U.S. patent application number 11/894677 was filed with the patent office on 2008-08-07 for method for preventing hiv-1 infection of cd4 cells.
This patent application is currently assigned to Progenics Pharmaceuticals, Inc.. Invention is credited to Graham P. Allaway, Virginia M. Litwin, Paul J. Maddon, William C. Olson.
Application Number | 20080187539 11/894677 |
Document ID | / |
Family ID | 26692524 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080187539 |
Kind Code |
A1 |
Allaway; Graham P. ; et
al. |
August 7, 2008 |
Method for preventing HIV-1 infection of CD4 cells
Abstract
This invention provides methods for inhibiting fusion of HIV-1
to CD4.sup.- cells which comprise contacting CD4.sup.- cells with a
non-chemokine agent capable of binding to a chemokine receptor in
an amount and under conditions such that fusion of HIV-1 to the
CD4.sup.+ cells is inhibited. This invention also provides methods
for inhibiting HIV-1 infection of CD4.sup.- cells which comprise
contacting CD4.sup.+ cells with a non-chemokine agent capable of
binding to a chemokine receptor in an amount and under conditions
such that fusion of HIV-1 to the CD4.sup.+ cells is inhibited,
thereby inhibiting the HIV-1 infection. This invention provides
non-chemokine agents capable of binding to the chemokine receptor
and inhibiting fusion of HIV-1 to CD4.sup.+ cells. This invention
also provides pharmaceutical compositions comprising an amount of
the non-chemokine agent capable of binding to the chemokine
receptor and inhibiting fusion of HIV-1 to CD4.sup.+ cells
effective to prevent fusion of HIV-1 to CD4.sup.+ cells and a
pharmaceutically acceptable carrier.
Inventors: |
Allaway; Graham P.;
(Mohegan, NY) ; Litwin; Virginia M.;
(Fayetteville, NY) ; Maddon; Paul J.; (Elmsford,
NY) ; Olson; William C.; (Ossining, NY) |
Correspondence
Address: |
John P. White;Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
Progenics Pharmaceuticals,
Inc.
|
Family ID: |
26692524 |
Appl. No.: |
11/894677 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09888938 |
Jun 25, 2001 |
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11894677 |
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08831823 |
Apr 2, 1997 |
6344545 |
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09888938 |
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60019715 |
Jun 14, 1996 |
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60014532 |
Apr 2, 1996 |
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Current U.S.
Class: |
424/143.1 ;
530/387.1; 530/388.22 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/76 20130101; G01N 2333/70514 20130101; A61P 37/00
20180101; G01N 2333/715 20130101; G01N 33/566 20130101; G01N
33/56988 20130101; G01N 2333/162 20130101; C07K 16/24 20130101;
G01N 2500/20 20130101; C07K 16/2866 20130101 |
Class at
Publication: |
424/143.1 ;
530/388.22; 530/387.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; A61P 37/00 20060101
A61P037/00 |
Claims
1-50. (canceled)
51. A monoclonal antibody, or a portion of such antibody, prepared
against a human CCR5 chemokine receptor expressed in a mammalian
cell line, which receptor binds RANTES, MIP-1.alpha. and
MIP-1.beta., and wherein the antibody is capable of inhibiting
infection of a human CD4.sup.+ cell by a HIV-1 virus.
52. The monoclonal antibody, or a portion of such antibody, of
claim 1, which inhibits fusion of the HIV-1 to CD4+ cells, and the
HIV-1 is macrophage-tropic.
53. The monoclonal antibody of claim 1.
54. The monoclonal antibody portion of claim 1.
55. A composition comprising the monoclonal antibody of claim 53
and a carrier.
56. The composition of claim 55, wherein the monoclonal antibody is
present in an amount effective to inhibit HIV-1 infection.
57. A composition comprising the monoclonal antibody portion of
claim 54 and a carrier.
58. The composition of claim 57, wherein the monoclonal antibody
portion is present in an amount effective to inhibit HIV-1
infection.
59. An antibody, or a portion of such antibody, which binds to a
chemokine receptor which binds RANTES, MIP-1.alpha. and
MIP-1.beta., which antibody inhibits infection of a human CD4+ cell
by a HIV-1 virus.
60. The antibody according to claim 59 which is capable of
inhibiting fusion of HIV-1 to CD4+ cells thereby inhibiting HIV-1
infection, and the HIV-1 is macrophage-tropic.
61. The antibody according to claim 59, obtainable by i) making a
cDNA library in a mammalian expression vector, using mRNA prepared
from CD4+ T-lymphocytes or macrophages; ii) identifying members of
the cDNA library encoding members of the chemokine receptor family
using appropriate degenerate oligonucleotide probes; iii)
expressing vectors containing chemokine receptor cDNAs in a
mammalian cell line which expresses human CD4 but does not fuse
with HeLa cells expressing gp120/gp41 from the macrophage-tropic
strain HIV.sub.JR-FL; iv) identifying clones which thereby gain the
ability to fuse with HeLa-env.sub.JR-FL; v) preparing monoclonal or
polyclonal antibodies to the thus-expressed receptor; vi) testing
the monoclonal or polyclonal antibodies for ability to inhibit
infection by a panel of HIV-1 isolates.
62. The antibody of claim 61 which is monoclonal.
63. A composition comprising the monoclonal antibody of claim 62
and a carrier.
Description
[0001] This application claims priority of U.S. Provisional
Application No. 60/019,715, filed Jun. 14, 1996, and U.S.
Provisional Application No. 60/014,532, filed April 2, 199, the
content of which are incorporated by reference into this
application.
[0002] Throughout this application, various references are referred
to within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citation for these
references may be found at the end of each series of
experiments.
BACKGROUND OF THE INVENTION
[0003] Chemokines are a family of related soluble proteins of
molecular weight between 8 and 10 KDa, secreted by lymphocytes and
other cells, which bind receptors on target cell surfaces resulting
in the activation and mobilization of leukocytes, for example in
the inflammatory process. Recently, Cocchi et al. demonstrated that
the chemokines RANTES, MIP-1.alpha. and MIP-1.beta. are factors
produced by CD8.sup.+ T lymphocytes which inhibit infection by
macrophage-tropic primary isolates of HIV-1, but not infection by
laboratory-adapted strains of the virus (1). These chemokines are
members of the C-C group of chemokines, so named because they have
adjacent cysteine residues, unlike the C-X-C group which has a
single amino acid separating these residues (2). While Cocchi et
al. found that expression of HIV-1 RNA was suppressed by treatment
with the chemokines, they did not identify the site of action of
these molecules.
[0004] A resonance energy transfer (RET) assay of HIV-1 envelope
glycoprotein-mediated membrane fusion was used to determine whether
fusion mediated by the envelope glycoprotein from the primary
macrophage-tropic isolate of HIV-1.sub.JR-FL would be specifically
inhibited by chemokines, when compared with fusion mediated by the
envelope glycoprotein from the laboratory-adapted T lymphotropic
strain HIV-1.sub.LAI. As described below, it was demonstrated that
this is indeed the case. This demonstrates that some chemokine
receptors are fusion accessory molecules required for HIV-1
infection. Previous studies have indicated that unidentified cell
surface molecules are required for virus entry in addition to the
HIV-1 receptor, CD4. While CD4 is required for HIV-1 attachment,
the accessory molecules are required for the membrane fusion step
of entry. These accessory molecules are generally expressed only on
human cells, so HIV-1 does not infect non-human CD4 cells (3-6).
Moreover it is possible to complement non-human CD4.sup.+ cells by
fusing them (using polyethylene glycol) with CD4.sup.- human cells,
resulting in a heterokaryon which is a competent target for HIV-1
envelope-mediated membrane fusion (7,8). These studies have been
performed using laboratory-adapted T lymphotropic strains of the
virus.
[0005] In some cases, it appears that fusion accessory molecules
are found on a subset of human CD4.sup.+ cells and are required for
infection by HIV-1 isolates with particular tropisms. For example,
macrophage-tropic primary strains of HIV-1 such as HIV-1.sub.JR-FL
may have different requirements for accessory molecules compared
with laboratory-adapted T lymphotropic strains such as
HIV-1.sub.LAI. This phenomenon may explain differences in tropism
between HIV-1 strains.
[0006] The current invention comprises a series of new therapeutics
for HIV-1 infection. It was demonstrated for the first time that
chemokines act at the fusion step of HIV-1 entry and specifically
inhibit membrane fusion mediated by the envelope glycoprotein of
primary macrophage-tropic primary, viral isolates, not
laboratory-adapted T lymphotrophic strains of the virus. Primary
macrophage-tropic isolates of the virus are of particular
importance since they are the strains usually involved in virus
transmission, and may have particular importance in the
pathogenesis of HIV-1 infection.
[0007] These results were obtained using a resonance energy
transfer (RET) assay of HIV-1 envelope-mediated membrane fusion.
Moreover, this assay is used to identify non-chemokines, including
fragments of chemokines and modified chemokines, that inhibit HIV-1
envelope glycoprotein-mediated membrane fusion and thereby
neutralize the virus, yet do not induce an inflammatory
response.
SUMMARY OF THE INVENTION
[0008] This invention provides a method for inhibiting fusion of
HIV-1 to CD4 cells which comprises contacting CD4.sup.+ cells with
a non-chemokine agent capable of binding to a chemokine receptor in
an amount and under conditions such that fusion of HIV-1 to the
CD4.sup.- cells is inhibited.
[0009] This invention also provides a method for inhibiting HIV-1
infection of CD4.sup.+ cells which comprises contacting CD4.sup.+
cells with a non-chemokine agent capable of binding to a chemokine
receptor in an amount and under conditions such that fusion of
HIV-1 to the CD4' cells is inhibited, thereby inhibiting the HIV-1
infection.
[0010] This invention further provides non-chemokine agents capable
of binding to the chemokine receptor and inhibiting fusion of HIV-1
to CD4.sup.+ cells.
[0011] This invention provides an agent which is capable of binding
to fusin and inhibiting infection. In an embodiment, the agent is
an oligopeptide. In another embodiment, the agent is an
polypeptide. In still another embodiment, the agent is an antibody
or a portion of an antibody. In a separate embodiment, the agent is
a nonypeptidyl agent.
[0012] In addition, this invention provides pharmaceutical
compositions comprising an amount of the above non-chemokine agents
or agents capable of binding to fusin effective to inhibit fusion
of HIV-1 to CD4.sup.+ cells and a pharmaceutically acceptable
carrier.
[0013] This invention provides a composition of matter capable of
binding to the chemokine receptor and inhibiting fusion of HIV-1 to
CD4.sup.- cells comprising a non-chemokine agent linked to a ligand
capable of binding to a cell surface receptor of the CD4.sup.-
cells other than the chemokine receptor such that the binding of
the non-chemokine agent to the chemokine receptor does not prevent
the binding of the ligand co the other receptor.
[0014] This invention also provides a pharmaceutical composition
comprising an amount of the above-described composition of matter
effective to inhibit fusion of HIV-1 co CD4.sup.- cells and a
pharmaceutically acceptable carrier.
[0015] This invention provides a composition of matter capable of
binding to the chemokine receptor and inhibiting fusion of HIV-1 to
CD4.sup.+ cells comprising a non-chemokine agent linked to A
compound capable of increasing the in vivo half-life of the
non-chemokine agent.
[0016] This invention also provides a pharmaceutical composition
comprising an amount of a composition of matter comprising a
non-chemokine agent linked to a compound capable of increasing the
in vivo half-life of the non-chemokine agent effective to inhibit
fusion of HIV-1 to CD4.sup.+ cells and a pharmaceutically
acceptable carrier.
[0017] This invention provide methods for reducing the likelihood
of HIV-1 infection in a subject comprising administering an
above-described pharmaceutical composition to the subject.
[0018] This invention also provides methods for treating HIV-1
infection in a subject comprising administering an above-described
pharmaceutical composition to the subject.
[0019] This invention also provides methods for determining whether
a non-chemokine agent is capable of inhibiting the fusion of HIV-1
to a CD4.sup.- cell which comprise: (a) contacting (i) a CD4.sup.+
cell which is labeled with a first dye and (ii) a cell expressing
the HIV-1 envelope glycoprotein on its surface which is labeled
with a second dye, in the presence of an excess of the agent under
conditions permitting the fusion of the CD4.sup.- cell to the cell
expressing the HIV-1 envelope glycoprotein on its surface in the
absence of the agent, the first and second dyes being selected so
as to allow resonance energy transfer between the dyes; (b)
exposing the product of step (a) to conditions which would result
in resonance energy transfer if fusion has occurred; and (c)
determining whether there is a reduction of resonance energy
transfer, when compared with the resonance energy transfer in the
absence of the agent, a decrease in transfer indicating that the
agent is capable of inhibiting fusion of HIV-1 to CD4.sup.+
cells.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1. Membrane fusion mediated by the HIV-1.sub.JR-FL
envelope glycoprotein is inhibited by RANTES, MIP-1.alpha. and
MIP-1.beta.. [0021] % RET resulting from the fusion of PM1 cells
and HeLa-env.sub.JR-FL (.box-solid.) or HeLa-env.sub.LAI
(.diamond-solid.) was measured in the presence and absence of
recombinant human chemokines at a range of concentrations: RANTES
(80-2.5 ng/ml), MIP-1.alpha. (400-12.5 ng/ml) and MIP-1, (200-6.25
ng/ml), as indicated. Chemokines were added simultaneously with the
cells at the initiation of a four hour incubation. Data are
representative of more than three independent experiments which
were run in duplicate. The percent inhibition of RET is defined as
follows:
[0021] % Inhibition=100[(Max RET-Min RET)-(Exp RET-Min RET)]/(Max
RET-Min RET) where Max RET is the FRET value obtained at four hours
with HeLa-env cells and CD4-expressing cells in the absence of an
inhibitory compound; Exp RET is the FRET value obtained for the
same cell combination in the presence of an inhibitory compound and
Min RET is the background % RET value obtained using HeLa cells in
place of HeLa envelope-expressing cells.
[0022] FIG. 2. CD4:HIV-1 gp120 binding in the presence of human
chemokines. [0023] The binding of soluble human CD4 to
HIV-1.sub.LAI and HIV-1.sub.JR-FL gp120 was determined in an ELISA
assay in the presence and absence of the monoclonal antibody OKT4A
or recombinant human chemokines at a range of concentrations,
identical to those used in the RET inhibition studies of FIG. 1:
OKT4A (62-0.3 nM), RANTES (10.3-0.3 nM), MIP-1.alpha. (53.3-2.9
nM), and MIP-10 (25.6-0.8 nM). Inhibitors were added simultaneously
with biotinylated HIV-1 gp120 to soluble CD4 coated microtiter
plates (Dynatech Laboratories, Inc., Chantilly, Va.). Following a
two hour incubation at room temperature and extensive washing, an
incubation with streptavidin-horseradish peroxidase was performed
for one hour at room temperature. Following additional washes,
substrate was added and the OD at 492 nm determined in an ELISA
plate reader. Data are representative of two independent
experiments which were run in quadruplicate.
[0024] FIG. 3. Specificity. time course and stage of i-chemokine
inhibition of HIV-1 replication. [0025] (a) PM1 cells
(1.times.10.sup.6) were preincubated with
RANTES.alpha.MIP-1.alpha.+MIP-1.beta.(R/M.alpha./M.beta.; 100 ng/ml
of each) for 24 h (-24 h) or 2 h (-2 h), then washed twice with
phosphate buffered saline (PBS). HIV-1 (BaL env-complemented) virus
(50 g of p24; see legend to Table 1) was added for 2 h, then the
cells were washed and incubated for 48 h before measurement of
luciferase activity in cell lysates as described previously
(10,11). Alternatively, virus and R/M.alpha./M.beta. were added
simultaneously to cells, and at the indicated time points (1 h, 3
h, etc) the cells were washed twice in PBS, resuspended in culture
medium and incubated for 48 h prior to luciferase assay. Time 0
represents the positive control, to which no .beta.-chemokines were
added. +2 h represents the mixture of virus with cells for 2 h
prior to washing twice in PBS, addition of R/M.alpha./M.beta. and
continuation of the culture for a further 48 h before luciferase
assay. [0026] (b) PM1 cells (1.times.10.sup.6) were infected with
HIV-1 (500 pg p24) grown in CEM cells (NL4/3; lanes 1-4) or
macrophages (ADA; lanes 5-8), in the presence of 500 ng/ml of
RANTES (lanes 1 and 5) or MIP-1.beta. (lanes 2 and 6), or with no
9-chemokine (lanes 4 and 8). Lanes 3 and 7 are negative controls
(no virus). All viral stocks used for the PCR assay were treated
with DNAse for 30 min at 37.degree. C., and tested for DNA
contamination before use. After 2 h, the cells were washed and
resuspended in medium containing the same .beta.-chemokines for a
further 8 h. DNA was then extracted from infected cells using a
DNA/RNA isolation kit (US Biochemicals). First round nested PCR was
performed with primers: 3+,
5,-CAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGG-3'(SEQ ID NO:1) preGag,
5'-AGCAAGCCGAGTCCTGCGTCGAGAG-3' (SEQ ID NO: 2) and the second round
with primers: LTR-test, 5'-GGGACTTTCCGCTGGGGACTTTC 3, (SEQ ID NO:3)
LRC2, 5'-CCTGTTCGGGCGCCACTGCTAGAGATTTTCCAC 3' (SEQ ID NO:4) in a
Perkin Elmer 2400 cycler with the following amplification cycles:
94.degree. C. for 5 min, 35 cycles of 94.degree. C. for 30 s,
55.degree. C. for 30 s, 72.degree. C. for 30 s, 72.degree. C. for 7
min. M indicates 1 kb DNA ladder; 1, 10, 100, 1000 indicate number
of reference plasmid (pAD8) copies. The assay can detect 100 copies
of reverse transcripts.
[0027] FIG. 4: HIV-1 env-mediated membrane fusion of cells
transiently expressing C-C CKR-5. [0028] Membrane fusion mediated
by .beta.-chemokine receptors expressed in HeLa cells was
demonstrated as follows: Cells were transfected with control
plasmid pcDNA3.1 or plasmid pcDNA3.1-CKR constructs using
lipofectin (Gibco BRL). The pcDNA3.1 plasmid carries a
T7-polymerase promoter and transient expression of .beta.-chemokine
receptors was boosted by infecting cells with 10.times.10.sup.7 pfu
of vaccinia encoding the T7-polymerase (vFT7.3) 4 h
post-lipofection (9). Cells were then cultured overnight in
R18-containing media and were tested for their ability to fuse with
HeLa-JR-FL cells (filled columns) or HeLa-BRU cells (hatched
column) in the RET assay. The FRET with control HeLa cells was
between 3% and 4% irrespective of the transfected plasmid.
[0029] FIG. 5 Membrane fusion mediated by the HIV.sub.LAI envelope
glycoprotein is inhibited by SDF-1. [0030] % RET resulting from the
fusion of PM1 cells and HeLa-env.sub.JR-FL or HeLa-env.sub.LAI
cells (as indicated on the graph) was measured in the presence of
recombinant SDF-1.alpha. (Gryphon Science, San Francisco) at the
indicated concentrations. Experimental method as described in the
legend to FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
[0031] This invention provides a method for inhibiting fusion of
HIV-1 to CD4 cells which comprises contacting CD4 cells with a
non-chemokine agent capable of binding to a chemokine receptor in
an amount and under conditions such that fusion of HIV-1 to the CD4
cells is inhibited.
[0032] This invention also provides a method for inhibiting HIV-1
infection of CD4 cells which comprises contacting CD4.sup.- cells
with a non-chemokine agent capable of binding to a chemokine
receptor in an amount and under conditions such that fusion of
HIV-1 to the CD4.sup.- cells is inhibited, thereby inhibiting the
HIV-1 infection.
[0033] In this invention, a chemokine means RANTES, MIP-1-.alpha.,
MIP-1-.beta. or another chemokine which blocks HIV-1 infection. A
chemokine receptor means a receptor capable of binding RANTES,
MIP-1-.alpha., MIP-1-.beta. or another chemokine which blocks HIV-1
infection.
[0034] Throughout this application, the receptor "fusin" is also
named CXCR4 and the chemokine receptor C-C CKR5 is also named
CCR5.
[0035] The HIV-1 used in this application unless specified will
mean clinical or primary or field isolates or HIV-1 viruses which
maintain their clinical characteristics. The HIV-1 clinical
isolates may be passaged in primary peripheral blood mononuclear
cells. The HIV-1 clinical isolates may be macrophage-trophic.
[0036] The non-chemokine agents of this invention are capable of
binding to chemokine receptors and inhibiting fusion of HIV-1 to
CD4.sup.- cells. The non-chemokine agents include, but are not
limited to, chemokine fragments and chemokine derivatives and
analogues, but do not include naturally occurring chemokines. The
non-chemokine agents include multimeric forms of the chemokine
fragments and chemokine derivatives and analogues or fusion
molecules which contain chemokine fragments, derivatives and
analogues linked to other molecules.
[0037] In an embodiment of this invention, the non-chemokine agent
is an oligopeptlde. In another embodiment, the non-chemokine agent
is a polypeptide. In still another embodiment, the non-chemokine
agent is an antibody or a portion thereof. Antibodies against the
chemokine receptor may easily be generated by routine experiments.
It is also within the level of ordinary skill to synthesize
fragments of the antibody capable of binding to the chemokine
receptor. In a further embodiment, the non-chemokine agent is a
nonpeptidyl agent.
[0038] Non-chemokine agents which are purely peptidyl in
composition can be either chemically synthesized by solid-phase
methods (Merrifield, 1966) or produced using recombinant technology
in either prokaryotic or eukaryotic systems. The synthetic and
recombinant methods are well known in the art.
[0039] Non-chemokine agents which contain biotin or other
nonpeptidyl groups can be prepared by chemical modification of
synthetic or recombinant chemokines or non-chemokine agents. One
chemical modification method involves periodate oxidation of the
2-amino alcohol present on chemokines or non-chemokine agents
possessing serine or threonine as their N-terminal amino acid
(Geophegan and Stroh, 1992). The resulting aldehyde group can be
used to link peptidyl or non-peptidyl groups to the oxidized
chemokine or non-chemokine agent by reductive amination, hydrazine,
or other chemistries well known to those skilled in the art.
[0040] As used herein, a N-terminus of a protein should mean the
terminus of the protein after it has been processed. In case of a
secretory protein which contains a cleavable signal sequence, the
N-terminus of a secretary protein should be the terminus after the
cleavage of a signal peptide.
[0041] This invention provides a method of identifying these
non-chemokine agents. One way of identifying such agents, including
non-peptidyl agents, that bind to a chemokine receptor and inhibit
fusion of HIV-1 to CD4.sup.- cells is to use the following assay:
1) Incubate soluble CD4 with biotinylated gp120 from
HIV-1.sub.JR-FL or HIV-1.sub.LAI; 2) Incubate this complex with
CCR5 or CXCR4-expressing cells (for HIV-1.sub.JR-FL or
HIV-1.sub.LAI gp120 s, respectively) that do not express CD4, in
the presence of absence of a candidate inhibitor; 3) Wash and then
incubate with streptavidin-phycoerythrin; and 4) Wash and then
measure the amount of bound gp120 using a flow cytometer or
fluorometer and calculate the degree of inhibition of binding by
the inhibitor.
[0042] Alternative methods to detect bound gp120 can also be used
in place of the biotinylated gp120-streptavidin-phycoerythrin
method described above. For example, peroxidase-conjugated gp120
could be used in place of the biotinylated gp120 and binding
detected using an appropriate calorimetric substrate for
peroxidase, with a spectrometric readout.
[0043] This invention further provides the non-chemokine agents
identified by the above methods.
[0044] This invention provides a non-chemokine agent capable of
binding to the chemokine receptor and inhibiting fusion or HIV-1 to
CD4.sup.+ cells. In an embodiment, the non-chemokine is a
polypeptide. In a further embodiment, this polypeptide is a
fragment of the chemokine RANTES (Gong et al., 1996). Ir a still
further embodiment, the polypeptide may also comprise the RANTES
sequence with deletion of the N-terminal amino acids of said
sequence. The deletion may be the first eight N-terminal amino
acids of the RANTES sequence (SEQ ID NO:5).
[0045] In a separate embodiment, the polypeptide may comprise the
MIP-1.beta. sequence with deletion of the N-terminal amino acids of
said sequence. The deletion may be the first seven, eight, nine or
ten N-terminal amino acids of the MIP-1.1 sequence.
[0046] In another embodiment of non-chemokine agent, the
polypeptide comprises the MIP-1.beta. sequence with the N-terminal
sequence modified by addition of an amino acid or oligopeptide. In
a separate embodiment, the polypeptide comprises the MIP-1.beta.
sequence with the N-terminal sequence modified by removing the
N-terminal alanine and replaced it by serine or threonine and
additional amino acid or oligopeptide or nonpeptidyl moiety. In a
further embodiment, the additional amino acid is methionine.
[0047] As described infra in the section of Experimental Details, a
cofactor for HIV-1 fusion and entry was identified and designated
"fusin" (Feng et al., 1996). This invention provides an agent which
is capable of binding to fusin and inhibiting infection. In an
embodiment, the agent is an oligopeptide. In another embodiment,
the agent is an polypeptide.
[0048] In a further embodiment, the polypeptide comprises SDF-1
with deletion of the N-terminal amino acids of said sequence. The
deletion may be the first six, seven, eight, or nine N-terminal
amino acids of the SDF-1 sequence.
[0049] This invention also provides the above non-chemokine agent,
wherein the polypeptide comprises SDF-1 sequence with the
N-terminal sequence modified to produce antagonistic effect to
SDF-1. One modification is to replace the N-terminal glycine of
SDF-1 by serine and derivatized with biotin. Another modification
is to replace the N-terminal glycine of SDF-1 by serine and
derivatized with methionine. A further modification is to add the
N-terminus of SDF-1 with a methionine before the terminal
glycine.
[0050] In still another embodiment, the agent is an antibody or a
portion of an antibody. In a separate embodiment, the agent is a
nonpeptidyl agent.
[0051] The agents capable of binding to fusin may be identified by
screening different compounds for their capability to bind to fusin
in vitro.
[0052] A suitable method has been described by Fowlkes, et al.
(1994), international application number: PCT/US94/03143,
international publication number: WO 94/23025, the content of which
is incorporated by reference into this application.
[0053] Briefly, yeast cells having a pheromone system are
engineered to express a heterologous surrogate of a yeast pheromone
system protein. The surrogate incorporates fusin and under some
conditions performs in the pheromone system of the yeast cell a
function naturally performed by the corresponding yeast pheromone
system protein. Such yeast cells are also engineered to express a
library of peptides whereby a yeast cell containing a peptide which
binds fusin exhibits modulation of the interaction of surrogate
yeast pheromone system protein with the yeast pheromone system and
this modulation is a selectable or screenable event. Similar
approaches may be used to identify agents capable of binding to
both fusin and the chemokine receptor C-C CKR-5.
[0054] This invention also provides pharmaceutical compositions
comprising an amount of such non-chemokine agents or agents capable
of binding to fusin effective to inhibit fusion of HIV-1 to
CD4.sup.+ cells and a pharmaceutically acceptable carrier.
[0055] Pharmaceutically acceptable carriers are well known to those
skilled in the art. Such pharmaceutically acceptable carriers may
be aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, saline and
buffered media. Parenteral vehicles include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's or fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers such as those based
on Ringer's dextrose, and the like. Preservatives and other
additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases and the
like.
[0056] This invention provides a composition of matter capable of
binding to the chemokine receptor and inhibiting fusion of HIV-1 to
CD4 cells comprising a non-chemokine agent linked to a ligand
capable of binding to a cell surface receptor of the CD4.sup.+
cells other than the chemokine receptor such that the binding of
the non-chemokine agent to the chemokine receptor does not prevent
the binding of the ligand to the other receptor. In an embodiment,
the cell surface receptor is CD4. In another embodiment, the ligand
is an antibody or a portion of an antibody.
[0057] This invention also provides a pharmaceutical composition
comprising an amount of an above-described composition of matter
effective to inhibit fusion of HIV-1 to CD4.sup.- cells and a
pharmaceutically acceptable carrier.
[0058] This invention provides a composition of matter capable of
binding to the chemokine receptor and inhibiting fusion of HIV-1 to
CD4.sup.+ cells comprising a non-chemokine agent linked to a
compound capable of increasing the in vivo half-life of the
non-chemokine agent. In an embodiment, the compound is polyethylene
glycol.
[0059] This invention also provides a pharmaceutical composition
comprising an amount of a composition of matter comprising a
non-chemokine agent linked to a compound capable of increasing the
in vivo half-life of the non-chemokine agent effective to inhibit
fusion of HIV-1 to CD4' cells and a pharmaceutically acceptable
carrier.
[0060] This invention provide methods for reducing likelihood of
HIV-1 infection in a subject comprising administering the
above-described pharmaceutical compositions to the subject. This
invention also provides methods for treating HIV-1 infection in a
subject comprising administering the above-described pharmaceutical
compositions to the subject.
[0061] This invention also provides methods for determining whether
a non-chemokine agent is capable of inhibiting the fusion of HIV-1
to a CD4 cell which comprise: (a) contacting (i) a CD4.sup.+ cell
which is labeled with a first dye and (ii) a cell expressing the
HIV-1 envelope glycoprotein on its surface which is labeled with a
second dye, in the presence of an excess of the agent under
conditions permitting the fusion of the CD4.sup.+ cell to the cell
expressing the HIV-1 envelope glycoprotein on its surface in the
absence of the agent, the first and second dyes being selected so
as to allow resonance energy transfer between the dyes; (b)
exposing the product of step (a) to conditions which would result
in resonance energy transfer if fusion has occurred; and (c)
determining whether there is a reduction of resonance energy
transfer, when compared with the resonance energy transfer in the
absence of the agent, a decrease in transfer indicating that the
agent is capable of inhibiting fusion of HIV-1 to CD4.sup.+
cells.
[0062] HIV-1 only fuses with appropriate CD4.sup.+ cells. For
example, laboratory-adapted T lymphotropic HIV-1 strains fuse with
most CD4.sup.+ human cells. Clinical HIV-1 isolates do not fuse
with most transformed CD4.sup.+ human cell lines but do fuse with
human primary CD4.sup.+ cells such as CD4.sup.+ T lymphocytes and
macrophages. Routine experiments may be easily performed to
determine whether the CD4.sup.+ cell is appropriate for the above
fusion assay.
[0063] As described in this invention, HIV-1 membrane fusion is
monitored by a resonance energy transfer assay. The assay was
described in the International Application Number, PCT/US94/14561,
filed Dec. 16, 1994 with International Publication Number WO
95/16789. This assay is further elaborated in a United States
co-pending application Ser. No. 08/475,515, filed Jun. 7, 1995. The
contents of these applications are hereby incorporated by reference
into this application.
[0064] In an embodiment of the above method, the non-chemokine
agent is an oligopeptide. In another embodiment, the non-chemokine
agent is a polypeptide. In still another embodiment, the agent is
an antibody or a portion thereof. In a further embodiment, the
non-chemokine agent is a nonpeptidyl agent.
[0065] In a separate embodiment, the CD4.sup.+ cell is a PM1 cell.
In another embodiment, the cell expressing the HIV-1 envelope
glycoprotein is a HeLa cell expressing HIV-1.sub.JR-FL
gp120/gp41.
[0066] This invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific experiments detailed are
only illustrative of the invention as described more fully in the
claims which follow thereafter.
EXPERIMENTAL DETAILS
First Series of Experiments
[0067] 1) Chemokines Inhibit Fusion Mediated by the Envelope
Glycoprotein from a Macrophage-tropic primary isolate of HIV-1 but
not from a Laboratory-Adapted T-Lymphotrophic Strain of the
Virus
[0068] The chemokines RANTES, MIP-1.alpha. and MIP-1.beta. were
obtained from R & D systems (Minneapolis, Minn.). They were
tested in the RET assay for ability to inhibit fusion between
HeLa-env.sub.JR-FL cells (expressing gp120/gp41 from the macrophage
tropic isolate HIV-1.sub.JR-FL) and PM1 cells, or for inhibition of
fusion between HeLa-env.sub.LAI cells (expressing gp120gp41 from
the laboratory-adapted strain HIV-1.sub.LAI) and various CD4.sup.-
T lymphocyte cell lines. As shown in FIG. 1, all three chemokines
inhibited fusion mediated by the macrophage tropic virus envelope
glycoprotein, but not that mediated by the laboratory-adapted
strain envelope glycoprotein.
[0069] The ability of the chemokines to block the interaction
between CD4 and HIV-1 gp120 which occurs at virus attachment was
then tested. It was found that the chemokines did not inhibit this
interaction (FIG. 2), demonstrating that their blockade of HIV-1
envelope glycoprotein-mediated membrane fusion occurs at the
membrane fusion event itself, rather than the initial CD4-gp120
interaction which precedes fusion.
2) Non-Chemokine Peptides and Derivatives that Inhibit HIV-1
Fusion
[0070] The non-chemokines include chemokine fragments and chemokine
derivatives that are tested in the RET assay to determine which are
active in inhibiting HIV-1 membrane fusion. Particular attention is
focused on fragments or derivatives that inhibit HIV-1 fusion but
do not activate leukocyte responses. These non-chemokines
include:
[0071] a) N-terminal derivatives of the chemokines. Addition of
residues to the N-terminus of chemokines inhibits the function of
these proteins without significantly reducing the r ability to bind
chemokine receptors. For example, Met-RANTES (RANTES with an
N-terminal methionine) has been shown to be a powerful antagonist
of native RANTES and is unable to induce chemotaxis or calcium
mobilization in certain systems. The mechanism of antagonism
appears to be competition for receptor binding (9). Similar results
were found using other derivatives of the N terminus of RANTES (9)
and also by N-terminal modification of other chemokines, such as
IL-8 (a member of the C-X-C chemokines) (10). The current invention
includes Met-RANTES and other chemokines derivatised by the
addition of methionine, or other residues, to the N-terminus so
that they inhibit fusion mediated by the envelope glycoprotein of
HIV-1.sub.JR-FL, and inhibit infection by many isolates of HIV-1,
yet do not activate the inflammatory response.
[0072] b) Chemokines with N-terminal amino acids deleted: Chemokine
antagonists have been generated by deleting amino acids in the
N-terminal region. For example, deletion of up to 8 amino acids at
the N-terminus of the chemokine MCP-1 (a member of the C-C
chemokine group), ablated the bioactivity of the protein while
allowing it to retain chemokine receptor binding and the ability to
inhibit activity of native MCP-1 (11,12).
[0073] The current invention includes N-terminal deletants of
RANTES, MIP-1.alpha. and MIP-1.beta., lacking the biological
activity of the native proteins, which inhibit HIV-1 fusion and
HIV-1 infection.
[0074] c) Other peptides: A series of overlapping peptides (e.g. of
20-67 residues) from all regions of RANTES, MIP-1.alpha. and MIP-1,
are screened by the same approaches to identify peptides which
inhibit HIV-1 fusion most potently without activating leukocytes.
Activation of leukocyte responses is measured following routine
procedures (9, 10, 1, 12).
3) Cloning the Chemokine Receptors
[0075] Chemokine receptors required for HIV-1 fusion are cloned by
the following strategy. First a cDNA library is made in a mammalian
expression vector (e.g. pcDNA3.1 from Invitrogen Corp. San Diego,
Calif.) using mRNA prepared from the PM1 cell line or CD4.sup.-
T-lymphocytes or macrophages. Degenerate oligonucleotide probes are
used to identify members of the cDNA library encoding members of
the chemokine receptor family, for example following previously
published methods (2). The vectors containing chemokine receptor
cDNAs are then individually expressed in one of several mammalian
cell lines which express human CD4 but do not fuse with
HeLa-env.sub.JR-FL cells (e.g. HeLa-CD4, CHO-CD4 or COS-CD4) or
HeLa-env.sub.LAI cells (e.g. CHO-CD4 or COS-CD4). Following
analysis in the RET assay, clones which gain the ability to fuse
with HeLa-env.sub.JR-FL or HeLa-env.sub.LAI are identified and the
coding sequences recovered, for example by PCR amplification,
following procedures well known to those skilled in the art. DNA
sequencing is then performed to determine whether the cDNA
recovered encodes a known chemokine receptor. Following expression
of the receptor, monoclonal and polyclonal antibodies are prepared
and tested for ability to inhibit infection by a panel of HIV-1
isolates.
REFERENCES OF THE FIRST SERIES OF EXPERIMENTS
[0076] 1. Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K.,
Gallo, R. C., Lusso, P. 1995. Science. 270:1811-1815. [0077] 2.
Raport, C. J., Schweickart, V. L., Chantry, D., Eddy Jr., R. L.,
Shows, T. B., Godiska, R., Gray, P. W. 1996. Journal of Leukocyte
Biology. 59: 18-23. [0078] 3. Maddon P J., Dalgleish A G., McDougal
J S., Clapham P R., Weiss R A., Axel R. 1986. Cell. 47:333-348.
[0079] 4. Ashorn P A., Berger E A., Moss B. 1990. J. Virol.
64:2149-2156. [0080] 5. Clapham P R., Blanc D., Weiss R A. 1991.
Virology. 181:703-715. [0081] 6. Harrington R D., Geballe A P.
1993. J. Virol. 67:5939-5947. [0082] 7. Broder C C., Dimitrov D S.,
Blumenthal R., Berger E A. 1993. Virology. 193:483-491. [0083] 8.
Dragic T., Charneau P., Clavel F., Alizon M. 1992. J. Virol.
66:4794-4802. [0084] 9. Wells, T. N., Power, C. A.,
Lusti-Narasimhan, M., Hoogewerf, A. J., Cooke, R. M., Chung, C. W.,
Peitsch, M. C., Proudfoot, A. E. 1996. Journal of Leukocyte
Biology. 59:53-60. [0085] 10. Moser, B., Dewald, B., Barella, L.,
Schumacher, C., Baggiolini, M., Clark-Lewis, I. 1993. Journal of
Biological Chemistry. 268:7125-7128. [0086] 11. Gong, J. H.,
Clark-Lewis, I. 1995. J. Exp. Med. 181:631-640. [0087] 12. Zhang,
Y. J., Rutledge, B. J., Rollins, B. J. 1994. Journal of Biological
Chemistry. 269:15918-15924. [0088] 13. Merrifield, R. B. (1963) J.
Am. Chem. Soc. 85: 2149-2154. [0089] 14. Goeghegan, K. F. Stroh, J.
F. (1992) Biocorjugate Chem. 3: 138-146.
Second Series of Experiments
[0090] The replication of primary, non-syncytium-inducing (NSI)
HIV-1 isolates in CD4.sup.- T-cells is inhibited by the C-C
.beta.-chemokines MIP-1.alpha., MIP-1.beta. and RANTES (1,2), but
T-cell line-adapted (TCLA) or syncytium-inducing (SI) primary
strains are insensitive (2,3). The .beta.-chemokines are small (8
kDa), related proteins active on cells of the lymphoid and monocyte
lineage (4-8). Their receptors are members of the
7-membrane-spanning, G-protein-linked superfamily, one oL which
(the LESTR orphan receptor) has been identified as the second
receptor for TCLA HIV-1 strains, and is now designated fusin (9).
Fusin is not known to be a .beta.-chemokine receptor (7-9).
[0091] To study how .beta.-chemokines inhibit HIV-1 replication, a
virus entry assay based on single-cycle infection by an
env-deficient virus, NL4/3.DELTA.env (which also carries the
luciferase reporter gene), complemented by envelope glycoproteins
expressed in trans was used (10,11). Various env-complemented
viruses were tested in PM1 cells, a variant of HUT-78 that has the
unique ability to support replication of primary and TCLA HIV-1
strains, allowing comparison of envelope glycoprotein functions
against a common cellular background (2,12). MIP-1.alpha.,
MIP-1.beta. and RANTES are most active against HIV-1 in combination
(2,3), and strongly inhibited infection of PM1 cells by
complemented viruses whose envelopes are derived from the NSI
primary strains ADA and BaL (Table 1a).
TABLE-US-00001 TABLE 1 Inhibition of HIV-1 entry in PM1 cells and
CD4.sup.+ T-cells by .beta.-chemokines % luciferase activity a) BaL
ADA NL4/3 HxB2 MuLV PM1 cells control without virus 2 2 2 5 3
control with virus 100 100 100 100 100 +R/M.alpha./M.beta.
(50/50/50) 2 3 92 117 100 +RANTES (100) 1 1 nd nd nd +MIP-1.alpha.
(100) 54 54 nd nd nd +MIP-1.beta. (100) 1 6 nd nd nd +MCP-1 (100)
46 50 nd nd nd +MCP-2 (100) 28 26 nd nd nd +MCP-3 (100) 58 46 nd nd
nd b) JR-FL HxB2 MuLV LW4 CD4.sup.+ T-cells control without virus 1
1 1 control with virus 100 100 100 +R/M.alpha./M.beta.
(200/200/200) 14 68 nd LW5 CD4.sup.+ T-cells control without virus
1 1 1 control with virus 100 100 100 +R/M.alpha./M.beta.
(200/200/200) 15 73 nd
Table 1 Legend:
[0092] PM1 cells were cultured as described by Lusso et al (12).
Ficoll/hypaque-isolated PBMC from laboratory workers (LW)
stimulated with PHA for 72 h before depletion of CD8+ Lymphocytes
with anti-CD8 immunomagnetic beads (DYNAL, Great Neck, NY). CD4+
Lymphocytes were maintained in culture medium containing
interleukin-2 (100 U/ml; Hofmann LaRoche, Nutley, N.J.), as
described previously (3). Target cells (1-2.times.10.sup.5) were
infected with supernatants (10-50 ng of HIV-1 p24) from 293-cells
co-transfected with an NL4/3.DELTA.env-luciferase vector and a
HIV-1 env-expressing vector (10,11). .beta.-Chemokines (R & D
Systems, Minneapolis) were added to the target cells simultaneously
with virus, at the final concentrations (ng/ml) indicated in
parentheses in the first column. The .beta.-chemokine concentration
range was selected based on prior studies (2,3). After 2 h, the
cells were washed twice with PBS, resuspended in
.beta.-chemokine-containing media and maintained for 48-96 h.
Luciferase activity in cell lysates was measured as described
previously (10,11). The values indicated represent luciferase
activity (cpm)/ng p24/mg protein, expressed relative to that in
virus-control cultures Lacking .beta.-chemokines (100%), and are
the means of duplicate or sextuplicate determinations, and, not
done. R/M.alpha./M.beta., RANTES+MIP-1.alpha.+MIP-1.beta..
[0093] RANTES and MIP-1.beta. were strongly active when added
individually, while other .beta.-chemokines--M1P-1.alpha., MCP-1,
MCP-2 and MCP-3 (refs. 13-15)--were weaker inhibitors (Table 1a).
However, MIP-1.alpha., MIP-1.beta. and RANTES, in combination, did
not inhibit infection of PM1 cells by the TCLA strains NL4/3 and
HxB2, or by the amphotropic murine leukemia virus (MuLV-Ampho)
pseudotype (Table 1a). Thus, phenotypic characteristics of the
HIV-1 envelope glycoproteins influence their sensitivity to
.beta.-chemokines in a virus entry assay.
[0094] The env-complementation assay was used to assess HIV-1 entry
into CD4+ T-cells from two control individuals (LW4 and LW5).
MIP-1.alpha., MIP-1.beta. and RANTES strongly inhibited infection
by the NSI primary strain JR-FL infection of LW4's and LW5's CD4
T-cells, and weakly reduced HxB2 infection of UV cells (Table 1b),
suggesting that there may be some overlap in receptor usage on
activated CD4 T-cells by different virus strains. BaL env-mediated
replication in normal PBL was also inhibited by MIP-1.alpha.,
MIP-1.beta. and RATES, albeit with significant inter-donor
variation in sensitivity (data not shown).
[0095] It was determined when .beta.-chemokines inhibited HIV-1
replication by showing that complete inhibition or infection of PM1
cells required the continuous presence of .beta.-chemokines for up
to 5 h after addition of ADA or BaL env-complemented virus (FIG.
3a). Pre-treatment of the cells wish .beta.-chemokines for 2 h or
24 h prior to infection had no inhibitory effect if the cells were
subsequently washed before virus addition. Furthermore, adding
S-chemokines 2 h after virus only minimally affected virus entry
(FIG. 3a). A PCR-based assay was next used to detect HIV-1 early
DNA reverse transcripts in PM1 cells after 10 h of infection;
reverse transcription of ADA, but not of NL4/3, could not be
detected in the presence of MIP-1.beta. and RANTES (FIG. 3b). Thus,
inhibition by .beta.-chemokines requires their presence during at
least one of the early stages of HIV-1 replication: virus
attachment, fusion and early reverse transcription.
[0096] As described in part in the First Series of Experiments,
these sites of action were discriminated, first by testing whether
.beta.-chemokines inhibited binding of JR-FL or BRU (LAI) gp120 to
soluble CD4, or of tetrameric CD4-IgG2 binding to HeLa-JR-FL cells
expressing oligomeric envelope glycoproteins (17). No inhibition by
any of the .beta.-chemokines was found in either assay, whereas the
OKT4a CD4-MAb was strongly inhibitory in both (FIG. 2 and data not
shown). Thus, s-chemokines inhibit a step after CD4 binding, when
conformational changes in the envelope glycoproteins lead to fusion
of the viral and cellular membranes (18). Cell-cell membrane fusion
is also induced by the gp120-CD4 interaction, and can be monitored
directly by resonance energy transfer (RET) between fluorescent
dyes incorporated into cell membranes (17). In the RET assay, OKT4a
completely inhibits membrane fusion of PM1 cells with HeLa cells
expressing the envelope glycoproteins of either JR-FL (HeLa-JR-FL,
the same cell line referred to above as HeLa-env.sub.JR-FL) or BRU
(HeLa-BRU, the same cell line referred to above as
HeLa-env.sub.LAI), confirming the specificity of the process (17).
RANTES, MIP-1.beta. (and to a lesser extent, MIP-1.alpha.) strongly
inhibited membrane fusion of HeLa-JR-FL cells with PM1 cells,
whereas fusion between PM1 cells and HeLa-BRU cells was insensitive
to these S-chemokines (FIG. 1 and Table 2a).
TABLE-US-00002 TABLE 2 Effect of .beta.-chemokines on HIV-1
envelope glycoprotein-mediated membrane fusion measured using the
RET assay % Fusion HeLa-JR-FL HeLa-BRU a) PM1 cells no chemokines
100 100 +R/M.alpha./M.beta. (80/400/100) 1 95 +RANTES (80) 8 100
+MIP-1.alpha. (400) 39 100 +MIP-1.beta. (100) 13 93 +MCP-1 (100) 99
98 +MCP-2 (100) 72 93 +MCP-3 (100) 98 99 b) LW5 CD4.sup.+ cells no
chemokines 100 100 +R/M.alpha./M.beta. (106/533/133) 39 100 +RANTES
(106) 65 95 +MIP-1.alpha. (533) 72 100 +MIP-1.beta. (133) 44 92
+OKT4A (3 ug/ml) 0 0
Table 2 Legend:
[0097] CD4.sup.- target cells (mitogen-activated CD4.sup.-
lymphocytes or PM1 cells) were labeled with octadecyl rhodamine
(Molecular Probes, Eugene, Oreg.), and HeLa-JR-FL cells, HeLa-BRU
cells (or control HeLa cells, not shown) were labeled with
octadecyl fluorescein (Molecular Probes), overnight at 37.degree.
C. Equal numbers of labeled target cells and env-expressing cells
were mixed in 96-well plates and .beta.-chemokines (or CD4 MAb
OKT4a) were added at the final concentrations (ng/ml) indicated in
parentheses in the first column. Fluorescence emission values were
determined 4 h after cell mixing (17). If cell fusion occurs, the
dyes are closely associated in the conjoined membrane such that
excitation of fluorescein at 450 nm results in resonance energy
transfer (RET) and emission by rhodamine at 590 nm. Percentage
fusion is defined as equal to 100.times.[(Exp RET-Min RET)/(Max
RET-Min RET)], where Max RET=% RET obtained when HeLa-Env and CD4
cells are mixed, Exp RET=% RET obtained when HeLa-Env and CD4.sup.-
cells are mixed in the presence of fusion-inhibitory compounds, and
Min RET=FRET obtained when HeLa cells (lacking HIV-1 envelope
glycoproteins) and CD4 cells are mixed. The % RET value is defined
by a calculation described elsewhere (17), and each is the mean of
triplicate determinations. These values were, for HeLa-JR-FL and
HeLa-BRU cells respectively: PM1 cells 11.5%, 10.5%; LW5 CD4.sup.-
cells, 6.0%, 10.5%; R/M.alpha./M.beta.,
RANTES+MIP-1.alpha.+MIP-1.beta..
[0098] Similar results were obtained with primary CD4' T-cells from
LW5 (Table 2b), although higher concentrations of .beta.-chemokines
were required to inhibit membrane fusion in the primary cells than
in PM1 cells. Thus, the actions of the .beta.-chemokines are not
restricted to the PM1 cell line. The RET assay demonstrates that
.beta.-chemokines interfere with env-mediated membrane fusion.
[0099] The simplest explanation of these results is that the
binding of certain .beta.-chemokines to their receptor(s) prevents,
directly or otherwise, the fusion of HIV-1 with CD4.sup.- T-cells.
It has been known for a decade that HIV-1 requires a second
receptor for entry into CD4 cells (19-21). This function is
supplied, for TCLA strains, by fusin (9). Several receptors for
MIP-1.alpha., MIP-1.beta. and RANTES have been identified (6,7),
and .beta.-chemokines exhibit considerable cross-reactivity in
receptor usage (4-8). However, C-C CKR-1 and, especially, C-C CKR-5
were identified as the most likely candidates, based on tissue
expression patterns and their abilities to bind MIP-1.alpha.,
MIP-1.beta. and RANTES (4, 7, 8, 15, 22). C-C CKR-1, C-C CKR-5 and
LESTR are each expressed at the mRNA level in PM1 cells and primary
macrophages (data not shown). These and other .beta.-chemokine
receptors were therefore PCR-amplified, cloned and expressed.
[0100] The expression of C-C CKR-5 in HeLa-CD4 (human), COS-CD4
(simian) and 3T3-CD4 (murine) cells rendered each of them readily
infectible by the primary, NSI strains ADA and BaL in the
env-complementation assay of HIV-1 entry (Table 3).
TABLE-US-00003 TABLE 3 C-C CKR-5 expression permits infection of
CD4-expressing cells by primary, NSI HIV-1 strains
R/M.alpha./M.beta. pcDNA3.1 LESTR CKR-1 CKR-2a CKR-3 CKR-4 CKR-5
CKR-5 COS-CD4 ADA 798 456 600 816 516 534 153000 3210 BaL 660 378
600 636 516 618 58800 756 HxB2 5800 96700 5240 5070 5470 5620 4850
5000 HeLa-CD4 ADA 678 558 4500 912 558 600 310000 6336 BaL 630 738
1800 654 516 636 104000 750 HxB2 337000 nd nd nd nd nd nd 356000
3T3-CD4 ADA 468 558 450 618 534 606 28400 1220 BaL 606 738 660 738
534 558 11700 756 HxB2 456 24800 618 672 732 606 618 606
Table 3 Legend:
[0101] Chemokine receptor genes C-C CKR-1, C-C CKR-2a, C-C CKR-3,
C-C CKR-4 and C-C CKR-5 have no introns (4-8, 15, 22) and were
isolated by PCR performed directly on a human genomic DNA pool
derived from the PBMC of seven healthy donors. Oligonucleotides
overlapping the ATG and the stop codons and containing BamHI and
Xhol restriction sites for directional cloning into the pcDNA3.1
expression vector (Invitrogen Inc.) were used. LESTR (also known as
fusin or HUMSTR) (4, 9, 24) was cloned by PCR performed directly on
cDNA derived from PM1 cells, using sequences derived from the NIH
database. Listed below are the 5' and 3' primer pairs used in first
(5-1 and 3-1) and second (5-2 and 3-2) round PCR amplification of
the CKR genes directly from human genomic DNA, and of LESTR from
PM1 cDNA. Only a single set of primers was used to amplify
CKR-5.
TABLE-US-00004 LESTR: (SEQ ID NO: 6) L/5-1 = AAG CTT GGA GAA CCA
GCG GTT ACC ATG GAG GGG ATC; (SEQ ID NO: 7) L/5-2 = GTC TGA GTC TGA
GTC AAG CTT GGA GAA CCA; (SEQ ID NO: 8) L/3-1 = CTC GAG CAT CTG TGT
TAG CTG GAG TGA AAA CTT GAA GAC TC; (SEQ ID NO: 9) L/3-2 = GTC TGA
GTC TGA GTC CTC GAG CAT CTG TGT; CKR-1: (SEQ ID NO: 10) C1/5-1 =
AAG CTT CAG AGA GAA GCC GGG ATG GAA ACT CC; (SEQ ID NO: 11) C1/5-2
= GTC TGA GTC TGA GTC AAG CTT CAG AGA GAA; (SEQ ID NO: 12) C1/2-1 =
CTC GAG CTG AGT CAG AAC CCA GCA GAG AGT TC; (SEQ ID NO: 13) C1/3-2
= GTC TGA GTC TGA GTC CTC GAG CTG AGT CAG; CKR-2a: (SEQ ID NO: 14)
C2/5-1 = AAG CTT CAG TAC ATC CAC AAC ATG CTG TCC AC; (SEQ ID NO:
15) C2/5-2 = GTC TGA GTC TGA GTC AAG CTT CAG TAC ATC; (SEQ ID NO:
16) C2/3-1 = CTC GAG CCT CGT TTT ATA AAC CAG CCG AGA C; (SEQ ID NO:
17) C2/3-2 = GTC TGA GTC TGA GTC CTC GAG CCT CGT TTT; CKR-3: (SEQ
ID NO: 18) C3/S-1 = AAG CTT CAG GGA GAA GTG AAA TGA CAA CC; (SEQ ID
NO: 19) C3/5-2 = GTC TGA GTC TGA GTC AAG CTT CAG GGA GAA; (SEQ ID
NO: 20) C3/3-1 = CTC GAG CAG ACC TAA AAC ACA ATA GAG AGT TCC; (SEQ
ID NO: 21) C3/3-2 = GTC TGA GTC TGA GTC CTC GAG CAG ACC TAA; CKR-4:
(SEQ ID NO: 22) C4/5-1 = AAG CTT CTG TAG AGT TAA AAA ATG AAC CCC
ACG G; (SEQ ID NO: 23) C4/5-2 = GTC TGA GTC TGA GTC AAG CTT CTG TAG
AGT; (SEQ ID NO: 24) C4/3-1 = CTC GAG CCA TTT CAT TTT TCT ACA GGA
CAG CAT C; (SEQ ID NO: 25) C4/3-2 = GTC TGA GTC TGA GTC CTC GAG CCA
TTT CAT; CKR-5: (SEQ ID NO: 26) C5/5-12 = GTC TGA GTC TGA GTC AAG
CTT AAC AAG ATG CAT TAT CAA; (SEQ ID NO: 37) C5/3-12 = GTC TGA GTC
TGA GTC CTC GAG TCC GTG TCA CAA GCC CAC.
[0102] The human CD4-expressing cell lines HeLa-CD4 (P42), 3T3-CD4
(sc6) and COS-CD4 (Z28T1) (23) were transfected with the different
pcDNA3.1-CKR constructs by the calcium phosphate method, then
infected 48 h later with different reporter viruses (200 ng of
HIV-1 p24/10.sup.6 cells) in the presence or absence of
.beta.-chemokines (400 ng/ml each of RANTES, MIP-1.alpha. and
MIP-1.beta.). Luciferase activity in cell lysates was measured 48 h
later (10, 11). .beta.-Chemokine blocking data is only shown for
C-C CKR-5, as infection mediated by the other C-C CKR genes was too
weak for inhibition to be quantifiable. In PCR-based assays of
HIV-1 entry, a low level of entry of NL4/3 and ADA into C-C CKR-1
expressing cells (data not shown) was consistently observed.
[0103] Neither LESTR nor C-C CKR-1, -2a, -3 or -4 could substitute
for C-C CKR-5 in this assay. The expression of LESTR in COS-CD4 and
3T3-CD4 cells permitted HxB2 entry, and HxB2 readily entered
untransfected (or control plasmid-transfected) HeLa-CD4 cells
(Table 3). Entry of BAL and ADA into all three C-C CKR-5-expressing
cell lines was almost completely inhibited by the combination of
MIP-1a, MIP-1.beta. and RANTES, whereas HxB2 entry into
LESTR-expressing cells was insensitive to .beta. chemokines (Table
3). These results suggest that C-C CKR-5 functions as a
.beta.-chemokine-sensitive second receptor for primary, NSI HIV-1
strains.
[0104] The second receptor function of C-C CKR-5 was confirmed in
assays of env-mediated membrane fusion. When C-C CKR-5 was
transiently expressed in COS and HeLa cell lines that permanently
expressed human CD4, both cell lines fused strongly with HeLa cells
expressing the JR-FL envelope glycoproteins, whereas no fusion
occurred when control plasmids were used (data not shown).
Expression of LESTR instead of C-C CKR-5 did not permit either
COS-CD4 or HeLa-CD4 cells to fuse with HeLa-JR-FL cells, but did
allow fusion between COS-CD4 cells and HeLa-BRU cells (data not
shown).
[0105] The fusion capacity of .beta.-chemokine receptors was also
tested in the RET assay. The expression of C-C CKR-5, but not of
C-C CKR-1, -2a, -3 or -4, permitted strong fusion between HeLa-CD4
cells and HeLa-JR-FL cells. The extent of fusion between HeLa-JR-FL
cells and C-C CKR-5-expressing HeLa-CD4 cells was greater than the
constitutive level of fusion between HeLa-BRU cells and HeLa-CD4
cells (FIG. 4). The fusion-conferring function of C-C CKR-5 for
primary, NS HIV-1 strains has therefore been confirmed in zwo
independent fusion assays.
EXPERIMENTAL DISCUSSION
[0106] Together, the above results establish that M1P-1.alpha.,
MIP-1.beta. and RANTES inhibit HIV-1 infection at the entry stage,
by interfering with the virus-cell fusion reaction subsequent to
CD4 binding. It was also shown that C-C CKR-5 can serve as a second
receptor for entry of primary NSI strains of HIV-1 into CD4+
T-cells, and that the interaction of J-chemokines with C-C CKR-5
inhibits the HIV-1 fusion reaction.
REFERENCES OF THE SECOND SERIES OF EXPERIMENTS
[0107] 1. Levy, J. A., Mackewicz, C. E. & Barker, E. Immunol.
Today 17, 217-224 (1996). [0108] 2. Cocchi, F. et al. Science 270,
1811-1815 (1995). [0109] 3. Paxton, W. A. et al. Nat. Med. 2,
412-417 (1996). [0110] 4. Neote, K., DiGregorio, D., Mak, J. Y.,
Horuk, R., & Schall, T. J. Cell 72, 415-425 (1993). [0111] 5.
Gao, J. -L. et al. J . Exp. Med. 177, 1421-1427 (1993). [0112] 6.
Bacon, K. B., Premack, B. A., Gardner, P. & Schall, T. J.
Science 269, 1727-1729 (1995). [0113] 7. Raport, C. J. et al. J.
Leukoc. Biol. 59, 18-23 (1996). [0114] 8. Wells, T. N. C. et al. J.
Leukoc. Biol. 59, 53-60 (1996). [0115] 9. Feng, Y., Broder, C. C.,
Kennedy, P. E. & Berger, E. A. Science 272, 872-877 (1996).
[0116] 10. Chen, B. K., Saksela, K., Andino, R. & Baltimore, D.
J. Virol. 68, 654-660 (1994). [0117] 11. Connor, R. I., Chen, B.
K., Choe, S., & Landau, N. R. Virology 206, 935-944 (1995).
[0118] 12. Lusso, P. et al. J. Virol. 69, 3712-3720 (1995). [0119]
13. Charo, I. F. et al. Proc. Natl. Acad. Sci. USA 91, 2752-2756
(1994). [0120] 14. Ben-Baruch, A. et al. J. Biol. Chem. 270,
22123-22128 (1995). [0121] 15. Combadiere, C et al. J. Biol. Chem.
270, 29671-29675 (1995). [0122] 16. Lip, J. P., D'Andrea, A. D.,
Lodish, H. F. & Baltimore, D. Nature 343, 762-764 (1990).
[0123] 17. Litwin, V. et al. J. Virol. (submitted for publication)
[0124] 18. Moore, J. P., Jameson, B. A., Weiss, R. A. &
Sattentau, Q. J. in Viral Fusion Mechanisms (ed Bentz, J.) 233-289
(CRC Press Inc, Boca Raton, USA, 1993). [0125] 19. Maddon, P. J. et
al. Cell 47, 333-348 (1986). [0126] 20. Ashorn, P. A., Berger, E.
A. & Moss, B. J. Virol. 64, 2149-2156 (1990). [0127] 21.
Clapham, P. R., Blanc, D. & Weiss, R. A. Virology 181, 703-715
(1991). [0128] 22. Samson, M., Labbe, O., Mollereau, C., Vassart,
G. & Parmentier, M. Biochemistry 11, 3362-3367 (1996). [0129]
23. Dragic, T., Charneau, P., Clavel, F. & Alizon, M. J. Virol.
66, 4794-4802 (1992) [0130] 24. Loetscher, M. et al. J. Biol. Chem.
269, 232-237 (1994). [0131] 25. Moore, J. P. & Ho, D. D. AIDS 9
(suppl A), S117-S136 (1995). [0132] 26. Trkola, A. & Moore, J.
P. (unpublished data). [0133] 27. Chaudhuri, A., et al. 1994. J.
Biol. Chem. 269, 7835-7838 (1994). [0134] 28. Neote, K., Mak, J.
Y., Kolakowski Jr., L. F. & Schall, T. J. Blood 84, 44-52
(1994). [0135] 29. Dragic, T., Picard, L. & Alizon, M. J.
Virol. 69, 1013-1018 (1995). [0136] 30. Puri, A., Morris, S. J.,
Jones, P., Ryan, M. & Blumenthal, R. Virology 219, 262-267
(1996).31
Third Series of Experiments
[0137] The chemokine SDF-1 (stromal cell-derived factor 1) is the
natural ligand for Fusin/CXCR4 and blocks infection by
laboratory-adapted strains of HIV-1 (Ref. 1 and 2). SDF-1 exists as
at least two forms, SDF-1.alpha. and SDF-1.beta. based on variable
splicing of the SDF-1 gene (Ref. 1 and 3) in the RET assay, this
chemokine specifically inhibits membrane fusion mediated by
gp120/gp41 form the laboratory-adapted strain HIV.sub.LAI but not
by gp120/gp41 from the macrophage-tropic isolate HIV-1.sub.JR-FL as
shown in FIG. 5.
REFERENCES OF THE THIRD SERIES OF EXPERIMENTS
[0138] 1. Bleul, C. C., et al. (1996) Nature 382:829-833 [0139] 2.
Oberlin, E., et al. (1996) Nature 382:833-835 [0140] 3. Shirozu,
M., et al. (1995) Genomics 28:495-500
Sequence CWU 1
1
27138DNAArtificial SequencePrimer 1caaggctact tccctgattg gcagaactac
acaccagg 38225DNAArtificial SequencePrimer 2agcaagccga gtcctgcgtc
gagag 25323DNAArtificial SequencePrimer 3gggactttcc gctggggact ttc
23433DNAArtificial SequencePrimer 4cctgttcggg cgccactgct agagattttc
cac 33560PRTHomo sapiens 5Pro Cys Cys Phe Ala Tyr Ile Ala Arg Pro
Leu Pro Arg Ala His Ile1 5 10 15Lys Glu Tyr Phe Tyr Thr Ser Gly Lys
Cys Ser Asn Pro Ala Val Val 20 25 30Phe Val Thr Arg Lys Asn Arg Gln
Val Cys Ala Asn Pro Glu Lys Lys 35 40 45Trp Val Arg Glu Tyr Ile Asn
Ser Leu Glu Met Ser 50 55 60636DNAArtificial SequencePrimer
6aagcttggag aaccagcggt taccatggag gggatc 36730DNAArtificial
SequencePrimer 7gtctgagtct gagtcaagct tggagaacca 30841DNAArtificial
SequencePrimer 8ctcgagcatc tgtgttagct ggagtgaaaa cttgaagact c
41930DNAArtificial SequencePrimer 9gtctgagtct gagtcctcga gcatctgtgt
301032DNAArtificial SequencePrimer 10aagcttcaga gagaagccgg
gatggaaact cc 321130DNAArtificial SequencePrimer 11gtctgagtct
gagtcaagct tcagagagaa 301232DNAArtificial SequencePrimer
12ctcgagctga gtcagaaccc agcagagagt tc 321330DNAArtificial
SequencePrimer 13gtctgagtct gagtcctcga gctgagtcag
301432DNAArtificial SequencePrimer 14aagcttcagt acatccacaa
catgctgtcc ac 321530DNAArtificial SequencePrimer 15gtctgagtct
gagtcaagct tcagtacatc 301631DNAArtificial SequencePrimer
16ctcgagcctc gttttataaa ccagccgaga c 311730DNAArtificial
SequencePrimer 17gtctgagtct gagtcctcga gcctcgtttt
301829DNAArtificial SequencePrimer 18aagcttcagg gagaagtgaa
atgacaacc 291930DNAArtificial SequencePrimer 19gtctgagtct
gagtcaagct tcagggagaa 302033DNAArtificial SequencePrimer
20ctcgagcaga cctaaaacac aatagagagt tcc 332130DNAArtificial
SequencePrimer 21gtctgagtct gagtcctcga gcagacctaa
302234DNAArtificial SequencePrimer 22aagcttctgt agagttaaaa
aatgaacccc acgg 342330DNAArtificial SequencePrimer 23gtctgagtct
gagtcaagct tctgtagagt 302434DNAArtificial SequencePrimer
24ctcgagccat ttcatttttc tacaggacag catc 342530DNAArtificial
SequencePrimer 25gtctgagtct gagtcctcga gccatttcat
302639DNAArtificial SequencePrimer 26gtctgagtct gagtcaagct
taacaagatg gattatcaa 392739DNAArtificial SequencePrimer
27gtctgagtct gagtcctcga gtccgtgtca caagcccac 39
* * * * *