloned into a pBR322 at a unique Eco RI site. Sequencing of the
genome (by the Sanger chain termination technique) was effected in
collaboration with Dr. John Sundberg and Dr. Hijo Delius
(Heidelberg, Germany). The L1 ORF extends from bp 5277 to bp 6785
(not including the termination codon which begins at 6786). For
brevity, the amino acid translation of the DNA sequence is
presented below and compared with the amino acid sequence of the
HPV-1 L1 protein. Stars (*) indicate amino acid identities. The
COPV L1 was compared with that of HPV-1 because the latter virus
was the prototype HPV virus which we chose for expression using the
pSVL vector (described in Example 3). However, given that the L1
protein is the most highly conserved protein of all the
papillomavirus proteins, a comparison of the COPV L1 sequences with
other papillomavirus L1 proteins would yield similar results.
[0169] This sequence analysis clearly indicates a high degree of
homology between the HPV-1 and COPV L1 proteins, including the
conservation of certain structural domains such as the positively
charged carboxyl-terminus which has recently been shown to mediate
nuclear translocation. The COPV L1 protein is predicted to consist
of 503 amino acids compared to the HPV-1 L1 protein of 508 amino
acids.
6 L1 Protein Sequences: HPV-1
MYNVFQMAVWLPAQNKFYLPPQPITRILSTDEYVTRTNLFYHATSERLLL *
**************** * ******* *** **** ***** COPV
M---AVWLPAQNKFYLPPQPSTKVLSTDEYVSRTNIFYHASSERLLT HPV-1
VGHPLFEI---SSNQTVTIPKVSPNAFRVFRVRFADPNRFAFGDKAIFNP **** ** * ******
**** *** ****** * * COPV VGHPFYEIYKEERSEEVIVPKVSPNQYRV-
FRLLLPDPNNFAFGDKSLFDP HPV-1 ETERLVWGLRGIEIGRGQPLGIGITGHPLL-
NKLDDAENPTNYINTHANG- * ********* ********** **** * *** * * COPV
EKERLVWGLRGLEIGRGQPLGISVTGHPTFDRYNDVENPNKNLAGHGGGT HPV-1
DSRQNTAFDAKQTQMFLVGCTPASGEHWT-SRRCPGEQVKLGDCPRVQMI *** * * ******
** ** **** * * * * ** COPV
DSRVNMGLDPKQTQMFMIGCKPALGEHWSLTRWCTGQVHTAGQCPPIELR HPV-1
ESVIEDGDMMDIGFGAMDFAALQQDKSDVPLDVVQATCKYPDYIRMNHEA ****** *********
*** ** ** * * ****** * * COPV
NTTIEDGDMVDIGFGAMDFKALQHYKSGVPIDIVNSACKYPDYLKMANEP HPV-1
YGNSMFFFARREQMYTRHFFTRGGSVGDKEAVPQSLYLTADAEPRTTLAT ** *** **** * **
* * * * ** * * * COPV YGDRCFFFVRREQLYARHIMSRSGTQG-LEPVPK-
DTYATREDN---NIGT HPV-1 TNYVGTPSGSMVSSDVQLFNRSYWLQRGQGQNNGI-
GWRNQLFITVGDNTR ** ***** *** ***** ** ** ** **** * **** * **** COPV
TNYFSTPSGSLVSSEGQLFNRPYWIQRSQGKNNGIAWGNQLFLTVVDNTR HPV-1
GTSLSIS---MKNNASTTYSNANFNDFLRHTEEFDLSFIVQLCKVRLTPE ** * * * *** **
* ********* ** COPV GTPLTINIGQQDKPEEGNYVPSSYR-
TYLRHVEEYEVSIIVQLCKVKLSPE HPV-1 NLAYIHTMDPNILEDWQLSVSQPPTN-
PLEDQYRFLGSSLAAKCPEQAPPE *** ******** *** * ** * * ** *** *** **
COPV NLAIIHTMDPNIIEDWHLNVT-PPSGTLDDTYRYI-NSLATKCPTNIPPK HPV-1
PQTDPYSQYKFWEVDLTERMSEQLDQFPLGRKFLYQSGMTQRTATSSTTK ** ******* *
***** ******* * * COPV TNVDPFRDFKFWEVDLKDKMTEQ-
LDQTPLGRKFLFQTN-VLRPRSVKVRS HPV-1 RKTVRVSTSAKRRRKA * * * * COPV
TSHVSVKRKAVKRKRK
[0170] In this experiment, 40 dogs were vaccinated at 8 and 10
weeks of age with 0.2 ml of several vaccine formulations. The
injections were performed in the foot pad as described previously
in Example 4. The recombinantly-expressed L1 protein was examined
in the electron microscope and found to assemble into virus-like
particles and, more importantly, to react with antiserum that was
specific for COPV conformational capsid surface epitopes. The first
control group of dogs was mock-vaccinated with phosphate buffered
saline (Group I), and the second group of dogs was vaccinated with
formalin-fixed wart homogenates (Group II) as described in Example
4. The third group was vaccinated with 20 .mu.g L1 protein
contained in phosphate buffered saline (Group III), the fourth
group with 20 .mu.g of L1 protein in PBS containing alum (Group
IV), and the fifth group with 20 .mu.g L1 protein in QS21 adjuvant
(Group V).
[0171] Two weeks after completing the second administration of
vaccine, all the animals were challenged with live, infectious COPV
by scarification with a wire brush as in Example 4. Dogs were then
evaluated weekly after challenge to detect oral papillomas for 10
weeks.
Results
[0172] In the control group of beagles (given phosphate buffered
saline for vaccination), six of eight animals (Group I) developed
oral tumors. By contrast, none (zero of thirty-two) of the dogs
which were injected with formalin-fixed wart extract or any of the
recombinant L1 protein-containing compositions showed any signs of
oral tumors after challenge.
[0173] These results are summarized in FIG. 6 and establish that
recombinant conformationally correct L1 proteins may be used as an
effective vaccine against COPV in Beagle dogs. This experiment also
indicates that COPV L1 protein is sufficient (in the absence of
viral L2 capsid protein as well as other cellular proteins in the
wart extract) to completely protect against infectious COPV
challenge. Moreover, given the substantial similarities between
COPV and human papillomavirus, these results provide further
evidence that conformationally correct human papillomavirus L1
proteins may be used as an effective vaccine against human
papillomavirus infection.
[0174] To further establish the importance of L1 conformation, the
antibody response against both linear and conformational COPV L1
epitopes was compared after the first vaccination, after the second
vaccination, and after challenge with infectious COPV. These
results are summarized in FIG. 7 and FIG. 8. It can be seen from
these figures that the Beagle dogs which were inoculated with the
wart extract or with the recombinant conformationally correct COPV
L1 proteins exhibit a substantial antibody response against COPV
conformational epitopes. By contrast, the control group exhibited
virtually no change in the antibody response to conformational
epitopes after challenge.
[0175] While vaccinated animals clearly developed an immune
response to conformational L1 epitopes, they failed to develop a
significant response to linear (non-conformational epitopes) as
demonstrated in FIG. 7. This provides further evidence that
antibodies to linear epitopes are not involved in protection.
[0176] Group 4 animals, which were inoculated with the recombinant
L1 protein in alum, had the highest linear epitope antibody
response. This suggests that the alum adjuvant may partially affect
the L1 protein's conformational structure, thereby exposing linear
L1 epitopes to the dog's immune system.
EXAMPLE 7
[0177] Example 3 of this application demonstrates that the HPV-1 L1
protein (when expressed by an SV40 vector in COS cells) reproduces
conformational epitopes of intact HPV-1 virions. In addition, the
L1 capsid protein was translocated into the cell nucleus, was of
appropriate size (57 kb), and could be isolated as a conformational
protein by immunoprecipitation techniques. As discussed supra,
based on these results and because of the similarities between
different papillomaviruses with respect to genetic organization,
virion structure, and amino acid sequence of capsid proteins, these
results have direct applicability to other PV L1 proteins,
including HPVs such as HPV-6, HPV-11, HPV-16, and HPV-18 as well as
L1 sequences of other species origin including, e.g., COPV and
equine papillomavirus.
[0178] Therefore, using the exact same methodology as above, the
COPV L1 sequence and fragments thereof were expressed by an SV40
vector in COS cells. It was found that the COPV L1 sequence was
expressed in proper conformation as demonstrating by reactivity
with conformationally-depende- nt antibodies.
[0179] By contrast, it was found that expression of COPV L1
sequences containing deletions in the amino-terminus portion of the
DNA resulted in L1 proteins which did not exhibit the correct
conformation. These results suggest that the amino-terminus portion
of the L1 sequence may be involved in the folding of the L1 protein
and its proper conformation.
[0180] However, expression of a COPV L1 DNA lacking the codons
encoding the 26 amino acids at the carboxy-terminus of the L1
protein and which were replaced by a 5 amino acid nuclear signal
sequence of a nonstructural viral protein of SV40, in COS cells was
found to result in COPV L1 proteins which were translocated into
cell nuclei and exhibited conformational epitopes. Therefore, these
results provide convincing evidence that PV L1 DNA fragments may
also be used to produce conformational L1 proteins. More
specifically, these results substantiate that PV L1 DNA sequences
which lack a portion of the carboxy-terminal portion of the L1
sequence result in conformational L1 proteins. Moreover, other L1
fragments which upon expression give rise to conformational L1
proteins may be identified by immunodetection, e.g., by screening
for reactivity with antibodies specific to conformational L1
epitopes.
EXAMPLE 8
[0181] To further confirm that it is not necessary for the L1
protein to assemble into virus-like particles to elicit a
protective response against PV infection and the subsequent
formation of papillomas, an additional experiment was conducted
using L1 protein expressed using the above-described SV40
vector/COS cell expression system. This system was selected because
the results of previous multiple electron microscopy studies have
indicated that PV pseudocapsids are not produced by COS cells.
[0182] The entire COPV L1 sequence was expressed using an SV40
vector in COS cells. Using substantially the same protocol as
described supra, naive beagle dogs were inoculated intradermally
with a formalin-fixed crude extract of COS cells expressing the
entire COPV L1 protein.
[0183] The results of this experiment demonstrated that of five
beagles inoculated with the formalin-fixed crude extract of COS
cells expressing COPV L1 protein, four were completely protected
upon intraoral challenge with infectious COPV. Therefore, this
provides convincing in vivo evidence that COPV L1 proteins
expressed in COS cells can be used as an effective vaccine for
providing immunity against COPV infection.
[0184] These results also indicate that PV conformationally correct
L1 proteins alone or linked together as capsomeres, but not
self-assembled as a pseudovirus particle are capable of eliciting
neutralizing antibodies that protect against the formation of
papillomas. Also, these results provide further evidence that it is
not necessary for the L1 protein to assemble into virus-like
particles to elicit a protective response against PV infection and
the subsequent formation of papillomas.
EXAMPLE 9
[0185] To demonstrate the exquisite specificity of canine
antibodies produced against conformational epitopes of COPV L1
protein, a second large vaccine study was conducted in weanling
beagles. Forty-two beagles were inoculated intradermally at 8 and
weeks of age with the following vaccine preparations: 14 received
COPV L1 VLPs alone; 7 received formalin-inactivated oral COPV L1
proteins; 7 received an extract of the vector alone, and 7 received
saline solution. All beagles were challenged orally with infectious
COPV at 12 weeks of age and followed for development of oral cavity
papillomas. None of 21 beagles receiving either COPV L1 VLPs or
formalin-inactivated papillomas developed oral cavity papillomas.
Twenty-seven of 28 of the remaining beagles developed florid oral
papillomatosis. All of the beagles receiving either denatured COPV
L1 proteins (nonconformational L1 proteins; 7 of 7) or HPV-11 L1
VLPs (7 of 7) developed extensive, large oral papillomas. This is
highly significant scientific evidence that conformationally
correct COPV-L1 protects against COPV-induced oral papillomas, and
that this protection is COPV L1 type specific since there was no
protection conferred by vaccination with the HPV-11 VLP (HPV-11 is
the prototype oral human papillomavirus that infects and causes
papillomas in the oral cavity).
[0186] Now having fully described this invention, it will be
understood by those with skill in the art that this invention may
be performed within a wide and equivalent range of conditions,
parameters, and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
[0187] All references cited herein are incorporated by reference in
their entirety as if individually incorporated by reference.
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