Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2000-03-16
2003-03-04
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
Reexamination Certificate
active
06528308
ABSTRACT:
TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. THE CD8
+
SUPPRESSOR MOLECULE INHIBITS HIV-1 VIRAL TRANSCRIPTION
5.2. SUBSETS OF CD8
+
CELLS EXPRESS ANTI-VIRAL ACTIVITY
5.3. GENERATION OF CD8
+
PERMANENTLY ESTABLISHED CELL LINES THAT EXPRESS ANTI-VIRAL ACTIVITY
5.4. PURIFICATION AND CHARACTERIZATION OF CD8
+
SUPPRESSOR MOLECULES
5.5. CLONING OF CD8
+
SUPPRESSOR MOLECULE
5.6. USES OF CD8
+
SUPPRESSOR MOLECULES
6. EXAMPLE: CD8
+
SUPPRESSOR ACTIVITY INHIBITS HIV-1 REPLICATION
6.1. MATERIALS AND METHODS
6.2. RESULTS
7. EXAMPLE: ISOLATION OF CD8
+
CLONAL CELLS EXPRESSING THE ANTI-HIV-1 SUPPRESSOR MOLECULES
7.1. MATERIALS AND METHODS
7.2. RESULTS
8. EXAMPLE: OLIGO CLONAL CD8
+
LYMPHOCYTES FROM ASYMPTOMATIC HIV-INFECTED INDIVIDUALS INHIBIT HIV-1 REPLICATION
8.1. MATERIALS AND METHODS
8.2. RESULTS
9. EXAMPLE: GENERATION OF TRANSFORMED CD8
+
SUPPRESSOR CD8
+
CELLS
9.1. MATERIALS AND METHODS
9.2. RESULTS
10. EXAMPLE: THE CD8
+
SUPPRESSOR MOLECULE INHIBITS HIV AFTER VIRAL ENTRY
10.1. MATERIALS AND METHODS
10.2. RESULTS
11. DEPOSIT OF MICROORGANISMS AND REFERENCES CITED
1. INTRODUCTION
The present invention relates to bioactive molecules, herein referred to as CD8
+
suppressor molecules, that are produced by the CD8
+
subset of human T-lymphocytes and suppress human immunodeficiency virus (HIV) replication through inhibition of viral transcription. The invention relates to isolation of clonal CD8
+
cell lines and/or the generation of permanently established transformed CD8
+
cell lines that produce antiviral activity. The invention also relates to the development of assay systems for detection of the antiviral activity. The cell lines and assay systems, described herein, may be utilized, e.g., to purify, characterize and clone a CD8
+
suppressor molecule. The CD8
+
suppressor molecules may be used, e.g., in therapeutic applications for treatment of diseases associated with HIV infection.
2. 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) (Barré-Sinoussi, F. et al., 1983 Science 220:868-70; Gallo, R. et al. 1984, Science 224:500-3). 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.
HIV-1 is a member of the lentivirus family of retroviruses (Teich, N. et al., 1984 In RNA Tumor Viruses ed. R. Weiss, N. Teich, H. Varmus, J. Coffin CSH Press, pp. 949-56). The life cycle of HIV-1 is characterized by a period of proviral latency followed by active replication of the virus. The primary cellular target for the infectious HIV-1 virus is the CD4
+
subset of human T-lymphocytes. Targeting of the virus to the CD4
+
subset of cells is due to the fact that the CD4
+
cell surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al., 1984, Nature 312:763-67; Klatzmann et al. 1984, Nature 312:767-68; Maddon et al. 1986 Cell 47:333-48).
In more detail, HIV-1 infection of susceptible cells is initiated via interactions between the virus envelope glycoprotein (gp120) and the CD4
+
cell surface receptor. Fusion of the viral and cell membranes then proceeds through subsequent interaction of this complex with a specific chemokine receptor, primarily the CCR5 or the CXCR4 chemokine receptor (Bieniasz & Cullen, 1998
, Front. Biosci
. 3:D44-D58; Moore et al., 1997
, Curr. Opin. Immunol
. 9:551-562). HIV-1 isolates that can infect T-cell lines and induce syncytia (SI) use the CXCR4 receptor and are termed X4 HIV-1. Such isolates are typically recovered late in the course of HIV progression and differ from the non-syncytia inducing (NSI) strains which predominate in the early stages of HIV infection. NSI strains gain entry to target cells through use of the CCR5 receptor and are referred to as R5 HV-1.
After binding to the cell surface and fusion of the virus and cell membrane, the HIV-1 virion becomes internalized and the virus's RNA genome is converted into linear double-stranded DNA molecules. This process is dependent on the action of the virally encoded reverse transcriptase. Following replication of the viral genome, the linear DNA molecule integrates into the host genome through the action of the viral integrase protein, thus establishing the proviral form of HIV-1. During the early phase of proviral expression, transcription of the viral genome results in expression of regulatory proteins such as Tat, Nef and Rev. Transcriptional activation of the proviral DNA is mediated through the viral 5′ LTR sequences (long terminal repeats). The initial low level of viral transcription is dramatically increased by the HIV encoded transactivator protein termed tat (transactivator protein) (Cullen, B. R. et al. 1989, Cell 58:423-26). The Rev protein promotes the transition from the early phase expression of regulatory proteins to late phase expression of structural proteins. Assembly of newly synthesized viral particles is followed by budding of virus particles from the cell membrane allowing the virus to infect new cells.
The HIV-1 virus is capable of establishing a latent state of infection for prolonged periods of time. Individuals infected with the human immunodeficiency virus may remain clinically healthy for long periods of time, with the estimated average length of the asymptomatic period between primary HIV infection and the progression to AIDS and increase in viral replication being approximately 8 to 10 years. It is generally believed that the humoral immune response to HIV-1 is not sufficiently protective against progression of the disease. Therefore, attention has turned to the possibility that the T-lymphocyte population of cells may maintain the period of latency by directly inhibiting HIV-1 replication.
A number of groups have recently noted that the CD8
+
subset of T-lymphocytes have the ability to inhibit the replication of HIV-1 in vitro (Walker, C. M. et al., 1989, Cellular Immunology 119:470-475; Kannagi, M. et al. 1990, J. Virology 64:3399-3406; Walker, C. M. et al., 1991 J. Virology 65:5921-5927). For example, addition of CD8
+
cells to naturally HIV-1 infected CD4
+
cell cultures has been found to inhibit the replication of HIV-1 in the infected cultures in a dose dependent manner. (Ref. supra). Furthermore, the inhibitory effect is not entirely dependent on cell-cell contact as an inhibitory effect is observed across a semi-permeable membrane suggesting that at least a portion of the CD8
+
suppressor activity is due to a soluble inhibitor of HIV-1 replication. (Ref. sra). To date, the molecular identity of the CD8
+
suppressor molecule, or a combination of factors, as well as the mechanism by which it exerts its antiviral effect remains undefined.
Because such CD8
+
suppressor molecules would be useful, e.g., in the treatment and/or inhibition of HIV infection, there is a need in the art to identify and characterize such molecules and their HIV suppression activity. There is also a need for methods, particularly screening methods, which may be used, e.g., to screen CD8
+
cells for such suppression activity and to isolate such CD8
+
suppressor molecules.
3. SUMMARY OF THE INVENTION
The present invention relates to molecules produced by the CD8
+
subclass of T-lymphocytes that are able to inhibit HIV viral replication. The molecules can be e.g., soluble molecules produced and/or secreted by the CD8
+
Bolognesi Dani P.
Greenberg Michael L.
Lacey Simon F.
Tomaras Georgia D.
Weinhold Kent J.
Duke University
Parkin Jeffrey S.
Pennie & Edmonds LLP
Scheiner Laurie
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