Note: Descriptions are shown in the official language in which they were submitted.
Wo 95/16789 2 1 7 8 ~ 9 5 pcr/uss4/l~56l
.
--1--
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
--2--
(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
--5--
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
--6--
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
--10-
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
-11-
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
-12 -
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
--14--
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
-15--
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
-18-
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
-19-
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
--20 -
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
-21-
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
--22--
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
.
-23--
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
WO 95/16789 2 1 7 g ~ ~ 5 PCTIUS94/14561
-24--
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
.
-26-
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
--27-
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
-28 -
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
--29 -
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
.
--30--
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
.
--31--
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
--32--
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
--34--
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
.
--36 -
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
-38-
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
.
-39-
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
--40 -
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
-41 -
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
--42 -
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
--43--
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
--44--
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
--46 -
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.
