Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2000-07-21
2002-12-17
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S005000, C435S004000, C435S007100, C435S236000, C435S239000, C435S173600
Reexamination Certificate
active
06495361
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an improved method of producing infectious papillomavirus in vitro, with a method comprising (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and (b) providing conditions that produce papillomavirus, wherein the conditions comprise not contacting the epithelial cell a fibroblast or an organotypic raft culture or dermal equivalent.
In earlier studies it was found that HPV infection is three fold more prevalent in spontaneous aborted products of conception compared to elective abortions (60% vs 20%), and that the HPVs were preferentially infecting the trophoblasts within the placenta (1,2). Other studies are supportive of our findings (3-5), that HPVs are present in spontaneous abortions or in trophoblasts. Malhomme et al. (1997)(3) have found that 70% of spontaneous abortion specimens contain HPV by PCR analysis. In addition, Sikstrom et al. (1995) found that women with a history of HPV infection had a higher risk of spontaneous abortion (odds ratio of 3) (6). Pao et al. (1995) found that HPV-18 was present in 50% of choriocarcinomas (malignant trophoblasts) and in some placentas (7). Also related to this issue, HPV DNA has been detected by PCR amplification of DNA from cells taken from amniotic fluids of pregnant women with evidence of HPV genital infection (60% HPV positive), indicating that HPV has the capacity to cross the placental barrier when already present in the cervix (8). In the same report the investigators were unable to detect HPV DNA sequences in amniotic fluid cells from women with no cervical HPV infection.
Trophoblasts are the cells of the placenta that are in direct contact with the maternal tissues. These cells are critical for anchoring the placenta to the maternal tissues. It is also through the trophoblasts that all nutrient exchange and waste exchange takes place. Thus, the disruption of the trophoblastic layer, by HPV or any other infectious or chemical agent, could likely result in abnormal plantation and subsequent expulsion of the gestation (5,9).
The present invention demonstrates that HPV-16 is fully active in trophoblasts. HPVs are already well known to be pathogenic viruses, and are the largest risk factor in the development of cervical cancer (18). Thus, trophoblasts become the second host cell type, in addition to the well studied replication of HPVs in differentiating keratinocytes. These data also support the hypothesis that HPV infection of trophoblasts may be linked to some spontaneous abortions. The search for factors that may be implicated in spontaneous abortion has led to the examination of numerous factors, including maternal age, environmental exposures, tobacco use, and nutritional factors (19-29). However, the studies exploring these relationships have been inconsistent in their findings. For every study supporting an association with spontaneous abortions, there is another study that does not support a relationship. The only uncontested factor thus far is maternal age. If the link between HPV infection and gestational loss can be further substantiated then specific treatment protocols might be developed. Finally, these data represent a technological advance in the ease of studying or generating HPVs, over the tedious and expensive organotypic raft culture system as described in (15) and in U.S. Pat. No. 5,994,115.
SUMMARY OF THE INVENTION
The present invention is directed to an improved method of producing infectious papillomavirus in vitro, with a method comprising (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and (b) providing conditions that produce papillomavirus, wherein the conditions comprise not contacting the epithelial cell a fibroblast.
Further, the present invention is directed to a method of producing infectious papillomavirus in vitro, comprises (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and (b) providing conditions that produce infectious papillomavirus, wherein the conditions comprise the epithelial cell not contacting an organotypic raft culture or a dermal equivalent.
Additionally, the present invention also is directed to a papillomavirus infected non-keratinocyte epithelial cell produced by the method comprising (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and (b) providing conditions that produce papillomavirus, wherein the conditions comprise not contacting the epithelial cell a fibroblast.
The present invention is further directed to a method of detecting the presence of papillomavirus in a subject comprising: (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; (b) providing conditions that produce infectious papillomavirus in the epithelial cell, where the conditions comprise the epithelial cell not contacting a fibroblast; and (c) detecting the presence of papillomavirus in the epithelial cell.
The present invention further is directed to a method of evaluating the inhibition of a papillomavirus comprising (a) introducing papillomavirus or papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; (b) providing conditions that produce infectious papillomavirus in the epithelial cell, wherein the conditions comprise the epithelial cell not contacting a fibroblast; (c) contacting a potential inhibitor of papillomavirus with the infectious papillomavirus produced in the epithelial cells; and (d) evaluating the presence or absence of the papillomavirus to determine the effectiveness of potential inhibitors.
The present invention also is directed to a method of producing a mutant or recombinant papillomavirus, wherein the method comprises: (a) introducing mutant or recombinant papillomavirus DNA or portions thereof necessary for replication, into an epithelial cell; and (b) providing conditions that produce infectious mutant or recombinant papillomavirus in the epithelial cell, wherein the conditions comprise epithelial cell not contacting a fibroblast.
The present invention further is directed to a method of producing a non-keratinocyte epithelial helper cell for production of mutant or recombinant papillomavirus comprising: (a) introducing papillomavirus DNA or portions thereof, into a non-keratinocyte epithelial cell, wherein the DNA or portions thereof, supplements the replication of the mutant or recombinant papillomavirus; and (b) providing conditions that produce said non-keratinocyte epithelial helper cell containing said papillomavirus DNA or portions thereof, that supplements the replication of the mutant or recombinant papillomavirus, wherein the conditions comprise the epithelial cell not contacting a fibroblast.
A method of reducing the risk of spontaneous abortion caused by papillomavirus comprising administering to a subject at risk of spontaneous abortion a drug that inhibits papaillomavirus.
REFERENCES:
patent: 5994115 (1999-11-01), Meyers
M.G. Frattini et al., “In vitro synthesis of oncogenic human Papillomaviruses Requires Episomal Genomes For differentiation-dependent late expression”,Proc. Nat. Acad. Sci., vol. 93, pp. 3062-3067, (1996).
P.L. Hermonat et al., “Human Papillomavirus is More Prevalent in First Trimester Spontaneously Aborted Products Of Compared to Elective Specimens”,Virus Genes, Kluwer Academic Press, vol. 14:1, pp. 13-17, (1997).
P.L. Hermonat et al., “Trophoblasts are the Preferential Target for Human Papilloma Virus Infection In Spontaneously Aborted Products of Conception”, Human Pathology, W.B. Saunders Company, vol. 29:2, pp. 170-174, (1998).
M.Melbye et al., “The role of human papillomaviruses in anogenital cancers”,Cancer Biology, The Academic Press, vol. 8, pp. 307-313, (1998).
C. Meyers, “Organotypic (raft) epithelial tissue culture system For The Differentiation-Dependent replication of
Hermonat Paul L.
Liu Yong
Foley & Lardner
Salimi Ali R.
University of Arkansas
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