PCR-hybridization assays specific for integrated retroviruses

Details PCR-hybridization assays specific for integrated retroviruses PCR-hybridization assays specific for integrated retroviruses

2001-04-18

2002-09-10

Fredman, Jeffrey (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

49, C536S024330

Reexamination Certificate

active

06448014

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of virology and diagnostics. More specifically, the present invention relates to the development of PCR-hybridization assays specific for integrated retroviruses and their use in the determination of latent infection in patients.
2. Description of the Related Art
It has been known for many years that latent HIV infection can occur in infected cell lines in vitro and nearly all of the early studies of the molecular mechanisms underlying HIV latency examined what is occurring in latently infected clonally derived T-cell lines (2-4, 10, 13, 15). It was also demonstrated many years ago that there is a considerably greater frequency (~100-fold more) of lymphocytes in HIV patients which contain HIV DNA than cells which are productively infected (i. e., possess HIV DNA as well as making HIV RNA, viral proteins, and progeny virions) (6, 7). Studies examining HIV infection of resting or activated CD4 lymphocytes in vitro clearly showed that only activated cells are primarily permissive for virus replication and allow progeny virus production, and infection of resting cells usually leads an abortive infection, in which the virus binds, enters, and completely or partially reverse transcribes proviral DNA. This viral DNA, however, does not integrate until the cell is activated into division within a few days (9, 17). Most of the relatively higher frequency of HIV DNA
+
/RNA
−
lymphocytes in HIV patients (~0.8%) (5), therefore, are very likely recently infected (abortively) resting lymphocytes. This should be expected since 99% of all lymphocytes are resting (16). However, some of these HIV DNA
+
/RNA
−
cells found in infected subjects may also be latently infected cells, and these cells would have integrated proviral DNA but are not making progeny virus. The frequency of latently infected cells in HIV
+
individuals is not clear.
Studies in vitro examining acute and chronic infection of both human T-cell lines and PHA-stimulated CD4 PBLs has shown that gradual shutdown of the virus into latency is routine for the majority of acutely infected cells, and shutdown into latency should be considered a normal part of the virus life cycle (11). Thus, in an HIV patient, any productively infected cells, which are not eliminated by the immune system or by replication of cytopathic HIVs, should become latently infected cells. Thus, it is possible that reliable quantitative data on the frequencies of latently infected cells in patients may reveal that there exists a large pool of such cells.
One study attempted to directly quantitate the frequency of latently infected cells in HIV patients using an inefficient re-ligation technique of PCR-amplified HIV-cellular DNA and found that the frequency was low: at approximately the same frequency as productively-infected cells (~7 cells per 10
6
) (5). More recent studies have used limiting dilution culturing of lymphocytes from patients, which are activated by PHA to reactivate latent virus. The frequencies of these cells are much higher (8). Unfortunately, this procedure also allows any recently abortively infected resting cells to complete the virus replication cycle and become HIV-producing cells, and thus those studies do not discriminate completely between the abortively infected cells and latently infected cells.
Understanding the extent of latent infection has become of great importance now because HAART therapy can reduce plasma viral loads for long periods of time, yet when the drugs are removed, virus usually returns. It seems reasonable to conjecture that a great deal of this return is due to reactivation of HIV from latently infected cells.
The prior art is deficient in the lack of effective means o f detecting latent HIV infection in patients. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention describes an in situ PCR assay that only detects integrated retroviruses, and this should allow accurate assessment of the frequency of productively and latently infected cells together in patients. Since the frequencies of productively infected cells at any given time in patients is known to be very low, any larger frequencies would have to be measuring latent infection. Through the use of PCR and hybridization, a new way to score for integrated retroviruses in cellular chromosomal DNA is possible. This method may be adapted in many forms of quantitative assays of latent HIV infection in infected subjects undergoing anti-retroviral drug therapy and should allow physicians to monitor the presence of latently infected cells so that they can determine whether treatment should be discontinued or not.
The invention utilizes PCR and employs one primer for a n Alu sequence in the human chromosomal DNA and the other primer near the end of the viral LTR. PCR-amplified DNA is then hybridized with a probe which only recognizes the amplified LTR sequence. The probe could be labeled in different ways, and numerous forms of this test could be developed, many of which could be quantitative.
In one aspect of the present invention, there is provided a quantitative PCR assay to measure HIV latent infection.
In another aspect of the present invention, there is provided a kit for detecting integrated retroviruses in a human sample.
In another embodiment, the invention can be used t o monitor latent infection by other retroviruses.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.


REFERENCES:
patent: 5538871 (1996-07-01), Nuovo et al.
Fritsch et al. Production of HIV-1 by huamn B cells infected in vitro: Characterization of an EBV genome-negative B cell line chronically synthesizing a low level of HIV-1 after infection. Virology, vol. 244: 542-551, 1998.*
Carteau et al. Chromosome structure and human Immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target. J. Virol., vol. 72(5): 4005-4014, 1998.*
Carteau S et al. Chromosome structure and human Immunodeficiency virus type 1 cDNa integration: centromeric alphoid repeats are a disfavored target. J Viorology, 72(5): 4005-4014, 1998.*
Embretson J et al. Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution. Proc.Natl.Acad.Sci.USA., 90: 357-361, 1993.*
Statagene Catalog.Gene characterization kits. p. 39, 1998.

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