U.S. patent application number 10/331395 was filed with the patent office on 2003-12-04 for graft animal model for high induction of papillomas, the propagation of papillomavirus and evaluation of candidate therapeutic agents.
This patent application is currently assigned to Boehringer Ingelheim (Canada) Ltd.. Invention is credited to Jianmin, Duan.
Application Number | 20030226157 10/331395 |
Document ID | / |
Family ID | 22356523 |
Filed Date | 2003-12-04 |
United States Patent
Application |
20030226157 |
Kind Code |
A1 |
Jianmin, Duan |
December 4, 2003 |
Graft animal model for high induction of papillomas, the
propagation of papillomavirus and evaluation of candidate
therapeutic agents
Abstract
The present invention relates to a graft animal model for
propagating papilloma virus and for evaluating and testing
candidate therapeutic agents against papilloma virus. The animal
model comprises, a recipient animal engrafted with injured skin
graft infected with a host-specific papilloma virus (PV). The
grafted skin, having demonstrable papillomas supports the
propagation of its host-specific PV. The invention particularly
relates to a xenograft animal model for hosting and propagating
human papillomavirus (HPV), thereby providing a means for
generating infectious and passaging HPV suspensions, and for
screening candidate therapeutic agents against HPV. The invention
additionally relates to a novel method for generating the xenograft
human animal model.
Inventors: |
Jianmin, Duan; (Chomedey,
CA) |
Correspondence
Address: |
BOEHRINGER INGELHEIM CORPORATION
900 RIDGEBURY RD
P O BOX 368
RIDGEFIELD
CT
06877
US
|
Assignee: |
Boehringer Ingelheim (Canada)
Ltd.
Laval
CA
|
Family ID: |
22356523 |
Appl. No.: |
10/331395 |
Filed: |
December 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10331395 |
Dec 30, 2002 |
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09465666 |
Dec 17, 1999 |
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6596924 |
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60114642 |
Jan 4, 1999 |
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Current U.S.
Class: |
800/9 ;
435/235.1; 800/18 |
Current CPC
Class: |
A61K 49/0008 20130101;
A01K 67/0271 20130101 |
Class at
Publication: |
800/9 ; 800/18;
435/235.1 |
International
Class: |
A01K 067/027; C12N
007/00 |
Claims
1. A graft mouse model for improving the rate of induction and
formation of human papillomas comprising: (a) a recipient mouse
selected from the group consisting of: severe combined
immuno-deficient (SCID) mice, SCID/beige mice, nude mice, and
NIH-nu-bg-xid mice, said mouse being grafted with human foreskin
tissue, said foreskin tissue having been meshed prior to said
grafting; and (b) inoculating said grafted foreskin tissue prior to
healing with an inoculum of human papillomavirus, wherein said
grafted foreskin is supported by said recipient mouse and is
capable of inducing and sustaining growth of said human
papillomavirus and harboring at least one papilloma containing
infectious viral particles.
2. The model according to claim 1, wherein said meshing is carried
out manually.
3. The model according to claim 1, wherein said meshing of the
foreskin tissue is accomplished with the use of a meshing
machine.
4. The model according to claim 1, wherein said mouse is the
NIH-nu-bg-xid mouse.
5. The model according to claim 1, wherein said human
papillomavirus is HPV low-risk or high-risk.
6. The model according to claim 5, wherein said low risk human
papillomavirus is selected from the group consisting of: type 6,
type 11 and type 13.
7. The model according to claim 5, wherein said high risk human
papillomavirus is selected from the group consisting of: type 16,
type 18, type 35, type 45, type 52 and type 58.
8. The model according to claim 6, wherein said human
papillomavirus is low risk type 6 or type 11.
9. A method for producing a graft mouse model for propagating
infectious human papilloma viral particles, said method comprising
the following steps: (a) obtaining foreskin tissue from a human
donor and meshing said foreskin; (b) grafting said meshed foreskin
tissue onto a recipient mouse selected from the group consisting
of: severe combined immuno-deficient (SCID) mice, SCID/beige mice,
nude mice, and NIH-nu-bg-xid mice (c) inoculating said grafted
foreskin tissue prior to healing with an inoculum of human
papillomavirus; and (a) providing sufficient time for said
papillomavirus to form in said grafted tissue and to harbor at
least one papilloma containing infectious viral particles.
10. The method according to claim 9, wherein said meshing is
carried out manually.
11. The method according to claim 9, wherein said meshing of the
foreskin tissue is accomplished with the use of a meshing
machine.
12. The method according to claim 9, wherein said human
papillomavirus is HPV low-risk or high-risk.
13. The method according to claim 12, wherein said low risk human
papillomavirus is selected from the group consisting of: type 6,
type 11 and type 13.
14. The method according to claim 12, wherein said high risk human
papillomavirus is selected from the group consisting of: type 16,
type 18, type 35, type 45, type 52 and type 58.
15. The method according to claim 13, wherein said human
papillomavirus is low risk type 6 or type 11.
16. The method according to claim 9, wherein said foreskin tissue
is inoculated with a papillomavirus suspension, in-situ immediately
post-grafting.
17. The method according to claim 16, wherein said post-grafting
inoculation in-situ is carried out by overlaying said grafted
tissue with a viral suspension, or by injecting the grafted tissue
with a viral suspension or a combination thereof.
18. The method according to claim 9, wherein said graft tissue is
inserted cutaneously onto said recipient mouse.
19. The method according to claim 9, wherein said graft tissue is
inserted subcutaneously onto said recipient mouse such as to form a
subcutaneous papilloma.
20. The method according to claim 19, wherein said subcutaneous
papilloma is exposed whereby skin covering the apex of the
subcutaneous papilloma is cut with a straight incision using
surgical scissors, said skin being gently retracted and held back
allowing the papilloma to grow outwardly and form a cutaneous
papilloma.
21. A method for evaluating the efficacy of a therapeutic agent
useful against papilloma virus infection comprising the steps of:
(a) providing a papillomavirus-infected mouse model according to
claim 1; (b) treating said papillomavirus-infected xenografted
mouse by administering a candidate therapeutic agent in an
appropriate pharmaceutical carrier; and (c) evaluating the efficacy
of said therapeutic agent in preventing the appearance, reducing
the physiological symptoms or reducing the evidence of said
infection in said infected mouse.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/465,666, filed Dec. 17, 1999, which claims, as does the
present application priority to U.S. provisional application No.
60/114,642, filed Jan. 4, 1999, the disclosures of all of which are
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a graft animal model for
propagating HPV and for evaluating and testing candidate
therapeutic agents against HPV. The animal model comprises, a
recipient animal engrafted with injured skin graft infected with a
host-specific papilloma virus (PV). The grafted skin, having
demonstrable papillomas supports the propagation of its
host-specific PV. The invention particularly relates to a highly
reproducible xenograft animal model for hosting and propagating
human papillomavirus, thereby providing a means for generating
infectious human PV suspensions and for passaging papillomavirus.
The invention additionally relates to a novel method for generating
the xenograft human animal model.
BACKGROUND OF THE INVENTION
[0003] Papillomaviruses (PV) are non-enveloped DNA viruses that
induce hyperproliferative lesions of the epithelia. The
papillomaviruses are widespread in nature and have been recognized
in higher vertebrates. Viruses have been characterized, amongst
others, from humans, cattle, rabbits, horses, and dogs. The first
papillomavirus was described in 1933 as cottontail rabbit
papillomavirus (CRPV). Since then, the cottontail rabbit as well as
bovine papillomavirus type 1 (BPV-1) have served as experimental
prototypes for studies on papillomaviruses. Most animal
papillomaviruses are associated with purely epithelial
proliferative lesions, and most lesions in animals are cutaneous.
In the human there are more than 75 types papillomavirus (HPV) that
have been identified and they have been catalogued by site of
infection: cutaneous epithelium and mucosal epithelium (oral and
genital mucosa). The cutaneous-related diseases include flat warts,
plantar warts, etc. The mucosal-related diseases include laryngeal
papillomas and anogenital diseases comprising cervical carcinomas
(Fields, 1996, Virology, 3rd ed. Lippincott-Raven Pub.,
Philadelphia, N.Y.).
[0004] There are more than 25 HPV types that are implicated in
anogenital diseases, these are grouped into "low risk" and "high
risk" types. The low risk types include HPV type 6, type 11 and
type 13 and induce mostly benign lesions such as condyloma
acuminata (genital warts) and low grade squamous intraepithelial
lesions (SIL). In the United States there are 5 million people with
genital warts of which 90% is attributed to HPV-6 and HPV-11. About
90% of SIL are also caused by low risk types 6 and 11. The other
10% of SIL are caused by high risk HPVs.
[0005] The high risk types papillomaviruses are associated with
high grade SIL and cervical cancer and include most frequently HPV
types 16, 18, 31, 33, 35, 45, 52, and 58. The progression from
low-grade SIL to high-grade SIL is much more frequent for lesions
that contain high risk HPV-16 and -18 as compared to those that
contain low risk HPV types. In addition, only four HPV types are
detected frequently in cervical cancer (types 16, 18, 31 and 45).
About 500,000 new cases of invasive cancer of the cervix are
diagnosed annually worldwide (Fields, 1996, supra).
[0006] Treatments for genital warts include physical removal such
as cryotherapy, CO.sub.2 laser, electrosurgery, or surgical
excision. Cytotoxic agents may also be used such as trichloroacetic
acid (TCA), podophyllin or podofilox. Immunotherapy is also
available such as Interferon or Imiquimod. These treatments are not
completely effective in eliminating all viral particles and there
is either a high cost incurred or uncomfortable side effects
related thereto. In fact, there are currently no effective
antiviral treatments for HPV infection, since with all current
therapies recurrent warts are common (Beutner & Ferenczy, 1997,
Amer. J. Med., 102(5A): 28-37).
[0007] The life cycle of HPV is closely coupled to keratinocyte
differentiation. Infection is believed to occur at a site of tissue
disruption in the basal epithelium. Unlike normal cells, cellular
division continues as the cell undergoes vertical differentiation.
As the infected cells undergo progressive differentiation the viral
copy number and viral gene expression increase, with the eventual
late gene expression and virion assembly in terminally
differentiated keratinocytes and the release of viral particles
(Fields, 1996, supra).
[0008] Papillomaviruses are fastidious viruses that cannot be
propagated in vitro. As such, the virus requires a host-specific
animal for growth. The ineffectiveness of the current methods to
treat PV infections has demonstrated the need to identify new
therapeutic agents as a means to prevent and treat HPV infections.
The success of developing candidate therapeutic agents to combat
papillomavirus has been limited in part due to difficulties
including, propagating the virus, obtaining sufficient infectious
viral particles and the lack of a good in-vivo model to evaluate
the effectiveness of candidate therapeutic agents. Attempts to
overcome these difficulties have been addressed by generating
xenograft animal models for human papillomavirus. However, all the
models known in the prior art have had limited success in
overcoming these difficulties.
[0009] The ideal animal model is described as having the following
attributes: being widely available, easy to handle and maintain in
a laboratory, large enough to provide tissue samples, able to
induce and form papilloma lesions that are comparable to those in
humans, the papillomas should be readily accessible for treatment,
and able to yield a large amount of infectious viral particles
(Stanley, et al., 1997, Antiviral Chemistry & Chemotherapy,
8(5):381-400).
[0010] In U.S. Pat. Nos. 4,814,268 and 5,071,757 (Kreider et al.),
human skin tissue subjected to human papillomavirus was grafted
under the renal capsule of athymic mice. This is a complex
procedure which requires surgical refinement. The graft is allowed
to remain in the animal until recoverable quantities of the virus
are produced. Examination of the graft site and recovery of viral
particles requires the animals to be killed. The infectivity of the
recovered viral particles from the graft site was reported to be
only at a 10.sup.-2 dilution. More importantly, since the
papillomas formed are not visible, evaluation of therapeutic agents
necessitates sacrificing the animal. Subsequent attempts by this
group (Kowett et al., 1990, Int. Virology, 31:109-115) to replicate
these published results, harvesting infectious viral stock capable
of infecting other animal models, have failed. The authors
hypothesized that the first wart tissue collected from patients and
used to infect an animal model probably contains more infectious
virions and is thus successful in initiating papilloma infection in
the xenograft animal.
[0011] Bonnez W. et al. (1993, Virology 197:455-458) described
human foreskin infected in vitro with HPV type 11, implanted under
the renal capsule, peritoneum and subcutaneous in SCID mice. Only
58% of the grafts showed signs of HPV infections. In the
subcutaneous implanted grafts, only 25% were positive for HPV by
immunocytochemistry and RT-PCR. The resultant subcutaneous
papillomas were not serially passaged or harvested.
[0012] Brandsma J. L. et al. (1995, J. of Vir. 69:2716-2721) and
U.S. Pat. No. 5,811,632, describe the delivery of HPV type 16
genomic DNA to human foreskin engrafted onto SCID mice. In total 16
grafts were inoculated with naked HPV DNA, eight inoculated
pre-engrafting and eight post-engrafting. Only two grafts
inoculated post-grafting appeared to develop signs of HPV
infection. However these two prior art documents do not teach
harvesting infectious viral particles or the passaging of
papillomavirus.
[0013] Sexton C. J. et al. (1995, J. of Gen. Vir. 76:3107-3112)
described a grafting method whereby a glass cover slip was first
inserted into the graft site of a SCID mouse for one to two weeks.
This is replaced with a silicone grafting chamber in which benign
wart tissue was placed. After five weeks, macroscopic warts
developed. Attempts to graft the wart tissue resulted in
hyperproliferative human epithelium devoid of viral infection. Thus
serial passaging of these warts and harvesting infectious particles
are not taught.
[0014] Bonnez W. et al. (1998, J. Virol. 72:5256-5261) reported the
isolation and propagation of HPV-16. The virus was isolated from
clinical samples and used to infect human foreskin prior to
subcutaneous implantation into SCID mice. The sites were prepared
by inserting glass cover slips at the graft sites two weeks prior
to engrafting the infected foreskin. The lesions at the graft sites
were exposed four weeks after engrafting and the animals sacrificed
24 weeks after engrafting. Only three of the five grafts showed
small papillomas. The virions from these papillomas were harvested
and used to inoculate a second set of xenografted human tissue. In
this second set of animals 60 grafts were attempted, the resultant
lesions were not exposed and the animals were sacrificed 16 weeks
after engrafting. Of the 60 grafts, 34 were positive for the
presence of HPV DNA and only 1 was positive for HPV capsid by
immunochemistry. This prior art does not teach passaging of
papillomas or the potential to harvest virulent infectious viral
particles to generate an infectious viral suspension. In this model
it took 40 weeks to produce one graft site in which potentially
infectious viral particles could be detected. In an improved animal
model it would be desirable to markedly decrease the incubation
time for inducing papillomas having infectious viral particles and
more importantly to increase the success rate of papilloma
formation evaluated by an increase in size and number of
papillomas.
[0015] To date there are no animal models for human papillomavirus
infections that are easy to generate, dependable, reliable and
reproducible and which allow for serial passaging of papillomas and
harvesting of infectious viral particles. There thus remains a need
to develop an animal model in which a human papillomavirus can be
easily propagated and serially passaged without requiring complex
surgical procedure, and which produces a great number of papillomas
and infectious viral particles suspension.
[0016] The animal model of this invention is particularly useful
for supporting the complete cycle of viral infection and vegetative
growth, and, for selecting and testing candidate agents for the
treatment or prevention of papillomavirus infections that would
have physiological and pharmacological relevance in humans.
[0017] The model of the present invention produces highly reliable
and reproducible papillomas from which infectious viral particles
can be harvested. The animal model of this invention can further be
used for screening and selecting candidate agents for the treatment
or prevention of human papillomavirus infections and any conditions
caused thereof.
[0018] It is a critical feature of the present invention, to
provide a method for producing a xenograft animal model wherein
injuring the host skin prior to grafting advantageously provides
wound healing that fosters papilloma induction. It is a specific
advantage of this invention to provide this injury by way of
meshing, additionally providing stretching of the host skin to
cover a larger graft area, thus reducing the demand for host skin
tissue. Further, meshed engrafted tissue improves the survival and
health of the engrafted skin tissue.
[0019] Therefore, it is a feature of the present invention, to
provide a xenograft animal model, which may be used for the growth
and propagation of papillomavirus. Particularly, these xenografted
animals when infected with a papillomavirus form papillomas as an
indication of papillomavirus infection. These animals are a
superior model for induction of papillomavirus infection that is
reliable and reproducible when compared with other known xenograft
animal models.
[0020] It is a specific feature of the present invention, to
provide human xenografted animals which may be used for the
induction, growth and propagation of human papillomavirus, and from
which infectious viral particles can be harvested thereby providing
infectious viral stock suspension.
[0021] It is a further feature of the present invention, to provide
such a viral stock suspension to be serially passaged to
papillomavirus-free animals in order to induce papillomavirus
infections in subsequent xenografted animals.
[0022] It is still another feature of the present invention to
provide a method for the production of these xenografted animals in
order to induce papillomavirus infections in these xenografted
animals and in which papillomavirus can be harvested and
propagated, and can be passaged to papilloma-free xenografted
animals.
[0023] A further feature of the present invention is to provide a
xenograft animal model to test potential therapeutic agents against
papillomavirus infection.
[0024] The present description refers to a number of documents, the
content of which is incorporated herein by reference.
SUMMARY OF THE INVENTION
[0025] Thus, the present invention is directed to a graft animal
model for reproducible papilloma induction, and propagation of
papillomavirus. This model serves also for screening and selecting
a therapeutic agent against papillomavirus infection. The invention
further provides a method for producing the grafted animal and the
model thereby produced.
[0026] Therefore and in accordance with a first embodiment of the
present invention there is provided a graft animal model for the
induction and formation of papillomas, and for the propagation of
human papillomavirus which is characterized by:
[0027] a recipient animal grafted with host skin tissue, said skin
tissue having been injured prior to said grafting,
[0028] inoculating said grafted skin tissue with an inoculum of a
host-specific papillomavirus,
[0029] wherein said grafted skin is supported by said recipient
animal and is capable of inducing and sustaining growth of
host-specific papillomavirus and harboring at least one papilloma
containing infectious viral particles.
[0030] The success of the model of the present invention is based
on the realization by the Applicant that the process of tissue
healing following injury in the donor skin improves the tissue's
susceptibility to PV infection and favors wart formation.
[0031] Within the model according to this first embodiment, there
is comprised a recipient animal grafted with host skin tissue,
wherein said skin tissue has been injured prior to said grafting,
whereby said grafted skin is capable of inducing and sustaining
growth of host-specific papillomavirus and harboring at least one
papilloma containing infectious viral particles.
[0032] In accordance with a second embodiment of the present
invention, there is provided a method for producing a graft animal
model for propagating infectious papilloma viral particles, said
method comprising the following steps:
[0033] obtaining skin tissue from a host donor and injuring said
skin,
[0034] grafting said injured skin tissue onto a recipient animal
capable of accepting said skin tissue,
[0035] inoculating said grafted tissue with an inoculum of a
host-specific papillomavirus, and
[0036] providing sufficient time for said papillomavirus to
propagate in said grafted tissue and to form papillomas as an
indication of papillomavirus infection.
[0037] An important aspect of this second embodiment is provided in
the step of inducing tissue healing following injury in the host
skin tissue to be grafted.
[0038] In a particular aspect of this second embodiment,
inoculation of the injured donor skin tissue with a papillomavirus
inoculum can be accomplished using for example papillomavirus
suspension that can be applied either in-vitro or in-situ. Injured
donor skin tissue inoculated in-vitro, pre-grafting can be
engrafted cutaneously or subcutaneously onto the immuno-deficient
recipient animal. Injured donor skin tissue that is engrafted
cutaneously can also be inoculated in-situ post-grafting.
[0039] In a further aspect of the present embodiment, the
subcutaneous papillomas formed in the infected grafted animal, can
be exposed by cutting open the subcutaneous papillomas with an
incision to the skin at the site of the subcutaneous papilloma
growth. The exposed papilloma develops a morphology that is similar
to cutaneous papilloma and can be observed and evaluated without
having to anesthetize or kill the grafted animal.
[0040] In accordance with a third embodiment of the present
invention, there is provided an graft animal model for screening
candidate therapeutic agents for protecting, preventing or treating
papillomavirus infection. Accordingly, a candidate agent (in a
therapeutically effective amount and in admixture with a
pharmaceutical carrier) is administered to the graft animal model
of the present invention. The efficacy of the candidate agent is
evaluated by means comprising; a change in size, growth and
morphology of the papillomas, and/or a decrease in viral load and
infectivity, when compared to a control papilloma from an untreated
grafted animal.
[0041] Therefore, in accordance with a fourth embodiment of the
present invention there is provided a method for evaluating the
efficacy of a therapeutic agent useful against papilloma virus
infection comprising the steps of:
[0042] providing a grafted animal model according to the present
invention,
[0043] inoculating said grafted host skin tissue with an inoculum
of host-specific papilloma virus,
[0044] treating said papillomavirus-infected animal by
administering a candidate therapeutic agent in an appropriate
pharmaceutical carrier, and
[0045] evaluating the efficacy of said therapeutic agent in
preventing the appearance, reducing the physiological symptoms or
reducing the evidence of said infection in said infected
animal.
[0046] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of the preferred embodiments with
reference to the accompanying drawings which is exemplary and
should not be interpreted as limiting the scope of the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] Having thus generally described the invention, reference
will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which: all
figures represent a preferred embodiment of the invention
consisting of a model of NIH-nu-bg-xid mice xenografted with meshed
human foreskin tissue infected with low-risk HPVs.
[0048] FIG. 1 shows the amplification products of DNA extracted
from clinical samples of excised human papilloma tissue. These
extracts were used as initial HPV stocks to induce first generation
papillomas.
[0049] Panel A shows a mixture of HPV-6 and -11. In lane 1, primer
pair VdB-6-U/D which comprise HPV-6 L2 open reading frame described
by Mant et al. (1997, J. Virol. Meth. 66:169-178) amplified the
expected 280 BP product. In lane 2, primer pair VdB-11-U/D which
comprise HPV-11 L1 open reading frame (Mant et al., supra)
amplified the expected 360 bp product. In lane 3, a positive
control, primer pair KM29/RS42 specific for human .beta.-globin
described by Saiki, in PCR Protocols (1990, Ed. Innis et al., pg:
13-20) amplified the expected 536 bp product. Lane M, represents a
DNA molecular weight ladder. The arrows on the right indicate the
molecular weights of the amplification products.
[0050] Panel B shows single type HPV-6 or -11 extracts. Using
primer pair VdB-6U/D which comprise HPV-6 L2 open reading frame,
the HPV-6 extract gave the expected 280 base pair product in lane
1, HPV-11 (lane 2) extract remained negative with the HPV-6 primer.
When primer pair VdB-11 U/D which comprise HPV-11 L1 open reading
frame, were used for PCR amplification, the expected 360 bp product
was observed for HPV-11 extract (lane 3), but not for HPV-6 extract
(lane 4).
[0051] FIG. 2A shows the appearance of a site engrafted with human
skin tissue injured by meshing that was not inoculated with human
wart extract inoculum. There were no visible warts at the site.
[0052] FIG. 2B shows a first generation wart formed at the site
engrafted with human skin tissue injured by meshing and inoculated
with an inoculum containing both HPV-6 and -11 extracted from
clinical samples of human wart tissue.
[0053] FIG. 2C shows a typical HPV wart induced by HPV-11 single
type virus. A first generation wart formed at the site engrafted
with human skin tissue injured by meshing and inoculated with an
inoculum containing HPV-11 single-type extracted from clinical
samples of human wart tissue.
[0054] FIG. 3 shows an exposed subcutaneous papilloma. Human skin
tissue was physically wounded by meshing, inoculated with an
inoculum of human wart tissue extracted from clinical samples and
engrafted subcutaneously. At about the 10.sup.th week post-grafting
the skin covering the apex of the subcutaneous papilloma is cut
with an incision, the skin is gently retracted and is fixed to the
engrafted tissue using sutures, allowing the papillomas to grow
outward and to protrude through the skin. These exposed
subcutaneous papillomas develop a similar morphological and
histological appearance as cutaneously growing papillomas.
[0055] FIG. 4 shows the growth rate of first generation single type
HPV-11 or -6 infected xenograft human foreskin tissues grafted
sub-cutaneously. The volume was measured as the product of
length.times.width.times.height- . HPV-11 induced warts were
collected at 20 weeks post-grafting for virus collection. Only a
few HPV-6 infected grafts had moderate growth after 7 months.
Successful single type HPV-11 or -6 infection and viral propagation
were verified by PCR analysis (FIG. 9), typical histology, in situ
hybridization, immunohistochemistry (FIG. 5), and infectivity of
harvested virus in subsequent passages (FIGS. 12, 13).
[0056] FIG. 5 shows a typical histology, in situ hybridization and
immumohistology of HPV-induced xenograft warts.
[0057] Panel A. Wart tissues were fixed with formalin immediately
upon collection. The samples were trimmed across epidermal to
subcutis, desiccated, processed through xylene, and perfused with
paraffin. Sections were cut at 5 .mu.m, stained with hematoxylin
and eosin for histology.
[0058] Panel B. For in situ hybridization, biotinylated DNA probes
specific for HPV-6 or -11 were obtained from DAKO Corporation.
Tissue sections were de-paraffined in xylene and re-hydrated
through graded ethanol and water. Following protease digestion,
probe solution was added to the slide. The slide were covered
without sealing, and incubated for 6 min at 92.degree. C. to
denature HPV and probe DNA. Slides were then placed in a humid
chamber for 1 hour at 37.degree. C. Following hybridization, slides
were subjected to a high stringency wash to reduce nonspecific
hybridization. Specific hybridization was visualized by catalyzed
reporter deposition using a tryamide signal amplification kit
(GenPoint, DAKO Corporation), brown intracellular staining was
identified as positive signals.
[0059] Panel C. For immunohistochemistry study, murine IgG1
monoclonal antibody (Novocastra Laboratories Ltd, UK) directed
against HPV-6 L1 coat fusion protein (amino acids 40-233) common to
HPV types 6, 11, and 18, was used to detect HPV-6 or -11 L1
expression in the wart tissues. Biotinylated goat anti-mouse IgG1
was added to react with the antibody followed by immunoperoxidase
staining which labels positive cells in brown.
[0060] FIG. 6 demonstrates the presence of HPV DNA extracted from
swab samples of 1.sup.st generation papillomas induced by a mixture
of HPV-6 and -11. DNA was extracted from swab samples taken from
sites engrafted with: non-inoculated meshed human skin tissue
(lanes 1-3), and meshed and inoculated human foreskin tissue (lanes
4-6). The DNA from these swabs was extracted and digested with Hind
III. An aliquot of the digested DNA was co-amplified with the
following primer pairs: MY09/MY011 which amplify the ORF region of
the L1 gene in HPVs non-specifically, and S-GH20/SPCO04 which
amplify a region in the human .beta.-globin gene (primer sequences
are as described in Mant et al., supra). The amplification products
of the DNA derived from 1.sup.st generation wart tissue show the
expected 450 bp and 286 bp bands corresponding to HPV L1 gene and
.beta.-globin DNA, respectively.
[0061] FIG. 7 shows the results of the amplification products of
DNA isolated from 1.sup.st generation cutaneous wart tissue induced
by a mixture of HPV-6 and -11. The isolated DNA was amplified with
HPV type specific primers as described in Mant et al. (supra). The
lanes in group A, B, C, D, and E correspond to the amplification
products of primers specific for HPV-6, -11, -16, -18, and -31,
respectively. In each group, lane 1 corresponds to the
amplification product of DNA extracted from 1.sup.st generation
cutaneous wart tissue, lane 2 corresponds to a positive control and
lane 3 to a negative control (no DNA in the amplification
reaction). The positive controls are standard HPV plasmids
pUC19-HPV6, pBR322-HPV11, pBluescript-HPV16, pBR322-HPV18 and
pBR322-HPV31 containing HPV-6, -11, -16, -18 and -31 DNA,
respectively, obtained from American Type Culture Collection
(Manassas, Va., U.S.A.). Amplification of these plasmids produces
amplification products greater than 3 Kb for the control plasmids
of HPV-6, -11, -16 and -31. Lane M represents the molecular weight
ladder. The amplification products demonstrated the presence of
HPV-6 (lane A1) and 11 (lane B1) in the 1.sup.st generation
cutaneous papillomas, but not the high risk types HPV-16, -18, and
-31.
[0062] FIG. 8 shows the presence of HPV types 6 and 11 in
subcutaneously engrafted sites infected with a mixture of HPV-6 and
-11. DNA isolated from swab samples obtained from the surface of
four distinct 1.sup.st generation exposed subcutaneous papillomas
are analyzed by amplification. Specific primers to HPV types 6 and
11 confirm the presence of HPV-6 (lanes 1 to 4) and HPV-11 (lanes 6
to 9). Lanes 5 and 10 are positive controls as described in FIG.
7.
[0063] FIG. 9 represents a PCR analysis of single type HPV-6 or -11
induced xenograft warts. Primer pair VdB-6U/D which comprise HPV-6
L2 open reading frame amplified the expected 280 bp products from
both HPV-6 induced sub-cutaneous warts (lanes 3 and 4), similar to
that obtained with control plasmid pU19-HPV-6 (lane 5). In
contrast, HPV-11 warts did not have positive signals when probed
for HPV-6 (lanes 1 and 2). In lanes 6 and 7, primer pair VdB-11 U/D
which comprise HPV 11 L1 open reading frame amplified the expected
360 bp products from both HPV-11 induced cutaneous and subcutaneous
warts, similar to that observed with control plasmid pUC19-HPV-11
(lane 10). HPV-6 warts gave negative signal when probed for HPV-11
(lanes 8, 9).
[0064] FIG. 10 shows the morphology at 9 weeks post-grafting of a
2.sup.nd generation cutaneous papilloma passaged from a 1.sup.st
generation papilloma induced with mixed-type HPV-6 and -11.
[0065] FIG. 11 summarizes of the % wart induction in the cutaneous
model (day 77 post-grafting), in 2.sup.nd generation xenografted
animals inoculated with HPV harvested from 1.sup.st generation
papillomas induced with mixed-type HPV-6 and -11. Subcutaneous
papillomas were subjected to two sequential viral particle
extractions, viral stock from the first extraction (SC1) and second
extraction (SC2) induced 80% (8 out of 10 engrafted sites) and 33%
(1 out of 3 engrafted sites) papillomas, respectively. A 1:10
dilution of SC1 did not induce any papillomas (0 out of 5 engrafted
sites). Viral particles harvested from cutaneous (cut) papillomas
induced 33% (2 out of 6 engrafted sites) papillomas in the 2.sup.nd
generation animals. This result suggested that viral stock
extracted from subcutaneous warts may be more effective in
generating infectious particles for subsequent infections.
[0066] FIG. 12 shows single type HPV-11 wart induction in the
cutaneous model by viral stock originally generated from
sub-cutaneous xenograft warts.
[0067] Panel A shows 3 individual experiments with highly
reproducible papilloma induction frequency and growth rate in the
2.sup.nd generation.
[0068] Panel B shows that the 3.sup.rd passage papilloma induction
is highly reproducible. Papilloma induction, passage and scoring
criteria were as described in the text.
[0069] FIG. 13 shows subcutaneous growth of single type HPV-11
infected xenograft in the 2.sup.nd and 3.sup.rd generation.
[0070] Panel A. Experiments 1 to 4 show 4 individual experiment in
the 2nd generation.
[0071] Panel B. Experiment 5 shows a 3.sup.rd passage in the
subcutaneous model. Papilloma induction, passage and volume
measurement was as described in the text.
[0072] FIG. 14 shows comparative growth rates of 1.sup.st and
2.sup.nd generation warts induced by single type HPV-6 in the
cutaneous model. The 2.sup.nd generation warts were induced by a
small stock prepared from 24 small sub-cutaneous and 1 small
cutaneous 1.sup.st generation warts collected s described in FIG.
4. Cutaneous wart scoring was as described in the text.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0073] Definitions
[0074] Unless defined otherwise, the scientific and technological
terms and nomenclature used herein have the same meaning as
commonly understood by a mammal of ordinary skill to which this
invention pertains. Generally, the procedures for cell culture,
infection, molecular biology methods and the like are common
methods used in the art. Such standard techniques can be found in
reference manuals such as for example Sambrook et al. (1989,
Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratories) and Ausubel et al. (1994, Current Protocols in
Molecular Biology, Wiley, New York).
[0075] "Sequence amplification" is a method for generating large
amounts of a target sequence. In general, one or more amplification
primers are annealed to a nucleic acid sequence. Using appropriate
enzymes, sequences found adjacent to, or in between the primers are
amplified. An amplification method used herein is the polymerase
chain reaction (PCR).
[0076] "Amplification primer" refers to an oligonucleotide capable
of annealing to a DNA region adjacent to a target sequence and
serving as the initiation primer for DNA synthesis under suitable
conditions well known in the art. The synthesized primer extension
product is complementary to the target sequence.
[0077] "Grafted animal" is a recipient animal onto which is grafted
a graft tissue.
[0078] "Allograft" is the engrafting of tissue obtained from a
donor animal onto a recipient animal that is of the same
species.
[0079] "Xenograft" is the engrafting of tissue obtained from a
donor animal onto a recipient animal that is a different species
from the donor animal.
[0080] "Xenograft animal" is a recipient animal onto which is
grafted a xenograft.
[0081] The terms "papilloma" and/or "warts" are used
interchangeably herein and define a highly proliferative tissue
growth caused by papillomavirus infections. These have gross
anatomical and histological features well known in the art and
occur cutaneously and subcutaneously.
[0082] The term "in-situ" refers to inoculating a living animal.
That is, an inoculum is applied to a living animal by any means
well known in the art. Specifically and for use herein, the term
describes inoculating donor tissue after engrafting onto a
recipient animal. It will be understood that in situ inoculation is
preferably performed on a cutaneous graft.
[0083] "Passaging" refers to collecting papillomavirus from a
previous generation of grafted warts from an infected-graft donor
animal and inducing warts to a recipient animal that is apparently
free of papillomavirus infection. The recipient animal can be
selected from a group consisting of immuno-compromised animals or a
natural host to the papillomavirus. The animal receiving the
passaged papillomavirus is referred to herein as a "subsequent
animal" or a "subsequent recipient animal". A preferred means of
passaging the virus consist in infecting meshed graft tissue prior
to its grafting onto the subsequent recipient animal.
[0084] The term "injuring" refers to any means of causing profound
injury or wound to a tissue, which would result in tissue healing
activity. Tissue injury can be induced by physical wounding or
chemical damage. Non-limiting examples of physical wounding
include: perforating, slitting, cutting, punching holes, burning
and meshing using appropriate tools known in the art (e.g.
scalpels, needles, pins, hole borers, meshers, etc.). Non-limiting
examples of chemical damage include; enzymatic treatment and
chemical burning. Tissue healing activity comprises new cell growth
and increases in cellular growth factors and adhesion factors such
as kinins and integrins. In addition, tissue healing activity may
also be induced by other means such as electrical and chemical
stimulation, chemical stimulation may include application of growth
factors and/or enzymes to the tissue.
[0085] The term "meshing" refers to a means of treating tissue,
specifically skin tissue whereby small openings or holes are poked
throughout the tissue. Meshing can be accomplished manually or by
use of a machine designed for this purpose. Meshing (Pope et al.,
1990, 20:177-187), is a procedure mostly used for medical and
veterinary applications to expand graft skin tissue to encompass
greater surface area to cover large skin wounds. Rob et al.
(Journal of Burn Care and Rehabilitation, 1987, 8(5):371-375),
describe an animal model in which meshed human skin is grafted onto
nude mice to investigate problems of scarring in the allografting
of severely burned patients. The failure to generate a human
xenograft model for propagating HPV has prompted the Applicant to
attempt the procedure of meshing human skin to generate a human
xenograft model. This is the first time this technique is applied
for the purpose of producing a graft model for use in propagating a
fastidious organism causing viral infections.
[0086] Preferred Embodiments
[0087] Recipient Animal:
[0088] In a particular aspect of this invention, the recipient
animal is a non-human mammal capable of receiving and supporting a
graft. Particularly, the recipient animal is capable of receiving a
xenograft by being immuno-compromised and is mostly incapable of
mounting a graft-rejection immune response thereby accepting the
foreign tissue as self. Preferably, the recipient animal is
immuno-compromised either by being immuno-deficient or
immuno-suppressed by biological or chemical means. Such biological
or chemical means include immuno-suppression by repeated treatment
with cyclosporin or other immuno-suppressive agents well known in
the art. More preferably, the immuno-compromised animal is
immuno-deficient. The term immuno-deficient is used to describe a
recipient animal in which the immune system has been partly or
completely compromised in order to allow engrafted foreign cells or
tissue to grow with minimal chance of rejection by the recipient
animal.
[0089] Even more preferably, the non-human mammal is a rodent, more
preferably a mouse, rat, rabbit, guinea-pig, or hamster. More
particularly, this mammal is a rodent such as a rat or a mouse
having no functional T-cell immunity, non-limiting examples being
severe combined immuno deficient (SCID) mice, SCID/beige mice, nude
mice, or NIH-nu-bg-xid mice. Specifically the recipient animal has
no or little functional T-cell immunity or B cells or NK cells.
Most preferably, the recipient animal is hairless to facilitate
grafting procedures such as the NIH-nu-bg-xid mouse.
[0090] Donor Tissue/Animal:
[0091] In a further aspect of the present invention, the donor
tissue can be taken from any animal that is a natural host to
papillomavirus. These animals are listed in Olson, C. (1987, The
Papaviridae, volume 2, pages 39-66). Non-limiting examples of a
donor animal can be selected from: dog, cattle, horse, swine,
rabbit (cottontail, domestic and New-Zealand White (NZW)), deer,
non-human primates and humans. These donor animals are hosts to the
corresponding papillomavirus such as: canine oral papillomavirus,
bovine papillomavirus, equine papilloma virus, swine
papillomavirus, cottontail rabbit papillomavirus (CRPV), deer
fibroma virus, primate papillomavirus and human papillomavirus
(HPV), respectively. In a preferred aspect of this invention, the
donor animal is human. In a preferred aspect of this invention the
human tissue is foreskin tissue obtained from infant circumcision
from a medical clinic or hospital.
[0092] Allografting or Xenografting:
[0093] In a particular embodiment of the present invention, there
is provided a model for grafting tissue from a donor animal that is
the same species (allograft) or of foreign species (xenograft). As
stated above, when the graft is foreign (xenograft) the recipient
animal must be immuno-compromised to be able to support such graft
without rejecting it as non-self.
[0094] Therefore, in a preferred embodiment of the invention there
is provided a xenograft animal model capable of forming human
papillomavirus infection. The model is reliable and easily
reproduced, and is particularly useful for the induction and
formation of demonstrable human papillomas. More particularly, the
xenograft animal model is useful for the propagation of human
papillomavirus and the harvesting of infectious viral particles of
HPV low-risk and high-risk.
[0095] Graft Tissue Injury:
[0096] The success of the model of the present invention is based
on the realization that profound tissue injury of skin obtained
from donors is important in the induction of human papillomavirus
infection in the graft model. Preferably, there is provided the
means for injuring skin tissue for use in generating a graft animal
model having increased susceptibility to PV infection. In an
important aspect of this invention, the induction of tissue healing
in the grafted tissue is as a consequence of tissue injury. Tissue
injury can be achieved using physical wounding and chemical damage.
Physical wounding can be achieved with slitting, cutting, burning,
perforating, poking holes, meshing, etc. by using any tools known
in the art such as needles, scalpels, forceps, pins, hole punchers,
meshing machine, etc. Chemical damage may comprise enzymatic
treatment. More preferably, the induction of tissue healing as a
consequence of injury is particularly due to physical wounding and
most preferably meshing. Physical wounding enhanced the success
rate in initiating infection by a fastidious organism, specifically
human PV in the xenograft human animal model. The success of
physical wounding, particularly meshing in improving the induction
of human papillomas may be attributed to several mechanisms.
Wounding, particularly meshing may stimulate neoepithelization
(Harries et al. 1995, Aust NZ J Surg, 65:600-603) thus increasing
the population of basal cells which are the target cells for HPV.
Further, tissue wounded by meshing may lead to pronounced healing
process. During this healing process, integrins such as
.alpha.6.beta.4, become widely expressed. A recent study (Evander
et al. 1997, J. Virol 71:2449) suggests that integrin
.alpha.6.beta.4, may be a receptor for papillomavirus binding and
entry into the host cells. Therefore, healing of human skin tissue
after physical wounding appears to be an important factor in the
development of graft model for PV infection.
[0097] Therefore, in a preferred embodiment of this invention,
human skin tissue for xenografting is physically wounded and
inoculated with a HPV inoculum. The Applicant reproducibly induced
demonstrable cutaneous and subcutaneous papillomas by meshing the
skin prior to grafting. Parallel experiments without skin meshing,
using the same viral stock failed to induce papillomas in
cutaneously and subcutaneously engrafted tissue.
[0098] Therefore in a preferable aspect of the invention, the
physical wounding of human skin tissue is accomplished by meshing,
either manually or with the use of a meshing machine. More
preferably, meshing of the skin tissue is accomplished with the use
of a meshing machine. Meshing advantageously provides stretching
human skin to cover a larger graft area, thus reducing the demand
for human skin tissue. Further, meshing of the engrafted tissue
facilitates the transudation of extrudates, thus improving the
survival and health of the engrafted skin tissue (Pope et al.,
supra).
[0099] Inoculum:
[0100] In an additional preferred aspect of the invention, the
human papillomavirus used for preparing the inoculum is selected
from HPV low risk or high risk type. Low risk type consisting of
types 6, 11 and 13. High risk types consisting of types 16, 18, 35,
45, 52 and 58. Preferably the papillomavirus is low risk consisting
of types 6, 11 and 13.
[0101] In a particular aspect, the initial HPV inoculum is derived
from clinically excised human papillomas obtained from a medical
clinic. The viral particles obtained from these clinical samples
comprise a mixture of HPV types including 6 and 11, or single type
HPV-6 or -11. Therefore, the xenografted human skin tissue is
inoculated with a mixed inoculum comprising HPV types 6 and 11, or
single type HPV-6 or -11.
[0102] In an additional aspect of the invention, there is provided
a viral suspension that is infectious to human skin tissue,
specifically xenografted human skin tissue. Advantageously, the
viral suspension can therefore be passaged to the animal model of
the present invention for generating subsequent HPV xenograft
animals. Preferably, the viral suspension harvested from the human
xenograft animal model of the present invention can be used to
isolate a viral suspension containing either single type or mixture
of HPV types. Most preferably, the viral suspension contains a
single viral type.
[0103] Advantageously, a pure viral isolate is useful for genotypic
and phenotypic characterization of pure HPV types. On the other
side, the infection with mixed types would allow to address
questions related to viral type predominance and interactions.
[0104] Inoculation:
[0105] The term "inoculation" refers to a means for introducing
infectious virus, virions or viral particles to a non-infected
tissue. The inoculum can be a clinical sample, a suspension which
is derived from a clinical sample or cultured sample, or an
isolated strain. The inoculum can be comprised of a single pure
viral strain or type, or a mixture of more than one viral strain or
type.
[0106] In a particular aspect of inoculation, the donor skin tissue
is scarified prior to or during inoculation. Scarification of the
donor skin tissue can be accomplished using an instrument for
wounding the tissue, such as a knife, scalpel, needle, etc.
Preferably the instrument used for scarification is dipped in a
papillomavirus suspension thereby inoculating papillomavirus
particles at the same time as scarifying the skin tissue (in-vitro,
pre-grafting). This pre-grafting inoculation (in-vitro) may also be
accomplished by means such as soaking the scarified tissue in a
papillomavirus suspension or by overlaying the pre-grafted tissue
with a viral suspension.
[0107] In an alternative aspect of the invention, the scarified
skin tissue is later inoculated with a papillomavirus suspension,
in-situ immediately post-grafting; or, in-situ a few weeks
post-grafting. This post-grafting inoculation (in-situ) may also be
accomplished by overlaying the grafted tissue with a viral
suspension, or by injecting the grafted tissue with a viral
suspension or a combination thereof.
[0108] Cutaneous or Subcutaneous Grafting:
[0109] In an additional aspect of the present invention the graft
tissue is inserted subcutaneously and/or cutaneously onto the
recipient animal. In the cutaneously grafted tissue the papillomas
are formed on the surface of the xenografted human tissue. In the
subcutaneously xenografted tissue the papillomas are formed
subcutaneously at the site of the grafted human tissue. Thus, the
present invention provides human xenografted animal models capable
of having demonstrable cutaneous and subcutaneous papillomas.
[0110] Exposing the Wart:
[0111] In a preferred aspect, the subcutaneous papillomas can be
exposed and allowed to develop into cutaneous papillomas.
Accordingly, the subcutaneous papillomas can be exposed by any
means known in the art and allowed to develop into cutaneous
papillomas. In a non-limiting example, the skin covering the apex
of the subcutaneous papilloma is cut with an incision, the skin is
gently retracted and held back allowing the papilloma to grow
outwardly and protrude through the skin. These exposed subcutaneous
papillomas develop similar morphological and histological
appearance as cutaneous papillomas. Preferably the cut skin is held
back by suturing the host skin to the grafted tissue.
[0112] Harvesting:
[0113] In a further aspect of this invention, HPV can be extracted
from the infected tissue of the xenografted animal model of this
invention, thus providing a viral suspension. Particularly,
papilloma tissue obtained from the xenografted animal model of the
present invention provide infectious particles to be comprised in a
viral suspension. The virions are harvested from the papilloma
tissue by means well known to a mammal skilled in the art. In a
non-limiting example, the excised papilloma tissue is homogenized
in an appropriate homogenizing buffer that is known in the art and
centrifuged. The supernatant comprising the virions is collected
either for immediate use or stored for later use. Preferably the
viral suspension is stored in phosphate buffered saline
supplemented with antibiotics.
[0114] Screening Therapeutic Agents:
[0115] In a preferred embodiment of the present invention, there is
provided an animal model for screening candidate therapeutic agents
against human papillomavirus. Particularly, the animal model has
been demonstrated to be highly reproducible and "propagatable" from
generation to generation providing visible human papillomas and
infectious viral particles as an indication of human papillomavirus
infection. Preferably, the candidate agent in a therapeutically
effective amount and in admixture with a pharmaceutical carrier, is
administered to the animal model of the present invention. The
therapeutic agent is selected for the purpose of antagonizing human
papillomavirus infection including; protecting, preventing and
treating human papillomavirus infection in an individual in need of
such medication. The therapeutic agent may be used singly or in
combination with other means for intervening in HPV infection.
Particularly, the therapeutic candidate agent is a compound capable
of preventing the progression of human papillomavirus infection in
an individual. Most preferably, the therapeutic candidate agent is
a compound capable of eliminating a human papillomavirus infection
or a recurrence thereof in an individual.
[0116] Alternatively, the therapeutic candidate agent is a compound
capable of eliciting an immune reaction in a mammal against human
papillomavirus infection thereby providing immunizing agents
against papillomavirus infections. Potential immunizing agents are
administered in a therapeutically effective amount and with any
suitable carrier, with the purpose of eliciting antibodies against
human papillomavirus. Such agents may provide partial or complete
immunity in an individual.
[0117] In a particular aspect of this embodiment, the therapeutic
candidate agent is administered to the animal model of the present
invention and to an individual in need by any means known in the
art including topical, oral or systemic.
[0118] Accordingly, the means to administer such an agent/s to an
animal includes oral, topical or systemic, in a vehicle comprising
one or more pharmaceutically acceptable carriers, the proportion of
which is determined by the solubility and chemical nature of the
compound, chosen route of administration and standard biological
practice.
[0119] For oral administration, the agent/s or a therapeutically
acceptable salt thereof can be formulated in unit dosage forms such
as capsules or tablets each containing a predetermined amount of
the active ingredient, in a pharmaceutically acceptable carrier.
For oral administration, the compound or a therapeutically
acceptable salt is administered in accordance with the body weight
of an animal per day, in order to achieve effective results.
[0120] For topical administration, the agent/s can be formulated in
pharmaceutically accepted vehicles containing the active agent/s.
Such formulations can be in the form of a solution, cream or
lotion. With reference to topical application, the agent/s is
administered topically in a suitable formulation to the infected
area of the body such as skin and genitalia in an amount sufficient
to cover the infected area. The treatment should be repeated, for
example, every four to six hours until lesions heal.
[0121] For parenteral administration, the agent/s may be
administered either intravenously, subcutaneous or intramuscular
injection, in compositions with pharmaceutically acceptable
vehicles or carriers. For administration by injection, it is
preferred to use the agent/s in solution in a sterile aqueous
vehicle which may also contain other solutes such as buffers or
preservatives as well as sufficient quantities of pharmaceutically
acceptable salts or of glucose to make the solution isotonic. With
reference to systemic administration, the agent/s is administered
at a dosage in accordance with the body weight of an animal or
individual per day in order to achieve effective results. Although
the selected agents comprised in the formulations disclosed
hereinabove may be effective and relatively safe medications for
treating papilloma viral infections, the possible concurrent
administration of these formulations with other treatments against
papilloma virus infections are also included. These treatments
include the use of cytotoxic agents such as, trichloroacetic acid,
podofilox and podophyllin; immunotherapy agents such as, interferon
and imiquinod, and, physical methods such as, electrosurgery,
cryotherapy, excision surgery and CO.sub.2 laser.
[0122] Suitable vehicles or carriers for the above noted
formulations are described in standard pharmaceutical texts, e.g.
in Remington's "The Science and Practice of Pharmacy", 19th ed.,
Mack Publishing Company, Easton, Pa., 1995, or in "Pharmaceutical
Dosage Forms And Drugs Delivery Systems", 6th ed., H. C. Ansel et
al., Eds., Williams & Wilkins, Baltimore, Md., 1995.
[0123] The dosage of the agent/s will vary with the form of
administration and the particular active agent/s chosen. In
general, the agent/s is most desirably administered at a
concentration level that will generally afford antiviral effective
results without causing any harmful or deleterious side effects to
the animal.
[0124] The efficacy of the candidate agent can be determined by
means including: effectiveness of the agent on the papillomas' size
and growth, viral load and infectivity of viral particles, and
other molecular and cellular parameters such as histology, in situ
hybridization, PCR and immuno-histochemistry.
EXAMPLES
[0125] Materials and Methods The immuno-deficient animals,
NIH-nu-bg-xid mice were purchased from Charles River Laboratories;
Wilmington, Mass., USA; Taconic, N.Y., USA and St. Constance,
Quebec, Canada. Animals were housed in microisolator cages inside
semi-rigid isolators with sterile food, water and bedding. All
experiments were conducted in class II-type safety cabinets
(NuAire, Plymouth, Minn., USA), and according to protocols approved
by the Canadian Council for Animal Care (Ottawa, Ontario,
Canada).
[0126] Grafting surgeries were carried out in mice anesthetized
with halothane. All incisions and grafted areas in the animals were
treated with an antibiotic cream such as Polysporin.TM. and
Sofra-tulle TM antibiotic dressing (Hoechst-Roussel Canada Inc.,
Montreal, Quebec, Canada). These were then covered with a layer of
petroleum jelly impregnated gauze and kept in position with a
flexible adhesive strip.
[0127] Cell and tissue culture media, .alpha.-modified Eagle's with
Earl's salts was purchased from Cellgro. This media is supplemented
with the following antibiotics: 0.05 mg/ml gentamycin, 100 .mu.g/ml
streptomycin and 100 U/ml penicillin, purchased from Gibco,
Ontario, Canada.
Example 1
Initial Viral Extraction from Clinically Excised Human Warts
[0128] Clinically excised human anogenital wart tissues were
obtained from a local hospital (kindly supplied by Dr. Ferenczy,
Jewish General Hospital, Montreal, Quebec, Canada). The collected
warts were placed in plastic test tubes, kept on dry ice and
transported to our laboratories. The clinical samples were,
weighed, minced into small pieces (.about.1-2 mm squares) and
homogenized with a Polytron.TM. in cold phosphate buffered saline
(4.degree. C.) to a final volume of about 5 ml/g tissue. The
homogenate was centrifuged at 3000 g (4.degree. C.) for 30 min. The
resulting pellet was optionally subjected to a second extraction
using the same procedure. The collected (1.sup.st and/or 2.sup.nd)
supernatant was supplemented with 1% (v/v) stock antibiotics;
gentamicin, 50 mg/ml, penicillin, 10,000 units/ml, and
streptomycin, 10,000 .mu.g/ml (obtained from Gibco, Ontario,
Canada), and stored at -80.degree. C. The extracted supernatants
were the initial HPV stock for infecting xenografted human skin
tissue. For samples too small to be extracted separately, the warts
were swabbed (as in Example 5) and their DNA was typed using PCR
assay as described in Example 2. Small warts with the same HPV type
were pooled. All manipulations of infected human tissue were
carried out according Biosafety Level 2 guidelines.
Example 2
HPV Typing of Clinically Excised Human Warts
[0129] HPV DNA was isolated from each sample and then used for
typing by PCR amplification using HPV type specific primers. The
viral supernantant was digested by adding SDS and proteinase K to a
final concentration of 0.5% and 0.2 mg/ml respectively and
incubated overnight at 55.degree. C. The DNA was extracted from the
digested supernatant using an equal volume of Tris-buffered phenol,
followed by one extraction with phenol:chloroform:isoamyl alcohol
(25:24:1) and one with chloroform:isoamyl alcohol (24:1). The DNA
was precipitated with sodium acetate (3M) and cold absolute
ethanol. The resultant pellet was washed in 70% ethanol, dried and
resuspended in 0.01 M Tris-HCl buffer (pH 8.0). Amplification
reactions were performed using 200 ng of the isolated DNA.
[0130] All amplification primers used herein are as described in
Mant et al. (J. Vir. Meth., 1997, 66:169-178). Specific primers to
HPV-6, VdB-6-U/D, amplified a 280 bp fragment including the HPV-6
L2 open reading frame, and specific primers to HPV-11, vdB-11-U/D
amplified a 360 bp fragment including the HPV-11 L1 ORF. As
positive control for the amplification reactions, primer pair
specific to human .beta.-globin DNA were used in each amplification
reaction. The amplification reactions were carried out in a Perkin
Elmer GeneAmp PCR System 9600 (Perkin Elmer, Norwalk, Conn.), in a
50 .mu.l volume containing, 5 .mu.l of 10.times.PCR buffer, 6 .mu.l
of 25 mM MgCl.sub.2, 1 .mu.l 12.5 dNTP mix, 2 .mu.l of each primer
at 10 .mu.M and 5U/.mu.l of AmpliTaq Gold.TM. (Applied Biosystems,
Mississauga, ONT) using the following conditions: denaturation at
95.degree. C. for 10 min, followed by 40 cycles of denaturation at
95.degree. C. for 30 sec, annealing at 58.degree. C. for 30 sec and
extension at 72.degree. C. for 1 min, with a final extension at
72.degree. C. for 5 min. The amplification products were analyzed
by electrophoresis on a 1% agarose gel and visualized with 0.5%
ethidium bromide.
[0131] The electrophoresed amplification products are shown in FIG.
1, bands of 280 bp and 360 bp corresponding to the vdB-6 and vdB-11
primers confirm the presence of both HPV types 6 and 11 in the
clinical wart extract analyzed in FIG. 1A whereas only single type
HPV-6 or -11 were present in individual extracts analyzed in FIG.
1B.
Example 3
Preparation of Human Foreskin
[0132] Neonatal foreskins from routine circumcisions were collected
at the Tiny Tots Clinic (Kindly supplied by Dr. Katz,
Dollard-des-Ormeaux, Quebec, Canada). The samples were placed in
alpha-modified Eagle's medium with Earle's salt (obtained from
Cellgro), supplemented with antibiotics (0.05 mg/ml gentamycin, 100
U/ml penicillin and 100 .mu.g/ml streptomycin) and transported to
our laboratories. All manipulations of human tissue were conducted
under a class II Bio-safety cabinet (NuAire.TM., Plymouth, Minn.,
U.S.A.,).
[0133] The foreskins were processed by first removing occluded
tissue and part of the underlying dermis, the split-thickness
foreskin tissue samples were prepared using one of the following
means:
[0134] A. The foreskin tissue was cut into squares of 1.times.1 cm
without being scarified or meshed.
[0135] B. The foreskin tissue was scarified using an aliquot of 70
.mu.l/cm.sup.2 of initial HPV inoculum and then cut into 1.times.1
cm squares. The scarified tissue was soaked in an additional
aliquot of 30 .mu.l/cm.sup.2 of the initial inoculum and incubated
at 37.degree. C. for 1 hour.
[0136] C. The foreskin tissue was scarified as above, meshed in a
meshing machine (The Zimmer Skin Graft Mesher.TM., Zimmer Bureau
Regional, Montreal, QC, Canada) then cut into 1.times.1 cm sizes.
The scarified and meshed tissue was soaked in an additional 30
.mu.l/cm.sup.2 aliquot of the initial inoculum and incubated at
37.degree. C. for 1 hour.
[0137] Tissue scarification is a technique well known in the art.
Briefly, the tissue is scraped with an instrument dipped in a viral
suspension, thereby introducing viral particles to non-infected
tissue. For the purpose of this invention 70 .mu.l/cm.sup.2 of the
initial inoculum prepared from clinical warts as described
hereinabove were used for tissue scarification.
Example 4
Cutaneous and Subcutaneous Grafting of Human Skin Tissue
[0138] NIH-bg-nu-xid mice obtained from Charles River Laboratories
(Wilmington, Boston, USA) or Taconic (N.Y. USA), were housed in
microisolator cages inside semi-rigid isolators and provided with
sterile food, water and bedding. All experiments were carried
within class II-type safety cabinets (NuAire, Plymouth, Minn.,
USA), according to protocols approved by the Canadian Council for
Animal Care (Ottawa, Ont. Canada).
[0139] All graft surgeries were carried out on recipient animals
anesthetized with halothane. For cutaneous grafting, a 1 cm.sup.2
area of skin from the laterodorsal area from the recipient animal
was carefully removed so as to preserve the underlying fascia and
minimize bleeding. The graft was fitted into the receiving bedding
and fixed in position with size 6-0 silk suture. The grafted areas
were dressed with Polysporin.TM. antibiotic cream and
Sofra-tulle.TM. antibiotic dressing (Hoechst-Roussel Canada Inc.,
Montreal, QC, Canada). These were then covered with a layer of
petroleum jelly impregnated gauze and kept in position with a
flexible adhesive strip. The dressings were kept for 3 weeks with
changing every 3-4 days or as necessary. For subcutaneous grafting,
the processed foreskin tissues were further cut into squares of
5.times.5 mm sizes and introduced into subcutaneous space via a
small opening in the central dorsal area. These incisions were
closed with sterile wound clips.
[0140] Starting from day 0 post-grafting, all xenografted animals
were given the antibiotic Septra.TM. in their drinking water at a
concentration of 1:800 (v/v). The graft sites were observed for the
first sign of wart formation and monitored weekly for growth.
[0141] In some experiments, subcutaneous engrafted tissues were
exposed 10 weeks after grafting by cutting the recipient animal's
skin formed over the graft site and suturing the edges of the
recipient animal's skin to the engrafted tissue. This surgical
procedure was performed with the xenografted animals under
halothane anesthesia. The surgical sites were dressed the same way
as for the cutaneously engrafted skin tissue (described
hereinabove) until the wounds were securely rejoined.
[0142] Grafts sites were examined daily for the development of
papillomas or other infections. At the onset of visible papillomas,
papilloma size was measured as the product of the length, width and
height or its cubic root [geometric mean diameter (GMD)]. For
cutaneous papillomas, we have developed a scoring system as
follows: (0) normal; (1) roughness; (2) small warts with 1-2 mm in
each dimension; (3) large warts >2 mm in any 2 dimension; (4)
semi-confluent papillomas covering up to half of the graft surface;
(5) confluent papillomas covering >{fraction (1/2)} of the graft
surface; (6) confluent papilloma with dense keratinization.
Example 4A
Papilloma Induction With Mixed HPV in Cutaneous Xenografts (FIG.
2B)
[0143] From the 8 sites engrafted cutaneously with meshed human
skin tissue inoculated with the initial inoculum containing HPV-6
and -11 (as prepared in Example 1), 6 formed visible papillomas
having an estimated GMD of 2.3.+-.0.4 mm (scoring between 2 and 5).
The appearance of a cutaneous papilloma is shown in FIG. 2B. No
visible papillomas were observed up to 6 months at the 4 sites
engrafted cutaneously with non-meshed human skin tissue using the
same initial inoculum and experimental conditions.
Example 4B
Papilloma Induction With Single-Type HPV in Cutaneous Xenografts
(FIG. 2C)
[0144] Table 1 summarizes the frequency of cutaneous induction with
single type HPV-11 or -6 prepared from clinical condylomas.
1 type Mouse survival Graft survival Induction frequency HPV-11 5/7
10 10/10 (7 wks) HPV-6 2/12 4 1/4 (14 wks)
[0145] The premature deaths of immunodeficient mice were caused by
contamination from the clinical extracts not related to HPV
infection. In subsequent experiments, we introduced a 30 min
centrifugation at 15000 g that minimized premature deaths to less
than 30%.
Example 4C
Papilloma Induction With Mixed HPV in Sub-Cutaneous Xenografts
(FIG. 3)
[0146] The 16 sites engrafted subcutaneously with meshed human
tissue inoculated with mixed HPV-6 and -11 (as prepared in Example
1) were exposed as described above. Seven weeks post-exposure a
total of 5 engrafted sites had visible papillomas. Of these, 4
papillomas demonstrated significant growth within the first 10
weeks of grafting and another one formed papilloma within 7 weeks
post-exposure. These exteriorized subcutaneous papillomas showed
similar morphology as cutaneous papillomas (FIG. 3). No apparent
growth was observed in 10 sites engrafted subcutaneously with
non-meshed human skin tissue, in the same time period (a total of
17 weeks). However, with repeated surgery for exposure, limited
growth was observed in 3 of these 10 sites, 10 weeks after exposure
(20 weeks post-grafting). A possible explanation for this limited
growth, is that during surgical exposure, wounding of the tissue
occurs. This process probably facilitates papillomavirus infection
by mimicking the effect of meshing.
Example 4D
Papilloma Induction With Single-Type HPV in Sub-Cutaneous
Xenografts (FIG. 4)
[0147] With single type HPV-11-infected sub-cutaneous xenografts,
papillomas grow significantly. In comparison, 1.sup.st generation
single type HPV-6 infected tissues do not have obvious growth in
most of the grafts.
[0148] In addition to their gross morphological similarity to
clinical papillomas, these cutaneous and sub-cutaneous warts
induced by either mixed or single type virus share the same
histological, cellular and immuno-histochemistry characteristics as
shown in FIG. 5.
Example 5
HPV Typing of Xenograft Warts
[0149] HPV typing was performed by analyzing the DNA of HPV
particles sloughed off from the uppermost layer of the graft site
or the visible papillomas, or from extracts of the xenografted
warts.
[0150] In the case of visible papillomas and non-inoculated
engrafted sites (used for negative control), the uppermost layer
was swabbed first with a cotton swab moistened with PBS followed by
gentle rubbing with a dry swab. DNA was isolated by incubating the
swabs overnight at 55.degree. C. in a volume of 0.5 ml of digestion
buffer (100 mM NaCl, 10 mM Tris-HCl, 25 mM EDTA, and 0.5% SDS) and
proteinase K to a final concentration of 0.2 mg/ml. At the end of
the incubation period the swabs were squeezed to remove excess
liquid and discarded. DNA was extracted from the digested swab
samples using classical methods. Briefly, to the digested swab
sample an equal volume of phenol:chloroform:isoamyl alcohol
(25:24:1) was added followed by centrifugation at 16000 g for 1 min
to separate the phases. The aqueous phase was passed through a
Microcon-50 microconcentrator (purchased from Millipore Canada
Ltd., following manufacturer's instructions) and the DNA was eluted
in 25 .mu.l of 0.25X Tris-EDTA pH 7.4 buffer. The eluted DNA was
digested with the restriction enzyme HindIII (100 U/ul) (purchased
from New England Biolabs, following manufacturer's instructions).
In the case of wart extracts, the DNA was extracted using the same
procedure presented in Example 1 for clinical samples.
[0151] Aliquots of 5 .mu.l of the Hind III digested DNA were
co-amplified with the primer pair MY09/MY011 which amplify the ORF
region of the L1 gene in HPVs non-specifically, and S-GH20/SPCO04
which amplify a region in the human p-globin gene that serves as
positive control (primer sequences are as described in Mant et al.,
supra). The results of the visualized amplification products are
shown in FIG. 6. Lanes 1 to 3, in which DNA derived from
non-infected graft sites is amplified, have an amplification
product corresponding only to .beta.-globin (286 bp). Lanes 4 to 6,
in which DNA derived from wart tissue is amplified, show
amplification products corresponding to HPV (450 bp) and
.beta.-globin (286 bp).
[0152] To identify the HPV type present in swab samples derived
from wart tissue, the Hind III digested DNA was amplified using
primer pairs specific to HPV types 6, 11, 16, 18 and 31 in
different amplification reactions (Mant et al., supra). The results
shown in FIG. 7 confirm the presence of HPV types 6 and 11, and the
absence of HPV types 16, 18 and 31 in the DNA isolated from
cutaneous wart tissue. Therefore amplification products of the DNA
extracted from the swab samples demonstrate the presence of HPV-6
and -11 in the cutaneous papillomas.
[0153] DNA isolated from swab samples obtained from the surface of
4 exposed subcutaneous grafts are analyzed by amplification using
HPV types 6 and 11 specific primers. The results show the presence
of both HPV types 6 and 11 (FIG. 8), and the absence of high-risk
HPV-16, -18 and -31 at all 4 sites (data not shown). The results
presented in FIG. 9 confirm the propagation of single type HPV-6 or
-11 separately.
Example 6
Harvesting of Tissue from 1.sup.st Generation Papillomas and
Producing HPV Stock for Passaging to 2.sup.nd and 3.sup.rd
Generation Xenografted Animals
[0154] The xenograft papillomas were surgically excised and the
tissue treated according to the method used for clinical samples
(described in Example 1). HPV stock was collected from these
tissues and the HPV typed (as in Example 1). The harvested stock
was used to inoculate meshed human skin tissue for engrafting onto
immuno-deficient recipient animals. This produced 2.sup.nd
generation HPV papillomas. The same procedure was repeated serially
to produce 3.sup.rd generation papillomas.
[0155] The viral stock can be stored in phosphate buffered saline
supplemented with antibiotics at 1% v/v of gentamycin (50 mg/ml),
penicillin (10,000 U/ml) and streptomycin (10,000 .mu.l/ml) at a
temperature of -80.degree. C.
[0156] FIG. 10 shows the gross morphology of a cutaneous papilloma
from a second generation xenografted animal, in the early stage of
wart growth formation. This demonstrates the successful propagation
of human papilloma viral particles isolated from a clinical sample
through at least two generations of human xenografted animal
models.
[0157] HPV-6 and -11 mixture stock extracted from 1.sup.st
generation cutaneous papillomas (cut) or 2 sequential extracts from
sub-cutaneous warts (SC1 and SC2), were used to inoculate meshed
human skin tissue for engrafting onto immuno-deficient recipients.
The resultant animals are referred to herein as 2.sup.nd generation
animals. The results are summarized in FIG. 11. The first extract
of subcutaneous wart (SC1) and the cutaneous harvested virus (cut)
induced papillomas in 80% and 33% of the graft sites respectively
(day 77 post-grafting), indicating that the SC1 stock was more
infectious. The SC2 stock only induced papillomas at 33%
inoculation sites, indicating that most of the infectious viral
particles were already collected from the first extraction (SC1). A
1:10 dilution of SC1 failed to induce visible papillomas,
suggesting that the model is very sensitive to detect low threshold
of infection.
[0158] FIG. 12 demonstrates the high frequency of papilloma
induction with single type HPV-11 in 2.sup.nd and 3.sup.rd
generation using papillomavirus collected from serial xenograft
passage. The reproducibility of sub-cutaneous passage of HPV-11 is
shown in FIG. 13. Although HPV-6 single type induced wart formation
at lower frequency than HPV-11, it seems that a second passage
shows improvement in the induction rate (FIG. 14).
DISCUSSION
[0159] The Applicant is the first to provide a highly reproducible
xenograft animal model for inducing and forming cutaneous and
subcutaneous human papillomas, propagating mixed or single type
human papillomavirus, harvesting infectious human papilloma virions
and advantageously passaging papilloma virions to papilloma-free
human xenografted animals.
[0160] This study presents a novel human xenograft animal model for
propagating HPV. The invention presents a model in which profound
tissue injury and in particular by meshing of human skin tissue
prior to engrafting plays a significant role. In the cutaneous
engrafted model, visible papillomas were induced only in the meshed
grafts. In the subcutaneous engrafted model, subcutaneous growth
prior to exposure resulted only in the meshed human tissue. With
exposure minimal papilloma growth was observed in all the
subcutaneous graft sites and only in some non-meshed engrafted
tissue following repeated exposure.
[0161] The mechanisms underlying the successful induction of human
papillomas in injured tissue, particularly meshed tissue are not
known but may be due to one or a combination of factors. First,
injuring and particularly meshing, may stimulate neoepithelization
(Harries et al., 1995, Aust NZ J. Surg., 65:600-603) thus,
increasing the population of basal cells which are the target cells
for HPV. Second, meshed grafts have a pronounced wound healing
process. It is known that during the process of wound healing, an
integrin, .alpha.6.beta.4 becomes over-expressed. A recent study
(Evander et al., 1997, J. Virol., 71:2449) suggests that this
integrin may be a receptor for papillomavirus binding and entry
into the host cells and may be an important factor in initiating
HPV infection. In addition, this over-expressed integrin may have
some other unidentified functions for cellular proliferation and
differentiation that may play an important role in papilloma
induction.
[0162] Therefore, wound healing in response to an injury appears to
be a significant factor in the induction of papillomas in human
skin tissue and may be an important component in generating animal
models for HPV infections. The results presented herein, provide
for the first time means to generate a highly reproducible
xenografted cutaneous and subcutaneous animal model for HPV. This
animal model is useful for propagating and harvesting highly
infectious viral particles and passaging human papillomavirus, and
for screening potential therapeutic agents.
[0163] This work provides a reproducible model of subcutaneous and
cutaneous HPV infection in NIH-nu-bg-xid mice, with the capability
of forming papillomas having infectious virions in the engrafted
wart tissue. The effect of potential candidate agents can be
assessed not only in terms of wart growth, but also with respect to
viral replication. The ability to propagate the virus through
subsequent generations of graft infection obviates the need for
clinical human papilloma tissue and provides a means of a
continuous and standardized supply of HPV stock for screening
purposes.
[0164] The selection of the NIH-nu-bg-xid mice has certain
advantages over both nu/nu mice and scid mice. As suggested by
Stanley, et al. (1997, Antiviral Chem. Chemother, 8:381-400), nu/nu
mice may be less immunodeficient than scid and NIH-nu-bg-xid mice
and thus not easily allow xenograft tissue to survive and grow. The
scid mice are covered in fur thus necessitating removal of the hair
before surgery and before evaluating and measuring experimental
endpoints. The NIH-nu-bg-xid mouse is essentially hairless and lack
functional T-cell, B-cell and NK-cells, thus making it the ideal
recipient in this animal model.
[0165] All references cited herein, including journal articles or
abstracts, published or corresponding U.S. or foreign patent
applications, issued U.S. or foreign patents, or any other
references, are entirely incorporated by reference herein,
including all data, tables, figures, and text presented in the
cited references. Additionally, the entire contents of the
references cited within the references cited herein are also
entirely incorporated by reference.
[0166] Reference to known method steps, conventional methods steps,
known methods or conventional methods is not in any way an
admission that any aspect, description or embodiment of the present
invention is disclosed, taught or suggested in the relevant
art.
[0167] The foregoing description of the specific embodiments will
fully reveal the general nature of the invention that others can,
by applying knowledge within the skill of the art (including the
contents of the references cited herein) readily modify and/or
adapt for various applications such specific embodiments, without
undue experimentation, without departing from the general concept
of the present invention. Therefore, such adaptations and
modifications are intended to be within the meaning and range of
equivalents of the disclosed embodiments, based on the teaching and
guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance presented herein, in
combination with the knowledge of one skilled in the art.
* * * * *