JP2008069148A - Method for preventing hiv-1 infection of cd4+ cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide methods for inhibiting the fusion of HIV-1 to CD4<SP>+</SP>cells. <P>SOLUTION: Methods, which comprise contacting CD4<SP>+</SP>cells with a non-chemokine agent capable of binding to a chemokine receptor of such amount and under such conditions as fusion of HIV-1 to the CD4<SP>+</SP>cells is inhibited, are provided. Methods for inhibiting HIV-1 infection of CD4<SP>+</SP>cells, which comprise contacting CD4<SP>+</SP>cells with a non-chemokine agent capable of binding to a chemokine receptor of such amount and under such conditions as fusion of HIV-1 to the CD4<SP>+</SP>cells is inhibited and thereby inhibit the HIV-1 infection, are provided. Non-chemokine agents capable of binding to the chemokine receptor and inhibiting fusion of HIV-1 to CD4<SP>+</SP>cells, are provided. Pharmaceutical compositions comprising an effective amount to prevent fusion of HIV-1 to CD4<SP>+</SP>cells and of the non-chemokine agent capable of binding to the chemokine receptor and inhibiting fusion of HIV-1 to CD4<SP>+</SP>cells and a pharmaceutically acceptable carrier, are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

Detailed Description of the Invention

  This application is a continuation-in-part of US application number 08 / 673,682, the parent application filed on June 25, 1996, which is also a grandfather application filed on June 14, 1996. This is a continuation-in-part of US Application No. 08 / 663,616, which is also a continuation-in-part of US Application No. 08 / 627,684, filed April 2, 1996. The contents of these earlier applications are hereby incorporated by reference herein as part of this specification.

  Throughout this application, various references are cited in parentheses. The disclosures of these publications are hereby incorporated by reference in their entirety in order to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these citations are listed at the end of each experimental series.

BACKGROUND OF THE INVENTION
Chemokines are a family of 8-10 KDa related soluble proteins secreted by lymphocytes and other cells that bind to receptors on the surface of target cells, eg, leukocytes in the inflammatory process Is activated and fluidized. Recently, by Cocchi et al., The chemokines RANTES, MIP-1α and MIP-1β are factors produced by CD8 ⁺ T lymphocytes, which are HIV-1 macrophage tropisms. It has been demonstrated that it inhibits infection by isolated substances, but does not inhibit infection by strains applied to laboratories of HIV-1 virus (1). These chemokines are members of the CC group of chemokines. The reason for this is because they carry adjacent cysteine residues, which is different from the CXC group in which cysteine residues are separated by one amino acid (2). Kotchi et al. Found that HIV-1 RNA expression was suppressed by treatment with chemokines, but did not identify the active site of these molecules.

Fusion mediated by envelope glycoproteins derived from primary macrophage tropic isolates of HIV-1 _JR-FL using resonance energy transfer (RET) analysis of membrane fusion mediated by HIV-1 envelope glycoproteins Was specifically inhibited by a chemokine compared to a fusion mediated by an envelope glycoprotein from the laboratory-adapted T lymphocyte affinity strain HIV-1 _LAI . As explained below, this proved to be exactly the case. This demonstrated that several chemokine receptors are fusion auxiliary molecules required for HIV-1 infection. Previous studies have pointed out that in order for the virus to enter, unidentified cell surface molecules are required in addition to the HIV-1 receptor CD4. This accessory molecule is required in the invading membrane fusion process, whereas CD4 is required for HIV-1 attachment. These auxiliary molecules are generally expressed only on human cells. Therefore, HIV-1 does not infect non-human CD4 ⁺ cells (3-6). Furthermore, by fusing them with human CD4 ⁺ cells (using polyethylene glycol), heterokaryon is a target that complements non-human CD4 ⁺ cells and is responsive to HIV-1 envelope-mediated membrane fusion. (Heteronuclear coexistents) can be generated (7, 8). These studies have been performed using laboratory adapted T lymphocyte affinity strains of the virus.

In some cases, fusion aid molecules are found on a subset of human CD4 ⁺ cells and appear to be necessary for infection by HIV-1 isolates with special tropism. For example, primary macrophage-tropic strains of HIV-1 such as HIV-1 _JR-FL have different requirements for auxiliary molecules compared to laboratory-adapted T lymphocyte affinity strains such as HIV-1 _LAI. May have sex. This phenomenon may explain the tropism difference between HIV-1 strains.

  The present invention encompasses a series of new treatments for HIV-1 infection. Chemokines work in the fusion process of HIV-1 entry and specifically target membrane fusion mediated by envelope glycoproteins of macrophage-tropic primary virus isolates, but not by laboratory-adapted T lymphocyte affinity strains of the virus For the first time. The primary macrophage tropic isolate of the virus is particularly important because it is a strain that is normally involved in viral transmission and can be particularly important in the pathogenesis of HIV-1 infection.

  These results were obtained using a resonance energy transfer (RET) analysis of membrane fusion mediated by the envelope of HIV-1. In addition, this assay is used to inhibit non-chemokines, including chemokine fragments and modified chemokines, that inhibit envelope glycoprotein-mediated membrane fusion of HIV-1, thereby neutralizing the virus and inducing an inflammatory response. Identified.

[Summary of the Invention]
The present invention provides a method of inhibiting fusion of HIV-1 with CD4 ⁺ . The method comprises contacting a CD4 ⁺ cell with a non-chemokine agonist capable of binding to a chemokine receptor under conditions and in an amount such that HIV-1 fusion to CD4 ⁺ cells can be inhibited.

The present invention also provides a method for inhibiting HIV-1 infection of CD4 ⁺ cells. The method comprises contacting a CD4 ⁺ cell with a non-chemokine agonist capable of binding to a chemokine receptor in an amount and under conditions such that HIV-1 fusion to CD4 ⁺ cells can be inhibited, thereby causing HIV-1 infection. It consists of obstructing.

The present invention further provides non-chemokine agents capable of binding to chemokine receptors and inhibiting HIV-1 fusion to CD4 ⁺ cells.

  The present invention provides an agent capable of binding to fusin and inhibiting infection. In one embodiment, the agent is an oligopeptide. In another embodiment, the agent is a polypeptide. In yet another embodiment, the agent is an antibody or part of an antibody. In another embodiment, the agent is a non-peptidyl agent.

In addition, the present invention contains an amount of an agent capable of binding to the non-chemokine agonist or fusin in an amount effective to inhibit fusion of HIV-1 to CD4 ⁺ cells and a pharmaceutically acceptable carrier. A pharmaceutical composition is provided.

The present invention provides substances that can bind to chemokine receptors and inhibit the fusion of HIV-1 to CD4 ⁺ cells. The composition comprises a non-chemokine agent linked to a ligand capable of binding to a CD4 ⁺ cell surface receptor other than a chemokine receptor, and binding of the ligand to other receptors even if the non-chemokine agent binds to a chemokine receptor Is not inhibited.

The present invention also provides a pharmaceutical composition comprising an amount of the above composition effective to inhibit fusion of HIV-1 to CD4 ⁺ cells and a pharmaceutically acceptable carrier.

The present invention is a substance that can bind to a chemokine receptor and inhibits the fusion of HIV-1 to CD4 ⁺ cells, and is linked to a compound that can increase the in vivo half-life of a non-chemokine agonist. A non-chemokine agent-containing substance is provided.

The invention also provides a non-chemokine agent linked to a compound capable of increasing the in vivo half-life of an effective amount of a non-chemokine agent to inhibit fusion of HIV-1 to CD4 ⁺ cells, and a pharmaceutical A pharmaceutical composition is provided that contains a carrier that can be used as a target.

  The present invention provides a method for reducing the likelihood of HIV-1 infection in a subject comprising administering to the subject the pharmaceutical composition described above. The present invention also provides a method of treating HIV-1 infection in a subject comprising administering the above pharmaceutical composition.

The invention also provides a method of determining whether a non-chemokine agent can inhibit the fusion of HIV-1 to CD4 ⁺ cells. The method comprises (a) CD4 ⁺ cells (i) labeled with a first dye and cells (ii) labeled with a second dye that expresses HIV-1 envelope glycoprotein on the cell surface, Contacting in the presence of an excess of the agent under conditions that allow fusion of cells expressing the HIV-1 envelope glycoprotein on the surface with CD4 ⁺ cells in the absence of the agent (here Wherein the first and second dyes are selected to allow transfer of resonance energy between the dyes); and (b) when the product of step (a) is fused (C) determining whether there is a decrease in resonance energy transfer as compared to resonance energy transfer in the absence of the agent; and , Showing that the agent can inhibit the fusion of HIV-1 to CD4 ⁺ cells The process is comprised.

Detailed Description of the Invention
The present invention provides a method of inhibiting fusion of HIV-1 and CD4 ⁺ cells, relative to CD4 ⁺ cells, the non-chemokine agent capable of binding to a chemokine receptor, for HIV-1 to CD4 ⁺ cells There is provided a method comprising contacting under an amount and under conditions in which fusion is inhibited.

The present invention also provides a method of inhibiting HIV-1 infection of CD4 ⁺ cells, relative to CD4 ⁺ cells, the non-chemokine agent capable of binding to a chemokine receptor, is fused against CD4 ⁺ cells of HIV-1 Provided is a method comprising inhibiting HIV-1 infection by contacting under an inhibited amount and under conditions.

  In the present invention, chemokine means RANTES, MIP-1α, MIP-1β, or other chemokines that block HIV-1 infection. A chemokine receptor means a receptor capable of binding RANTES, MIP-1α, MIP-1β, or other chemokines that block HIV-1 infection.

  Throughout this application, the receptor “fusin” is also named CXCR4, and the chemokine receptor C-C CKR5 is also named CCR5.

  HIV-1 as used in this application means, unless specified, a clinical isolate of HIV-1 virus or primary or field isolate that retains its clinical characteristics. Clinical isolates of HIV-1 may be passaged in primary peripheral blood mononuclear cells. The clinical isolate of HIV-1 is tropic for macrophages.

The non-chemokine agonists of the present invention can bind to chemokine receptors and inhibit the fusion of HIV-1 to CD4 ⁺ cells. Such non-chemokine agonists include, but are not limited to, chemokine fragments, derivatives and analogs. However, natural chemokines are not included. The non-chemokine agonists include various forms of fusion molecules comprising chemokine fragments, derivatives and analogs, or chemokine fragments, derivatives and analogs linked to other molecules.

  In one embodiment of the invention, the non-chemokine agent is an oligopeptide. In another embodiment, the non-chemokine agent is a polypeptide. In another aspect, the non-chemokine agent is an antibody or part of an antibody. Antibodies to chemokine receptors can be easily generated by routine experimentation. It is also within the skill in the art to synthesize antibody fragments capable of binding to chemokine receptors. In yet another aspect, the non-chemokine agent is a non-peptidyl agent.

  Non-chemokine agents with a pure peptidyl composition can be chemically synthesized by the solid phase method (Merrifield, 1966) or can be produced using recombinant techniques in either prokaryotic or eukaryotic systems. Such synthesis and recombination methods are known in the prior art.

  Non-chemokine agents containing biotin or other peptidyl groups can be prepared by chemically modifying synthetic or recombinant chemokine agents or non-chemokine agents. One method of chemical modification includes periodate oxidation of 2-aminoalcohols present in chemokines or non-chemokine agents that have serine or threonine as their N-terminal amino acid (Geophegan and Stroh, 1992). . The resulting aldehyde group is used to link a peptidyl or non-peptidyl group to an oxidized or non-chemokine agent by reductive amination, hydrazination or other chemical techniques known to those skilled in the art. be able to.

  As used herein, the N-terminus of a protein means the end of the protein after being processed. In the case of a secretory protein containing a cleavable signal sequence, the N-terminus of the secreted protein should be the end after cleavage of the signal peptide.

The present invention provides methods for identifying these non-chemokine agents. One method of identifying such agents, including non-peptidyl agents that bind to chemokine receptors and inhibit fusion of HIV-1 to CD4 ⁺ cells, includes the use of the following analytical methods: 1) Soluble CD4 is incubated with biotinylated gp120 obtained from HIV-1 _JR-FL or HIV-1 _LAI ; 2) this complex is expressed as CCR5 or CXCR4 that does not express CD4 (respectively HIV-1 _JR-FL Or against HIV-1 _LAI gp120s) in the presence or absence of candidate inhibitors; 3) washed, then incubated with streptavidin phycoerythrin, 4) washed, and then with gp120 The amount bound is measured using flow cytometry or a fluorometer and the degree of binding inhibition by the inhibitor is calculated.

  Instead of the biotinylated gp120-streptavidin phycoerythrin method described above, several alternative methods for detecting bound gp120 can also be used. For example, instead of biotinylated gp120, peroxidase-conjugated gp120 can be used, and binding can be detected by spectrometer readout using a suitable chromogenic substrate for peroxidase.

  The present invention further provides non-chemokine agonists identified by the several methods described above.

The present invention provides non-chemokine agonists that can bind to chemokine receptors and inhibit the fusion of HIV-1 to CD4 ⁺ cells. In one embodiment, the non-chemokine agent is a polypeptide. In yet another embodiment, the polypeptide is a fragment of the chemokine RANTES (Gong et al., 1996). In yet another embodiment, the polypeptide may consist of a RANTES sequence that lacks the N-terminal amino acid of the sequence. The deletion may be the first 8 N-terminal amino acids of the RANTES sequence (SEQ ID NO: 5).

  In another embodiment, the polypeptide may consist of a MIP-1β sequence lacking the N-terminal amino acid of the sequence. The deletion may be the first 7, 8, 9, 10 N-terminal amino acids of the MIP-1β sequence.

  In another embodiment of the non-chemokine agonist, the polypeptide consists of a MIP-1β sequence with an N-terminal sequence modified by the addition of amino acids or oligopeptides. In another embodiment, it consists of a MIP-1β sequence having an N-terminal sequence modified by removing the N-terminal alanine and replacing it with serine or threonine, and another amino acid, or oligopeptide or non-peptidyl component . In yet another aspect, the other amino acid is methionine.

  As described in the experimental details section below, a cofactor of HIV-1 fusion and invasion has been identified and named “Fujin” (Feng et al, 1996). The present invention provides an agent capable of binding to fudin and inhibiting infection. In one embodiment, the agent is an oligopeptide. In another aspect, the agent is a polypeptide.

  In a further aspect, the polypeptide consists of SDF-1 with a deletion of the N-terminal amino acid of the sequence. The deletion may be the first 6, 7, 8, 9 of the N-terminal amino acid of the SDF-1 sequence.

  The present invention also provides an agent comprising the above SDF-1 sequence which is a non-chemokine agonist and which has an N-terminal sequence modified so as to produce an anti-antagonistic effect on SDF-1. One modification is to substitute the N-terminal glycine of SDF-1 with serine and derivatize with biotin. Another modification involves replacing the N-terminal glycine of SDF-1 with serine and derivatizing with methionine. Yet another modification is to add methionine before the terminal glycine to the N-terminus of SDF-1.

  In yet another embodiment, the agent is an antibody or part of an antibody. In another embodiment, the agent is a non-peptidyl agent.

  The agent capable of binding to fusin can be identified by screening various compounds for its ability to bind to fusin in vitro.

  Fowlkes et al. 1994 et al. Describe suitable methods in International Application: PCT / US94 / 03143, International Publication WO94 / 23025. The contents of which are hereby incorporated by reference herein as part of the present specification. Briefly, yeast cells having a pheromone system are processed so as to express a heterologous substitute for a yeast pheromone protein. The surrogate takes up fudin and under certain conditions performs the function naturally performed by the corresponding yeast pheromone protein in the yeast cell pheromone system. Such yeast cells are also engineered to express a library of peptides. As a result, yeast cells containing peptides that bind fusin regulate the interaction between the surrogate yeast pheromone protein and the yeast pheromone system, and this regulation is a phenomenon that can be selected or screened. A similar approach can be used to identify agents that can bind to both fudin and the chemokine receptor C-C CKR5.

The invention also contains an amount of such a non-chemokine agent or agent capable of binding to fusin and a pharmaceutically acceptable carrier in an amount effective to inhibit fusion of HIV-1 to CD4 ⁺ cells. A pharmaceutical composition is provided.

  Pharmaceutically acceptable carriers are well known to those skilled in the art. Such pharmaceutically acceptable carriers include aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (eg olive oil), and injectable organic esters (eg ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or immobilized oil. Intravenous vehicles include liquids, nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, inert gases and the like may be present.

The present invention is a substance capable of binding to a chemokine receptor and inhibiting the fusion of HIV-1 to CD4 ⁺ cells, and is linked to a ligand capable of binding to a CD4 ⁺ cell surface receptor other than the chemokine receptor. A non-chemokine agent, wherein the binding of the ligand to the other receptor is not inhibited when the non-chemokine agent binds to the chemokine receptor. In one embodiment, the cell surface receptor is CD4. In another embodiment, the ligand is an antibody or part of an antibody.

The present invention also provides a pharmaceutical composition comprising an amount of the above-described substance effective to inhibit fusion of HIV-1 to CD4 ⁺ cells and a pharmaceutically acceptable carrier.

The present invention is a substance that binds to a chemokine receptor and can inhibit the fusion of HIV-1 to CD4 ⁺ cells, and can increase the in vivo half-life of non-chemokine agonists. Substances comprising a non-chemokine agent linked to a compound are provided. In one embodiment, the compound is polyethylene glycol.

The present invention also provides a non-chemokine agent linked to a compound capable of increasing the in vivo half-life of an amount effective to inhibit fusion of HIV-1 to CD4 ⁺ cells. A pharmaceutical composition is provided that includes a substance comprising and a pharmaceutically acceptable carrier.

  The present invention also provides a method for reducing the likelihood of HIV-1 infection in a subject comprising administering to the subject a pharmaceutical composition as described above. The present invention also provides a method of treating HIV-1 infection, comprising administering the pharmaceutical composition described above.

The invention also provides a method for determining whether a non-chemokine agonist can inhibit the fusion of HIV-1 to CD4 ⁺ cells, comprising: (a) CD4 ⁺ cells labeled with a first dye ( i) and cell (ii) labeled with a second dye that expresses HIV-1 envelope glycoprotein on the cell surface, and HIV-1 envelope glycoprotein is expressed on the surface in the presence of excess of the aforementioned agent. Contacting the cells to be fused with CD4 ⁺ cells under conditions that allow the fusion of the cells in the absence of the agent (wherein the first and second dyes are resonance energies between these dyes). (B) exposing the product of step (a) to conditions that cause resonance energy transfer when fusion occurs; and (c) Is there a decrease in resonance energy transfer compared to resonance energy transfer in the absence? Determining that the reduction in energy transfer indicates that the agent is capable of inhibiting fusion of HIV-1 to CD4 ⁺ cells.

HIV-1 only fuses with appropriate CD4 ⁺ cells. For example, the laboratory-adapted T lymphocyte affinity HIV-1 strain fuses with most CD4 ⁺ human cells. Clinical HIV-1 isolates do not fuse with most transformed CD4 ⁺ human cell lines, but do fuse with human primary CD4 ⁺ cells such as CD4 ⁺ T lymphocytes and macrophages. By conducting routine experiments, it can be easily determined whether the CD4 ⁺ cells are suitable for the fusion analysis described above.

  As described in the present invention, HIV-1 membrane fusion is monitored by resonance energy transfer (RET) analysis. The analysis is described in International Publication WO 95/16789 as well as in International Application PCT / US94 / 14561 filed on December 16, 1994. This analysis is described in further detail in US co-pending application number 08 / 476,515, filed June 7, 1995. The contents of these applications are hereby incorporated by reference.

  In one embodiment of the above method, the non-chemokine agent is an oligopeptide. In another embodiment, the chemokine agent is a polypeptide. In yet another embodiment, the agent is an antibody or part of an antibody. In another embodiment, the chemokine agent is a non-peptidyl agent.

In another embodiment, the CD4 ⁺ cell is a PM1 cell. In another embodiment, the cell that expresses HIV-1 envelope glycoprotein is a HeLa cell that expresses HIV-1 _JR-FL gp120 / gp41.

  The invention will be better understood by reference to the experimental details that follow. However, one skilled in the art will readily appreciate that the specific experiments detailed are merely illustrative of the invention, which is fully described in the claims below.

Experimental Details 1st Experimental Series 1) Chemokines are fusions mediated by envelope glycoproteins derived from primary tropic isolates of HIV-1 rather than HIV-1 laboratory-adapted T lymphocyte affinity strains Inhibits.
The chemokines RANTES, MIP-1α and MIP-1β were obtained from the R & D system (Minneapolis, Minn.). These have the ability to inhibit fusion between Hela-env _JR-Fl cells (expressing gp120 / gp41 obtained from macrophage isolate HIV-1 _JR-Fl ) and PM1 cells, or Hela-env _LAI The ability to inhibit fusion between cells (expressing gp120 / gp41 obtained from laboratory application strain HIV-1 _LAI ) and various CD4 ⁺ T lymphocyte cell lines was tested by RET analysis. As shown in FIG. 1, all three chemokines inhibited fusion mediated by macrophage virus envelope glycoproteins, but did not inhibit fusion mediated by laboratory applied envelope glycoproteins.

  Next, the ability of the chemokine to inhibit the interaction between CD4 and HIV-1 gp120 that occurs upon virus attachment was tested. The chemokines were found not to inhibit this interaction (Figure 2). This suggests that the inhibition of membrane fusion mediated by HIV-1 envelope glycoprotein occurs when membrane fusion itself occurs rather than the initial CD4-gp120 interaction that occurs prior to fusion. Proved.

2) Non-chemokine peptides and derivatives thereof that inhibit HIV-1 fusion Non-chemokines include chemokine fragments and chemokine derivatives that have been tested by RET analysis and which are active in inhibiting HIV-1 membrane fusion. Decided if there is. In particular, attention has been focused on fragments or derivatives that inhibit HIV-1 fusion but do not activate the leukocyte response. These non-chemokines include the following:
a) N-terminal derivatives of the class of chemokines. Adding residues to the N-terminus of chemokines inhibits the function of these proteins without seriously reducing the ability of chemokines to bind to chemokine receptors. For example, Met-RANTES (RANTES with N-terminal methionine) is known to be a potent anti-antagonist of natural RANTES and in some systems cannot induce chemotaxis or calcium metabolism. . The mechanism of antagonism appears to be competition to bind to the receptor (9). Similar results were seen using another derivative of the N-terminus of RANTES (9) and by modifying the N-terminus of other chemokines, such as IL8 (a member of the CXC chemokine) (10). The present invention includes Met-RANTES and other chemokines induced by the addition of methionine or other residues to the N-terminus, resulting in fusion mediated by the HIV-1 _JR-FL envelope glycoprotein And many isolates of HIV-1 inhibit infection and do not activate inflammatory responses.

b) Chemokines with N-terminal amino acid deletion:
Chemokine anti-antagonists have been generated by deleting amino acids in the N-terminal region. For example, by deleting up to 8 amino acids at the N-terminus of the chemokine MCP-1 (a member of the CC chemokine group), the ability to inhibit chemokine receptor binding and natural MCP-1 activity is maintained. The biological activity of the protein was removed (11, 12).

  The present invention includes N-terminal deletions of RANTES, MIP-1α and MIP-1β that lack the biological activity of the native protein to inhibit HIV-1 fusion and HIV-1 infection. C) Other peptides: Overlapping series of peptides (eg 20-67 residues) obtained from the entire region of RANTES, MIP-1α and MIP-1β are screened by the same approach and do not activate leukocytes Thus, the most potent peptides for inhibiting HIV-1 fusion were identified. Activation of the leukocyte response is measured by a routine method of the following procedure (9, 10, 11, 12).

3) Cloning of chemokine receptors
The chemokine receptor required for HIV-1 fusion is cloned by the following method. First, a cDNA library is constructed using mRNA prepared from PM1 cell lines or CD4 ⁺ T lymphocytes or macrophages in a mammalian expression vector (eg, pcDNA3.1 obtained from Invitrogen Corp. Sna Diego, CA). create. Using degenerate oligonucleotide probes, members of the cDAN library encoding members of the chemokine receptor family are identified using, for example, the following recently published method (2). The vector containing chemokine receptor cDNAs then expresses human CD4 but HeLa-env _JR-FL cells (eg, HeLa-CD4, CHO-CD4 or COS-CD4) or HeLa-env _LAI cells (eg, CHO- It is expressed individually in several mammalian cell lines that do not fuse with CD4, COS-CD4). Following analysis in the RET analysis, a clone that has acquired the ability to fuse with HeLa-env _JR-FL cells or HeLa-env _LAI cells is identified, and its coding sequence is regenerated by PCR amplification according to methods known to those skilled in the art. It was done. DNA was then sequenced to determine if the regenerated cDNA encodes a known chemokine receptor. Following receptor expression, monoclonal and polyclonal antibodies were prepared and tested for their ability to inhibit infection by a panel of HIV-1 isolates.

Cited reference 1

第二実験シリーズ
CD4^＋Ｔ細胞において、一次非シンシチウム誘導性（NSI）HIV-1単離物の複製物は、C-CβケモカインMIP-1α、MIP-1β及びRANTES（1、２）によって阻害されるが、T細胞系適用性（TCLA）又はシンチウム誘導性（SI）の初代株は感受性を示さない（2、3）。該βケモカインは、リンパ球及び単核球細胞形（4−8）の細胞上で活性な、小さい（8kDa）関連蛋白である。それらのレセプターは、７つの膜にかかるG蛋白連結スーパーファミリーのメンバーであり、そのうちの一つ（LESTRオーファンレセプター）はTCLA HIV-1株の第二レセプターとして特定されてきていたが、現在はフュージンと命名されている（９）。フュージンがβケモカイン・レセプターである事は知られていない（７−９）。

Second experiment series
In CD4 ⁺ T cells, replicas of primary non-syncytium-inducible (NSI) HIV-1 isolates are inhibited by C-Cβ chemokines MIP-1α, MIP-1β and RANTES (1, 2), but T Cell line applicability (TCLA) or syncytium-induced (SI) primary strains are not sensitive (2, 3). The β-chemokine is a small (8 kDa) related protein that is active on cells of lymphocyte and mononuclear cell shape (4-8). These receptors are members of the G protein-linked superfamily that spans seven membranes, one of which (the LESTR orphan receptor) has been identified as the second receptor of the TCLA HIV-1 strain. It is named fujin (9). It is not known that fudin is a β chemokine receptor (7-9).

To study how β-chemokines inhibit HIV-1 replication, a single env deletion virus NL4 / 3Δenv (which also carries a luciferase receptor gene) supplemented with an envelope glycoprotein expressed in trans Virus intrusion analysis based on cycle infection was employed (10, 11). Various env supplemented viruses were tested in PM1 cells (HUT-78 variants with specific ability to help replicate primary and TCLA HIV-1 strains) and tested for envelope glycoprotein function against a common cellular background. Allows comparison (2, 12). MIP-1α, MIP-β and RANTES, when combined, are most active against HIV-1 (2, 3) and have envelopes derived from NSI primary strains ADA and BaL (Table 1a). It strongly inhibits infection of PM1 cells by supplemented viruses.

Description of Table 1 PM1 cells were cultured as described by Lusso et al. (12). Ficoll / Hypaque isolated PBMCs obtained from laboratory workers (LW) were stimulated with PHA for 72 hours prior to removal of CD8 ⁺ lymphocytes with anti-CD8 immunomagnetic beads (DYNAL, Great Neck NY) . CD4 ⁺ lymphocytes were maintained in culture medium containing interleukin 2 (100 U / ml, Hoffmann laRoche, Nutley, NJ) as previously described (3). The target cell (1-2X10 ⁵ ) is infected with the supernatant (10-50 ng of HIV-1 p24) obtained from 293 cells transfected with the NL4 / 3Δenv luciferase vector and the HIV-1 env expression vector. It was. β-chemokine (R & D Systems, Minneapolis) was added to the target cells at the same time as the virus at the final concentration (ng / ml) indicated in brackets in the first column. The β-chemokine concentration range was selected based on previous studies (2, 3). Two hours later, the cells were washed twice with PBS, resuspended in β-chemokine conditioned medium and maintained for 48-96 hours. Luciferase activity in the cell melt was measured as described previously (10, 11). The values shown represent luciferase activity (cpm) / ng p24 / mg protein, expressed relative to the luciferase activity in the control medium of the virus lacking β-chemokine (100%), in double or hexafold. It is the average of the measurements made. nd is not measured. R / Mα / Mβ, RANTES + MIP-1α + MIP-1β.
RANTES and MIP-1β were very active when added individually, whereas MIP-1α, MCP-1, MCP-2, MCP-3 (reference numbers 13-15) were weak inhibitors (Table 1a). However, MIP-1α, MIP-1β and RANTES, when combined, inhibit infection of PM1 cells with TCLA strains NL4 / 3 and HxB2 or non-lipophilic murine leukocyte virus (MuLV-Ampho) pseudotypes. None (Table 1a). Thus, in the virus invasion analysis, the phenotypic characteristics of the HIV-1 envelope glycoprotein affect the sensitivity of β-chemokines.

Env supplementation analysis was used to assess HIV-1 entry into CD4 ⁺ T cells obtained from two control solids (LW4, LW5). MIP-1α + MIP-1β and RANTES strongly inhibit infection by infecting LW4 ′ and LW5 ′ CD4 ⁺ T cells with the primary NSI strain JR-FL, and slightly reduce HxB2 infection of LW cells. (Table 1b). This suggests that receptors on CD4 ⁺ T cells activated by different virus strains can be used somewhat redundantly. BaL env mediated replication in normal PBL was also inhibited by MIP-1α, MIP-1β and RANTES, but in terms of sensitivity, significant mutations (data not shown) were seen between donors.

  When β-chemokines inhibit HIV-1 replication, β-chemokines continue to be up to 5 hours after addition of ADA or BaL env complement virus (Figure 3a) to completely inhibit infection of PM1 cells. It was decided by proving that it is necessary to exist. Prior to infection, cells were pretreated with β-chemokines for 2 or 24 hours, and the cells were subsequently washed before addition of virus with no inhibitory effect. Furthermore, the addition of β-chemokine 2 hours after the addition of virus had a negligible effect on virus entry (Fig. 3a). Next, PCR-based analysis was used to detect early DNA reverse transcripts in PM1 cells 10 hours after infection. A reverse transcript of ADA but not NL4 / 3 could not be detected in the presence of MIP-1β and RANTES (FIG. 3b). Thus, inhibition by β-chemokines is present in at least one of the early stages of HIV-1 replication; that is, during at least one stage of viral attachment, fusion and early reverse transcription There is a need.

As described in part in the first experimental series, β-chemokines inhibit the binding of JR-Fl or BRU (LAI) gp120 to soluble CD4, or the tetrameric CD4-IgG2 oligomer By first testing whether binding to HeLa-JR-Fl cells expressing envelope glycoprotein (17) was inhibited, the site of action of β-chemokines was identified. In either analysis, no inhibition was observed by any β-chemokine, but OKT4a CD4-MAb showed strong inhibition in both analyzes (FIG. 2, data not shown). Thus, β-chemokines inhibit the process after CD4 binding, that is, when morphological changes in the envelope glycoprotein lead to fusion of the virus with the cell membrane (18). Cell-cell fusion is also induced by the interaction of gp120-CD4 and can be directly monitored by resonance energy transfer (RET) between fluorescent dyes incorporated into the cell membrane (17). In the RET analysis, OKT4a is expressed as PM1 cells and JR-FL (HeLa-JR-Fl, the same cell line referred to above as HeLa env _JR-FL ) or BRU (HeLa-BRU, HeLa-env _LAI as described above). It completely inhibits membrane fusion with HeLa cells expressing the envelope glycoprotein of any of the same cell lines mentioned). This confirmed that it was process specific (17). RANTES, MIP-1β (and to a lesser extent MIP-1α) strongly inhibits membrane fusion between HeLa-JR-FL cells and PM1 cells, but the fusion between PM1 cells and HeLa-BRU cells The sensitivity to these β-chemokines was not shown (FIG. 1 and Table 2a).

Explanation of Table 2: CD4 ⁺ target cells (CD4 ⁺ lymphocytes or PM1 cells activated by mitogens) are labeled with ocradecyl rhodamine (Molecular Probes Eugene, OR) and HeLa-JR-Fl cells HeLa- BRU cells (or control HeLa cells, not shown) were labeled with octadecyl fluorescein (Molecular Probes) overnight at 37 ° C. The same number of labeled target cells and env-expressing cells were mixed in a 96-well plate and β-chemokine was added at the final concentration (ng / ml) indicated in parenthesis of the first column. The fluorescence emission value was measured 4 hours after cell mixing (17).

If cell fusion occurs, the dye associates tightly with the bound membrane. Then, fluorescence is excited at 450 nm, resulting in resonance energy transfer (RET), and light emission by rhodamine occurs at 590 nm. The percent fusion is defined as equal to 100 × [(experimental RET−minimum RET)] / (maximum RET−minimum RET). Here, the maximum RET is a% RET value obtained when Hela-Env cells and CD-expressing cells are mixed. Experimental RET is the% RET value obtained when Hela-Env cells and CD4 ⁺ cells are mixed in the presence of a fusion inhibitor compound. The minimum RET is the% RET value obtained when mixing Hela cells (which lack HIV-1 envelope glycoprotein) and CD4 ⁺ cells. The% RET values are specified in calculations described elsewhere, (17) hand woven, each of which is the average of triplicate measurements. These values for Hela-JR-Fl and HeLa-BRU cells are PM1 cells 11.5%, 10.5%; LW5CD4 ⁺ cells 6.0%, 10.5%; R / Mα / Mβ, RANTES + MIP-1α + MIP-1β .
Similar results were obtained with primary CD4 ⁺ T cells derived from LW5 (Table 2b), but higher concentrations of β-chemokine in primary cells are required to inhibit membrane fusion than in PM1 cells. Met. Thus, β-chemokine activity is not limited to the PM1 cell line. RET analysis demonstrates that β-chemokines inhibit env-mediated membrane fusion.

To more simply explain these results, the binding of certain β-chemokines to their receptors prevents fusion of HIV-1 with CD4 ⁺ T cells, either directly or otherwise. It has been known for decades that HIV-1 requires a second receptor to enter CD4 ⁺ cells (19-21). This function is provided by fujin (9) for the TCLA strain. Several receptors have been identified for MIP-1α, MIP-1β and RANTES (6, 7), and β-chemokines show considerable cross-reactivity when utilizing the receptors (4-8). However, based on the tissue expression pattern and its ability to bind to MIP-1α, MIP-1β and RANTES (4, 7, 8, 15, 22), CC CKR-1, especially CC CKR-5, is most likely Identified as a candidate. CC CKR-1, CC CKR-5 and LESTR are each expressed at the mRNA level in PM1 cells and primary macrophages (data not shown). These and other β chemokine receptors were thus PCR amplified, cloned and expressed.

When CC CKR-5 is expressed in HeLa-CD4 (human), COS-CD4 (monkey), and 3T3-CD4 (murine) cells, each of these cells is identified as the primary NSI in the env complementation analysis of HIV-1 entry. It became easily infected by strains ADA and BaL (Table 3).

Description of Table 3 Chemokine receptor genes CC CKR-1, CC CRK-2a, CC CKR-3, CC CKR-4, CC-CKR-5 have no introns (4-8, 15, 22), 7 Isolated by PCR performed directly on RBMC-derived human genomic DNA pools of healthy human donors. Oligonucleotides were used that overlap with the ATG and stop codons and contain BamHI and Xhol restriction sites for directional cloning into pcDNA3.1 expression vector (Invitrogen Inc.). LESTR (also known as fusin or HUMSTR) (4, 9, 24) was cloned using sequences derived from the NIH database by PCR performed directly on cDNA derived from PM1 cells. The following list shows PCR amplification of LESTR obtained from CKR gene and PM1 cDNA obtained from direct human genomic DNA in the first round (5-1 and 3-1) and the second round (5-2, 3-2). The 5′-3 ′ plumer pair used in Only one set of primers was used to amplify CKR-5.

LESTR: L / 5-1 = AAG CTT GGA GAA CCA GCG GTT ACC ATG GAG GGG ATC (sequence recognition number: 6);
L / 5-2 = GTC TGA GTC TGA GTC AAG CTT GGA GAA CCA (sequence recognition number: 7);
L / 3-1 = CTC GAG CAT CTG TGT TAG CTG GAG TGA AAA CTT GAA GAC TC (sequence recognition number: 8);
L / 3-2 = GTC TGA GTC TGA GTC CTC GAG CAT CTG TGT (sequence recognition number: 9);
CKR-1: C1 / 5-1 = AAG CTT CAG AGA GAA GCC GGG ATG GAA ACT CC (sequence recognition number: 10);
C1 / 5-2 = GTC TGA GTC TGA GTC AAG CTT CAG AGA GAA (sequence recognition number: 11);
C1 / 3-1 = CTC GAG CTG AGT CAG AAC CCA GCA GAG AGT TC (sequence recognition number: 12);
C1 / 3-2 = GTC TGA GTC TGA GTC CTC GAG CTG AGT CAG (sequence recognition number: 13);
CKR-2a: C2 / 5-1 = AAG CTT CAG TAC ATC CAC AAC ATG CTG TCC AC (sequence recognition number: 14);
C2 / 5-2 = GTC TGA GTC TGA GTC AAG CTT CAG TAC ATC (sequence recognition number: 15);
C2 / 3-1 = CTC GAG CCT CGT TTT ATA AAC CAG CCG AGA C (sequence recognition number: 16);
C2 / 3-2 = GTC TGA GTC TGA GTC CTC GAG CCT CGT TTT (sequence recognition number: 17);
CKR-3: C3 / 5-1 = AAG CTT CAG GGA GAA GTG AAA TGA CAA CC (sequence recognition number: 18);
C3 / 5-2 = GTC TGA GTC TGA GTC AAG CTT CAG GGA GAA (sequence recognition number: 19);
C3 / 3-1 = CTC GAG CAG ACC TAA AAC ACA ATA GAG AGT TCC (SEQ ID NO: 20);
C3 / 3-2 = GTC TGA GTC TGA GTC CTC GAG CAG ACC TAA (sequence recognition number: 21);
CKR-4 = C4 / 5-1 = AAG CTT CTG TAG AGT TAA AAA ATG AAC CCC ACG G; (SEQ ID NO: 22);
C4 / 5-2 = GTC TGA GTC TGA GTC AAG CTT CTG TAG AGT (sequence recognition number: 23);
C4 / 3-1 = CTC GAG CCA TTT CAT TTT TCT ACA GGA CAG CAT C (sequence recognition number: 24);
C4 / 3-2 = GTC TGA GTC TGA GTC CTC GAG CCA TTT CAT (sequence recognition number: 25);
CKR-5, C5 / 5-12 = GTC TGA GTC TGA GTC AAG CTT AAC AAG ATG GAT TAT CAA (SEQ ID NO: 26);
C5 / 3-12 = GTC TGA GTC TGA GTC CTC GAG TCC GTG TCA CAA GCC CAC (sequence recognition number: 37).

Human CD4 expressing cell lines HeLa-CD4 (P42), 3T3-CD4 (sc6) and COS-CD4 (Z28T1) (23) were transfected with different pcDNA3.1-CKR constructs by the calcium phosphate method for 48 hours. Later, different reporter viruses (200 ng HIV-1p24 / 10 ⁶ cells) were infected in the presence or absence of β-chemokines. Luciferase activity in the cell melt was measured after 48 hours (10, 11). Inhibition data for β-chemokines was shown only for CC CKR-5, as the degree of inhibition of infection mediated by other CC CKR genes was too low to be quantified. In PCR-based analysis of HIV-1 entry, it was observed that the level of entry of NL4 / 3 and ADA into CC-CKR-1-expressing cells was consistently low (data not shown).

  Neither LESTR nor C-C-CKR-1, -2a, -3 or -4 could be substituted for C-C CKR-5 during this analysis. Expression of LESTR in COS-CD4 and 3T3-CD4 cells allowed entry of HxB2, which readily entered into untransfected (control plasmid transfected) HeLa-CD4 cells (Table 3). The entry of BAL and ADA into all three C-C CKR-5 expressing cell lines was almost completely inhibited by the combination of MIP-1α, MIP-1β and RANTES. However, entry of HxB2 into LESTR expressing cells was insensitive to β-chemokines (Table 3). These results suggested that C-C CKR-5 functions as a β-chemokine sensitive second receptor for the primary NSI HIV-1 strain.

  The second receptor function of C-C CKR-5 was confirmed in membrane fusion analysis mediated by env. When CC CKR-5 was transiently expressed in COS and HeLa cell lines that permanently express human CD4, both cell lines were strongly fused with HeLa cells that express the JR-FL envelope glycoprotein. However, no fusion occurred when using the control plasmid (data not shown). Even when LESTR was expressed instead of C-C CKR-5, neither COS-CD4 nor HeLa-CD4 cells could fuse with HeLa-JR-FL cells. However, fusion between COS-CD4 cells and HeLa-BRU cells was possible (data not shown).

  The fusion ability of the β chemokine receptor was also tested by RET analysis. Expression of C-C CKR-5 but not C-C CKR-1, -2a, -3, or -4 allowed strong fusion between HeLa-CD4 cells and HeLa-JR-FL cells. The degree of fusion between HeLa-JR-FL cells and C-C CKR-5 expressing HeLa-CD4 cells was greater than that between constitutive levels of HeLa-BRU and HeLa-CD4 cells (FIG. 4). Thus, the C-C CKR-5 fusion-conferring function for the first NSI HIV-1 strain was confirmed in two independent fusion analyses.

Experimental considerations Based on the above results, MIP-1α, MIP-1β and RANTES inhibit HIV-1 infection at the entry stage by blocking the virus-cell fusion reaction following CD4 binding. Was proved. CC CKR-5 can act as a second receptor for the primary NSI strain of HIV-1 to enter CD4 + T cells, and the interaction between β-chemokine and CC CKR-5, It was proved that HIV-1 fusion reaction was inhibited.

Cited reference 2

第三実験シリーズ
ケモカインSDF-1（間質細胞派生因子１）はフュージン/CXCR4に対する天然リガンドで、HIV-1の実験室適用株による感染を阻止する（参照1及び２）。SDF-1は、SDF-1遺伝子（参照1及び３）の可変スプライシングで少なくとも二つの型：SDF-1α及びSDF-2β、で存在する。RET分析においては、このケモカインは、図５に示したようにマクロファージ性単離物HIV-１_{ＪＲ−ＦＬ}から得られる gp120/gp41によってではなく、実験室適用株HIV_ＬＡＮから得られる gp120/gp41によって媒介される膜融合を特異的に阻害する。

Third Experimental Series Chemokine SDF-1 (stromal cell-derived factor 1) is a natural ligand for fudin / CXCR4 and blocks infection by laboratory-applied strains of HIV-1 (Refs 1 and 2). SDF-1 exists in at least two forms: SDF-1α and SDF-2β with variable splicing of the SDF-1 gene (references 1 and 3). In RET analysis, this chemokine is obtained from the macrophage isolate HIV-1 _JR-FL as shown in FIG. Obtained from lab-applicable strain HIV _LAN , not by gp120 / gp41 It specifically inhibits membrane fusion mediated by gp120 / gp41.

Citation of the third experimental series
1. Bleul, CC, et al. (1996) Nature 382: 829-833
2. Oberlin, E., et al. (1996) Nature 382: 833-835
3. Shirozu, M., et al. (1995) Genomics 28: 495-500

Membrane fusion mediated by HIV-1 _JR-FL envelope glycoprotein is inhibited by RANTES, MIP-1α and MIP-1β PM1 cells and HeLa-env _JR-FL (solid squares) or HeLa-env _LAI % RET obtained from fusion with (solid diamonds) in the presence and absence of recombinant human chemokines, concentration range: RANTS (80-2.5 ng / ml), MIP-1α (400-12.5 ng / ml), MIP-1β (200-6.25 ng / ml) and measured as indicated.

Chemokines and cells were added simultaneously at the start of the 4 hour incubation. Data show 3 or more independent experiments performed in duplicate. The% inhibition of RET is defined as described below.

% Inhibition = 100 · [(maximum RET−minimum RET) − (experimental RET−minimum RET)]
/ (Maximum RET-minimum RET)
In this case, the maximum RET is the% RET value obtained at 4 hours using HeLa-env cells and CD-expressing cells in the absence of inhibitory compounds. Experimental RET is the% RET value obtained for the same cell combination in the presence of inhibitory compounds. The minimum RET is the background% RET value obtained using Hela cells instead of HeLa envelope expressing cells.
CD4: HIV-1 gp120 binding in the presence of human chemokines Binding of soluble human CD4 to HIV-1 _LAI and HIV-1 _JR-FL gp120 in the presence and absence of monoclonal antibody OKT4A or recombinant human chemokine , Concentration ranges similar to those used in the RET inhibition study of Figure 1: OKT4A (62-0.3 nM), RANTS (10.3-0.3), MIP-1α (53.3-2.9 nM), MIP-1β (25.6-0.8 nM ) And determined by Eliza analysis. Inhibitors were added simultaneously with biotinylated HIV-1 gp120 to soluble CD4 applied to microtiter dishes (Dynatech Laboratories, Inc., Chantilly, VA). After incubation at room temperature for 2 hours and extensive washing, incubation at room temperature for 1 hour with streptavidin horseradish peroxidase was performed. After further washing several times, the substrate was added, and the optical density (OD) at 492 nm was measured with an Eliza plate reader. The data shows two independent experiments performed in quadruplicate. Specificity: Time change and stage of inhibition of β-chemokine of HIV-1 replication (a) PM1 cells (1 × 10 ⁶ ) were treated with RANTES + MIP-1α + MIP-1β (R / Mα / Mβ, 100 ng / ml each) for 24 hours ( -24h) or 2 hours (-2h) and washed twice with phosphate buffered saline (PBS). Add HIV-1 (BaL env-complement) virus (p24 50 ng, see description in Table 1) for 2 hours, wash cells, and before measuring luciferase activity in cell melt as described above Incubated for 48 hours (10, 11).

Alternatively, add virus and R / Mα / Mβ simultaneously to the cells, wash the cells twice in PBS at the indicated time point (1 hour, 3 hours, etc.), resuspend in culture medium and before luciferase analysis For 48 hours. Time 0 represents a positive control where no beta chemokine was added. +2 hours indicates the virus and cell mixture for 2 hours before washing twice with PBS. Incubation is continued for an additional 48 hours before addition of R / Mα / Mβ and luciferase analysis.
Specificity: Time course and stage of inhibition of β-chemokine in HIV-1 replication (b) HIV-1 (500 pg) grown in PM1 cells (1x10 ⁶ ) in CEM cells (NL4 / 3, lanes 1-4) p24) or macrophages (ADA; lanes 5-8) in the presence of 500 ng / ml RANTES (lanes 1 and 5), MIP-1β (lanes 2 and 6) or β-free chemokines (lanes 4 and 8) Infected. Lanes 3 and 7 are negative controls (no virus). All virus stocks used for PCR analysis were treated with DNAse for 30 minutes at 37 ° C. and tested for DNA contamination prior to use. After 2 hours, the cells were washed and resuspended for another 8 hours in medium containing the same β-chemokine. Next, DNA was extracted from the infected cells using a DNA / RNA isolation kit (US biochemical). The first nested PCR uses primers: U3 +, 5'-CAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGG-3 '(sequence recognition number 1) preGag, 5'-AGCAAGCCGAGTCCTGCGTCGAGAG-3' (sequence recognition number 2), and the second round is primer : LTR test, 5'-GGGACTTTCCGCTGGGGACTTTC-3 '(sequence recognition number 3) LRC2, 5'-CCTGTTCGGGCGCCACTGCTAGAGATTTTCCAC 3' (sequence recognition number 4) using Perkin Elmer 2400 cycler with the following amplification cycle: 94 ° C 5 Minutes, 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds, and 72 ° C. for 7 minutes were performed in 35 cycles. M represents a 1 Kb DNA ladder, and 1, 10, 100, and 1000 represent the copy number of the reference plasmid (pAD8). This analysis detected 100 copies of reverse transcript. HIV-1 env-mediated membrane fusion transiently expressing CC CKR-5 Membrane fusion mediated by β-chemokine receptors expressed in HeLa cells was demonstrated as follows. Control plasmid pcDNA3.1 or plasmid pcDNA3.1-CKR construct was transfected into cells using Lipofectin (Gibco BRL). The pcDNA3.1 plasmid carries the T7 polymerase promoter, and transient expression of the β chemokine receptor is achieved by infecting cells with a 1x10 ⁷ pfu vaccine encoding T7 polymerase (vFT7.3). It increased at 4 hours after lipofectin administration (9). The cells were then cultured overnight in medium containing R18 and tested for their ability to fuse with HeLa-JR-FL cells (black columns) or HeLa-BRU cells (shaded columns). The% RET for control HeLa cells was 3% and 4% regardless of the transfected plasmid. Membrane fusion mediated by HIV _LAI envelope glycoproteins is% RET obtained from fusion of PM1 cells inhibited by SDF-1 with Hela-env _JR-FL or Hela-env _LAI cells (as shown on the graph) ) Were measured at the indicated concentrations in the presence of recombinant SDF-1α (Gryphon Science, San Francisco). The experimental method is as described in the explanation for FIG.

Claims (50)

A method of inhibiting fusion of HIV-1 and CD4 ⁺ cells, relative to CD4 ⁺ cells, the non-chemokine agent capable of binding to a chemokine receptor, inhibited the fusion to CD4 ⁺ cells of HIV-1 A method comprising contacting under a predetermined amount and condition. A method of inhibiting HIV-1 infection of CD4 ⁺ cells, relative to CD4 ⁺ cells, the non-chemokine agent capable of binding to a chemokine receptor, an amount fusion to CD4 ⁺ cells of HIV-1 is inhibited and A method comprising inhibiting HIV-1 infection by contacting under conditions.   3. The method of claim 1 or 2, wherein the non-chemokine agent is an oligopeptide.   3. The method of claim 1 or 2, wherein the non-chemokine agent is a polypeptide.   3. The method of claim 1 or 2, wherein the non-chemokine agonist is an antibody or part of an antibody.   3. The method of claim 1 or 2, wherein the non-chemokine agent is a non-peptidyl agent. A non-chemokine agonist capable of binding to a chemokine receptor and inhibiting fusion of HIV-1 to CD4 ⁺ cells.   The non-chemokine agent of claim 7, wherein the non-chemokine agent is an oligopeptide.   The non-chemokine agent of claim 7, wherein the non-chemokine agent is a non-peptidyl agent.   8. The non-chemokine agent of claim 7, wherein the non-chemokine agent is a polypeptide.   11. The non-chemokine agent of claim 10, wherein the polypeptide is an antibody or part of an antibody.   11. The non-chemokine agent of claim 10, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.   11. The non-chemokine agent of claim 10, wherein the polypeptide comprises a MIP-1β sequence that lacks the first seven N-terminal amino acids of the sequence.   11. The non-chemokine agent of claim 10, wherein the polypeptide comprises a MIP-1β sequence lacking the first 8 N-terminal amino acids of the sequence.   11. The non-chemokine agent of claim 10, wherein the polypeptide comprises a MIP-1β sequence lacking the first nine N-terminal amino acids of the sequence.   11. The non-chemokine agent of claim 10, wherein the polypeptide comprises a MIP-1β sequence lacking the first 10 N-terminal amino acids of the sequence.   11. The non-chemokine agent of claim 10, wherein the polypeptide comprises an N-terminal sequence MIP-1β sequence modified by the addition of amino acids or oligopeptides.   11. The non-chemokine agent of claim 10, wherein the polypeptide is modified by removing the N-terminal alanine and replacing it with serine or threonine and another amino acid or oligopeptide or non-peptidyl moiety. An agent comprising a MIP-1β sequence having   19. The non-chemokine agonist of claim 17 or 18, wherein said another amino acid is methionine.   An agent that can bind to CXCR4 and inhibit HIV-1 infection.   21. The agent according to claim 20, wherein the agent is an oligopeptide.   21. The agent of claim 20, wherein the agent is a polypeptide.   23. The non-chemokine agent of claim 22, wherein the polypeptide comprises an SDF-1 sequence that lacks the first six N-terminal amino acids of the sequence.   23. The non-chemokine agent of claim 22, wherein the polypeptide comprises an SDF-1 sequence that lacks the first seven N-terminal amino acids of the sequence.   23. The non-chemokine agent of claim 22, wherein the polypeptide comprises an SDF-1 sequence that lacks the first 8 N-terminal amino acids of the sequence.   23. The non-chemokine agent of claim 22, wherein the polypeptide comprises an SDF-1 sequence that lacks the first nine N-terminal amino acids of the sequence.   23. The non-chemokine agent of claim 22, wherein the N-terminal glycine of SDF-1 is substituted with serine and induced with biotin.   23. The non-chemokine agent of claim 22, wherein the N-terminal glycine of SDF-1 is substituted with serine and induced with methionine.   23. The non-chemokine agonist according to claim 22, wherein the N-terminus of the SDF-1 is modified by adding methionine before the terminal glycine.   23. The agent of claim 22, wherein the agent is an antibody or part of an antibody.   21. The agent of claim 20, wherein the agent is a non-peptidyl agent. A pharmaceutical composition comprising an amount of the non-chemokine agent of claim 7 effective to inhibit fusion of HIV-1 to CD4 ⁺ cells and a pharmaceutically acceptable carrier. 21. A pharmaceutical composition comprising an amount of the non-chemokine agent of claim 20 effective to inhibit fusion of HIV-1 to CD4 ⁺ cells and a pharmaceutically acceptable carrier. Non-chemokines linked to ligands that can bind to chemokine receptors and inhibit the fusion of HIV-1 to CD4 ⁺ cells and bind to CD4 ⁺ cell surface receptors other than chemokine receptors A substance comprising an agent, wherein binding of the ligand to the other receptor is not inhibited when the non-chemokine agent binds to a chemokine receptor.   35. The substance of claim 34, wherein the cell surface receptor is CD4.   35. The substance of claim 34, wherein the ligand is an antibody or part of an antibody. 35. A pharmaceutical composition comprising an amount of the substance of claim 34 effective to inhibit fusion of HIV-1 to CD4 ⁺ cells and a pharmaceutically acceptable carrier. A compound capable of binding to a chemokine receptor and inhibiting the fusion of HIV-1 to CD4 ⁺ cells, which can increase the in vivo half-life of a non-chemokine agonist A substance comprising a non-chemokine agent linked to   40. The material of claim 38, wherein the compound is polyethylene glycol. 40. A pharmaceutical composition comprising an amount of the substance of claim 38 effective to inhibit fusion of HIV-1 to CD4 ⁺ cells and a pharmaceutically acceptable carrier.   41. A method of reducing the likelihood of HIV-1 infection in a subject comprising administering to the subject a pharmaceutical composition of claim 32, 33, 37 or 40.   42. A method of treating HIV-1 infection in a subject comprising administering to the subject the pharmaceutical composition of claim 32, 33, 39 or 40. A method for determining whether a non-chemokine agonist can inhibit the fusion of HIV-1 to CD4 ⁺ cells:
(A) a CD4 ⁺ cell (i) labeled with a first dye and a cell (ii) labeled with a second dye that expresses HIV-1 envelope glycoprotein on the cell surface; In the presence of excess, a step of contacting the cells expressing the HIV-1 envelope glycoprotein on the surface with CD4 ⁺ cells under conditions that allow fusion in the absence of the agent (wherein The first and second dyes are selected to allow transfer of resonance energy between the dyes);
(B) exposing the product of step (a) to conditions that cause resonance energy transfer when fusion occurs;
(C) determining whether there is a decrease in resonance energy transfer compared to resonance energy transfer in the absence of the agent, whereby the agent is HIV-1 CD4 ⁺ cells And a step shown to be capable of inhibiting the fusion with.   44. The method of claim 43, wherein the agent is an oligopeptide.   44. The method of claim 43, wherein the agent is a polypeptide.   44. The method of claim 43, wherein the agent is an antibody or part of an antibody.   44. The method of claim 43, wherein the agent is a non-peptidyl agent. 44. The method of claim 43, wherein the CD4 ⁺ cells are PM1 cells. 44. The method of claim 43, wherein the cells that express HIV-1 envelope glycoprotein are HIV-1 _JR-FL gp120 / gp41 cells that express HeLa cells. 44. The method of claim 43, wherein the HIV-1 envelope glycoprotein-expressing cell is HIV-1 _LAI gp120 / gp41 that expresses HeLa cells.

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