Cells expressing both human CD4 and a human fusion accessory...

Details Cells expressing both human CD4 and a human fusion accessory... Cells expressing both human CD4 and a human fusion accessory...

1997-01-30

2001-03-06

Priebe, Scott D. (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C435S069100, C435S320100, C435S455000, C435S456000

Reexamination Certificate

active

06197578

ABSTRACT:

FIELD OF THE INVENTION
The present invention pertains to in vitro and in vivo models for the study of human immunodeficiency virus (HIV) infection and the effectiveness of anti-HIV therapeutics.
The susceptibility to HIV infection depends on the cell surface expression of the human CD4 molecule and a heretofore unidentified human fusion accessory factor. The functional assays described herein identified a molecule, designated CXCR4. The term CXCR4 is preferred, however, the terms fusin or HFAF have also been used to refer to the same molecule. Comparison of the nucleotide sequence of the cDNA encoding CXCR4 against a computer database revealed that CXCR4 is a member of the 7-transmembrane segment superfamily of G-protein-coupled cell surface molecules. Many of the superfamily members function as ligand receptors in relation, for example, to peptide hormones, neurotransmitters, and chemokines. CXCR4 has no known ligand, however, and its function is unknown.
A key aspect of the present invention is the discovery that CXCR4 plays an essential role in the membrane fusion step of HIV infection. The establishment of stable, nonhuman cell lines and transgenic mammals having cells that coexpress human CD4 and CXCR4 provides valuable tools for the continuing research of HIV infection and the development of more effective anti-HIV therapeutics. In addition, antibodies against CXCR4, isolated and purified peptide fragments of CXCR4, and CXCR4-binding biologic agents, capable of blocking membrane fusion between HIV and target cells represent potential anti-HIV therapeutics.
BACKGROUND OF THE INVENTION
The HIV infection cycle begins with the entry of the virus into the target cell. The human CD4 molecule is the primary receptor recognized by HIV. The binding of the HIV envelope glycoprotein (env) to the CD4 receptor results in the fusion of virus and cell membranes, which in turn facilitates virus entry into the host. The eventual expression of env on the surface of the HIV-infected host cell enables this cell to fuse with uninfected, CD4-positive cells, thereby spreading the virus.
Recent studies have shown that this HIV fusion process occurs with a wide range of human cell types that either express human CD4 endogenously or have been engineered to express human CD4. The fusion process, however, does not occur with nonhuman cell types engineered to express human CD4. Although such nonhuman cells can still bind env, membrane fusion does not follow. The disparity between human and nonhuman cell types exists apparently because membrane fusion requires the coexpression of human CD4 and an accessory factor specific to human cell types. Because they lack this accessory factor, nonhuman cell types engineered to express only human CD4 are incapable of membrane fusion, and are thus nonpermissive for HIV infection. To date there has been no report of any stable, nonhuman cell line that is permissive for HIV infection as a result of human CD4 and CXCR4 coexpression.
The importance of human CD4 and CXCR4 coexpression also impacts the establishment of a successful small animal model. The development of a small animal model is crucial to the study of HIV infection and the effectiveness of anti-HIV therapeutics. In recent years, researchers have bred transgenic animals having cells that express human CD4. See, for example, Dunn et al.,
Human immunodeficiency virus type
1
infection of human CD
4-
transgenic rabbits,
J. Gen. Vir. 76:1327-1336 (1995); Snyder et al.,
Development and Tissue
-
Specific Expression of Human CD
4
in Transgenic Rabbits,
Mol. Reprod. & Devel. 40:419-428 (1995); Killeen et al.,
Regulated Expression of Human CD
$
Rescues Helper T
-
Cell Development in Mice Lacking Expression of Endogenous CD
4, EMBRO J. 12:1547-1553 (1993); Forte et al.,
Human CD
4
Produced in Lymphoid Cells of Transgenic Mice Binds HIV p
120
and Modifies the Subsets of Mouse T
-
Cell Populations,
Immunogenetics 38:455-459 (1993). These animals, however, have low susceptibility to HIV infection, presumably because of the lack of CXCR4 expression. To date, there has been no report of any transgenic animal that is significantly susceptible to HIV infection as a result of human CD4 and CXCR4 coexpression.
Without an effective vaccine, the number of individuals infected with HIV will likely increase substantially. Furthermore, in the absence of effective therapy, most individuals infected with HIV will develop acquired immune deficiency syndrome (AIDS) and succumb to either opportunistic infections and malignancies that result from the deterioration of the immune system, or the direct pathogenic effects of the virus. Despite the present availability of some anti-HIV agents that slow disease progression, a pressing need remains for more effective therapeutics and drug combinations. To date, there has been no report of any anti-HIV therapeutic that relates to CXCR4.
It is apparent from the foregoing that a need exists for in vitro and in vivo models suitable to the study of HIV infection and the effectiveness of anti-HIV therapeutics. By the same token, the need remains for more effective anti-HIV therapeutics. Although CXCR4 is a member of the known 7-transmembrane segment superfamily of G-protein-coupled cell surface molecules, the essential role of CXCR4 in the membrane fusion step of HIV infection was not elucidated heretofore.
SUMMARY OF THE INVENTION
Accordingly, it is an objective of the present invention is the establishment of stable, nonhuman cell lines, the cells of which contain DNA encoding CXCR4 and express both human CD4 and CXCR4.
Another objective of the present invention is the establishment of transgenic mammals having cells that coexpress human CD4 and CXCR4.
A further objective of the present invention is the production of antibodies, preferably monoclonal antibodies, against CXCR4 that block membrane fusion between HIV and a target cell or between an HIV infected cell and an uninfected CD4 positive cell.
Yet another objective is the isolation and purification of peptide fragments of CXCR4 that block membrane fusion between HIV and a target cell. Also included are fragments of HIV env polypeptide that block membrane fusion between HIV and target cell or between an HIV infected cell and an uninfected CD4 positive cell.
It also is an objective of the present invention to isolate and purify CXCR4-binding agents, both biologic and chemical compounds, that block membrane fusion between HIV and a target cell or between an HIV infected cell and an uninfected CD4 positive cell. A biologic agent of the invention includes stromal cell derived factor 1 (SDF-1), which is a natural ligand for CXCR4.
In accomplishing these and other objectives, there is provided a stable, nonhuman cell line, the cells of which contain DNA encoding a human accessory fusion factor associated with HIV infection (CXCR4), and coexpress human CD4 and CXCR4; a transgenic non-human mammal comprised of cells that coexpress human CD4 and CXCR4; an antibody against CXCR4 that blocks membrane fusion between HIV and a target cell; a monoclonal antibody against CXCR4 that blocks membrane fusion between HIV and a target cell; an isolated and purified peptide fragment of CXCR4, wherein said peptide fragment blocks membrane fusion between HIV and a target cell; and an isolated and purified CXCR4-binding biologic agent, wherein said biologic agent blocks membrane fusion between HIV and a target cell.
Also included in the invention are methods of treating a subject having or at risk of having an HIV-related disorder associated with expression of CXCR4 comprising administering to an HIV infected or susceptible cell of the subject, a reagent that suppresses CXCR4. Therapeutic methods of the invention using an anti-CXCR4 antibody are described. Further, the invention also includes methods of gene therapy wherein an antisense nucleic acid that hybridizes to a CXCR4 nucleic acid is administered to a subject. The reagent is introduced into the cell using a carrier, such as a vector. Administration of the reagent can be in vivo or ex vivo.
In

Affiliated with

Also associated with

Rating

Cells expressing both human CD4 and a human fusion accessory... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Cells expressing both human CD4 and a human fusion accessory..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cells expressing both human CD4 and a human fusion accessory... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2514157