Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-08-03
2001-12-25
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S325000, C435S335000, C435S339100, C435S358000, C435S361000, C435S363000, C435S354000, C435S343100, C530S350000
Reexamination Certificate
active
06333163
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel chemoattractant receptor and its use. More particularly, this invention relates to a novel heptahelix-type chemokine receptor which naturally binds leukotriene B4 and also serves as a coreceptor facilitating entry of human immunodeficiency virus type 1 (HIV-1) into cells expressing CD4, and methods for using this novel receptor in HIV infectivity and drug screening studies.
2. Description of Related Art
It is now well understood that HIV-1 infection is initiated by interaction of the virion envelope glycoproteins (gpl120/41) with at least two classes of cell membrane receptors. First, the virus associates with the CD4 receptor (1-3), which induces conformational changes in the glycoprotein envelope (4, 5), allowing the virus to subsequently bind to a seven-transmembrane envelope with the cell membrane leading to viral entry. Two major coreceptors have been identified, both belonging to the chemokine family of G-protein coupled receptors.
Macrophage-tropic (M-tropic) strains of HIV, which replicate in macrophages and CD4+T-cells use the &bgr;-chemokine receptor CCR5 (6-10). T-tropic isolates of HIV, which replicate in primary CD4+T cells, established CD4+T cell lines, as well as macrophages, use the &agr;-chemokine receptor CXCR4 (11).
A key observation leading to the recent discovery of the viral entry cofactors was the finding that certain &bgr;-chemokines have a strong suppressive effect on the HIV-1 infection in vitro (12). Analysis of viral isolates obtained sequentially from infected individuals has shown a loss of sensitivity to inhibition by &bgr;-chemokines along with a shift in virus phenotype from a non-syncytium-inducing (NSI) to a syncytium-inducing (SI) phenotype (13-17). This suggests that there is a shift in chemokine receptor usage from CCR5 to CXCR4 as the infection progresses. This is in accordance with the findings that dual-tropic virus strains utilize both types of coreceptors, and that additional coreceptors exist for certain subsets of primary viruses, in addition to their primary usage of CCR5 or CXCR4 (9, 10, 18-20). Hence, the usage of the two major cofactors may be viewed as extremes in an adaption process, along which the virus expands its coreceptor usage to include several different receptors.
Among the superfamily of G-protein coupled, or heptahelix, receptors the chemokine ones form a structurally related group that belongs to the subfamily of leukocyte chemoattractant receptors, which also includes receptors for the so-called classical chemoattractants (24).
Recently, a novel chemoattractant-like receptor, CMKRL1, was cloned (25) whose natural ligand subsequently was shown to be leukotriene B4 (26, 27). This is the first cloned leukotriene receptor, although the leukotrienes themselves, formed from arachidonic acid through the lipooxygenase pathway, have been known for more than two decades (28). The leukotriene B4 receptor, or BLTR, is widely expressed in the immune system (25, 27)—including thymus, spleen, lymph nodes and PBMC—and it shows approximately 30% identity with CCR5 and CXCR4 which, in turn, exhibit the same degree of homology when compared to each other.
Because of the continuing interest in understanding the biology of HIV infection and ways to block such infections, there exists a need in the art for identifying additional HIV coreceptors. In particular, there exists a need in the art for information on the identity, characterization, and efficacy of additional HIV coreceptors. The identification and characterization of additional coreceptors would be particularly advantageous in developing specific therapies for preventing primary HIV infection and limiting the spread of such infections once they have occurred.
Therefore, it is important to search for additional G-protein-coupled receptors that might facilitate the entry of various primary HIV-1 isolates into suitable target cells during the course of the infection. Further, it is important to identify pharmacologically active compounds that inhibit the interaction between the initial HIV-CD4 complex and the coreceptor.
SUMMARY OF THE INVENTION
The present invention provides data showing that CMKRL1 supports the entry of select primary isolates of mainly the SI phenotype into CD4-positive murine host cells. Since, within the group of leukocyte chemoattractant receptors, CMEKRL1/BLTR can be viewed as a “cousin” of the previously identified chemokine-type coreceptors, this hereby introduces an essentially new class of coreceptors that is required for the cellular entry of HIV-1.
Accordingly, this invention aids in fulfilling existing needs in the art. More particularly, this invention provides a B-cell lymphoblast derived heptahelix receptor which naturally binds leukotriene B4 (26, 27) and also acts as a coreceptor for primary isolates of HIV-1. This novel receptor, BLTR, is strongly expressed in lymphoid cells and tissues, including leukocytes, lymph nodes, thymus, spleen, and bone marrow.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the means of the elements and combinations particularly pointed out in the written description and claims hereof, as well as the appended drawings.
To achieve these and other advantages, and in accordance with the purpose of the invention as embodied and broadly described, the invention comprises a cell line which expresses the human leukotriene B4 receptor on its surface as a result of transfection with DNA sequences encoding BLTR. When CD4 is coexpressed in these cells they are susceptible to infection with certain isolates of HIV-1. Thus, these cells may be useful in characterizing primary HIV infection and coreceptor selection studies.
A cell line capable of surface expression of the human leukotriene B4 receptor may be created by transfecting cells which lack this receptor with DNA fragments or a vector expressing BLTR. The resulting cell line may be stably or transiently transformed, resulting in the expression of human leukotriene B4 receptor.
The present invention also provides recombinant expression vectors comprising the DNA sequences encoding the human leukotriene B4 receptor, recombinant human leukotriene B4 receptor molecules produced using the recombinant expression vectors, and processes for using the expression vectors.
In another embodiment the present invention provides an antibody which specifically binds the human leukotriene B4 receptor and inhibits its ability to function as a viral coreceptor. In addition to blocking membrane fusion, this antibody is useful in a number of diagnostic applications.
In yet another embodiment, the present invention provides a method for screening a drug or other pharmacologically-active agent for its ability to prevent viral infection of cell lines expressing the human leukotriene B4 receptor on its surface.
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in, and constitute a part of, this specification. They are included to illustrate one/several embodiment(s) of the invention and, together with the description, serve to explain the principles of the invention.
REFERENCES:
Owman et al. Proc. natl. Acad. Sci. USA , Aug. 4th, 1998, vol. 95, pp. 9530-9534.*
Gifford et al. J. Immunol. Feb. 15, 1987, vol. 138, pp. 1184-1189.*
G. Alkhatib et al., “CC CKR5: A RANTES, MIP-1&agr;, MIP-1&bgr; Receptor as a Fusion Cofactor for Macrophage-Tropice HIV-1,”Science,vol. 272, pp. 1955-1958, Jun. 28, 1996.
H. Deng et al., “Expression cloning of new receptors used by simian and human immunodeficiency viruses,”Nature,vol. 388, pp. 296-300, Jul. 17, 1997.
T. Dragic et al., “HIV-1 entry into CD4+cells is mediated by the chemokine receptor CC-CKR-5,”Nature,vol. 381, pp. 667-673, Jun. 20, 1996.
H. Choe
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Housel James
Li Bao-Qun
Owman Invest, Ltd.
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