Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-11-19
2001-03-13
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S455000, C435S456000, C435S325000, C435S371000
Reexamination Certificate
active
06200745
ABSTRACT:
FIELD OF INVENTION
This invention relates to a novel in vitro method of testing antiviral activity of various agents. More specifically, it describes a method of testing effectiveness of anti-papillomavirus agents which act early in the infection process. The method is useful in testing effectiveness of existing and potential antiviral drugs, in particular, future drugs directed to treatment of Human Papillomavirus infections.
BACKGROUND OF THE INVENTION
Testing of the antiviral effectiveness of the new and existing agents against Human Papillomavirus are still performed in the in vivo testing involving use of laboratory animals and human subjects. These studies are expensive, time consuming and altered by individual differences among subjects.
A demonstration of efficacy prior to in vivo animal model testing would limit the candidate in vivo agents to the ones with increasing potential for in vivo effectiveness. This is especially important for those newer, more speculative agents for which purer antiviral effects are lacking. In vitro demonstration of efficacy can support a decision for expensive testing in animal model systems. In vitro studies are also useful for exploring drug-virus interactions which are awkward or infeasible in whole animal systems. In vitro testing offers the following advantages: 1) preliminary data on efficacy; 2) rapid turn around time; 3) economy; 4) ability to precisely control environmental conditions; 5) elimination of pharmacokinetics and variability of whole animal systems; and 6) small amounts of drugs are required.
There are no established systems for in vitro papillomavirus testing. There have been some recent, encouraging developments, elsewhere and in our laboratory. Broker's laboratory has recently suggested that the xenograft system, which we originated, might be useful for antiviral testing (S. Dollard, et al., 1992, Gene Dev., 6:1131-1142).
In that one approach, fragments of HPV-11 infected human foreskin tissue is excised from the papillomatous cysts, growing beneath the renal capsule, and the fragments are placed onto a collagen gel “raft” culture. HPV-11 replication continues in the tissue fragment, as cells migrate laterally across the surface of the gel. We have explored the use of this system as a possible target for antiviral testing, and we have found that there is a high degree of regional variability in the extent of cell migration, tissue growth, and HPV-11 replication. We do not believe that this in vitro system is sufficiently consistent or precise to form a basis for tests. Further, since preliminary xenografts are required, the cost of the test includes their preliminary growth for three months, so some of the theoretical advantages of in vitro tests, economy and rapid turn-around are lacking.
Another in vitro system with potential was recently described by Laimins' group (Meyers, et al., 1992, Science, 257:971-973). In this system, human cervical cells, bearing HPV-31b episomal DNA are placed on collagen gel raft cultures and biosynthesis of complete virions occurs in the differentiating cells. It seems likely that this system may also be affected by regional variability.
Many of the disadvantages of the prior art methods of testing antiviral activity are overcome by the method of the present invention which precisely measures antiviral activity without the interferences of the regional variability, since the cell cultures are evenly dispersed monolayers.
SUMMARY OF THE INVENTION
In accordance with the present invention, a new in vitro method of testing of the antiviral activity of the potential agents in the initial stages of papillomavirus infection is presented.
We have recently developed a monolayer cell culture system in which we have conducted antiviral testing. The monolayer is a uniform cell sheet with no regional variability. The system can use, alternatively, two rabbit epithelial cell lines: RK-13, derived from domestic rabbit kidney, and SF1Ep, derived from cottontail epidermis. The cells are planted in vitro, infected with cottontail rabbit papillomavirus CRPV virion, and this is soon followed by a wave of CRPV DNA replication and mRNA synthesis, probably ORFs E6 and E7. Under these conditions, the epithelial cells do not complete cytodifferentiation, a requisite for complete virion synthesis, so the papillomavirus infection is abortive, and the CRPV DNA is lost after 3-5 passages. However, only a few days post-infection is sufficient for antiviral testing.
Testing of the antiviral activity involves exposing cells to the various amount of the agent and measuring the effect on the level of CRPV transcription and cell proliferation/viability.
CRPV infection of an established cottontail epidermal cell line (Sf1Ep) results in the production of CRPV-specific transcripts without concomitant morphological transformation (M. Angell, et al., J. Vir. Meth., 1992, 39:207-216). The most abundant transcripts correspond in size to those of the E6 and E7 open reading frames (ORFs) which are also among the most abundant in domestic and cottontail rabbit papillomas. CRPV RNA production was both time and dose-dependent with RNA production diminishing with decreasing viral dose and increasing culture passage. Infected cultures contain episomal CRPV DNA which did not appreciably change in abundance with time but are significantly reduced with culture passage. All features of in vitro infection, especially RNA production, are inhibited by CRPV-neutralizing but not HPV-11-neutralizing monoclonal antibodies. Much of this inhibition can be attributed to a blockage of viral penetration as indicated by the reduction of CRPV DNA within virus-neutralized cultures. Our data indicate that although CRPV infection of Sf1Ep cells was abortive, it serves as a useful model for analysis of early infection events.
Substituting the neutralizing antibody with an antiviral agent has proven to be a useful way of measuring the effectiveness of the antiviral agents and formed the basis for the novel method of testing the antiviral activity of unknown agents.
OBJECTS OF THE INVENTION
An object of this invention is to provide a novel method of in vitro testing of the antiviral activity of potential antiviral agents.
This and other objects and advantages of the invention over the prior art and a better understanding of its use will become readily apparent from the following description and are particularly delineated in the appended claims.
REFERENCES:
Khan et al., J. Virol., vol. 67, No. 6, pp. 3396-3403, Jun. 1993.
Angell Michael G.
Foster Chris
Hitchcock Margaret
Isseroff Roslyn Rivkah
Kreider John W.
Guzo David
Monahan Thomas J.
The Penn State Research Foundation
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