Entry of HIV-1 into target cells requires cell surface CD4 as well as additional host cell cofactors. A cofactor required for infection with virus adapted for growth in transformed T cell lines was recently identified and named fusin. Fusin, however, does not promote entry of macrophage-tropic viruses that are believed to be the key pathogenic strains in vivo. It has now been determined that the principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR5, a receptor for the .beta.-chemokines RANTES, MIP-1.alpha., and MIP-1.beta..

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention relates to the identification and application of an agent capable of promoting the translocation of macrophage-trophic HIV through the membrane of a target CD4.sup.+ cell, which agent exhibits certain of the following characteristics and activities: A. It is present in, on, or proximal to the cell membrane of the target CD4.sup.+ cell; B. It acts in tandem with CD4, in connection with the translocation; and C. It is capable of interacting with associated G-proteins to thereby transduce an intracellular signal.

A further characteristic attendant to the activity of the translocation promoting agent of the present invention is an observed increase in the concentration of intracellular calcium. The present agent may also be described as a mediator of the entry of envelope glycoproteins of macrophage-trophic strains of HIV-1 into target cells.

In a further aspect of the invention, the present translocation promoting agent appears to act in conjunction with CD4 in facilitating the penetration of the macrophage-trophic virus into the target cell to establish HIV infection. A particular family of receptors known as C--C (or .beta.) chemokine receptors (CKRs) has been identified as defining certain of the activities and characteristics set forth above, and a specific such receptor, CC-CKR5, is exemplified herein.

Other analogous receptors, such as those encoded by some viruses, particularly members of the Herpes virus family (CMV, HHV-6, HHV-8), serve to broaden the host range of HIV in individuals infected with both HIV and these viruses. This may therefore increase the range of tissues infected or provide a ligand for HIV envelope that may result in deleterious signal transduction in various tissues. Such information could lead to novel approaches to block the synergy between HIV and viral cofactors.

The present invention also relates to the use of a recombinant DNA molecule or cloned gene, or a truncated or degenerate variant thereof, which encodes a translocation promoting agent or the active portion thereof; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene. In another embodiment, the human and murine DNA sequences of the translocation promoting agent of the present invention, or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the translocation promoting agent. For example, the probes may be prepared with a variety of known vectors, such as the phage .lamda. vector. The present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences so prepared or constituted. Correspondingly, the preparation of antisense RNA and ribozymes are included herein.

The present invention also includes translocation promoter agents having the activities noted herein. In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present translocation promoter agent(s).

According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human translocation promoter agent.

The present invention also includes animal models. In one aspect of the invention a non-human animal model is used in the study of HIV infection and HIV disease in order to develop modes of diagnosis, prevention, treatment and/or cures. In some embodiments, a transgenic animal is produced containing the CD4 enhancer/promoter/silencer and the CD4 coding sequence as described by Killeen et al., The EMBO J. 12, 1547 1553 (1993) along with a translocation promoting agent. In one such embodiment, a transgenic animal has a translocation promoting agent regulated by the CD4 enhancer/promoter/silencer. More particularly, the regulation of the translocation promoting agent may include the CD4 enhancer/promoter/silencer plus a macrophage-specific enhancer. Yet further, the macrophage-specific enhancer can be all or a functional portion of the first intron of the human CD4 gene.

In a further aspect of the invention, a transgenic non-human animal is produced with the gene for the animal homolog of the translocation promoting agent replaced by its human counterpart. In this embodiment, the translocation promoting agent may be selected from CC-CKR-5, fusin, CC-CKR-2B and CC-CKR-3. In a preferred embodiment the translocation promoting agent is CC-CKR-5.

In a variant embodiment, a transgenic animal is produced with the genes for two such animal homologs of two translocation promoting agents replaced by their human counterparts. In one such specific embodiment, one of the translocation promoting agents is CC-CKR-5 and the other is fusin. In other embodiments more than two such animal homologs are replaced by their human counterparts. In a more preferred embodiment the transgenic animal also contains human CD4.

In a more generalized application of this aspect of the invention the replacement of the animal homolog gene is performed in the animal germ line. Preferably as a knockin as generally described in Hanks et al., (1995). A more focussed construct may be prepared by the replacement of the gene in T-cells and macrophages. In variant preparations, human CD4 may also be present in the animal T-cells and/or macrophages. A particular replacement gene that may be used comprises a nucleic acid that encodes a human translocation promoting agent expressed under the control of a gene naturally expressed in macrophages and/or T-Cells e.g. lysozyme. In a specific embodiment, the human translocation promoting agent placed between the 5' end and the 3' prime end of the lysozyme gene is CC-CKR-5.

The non-human animal prepared in accordance herewith may be any animal that is amenable to transgenic technology. In a preferred embodiment the non-human animal is selected from the group consisting of a mouse, a rabbit, a sheep, a goat, and a pig. In another preferred embodiment, the non-human animal is a primate.

The concept of the translocation promoter agent contemplates that specific factors exist for correspondingly specific ligands, such as CD4 and the like, as described earlier. Accordingly, the exact structure of each translocation promoter agent will understandably vary so as to achieve this ligand and activity specificity. It is this specificity and the direct involvement of the translocation promoting agent in the chain of events leading to HIV infection, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.

In a related aspect, the mutability of retroviruses in general, and specifically HIV-1, is consistent with the identity of the translocation promoting agent changing as the viral infection progresses. Such changes are due to changes in HIV-1 that lead to the emergence and activity of different translocation promoting agents in different populations (e.g. geographically separated groups of people.) Furthermore, changes in the virus and its choice of translocation promoting agent can be indicative of different stages of the infection in an infected individual. Information regarding the progression of the infection is extremely valuable to that individual and their health care provider, e.g., such as in the treatment of that individual.

The present invention includes a method of monitoring the progression of the HIV infection by determining the identity of the translocation promoting agent as the infection progresses. The method includes the use of the assorted cell lines disclosed herein (and others prepared in the same manner) which express the different .beta.-chemokine receptors along with CD4. .beta.-Chemokine receptors are prime candidates for being translocation promoting agent agents.

The first step entails quantifying the amounts of the particular translocation promoting agents that are utilized by the virus during the specific stages of the infection, in a relevant population of HIV-1 infected people. A proportional relationship between these quantified amounts may then be correlated with the specific stages of HIV-1 infection. The second step is to quantify the amounts of the particular translocation promoting agents that are utilized by the virus in a biological sample obtained from an infected individual. The third step is to correlate the proportions determined for the individual with those determined for the group and thereby determine the stage of the HIV-1 infection for the individual.

The amount of each .beta.-chemokine used as a translocation promoting agent by HIV-1 in a given biological sample can be determined with the assorted cell lines disclosed herein. These cell lines express different .beta.-chemokine receptors along with CD4 and may be detectably labeled so as to allow their relative sensitivity to the virus be determined. Only the HIV-1 strain that uses the particular .beta.-chemokine as the translocation promoting agent, can successfully infect a test cell with that particular .beta.-chemokine. A detectable label inside the cell that is activated by successful infection will allow quantitation of the amount of the particular .beta.-chemokine. Detectable labels may be either a genetic marker or linked to a genetic marker that is only expressed upon HIV-1 infection. Appropriate detectable labels include luciferase green fluorescent protein, as well as the others described herein.

The invention also includes an assay system for the screening of potential drugs effective to modulate translocation of macrophage-trophic HIV into target mammalian cells by interrupting or potentiating the translocation promoting agent. In one instance. the test, drug could be administered to a cell (such as a transformal cell) with the ligand that activates the translocation promoting agent, or an extract containing the activated translocation promoting agent, to determine its effect upon the binding activity of the translocation promoting agent to any chemical sample (including DNA), or to the test drug, by comparison with a control.

The assay system could more importantly, be adapted to identify drugs or other entities that are capable of binding to the translocation promoting agent and/or to corresponding factors or proteins, either in the cytoplasm or in the nucleus, and inhibit or potentiate the translocation promoting activity. Such an assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity. For example, such drugs might be used to inhibit penetration of HIV into the target cell, or to treat other pathologies.

One embodiment includes an in vitro method for identifying a viral envelope glycoprotein that binds to a particular translocation promoting agent comprising contacting a labeled viral envelope glycoprotein with a translocation promoting agent attached to a solid support, washing the solid support, and then detecting the labeled viral envelope glycoproteins associated with the solid support. A viral envelope glycoprotein that binds the translocation promoting agent is identified when the labeled viral envelope glycoprotein is detected associated with the solid support. Any translocation promoting agent of the present invention may be used in this assay. In a related embodiment, the cytoplasmic domain of a translocation promoting agent is attached to the solid support rather than the full length protein. In one embodiment, the viral envelope glycoprotein is an HIV envelope glycoprotein. In a preferred embodiment the translocation promoting agent is CC-CKR5.

In yet a further embodiment, the invention contemplates the identification and use of antagonists of the activity of a translocation promoting agent. In particular, an agent or molecule that inhibits the HIV-translocating activity of the translocation promoting agent. In a specific embodiment, the antagonist can be a peptide having the sequence of a portion of the active domain of the translocation promoting agent.

The utility of the present invention extends to the use of the present invention in assays to screen for drugs and small molecules that would function as inhibitors of translocation promoting agent activity. A particular such assay may be constituted in a transgenic non-human mammal that comprises a DNA construct containing a human CD4 gene and a DNA construct containing human CC-CKR-5 gene, wherein both CD4 protein and CC-CKR-5 protein are expressed by said non-human mammal.

The reporter gene can encode any detectable gene product but is more preferably either luciferase, or green fluorescent protein. The HIV LTR is preferably HIV-2 LTR. The human chemokine receptor is preferably CC-CKR1, CC-CKR2A, CC-CKR2B, CC-CKR-3, CC-CKR-4, CXC-CR4, or CC-CKR5.

The present invention also includes a method of using such a cell for identifying a human chemokine receptor that facilitates the infection of a particular HIV strain into the transformed mammalian cell. One embodiment of this type comprises infecting the cell with a primary HIV strain, and then detecting the reporter gene. The human chemokine receptor is identified when the reporter gene is detected above the background value determined in the absence of HIV infection. In one embodiment the reporter gene encodes green fluorescent protein. In one particular embodiment of this type, the detecting is performed by FACS analysis. The human chemokine receptor is preferably CC-CKR1, CC-CKR2A, CC-CKR2B, CC-CKR-3, CC-CKR-4, CC-CKR5 or CXC-CR4. The particular HIV strain is a preferably a primary HIV-1 strain.

The present invention also includes a method of using such cells for identifying a drug that interferes with the translocation of HIV. One such embodiment comprises administering a potential drug to the cell; infecting the cell with a primary HIV strain; and then detecting the reporter gene. In this case, the reporter gene is detected in the absence of the drug, indicating that the HIV strain is translocated into the cell. A potential drug is identified as a drug when the reporter gene is either not detected, or is detected in a lesser amount in the presence of the drug. In one embodiment, the reporter gene encodes green fluorescent protein. In one particular embodiment of this type, the detecting is performed by FACS analysis. The human chemokine receptors are preferably CC-CKR1, CC-CKR2A, CC-CKR2B, CC-CKR-3, CC-CKR-4. CC-CKR5, and CXC-CR4.

The present invention also includes a method of using such cells for identifying an antibody that interferes with the translocation of HIV into the cell. One embodiment comprises administering an antibody to the cell; infecting the cell with a primary HIV strain; and then detecting the reporter gene. The reporter gene is detected in the absence of the antibody, indicating that the HIV strain is translocated into the cell. The potential antibody is identified as an antibody that interferes with the translocation of HIV, when the reporter gene is either not detected, or is detected in a lesser amount in the presence of the antibody. The antibody can be an antibody to HIV, an antibody to CD4, an antibody to the translocation promoting agent, an antibody to a purified HIV envelope protein, or an antibody raised specifically against a complex of two or more the above. In one such embodiment the reporter gene encodes green fluorescent protein. In a particular embodiment of this type, the detecting is performed by FACS analysis. The human chemokine receptor is preferably CC-CKR1, CC-CKR2A, CC-CKR2B, CC-CKR-3, CC-CKR-4, CC-CKR-5, or CXC-CR4.

The present invention likewise extends to the development of antibodies to the translocation promoting agent(s), including naturally raised and recombinantly prepared antibodies. Antibodies can be used for various purposes including to evaluate the relative resistance or permissiveness of CD4.sup.+ cells to HIV infection to block HIV translocation, and to identify such proteins that function as macrophage-tropic HIV translocation receptors. For example, the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the translocation promoting agent(s). Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating translocation promoting agent activity.

In a specific embodiment, an antibody is raised against CC-CKR-5 and inhibits HIV binding. Such an antibody may be used for permissive immunotherapy and protects against host cell invasion by a number of viral isolates. Such an antibody was raised against fusin and has been shown to inhibit HIV-1 infection [see Dimitrov, D. S. Nature Medicine 2 640 641 (1996)].

In another embodiment, the antibody is specific for a conformational epitope on the translocation promoting agent that becomes accessible upon binding CD4 and/or an envelope protein of HIV-1. In still another embodiment, the antibody reacts with a shared epitope of the translocation promoting agent and HIV or CD4 or both. In preferred embodiments of this aspect of the invention the translocation promoting agent is CC-CKR-5. In yet another embodiment, a chimeric antibody is prepared that specifically binds to the gp120-gp41 molecules of HIV-1 and CC-CKR-5.

The present invention also includes chimeric proteins containing the translocation promoting agent and an additional protein or peptide. In one such chimeric protein, the translocation promoting agent includes an epitope tag inserted into its amino-terminal extracellular domain. The chimeric protein can be encoded by a translocation promoting agent expression vector. In a preferred embodiment of this type, the chimeric protein is expressed in a mammalian cell as a transmembrane receptor which can be detected with an antibody that recognizes the epitope tag.

In one such embodiment the translocation promoting agent contains an influenza (HA) epitope tag in its N-terminal extracellular domain. In an embodiment of this type the HA epitope tag is a peptide having an amino acid sequence of SEQ ID NO:10 or SEQ ID NO:12. In a related embodiment, the N-terminal DNA sequence of the HA epitope tag comprises SEQ ID NO:9 or SEQ ID NO:11. In one particular embodiment, the translocation promoting agent is CC-CKR5. In a preferred embodiment of this type, the insertion of the HA epitope tag also allows for very sensitive detection of the translocation promoting agent at the cell surface with the use of an anti-HA monoclonal antibody. In a specific embodiment of this type, the amino-terminal amino acid sequence of the chimeric protein comprises SEQ ID NO: 6. In a related embodiment, the N-terminal DNA sequence of the chimeric protein comprises SEQ ID NO:5.

Thus, the translocation promoting agent(s), analogs, thereof, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays. Such a radioimmunoassay, for example, can use an antibody to the translocation promoting agent that has been labeled by either radioactive addition, or radioiodination.

In an immunoassay, a control quantity of a solubilized translocation promoting agent or antibodies thereto, or the like may be prepared and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.

The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the translocation promoting agent, or to identify drugs or other agents that may mimic or block their activity. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the translocation promoting agent, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).

In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the translocation promoting agent(s), its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the binding activity of the translocation promoting agent or its subunits, and comprises administering an agent capable of modulating the production and/or activity of the translocation promoting agent or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host. For example, drugs or other binding partners to the translocation promoting agent or proteins may be administered to inhibit or potentiate translocation promoting agent activity.

More specifically, such therapeutic methods could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of the HIV translocating promoter activity of the translocation promoting agent or its subunits, such as antibodies, or other drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention. For example, drugs, antibodies or other binding partners to proteins such as CC-CKR5, may be administered to inhibit the activity of the translocation promoting agent. Particular agents contemplated herein include proteins such as CC-CKR5 modified by the binding of the extracytoplasmic domain to a nonfunctional transmembrane domain, or to a lipid, to serve as a competitive inhibitor. Such modified translocation promoting agents may be inserted into macrophages by gene therapy techniques and placed into subject animals, including humans to prevent HIV infection or combat HIV infection at its early stage of infection.

Further, the invention extends to the soluble form(s) of the translocation promoting agent, that may function in similar fashion to antagonists to the agent, to inhibit HIV ingress to the target cell. All of the aforementioned agents, including small molecules and other cognates, may be formulated for use in the treatment of fluids such as the blood, and in the preparation, for example, of a spermicidal composition or like formulation, to treat the development of macrophage-trophic HIV that is known to be present in infected blood samples, and that is particularly known to be sexually transmitted.

In particular, proteins corresponding to translocation promoter agents, such as, for example, the chemokine receptors set forth herein, their antibodies, agonists. antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein inhibitory therapy is appropriate. The application of the therapeutic compositions and methods of the invention will, it is believed, dramatically reduce the incidence of primary HIV infection.

Yet another aspect of the invention includes the identification of a ligand for fusin. Supernatents and extracts of various cell lines and populations (e.g. CD8-lineage cells) are used to assay for the inhibition of infection by a fusin-tropic virus.

In a related aspect the identified ligand for fusin is isolated by standard column chromatography and gel electrophoresis, with the use of the assay described above. In one embodiment of this aspect of the invention, a fusin affinity column is used.

Accordingly, it is a principal object of the present invention to provide antagonists including antibodies, to the translocation promoter agent and its subunits, and methods for their preparation, including recombinant means.

It is a further object of the present invention to provide a method for detecting the presence of the translocation promoter agent and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.

It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or combating the adverse effects of the translocation promoter agent and/or its subunits in mammals.

It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the translocation promoter agent or subunits thereof, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.

It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the translocation promoter agent or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.

It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the translocation promoter agent, its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of the translocation promoter agent.

 

Claim 1 of 1 Claim

1. A method of identifying an agent that inhibits entry of a macrophage-tropic HIV virus into a target cell, wherein said entry of the macrophage-tropic HIV into said target cell is a fusion process mediated by CC-CKR5 and CD4 expressed on the surface of said target cell, the method comprising the steps of: (a) contacting said target cell with a virus pseudotyped with a macrophage-tropic HIV envelope in the presence or absence of said agent; (b) measuring the fusion between the virus pseudotyped with a macrophage-tropic HIV envelope and said target cell, wherein the measurement of said fusion is performed by a fluorescence intensity detection or by a fluorescence activated cell sorting (FACS) analysis, and (c) determining whether said fusion of the virus pseudotyped with a macrophage-tropic HIV envelope is inhibited in the presence of the agent but not in the absence of the agent.

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