Determining viral load in double negative T cells

Details Determining viral load in double negative T cells Determining viral load in double negative T cells

2001-11-23

2003-12-16

Stucker, Jeffrey (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S007100, C435S007240, C435S007900, C435S007920, C435S974000, C435S975000, C436S518000, C436S536000, C530S388730, C530S388750

Reexamination Certificate

active

06664042

ABSTRACT:

BACKGROUND OF THE INVENTION
The type-1 human immunodeficiency virus (HIV-1) has been implicated as the primary cause of the slowly degenerate disease of the immune system termed acquired immune deficiency syndrome (AIDS). Infection of the CD4+subclass of T-lymphocytes with the HIV-1 virus leads to depletion of this essential lymphocyte subclass which inevitably leads to opportunistic infections, neurological disease, neoplastic growth and eventually death. HIV-1 infection and HIV-1 associated diseases represent a major health problem and considerable attention is currently being directed towards the successful design of effective therapeutics.
Like other retroviruses, HIV down-regulates its own receptor (
51
,
52
). CD4 down-regulation may alter signaling pathways because of the association of CD4 with p56
lck
(
53
), protect from glycoprotein (gp120)-induced apoptosis (
53
), or prevent superinfection (
54
). However, evidence for CD4 down-regulation in HIV-positive subjects is still lacking and most prior work was performed with transformed cell lines with unknown relevance in vivo. CD4 down-regulation occurs in primary human peripheral blood lymphocytes (PBL) infected with a reporter virus (
27
,
55
). For example, in PBL infected with HIV-HAS (HIV with a murine heat stable antigen reporter gene), HSA
+
cells progressively lost CD4 expression, while maintaining high levels of CD4 mRNA and unaltered levels of T cell receptor (TCR) and CD8 expression (
57
). Therefore, infected double-negative (DN) T cells are generated from CD4
+
precursors. In normal subjects, DN cells are a heterogeneous population. They include natural killer T cells implicated in initiation of a T helper-2 response (
56
). Whether such normal DN subsets can become HIV infected is not known.
In earlier ex vivo studies, HIV DNA seemed to be exclusively in the CD4
+
population of peripheral T lymphocytes (
57
,
58
). It is known now that CD8
+
cells can also be infected (
59
,
60
), but there are no data on DN T cells obtained ex vivo.
In the past few years, a number of drugs, including protease inhibitors, have been developed which greatly decrease the viral load of HIV in infected patients. The discovery of these various drugs has led to cocktail therapies such as the successful highly active antiretroviral therapy (HAART). Current medical cocktails are so potent that the viral load becomes “undetectable” in treated patients using standard viral load tests (usually with a lower limit of detection between 25-400 virions/ml of plasma). Even though the viral load may be non-detectable in plasma, discontinuing therapy leads to a resurgence of the infection. Therefore, HIV drug therapy must be maintained indefinitely. Thus a need exists for an improved method of detecting HIV in patients. Furthermore, current tests do not distinguish functional (infectious) virus from inactivated or non-infectious virus. Thus, there is a need for a method which preferentially identifies viable HIV virus in patients.
SUMMARY OF THE INVENTION
The present invention provides a method for determining viral load in a patient infected with human immunodeficiency virus. The levels of human immunodeficiency virus are measured in CD4
−
CD8
−
double negative cells.
The invention also provides a kit for determining viral load in a patient infected with human immunodeficiency virus. The kit contains a component for separating T cells from a biological sample; a component for separating CD4
−
CD8
−
cells from CD4
+
or CD8
+
cells; and a component for detecting human immunodeficiency virus.


REFERENCES:
patent: 5420264 (1995-05-01), Seed et al.
patent: 0 230 768 (1987-05-01), None
McSharry et al. “Detection and quantitation of human immunodeficiency virus-infected peripheral blood momonuclear cells by flow cytometry”, J. of Clinical Microbiology, vol. 28, No. 4(Apr. 1990). QR46.J87.*
Patterson et al. “Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry”, Science, vol. 260(1993).

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