CA2178495A1 - Methods for using resonance energy transfer-based assay of hiv-1 envelope clycoprotein-mediated membrane fusion, and kits for practicing same - Google Patents

Abstract

The subject invention provides methods for determining whether an agent is capable of either inhibiting or specifically inhibiting the fusion of CD4+cell with SSD 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 . This imvention also provides methods for quantitatively determining the ability of an antibody containing sample to either inhibit or specifically inhibit the fusion of CD4+cell with an HIV-1 envelope glycoprotein+cell.

Description

Wo 95/16789 2 1 7 8 ~ 9 5 pcr/uss4/l~56l .

NETHQDS FOR USING ._E 1- ENERGY TRANSFER-BASED ASSAY
OF T~TV-l ENVETnP~ GL~:u~KuL~lN-NEDIATED M~D~r~ FUSION
5 ~D ~ITS FOR PRACTICING SAME
Backqround of the Invention Throughout this application, various publications are 10 referenced. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains .
15 HIV infects primarily helper T lymphocytes and monocytes/
macrophages--cells that express surface CD4--leading to a gradual loss of immune function which results in the dev~ , -nt of the human acquired immune deficiency syndrome (AIDS) . The initial phase of the HIV
20 replicative cycle involves the high affinity interaction between the HIV exterior envelope glycoprotein gpl2 o and the cellular receptor CD4 ~ t7~-nn, D.R., et al., T n~, Rev. 2, 43-66 (1990) ) . Following the attachment of HIV to the cell surf ace, viral and target 25 cell membranes fuse, resulting in the introduction of the viral genome into the cytoplasm. Several lines of evidence demonstrate the requirement of this interaction for viral infectivity. In vitro, the introduction of a functional cDNA encoding CD4 into human cells which do 30 not normally express CD4 is sufficient to render these otherwise resistant cells susceptible to HIV infection (Maddon, P.J., et al, Cell 47, 333-348 ~1986) ) .
Characterization of the interaction between HTV gpl20 and 35 CD4 has been facilitated by the isolation of cDNA clones encoding both molecules (Maddon, P.J., et al., Cell 42, 93-104 (1985), Wain-E~obson, S., et al., Cell 40, 9-17 WO 95/16789 ~ 8 i~ ~ ~ PCT/US94/14561 (1985) ) . CD4 is a nonpolymorphic, lineage-restricted cell surface glycoprotein that is a member of the immunoglobulin gene suRerfamily. High-level Pxpre6sion of both full-length and truncated, soluble versions of 5 CD4 (sCD4) have been described in stable expression systems. The availability of large ~uantities of purif ied sCD4 has permitted a detailed understanding of the structure of this complex glycoprotein. Mature CD4 has a relative molecular weight of 55,000 and consists of 10 an amino-terminal 372 amino acid extracellular domain rnnt;~; n; n~ four tandem immunoglobulin-like regions denoted V1-V4, followed by a 23 amino acid tr~nr ~"dlle domain and a 38 amino acid cytoplasmic segment.
Experiments using truncated sCD4 proteins demonstrate 15 that the determinants of high-affinity binding to HIV
gpl20 lie within the amino-terminal immunoglobulin-like domain V1 (Arthos, J., et al., Cell 57, 469-481 (1989) ) .
Mutational analysis of V1 has defined a discrete gpl20-binding site (residues 38-52 of the mature CD4 protein) 20 that comprises a region structurally homologous to the second complementarity-detPrmin;n~ region (CDR2~ of n~lobulins (Arthos, J., et al., Cell 57, 469-481 (1989) ) .
25 The HIV-1 envelope gene env encodes an envelope glycoprotein precursor, gpl60, which is cleaved by cellular proteases before trarLsport to the plasma membrane to yield gpl20 and gp41. The membrane-sr~nn;ng glycoprotein, gp41, is non-covalently associated with 3 0 gpl2 0, a purely extracellular glycoprotein . The mature gpl20 molecule is heavily glycosylated (approximately 24 N-linked oligosaccharides), t nn~;nc approximately 480 amino acid residues with 9 intra-chain disulfide bonds (Leonard, C.K., et. al., J. Biol. Chem. 265, 10373-10382 35 (1990) ), and projects from the viral membrane as a Wo 95/16789 ~ ~ ~ 3 ~ 9 ~ PCTr[JS94rl4561 dimeric or multimeric molecule (Earl, P.L., et. al. Proc.
Natl. Acad. Sci. U.S.A. 87, 648-652 ~1990) ) .
~ llt~t;~n~l studies of HIV-1 gpl20 have delineated 5 important functional regions of the molecule. The regions of gpl20 that interact with gp41 map primarily to the N- and C- termini ~Helseth, E., et. al., J. Virol.
65, 2119-2123 ~1991) ) . The pr~l( n~nt strain-specific neutralizing epitope on gpl20 is located in the 32-34 10 amino acid residue third variable loop, herein referred to as the V3 loop, which resides near the center of the gpl20 se~uence ~Bolognesi, D.P. TIBTech 8, 40-45 ~1990) ) .
The CD4-binding site maps to discontinuou3 region6 of gpl20 that include highly conserved or invariant amino 15 acid re6idues in the second, third, and fourth conserved domains ~the C2, C3 and C4 domains) of gpl20 ~Olshevsky, U., et al. J. Virol. 64, 5701-5707 ~1990) ) . It has been po3tulated that a small pocket formed by these conserved residues within gpl20 could ~ ' te the CDR2 loop of 20 CD4, a region defined by mutational analyses as important in interacting with gpl20 ~Arthos, J., et al., Cell 57, 469-481 ~1989) ) .
Following the binding of HIV-l gpl20 to cell surface CD4, 25 viral and target cell membranes fuse, resulting in the introduction of the viral capsid into the target cell cytoplasm ~Maddon, P.J. et al ., Cell 54 :865 ~1988) ) .
Most evidence to date indicates that HIV-1 fusion is pH-ln~l~r~nrl,ont and occurs at the cell surface. The HIV-1 30 fusion protein is gp41, the tr~n~ le component of the envelope glycoprotein. This protein has a hydrophobic fusion peptide at the amino-terminus and mutations in this peptide inhibit fusion ~owalski, M. et al., Science 237:1351 ~1987)). In addition to gp41, 35 r~cent observ~tions suggest that gpl20 plays a role in Wo 95/16789 ~ ~ ~ 8 4 9 ~ PCTIUS94/14561 membrane fusion distinct from its function in attachment.
For example, ~nt;ho~;es to the principle neutralizing epitope on gpl2 0, the V3 loop, can block inf ection without inhibiting att~ t (Skinner, M.A. et al., J.
Virol. 62 :4195 (1988) ) . in addition, mutations in the tip of this loop reduce or prevent syncytia formation in HeLa-CD4 cells expressing the mutated gpl20/gp41 molecules ~Freed, E.O. et al., J. Virol. 65:190 (1991)) .
Several lines of evidence have implicated molecules in addition to CD4 and gpl20/gp41 in HIV-l induced membrane fusion. For example, recent studies have indicated that human cells may contain an accessory molecule, not present in non-primate cells, which is re~uired for HIV-l fusion (Dragic, T. et al_, J. Virol. 66:4794 (1992) ) .
The nature of this accessory molecule or molecules is unknown. While some studies have postulated it might be a cell surface protease (Hattori, T. et al, Febs. Lett.
248:48 (1989) ), this has yet to be confirmed.
Fusion of the HIV- l virion with the host cell plasma membrane is mimicked in many ways by the fusion of HIV-l infected cells expressing gpl20/gp41 with uninfected cells expressing CD4. Such cell-to-cell fusion results in the formation of multinucleated giant cells or syncytia, a phPnl observed with many viruses which fuse at the cell surface. Much of our current understanding of HIV-l-induced membrane fusion is derived from studies of syncytium formation. For example, thls approach was used to demonstrate that expression of HIV-l gpl20/gp41 in a membrane, in the absence of any other viral protein, iæ necessary and sufficient to induce fusion with a CD4 membrane (Lifson, J.D. et al., Nature 323 :725 (1986) ) .

Wo 95116789 ~ PCT/US94/14561 Compared with virion fusion to cells, syncytium formation induced by HIV-1 appears to involve an additional step.
First, the gpl20/gp41-bearing membrane fuses with the CD4- bearing membrane. This i5 a rapid and reversible 5 process which connects the membranes at localized sites and allows membrane-bound dyes to flow from one cell to the other (Dimitrov, D. et al., AIDS Res. Human Retroviruses 7:799 (1991) ) . This step presumably parallels the att~ t, of a virion to a CD4~ cell and 10 the fusion therebetween. The second stage in cells fusion is the irreversible fusion of cells to form syncytia. The efficiency of this process i5 increased by the interaction of cellular adhesion molecules such as ICAM-l and LFA-l, although these molecules are not 15 absolutely re~auired for syncytium formation to proceed (Golding, H. et al ., AIDS Res. Human Retroviruses 8 :1593 (1992) ) .
Most of the studies of ~IIV-l fusion, including those 20 discussed above, have been performed with strains of HIV-1 which have been extensively propagated in transformed human T cell lines. These strains, known as laboratory-adapted strains, differ in several important characteristics from primary or clinical isolates of the 25 virus obtained from HIV-l infected individuals (O' ~rien, W.A. et al., Nature 348:69 (1990)) . Some examples of these dif f erences are listed in the table below .

W0 9S/16789 2 ~ 9 ~ PCrNS94/14561 Laboratory adapted Primary Isolates Strains tropic for transformed many are tropic for ~
T cell lines, do not primary monocytes and do inf ect primary monocytes not inf ect transf ormed T cell lines very sensitive to relatively insensitive to neutralization by neutrali2ation by sCD4 soluble CD4 gpl20 spr,nt~n,,Q-lcly little spontaneous stripping dissociates from gp41, and sCD4 only causes and this stripping is stripping at 4C, not increased by sCD4 at 37C
These differences are mirrored by differences in the primary sequence of the viral proteins, and in particular of the envelope glycoproteins. In some cases, the dif f erent tropisms of primary isolates and laboratory-adapted strains of HIV-1 have been mapped to regions on gpl20 such as the V3 loop (0' Brien, W.A. et al., Nature 348:69 (1990)). It is possible that different V3 loops interact with different accessory molecules on ~T cell lines or monocytes, thereby mediating tropism.
HIV-1 envelope-mediated cell fusion is a model for the early stages of XIV- 1 i~f ection and can be used as an assay for anti-viral molecules which block HIV-1 att~rl -n~ and fusion (Sodroski, J. et al., Nature 322-470 (1986), Lifson, J.D. et al., Nature 323 :725 (1986) ) .
Moreover, HIV-1 induced cell fusion is important in its own right as a potential mechanism for the pathogenesis of HIV-l infections. It is a mode of transmission of HIV-l from infected to uninfected cells ~Gupta, P. et al., J. Virol. 63:2361 (1989), Sato, H. et al., Virology 186:712 (1992) ) and by this mechanism, it could contribute to the spread of HIV-1 throughout the body of Wo95/16789 ~ 7~ PCr/US94/14561 the infected individual. Cell fusion is also a direct -h~ni ~m of HIV-l-induced cell death ~Sodroski, J. et al., Nature 322:470 (1986), Lifson, J.D. et al., Nature 323:725 (1986) ) . Syncytia are seen in vivo, notably in 5 the brains of AIDS patient6 suffering from neurological 7;rationS guch as AIDS 1 t;~ complex (Pumarola-Sune, T. et al., Ann. Neurol. 21 :4gO (1987) ) . In addition, syncytia have been observed in the spleen~ of HIv-l-infected individuals (Byrnes, R.K. et al., JAMA
250:1313 (1983) ) . It is ~ossible that cell fusion may play a role in the depletion of CD4 T lymphocyte6 that is characteristic of the pathogenic process leading to AIDS (Haseltine, W.A. in AIDS and the new viruses, Dalgleish, A.G. and Weiss, R.A. ed6. (1990) ) .
In this context, it may be signif icant that ~IV- 1 isolates from asymptomatic HIV-l-infected individuals often infect cells in vitro without inrlll;nJ syncytia.
In contrast, clinical isolates from patients with ARC and AIDS are commonly highly virulent, gyncytia-;n~l-lr;n, strains (Tersmette, M. et al ., J. Virol . 62 :2026 (1988) ) .
In addition, there is often a switch from non-syncytium ;nf~llr;nJr (NSI) to syncytium-inducing (SI) isolates within patients as the disease progresses and symptoms appear (Tersmette, M. et al ., J. Virol. 63 :2118 (1989), Cheng-Mayer, C. et al., science 240:80 (1988) ) . It is not clear why some HIV-1 strains do not induce syncytia, although it is possible that cells infected with these strains do not express sufficient levels of gpl20/gp41 for cell fusion to occur, by analogy with some other fusogenic viruses. However, it is believed that this switch from NSI to SI HIV-1 strains influences the clinical course of HIV-1 infection. The presence of naturally occurring anti-syncytia antibodies in some subjects may delay the dev~lnF-^nt of HIV-1 related WO 95116789 ~ 9 ~ PCTIUS94114561 diseases in these subjects (Brenner, T.J. et al., Lancet 3 3 7 : 1 0 01 ( 1991 ) ) .
The development of methods for measuring HIV-l envelope 5 glycoprotein-mediated membrane fusion serves a useful role in further ~ ;n~ the I ~h~n;~m of: HIV-l infection, and ~nahl;n~ the ;r~n~;f;r~tion of agents which alter HIV-l envelope glycoprotein-mediated cell fusion. At present there exist several potential methods 10 for measuring such fusion.
The first is an assay of HIV-l envelope glycoprotein-mediated cell fusion in which fusion is measured microscopically by measuring the transfer of fluorescent 15 dyes between cells (Dimitrov, D.S., et al., AIDS Res.
Human Retroviruses 7: 799-~05 (1991) ) . This technique measures dye distribution rather tha~ fluorescence intensity and as such cannot be performed using f luorometer . The assay would not be easily automated 20 and has not been performed with cells which stably express the HIV- 1 envelope glycoprotein.
The second is an assay for HIV-l envelope-mediated cell fusion. measured between (a) cells which stably express 25 the HIV-l tat gene product in addition to gpl20/gp41, and (b) CD4~ cells which contain a construct consisting of the lac~qidase gene under the control of the HIV-l LTR
promotor. When these cells fuse, ,B-galactosidase is expressed a~d can be measured using an cl~LU~Liate 30 soluble or insoluble ~:11L~ ,_lliC substrate (Dragic, T., et al., Journal of Virology 66:4794 (1992)) . This assay takes at least 1 day to perform and cannot easily be adapted to new target cells such as primary macrophage cells. This assay also does not measure cell fusion in 35 real time and is thus not amenable to use in analyzing W0 95~16789 ~ PCT/US94/14561 fusion kinetics.
Finally, the third is a fluorescence deql~Pn~-h-ns assay for the fusion of HIV-1 virions to cells (Sinangil, F., et al., FEBS Letters 239:88-92 ~1988) ) . This assay requires the use of purified HIV-1 virions, and both the purification of HIV-1 virions and the assay must be performed in a r~nt~1 facility. It would be difficult to readily isolate sufficient quantities of clinical virus isolates to perform the assay.
Furth~ e, this assay i~ more complicated and less reproducible than a RET assay using cells which qtably express HIV- 1 envelope glycoproteins and CD4 .
lS The methods of the sub; ect invention employ a resonance energy transfer (RET) based assay which ~JV~ C~ -'' the problems inherent in the above-identified methods for measuri~g HIV-1 envelope glycoprotein-mediated membrane fusion. Specifically, the methods of the subject invention employ a RET assay which is rapid, reproducible, quantitative, adaptable to various cell types, and relatively safe, and can be automated.

Wo 95/16789 2 ~ ~ ~ q 9 5 PCT/US94/14561 r - of the Invention The subject invention provides a method for r~f~t~rminin~
5 whether an agent is capable of specifically inhibiting the fusion of a CD4+ cell with an HIV-l envelope glycoprotein~ cell which comprises: (a) ~nnt~ctin~ a sample c~nt~ining a suitable amount of ~the agent with a suitable amount of the CD4 ' cell and a suitable amount of 10 the HIV-l envelope glycoprotein+ cell under conditions which would permit the fusion of the SD4+ cell with the HIV- l envelope glycoprotein+ cell in the absence of the agent, the cell membranes of the CD4t cell and the HIV-l envelope glycoprotein' cell being labeled with a first dye 15 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 transf er value of the resulting sample after a suitable period of time; (c) 20 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-l envelope glycoprotein+ cell; and (d) determining whether the agent inhibits the fusion of 25 a first control cell with a second control cell under conditions which would permit non-XIV-l 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 speci~ically 30 inhibiting the fusion of the CD4+ cell with the XIV-l envelope glycoprotein+ cell.
The subject invention also provides a method for determining whether an agent is capable of specif ically 35 inhibiting the infection ~of a CD4+ cell with HIV-l Wo 95/~6789 ~ j PCr/US94/14561 which comprises ~l~t~rTr;nin~ whether the agent is capable of specifically inhibiting the fusion of a CD4' cell with an HIV-l envelope glycoprotein+ cell by the method of the subject invention, so as to thereby determine whether the 5 agent is capable of spe~~;f;c~lly inhibiting the infection of a CD4+ cell with HIV-l.
The 8ubject invention further provides a method for det~ n;n~ whether an agent is capable of inhibiting the lO fusion of a CD4+ cell with an HIV-l envelope glycoprotein+
cell which comprises: (a) contacting a sample r~nt~ining a suitable amount of the agent with a suitable amount of the CD4 ' cell and a suitable amount of the ~IV- l envelope glycoprotein~ cell under conditions which would permit the 15 fusion of the CD4+ cell with the HIV-l envelope glycoprotein~ cell in the absence of the agent, the cell membranes of the CD4' cell and the HIV-l envelope glycoprotein ' cell being labeled with a f irst dye and a second dye, respectively, which fir8t and second dyes 20 permit r,~cr~n~n--e energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent r.o~f,n~n~ ~ energy transfer value of the resulting sample after a suitable period of time; and (c) comparing the percent resonance energy transfer value so determined 25 with a known standard, so as to determine whether the agent is capable of inhibiting fusion of the CD4+ cell with the HIV- l envelope glycoprotein~ cell .
This invention also provides an agent determined by the 30 above-described method.
The subject invention further provides a method for quantitatively determining the ability of an antibody-~ nt~in;n~ sample to specifically inhibit the fusion of 35 a CD4~ cell with an HIV-l envelope glycoprotein~ cell Wo 9~/16789 ~ t ~ ~ ~ 9 ~ PCr/US94/14561 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- l envelope glycoprotein~ cell under conditions which would 5 permit the fusion of the CD4 cell with the HIV-l envelope glycoprotein' cell in the absence of the antibody-contA;n;ng sample, the cell - d1~es of the CD4~ cell and the HIV- l envelope glycoprotein+ cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit resonance er,ergy 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 15 energy transfer value 80 determined with a known standard, 80 as to quantitatively determine the ability of the antibody-cnntA;n;ng sample to inhibit the fusion of the CD4 ' cell with the HIV- l envelope glycoprotein+
cell; and (d) det,orTn;n;n~ whether the antibody-cnntA;n;n~
20 sample inhibits the fusion of a first control cell with a second control cell under conditions which would permit non-HIV-l envelope glycoprotein-r-~ At.-~ fusion of the f irst and second control cells in the absence of the agent, 80 as to quantitatively determine the ability of 25 the antibody-~nntA;n;n~ sample to specifically inhibit the fusion of the CD4' cell with the HIV-l envelope glycoprotein~ cell.
The sub]ect invention further provides a method for 30 quantitatively det~orrn;n;n1 the ability of an antibody-cnntA;n;n~ sample to inhibit the fusion of a CD4 cell with an HIV-l envelope glycoprotein~ cell which comprises (a) contacting a predetermined amount of the antibody-cnntA;n;n~ sample with a suitable amount of the CD4~ cell 35 and a suitable amount o~ the HIV-l envelope glycoprotein~

WO95116789 _l3_ PCT/U594n4~61 cell under conditions which would permit the fusion of the CD4 ' cell with the HIV- l envelope glycoprotein~
cell in the absence of the antibody-rnnt~;n;n~ sample, the cell membranes of the CD4 ' cell and the HIV- l 5 envelope glycoprotein' cell being labeled with a first dye and a second dye, respectively, which first and second dyes permit r~nn~nre energy transfer therebetween only when juxtaposed within the same membrane; (b) determining the percent resonance energy transfer value of the lO 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-cnnt~;n;nr sample to inhibit the fusion of the CD4+ cell with the HIV-l 15 envelope glycoprotein' cell.
The subject invention further provides a method for determining the stage or clinical prognosis of an HIV-l infection in an HIV-l-infected subject which comprises:
20 (a) obtaining an antibody-cnnt~;n;n~ sample from the HIV-l-infected subject; (b) quantitatively detf-~n;nin~ the ability of the antibody-crnt~;n-nr sample so obtained to inhibit the fusion of a CD4t cell with an HIV-l envelope glycoprotein~ cell by the method of the subject invention;
25 and (c) comparing the ability of the antibody-cnnt~;n1ng sample to inhibit the fusion of the CD4~ cell with the HIV-l envelope glycoprotein' cell so determined with that of an antibody-cnnt~;n;n~ sample obtained from an HIV-l-infected subject having an HIV-l infection at a known 30 stage or having a known clinical prognosis, so as to determine the stage or clinical prognosis of the HIV-l inf ection in the HIV- l - inf ected sub j ect .
The subject invention further provides a method for 35 ~l~t~rm;n;nr the efficacy of an anti-HIV-l vaccination in W095/16789 ~1 7a495 PCT/US94/14561 a vaccinated, non-HIV-l-lnfected subject which comprises:
(a) obtaining an antibody-~ nt~;n;n~ sample from the vaccinated, non-HIV-l-infected subject; (b) ~uantitatively determining the ability of the antibody-5 r~ntA;n;n~ sample 80 obtained to inhibit the fusion of aCD4' cell with an HIV-l enYelope glycoprotein' cell by the method of the subject invention; and (c) comparing the ability of the antibody-c~7nt~;n;ng sample to inhibit the fusion of the CD4~ cell with the HIV-l envelope lO glycoprotein~ cell 80 determined with that of an antibody-,-nntA;n;ng sample obtained from a vaccinated, non-HIV-l-infected subject for whom the anti-HIV-l v~c;n;~ti- n has a known efficacy, so as to determine the efficacy of the anti-HIV-l vaccination in the VA~'~';n~te~, non-HIV-l-15 infected subject.
The subject invention further provides a kit fordetermining whether an agent is capable of specifically inhibiting the fusion of ~ a CD4~ cell with an E~IV-l 20 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; (b) a suitable amount of an HIV-l envelope glycoprotein~ cell whose cell membrane is labeled with a second dye, the HIV-l envelope 25 glycoproteint cell being capable of fusing with the CD4 cell of (a) under suitable r-~n~l; t; r~n.q 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 30 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-l envelope glycoprotein-mediated fusion with the 35 first control cell of (c) under suitable conditions in Wo 95116789 ~ 3 PCTiUS94/14561 the absence of the agent.
The subject invention further provides a kit for determining whether an agent is capable of inhibiting the 5 fusion of a CD4' cell with an HIV-l envelope glycoprotein~
cell which comprises, in 5eparate 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-l envelope glycoprotein' cell whose cell membrane is lO labeled with a 9econd dye, the HIV-l envelope glycoprotein' cell being capable of fusing with the CD4 ' cell of (a) under suitable conditions in the absence of the agent, and the f irst and second dyes permitting resonance energy transfer therebetween only when 15 juxtaposed within the same membrane.
The subject invention further provides a method for det~r~; n1 ng whether an HIV-l isolate is syncytium-; n~ ; ng which comprises: (a) obtaining a sample of an 20 HIV-l 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 CD~t cell under conditions which would permit the fusion of the CD4' cell with a syncytium-;n~l11c;n~ HIV-l strain 25 envelope glycoprotein~ cell, the cell membrane of the CD4' cell being labeled with a second dye which permits resonance energy tran9fer between the first dye only when the first and second dyes are juxtaposed within the same membrane; (c) cl~t~ n;n~ the percent r.oC~n~n~ energy 30 transfer value of the resulting sample after a suitable period of time; and (d) comparing the percent resonance ener~y transfer value so det--rm;n~-l with a known standard, so as to determine whether the HIV-l isolate is syncytiurn-; nr~ ; ng .

Wo 95/16789 2 1 ~ ~ ~ 9 5 PCT/US9411~561 Finally, the subject invention provides a method for determining the stage of an HIV-l infection in an HIV-l-infected Eubject which comprises determining by the method of the subject invention whether the HIV-l isolate 5 with which the HIV-l infected subject is infected is syncytium ;n~ll1c;n~, so as to thereby determine the stage of the HIV-l infection in the ~IIV-l-infected subject.

WO 95/16789 _ 1 7 _ PCrlUS94/14561 8~ief De~ori~tion o~ the Fiqure~
Fiaure 1 Time course of fusion between HeLa-env' cells and HeLa-5 CD4~ cells measured by the RET assay.
Fiaure 2 Blocking of fusion between HeLa-env' cells and HeLa-CD4+
cells by OKT4a, measured using RET.

Fiqure ~
Blocking of fusion between 160G7 cells and C8166 cells by sCD4, measured using RET.
15 Fiaure 4 A comparative analysis of results of blocking experime-~s by two methods using OKT4a to inhibit the fusion of HeLa-env' and ~IeLa-CD4' cells.

Wo 95/16789 ~ t ~ ~ ~ q 5 ~'CT/US94/14561 Detailed De~cri~tion of the Invention The plasmid designated pMA243 was deposited pursuant to, and in satisfaction of, the re~uirements of the Budapest 5 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, Maryland 20852 under ATCC
Accession No. 75626. The plasmid pMA243 was deposited with the ATCC on December 16, 1993.
The sub; ect invention provides a method f or determining whether an agent is capable of specif ically inhibiting the fusion of a CD4' cell with an HIV-1 envelope 15 glycoprotein' cell which comprises: (a) contacting a sample c~t~;nin~ 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 20 HIV-1 envelope glycoprotein~ cell in the absence of the agent, the cell ' ~lles 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 25 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 r~C-~n~n~ energy transfer value 80 detprTl~; nf~ with a known standard, so as to determine 30 whether the agent is capable of inhibiting fusion of the CD4t 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 35 glycoprotein-mediated fusion of the first and second Wo 9S116789 ~ 9 5 PCr/US94/14561 control cells in the absence of . the agent, 80 as to determine whether the agent is capable of specifically inhibiting the fusion of the CD4+ cell with the HIV- l envelope glycoprotein' cell This invention provides an agent determined to be capable of sp~ l l y inhibiting the fusion of a CD4+ cell with an HIV-l envelope glycoprotein' cell using the above-described method.

As used herein, the term "agent" ;n~ C both protein and non-protein moieties. In one embodiment, the agent is a small molecule. In another etnbodiment, the agent is a protein. The protein may be, by way of example, an 15 antibody directed against a portion of an HIV-l envelope glycoprotein, e.g., gpl20. The agent may be derived from a library of low 1 ec~ r weight ~ or a library of extracts from plants or other organisms.

20 As used herein, "capable of specifically inhibiting the fusion of a CD4+ cell with an HIV-l envelope glycoprotein~
cell" means (a) capable of reducing the rate of fusion of CD4 ' cell membrane with HIV- l envelope glycoprotein+ cell membrane by at least 596, but not capable of reducing the 25 rate of non-CD4/HIV-l envelope glycoprotein-mediated cell membrane fusion, or (b) capable of reducing by at least 5~ the total amount of fusion of CD4 ' cell membrane with HIV- l envelope glycoprotein' cell membrane occurring by the endpoint of fusion, but not capable of reducing the 30 total amount of non-CD4/HIV-l envelope glycoprotein-mediated cell membrane fusion occurring by the endpoint of fusion. As used herein, the rate of cell membrane fusion means the total ~uantity of cell membrane fused per unit of time. As used herein, the "endpoint of 35 fusion" mean5 the point in time at which all fusion of WO 95/16789 ~ t 7 8 ~ ~ ~ PC'r/US94/14561 CD4+ cell membrane with HIV-l envelope glycoprotein+ cell membrane capable of occurring has occurred.
An example of the method of the sub; ect invention is 5 provided ini~- A known amount of CD4' cell is contacted with a known amount of HIV-l envelope glycoproteint 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 eriual lO to the sum of the amounts of CD4' cell membrane and HIV-l envelope glycoprotein' cell membrane, e.g., 0.5 x Y CD4 cell membrane + 0 . 5 x Y HIV-l envelope glycoprotein` cell membrane . In the presence of the agent, 0 . 2 x Y amount of cell membrane fuses per unit of time. The agent is 15 shown not to reduce the rate of non-CD4/HIV-l envelope glycoprotein-mediated cell membrane fusion. Accordingly, the agent specifically inhibits the fusion of a CD4+ cell with an HIV-l envelope glycoprotein+ cell.
20 As used herein, the fusion of CD4' cell membrane with HIV-envelope glycoprotein' cell membrane means thehydrophobic joining and integration of CD4+ cell membrane with HIV-l envelope glycoprotein+ cell membrane to form a hybrid membrane comprising components of both cell 25 membranes, and does not mean the CD4/HIV-l envelope glycoprotein-mediated adhesion therebetween, which adhesion is a prereriuisite for the fusion.
As used herein, the term "CD4" lnrl~ c (a) native CD4 30 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 af native CD4 which is reriuired for native CD4 to form a complex with the HIV-l 35 gpl20 envelope glycoprotein. In one embodiment, the CD4-WO95116789 2 ~ PCT/US94/14561 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 re~uired for native CD4 to form a complex with the HIV-1 gpl20 5 envelope glycoprotein i5 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 HeBa cell, and a non-biological cell, e.g., 10 a lipid veeicle (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, where~n the CD4' cell is capable of specifically binding to and fusing with an 15 HIV-1 envelope glycoprotein~ cell exposed thereto. In the preferred embodiment, the suitable CD4 cell is a CD4+
HeLa celI.
As used herein, an HIV-1 envelope glycoprotein~ cell is 20 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 a CD4 cell exposed thereto. In one embodiment, the HIV-1 envelope glycoprotein' cell is an HIV-1 envelope 25 glycoprotein~ HeLa cell. I+n 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 30 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 CD4t cell. For example, 35 the HIV-1 isol-tes JR-FL, JR-CSF and BaL are tropic for Wo 95/16789 2 1 ~ ~ ~ 9 ~ PCTIUS94/l456l CD4' primary human macrophages, the HIV-1 isolates LAI
and IIIB are tropic for human CD4+ ~ 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 5 ~IV-1 isolates JR-FL, JR-CSF, BaL, LAI, IIIB, MN and SF-2 may also be tropic f or CD4~ cell types other than those enumerated su~a.
The suitable amounts of agent, CD4~ cell and HIV-1 10 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 CD4' cell with the HIV- 1 envelope glycoprotein~ cell in the absence 15 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 ~_ ; n~l ed with the lipid 20 molecules of its cell ' a.le.
Resonance energy transfer = is def ined as follows: For juxtaposed dyes D1, having excitation and emission ~pectra Exl and Eml, respectively, and D2, having 25 excitation and ~m; CF:I; nn spectra Ex2 and Em2, respectively, wherein (a) Eml has a higher average frequency than that of Em2 and (b) Eml and Ex2 overlap, r.~cnnz3nre ener~y transfer is the transfer of electromagnetic energy fr~m D1 to D2 at a frequency 30 within the Eml and Ex2 overlap, which resonance energy transfer (a) results from the electrr-~gnr~; c excitation of D1 at a frequency within the Exl spectrum and (b) causes the subsequent emission of electromagnetic energy from D2 at a frequency within the Em2 spectrum.
35 Accordingly, resonance energy transfer between D1 and D2 Wo95/16789 2 ~ PCr/US94/14561 .

can be detected by exciting Dl with electromagnetic energy at a frequency within Exl and measuring the subsequently emitted electromagnetic energy at a frequency within Em2, the emission of electromagnetic 5 energy at a frequency within Em2 indicating the O~ UL ' ence of resonance energy tran3fer between Dl and D2 .
The first and second dyes are ~ juxtaposed within the same lO membrane" if they are present within the 3ame lipid membrane at a suitably short distance ~rom each other, which suitably short distance may be readily determined by one skilled in the art.
15 In the 3ubject invention, detPrm;n;n~ the percent rP~nnAn~~P energy transfer value may be performed according to methods well known to those skilled in the art. In one . ' o-l;~~ , the percent resonance energy transf er value is determined by: ( l ) determining the 20 resonance energy transfer value (RET) by subtracting from the total emission from Dl and D2 at a frequency within Em2 the ele~:Ll~ ~nPtiC energy emission due to direct Dl and D2 emission following P~r-; tAtion at a frequency within Exl and emission at the frequency within Em2, 25 which Dl 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 (~6 RET value) by dividing the resonance energy transfer value obtained in 30 step (l) 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 35 is ~PtPrrn;np'l may be determined according to methods well 2 1 7 g ~ ~ 5 PCTIUS94/14561 known to those skilled in the art.
The known standard is a percent resonance energy transfer value obtained using the CD4 ' cell, the HIV- l envelcpe 5 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 lO via non-HIV-l envelope glycoprotein-mediated fusion both in the presence and absence of an agent capable of inhibiting HIV-l envelope glycoprotein-mediated fusion, and are not capable of fusing via HIV-l envelope glycoprotein-mediated fusion. Such cells are will known 15 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 37C with polyethylene glycol lO00 or with Sendai virus. These methods of inducing fusion of He~a cells are well known to those skilled in 2 o the art .
In one embodiment, the agent is an antibody. As used in the subject invention, the term "antibody~ includes, but i5 not limited to, both naturally occurring and non-25 naturally occurring antibodies . Specif ically, the term"antibody" includes polyclonal and monoclonal ~nt;hor~;es, and antigen-binding fragments thereof.
Furthermore, the term "antibody" includes chimeric antibodies, wholly synthetic antibodies, and antigen-3 0 binding ~ - _ t s thereof .~
In one embodiment, the f irst dye is a rhodamine moiety-cnnt~;n;nr~ molecule and the second dye is a fluorescein moiety-r~7ntA;n;nr~ molecule. Rh~ m;n,o moiety-containing 35 molecules and fluorescein moiety-containing molecules are Wo 95J16789 ~ 5 PCT/US94114561 well known to those skilled in the art.
In the preferred .omhn~ , the rhnri~m1n~ moiety-~ nt~;n;n~ molecule is octadecyl rhnri~m;n~ B
chloride and the fluorescein moiety-cnnt~in;n~ molecule i6 fluorescein octadecyl ester.
In another c-'~o~; ', the first dye is a fluorescein moiety-cnnt~;n;n~ molecule and the second dye is a lD rhodamine moiety-cnnt~; n; n~ molecule.
In one ~mhn~; t, the CD4' cell i8 a CD4~ HeLa cell. In another ~ ; t, the HIV-l envelope glycoprotein~ cell is an HIV-ll,~ gpl20/gp41' HeLa cell. HIV-lL1,I is a 15 laboratory-adapted strain that is tropic for phytnh ~glutinin (PHA)-stimulated peripheral blood lymphocytes ~PBLs) and immortalized human T-cell lines.
The sub; ect invention also provides a method f or 20 ~l~t~rm;n;ng whether an agent is capable of specifically inhibiting the infection of a CD4t cell with HIV-l which comprises ~ t~orm;n;n~ whether the agent is capable of specifically inhibiting the fusion of a CD4' cell with an HIV-l envelope glycoprotein' cell by the method o~ the 25 subject invention, 80 as to thereby determine whether the agent is capable of specifically inhibiting the infection of a CD4+ cell with HIV-l.
The subject invention further provides a method for 30 det~rm;n;n~ whether an agent is capable of inhibiting the fusion of a CD4' cell with an HIV-l envelope glycoprotein~
- cell which comprises: (a) contacting a sample ~nnt~;n;ng a suitable amount of the agent with a suitable amount o~
the CD4~ cell and a suitable amount of the HIV-l envelope 35 glycoprotein~ cell under conditions which would permit the ~ 1 7349~
Wo 95116789 PCrlUS94/14561 .

fusion of the CD4~ cell with the XIV-l envelope glycoprotein' cell in the absence of the agent, the cell membranes of the CD4 ' cell and the HIV-l envelope glycoproteint cell being labeled with a f irst dye and a 5 second dye, respectively, which first and second dyes permit resonance energy transf er therebetween only when juxtapo6ed 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 10 the percent rPA~m~nr P energy transfer value 80 determined with a known standard, 80 as to determine whether the agent is capable of inhibiting fusion of the CD4~ cell with the HIV-l envelope glycoprotein~ cell.
15 As used herein, "capable of inhibiting the fusion of 8 CD4 cell with an ~IIV-l envelope glycoprotein~ cell~ means capable of (a) reducing the rate of fusion of CD4 ' cell membrane with HIV-l envelope glycoprotein' cell membrane by at least 596, or (b) reducing by at least 59~ the total 20 amount of fusion of CD4' cell membrane with HIV-l envelope glycoprotein~ cell membrane occurring by the endpoint of fusion. An agent capable of inhibiting the fusion of a CD4~ cell with an HIV-l envelope glycoprotein' cell may also be capable of reducing the rate to non-CD4/HIV-l 25 envelope glycoprotein-mediated cell membrane ~usion.
This invention provides an agent determined to be capable of inhibiting the fusion of a CD4 ' cell with an ~IIV- 1 envelope glycoprotein' cell using the above-described 3 0 method In one embodiment, the first dye is a rhodamine moiety-,-r,ntA;ning molecule and the second dye is a fluorescein moiety-c~n~Ai ni n~ molecule.

~ WO95/16789 2 1 784 ~5 PCI/US94/14561 In the pre~erred embodiment, the rhodamine moiety-rnnt~;n;n~ molecule ig octadecyl rh~m;n,~ B chloride and the fluorescein moiety-r~nt~;n;n~ molecule is fluorescein octadecyl ester.

In another: ' ~'; ~, the first dye is a fluorescein moiety-rnnt~;n;n~ molecule and the second dye is a rhodamine moiety-r~nt~;nin~ molecule.
10 In one ~ ' ~,1; , the CD4 ' cell is a CD4 ' HeLa cell . In another embodiment of the subject invention, the HIV-1 envelope glycoprotein~ cell i8 an HIV-l~A~ gpl20/gp41~ Hel.a cell .
15 The subject invention further provides a method for quantitatively determining the ability of an antibody-rrmt::l;n;n~ sample to specifically inhibit the fusion of a CD4' cell with an HIV-l envelope glycoprotein~ cell which comprises: ~a) r~nt~t;nS a predetermined amount 20 of the antibody-~ nt~;n;n~ 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 CD4t cell with the HIV-l envelope glycoprotein cell in the absence of the 25 antibody-r~mlt~;n;n~ sample, the cell membranes of the CD4' cell and the HIV-l envelope glycoprotein~ cell being labeled with a first dye and a second dye, respectively, which f irst and second dyes permit resonance energy transfer therebetween only when juxtaposed within the 30 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 35 ability of the antibody-rr~nt~i ni n~ sample to inhibit the 21 7~q5 Wo 95/16789 PCrlUS94/14561 fusion of the CD4~ cell with the HIV-l envelope glycoprotein' cell; and (d) det/~rm;nin3 whether the antibody-rnnt~;n;nr~ sample inhibits the fusion of a first control cell with a second control cell under conditions 5 which would permit non-HIV- l envelope glycoprotein-mediated fusion of the first and second control cells in the absence of the agent, so as to ~uantitatively determine the ability of the antibody-rnntz~;n;nrJ sample to specif ically inhibit the f usion of the CD4+ cell with 10 the HIV-l envelope glycoprotein~ cell.
The antibody-rnnt~;n;ng sample may be any antibody-rnnt l;n;nS sample. In one embodiment, the antibody-rnnt~;n;n~ sample is a ~serum sample. In another 15 embodiment, the antibody-cnnt~;n;nrJ sample is an IgG
preparation. Methods of obtaining an antibody-rnnti~;n;ng sample are well known to those skilled in the art.
In one ' ~; t, the first dye is a rhnrl~m;n~ moiety-20 rnntA;n;nr, molecule and the second dye is a fluoresceinmoiety- cn~t ~; n; ng molecule .
In the preferred embodiment, the rhnrl~m;nP moiety-rnntA;n;nr~ molecule i3 octadecyl rhn~l~m;n~ B chloride and 25 the fluorescein moiety-rnnt~;n;n~ molecule is fluorescein octadecyl ester.
In another embodiment, the f irst dye is a f luorescein moiety-rnnt~;n;nr~ molecule and the second dye is a 30 rhodamine moiety-rnnt~;n;nrJ molecule.
In one pmhnrl;r~nt, the CD4~ cell is a CD4' XeLa cell. In another embodiment of the subject invention, the HIV-l envelope glycoprotein' cell is an HIV-lLAI gpl20/gp41~ He~a 35 cell.

095/16~89 2 ~ PCr/US9411456 The subject invention further provides a method for quantitatively determining the ability of an antibody-,-nnt~;n;ng sample to inhibit the fusion of a CD4' cell with an ~IIV-l envelope glycoprotein~ cell which comprises:
5 (a) cnnt~ctin~ a predetermined amount of the antibody-rnnt~;n;n~ sample with a suitable amount of the CD4+ cell and a suitable amount of the ~IIV-l envelope glycoprotein' cell under conditions which would permit the fusion of the CD4+ cell with the ~IV-l envelope ~lycoproteint cell lO in the absence of the antibody-cnnt~;n;ng sample, the cell membranes of the CD4' cell and the HIV-l envelope glycoprotein~ cell being labeled with a f irst dye and a second dye, respectively, which ~irst and second dyes permit resonance energy transfer therebetween only when 15 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 80 determined with a known standard, 80 as to quantitatively determine 20 the ability of the antibody-c~nt~;n;n~ sample to inhibit the fusion of the CD4~ cell with the ~IV-~ envelope glycoprotein' cell.
In one ~mho~; t, the first dye is a rhn-i~m;n~ moiety-25 cnnt~;n;n~ molecule and the second dye is a fluoresceinmoiety-~ nnt~; n; n~ molecule .
In the preferred ~mho~ t, the rhnrl~mi n,o moiety-containing molecule is octadecyl rhn~iAm;n~ B chloride and 30 the fluorescein moiety-t-nnt~;n;ng molecule is fluorescein octadecyl ester.
In another ~mhor~; t, the first dye is a fluorescein moiety-~nntA;n;n~ molecule and the second dye is a 35 rhnrl~m;n~ moiety-cnnt~;n;n~ molecule.

21 78~5 WO 95/~6789 PCrlUS94/14S61 .

In one ' ~ t, the CD4t cell is a CD4t HeLa cell. In another ~mhn~;- of the=subject invention, the HIV-l envelope glycoprotein~ cell is an HIV-l~ gpl20/gp4l HeLa cell .
The subject invention further provides a method for determining the stage of clinical prognosis of an HIV- l infection in an EIIV-l-infected subject which comprises:
(a) obtaining an antibody-rnnt~;n;nr sample from the HIV-lO l-infected subject; (b) riuantitatively determining the ability of the antibody-cnnt~;n;nr~ sample so obtained to inhibit the fusion of a CD4~ cell with an HIV-l envelope glycoproteint cell by the method of the subject invention;
and (c) comparing the ability of the antibody-containing 15 sample to inhibit the fusion of the CD4t cell with the HIV-l envelope glycoproteint cell so determined with that of an antibody-rnnt~in;nr, sample obtained from an HIV-l infected subject having an HIV-l infection at a known stage or having a known clinical prognosis, so as to 20 determine the stage or clinical prognosis of the HIV-l infection in the HIV-l-infected subject.
As used herein, an "HIV-infected subject~ means a subject having at least one of his own cells invaded by HIV-l.
25 In the preferred embodiment, the subject is a human.
The subject invention further provides a method for determining the efficacy of an anti-HIV-l v~rr;n~t;nn in a vaccinated, non-HIV-l-infected subject which comprises:
30 (a) obtaining an antibody-rnnt~;n;n~ sample from the vaccinated, non-HIV-l-infected subject; (b) s~uantitatively det~m;n;nr~ the ability of the antibody-rnnti~;n;ng sample so obtained to inhibit the fusion of a CD4t cell with an HIV-l envelope glycoprotein~ cell by the 35 method of the subject invention; and (c) comparing the wo 9S116789 2 1 7 8 4 9 5 PCT/US94114561 .

ability of the antibody-cnnt~in1nr~ sample to inhibit the fusion of the CD4 ' cell with the HIV-l envelope glycoprotein' cell 80 determined with that of an antibody-containing sample obtained from a vaccinated, non-HIV-l-5 infected subject for whom the anti-HIV-l vaccination has a known efficacy, 80 as to rl~t~ n~ the efiicacy of the anti-HIV-l v~r~r;n~tinn in the vaccinated, non-HIV-l-inf ected sub j ect .
10 As used herein, "anti-HIV-l vaccination~ means the administration to a subj ect 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-l surface envelope 15 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-l 20 vaccination" mean5 the degree to which the vaccination or successive vacclnAtinnc (i.e.,; ;~at;on1 causes the titre of E~IV-l-neutralizing Ant;ho~;es in the vaccinated sub; ect to increase . In other words, the higher the efficacy of an anti-HIV-l v~rr;nct;~-~, the higher the 25 titre of lIIV-l-neutralizing Ant;horl;es in the vaccinated sub; ect .
As used herein, a "non-HIV-l-infected subject" means a subject not having any of his own cells invaded by HIV-l.
30 In the preferred ~~; ~, 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 a CD4' cell with an HIV-l 3~ envelope glycoprotein' cell which comprises, in separate 2~ 9~
Wo 95/16789 Pcr~Sg4114561 compartments: (a) a suitable amount of a CD4+ cell whose cell membrane is labeled with a first dye; (b) a suitable amount of an XIV- l envelope glycoprotein~ cell whose cell ~ is labeled with a second dye, the HIV-l 5 envelope glycoprotein' cell being capable of fusing with the CD4 ' cell of (a) under suitable conditions in the absence of the agent, and the f irst and second dyes permitting r~ n~n~ ~ energy transfer therebetween only when juxtaposed within the same ' CL11~:; (c) a suitable lO 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-l envelope glycoprotein-mediated fusion with the 15 first control cell of (c) under suitable conditions in the absence o~ the agent.
The kit of the subject invention may further comprise additional buffers. Furthermore, the cells may either be 20 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 25 specifically inhibiting the fusion of a CD4' cell with an HIV-l envelope glycoprotein~ cell. Such amounts may be readily determined according to methods well known to those skilled in the art.
30 In one embodiment, the first dye is a rhn~l~m;n~ moiety-c ~nt~;n;ng molecule and the second dye is a fluorescein moiety-containing molecule In the pref erred ' '; ~, the rhodamine moiety-35 c~n~;n;ns molecule is octadecyl rho~rn;n~ B chloride and Wo 95/16789 ~ PCT/US94/14561 the fluorescein moiety-cnnt~in;ng molecule i5 fluorescein octadecyl ester.
In another embodiment, the f irst dye is a f luorescein 5 moiety-cnnt~;n;n~ molecule and the second dye i8 a rhn~l~m; n~ moiety-c~nt~; n; ns molecule .
In one ` _1~; r L, the CD4~ cell is a CD4~ He~a cell . In another: ' o~1;r t of the subject invention, the ~IV-l 10 envelope glycoproteinr cell is an HIV-l~ gpl20/gp41 HeLa cell .
The subject invention further provides a kit for determining whether an agent is capable of inhibiting the 15 fusion of a CD4~ cell with an HIV-l 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-l envelope glycoprotein' cell whose cell membrane is 2 0 labeled with a second dye, the HIV- l envelope glycoproteint 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 25 juxtaposed within the same membrane.
The kit of the subject invention may further comprise additional buffers. Furt~ e, 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 ~IV-l 35 envelope glycoprotein~ cell. Such amounts may be readily Wo 95/16789 ~ 1 7 8 ~ ~ 5 PCrlUS94/14561 determined according to methods well known to~= those skilled in the art.
In one embodiment, the first dye is a rhr,-l~m;n~ moiety-5 rr,nt;l;n;ng molecule and the second dye is a fluoresceinmoiety-r~nt~3;n;n~ molecule.
In the pref erred embodiment, the rhodamine moiety-rr,n~;n;n~ molecule is octadecyl rhodamine B chloride and lO the fluorescein moiety-containing molecule is fluorescein octadecyl ester.
In another embodiment, the f irst dye is a f luorescein moiety-rrnt;l;n;nr, molecule and the second dye is a 15 rhodamine moiety-rrn~;n;ng molecule.
In one: ` ~rl; , the CD4 ' cell is a CD4 He~a cell . In another embodiment of the subject invention, the XIV-l envelope glycoprotein~ cell is an HIV-lLAI gpl20/gp41' HeIa 20 cell.
The subject invention further provides a method for determining whether an HIV-l isolate is syncytium-; n~ ; n~ which comprises: (a) obtaining a sample of an 25 HIV-l 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 CD4t cell under conditions which would permit the fusion of the CD4' cell with a syncytium-inducing HIV-l strain 3 0 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 ~uxtaposed within the same membrane; (c) determining the percent resonance energy 35 transfer value of the resulting sample after a su~table Wo 95/16789 2 ~ 7 6 ~ 9 5 Pcr/USs4/l4s6l period of time; and (d) comparing the percent resonance energy transfer value 80 determined with a known standard, so as to determine whether the XIV-1 isolate i9 syncytium-; n~ll, 1 n~.

used herein, "syncytium-inducing" means capable of causing the formation of syncytia ~multi-nucleated cells resulting from XIV-1 envelope glycoprotein-mediated cell fusion) when contacted with a plurality of CD4 ' cells 10 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 f rom blood or any other bodily f luid known to contain HIV-1 isolate envelope glycoprotein' cells in HIV-infected subjects. ~lternatively, HIV-1 isolate envelope 20 glycoprotein cells may be obtained by culturing cells n vitro with blood or= other bodily fluids t-nnt~;ning 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 method3 well known to those skilled in the art.
30 In one embodiment, the first dye is a rhn/1~minl~ moiety-, nn~i~in;ng molecule and the second dye is a fluorescein moiety-containing molecule.
In the preferred embodiment, the rhnrl~m;n~ moiety-35 containing molecule is octadecyl rhodamine B chloride and WO 95116789 2 1 7 8 ~ ~ 5 PCrlUS941~4561 .

the fluorescein moiety-cnrt~inin~ molecule is fluorescein octadecyl ester.
In another lomho~; - , the f irst dye is a f luorescein 5 moiety-~nnt~;n;n~ molecule and the eecond dye is a rhnr~m;nf~ moiety-l-nnt~;n;n~ molecule.
In one r--I o~;r ~, the CD4t cell is a CD4' HeLa cell .
lO The subject invention further provides a method for determining the 5tage of an HIV-l infection in an HIV-l-infected bubject which comprises determining by the method of the subject invention whether the HIV-l isolate with which the HIV-l-infected subject is infected is 15 syncytium-;n~1/~;n~, so as to thereby determine the stage of the HIV-l infection in the HIV-l-infected subject.
Finally, the subject invention provides a method for quantitatively measuring the fusion of a CD4 ' cell with 20 an HIV-l envelope glycoprotein~ cell which comprises: (a) contacting a sample of the CD4 cell with the HIV- l envelope glycoprotein~ cell under conditions permitting fusion therebetween, the cell membranes of the CD4~ cell and the HIV- l envelope glycoprotein' cell being labeled 25 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 30 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-l envelope glycoproteint cell.
35 This invention will be better understood by reference to 21 7~49~
WO 9511678g PCT/Uss4/l456l the Experimental Details which follow, but those skilled in the art will readily appreciate that the specif ic experiments detailed are only illustrative of the invention as described more fully in the claims which 5 follow thereafter.

Wo 95/16789 2 1 ;7 ~ ~ 9 5 PCTIUS94/14561 iment~l Detail~
A - 3a~ u~d 5 The RET-based fusion assay of the subject invention measures fusion between cells which express the HIV-1 envelope glycoprotein (gpl20/gp41) and cells which express CD4. Such cell-cell fusion may lead to the production of multinucleated cells or syncytia.
10 Molecules which block HIV-1 att2.rl t or fusion to host cells also block syncytia formation. Syncytia assays have been used in many laboratories to detect virus or anti-virus molecules, and typically have a visual readout. In the dev~lt~ of the assay, permanent cell lines which stably express gpl20/gp41 or CD4 were used.
The rttFtnn~nrt~ energy transfer techllirue has been used in a variety of studies of membrane fusion including the fusion of nl~rl t' ttt~tl cells induced by viruses or 20 polyethylene glycol. IIowever, it has not previously been used to study ~IIV-1 envelope glycoprotein-mediated membrane fusion. The technique involves labeling one fusion partner ~e.g. a gpl20/gp41-expressing cel~ line) with a fluorescent dye such as octadecyl fluorescein 25 (F18) and the other fusio~ partner (e.g. a CD4-expressing cell line) with a dye such as octadecyl rhn~l~mint- ~R18).
The dyes are chosen such that the emission spectrum of one (F18) overlaps the excitation spectrum of the second (R18). When the cells fuse, the F18 and R18 associate 30 together closely enough that st; llAt;nn of F18 results in resonance energy transfer to R18 and emission at the R18 t-m; t~t~; nn wavelengths . The octadecyl versions of the fluors E~pnnt~nt~nusly insert into the plasma membranes of cells using the l ~h~l; nrJ protocol described below.

wo 95/16789 2 1 7 8 4 9 5 PCT/U594114561 .

B - C~ Te9ted (1) A Chinese Hamster Ovary (CHO) cell line which expre6ses HIV-l~ gpl20/gp41 (160G7) was mixed with a 5 human T lymphocyte cell line which expresses CD4 ~C8166) CD4' cells are commercially available 160G7 cells may be obtained at the MRC AIDS Directed Program (United Kingdom) . C8166 cells may be obtained at the MRC AIDS
Directed Program (United Kingdom) and the NIH AIDS
Research and Reference Reagent Program (Bethesda, Maryland). It was previously demonstrated that 160G7 cells and C8166 cells fuse to form multinucleated syncytia. This assay is a syncytium assay which requires visual ro~n~; ng of syncytia with the aid of a low power microscope. This assay is suitable for analyzing blocking agents such as CD4-based molecules and nf~u1-ri:ll;i;n~ antibodies directed against gpl20 and gp41 (2) Human epithelial carcinoma (HeLa) cells which express HIV~ gpl20/gp41 (HeLa-env) and HeLa cells which express CD4 (HeLa-CD4 ' ) were also used HeLa-CD4 ' cells may be obtained at the MRC AIDS Directed Program (United Kingdom) and the NIH AIDS Research and Reference Reagent Program (Bethesda, Maryland) HeLa-env' cells express much higher levels of gpl20/gp41 than do 160G7 cells, as demonstrated by the ability to easily detect gpl20 on the surface of HeLa-env~ cells but not 160G7 cells by flow cytometry using an anti-gpl20 antibody Visual analysis demonstrates that HeLa-env~ cells fuse readily with C8166 and HeLa-CD4' cells to form syncytia HeLa-env' cells may be obtained, for example, by transfecting HeLa cells with an enV-~nr~;n~ plasmid, such as pMA243, using the calcium phosphate precipitation 35 method and subsequent selection of transfectants with 2~M

Wo 95/16789 ~ 1 7 ~ ~ q ~ PCT/US94/14561 methotrexate. The plasmid pMA243 is designed to expreEs the HIV-lL~U gene5 env, tat, rev and vpu, in addition to the selectable marker DHFR*, with all genes under the control of the ~IIV-1 LTR (Dragic, T., et al., J. Virol.
66:4794-4802 (1992) ) . D~IFR* i8 a mutant dihydrofolate reductase gene that demonstrates a reduced af f inity f or methotrexate. In pMA243, the D~IFR* gene is expressed from the mRNA spliced transcript that normally encodes the HIV- 1 nef gene which is deleted in this vector~ The HIV-1-encoded tat and rev genes are required for high level expression of the env gene. The plasmid pMA243 also encodes an ampicillin resistance marker and bacterial origin of replication.
C - Cuvette Assav Method The cell labeling conditions were modified from those used in a previous study where RET was used to monitor polyethylene glycol-induced cell fusion (Wanda, P.E., and Smith, J.D., J. Histochem. Cytochem. 30 : 1297 (1982) ) .
F18 (fluorescein octadecyl ester; Molecular Probes Eugene, Oregon. Catalog No. F3857) or R18 (octadecyl rhn~;~mi nP B, chloride salt; Molecular Probes, Catalog No .
0246) were dissolved in ethanol at 5-lOmg/ml and diluted approximately 1000-fold into the appropriate growth medium. The exact concentration in the medium was adjusted to bring the OD to 0.34 at 506nm (F18) or 1.04 at 565nm (R18). Monolayers of cells were incubated with the appropriate medium overnight, then washed and counted. 100,000 cells of each type were mixed together in wells of a ~4-well tissue culture plate. At intervals after mixing, the cells were removed with EDTA, washed and placed in a fluorometer cuvette. Fluorescence was measured at three sets of excitation and emission wavelengths (see table below) using a Perkin-Elmer I,S50 W095/16789 2 ~ ~ 8 q ~ 5 PCT/US94/14561 f luorometer .
~xcitation Emission mea~ L.",~
wavelength wavelength obtained 5 450nm 530nm Total F18 f luorescence 557nm 590nm Total R18 f luorescence 450nm 590nm RET*
10 * The calculation of RET requires first subtracting the fluorescence due to direct F18 and R18 fluorescence following excitation at 450 and emission at 590. The fluorescence measurements are de~Prm;nP~ by measuring the fluorescence of cells labeled with each dye separately.
The RET value, calculated as described above, is divided by the total R18 fluorescence to give a ~ RET value. The results of initial experiments indicate that RET can be measured using both cell combinations listed above. A
20 greater signal was produced when the envelope glycoprotein-expressing cells were F18-labeled and the CDg-expressing cells were R18 labeled than when the envelope glycoprotein-expressing cells were R18-labeled and the CD4-expressing cells were F18~1abeled.
D - Reslll ts of time course RET studies ~n~l ex~eriments with co~trol cell lines, usin~ the cuvette assav method 30 Time course experiments were performed with the HeLa-env~
+ HeLa-CD4 combination (Pigure 1). A control cell line, HeLa-~env', waS used. HeLa-~env~ cells express HIV-1 envelope glycoprotein, with a 400 base pair deletion in Wo 95/l6789 2 1 7 ~ 4 9 5 PCrlUS94/14~61 the gpl20-~n~o~ing region of the env gene. These cells do not fuse with CD4 ' human cells .
The results demonstrate that fusion can be measured by the RET assay at 2 hours, but not at 1 hour, consistent with previous studies of HrV-1 envelope-mediated cell fusion using flUorescence microscopy. At 4 hours, massive cell fusion was evident by visual inspection of the culture, and this time point yielded reproducible RET
values in several experiments. In other experiments, the combination of 160G7 cells with C8166 cells gave a reproducible maximum RET value at about 4 hours but with lower values than those obtained using HeLa-env~ and HeLa-CD4' (data not shown). Presumably, this difference results from the much greater level of qpl20/gp41 expression on HeLa-env' cells as compared with 160G7 cells .
A number of control experiments were performed using combinations of cells which, based on previous studies, are known not to fuse. These combinations included HeLa cells ~mh;nf~ with HeLa-CD4~ cells, or HeLa-env' cells r-n-~; n~ with CHO-CD4 or the human glioma cell line U87.MG-CD4. CHO-CD4 cells, like other non-primate cells, do not fuse with cells expressing HIV-1 gpl20/gp41.
U87.MG-CD4 cells are one of the few CD4' human celL li~es which do ~ot fuse with HIV-1 envelope glycoprotein-expressing cells. RET values obtained with these combinations of cells (data not shown) were in general similar to those using the control HeLa-~env~ + HeLa-CD4 ( Figure 1 ) .
E - Results of RET ex~eriments with ~lockinq aqents usiilq the cuvette assav method Wo 95/16789 2 1 7 8 ~ 9 5 PCT/US94114561 It was next determined whether sCD4 (which interacts with gpl20/gp41~ cells) or the murine MAb OKT4a (which interacts with CD4~ cells) could block RET (Figures 2 and 3 ) . Both these molecules are known to inhibit HIV- l 5 infection and syncytium formation. The percent blocking was calculated as 96 RET at each concentration of blocking agent divided by ~ RET in the absence of blocking agent at 4 hours.
l0 ~s shown in Figures 2 and 3, both sCD4 and OKT4a block fusion as measured by RET. The concentrations of these agents required for 50~ inhibition are similar to those determined using other assays. For example. the ICs~ for sCD4 inhibition of fusion between 160G7 an C8166 was 15 approximately 4~g/ml measured using the RET assay, as compared with 5 . 511g/ml measured by a visual syncytium assay (i.e., an assay for measuring the inhibition of syncytium formation, wherein the syncytia are q-l~nt;ti:lt~d visually using a low-power microscope) using the same 20 , ' ;n~tion of cells. In summary, these results demonstrate that the RET method can be used to measure HIV-l envelope-r~ ii ;~t~d cell fusion in a rapid and reproducible fashion. When compared with data from the more conventional visual syncytium assay, the results are 25 in .~cF~ nt agreement.
F - Control blockina exrJeriment with IKT4 usinq cuvette i~ CSaV method 30 Control experiments were performed to examine inhibition of ~ RET by OKT4. OKT4 is a mouse monoclonal antibody that binds CD4 but does not inhibit the CD4-gpl20 interaction, HIV-l infection, or HIV-induced cell fusion.
Using the cuvette method and the HeLa-env~ + HeLa-CD4 35 combination, OKT4 gave 09~ inhibition of RET at 0.2 llg/ml Wo 95/16789 2 ~ 7 8 ~ 9 5 PCr/US94/14561 or 2 . O ~g/ml, compared with 65~ inhibition by OKT4a at O . 2 f~g/ml in the same experiment . These results demonstrate that inhibition of HIV-l envelope-mediated membrane fusion as measured by RET i9 specific for agents 5 that block HIV-l infection and HIV-induced cell fusion.
G - Automation of the RET assaY usina the ~late reader assav lO A fluorescent plate reader was used to analyze the RET
assay. This method has the advantage of reducing the manip~ t; ~nc~ reauired, notably the need to remove cells for mea~uL of fluorescence in a cuvette. The plate reader measures f luorescence of cells directly in a 15 multi-well tissue culture plate. Moreover, the speed of assay readout is dramatically increased (by approximately lOO-fold). The Millipore "Cytofluor" was used in this experiment. This is a dedicated plate reader which has been used in a variety of different cell-based 20 fluorescence assays and is suitable for use with a range of plate formats including 24-well and 96-well tissue culture plates. The Cytofluor also has the major advantages of speed and compatibility with IBM software analysis programs.
The results indicate that the assay can be readily performed in 24 or 96 well tissue culture plates using the fluorescence plate reader.
30 In one '~o~; , when performing the assay on a routine basis, two types of mea~u~ tc are done. In the first, RET is measured at a single time point following mixing of labeled cells and a candidatè blocking agent. In the second, the assay is adapted to measure changes in the 35 rate of cell fusion in the presence or absence of wo 95116789 2 1 7 8 ~ 9 ~ PCT/US94114561 blocking agents. One of the advantages of the RET as6ay i5 that it measures fusion in real time and thus is amenable to kinetic analysis.
For example, a method of using the plate reader assay and measuring RET at a single time point i5 provided below.
In this assay a 96-well flat bottom tissue culture plate is used. The method is a modification of the cuvette method described above.
Example of a single time-point plate reader assay method:
l. Prepare dyes:
Rl8: lO mg/ml in 100% EtOH (for HeLa-CD4' cells) Fl8: 5 mg/ml in 100% EtOH (for HeLa-env~ cells) 2. Add dyes to appropriate concentrations, in cell culture medium ~ nt~;nin~ 10% fetal calf serum, as determined by absorbance measurements:
Fl8' medium: 0.34 at 506 nm Rl8' medium: 0.52 at 565 nm 3. Add medium + dye to the appropriate cells as indicated above, then incubate overnight to stain cells.

4. Wash cells and count.

5. Plate out 20,000 cells of each line/well, some wells having one or other cell line separately, other wells with both cell lines, and other wells with various concentrations of ~n~ihor~ies or other inhibitory agents added in addition to both cell lines .

WO 95tl6789 2 i 7 ~ fi ~ :~3 PCT/US94/14561 6. 4 hours later, remove the media and wash all of the wells three times with PBS ( the cells remain adherent in the wells). Add 200 ,ul PBS to each well Read fluorescence in the wells using the Millipore Cytof luor plate reader with f ilter hinFIt; ong listed below:
F18: excitation 450 nm emission 530 nm (X) R18: excitation 530 nm emission 590 nm 10 (Y) F18 + R18: excitation 450 nm emission 590 nm (Z) The emission values, X, Y and Z (as indicated above) are 15 recorded for each cell combination:
A) HeLa - env~ + HeLa - CD4 B) HeLa-env~ alone C ) HeLa - CD4 ~ alone 20 For example, the F18 reading for HeLa-env~ cells alone is given by By~
Then 96 RET is calculated using this formula:
Az - (Ay . Bz/By) - (Ay . Cz/Cy) 9~ RET = =. 100 Ay Similar results were obtained in experiments comparing 30 inhibition of ~z RET using the cuvette method and the plate reader method. For example, Figure 4 illustrates the inhibition of fusion between HeLa-env~ and He~a-CD4 cells by the monoclonal anti-CD4 antibody, OKT4a, measured as a reduction in 9~ RET det~rmin~A by both 35 methods at 4 hours after mixing the cells.

Claims (19)

What is claimed is:

1. A method for determining whether an agent is capable of specifically inhibiting the fusion of a CD4+ cell with an HIV-1 envelope glycoprotein 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;
(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.

2. The method of claim 1, wherein the agent is an antibody.

3. 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 claim 1, so as to thereby determine whether the agent is capable of specifically inhibiting the infection of a CD4+ cell with HIV-1.

4. A method for determining whether an agent is capable of inhibiting the fusion of a CD4+ cell with an HIV-1 envelope glycoprotein+ 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 XIV-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 furion of the CD4+ cell with the HIV-1 envelope glycoprotein+ cell .

5. A method for quantitatively determining the ability of an antibody-containing sample to specifically inhibit the fusion of a CD4+ cell with an HIV-1 envelope glycoprotein+ 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.

6. A method for guantitatively determining the ability of an antibody-containing sample to inhibit the fusion of a CD4+ cell with an HIV-1 envelope glycoprotein+ 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.

7. 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 claim 6; 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 and 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 of clinical prognosis of the HIV-1 infection in the HIV-1-infected subject.

8. 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 a CD4+ cell with an HIV-1 envelope glycoprotein+ cell by the method of claim 6; 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.

9. A kit for determining whether an agent is capable of specifically 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;
(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.

10. 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.

11. 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.

12. A method for determining the stage of an HIV-1 inf ection in an HIV-1-infected subject which comprises determining by the method of claim 11 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.

13. The method of claim 1, 4, 5, 6, 9, 10 or 11, wherein the first dye is a rhodamine moiety-containing molecule and the second dye is a fluorescein moiety-containing molecule.

14. The method of claim 13, wherein the rhodamine moiety-containing molecule is octadecyl rhodamine B
chloride and the fluorescein moiety-containing molecule is fluorescein octadecyl ester.

15. The method of claim 1, 4, 5, 6, 9, 10 or 11, wherein the first dye is a fluorescein moiety-containing molecule and the second dye is a rhodamine moiety-containing molecule.

16. The method of claim 1, 4, 5, 6, 9, 10 or 11, wherein the CD4+ cell is a CD4+ HeLa cell.

17. The method of claim 1, 4, 5, 6, 9 or 10 wherein the HIV-1 envelope glycoprotein+ cell is an HIV-1LAI
gp120/gp41+ HeLa cell.

18. An agent determined to be capable of specifically inhibiting the fusion of a CD4+ cell with an HIV-1 envelope glycoprotein+ cell using the method of claim 1.

19. An agent determined to be capable of inhibiting the fusion of a CD4+ cell with an HIV-1 envelope glycoprotein+ cell using the method of claim 4.