Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-08-13
2003-09-09
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C530S350000
Reexamination Certificate
active
06617103
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the detection of Herpes Simplex type 2 (HSV-2), more particularly, the invention relates to a transforming fragment of HSV-2 and to the detection thereof in clinical specimens.
BACKGROUND OF THE INVENTION
Nearly one fifth of adults in the United States are infected with herpes simplex virus type 2 (HSV-2). Although HSV-2 is the most common cause of genital ulceration in developed countries, subclinical HSV-2 infections are suspected to affect a more important proportion of infected individuals. HSV-2 has also been proposed as a causative agent of genital cancer (Guibinga et al., 1995, Arch. STD/HIV Res. 9:163-179). However conflicting results from in vitro and in vivo studies have shed doubts on the role of this agent in cancer of the uterine cervix (Guibinga et al., 1995, Arch. STD/HIV Res. 9:163-179). A transforming region of the HSV-2 genome—the 7.6 kb BglII N (m.u. 0.58-0.63) segment—has been identified, using transfection experiments. This was further supported by studies showing that BglII N sequences can also cooperate with oncogenic papillomas viruses to transform cells (DiPaolo et al., 1990, Virol. 777-779). Initially, the transforming ability of HSV-2 was thought to be located on the left-end (Xho-3 subfragment) of the BglII N segment (Galloway et al., 1983, Nature 392:21-24; Ibid., 1984, Proc. Natl. Acad. Sci. USA 81:4736-4740). However, neither the presence of a viral protein (Galloway et al., 1982, J. Virol. 42:530-537; Vonka et al., 1987, Adv. Cancer Res. 48:149-191) nor the persistence or integration (Galloway et al., 1983; Vonka et al., 1987) of specific HSV sequences, seemed to be required for the maintenance of the transformed phenotype (Pilon et al., 1989, Biochem. Biophys. Res. Comm. 159:1249-1261). The transforming ability of HSV-2 was left unexplained. Transfection of NIH 3T3 cells with the right-end (Kessous-Elbaz et al., 1989, J. Gen. Virol. 70:2171-2177; Pilon et al., 1989; Saavedra et al., 1985, EMBO J. 4:3419-3426) of the BglII N fragment (the Xho-1 and Xho-2 subfragments) showed an increase in the number of transformed foci, and HSV-2 sequences were retained more efficiently in transformed cells (Kessous-Elbaz et al., 1989; Pilon et al., 1989; Saavedra et al., 1985).
A number of clinical and epidemiologic studies have concluded that high risk papillomaviruses, such as HPV-16 and HPV-18 are necessary for the development of cervical cancer, but the long delay following infection indicates the importance of other factors (Kessler, 1986, In: Viral Etiology of Cervical Cancer, Peto et al., Eds. Cold Spring Harbor, N.Y., 55-64; and, zur Hauzen, 1989, Cancer Research 49:46774681), particularly other sexually transmitted infections (Kaufman et al., 1986, Clin. Obstet. Gynecol. 29:678-698; Macnab et al., 1989, Biomed. and Pharmacother. 43:167-172; zur Hausen, 1982, Lancet 2:1370-1372), for the development of malignancy. Although the etiologic link between herpes simplex virus-2 (HSV-2) and cervical cancer was proposed over two decades ago, the significance of the importance of HSV-2 to cervical cancer has been rather recent. The role for HSV-2 infection has been based primarily on sero-epidemiological data (Nahmias et al., 1970, Am. J. Epidemiol. 91:547-552; Rawls et al., 1968, Am. J. Epidemiol. 87:647-656) and on observation of viral antigens in exfoliated cells from patients with cervical dysplasia and cancer (Royston et al., 1970, Proc. Nat. Acad. Sci. 67:204-212). The difficulty in establishing a strong association was compounded by the lack of persistence of HSV sequences in the neoplastic cervical lesions (Macnab et al., 1989, Biomed. and Pharmacother. 43:167-172). In fact, in a prospective case-control study (Vonka, 1984, Int. J. Cancer 33:61-65) the investigators failed to observe such an association, which was later suggested may have resulted from overmatching of the cohort of women for sexual activity that minimized the risk factor (Reeves et al., 1989, New Engl. J. Med. 320:1437-1441). In other studies the lack of correlation of HSV-2 with cervical cancer was attributed to the use of immunoglobulin G instead of immunoglobulin A as a marker for the presence of HSV-2 (Corbino et al., 1989, Eur. J. Gynaecol. Oncol. 10:103-108).
A recent case-control study, using confirmed histological cases of cervical cancer from Latin America, found that the presence of HSV-2 antibodies correlated with a nine-fold excess risk of cervical cancer compared to women negative for HSV-2 or HPV-16/18 (Hildesheim et al., 1991, Int. J. Cancer 49:335-340). In well controlled studies of cervical cancer, others have reported a two-to-four fold excess risk in HSV-2 seropositive women (Slattery et al., 1989, Amer. J. Epidemiol. 130:248-258) and in women with both HPV and HSV-2 present in cervical tumor biopsies (Di Luca et al., 1989, Int. J. Cancer 43:570-577; Ibid., 1987, Int. J. Cancer 40:763-768.). Finally, in the last case control study (Daling et al., 1996, Cancer Epidemiology, Biomarkers and Prevention 5:541-548), involving women from western Washington state, the potential cofactors with HPVs in the development of cervical cancer were analysed. A significant increase in risk associated with HSV-2, as measured by antibodies, was found only in women whose tumor biopsies were negative for HPV. One major problem in establishing a definite link between HSV-2 and cervical cancer has been the difficulty to consistently detect HSV-2-specific DNA in cervical cancer biopsy samples despite the fact that several investigators have reported the presence of herpes virus specific sequences in some of the carcinomas tissues they have analysed (Frenkel et al., 1972, Proc. Nat. Acad. Sci. 69:3784-3789; Park et al., 1983, EMBO J. 2:1029-1034; Royston et al., 1970, Proc. Nat. Acad. Sci. 67204-212).
Experimental support for a role of HSV-2 in cervical cancer has come from in vitro studies that demonstrated its transforming potential using the inactivated virus (Duff et al., 1971, Nature 233:48-50; Macnab, 1974, J. Gen. Virol. 24:143-153) or its fragments, BglII N (mtr II) and BglII C (mtrIII) (Galloway et al., 1981, J. Virol. 38:749-760; Jariwalla et al., 1980, Proc. Natl. Acad. Sci. (USA) 77:2279-83; Reyes et al., 1979, Cold Spring Harbor Symp. Quant. Biol. 44:629-641). However the elucidation of the mechanism(s) leading to the transformed phenotype has been complicated by the loss of the viral sequences, which suggested the hypothesis of hit and run mechanism (Galloway et al., 1983, Nature 302:21-24) and review (Macnab et al., 1987, J. Gen. Virol. 68:2525-2550). Our studies on the transforming potential and the retention of BglII N and its Xhol restricted subfragments have suggested that BglII N, when present in its entirety, might have a toxic effect resulting from either a high copy number or from specific function(s) expressed by its coding sequences (Saavedra et al., 1985, EMBO J. 4:3419-3426). These studies also demonstrated that two BglII N subfragments—Xho-1+2 and Xho-2—induced the tumorigenic conversion of NIH3T3 cells and were stably retained in the transformed cell lines and their derived tumors (Saavedra et al., 1985, EMBO J. 4:3419-3426). The role of HSV-2 in cervical cancer has been further supported by in vitro studies showing its oncogenic cooperation with HPV 16/18. HPV-16 immortalized human foreskin keratinocytes transfected with a recombinant plasmid bearing the HSV-2 fragment BglII N yielded tumorigenic clones, whereas the parental HPV-immortalized cell lines were incapable of inducing tumors. Southern blot analysis of the viral sequences present in the transformed cell lines indicated that HPV-16 genomes were maintained unchanged in their integrated state, but HSV-2 sequences were not found in the tumor-derived cell lines (DiPaolo et al., 1990, Virology 177:777-779). Using a similar approach, Dhanwada et al. (Dhanwada et al., 1993, J. Gen. Virol. 74:955-963; Ibid., 1992, J. Gen. Virol. 73791-799) showed that HSV-2 mtrIII (BglII C) region induced rearrangements of HPV-18 DNA sequences in the immortalized human
Coutlée François
Dipaolo Joseph A.
Kessous Allegria
Knobbe Martens Olson & Bear LLP
The United States of America as represented by the Department of
Wortman Donna C.
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