Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1994-03-10
2002-06-04
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S325000, C435S366000, C435S371000, C435S375000, C435S377000, C435S384000
Reexamination Certificate
active
06399353
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biosynthetic process of preparing papillomavirus and to processes for screening anti-papillomaviral agents, determining the papillomavirus infectivity of epithelial cells, detecting the presence of anti-papillomavirus antibodies, and vaccinating against papillomavirus infection.
BACKGROUND OF THE INVENTION
Papillomaviruses (PV) are important pathogens associated with a variety of neoplasias. Human PV (HPV) types 16, 18, 31, 33, 35, and 51 have been associated with malignant lesions of the anogenital area, and types 6 and 11 are found in benign genital lesions (Syijanen, et al., 1987; Salzman, et al., 1987). Study of the complete viral life cycle has been prevented by the lack of a cell culture system that will permit vegetative viral replication. Human papillomaviruses have been propagated in rodents by either grafting infected tissue under the renal capsule (Kreider, et al., 1987; Kreider, et al., 1990) or under the flank skin (Sterling, et al., 1990) of a nude mouse, but no reproducible permissive in vitro system has yet been described. This is probably a result of the evolution of a viral life cycle that is tightly coupled to the differentiation program of keratinocytes in which virion production is limited to differentiating suprabasal cells (Taichman, et al. 1987).
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides a process of biosynthesizing papillomavirus in an epithelial cell containing papillomavirus DNA comprising inducing complete differentiation of said epithelial cell. Preferably, inducing complete differentiation comprises the steps of:
a) providing a cell line of epithelial cells that contain papillomavirus DNA;
b) placing the epithelial cells onto a dermal equivalent in an epithelial culture medium to form an organotypic culture;
c) maintaining the organotypic culture under biological culture conditions and for a period of time sufficient for the epithelial cells to attach to the dermal equivalent;
d) placing the dermal equivalent on the surface of the epithelial culture medium so that the epithelial cells are not in direct contact with the medium;
e) inducing the expression of filaggrin or a differentiation-specific keratin in the epithelial cells;
f) maintaining the epithelial cells under differentiation conditions and for a period of time sufficient for the epithelial cells to stratify and differentiate; and
g) recovering the papillomavirus from the epithelial cells.
In a preferred embodiment, the papilloma virus is a human papillomavirus such as HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 or HPV-51.
Any epithelial cell that contains or can be infected or transformed to contain PV can be used in a process of the present invention. A preferred epithelial cell line whose cells contain papillomavirus DNA is designated CIN-612. CIN-612 cells are derived from a cervical intraepithelial neoplasia type 1 (CIN 1) lesion that maintains episomal copies of HPV type 31b DNA.
Preferably, filaggrin or a differentiation-specific keratin is induced by intermittently exposing epithelial cells to a protein kinase C inducer. A preferred protein kinase C inducer is a phorbol ester or a diacylglycerol. Even more preferably, a protein kinase C inducer is TPA.
Papillomavirus prepared in accordance with a process of the present invention can be used in assay to detect the presence of anti-papillomavirus antibodies in a sample. Such an assay comprises the steps of:
a) contacting a sample with papillomavirus prepared in accordance with a process of this invention to form a reaction mixture;
b) maintaining the reaction mixture under immunoreaction conditions and for a period of time sufficient for the papillomavirus to immunoreact with anti-papillomavirus and form an immunocomplex; and
c) detecting the presence of the immunocomplex and thereby the presence of the anti-papillomavirus antibodies.
In another aspect, the present invention provides a process of identifying a substance for its ability to modulate papillomavirus biosynthesis comprising the steps of:
a) preparing a model system of biosynthesizing papillomavirus;
b) selecting a substance suspected of having the ability to modulate papillomavirus biosynthesis; and
c) testing for the ability of said substance to modulate said papillomavirus biosynthesis is said model system.
In a preferred embodiment, the model system is a process of biosynthesizing papillomavirus as set forth above. In accordance with that preferred embodiment, papillomavirus is biosynthesized as set forth above in the presence and absence of a substance suspected of having the ability to modulate papillomavirus biosynthesis.
In another aspect, the present invention provides a process of determining the papillomavirus infectivity of epithelial cells comprising the steps of:
a) placing said epithelial cells onto a dermal equivalent in an epithelial culture medium to form an organotypic culture;
b) maintaining said organotypic culture under biological culture conditions and for a period of time sufficient for said epithelial cell to attach to said dermal equivalent;
c) placing said dermal equivalent on the surface of said epithelial culture medium so that the epithelial cells are not in direct contact with said medium;
d) exposing said epithelial cells to papillomavirus;
e) inducing the expression of filaggrin or a differentiation-specific keratin in said epithelial cells;
g) maintaining said epithelial cells under differentiation conditions and for a period of time sufficient for said epithelial cells to stratify and differentiate; and
h) detecting the presence of papillomavirus in said epithelial cells and thereby the papillomavirus infectivity of said epithelial cells.
Epithelial cells, papillomavirus, and means for inducing expression of filaggrin or a differentiation-specific keratin in those processes are the same as set forth above in relation to a process of preparing papilloma virus.
DETAILED DESCRIPTION OF THE INVENTION
I. A Process of Biosynthesizing Papillomavirus
In one aspect, the present invention provides a process of biosynthesizing papillomavirus in an epithelial cell containing papillomavirus DNA comprising inducing complete differentiation of said epithelial cell. Preferably, inducing complete differentiation comprises the steps of:
a) providing a cell line of epithelial cells that contain papillomavirus DNA;
b) placing the epithelial cells onto a dermal equivalent in an epithelial culture medium to form an organotypic culture;
c) maintaining the organotypic culture under biological culture conditions and for a period of time sufficient for the epithelial cells to attach to the dermal equivalent;
d) placing the dermal equivalent on the surface of the epithelial culture medium so that the epithelial cells are not in direct contact with the medium;
e) inducing the expression of filaggrin or a differentiation-specific keratin in the epithelial cells;
f) maintaining the epithelial cells under differentiation conditions and for a period of time sufficient for the epithelial cells to stratify and differentiate; and
g) recovering the papillomavirus from the epithelial cells.
Any papillomavirus (PV) can be biosynthesized by a process of this invention. Papillomavirus infections have been described in humans, cattle, horses, dogs, sheep, rabbits, swine, goats, hamsters, cats, rodents, deer, beaver, coyotes, wolves, bears, elephants, rhinoceros, opossum, armadillos, birds, reptiles, amphibians, and fish. Papillomaviruses have been characterized in cattle, deer, horses, rabbits, dogs, rodents, and birds. Papillomavirus Infections in Animals,
Papillomaviruses and Human Disease
, K. Sytjanen, L. Gissman, L. G. Koss (Eds.)
In a preferred embodiment, a papillomavirus that is biosynthesized by a process of the present invention is a human papillomavirus (HPV). Exemplary and preferred HPV include HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-51.
Epithelial cells containing papillomavirus DNA can be obtained from infected subjects, prepared by infecting ep
Laimins Laimonis A.
Meyers Craig M.
Fulbright & Jaworski LLP
Guzo David
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