Link:  Pharm/Biotech Resources

Title:  Methods for using resonance energy transfer-based assay of HIV-1 envelope glycoprotein-mediated membrane fusion, and kits for practicing same

United States Patent:  6,972,126

Issued:  December 6, 2005

Inventors:  Allaway; Graham P. (Moreton Merseyside, GB); Litwin; Virginia M. (Fayetteville, NY); Maddon; Paul J. (Elmsford, NY)

Assignee:  Progenics Pharmaceuticals, Inc. (Tarrytown, NY)

Appl. No.:  412284

Filed:  October 5, 1999

This invention provides: agents determined to be capable of specifically inhibiting the fusion of a macrophage-tropic primary isolate of HIV-1 to a CD4⁺ cell, but not a T cell-tropic isolate of HIV-1 to a CD4⁺ cell; and agents determined to be capable of specifically inhibiting the fusion of a T cell-tropic isolate of HIV-1 to a CD4⁺ cell, but not a macrophage-tropic primary isolate of HIV-1 to a CD4⁺ cell. This invention also provides: agents capable of specifically inhibiting the fusion of a macrophage tropic primary isolate of HIV-1 with a CD+ cell susceptible to infection by a macrophage-tropic primary isolate of HIV-1; and agents capable of specifically inhibiting the fusion of a T cell-tropic isolate of HIV-1 with a CD4⁺ cell susceptible to infection by a T cell-tropic isolate of HIV-1. The agents include but are not limited to antibodies. This invention further provides: methods of inhibiting fusion of a macrophage-tropic primary isolate of HIV-1 with a CD+ cell susceptible to infection by a macrophage-tropic primary isolate of HIV-1 which comprises contacting the CD4⁺ cell with an amount of an agent capable of specifically inhibiting such fusion so as to thereby inhibit such fusion; and methods of inhibiting fusion of a T cell-tropic isolate of HIV-1 with a CD4⁺ cell susceptible to infection by a T cell-tropic isolate of HIV-1 which comprises contacting the CD4⁺ cell with an amount of an agent capable of specifically inhibiting such fusion so as to thereby inhibit such fusion.

SUMMARY OF THE INVENTION

The subject invention provides a method for determining whether an agent is capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a sample containing a suitable amount of the agent with a suitable amount of the appropriate CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the agent, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to determine whether the agent is capable of inhibiting fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell; and (d) determining whether the agent inhibits the fusion of a first control cell with a second control cell under conditions which would permit non-HIV-1 envelope glycoprotein-mediated fusion of the first and second control cells in the absence of the agent, so as to determine whether the agent is capable of specifically inhibiting the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

The subject invention also provides a method for determining whether an agent is capable of specifically inhibiting the infection of a CD4⁺ cell with HIV-1 which comprises determining whether the agent is capable of specifically inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell by the method of the subject invention, so as to thereby determine whether the agent is capable of specifically inhibiting the infection of a CD4⁺ cell with HIV-1.

The subject invention further provides a, method for determining whether an agent is capable of inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a sample containing a suitable amount of the agent with a suitable amount of the CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the agent, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; and (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to determine whether the agent is capable of inhibiting fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

This invention also provides an agent determined by the above-described method.

The subject invention further provides a method for quantitatively determining the ability of an antibody-containing sample to specifically inhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a predetermined amount of the antibody-containing sample with a suitable amount of the CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the antibody-containing sample, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to quantitatively determine the ability of the antibody-containing sample to inhibit: the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell; and (d) determining whether the antibody-containing sample inhibits the fusion of a first control cell with a second control cell under conditions which would permit non-HIV-1 envelope glycoprotein-mediated fusion of the first and second control cells in the absence of the agent, so as to quantitatively determine the ability of the antibody-containing sample to specifically inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

The subject invention further provides a method for quantitatively determining the ability of an antibody-containing sample to inhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a predetermined amount of the antibody-containing sample with a suitable amount of the CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the antibody-containing sample, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; and (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to quantitatively determine the ability of the antibody-containing sample to inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

The subject invention further provides a method for determining the stage or clinical prognosis of an HIV-1 infection in an HIV-1-infected subject which comprises: (a) obtaining an antibody-containing sample from the HIV-1-infected subject; (b) quantitatively determining the ability of the antibody-containing sample so obtained to inhibit the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell by the method of the subject invention; and (c) comparing the ability of the antibody-containing sample to inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell so determined with that of an antibody-containing sample obtained from an HIV-1-infected subject having an HIV-1 infection at a known stage or having a known clinical prognosis, so as to determine the stage or clinical prognosis of the HIV-1 infection in the HIV-1-infected subject.

The subject invention further provides a method for determining the efficacy of an anti-HIV-1 vaccination in a vaccinated, non-HIV-1-infected subject which comprises: (a) obtaining an antibody-containing sample from the vaccinated, non-HIV-1-infected subject; (b) quantitatively determining the ability of the antibody-containing sample so obtained to inhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell by the method of the subject invention; and (c) comparing the ability of the antibody-containing sample to inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell so determined with that of an antibody-containing sample obtained from a vaccinated, non-HIV-1-infected subject for whom the anti-HIV-1 vaccination has a known efficacy, so as to determine the efficacy of the anti-HIV-1 vaccination in the vaccinated, non-HIV-1-infected subject.

The subject invention further provides a kit for determining whether an agent is capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises, in separate compartments: (a) a suitable amount of a CD4⁺ cell whose cell membrane is labeled with a first dye; (b) a suitable amount of an HIV-1 envelope glycoprotein⁺ cell whose cell membrane is labeled with a second dye, the HIV-1 envelope glycoprotein⁺ cell being capable of fusing with the CD4⁺ cell of (a) under suitable conditions in the absence of the agent, and the first and second dyes permitting resonance energy transfer therebetween only when juxtaposed within the same membrane; (c) a suitable amount of a first control cell whose cell membrane is labeled with the first dye; and (d) a suitable amount of a second control cell whose cell membrane is labeled with the second dye, the second control cell being capable of non-HIV-1 envelope glycoprotein-mediated fusion with the first control cell of (c) under suitable conditions in the absence of the agent.

The subject invention further provides a kit for determining whether an agent is capable of inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell which comprises, in separate compartments: (a) a suitable amount of a CD4⁺ cell whose cell membrane is labeled with a first dye; and (b) a suitable amount of an HIV-1 envelope glycoprotein⁺ cell whose cell membrane is labeled with a second dye, the HIV-1 envelope glycoprotein⁺ cell being capable of fusing with the CD4⁺ cell of (a) under suitable conditions in the absence of the agent, and the first and second dyes permitting resonance energy transfer therebetween only when juxtaposed within the same membrane.

The subject invention further provides a method for determining whether an HIV-1 isolate is syncytium-inducing which comprises: (a) obtaining a sample of an HIV-1 isolate envelope glycoprotein⁺ cell whose cell membrane is labeled with a first dye; (b) contacting a suitable amount of the sample with a suitable amount of a CD4⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with a syncytium-inducing HIV-1 strain envelope glycoprotein⁺ cell, the cell membrane of the CD4⁺ cell being labeled with a second dye which permits resonance energy transfer between the first dye only when the first and second dyes are juxtaposed within the same membrane; (c) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; and (d) comparing the percent resonance energy transfer value so determined with a known standard, so as to determine whether the HIV-1 isolate is syncytium-inducing.

Finally, the subject invention provides a method for determining the stage of an HIV-1 infection in an HIV-1-infected subject which comprises determining by the method of the subject invention whether the HIV-1 isolate with which the HIV-1 infected subject is infected is syncytium inducing, so as to thereby determine the stage of the HIV-1 infection in the HIV-1-infected subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Time course of fusion between HeLa-env⁺ cells and HeLa-CD4⁺ cells measured by the RET assay.

FIG. 2 Blocking of fusion between HeLa-env⁺ cells and HeLa-CD4⁺ cells by OKT4a, measured using RET.

FIG. 3 Blocking of fusion between 160G7 cells and C8166 cells by sCD4, measured using RET.

FIG. 4 A comparative analysis of results of blocking experiments by two methods using OKT4a to inhibit the fusion of HeLa-env⁺ and HeLa-CD4⁺ cells.

FIG. 5 RET time course analysis. The time course of fusion between HeLa-env_LAI⁺ and HeLa-CD4⁺ cells (open boxes) or HeLa-env_JR-FL⁺ and PM1 cells (closed boxes) was measured using the RET assay at various intervals after mixing the cells.

FIG. 6 Inhibition of RET using the anti-attachment monoclonal antibody OKT4A. % RET resulting from the fusion of HeLa-env_LAI⁺ and HeLa-CD4⁺ cells (open boxes) or HeLa-env_JR-FL⁺ and PM1 cells (closed boxes) was measured in the presence and absence of various concentrations of OKT4A. Percent inhibition of RET at each concentration of OTK4A was calculated from this formula:


Where A is the maximum % RET in the absence of antibody, B is the % RET following incubation with OKT4A and C is the background % RET determined using HeLa cells in place of HeLa-env_LAI⁺ or HeLa-env_JR-FL⁺ cells.

DETAILED DESCRIPTION OF THE INVENTION

The plasmid designated pMA243 was deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 under ATCC Accession No. 75626. The plasmid pMA243 was deposited with the ATCC on Dec. 16, 1993.

This invention provides a method for determining whether an agent is capable of inhibiting the fusion of a macrophage-tropic primary isolate of HIV-1 to a CD4⁺ cell which comprises: (a) contacting (i) an appropriate CD4⁺ cell, which is labeled with a first dye, with (ii) a cell expressing the HIV-1 envelope glycoprotein of the macrophage-tropic primary isolate of HIV-1 on its surface, which is labeled with a second dye, in the presence of an excess of the agent under conditions permitting the fusion of the CD4⁺ cell to the cell expressing the HIV-1 envelope glycoprotein on its surface in the absence of the agent, the first and second dyes being selected so as to allow resonance energy transfer between the dyes; (b) exposing the product of step (a) to conditions which would result in resonance energy transfer if fusion has occurred; and (c) determining whether there is a reduction of resonance energy transfer, when compared with the resonance energy transfer in the absence of the agent, a decrease in transfer indicating that the agent is capable of inhibiting fusion of HIV-1 to CD4⁺ cells.

The subject invention provides a method for determining whether an agent is capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a sample containing a suitable amount of the agent with a suitable amount of the appropriate CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the agent, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to determine whether the agent is capable of inhibiting fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell; and (d) determining whether the agent inhibits the fusion of a first control cell with a second control cell under conditions, which would permit non-HIV-1 envelope glycoprotein-mediated fusion of the first and-second control cells in the absence of the agent, so as to determine whether the agent is capable of specifically inhibiting the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

This invention provides an agent determined to be capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell using the above-described method.

This invention provides a therapeutic agent determined to be capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate cell using the above-described method.

As used herein, the term "agent" includes both protein and non-protein moieties. In one embodiment, the agent is a small molecule. In another embodiment, the agent is a protein. The protein may be, by way of example, an antibody directed against a portion of an HIV-1 envelope glycoprotein, e.g., gp120. The agent may be derived from a library of low molecular weight compounds or a library of extracts from plants or other organisms. In an embodiment, the agent is known. In a separate embodiment, the agent is not previously known.

As used herein, "capable of specifically inhibiting the fusion of-an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell" means (a) capable of reducing the rate of fusion of a CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cell membrane by at least 5%, but not capable of reducing the rate of non-CD4/HIV-1 envelope glycoprotein-mediated cell membrane fusion, or (b) capable of reducing by at least 5% the total amount of fusion of a CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cell mebrane occurring by the endpoint of fusion, but not capable of reducing the total amount of non-CD4/HIV-1 envelope glycoprotein-mediated cell membrane fusion occurring by the endpoint of fusion. As used herein, the rate of cell membrane fusion means the total quantity of cell membrane fused per unit of time. As used herein, the "endpoint of fusion" means the point in time at which all fusion of CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cell membrane capable of occurring has occurred.

An example of the method of the subject invention is provided infra. A known amount of HIV-1 envelope glycoprotein⁺ cell is contacted with a known amount of CD4⁺ cell together with an agent under conditions which would permit the fusion of Y amount of cell membrane per unit of time in the absence of the agent, wherein Y is equal to the sum of the amounts of CD4⁺ cell membrane and HIV-1 envelope glycoprotein⁺ cell membrane, e.g., 0.5 ×Y CD4⁺ cell membrane +0.5 ×Y HIV-1 envelope glycoprotein⁺ cell membrane. In the presence of the agent, 0.2 ×Y amount of cell membrane fuses per unit of time. The agent is shown not to reduce the rate of non-CD4/HIV-1 envelope glycoprotein-mediated cell membrane fusion. Accordingly, the agent specifically inhibits the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell.

As used herein, the fusion of CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cell membrane means the hydrophobic joining and integration of CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cell membrane to form a hybrid membrane comprising components of both cell membranes, and does not mean the CD4/HIV-1 envelope glycoprotein-mediated adhesion therebetween, which adhesion is a prerequisite for the fusion.

As used herein, the term "CD4" includes (a) native CD4 protein and (b) a membrane-bound CD4-based protein. As used herein, a membrane-bound CD4-based protein is any membrane-bound protein, other than native CD4, which comprises at least that portion of native CD4 which is required for native CD4 to form a complex with the HIV-1 gp120 envelope glycoprotein. In one embodiment, the CD4-based protein comprises a portion of a non-CD4 protein. If the CD4-based protein comprises a portion of a non-CD4 protein, then the portion of native CD4 which is required for native CD4 to form a complex with the HIV-1 gp120 envelope glycoprotein is the portion of native CD4 having the amino acid sequence from +1 to about +179.

As used herein, the word "cell" includes a biological cell, e.g., a HeLa cell, and a non-biological cell, e.g., a lipid vesicle (e.g., a phospholipid vesicle) or virion.

As used herein, a CD4⁺ cell is a cell having CD4 affixed to the surface of its cell membrane, wherein the appropriate CD4⁺ cell is capable of specifically binding to and fusing with an HIV-1 envelope glycoprotein⁺ cell exposed thereto. In one embodiment, the suitable CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment, the suitable CD4⁺ cell is a PM1 cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

As used herein, an HIV-1 envelope glycoprotein⁺ cell is a cell having HIV-1 envelope glycoprotein affixed to the surface of its cell membrane so as to permit the HIV-1 envelope glycoprotein⁺ cell to specifically bind to and fuse with an appropriate CD4⁺ cell exposed thereto. In one embodiment, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1 envelope glycoprotein⁺ HeLa cell. In another embodiment, the HIV-1 envelope glycoprotein⁺ cell is HIV-1.

Each HIV-1 isolate is tropic for a limited number of CD4⁺ cell types. Accordingly, in the subject invention, the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell means the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell, which HIV-1 envelope glycoprotein corresponds to an envelope glycoprotein from an HIV-1 isolate tropic for the CD4⁺ cell. For example, the HIV-1 isolates _JR-FL, JR-CSF and BaL are tropic for CD4⁺ primary human macrophages, the HIV-1 isolates LAI and IIIB are-tropic for human CD4⁺ T lymphocyte cell lines and HeLa-CD4 cells, and the HIV-1 isolates MN and SF-2 are tropic for human CD4⁺ T lymphocyte cell lines. The HIV-1 isolates _JR-FL, JR-CSF, BaL, LAI, IIIB, MN and SF-2 may also be tropic for CD4⁺ cell types other than those enumerated supra.

As used herein, an appropriate CD4⁺ cell line is a cell line that fuses with the HIV-1 envelope glycoprotein⁺ cell line, such that the % RET measurement obtained is at least 5 fold greater than the background level obtained using a combination of cells which do not fuse (e.g. HeLa cells mixed with the CD4⁺ cell line). Moreover, the % RET obtained using the CD4⁺ cell line and the HIV-1 envelope glycoprotein⁺ cell line should be inhibited to background levels using 1 ug/ml OKT4A.

The suitable amounts of agent, CD4⁺ cell and HIV-1 envelope glycoprotein⁺ cell may be determined according to methods well known to those skilled in the art.

Conditions which would permit the fusion of the appropriate CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the agent are well known to those skilled in the art.

As used herein, a cell "labeled" with a dye means a cell having a dye integrated into its cell membrane, i.e., a cell having dye molecules commingled with the lipid molecules of its cell membrane.

Resonance energy transfer is defined as follows: For juxtaposed dyes D1, having excitation and emission spectra Ex1 and Em1, respectively, and D2, having excitation and emission spectra Ex2 and Em2, respectively, wherein (a) Em1 has a higher average frequency than that of Em2 and (b) Em1 and Ex2 overlap, resonance energy transfer is the transfer of electromagnetic energy from D1 to D2 at a frequency within the Em1 and Ex2 overlap, which resonance energy transfer (a) results from the electromagnetic excitation of D1 at a frequency within the Ex1 spectrum and (b) causes the subsequent emission of electromagnetic energy from D2 at a frequency within the Em2 spectrum. Accordingly, resonance energy transfer between D1 and D2 can be detected by exciting D1 with electromagnetic energy at a frequency within Ex1 and measuring the subsequently emitted electromagnetic energy at a frequency within Em2, the emission of electromagnetic energy at a frequency within Em2 indicating the occurrence of resonance energy transfer between D1 and D2.

The first and second dyes are "juxtaposed within the same membrane" if they are present within the same lipid membrane at a suitably short distance from each other, which suitably short distance may be readily determined by one skilled in the art.

In the subject invention, determining the percent resonance energy transfer value may be performed according to methods well known to those skilled in the art. In one embodiment, the percent resonance energy transfer value is determined by: (1) determining the resonance energy transfer value (RET) by subtracting from the total emission from D1 and D2 at a frequency within Em2 the electromagnetic energy emission due to direct D1 and D2 emission following excitation at a frequency within Ex1 and emission at the frequency within Em2, which D1 and D2 emissions are measured by separately measuring the electromagnetic energy emission due to cells labeled with each dye; and (2) determining the percent resonance energy transfer value (% RET value) by dividing the resonance energy transfer value obtained in step (1) by the total D2 emission at the frequency within Em2.

The suitable period of time after which the percent resonance energy transfer value of the resulting sample is determined may be determined according to methods well known to those skilled in the art.

The known standard is a percent resonance energy transfer value obtained using the CD4⁺ cell, the HIV-1 envelope glycoprotein⁺ cell, and an agent having a known ability to inhibit the fusion thereof.

In the subject invention, the first control cell and second control cell are capable of fusing with each other via non-HIV-1 envelope glycoprotein-mediated fusion both in the presence and absence of an agent capable of inhibiting HIV-1 envelope glycoprotein-mediated fusion, and are not capable of fusing via HIV-1 envelope glycoprotein-mediated fusion. Such cells are will known to those skilled in the art, and include, by way of example, HeLa cells which can be induced to fuse with each other by incubation at 37° C. with polyethylene glycol 1000 or with Sendai virus. These methods of inducing fusion of HeLa cells are well known to those skilled in the art.

In one embodiment, the agent is an antibody. As used in the subject invention, the term "antibody" includes, but is not limited to, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal and monoclonal antibodies, and antigen-binding fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies, wholly synthetic antibodies, and antigen-binding fragments thereof.

In one embodiment, the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule. Rhodamine moiety-containing molecules and fluorescein moiety-containing molecules are well known to those skilled in the art.

In the preferred embodiment, the rhodamine moiety-containing molecule is octadecyl rhodamine B chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment of the subject invention, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_LAI gp120/gp4⁺ HeLa cell. In a separate embodiment, the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_JR-FL gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

The subject invention also provides a method for determining whether an agent is capable of specifically inhibiting the infection of a CD4⁺ cell with HIV-1 which comprises determining whether the agent is capable of specifically inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell by the method of the subject invention, so as to thereby determine whether the agent is capable of specifically inhibiting the infection of a CD4⁺ cell with HIV-1.

The subject invention further provides a method for determining whether an agent is capable of inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a sample containing a suitable amount of the agent with a suitable amount of the CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the appropriate CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the agent, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; and (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to determine whether the agent is capable of inhibiting fusion of the HIV-1 envelope glycoprotein⁺ cell with the CD4.

As used herein, "capable of inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell" means capable of (a) reducing the rate of fusion of CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cell membrane by at least 5%, or (b) reducing by at least 5% the total amount of fusion of CD4⁺ cell membrane with HIV-1 envelope glycoprotein⁺ cell membrane occurring by the endpoint of fusion. An agent capable of inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell may also be capable of reducing the rate to non-CD4/HIV-1 envelope glycoprotein-mediated cell membrane fusion.

This invention provides an agent determined to be capable of inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell using the above-described method.

In one embodiment, the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment of the subject invention, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_LAI gp120/gp41⁺ HeLa cell. In a separate embodiment, the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_JR-FL gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

In the preferred embodiment, the rhodamine moiety-containing molecule is octadecyl rhodamine B chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

The subject invention further provides a method for quantitatively determining the ability of an antibody-containing sample to specifically inhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a predetermined amount of the antibody-containing sample with a suitable amount of the appropriate CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the antibody-containing sample, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to quantitatively determine the ability of the antibody-containing sample to inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell; and (d) determining whether the antibody-containing sample inhibits the fusion of a first control cell with a second control cell under conditions which would permit non-HIV-1 envelope glycoprotein-mediated fusion of the first and second control cells in the absence of the agent, so as to quantitatively determine the ability of the antibody-containing sample to specifically inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.

The antibody-containing sample may be any antibody-containing sample. In one embodiment, the antibody-containing sample is a serum sample. In another embodiment, the antibody-containing sample is an IgG preparation. Methods of obtaining an antibody-containing sample are well known to those skilled in the art.

In-one embodiment, the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule is octadecyl rhodamine B chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment of the subject invention, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_LAI gp120/gp41⁺ HeLa cell. In a separate embodiment, the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_JR-FL gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for quantitatively determining the ability of an antibody-containing sample to inhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises: (a) contacting a predetermined amount of the antibody-containing sample with a suitable amount of the appropriate CD4⁺ cell and a suitable amount of the HIV-1 envelope glycoprotein⁺ cell under conditions which would permit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell in the absence of the antibody-containing sample, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; and (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to quantitatively determine the ability of the antibody-containing sample to inhibit the fusion of the HIV-1 envelope glycoprotein⁺ with the CD4⁺ cell.

In one embodiment, the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule is octadecyl rhodamine B chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment of the subject invention, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_LAI gp120/gp41 HeLa cell. In a separate embodiment, the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺- cell is an HIV-1_JR-FL gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for determining the stage of clinical prognosis of an HIV-1 infection in an HIV-1-infected subject which comprises: (a) obtaining an antibody-containing sample from the HIV-1-infected subject; (b) quantitatively determining the ability of the antibody-containing sample so obtained to inhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell by the method of the subject invention; and (c) comparing the ability of the antibody-containing sample to inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell so determined with that of an antibody-containing sample obtained from an HIV-1 infected subject having an HIV-1 infection at a known stage or having a known clinical prognosis, so as to determine the stage or clinical prognosis of the HIV-1 infection in the HIV-1-infected subject.

As used herein, an "HIV-infected subject" means a subject having at least one of his own cells invaded by HIV-1. In the preferred-embodiment, the subject is a human.

The subject invention further provides a method for determining the efficacy of an anti-HIV-1 vaccination in a vaccinated, non-HIV-1-infected subject which comprises: (a) obtaining an antibody-containing sample from the vaccinated, non-HIV-1-infected subject; (b) quantitatively determining the ability of the antibody-containing sample so obtained to inhibit the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell by the method of the subject invention; and (c) comparing the ability of the antibody-containing sample to inhibit the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell so determined with that of an antibody-containing sample obtained from a vaccinated, non-HIV-1-infected subject for whom the anti-HIV-1 vaccination has a known efficacy, so as to determine the efficacy of the anti-HIV-1 vaccination in the vaccinated, non-HIV-1-infected subject.

As used herein, "anti-HIV-1 vaccination" means the administration to a subject of a vaccine intended to elicit the production of antibodies by the vaccinated subject which are capable of specifically binding to epitopes present on an HIV-1 surface envelope glycoprotein. Vaccines in general are well known to those skilled in the art, and comprise an antigen, e.g., a protein, and an adjuvant.

As used herein, the "efficacy of an anti-HIV-1 vaccination" means the degree to which the vaccination or successive vaccinations (i.e., immunization) causes the titre of HIV-1-neutralizing antibodies in the vaccinated subject to increase. In other words, the higher the efficacy of an anti-HIV-1 vaccination, the higher the titre of HIV-1-neutralizing antibodies in the vaccinated subject.

As used herein, a "non-HIV-1-infected subject" means a subject not having any of his own cells invaded by HIV-1. In the preferred embodiment, the subject is a human.

The subject invention further provides a kit for determining whether an agent is capable of specifically inhibiting the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ cell which comprises, in separate compartments: (a) a suitable amount of an appropriate CD4⁺ cell whose cell membrane is labeled with a first dye; (b) a suitable amount of an HIV-1 envelope glycoprotein⁺ cell whose cell membrane is labeled with a second dye, the HIV-1 envelope glycoprotein⁺ cell being capable of fusing with the CD4⁺ cell of (a) under suitable conditions in the absence of the agent, and the first and second dyes permitting resonance energy transfer therebetween only when juxtaposed within the same membrane; (c) a suitable amount of a first control cell whose cell membrane is labeled with the first dye; and (d) a suitable amount of a second control cell whose cell membrane is labeled with the second dye, the second control cell being capable of non-HIV-1 envelope glycoprotein-mediated fusion with the first control cell of (c) under suitable conditions in the absence of the agent.

The kit of the subject invention may further comprise additional buffers. Furthermore, the cells may either be dried or suspended in liquid or gel.

The suitable amounts of cells are amounts which would permit one skilled in the art to determine, without undue experimentation, whether an agent is capable of specifically inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell. Such amounts may be readily determined according to methods well known to those skilled in the art.

In one embodiment, the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule is octadecyl rhodamine B chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment of the subject invention, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_LAI gp120/gp41⁺ HeLa cell. In a separate embodiment, the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺- cell is an HIV-1 _JR-FL gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

The subject invention further provides a kit for determining whether an agent is capable of inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelope glycoprotein⁺ cell which comprises, in separate compartments: (a) a suitable amount of a CD4⁺ cell whose cell membrane is labeled with a first dye; and (b) a suitable amount of an HIV-1 envelope glycoprotein⁺ cell whose cell membrane is labeled with a second dye, the HIV-1 envelope glycoprotein⁺ cell being capable of fusing with the CD4⁺ cell of (a) under suitable conditions in the absence of the agent, and the first and second dyes, permitting resonance energy transfer therebetween only when juxtaposed within the same membrane.

The kit of the subject invention may further comprise additional buffers. Furthermore, the cells may either be dried or suspended in a liquid or gel carrier.

The suitable amounts of cells are amounts which would permit one skilled in the art to determine, without undue experimentation, whether an agent is capable of inhibiting the fusion of a CD4⁺ cell with an HIV-1 envelope-glycoprotein⁺ cell. Such amounts may be readily determined according to methods well known to those skilled in the art.

In one embodiment, the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule is octadecyl rhodamine B chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment of the subject invention, the HIV-1 envelope glycoprotein⁺ cell is an HIV-1_LAI gp120/gp41⁺ HeLa cell. In a separate embodiment, the CD4⁺ cell is a PM1 cell and the HIV-1 envelope glycoprotein⁺- cell is an HIV-1_JR-FL gp120/gp41 HeLa cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for determining whether an HIV-1 isolate is syncytium-inducing which comprises: (a) obtaining a sample of an HIV-1 isolate envelope glycoprotein⁺ cell whose cell membrane is labeled with a first dye; (b) contacting a suitable amount of the sample with a suitable amount of a CD4⁺ cell under conditions which would permit: the fusion of the CD4⁺ cell with a syncytium-inducing HIV-1 strain envelope glycoprotein⁺ cell, the cell membrane of the CD4⁺ cell being labeled with a second dye which permits resonance energy transfer between the first dye only when the first and second dyes are juxtaposed within the same membrane; (c) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; and (d) comparing the percent resonance energy transfer value so determined with a known standard, so as to determine whether the HIV-1 isolate is syncytium-inducing.

As used herein, "syncytium-inducing" means capable of causing the formation of syncytia (multi-nucleated cells resulting from HIV-1 envelope glycoprotein-mediated cell fusion) when contacted with a plurality of CD4⁺ cells under suitable conditions.

Obtaining a sample of an HIV-1 isolate envelope glycoprotein⁺ cells may be performed according to methods well known to those skilled in the art.

HIV-1 isolate envelope glycoprotein⁺ cells may be obtained from blood or any other bodily fluid known to contain HIV-1 isolate envelope glycoprotein⁺ cells in HIV-infected subjects. Alternatively, HIV-1 isolate envelope glycoprotein⁺ cells may be obtained by culturing cells in vitro with blood or other bodily fluids containing the HIV-1 isolate or HIV-1 isolate-infected cells, and recovering the HIV-1 isolate envelope glycoprotein⁺ cells produced thereby.

The suitable amounts of sample and CD4⁺ cell may be determined according to methods well known to those skilled in the art.

In one embodiment, the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule.

In the preferred embodiment, the rhodamine moiety-containing molecule is octadecyl rhodamine B chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

In another embodiment, the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

In one embodiment, the CD4⁺ cell is a CD4⁺ HeLa cell. In another embodiment, the CD4⁺ cell is a PM1 cell. In a further embodiment, the CD4⁺ cell is a primary human T lymphocyte. In a still further embodiment, the CD4⁺ cell is a primary human macrophage.

The subject invention further provides a method for determining the stage of an HIV-1 infection in an HIV-1-infected subject which comprises determining by the method of the subject invention whether the HIV-1 isolate with which the HIV-1-infected subject is infected is syncytium-inducing, so as to thereby determine the stage of the HIV-1 infection in the HIV-1-infected subject.

Finally, the subject invention provides a method for quantitatively measuring the fusion of an HIV-1 envelope glycoprotein⁺ cell with an appropriate CD4⁺ which comprises: (a) contacting a sample of the appropriate CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell under conditions permitting fusion therebetween, the cell membranes of the CD4⁺ cell and the HIV-1 envelope glycoprotein⁺ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the resulting sample after a suitable period of time; and (c) comparing the percent resonance energy transfer value so determined with a known standard, so as to quantitatively measure the fusion of the CD4⁺ cell with the HIV-1 envelope glycoprotein⁺ cell.
 

Claim 1 of 3 Claims

1. A monoclonal antibody determined to be capable of specifically inhibiting the fusion of a macrophage-tropic primary isolate of HIV-1 to a CD4+ cell susceptible to infection by a macrophage-tropic primary isolate of HIV-1, but not a T cell-tropic isolate of HIV-1 to a CD4+ cell, using a method which comprises:

a) contacting (i) a first appropriate CD4+ cell, which is labeled with a first dye, with (ii) a cell expressing the HIV-1 envelope glycoprotein of the macrophage-tropic primary isolate of HIV-1 on its surface, which is labeled with a second dye, in the presence of an excess of the antibody under conditions which would normally permit the fusion of the CD4+ cell to the cell expressing the HIV-1 envelope glycoprotein on its surface in the absence of the antibody, the first and second dyes being selected so as to allow resonance energy transfer between the dyes;

b) exposing the product of step (a) to conditions which would result in resonance energy transfer if fusion has occurred; and

c) determining whether there is a reduction of resonance energy transfer, when compared with the resonance energy transfer in the absence of the antibody;

d) contacting (i) a second appropriate CD4+ cell, which is labeled with a first dye, with (ii) a cell expressing the HIV-1 envelope glycoprotein of a T-cell tropic isolate of HIV-1 on its surface, which is labeled with a second dye, in the presence of an excess of the antibody under conditions which would normally permit the fusion of the CD4+ cell to the cell expressing the HIV-1 envelope glycoprotein on its surface in the absence of the antibody, the first and second dyes being selected so as to allow resonance energy transfer between the dyes;

e) exposing the product of step (d) to conditions which would result in resonance energy transfer if fusion has occurred;

f) determining whether there is a reduction of resonance energy transfer, when compared with the resonance energy transfer in the absence of the antibody; and

g) comparing the determination made in step (c) with the determination made in step (f), wherein a decrease in transfer in step (c) but not in step (f) indicates that the antibody is capable of specifically inhibiting fusion of the macrophage-tropic primary isolate of HIV-1 to CD4+ cells, but not capable of specifically inhibiting the fusion of a T cell-tropic isolate of HIV-1 to the CD4+ cells,

wherein the monoclonal antibody is further capable under identical conditions of (a) specifically inhibiting 67% or greater of fusion of a CD4+ PM-1 cell to a HeLa cell expressing envelope glycoprotein from HIV-1_JR-FL, and (b) inhibiting 18% or less of fusion of a CD4+ SUP-T1 cell to a HeLa cell expressing envelope protein from HIV-1_LAI, wherein the antibody (i) does not cross-react with HIV-1 envelope glycoprotein or CD4, (ii) reacts with an antigen on the surface of a PM-1 cell, and (iii) does not react with an antigen on the surface of a SUP-T1 cell.

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