Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-01-07
2001-04-17
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S007200, C435S007210, C435S091200, C435S091320, C435S091330, C435S091510, C435S325000, C435S366000, C435S371000
Reexamination Certificate
active
06218105
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
This invention relates to a high-throughput in vitro method of testing antiviral activity of various agents. More specifically, it describes a method of testing effectiveness of anti-papilloma virus agents which act early in the infection process. The method is useful in testing effectiveness of existing and potential antiviral drugs, including the efficacy of vaccines, in particular, future drugs directed to treatment of human papilloma virus infections.
2. Description of the Background Art
Cervical cancer is strongly linked to human papilloma virus (HPV) infection. HPV types 6 and 11 are associated with condylomata and low grade dysplasia while HPV-16 and -18 are associated with high grade dysplasia and cervical cancer. Information on the human immune response against Human papilloma viruses (HPV) predominantly comes from serological studies that used enzyme-linked immunosorbent assays (ELISA) targeted against HPV antigens. The simple detection of anti-HPV antibodies, however, fails to identify neutralizing antibodies (N-Abs) against HPV. Thus it has been widely recognized that only the detection of specific N-Abs against HPV will identify an effective immune response (protective antibodies). See, e.g., Foster et al.,
Papillomavirus Report
8,127-131 (1997).
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 have been some encouraging developments, elsewhere and in our laboratory, for in vitro papillommavirus testing. 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 the Broker 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. This in vitro system, however, does not appear to be sufficiently consistent or precise to form a basis for tests. Further, since preliminary xenografts are required, the cost of the test includes the preliminary growth for three months, so some of the theoretical advantages of in vitro tests, economy and rapid turnaround are lacking.
Another in vitro system with potential was described by Laimins' group (Meyers, et al., 1992, Science, 257:971-973). In this system, human cervical cells, bearing HPV-31
b
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 disadvantageously 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.
Other in vitro methods have appeared in recent years but only one assay utilizes infectious virion. See, e.g., Smith et al.,
J Invest Dermatol
105, 438-444 (1995). Most vaccines and anti-viral agents to date prevent infection in the early stages of infection, i.e. binding to viral coat proteins, inhibition of viral nucleic to acids form entering the cellular nucleus or preventing RNA transcription. By using intact virion the infection of monolayers more closely mimics a true viral infection than the use of puesdo-virion. This in vitro PV assay relies on methods not conducive to high throughput screening. Past methodology required the isolation of RNA before RT-PCR could be performed to detect HPV mRNA expression within infected cells. Also nested set PCR was necessary for the detection of viral mRNA which is cumbersome in a high throughput method.
There is accordingly a need for an in vitro assay utilizing infectious virion which is amenable to use with high-throughput techniques. The present invention satisfies this need, as well as others, and generally overcomes the deficiencies found in the background art.
SUMMARY OF THE INVENTION
The present invention is a in vitro assay which allows for large scale screening of antiviral agents, in particular, human neutralizing antibodies against HPV-11. The invention can also be used for other viral HPV types such as -16, -18 and 31 as well as other viruses. This HT-RT-PCR HPV in vitro infectivity assay facilitates serologic studies on the response to HPV-11 infection and document the presence of neutralizing antibodies in the serum of and/or cervicovaginal secretions of patients with clinical HPV-related disease. The assay of the invention can also be utilized to screen numerous anti-viral candidates other than neutralizing antibodies. This invention can further be applied to the high throughput screening of specific cellular mRNA transcripts.
The invention, in general terms, comprises growing cells in a multi-well format, infecting the cells with intact virion incubated with a test agent, and measuring expression of at least one viral nucleic acid sequence in said cells. The method also preferably comprises incubating intact virion without test agent to define a control. The cells are preferably human keratinocyte cells grown in monolayers. The viral nucleic acid sequence will generally comprise viral mRNA. In one preferred embodiment, the intact virion comprise Human Papilloma Virus, and more preferably Human Papilloma Virus -11. Measuring expression is generally carried out by releasing the viral mRNA from the cells by lysis, amplifying the iRNA as cDNA via RT-PCR, and detecting amplicons with specific probes. Cell lysis may be carried out by heating or by treatment with detergent.
By way of example, and not necessarily of limitation, epithelial cells are grown in monolayers in multi-well tissue culture plates and then subjected to infectious HPV-11 virion. To detect neutralizing antibodies (antiviral effectiveness), test serum is added to HPV-11 virion prior to addition to the cells. Failure to detect HPV infection is indicative of neutralizing antibodies. The infected cells (with and without antiviral agents) are allowed to grow to confluency and then target mRNA is obtained by an extraction-less method.
The extraction-less method either involves trypsinization of the infected cells followed by cell lysis by heat or direct cell lyses by detergent solution. An aliquot of cell lysate are directly placed into multi-well PCR plates for one-tube RT-PCR.
The crude lysate is then subjected to RT-PCR under conditions which preferentially amplify the specific cDNA target rather than genomic DNA. The target mRNA contains a splice site which is utilized in primer and probe design. The
Hall Kathleen S.
Smith Lloyd H.
Bozicevic Field & Francis LLP
Guzo David
Hall Robert C.
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