WO1998015569A1 - Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih - Google Patents

Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih Download PDF

Info

Publication number
WO1998015569A1
WO1998015569A1 PCT/US1997/018397 US9718397W WO9815569A1 WO 1998015569 A1 WO1998015569 A1 WO 1998015569A1 US 9718397 W US9718397 W US 9718397W WO 9815569 A1 WO9815569 A1 WO 9815569A1
Authority
WO
WIPO (PCT)
Prior art keywords
hiv
ccr5
ccr3
cells
binding
Prior art date
Application number
PCT/US1997/018397
Other languages
English (en)
Other versions
WO1998015569A9 (fr
Inventor
Joseph Sodroski
Walter Newman
Lijun Wu
Norma Gerard
Craig Gerard
Original Assignee
Dana-Farber Cancer Institute
Leukosite Incorporated
Children's Medical Center Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dana-Farber Cancer Institute, Leukosite Incorporated, Children's Medical Center Corporation filed Critical Dana-Farber Cancer Institute
Priority to CA002267662A priority Critical patent/CA2267662A1/fr
Priority to EP97913670A priority patent/EP0956290A1/fr
Priority to AU50800/98A priority patent/AU731975B2/en
Publication of WO1998015569A1 publication Critical patent/WO1998015569A1/fr
Publication of WO1998015569A9 publication Critical patent/WO1998015569A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/162—HIV-1, HIV-2 env, e.g. gp160, gp110/120, gp41, V3, peptid T, DC4-Binding site
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the present invention is directed to gpl20 polypeptides having a conformational discontinous chemokine receptor binding site and methods for inhibiting HIV infectivity.
  • Human immunodeficiency viruses type 1 and type 2 HIV-1 and HIV-
  • AIDS acquired immunodeficiency syndrome
  • AIDS results from the depletion of CD4-positive T lymphocytes in HIV-infected individuals (Fauci et al, 1984).
  • HIV-1 infects T lymphocytes, monocytes /macrophage, dendritic cells and, in the central nervous system, microglia (Gartner et al, 1986; Koenig et al, 1986; Pope et al, 1994; Weissman et al, 1995).
  • CD4 glycoprotein which serves as the receptor for HIV- 1 and HIV-2 (Dalgleish et al, 1984; Klatzman et al, 1984; Maddon et al, 1986). Efficient entry of HIV- 1 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gpl20, to the CD4-arnino-terrninal domain (McDougal et al, 1986; Helseth et al, 1990). After virus binding, the HIV- 1 envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the entry process (Kowalski et al, 1987; Stein et al, 1987; Helseth et al, 1990).
  • Membrane fusion directed by HIV-1 envelope glycoproteins expressed on the infected cell surface leads to fusion with uninfected CD4-positive cells, resulting in syncytia (Lifson et al, 1986; Sodroski et al, 1986).
  • HIV- 1 envelope glycoprotein-mediated membrane fusion Host cell factors in addition to CD4 appear necessary for effective HIV- 1 envelope glycoprotein-mediated membrane fusion.
  • Some human and animal cells have been shown to be resistant to HIV- 1 infection and syncytium formation even when human CD4 was expressed on the cell surface (Maddon et al, 1986; Ashorn et al, 1990; Chesebro et al, 1990; McKnight et al, 1994).
  • somatic cell hybrids suggested the possibility that a positive factor expressed in cells susceptible to syncytium formation could complement the block to fusion in resistant cell types (Clapham et al, 1991; Dragic et al, 1992; Broder et al, 1993).
  • HIV- 1 variants exhibiting distinct differences in the ability to fuse with and to enter particular subsets of CD4-positive cells have been identified (Broder and Berger, 1995).
  • T cell line-tropic primary viruses These viruses will be referred to herein as T cell line-tropic primary viruses (sometimes referred to as "T")
  • T cell line-tropic primary viruses by virtue of their ability to replicate on some immortalized cell lines, serve as precursors to the laboratory-adapted isolates, which have been extensively passaged on such cell lines. Laboratory adaptation, however, results in a loss of the ability of HIV- 1 to replicate in primary monocyte/macrophage cultures (Schuitemaker et al, 1991 ; Chesebro et al, 1991 ; Westervelt et al, 1992; Valentin et al, 1994).
  • HIV- 1 isolates replicate on primary T lymphocytes
  • three groups of virus variants can be defined based on the ability to replicate in primary monocyte /macrophages or in immortalized T cell lines: (1) macrophage-tropic primary viruses that cannot infect T cell lines; (2) laboratory-adapted viruses that cannot infect primary monocytes/macrophages; and (3) T cell line-tropic primary viruses that exhibit dual-tropism for these cell types.
  • V3 region of the g l20 exterior envelope glycoprotein determine tropism-related phenotypes (Cheng-Mayer et al, 1990; O'Brien et al, 1990; Hwang et al, Westervelt et al, 1992; Chesebro et al, 1992; Willey et al,
  • V3 region which contains a surface-exposed, disulfide-linked loop (Leonard et al, 1990; Moore et al, 1994), might act in conjunction with target cell moieties to determine the efficiency of membrane fusion events.
  • HUMSTR human G protein-coupled seven transmembrane segment receptor
  • LCR-1 LCR-1 or LESTR
  • CXCR4 Creasppiel et al, 1993; Jazin et al, 1993; Loetscher et al, 1994
  • Antibodies to HUMSTR blocked cell fusion and infection by laboratory-adapted HIV- 1 isolates but not by macrophage-tropic primary viruses (Feng et al, 1996).
  • HUMSTSR While its natural ligand is currently unknown, HUMSTSR exhibits sequence similarity to the receptor for interleukin-8, an alpha (CXC) chemokine) (Probst et al, 1992).
  • CXC alpha
  • Chemokines are a family of structurally related peptides that recruit leukocytes to inflammatory lesions, induce release of granule contents from granulocytes, regulate integrin avidity, and in general exhibit proinflammatory properties.
  • the ⁇ chemokines, or CXC chemokines primarily activate neutrophils, while the ⁇ chemokines or CC chemokines, generally activate monocytes, lymphocytes, basophils and eosinophils (Baggiolini et al, 1994; Schall and Bacon, 1994). Receptors to these chemokines belong to the G protein-coupled receptor family.
  • the large family of G protein-coupled receptors responds to chemoattractants, neurotransmitters, peptide hormones, light and odorants.
  • Amino acid identity among receptors that bind functionally related ligands ranges from 20-80% (Probst et al, 1992; Gerard and Gerard, 1994). Seven transmembrane receptors that transduce their signals through heterotrimeric G proteins are used by leukocytes to respond to chemokines (Horuk et al, 1994).
  • chemokines Hast et al, 1994.
  • There are a number of closely related molecules in the CC chemokine receptor family but only six of these have been characterized in ligand binding assays. These are designated CCR1, CCR2A, CCR2B, CCR3, CCR4 and CCR5.
  • FIGS. 1A- 1D show CAT activity in transfected HeLa cells exposed to recombinant HIV- 1 viruses.
  • HeLa cells expressing human CD4 only are shown in Fig. 1A
  • CD4 and CCR1F are shown in Fig. IB
  • CD4 and CCR3 are shown in Fig. 1C
  • CD4 and CCR5 are shown in Fig. ID.
  • the cells were exposed to recombinant viruses containing either no envelope glycoproteins (None) or envelope glycoproteins of the ADA, YU2, Br20-4 or HXBc2 isolates.
  • the results of the CAT assay performed on the HeLa cell lysates are shown.
  • Figure 2 shows the effect of eotaxin on CCR3-mediated enhancement of YU2 recombinant virus.
  • HeLa-CD4 cells transfected with plasmids expressing CD2, CCR1F, CCR3, CCR3F or CCR5 were incubated for one hour at 37°C with increasing amounts of eotaxin.
  • Recombinant HIV- 1 viruses containing the envelope glycoprotein of the YU2 macrophage-tropic primary isolate were added to the cells.
  • CAT activity in the cell lysates was assessed 72 hours later.
  • FIGS 3A-3D show the effect of CCR3, CCR5 and HUMSTSR expression on HIV- 1 infection of Cf2Th canine thymocytes.
  • Cf2Th canine thymocytes expressing human CD4 only Fig 3A
  • CD4 and CCR3 Fig 3B
  • CD4 and CCR5 Fig 3C
  • CD4 and HUMSTSR Fig 3D
  • the CAT assay results are shown. The results of a single experiment are shown. Comparable results were obtained in a repeat experiment.
  • Figures 4A-D show the effect of chemokine receptor expression on HIV- 1 envelope glycoprotein-directed syncytium formation.
  • HeLa cells expressing either no envelope glycoprotein (None) or the ADA, YU2, HXBc2 (ADA-V3), and HXBc2) (YU2-V3) envelope glycoproteins were cocultivated with HeLa-CD4 expressing CCRl , CCR3 (Fig 4B) or CCR5 (Figs. 4C and 4D).
  • 2 ⁇ g/ml of the OKT4a antibody Ortho Pharmaceuticals, Inc.
  • Fig. 4D was added at the beginning of the cocultivation. After 12 hours, the syncytia in the wells were counted. The results of a single experiment are shown. The experiment was repeated with comparable results.
  • Fig. 5 shows the effect of eotaxin on CCR3-mediated enhancement of a recombinant virus containing amphotropic murine leukemia virus (A- MuLV).
  • A- MuLV amphotropic murine leukemia virus
  • HeLa-CD4 cells which were transfected with plasmids expressing CD2, CCR1F or CCR3 under the same conditions as described in Figure 2.
  • a chimeric HIV- 1 virus containing the A-MuLV envelope glycoproteins were added to the cells.
  • CAT activity in the cell lysate was assayed 72 hours later.
  • Figures 6A-C show the results of RT-PCR analysis of primary human brain cultures.
  • Figure 6A shows detection of CCR5, CXCR4 and CCR3 transcripts in primary brain culture.
  • Figure 6B shows detection of CCR3 in microglia but not other brain cell types by double immunofiuoresence staining.
  • Figure 6C shows detection of CCR3 in microglia.
  • Figure 7 shows identification of HIV- 1 infected cells (YU2 or ADA infected) using GFP fluorescence in combination with cell specific markers as indicated.
  • Figure 8A-D show the efficiency of early phase virus replication as determined by measuring luciferase activity in primary brain culture.
  • Figures 9A-E show inhibition of MIP- l ⁇ and MlP- l ⁇ binding to chemokine receptor- expressing cells by gp l20 glycoproteins in the absence and presence of sCD4.
  • Figure 9A shows the gpl20 glycoproteins used in the study, indicating the conserved (C 1-C5) and variable (V1-V5) regions of the native gpl20 glycoproteins included in each of the proteins.
  • the YU2 derivatives are chimeric molecules containing YU2 sequences (shown in white) sufficient for CCR5 utilization [Choe, H., et al., Cell 85: 1 135- 1 148 (1996)], as well as sequences (shown in black) derived from the HXBc2 gpl20 glycoprotein.
  • Figure 9B shows the gpl20 glycoprotein variants tested for ability to inhibit MIP- l ⁇ binding to CCR5F-L1.2 cells.
  • sCD4 was included (at 100 nM final concentration unless otherwise noted).
  • Figure 9C shows the effect of different doses of gpl20 variants on MIP- l binding to CCR5F-L1.2 cells, in the absence (broken lines and open symbols) or presence (solid lines and closed symbols) of 100 nM sCD4. Results are shown for the JR-FL (V ), BAL ( ⁇ ,A), YU2 ⁇ C1 ⁇ V1/2 ⁇ C5 9), HXBc2 (0), HXBc2 ⁇ Cl ⁇ Vl/2/3 ⁇ C5 (°) and YU2 ⁇ C 1 ⁇ V1 /2/3 ⁇ C5 ( ⁇ )glyco ⁇ roteins.
  • Figure 9D shows the g ⁇ l20 variants tested, in the presence of 100 nM sCD4, for ability to inhibit MIP- l ⁇ binding to CCR1-L1.2 cells.
  • Figure 9E shows gp l20 variants tested for the ability to inhibit MlP- l ⁇ binding to CCR5F-L1.2 cells in the absence and presence of 100 nM sCD4.
  • Figures 10A-C show comparison of two-domain and four-domain soluble CD4 proteins for ability to inhibit MIP- l ⁇ and MlP- l ⁇ binding in the absence and presence of gpl20 glycoproteins.
  • Figure 10A shows the ability of D 1D2 sCD4 ( ⁇ ,A), S CD4 (O,*), and soluble VCAM ( ⁇ ,") to inhibit MIP- l binding to CCR5F-L1.2 cells in the absence (open figures) and presence (closed figures) of 50 nM BAL gpl20.
  • Figure 10B shows the ability of D1D2 sCD4 ( ⁇ ,A) and sCD4 (0,#) to inhibit MlP- l ⁇ binding to CCR5F-L1.2 cells in the absence (open figures) and presence (closed figures) of 50 nM YU2 ⁇ C1 ⁇ V1 /2 ⁇ C5 glycoprotein.
  • Figure 10C shows the D 1D2 sCD4 ( ⁇ ) and sCD4 (O) proteins ability to inhibit MIP- l binding to CC41-L1.2 cells at the indicated concentrations, in the absence of gp l20 glycoproteins.
  • Figure 11 shows the effects of monoclonal antibodies on the inhibition of MIP- l binding to CCR5-expressing cells by gpl20-sCD4 mixtures.
  • Monoclonal antibodies (final concentration 500 nM) directed against gpl20 (black shading), against CD4 (white shading) or against hybrid gpl20-CD4 epitopes (grey shading) were tested for the ability to affect the inhibition of MIP- l ⁇ binding to CCR5F-L1.2 cells by a mixture of the JR- FL gpl20 glycoprotein (50 nM final concentration) and sCD4 (100 nM final concentration).
  • the inhibition of MIP- l binding to CCR5F-L1.2 cells by the JR-FL gpl20-sCD4 mixture in the absence of added antibody is also shown (hatched bar) .
  • Figures 12A and B show binding of a radiolabeled macrophage-tropic primary virus gpl20 derivative to CCR5-expressing cells.
  • Figure 12A shows binding in the presence of 100 nM sCD4 of iodinated YU2 ⁇ C1 ⁇ V1 /2 ⁇ C5 protein to CCR5F-L1.2 cells in the presence of increasing concentrations of either rYU2 ⁇ Cl ⁇ Vl/2 ⁇ C5 protein (O) or HXBc2 ⁇ Cl ⁇ C5 protein ( ⁇ ).
  • Figure 12A shows binding in the presence of 100 nM sCD4 of iodinated YU2 ⁇ C1 ⁇ V1 /2 ⁇ C5 protein to CCR5F-L1.2 cells in the presence of increasing concentrations of either rYU2 ⁇ Cl ⁇ Vl/2 ⁇ C5 protein (O) or HXBc2 ⁇ Cl ⁇ C5 protein ( ⁇ ).
  • 12B shows binding in the presence of 100 nM sCD4 and 100 nM HXBc2 ⁇ Cl ⁇ C5 protein, of iodinated YU2 ⁇ C 1 ⁇ V1/2 ⁇ C5 protein to CCRF- L1.2 cells in the presence of the indicated concentrations of YU2 ⁇ C 1 ⁇ V1 /2 ⁇ C5 protein (O), MIP- l ⁇ (0), MlP- l ⁇ (°), RANTES ( ⁇ ) or YU2 ⁇ C 1 ⁇ V1 /2/3 ⁇ C5 protein (X).
  • Figure 13 illustrates the results from a typical experiment for gp l20/sCD4/CCR-binding using different amounts of membranes.
  • the signal-to-noise ration (the ratio of total binding vs. non-specific binding) is shown on the top of each membrane concentration.
  • Figure 14 shows a Scatchard analysis using 5 ⁇ g membranes.
  • Figure 15 shows that as a positive control, anti-CCR5 mAb 2D7 can efficiently inhibit the binding of 125 I-gp 120/sCD4 to CCR5.
  • Figure 16 is a comparison of the amino-terminal sequence of gprl5, gprl , rCCR5 and CCR5 with the three conserved tyrosines (Y) shown in bold and reasonably conserved residues underlined.
  • MDPEETSVYLDYYYATSPN SEQ ID NO: l
  • MEDLEETLFEEFENYSYDLDYYSLESD SEQ ID NO:2
  • MDYQVSSPTYDIDYYTSEPC SEQ ID NO:3
  • MDYQVSSPIYDINYYTSEPC SEQ ID NO:4
  • ⁇ -chemokine receptors are the receptors that bind ⁇ -chemokines.
  • ⁇ - chemokines are a family of 8- 10 kD secreted proteins. These proteins are characterized as ⁇ -chemokines based on the absence of an intervening amino acid in the first of two conserved cysteine pairs (CC) as opposed to the ⁇ -chemokines that have an intervening amino acid in the first conserved cysteine pair (CXC).
  • the chemokines include macrophage inflammatory protein (MIP- l ⁇ and MlP- l ⁇ ), RANTES (regulated on activation T expressed and secreted), monocyte chemotactic protein (MCP- 1, MCP-2, MCP-3) and eotaxin.
  • MIP- l ⁇ and MlP- l ⁇ macrophage inflammatory protein
  • RANTES regulated on activation T expressed and secreted
  • MCP- 1, MCP-2, MCP-3 monocyte chemotactic protein
  • eotaxin eotaxin.
  • the class of surface proteins that bind certain of these chemokines have been identified and belong to the G-protein -coupled seven transmembrane segment receptor family.
  • the chemokine receptors (sometimes referred to as CXR- or CCR-) are characterized based on the specific chemokines they bind to. For example, CCRl for example binds chemokines MIP- l ⁇ and RANTES with high affinity
  • CCR2A and CCR2B for example bind both MCP- 1 and MCP-3.
  • CCR3 for example binds chemokines such as eotaxin, RANTES, and MCP-3 with high affinity.
  • CCR4 for example binds MIP- l ⁇ , RANTES and MCP- 1.
  • CCR5 for example binds to chemokines such as MlP- l ⁇ , MlP- l ⁇ and RANTES. These represent the six ⁇ -chemokine receptors that have currently been characterized.
  • the chemokine receptors share significant identity with each other.
  • CCR5 has significant identity to CCR2, sharing 71% identical amino acid residues. Its identity with other members of the family is about 50%.
  • CCR3 shares a 62% amino acid identity with CCRl and identity with the other characterized receptors that ranges between about forty and fifty percent.
  • CCR3 and CCR5 do not show as much identity to each other as they do to other chemokine receptors.
  • they do not show a chemokine affinity pattern that is as similar to each other as it is to other members of the family. Yet, these two receptors facilitate entry of primary HIV- 1 macrophage-tropic strains.
  • CCR3 In the initial assays where CCR3 was not as highly expressed as CCR5, CCR5 displayed a broader apparent host range than CCR3. However, in more sensitive assays we have found that CCR3 facilitate HIV infection for all primary macrophage-tropic strains tested. Thus, CCR3 can interact with macrophage-tropic strains.
  • CCR5 is particularly involved with the following isolates: ADA, YU2, Br20-4, Br25-9, Rw20-5, Th966, TN243 and 89.6 More preferably the strains are ADA, YU2 BR20-4 and RW20-5.
  • CCR3 is particularly effective with the ADA and YU2 viruses and, to a lesser extent, with the 89.6 and ELI.
  • CCR5 is expressed in primary monocyte/macrophage, primary T cells and granulocyte precursors [Deng, H.K., et al., 1996; G., et al., 1996] and CXCR4 is expressed in a broad range of tissues and cell types including the brain and T cell [Feng, Y., et al, 1996], CCR3 expression appears more restricted, typically eosinophils.
  • Microglia are the major targets for HIV infection of the central nervous system. Microglia express CCR3 as well as CCR5 Price, R.W., 1996; Watkins, B.A., et al., 1996; Takahashi, K., et al., 1990). Astrocytes are also infected, but only at a very low level (Takahashi, K., et al., 1996; Harouse, J.M., et al., 1989; Tornatore C., et al., 1991).
  • HIV- 1 entry into microglia is CD4-dependent, (Jordan, et al., 1991) while entry into astrocytes and some neurally-derived cell lines is CD4-independent (Harouse, J.M., et al., 1989; Tornatore C, et al., 1991).
  • HIV- 1 viruses that infect the CNS are M-tropic HIV- 1 isolates, which represent the majority of primary isolates (Watkins, B.A., et al., 1990; Korber, B.T.M., et al., 1994; Power, C, et al., 1994; Strizki, J.M., et al., 1996).
  • Particular env sequences have been suggested as being associated with brain infection or dementia (Jirberm , et al., 1994; Power, C, et al., although specific determinants of HIV- 1 neurotropism have not been identified.
  • macrophage tropic isolates use CCR5 and CCR3 as co-receptors to infect microglia efficiently, whereas T-tropic isolates use CXCR4.
  • blocking CCR5 or CCR3 can reduce microglia infection by M-tropic isolates by 70-80%.
  • chemokines had different inhibitory effects on HIV activity. For example, RANTES was reported as having greater inhibitory activity than the other chemokines identified. This indicates that the known chemokines receptors will not typically be solely responsible for enhancing infection. This is because CCR5 has a greater sensitivity to MIP- l ⁇ than RANTES, yet RANTES exhibits a greater inhibitory activity than MIP- l ⁇ . Similarly CCR3 is responsive to RANTES but not to MlP- l ⁇ .
  • CCR5 is primarily expressed in promyeloblastic cells, particularly KG- 1A, CD4-positive, and CD8-positive human PBMC and cells of the myeloid lineage.
  • CCR3 as discussed above is highly expressed in eosinophils with some expression in peripheral blood T lymphocytes. We have discovered that CCR3 is also expressed on dendritic cells, which is an important HIV reservoir. CCR3 is expressed at low levels on monocytes. The complete characterization of the full tissue and cell-type distribution for these molecules awaits further studies.
  • Enhanced effectiveness in facilitating infections appears to be dependent upon the number of receptors expressed.
  • Assays which measure receptor level can be used in monitoring HIV-infected and high risk individuals. Differences in the levels of expression of these receptors in different individuals can account for some of the differences observed in onset of AIDS in HIV-infected individuals. Thus, determining the level of these receptors in HIV-infected individuals can be an important tool in determining whether an individual is at a greater risk for enhanced risk of infection and onset of AIDS. This knowledge can be used in determining the type of treatment for that individual.
  • the determination of the number of receptors present on the cells of an individual can readily be accomplished by standard means, for example, using FACS analysis or analysis of RNA levels.
  • the level can be compared to a reference level, which can be determined by standard means. For example, one can prepare averages for individuals exhibiting early onset of AIDS, standard onset of AIDS and delayed onset of AIDS. This can also be done with respect to risk of HIV infection. Moreover, one can also take into account the presence of chemokines such as RANTES, MIP- l and/or MIP- l ⁇ in relationship to the level of CCR3 and/ or CCR5 present. These assays are further discussed below.
  • Viral variation particularly that found in the gpl20 glycoprotein sequences (28,29), dictates the specific chemokine receptor that can be utilized as an entry cofactor.
  • M-tropic HIV- 1 variants that use the chemokine receptor CCR5 as a coreceptor predominate during the asymptomatic stages of infection [Alkhatib, G., et al., Nature Med 2: 1244- 1247; Deng, H.K., et al., Nature 381:661-666 (1996); Doranz, B., et al., Cell 85: 1149-1158 (1996); Dragic, T., et al., Nature 381:667-673 (1996); Zhang, L., et al., Nature 383:768 (1996); Connor, R.I., et al., J Exp Med 185:621-628 (1997)].
  • CCR5 is expressed on T lymphocytes, monocytes/macrophages, brain microglia and dendritic cells Wu, L., et al., J Exp Med; Granelli- Piperno, A., et al., J Exp Med 184:2433-2438 (1996); Raport, C, et al., J Biol Ckem 271: 1761- 1766 (1996); He, J., et al., Nature 385:645-649 (1997)].
  • T- tropic HIV- 1 variants emerge that can use chemokine receptors, especially CXCR4, but also CCR3 and CCR2b, in addition to CCR5 [Zhang, L., et al., Nature 383:768 (1996); Connor, R.I., et al., J Exp Med 185:621-628 ( 1997); Simmons, G., et al., J Virol 70:8355-
  • Another preferred embodiment of this invention is in the diagnosis of susceptibility to HIV infection.
  • the receptors, nucleotide sequences encoding receptors and antibodies that bind to receptors can be particularly useful for diagnosis of susceptibility to infection where higher levels of the receptors indicate an increased risk of infection.
  • the nucleotide sequences are known, for example the sequence for CCR3 is available from GenBank/EMBL/DDB under Accession Nos. U 49727 and U51241.
  • the nucleotide sequences of the receptors or fragments thereof can be used to measure levels of chemokine receptor RNA expression.
  • the antibodies of the invention can be used in standard techniques such as Western blotting to detect the presence of cells expressing receptors and using standard techniques, e.g. FACS or ELISA, to quantify the level of expression.
  • One preferred approach is the use of antibodies to these receptors.
  • Antibodies to these receptors can be prepared by standard means. For example, one can use single chain antibodies to target these receptors.
  • An alternative strategy is to use CCR3 and CCR5 decoys. For example, one could prepare a decoy comprising the portion of these receptors present on the exterior of the cell membrane. Another strategy is to prepare soluble forms of these receptors using their known sequence.
  • gp l20 derivatives containing the chemokine binding site attached to a soluble CD4 molecule will have enhanced binding affinity to the chemokine receptors over the uncomplexed gpl20.
  • Molecules that preferentially bind to these binding sites on gp l20 will also prevent membrane fusion, for example, we have shown that the broadly neutralizing monoclonal antibody 17b can inhibit binding of gpl20, e.g. the binding of the glycoprotein to CCR5.
  • a direct binding assay such as exemplified in Figure 12.
  • a direct binding assay such as exemplified in Figure 12.
  • soluble CD4s There are various soluble CD4s known in the art including a two-domain (D 1D2 sCD4) and a four-domain version.
  • the labeled gpl20, or derivative e.g. a conformational deletion such as YU2 ⁇ C1 ⁇ V1/2 ⁇ C5 protein and soluble CD4 can be added to a medium containing a cell line expressing a chemokine receptor that that derivative will bind to. In this example, the derivative will bind to CCR5.
  • a derivative from a T cell tropic gp l20 when using a derivative from a T cell tropic gp l20 one would use a cell line that expresses CXCR4. Binding of the protein can then be directly measured.
  • the compound of interest can be added before or after the addition of the labeled gpl20 or derivative and the effect of the derivative on binding can be determined by comparing the degree of binding in that situation against a base line standard with that gpl20 or derivative, not in the presence of the compound.
  • a preferred assay uses the labeled gpl20, or derivative, for example a gpl20 protein derived from an M-tropic strain such as JR-FL, iodinated using for instance solid phase lactoperoxidase (in one example having a specific activity of 20 ⁇ Ci/ ⁇ g).
  • the cell line containing the chemokine receptor in this example would be a CCR5 cell line, e.g. LI.2 or membranes thereof. Soluble CD4 would be present.
  • cell membranes are preferable, although similar procedures work with whole cells.
  • Unlabeled gp 120 can serve as a control.
  • binding buffer e.g. 50mM HEPES, pH 7.2, 1 mM CaCb, 5 mM MgCb and 0.5% BSA
  • binding buffer e.g. 50mM HEPES, pH 7.2, 1 mM CaCb, 5 mM MgCb and 0.5% BSA
  • 25 ⁇ l of binding buffer (for total binding) unlabeled gpl20 at a final concentration of 100 nM (for non-specific binding), or test compounds at the desired concentrations are added.
  • 25 ⁇ l of membranes (or whole cells), detected in binding buffer at the desired concentration are added, followed by 25 ⁇ l of labeled (e.g.
  • the binding assay can be adapted depending upon precisely what is being tested.
  • the conformational derivative must contain a sufficient number of amino acid residues to define the binding site of the gpl20 to the chemokine receptor and a sufficient number of amino acids to maintain the conformation of the peptide in a conformation that approximates that of wild- type gpl20 bound to soluble CD4 with respect to the chemokine receptor binding site.
  • the derivative also contains a CD4 binding site (e.g. from the C3 region residues 368 and 370, and from the C4 region residues 427 and 457).
  • a CD4 binding site e.g. from the C3 region residues 368 and 370, and from the C4 region residues 427 and 457.
  • the chemokine binding site is a discontinuous binding site that includes portions of the C2, C3, C4 and V3 regions.
  • the gpl20 derivatives that contain the binding site.
  • the gp l20 derivative is designed to be permanently attached at the CD4 binding site to sufficient domains of CD4 to create a conformation of the chemokine binding site approximating that of the native gp 120 CD4 complex.
  • An alternative g ⁇ l20 derivative is one wherein the linkers used result in a conformation for the derivative so that the discontinuous binding site with the chemokine receptor approximates the conformation of the discontinuous binding site for the chemokine receptor in the wild-type gp 120 /CD4 complex.
  • Stabilized forms of these complexes can readily be made, for example, by conjugates such as a poly(alkylene oxide) conjugate.
  • the conjugate is preferably formed by covalently bonding the hydroxyl terminals of the poly(alkylene oxide) and a free amino group in the gpl20 derivative that will not affect the conformation of the discontinuous binding site.
  • Other art recognized methods of conjugating these materials include amide or ester linkages. Covalent linkage as well as non-covalent conjugation such as lipophilic or hydrophilic interactions can be used.
  • the conjugate can be comprised of non-antigenic polymeric substances such as dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinyl alcohols, polyacryl amides or other similar substantially non-immunogenic polymers.
  • Polyethylene glycol(PEG) is preferred.
  • Other poly(alkylenes oxides) include monomethoxy-polyethylene glycol polypropylene glycol, block copolymers of polyethylene glycol, and polypropylene glycol and the like.
  • the polymers can also be distally capped with C l-4 alkyls instead of monomethoxy groups.
  • the poly(alkylene oxides) used must be soluble in liquid at room temperature. Thus, they preferably have a molecular weight from about 200 to about 20,000 daltons, more preferably about 2,000 to about 10,000 and still more preferably about 5,000.
  • these compounds can be included in vaginal foams or gels that are used as preventives to avoid infection and applied before people have sexual contact.
  • the peptides when used for administration are prepared under aseptic conditions with a pharmaceutically acceptable carrier or diluent.
  • Doses of the pharmaceutical compositions will vary depending upon the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 ⁇ g/kg a day, more preferably 1 to 10,000 ⁇ g/kg. Routes of administration include oral, parenteral, rectal, intravaginal, topical, nasal, ophthalmic, direct injection, etc.
  • V3 region of the gpl20 exterior envelope glycoprotein determine tropism-related phenotypes (Cheng-Mayer et al, 1990; O'Brien et al, 1990; Hwang et al, Westervelt et al, 1992; Chesebro et al, 1992; Willey et al,
  • One preferred approach is the use of antibodies to the binding site for these chemokine receptors.
  • Antibodies to these receptors can be prepared by standard means using the g ⁇ l20 derivatives and gpl20/CD4 complexes. For example, one can use single chain antibodies to target these binding sites.
  • An alternative strategy is to use the stabilized gpl20/CD4 complexes as decoys.
  • the inhibition of HIV infection means that as compared to a control situation infection is reduced, inhibited or prevented.
  • Infection is preferably at least 20% less, more preferably at least 40% less, even more preferably at least 50% less, still more preferably at least 75% less, even more preferably at least 80% less, and yet more preferably at least 90% less than the control.
  • the isolated nucleotide sequences and isolated polypeptides of the invention encoding receptors can be mutagenized by any of several standard methods including treatment with hydroxylamine, passage through mutagenic bacterial strains, etc.
  • the mutagenized sequences can then be classified "wild type” or "non-wild type” depending whether it will still facilitate infectivity or not.
  • Mutagenized sequences can contain point mutations, deletions, substitutions, rearrangements etc.
  • Mutagenized sequences can be used to define the cellular function of different regions of the receptors they encode, and to define the portions of the receptor that facilitate HIV- 1 infection. This information can be used to assist in the design of small molecules or peptides mimicking the HIV-interactive part of the chemokine receptor.
  • small molecule /peptides can be used to inhibit HIV- 1 infection.
  • the inhibition of HIV-infection means that as compared to a control situation infection is reduced, inhibited or prevented. Infection is at least 20% less, preferably at least 40% less, more preferably at least 50% less, still more preferably at least 75% less, even more preferably at least 80% less, and yet more preferably at least 90% less than the control.
  • chemokines themselves (e.g. eotaxin, RANTES, MCP- 1 , MIP- l and/or MIP- l ⁇ ), fragments of chemokines, preferably surface fragments, and smaller molecules or peptides the mimic the chemokines.
  • chemokines themselves (e.g. eotaxin, RANTES, MCP- 1 , MIP- l and/or MIP- l ⁇ ), fragments of chemokines, preferably surface fragments, and smaller molecules or peptides the mimic the chemokines.
  • Such molecules and peptides can be synthesized by known techniques.
  • SIV primate immunodeficiency virus
  • CCR5 CCR5
  • gprl and gprl5 two orphan seven- transmembrane segment receptors, gprl and gprl5, serve as coreceptors for SIV, and are expressed in alveolar macrophages.
  • gprl 5 which is also expressed in CD4 + T lymphocytes is the more efficient.
  • the SIV coreceptors, gprl and gprl 5 are expressed in U87 and
  • CEMxl74 cells respectively.
  • CEMxl74 supports SIV entry but lacks CCR5 and does not support efficient entry of HIV- 1 viruses using CCR5.
  • the neuroglioma cell line U87, stably transfected with CD4 similarly supports entry of SIV ma c 239 but does not allow for efficient entry of known HIV- 1 viruses.
  • the HIV- 1 strains ADA and YU2 weakly use gprl5. This may be an inadvertent consequence of similarities in the amino-terminal regions of gprl 5 and CCR5, or it may indicate an adaptation to these or a related receptor that occurs in some HIV- 1 subsets.
  • gprl and gprl 5 resemble the angiotensin II receptor and the orphan receptors dez and apj more than they do any of the known chemokine receptors [Marchese, A., et al., Genomics 23:609-618 (1994); Heiber, M., et al., Genomics 32:462-465 (1996)].
  • Gprl5, like dez and gprl, lacks the cysteines in the amino-terminal region and the third extracellular loop that, in the chemokine receptors, are thought to be disulfide linked.
  • gprl 5 and gprl amino termini contain three tyrosines that align with similarly-positioned tyrosines in CCR5 (See Figure 15). Alteration of these tyrosines has been shown to decrease the efficiency with which CCR5 supports the entry of SIV and macrophage-tropic HIV- 1 isolates (M. Farzan, H. Choe and J. Sodroski, unpublished observations).
  • gpr 15 and gprl as SIV coreceptors suggests a greater range and complexity of coreceptors for the primate immunodeficiency viruses than heretofore described. Comparative studies of these divergent coreceptors with the known coreceptors for these viruses should assist the identification of common structural elements in 7-TMS proteins that serve as viral entry cofactors.
  • a molecule that binds to at least one of the tyrosine residues present in the amino terminus of the coreceptors is a preferred molecule for interfering with HIV entry.
  • One class of molecules are antibodies, for example a single chain antibody.
  • SEQ ID NO:4 or fragments thereof to generate an antibody by standard means.
  • Another class of molecules is a small molecule.
  • One preferred use of these compounds is to minimize the risk of HIV transmission.
  • These compounds can be included in ointments, foams, creams that can be used during sex. For example, they can be administered preferably prior to or just after sexual contact such as intercourse.
  • One preferred composition would be a vaginal foam containing one of the compounds.
  • the compound would be a decoy or blocker, for example, a small molecule that binds to the CCR3 receptor.
  • Another use would be in systemic administration to block HIV- 1 replication in the blood and tissues. The compound could also be administered in combination with other HIV treatments.
  • Another strategy is to express antibodies to these receptors in infected individuals intracellularly. This can be done by the method of Marasco and Haseltine set forth in WO94-02610 (PCT/US93/06735 filed July 16, 1993) published February 3, 1994.
  • additional compounds that bind to these receptors and thus interfere with their ability to facilitate HIV infection can readily be screened for. For example, one can select cells expressing high numbers of these receptors, plate them; e.g. add labeled g ⁇ l20 and CD4 and screen for compounds or combinations of compounds that will interact with, e.g. binding of, these receptors by standard techniques. Alternatively, one can use known techniques to prepare cells that will express these receptors and use those cells in drug screens.
  • the ability of drugs to block HIV- 1 infection or syncytium formation can be screened using assays similar to those showing in Figures 1-5.
  • the surface receptors would only be CD4 and either CCR3 and/ or CCR5.
  • Such a method can be used to select molecules that specifically affect the pathway. These molecules may be combined with other drugs, for example, for their combined or synergistic effects.
  • CD4 cells when comparing CD4 cells there can be a variety of other factors affecting such cells, thus, such a comparison does not provide the same data.
  • One of the problems that has been encountered in in vivo testing compounds that affect HIV- 1 is the relatively small number of animals that can be infected by HIV. While systems such as a chimeric virus comprising SIV and HIV (SHIV) have extended the number of animal models that can be used, this approach is primarily directed to systems that use other primates. Now one can prepare transgenic animals that have cells that express CD4 and at least CCR5 or CCR3 to further extend the range of animals susceptible to HIV- 1 infection. This permits one to create a much broader range of animal models.
  • a "transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the somatic cells and/ or the germ line of the animal that develops from that cell.
  • the preferred DNA contains nucleotide sequences that are homologous to human CD4, CCR3 and/or CCR5 genes. These sequences may be entirely foreign to the transgenic animal or may even be identical to the homologous gene of the animal, but which is inserted into the animal's genome at a location which differs from that of the natural copy.
  • Transgenic animals can provide good model systems for studying the development of AIDS, the effects of potential therapeutic reagents, and the safety (e.g. toxicity, carcinogenicity) of such agents administered to the animals.
  • An exemplary pharmaceutical composition is a therapeutically effective amount of a decoy, antibody etc. that affects the ability of the receptor to facilitate HIV infection optionally included in a pharmaceutically- acceptable and compatible carrier.
  • pharmaceutically-acceptable and compatible carrier includes (i) one or more compatible solid or liquid filler diluents or encapsulating substances that are suitable for administration to a human or other animal, and/ or (ii) a system, such as a retroviral vector, capable of delivering the molecule to a target cell.
  • carrier thus denotes an organic or inorganic ingredient, natural or synthetic, with which the molecules of the invention are combined to facilitate application.
  • terapéuticaally-effective amount is that amount of the present pharmaceutical compositions which produces a desired result or exerts a desired influence on the particular condition being treated.
  • concentrations may be used in preparing compositions incorporating the same ingredient to provide for variations in the age of the patient to be treated, the severity of the condition, the duration of the treatment and the mode of administration.
  • compatible means that the components of the pharmaceutical compositions are capable of being commingled with a small molecule, nucleic acid and/ or polypeptides of the present invention, and with each other, in a manner such that does not substantially impair the desired pharmaceutical efficacy.
  • compositions of the invention will vary depending on the subject and upon particular route of administration used.
  • Dosages can range from 0.1 to 100,000 ⁇ g/kg per day, more preferably 1 to 10,000 ⁇ g/kg.
  • an overall dose range of from about, for example, 1 microgram to about 300 micrograms might be used for human use.
  • This dose can be delivered at periodic intervals based upon the composition. For example on at least two separate occasions, preferably spaced apart by about 4 weeks. Other compounds might be administered daily.
  • Pharmaceutical compositions of the present invention can also be administered to a subject according to a variety of other, well-characterized protocols.
  • certain currently accepted immunization regimens can include the following: (i) administration times are a first dose at elected date; a second dose at 1 month after first dose; and a third dose at 5 months after second dose.
  • the small molecules and polypeptides of the invention may also be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of this invention.
  • Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulphonic.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • the present invention also provides pharmaceutical compositions, for medical use, which comprise nucleic acid and/ or polypeptides of the invention together with one or more pharmaceutically acceptable carriers thereof and optionally any other therapeutic ingredients.
  • compositions include those suitable for oral, rectal, intravaginal, topical, nasal, ophthalmic or parenteral administration, all of which may be used as routes of administration using the materials of the present invention.
  • Other suitable routes of administration include intrathecal administration directly into spinal fluid (CSF), direct injection onto an arterial surface and intraparenchymal injection directly into targeted areas of an organ.
  • Compositions suitable for parenteral administration are preferred.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing the active ingredients of the invention into association with a carrier which constitutes one or more accessory ingredients.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the nucleic acid and/ or polypeptide of tbje invention in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the molecule of the invention which is preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectibles.
  • Antibodies antibodies
  • antibodies is meant to include monoclonal antibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with polypeptides encoded by eukaryotic nucleotide sequences of the present invention.
  • “selectively reactive” refers to those antibodies that react with one or more antigenic determinants of CCR3 or CCR5, or gpl20 and/ or CD4 and do not react with other polypeptides.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Antibodies can be used for diagnostic applications or for research purposes.
  • antibodies may be raised against amino-terminal (N- terminal) or carboxyl-terminal (C-terminal) peptides of a polypeptide encoded by CCR3, CCR5. Most preferably one selects an exposed cell- surface epitope of one of these receptors.
  • One approach is to isolate a peptide sequence that contains an antigenic determinant for use as an immunogen.
  • This peptide immunogen can be attached to a carrier to enhance the immunogenic response.
  • the peptide immunogen can correspond to any portion of a polypeptide encoded by a eukaryotic nucleotide sequence of the invention, certain amino acid sequences are more likely than others to provoke an immediate response, for example, an amino acid sequence including the N- or C-terminus of a polypeptide encoded by a gene that contains nucleotide sequences of the invention.
  • cDNA clone encoding a CCR3, CCR5 or a fragment thereof may be expressed in a host using standard techniques (see above; see Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York: 1989) such that 5-20% of the total protein that can be recovered from the host is the desired protein. Recovered proteins can be electrophoresed using PAGE and the appropriate protein band can be cut out of the gel. The desired protein sample can then be eluted from the gel slice and prepared for immunization.
  • a protein of interest can be purified by using conventional methods such as, for example, ion exchange hydrophobic, size exclusion, or affinity chromatography.
  • mice can be immunized twice intraperitoneally with approximately 50 micrograms of protein immunogen per mouse. Sera from such immunized mice can be tested for antibody activity by immunohistology or immunocytology on any host system expressing such polypeptide and by ELISA with the expressed polypeptide.
  • active antibodies of the present invention can be identified using a biotin-conjugated anti-mouse immunoglobulin followed by avidin-peroxidase and a chromogenic peroxidase substrate. Preparations of such reagents are commercially available; for example, from Zymad Corp., San Francisco, California.
  • mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production. Positive supernatants of such hybridomas can be identified using the assays described above and by, for example, Western blot analysis.
  • amino acid sequence of polypeptides encoded by a eukaryotic nucleotide sequence of the present invention may be analyzed in order to identify portions of amino acid sequence which may be associated with increased immunogenicity.
  • polypeptide sequences may be subjected to computer analysis to identify potentially immunogenic surface epitopes.
  • Such computer analysis can include generating plots of antigenic index, hydrophilicity, structural features such as amphophilic helices or amphophilic sheets and the like.
  • any technique that provides for the production of antibody molecules by continuous cell lines may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (Nature, 256: 495-497, 1973), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies, and the like are within the scope of the present invention. See, generally Larrick et al., U.S. Patent 5,001,065 and references cited therein.
  • SCA single-chain antibody
  • the monoclonal antibodies may be human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
  • the present invention provides for antibody molecules as well as fragments of such antibody molecules.
  • moieties can be coupled to the resultant antibodies or to other molecules of the invention. See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.
  • Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities.
  • This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation.
  • the preferred binding is, however, covalent binding.
  • Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
  • Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules.
  • representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines.
  • Preferred linkers are described in the literature. See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, Umemoto et al. U.S. Patent 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker.
  • MBS M-maleimidobenzoyl-N-hydroxysuccinimide ester
  • linkers include: (i) EDC ( l-ethyl-3-(3-dimethylamino- propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl- alpha-methyl-alpha-(2-pyridyl-dithio) -toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem.
  • linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties.
  • sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
  • NHS-ester containing linkers are less soluble than sulfo-NHS esters.
  • the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability.
  • Disulfide linkages are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.
  • Sulfo-NHS in particular, can enhance the stability of carbodimide couplings.
  • Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
  • Antibodies of the present invention can be detected by appropriate assays, e.g., conventional types of immunoassays.
  • a sandwich assay can be performed in which the receptor or fragment thereof is affixed to a solid phase. Incubation is maintained for a sufficient period of time to allow the antibody in the sample to bind to the immobilized polypeptide on the solid phase. After this first incubation, the solid phase is separated from the sample. The solid phase is washed to remove unbound materials and interfering substances such as non-specific proteins which may also be present in the sample.
  • the solid phase containing the antibody of interest bound to the immobilized polypeptide of the present invention is subsequently incubated with labeled antibody or antibody bound to a coupling agent such as biotin or avidin.
  • Labels for antibodies are well- known in the art and include radionuclides, enzymes (e.g. maleate dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, and the like.
  • the labeled antibodies are incubated with the solid and the label bound to the solid phase is measured, the amount of the label detected serving as a measure of the amount of anti-urea transporter antibody present in the sample.
  • H. 1 Primary macrophage-tropic and laboratory-adapted human immunodeficiency viruses type 1 (HIV- 1) require particular chemokine receptors, CCR5 and CXCR4, respectively, in addition to the primary receptor, CD4, for efficient entry into target cells [Feng, Y., et al., Science 272:872-877 ( 1996); Choe, H., et al., Cell 85: 1 135- 1 148 (1996); Doranze, B.J., et al., Cell 85: 1 149- 1 158 ( 1996); Dragic, T., et al., Nature 381:661-666 (1996); Deng, H., et al., Nature 381:661-666 (1996)].
  • a gp l20 fragment retaining the CD4 binding site and overlapping epitopes was able to interact with CCR5 only if the V3 loop, which can specify HIV- 1 tropism and chemokine receptor choice [Choe, H., et al., Cell 85: 1 135- 1 148 ( 1996); Cheng-Mayer, C, et al., J Virol 64:4390-4398 ( 1990); Chesebro, B., et al., J Virol 65:5782- 5789 (1991); Hwang, S., et al., Science 253:71-74 ( 1991); Westervelt, P., et al., J Virol 66:2577-2582 (1992)], was also present on the molecule.
  • the murine pre-B lymphoma line, LI .2 was stably transfected with CCR5 cDNA, tagged at the N-terminus with a FLAG epitope (Kodak), in the pMRBlO l expression vector, as described [Ponath, P., et al., J Exp Med 183:2437-2448 (1996)].
  • the pMRBlOl plasmid is a derivative of Ee ⁇ hcmvbglii that contains the E. coli gpt gene and was kindly provided by Martin Robinson (CellTech).
  • CCR5 The cell surface expression of CCR5 was monitored by staining with an anti-FLAG antibody, and cells with a high level of CCR5 expression were selected by several rounds of limiting dilution and rescreening.
  • LI.2 cells stably expressing CCRl were kindly provided by James Campbell and Eugene Butcher (Stanford University).
  • Scatchard analysis of MIP- l ⁇ binding to CCRl -LI.2 cells revealed a dissociation constant of 8 nM and 2 x 10 4 binding sites per cell (S. Qin, unpublished results). Chemokine Binding and Competition Assays
  • 125 I-labeled human MIP- l ⁇ and MlP- l ⁇ and unlabeled chemokine were purchased from DuPont NEN (Boston, MA) and Peprotech (Rocky Hill, NJ), respectively.
  • CCR5F-L1.2 cells were washed and resuspended in binding buffer (50 mM HEPES, pH7.5, 1 mM CaCb, 5mM MgCb and 0.5% BSA) at a concentration of 5 x 10 8 cells/ml.
  • binding buffer 50 mM HEPES, pH7.5, 1 mM CaCb, 5mM MgCb and 0.5% BSA
  • For binding and competition studies which were conducted in a final volume of 100 ⁇ l, g ⁇ l20 glycoproteins were mixed with soluble CD4 on ice for 5-10 minutes, after which monoclonal antibodies were added if appropriate.
  • T-B where S is the test sample, B is the background, and T is the total binding.
  • the BAL g ⁇ l20 derivative contains an amino terminal deletion of 32 residues compared with the wild-type gp l20 glycoprotein.
  • the YU2 glycoproteins were produced from a chimeric env gene, containing YU2 sequences from Bgl II (nucleotide 6620)to BG1 II (nucleotide 7200) in an HXBc2 background.
  • the ⁇ C 1 proteins contain deletions up to and including residue 82.
  • the ⁇ V1 /2 and ⁇ V3 proteins contain deletions equivalent to the ⁇ 128- 194 and ⁇ 298-329 deletions, respectively, previously described for the HXBc2 glycoprotein [Wyatt, R., et al., J Virol 69:5723-5733 ( 1995)].
  • the ⁇ C5 proteins contain a deletion of the carboxy-terminal 19 residues of the mature gpl20 glycoprotein. All gpl20 proteins utilize the tissue plasminogen activator signal sequence for translocation into the endoplasmic reticulum.
  • Protein expression of recombinant YU2 and HXBc2 derivatives was induced by transfer of Drosophila lines into serum-free medium containing 750 mM CuSO for seven days at 25° C.
  • Recombinant proteins were purified by passage of cell supernatants over an F 105 monoclonal antibody column, which was extensively washed with PBS containing 500 mM NaCl and then reequilibrated in PBS containing 150 mM NaCl.
  • the gpl20 glycoproteins were eluted with 100 mM glycine-HCL, pH 2.8 and fractions were immediately neutralized with 1 M Tris base.
  • the gp l20 glycoproteins were concentrated using Centriprep 30 spin filters (Amicon), and resuspended in PBS containing protease inhibitors. Protein concentrations were determined by comparison with commercially available gpl20 (Agmed) on Coomassie blue-stained SDS-PAGE gels. All of the gpl20 preparations were homogenous, with the exception of the BAL gpl20 preparation, in which approximately 5 percent of the protein was proteolytically cleaved.
  • Soluble CD4 proteins [Arthos, J., et al., Cell 57:469-481 (1989)] were kindly provided by Dr. Raymond Sweet (SmithKline Beecham).
  • the soluble VCAM protein used in these studies is a chimera of human D 1D2 VCAM and the murine constant kappa chain.
  • the soluble VCAM was expressed in SF9 cells by a recombinant baculovirus and purified on a Protein A column.
  • the YU2 ⁇ C1 ⁇ V1/2 ⁇ C5 protein was iodinated to a specific activity of 900 Ci/mmol using solid-phase lactoperoxidase and glucose oxidase (Enzymobeads, BioRad, Richmond, CA) [Gerard, N.P., et al., J Biol Chem 264: 1760- 1766 (1989)].
  • the CCR5F-L1.2 cells were preincubated for 10-20 minutes at room temperature in phosphate-buffered saline.
  • the labeled protein was added (final concentration 0.1 nM to 5 x 10 s cells in duplicate in 100 ⁇ l of 50 mM HEPES, pH 7.2 containing ImM CaCb, 5 mM MgCb 0.5% BSA, 100 nM sCD4 and different concentrations of unlabeled YU2 ⁇ C1 ⁇ V1/2 ⁇ C5 or HXBc2 ⁇ Cl ⁇ C5 protein.
  • the iodinated YU2 ⁇ C 1 ⁇ V1 /2 ⁇ C5 protein was incubated with the CCR5F-L1.2 cells in the same buffer containing 100 nM sCD4, 100 nM HXBc2 ⁇ Cl ⁇ C5 and different concentrations of RANTES MIP- l ⁇ , MIP- l ⁇ or the
  • HXBC2 ⁇ C 1 ⁇ V 1 /2/ 3 ⁇ C5 protein After 30 minutes of incubation at 37° C, cells were washed in 50 mM HEPES, pH 7.2, l mM CaCb, 5 mM MgCb, 0.5% BSA and 0.5 M NaCl and bound radioactivity counted. The background (non-specific) binding was determined in the presence of 100 nM unlabeled YU2 ⁇ C 1 ⁇ V1/2 ⁇ C5 protein. The percent specific binding was calculated using the following formula:
  • T-B S represents the observed counts bound at a given concentration of unlabeled competitor protein
  • T represents the observed counts bound in the absence of competitor
  • B represents the background counts
  • CCR5F-L1.2 cells which are murine lymphocytes stably expressing an epitope-tagged CCR5 protein (5-8 x 10 4 binding sites/cell), with dissociation constants of 1.1 , 0.4 and 0.2 nM, respectively (data not shown).
  • the HIV- 1 gp l20 derivatives used in this study were derived from the JR-FL, BAL and YU2 macrophage- tropic primary viruses or from the HXBc2 laboratory adapted virus.
  • Some of the gpl20 glycoproteins contain deletions of the first (C I) and fifth (C5) conserved regions, which are important for the g ⁇ l20 interaction with the gp41 transmembrane glycoprotein [Helseth, E., et al., J. Virol 65:21 19-2123 (1991)], or deletions of the major V1/V2 or V3 variable loops [Wyatt, R., et al., J Virol 67:4557-4565 (1993); Wyatt, R., et al., J Virol 69:5723-5733 (1995)].
  • the F105 antibody recognizes a discontinuous HIV- 1 gpl20 epitope that overlaps the CD4 binding site [Posner, M., et al., J Immunol 146:4325-4332 (1991)], while the 17b antibody binds a discontinuous gpl20 epitope that is increased in exposure following CD4 binding [Thali, M., J Virol 67:3978-3988 (1993)].
  • CD4 glycoprotein Two soluble forms of the CD4 glycoprotein were included in the study, four-domain soluble CD4 (sCD4) and a protein consisting of the amino-terminal two domains of CD4 (D 1D2 sCD4) [Hussey, R., et al., Nature 331 :78-81 (1988);Arthos, J., et al., Cell 57:469-481 ( 1989)].
  • sCD4 the JR-FL, BAL and YU2 ⁇ C1 ⁇ V1 /2 ⁇ C5 envelope glycoproteins, which were derived from the macrophage-tropic primary viruses, significantly inhibited MIP- l ⁇ binding to CCR5F-L1.2 cells ( Figure 9B).
  • a dose-response curve indicated inhibitory concentrations (ICso) of 4,7,5.5 and 0.7 nM for the JR-FL, BAL and YU2 ⁇ C 1 ⁇ V1 /2 ⁇ C5 glycoproteins, respectively, in the presence of sCD4 ( Figure 9C).
  • ICso inhibitory concentrations of 4,7,5.5 and 0.7 nM for the JR-FL, BAL and YU2 ⁇ C 1 ⁇ V1 /2 ⁇ C5 glycoproteins, respectively, in the presence of sCD4
  • Figure 9C In the absence of sCD4, 500 nM concentrations of the JR-FL, BAL and YU2 ⁇ C1 ⁇ V1 /2 ⁇ C5 glycoproteins inhibited 32-45 percent of MIP- l binding to CCR5F-L1.2 cells.
  • the presence of sCD4 resulted in a 2- to 3- log increase in the efficiency of the observed inhibition.
  • the YU2 ⁇ C 1 ⁇ VI/2/3 ⁇ C5 glycoprotein which differs from the YU2 ⁇ C1 ⁇ V1 /2 ⁇ C5 glycoprotein by the absence of the gp l20 V3 loop, was dramatically reduced in the ability to inhibit MIP-l binding in the presence and absence of sCD4 ( Figure9B and 9C).
  • the V3 loop appears to be critical for the inhibition of MIP- l ⁇ binding to the CCR5F-L1.2 cells. No significant inhibition of MIP- l ⁇ binding was observed for any of the HXBc2 envelope glycoprotein derivatives over that seen with sCD4 alone. The latter inhibition was 12 percent or less and did not significantly increase at higher sCD4 concentrations.
  • the observed chemokine receptor specificity indicates that interaction with MIP- l ⁇ is not the basis for the observed inhibition of binding by the gpl20 glycoprotein-CD4 complexes.
  • MlP- l ⁇ and MlP- l ⁇ binding to CCR5-expressing cells can be specifically inhibited by gp l20 glycoproteins or some gpl20 fragments derived from macrophage-tropic primary HIV- 1 isolates, that the efficiency of this inhibition is dramatically increased in the presence of soluble CD4, and that an intact V3 loop appears to be important for the effect.
  • the two-domain soluble CD4 (D 1D2 sCD4) was compared to four- domain sCD4 for the ability to promote the high-affinity interaction with CCR5 when mixed with gp l20 glycoproteins from macrophage-tropic primary HIV- 1.
  • the D 1D2 sCD4 inhibited MIP- l ⁇ and MIP- l ⁇ binding with an ICso of 20-30 nM in the absence of the gp l20 glycoprotein.
  • the 17b neutralizing antibody which recognizes a discontinuous, conserved gpl20 epitope exposed better after CD4 binding [Thali, M., J Virol 67:3978-3988 ( 1993)], blocked the inhibition of MIP- l ⁇ binding by the gp l20-sCD4 mixture. Inhibition of MIP- l ⁇ binding was also decreased by the addition of CG10 antibody, which recognizes an epitope present only on gpl20-CD4 complexes [Gershoni J., et al., FASEB J 7: 1185- 1 187 (1993)].
  • CD4-mediated induction of CCR5 binding may contribute to the observed enhancement of primary HIV- 1 infection by sCD4 [Sullivan, N., et al., J Virol 69:4413-4422 ( 1995)].
  • a sequential, two-step process for viral attachment and entry allows conserved elements on the viral glycoproteins interacting with chemokine receptors to remain sequestered from antiviral antibodies, until such time as proximity to the target cell membrane is achieved by the virus.
  • the limited accessibility of antibodies to CD4-induced gpl20 moieties in the context of the membrane-anchored, oligomeric envelope glycoprotein- CD4 complex may then allow membrane fusion and virus entry to proceed in the face of the humoral immune response.
  • CCR5 A low affinity interaction with CCR5 can apparently occur in the absence of CD4 for gp l20 variants derived from macrophage-tropic primary HIV-1. These results indicate that a major site for CCR5 interaction is contained on the gp l20 glycoprotein.
  • the CCR5-interactive region must be reasonably well-conserved, since CCR5 can be used as a coreceptor by diverse HIV- 1 strains as well as by simian immunodeficiency viruses [Choe, H., et al., Cell 85: 1 135- 1 148 (1996); Marcon, L., et al. submitted]. While additional studies will be required to define this site absolutely precisely, our data provides general information concerning the CCR5-interactive region.
  • the CCR5-interactive region is preserved on a gpl20 fragment lacking the C I , VI /V2 and C5 regions.
  • some HIV- 1 -neutralizing antibodies that do not interfere with gpl20-CD4 binding blocked the interaction of soluble CD4-gpl20 complexes with CCR5.
  • One of these antibodies, 17b recognizes a discontinuous gp l20 epitope that is exposed better upon CD4 binding [Thali, M., J Virol 67:3978-3988 (1993)], a property shared by the CCR5-interactive region.
  • a component of the chemokine receptor binding site on gpl20 may reside in a V3 structure demonstrating moderate variability. Together these observations implicate a discontinuous gpl20 structure in the vicinity of the 17b epitope, with a probable contribution from V3 sequences, as the CCR5-interactive moiety on the gpl20 glycoprotein.
  • D 1D2 sCD4-CCR5 interaction is biologically relevant, it is probably not restricted to CCR5, since other chemokine receptors as discussed above can be used as coreceptors for HIV- 1 variants [Feng, Y., et al., Science 272:872-877 (1996); Choe, H., et al., Cell 85: 1 135- 1 148 (1996); Doranze, B.J., et al., Cell 85: 1 149- 1 158 (1996)].
  • the regions of gp l20 and CD4 that appear to contribute to CCR5 interaction are expected to be quite distant from the target cell membrane, at least upon initial attachment of the virus to CD4.
  • the SV-A-MLF-Env plasmid expressing the amphotropic murine leukemia virus envelope glycoproteins was obtained from Dan Littman (Landau et al, 1991).
  • the derivation and construction of the pSVIIIenv plasmids expressing the envelope glycoproteins from various strains of HIV- 1 have been described (Sullivan et al, 1995; Gao et al, 1996; Karlsson et al, 1996).
  • the chimeric HXBc2 (YU2-V3) and HXBc2 (ADA-V3) en ⁇ constructs were kindly supplied by Lee Ratner, and were designated HY (V3A + V3B) and HA (V3A + V3B) in a previous publication (Carrillo et al, 1993).
  • the chimeric HXBc2 (YU2- VI /V2) en ⁇ genes were created by substituting the Dra III Stu I fragment of the YU2 en ⁇ gene into the corresponding segment (nucleotides 6619 to 6901) of the HXBc2 en ⁇ gene.
  • the cDNAs encoding the chemokine receptors were cloned into the pcDNA3 vector (Invitrogen) for expression.
  • the CCRl, CCR3 and CCR5 proteins which are known sequences, were also expressed as fusion proteins containing an epitope tag (MDYKDDDDK)(SEQ ID NO:5) (FLAG tag, IBI-Kodak) at the amino terminus.
  • Cell lines HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics.
  • HeLa-CD4 (clone 1022) cells were obtained from Dr. Bruce Chesebro through the National Institutes of Health AIDS Research and Reference Reagent Program.
  • the Cf2Th canine thymocyte line was obtained from the American Type Culture Collection (ATCC CRL 1430) and was propagated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Env-complementation Assay
  • HeLa cells were cotransfected by the calcium phosphate method (Cullen, 1989) either with 15 ⁇ g pHXBHIO ⁇ envCAT alone or with 15 ⁇ g pHXBHIO ⁇ envCAT and 3 ⁇ g pSVIIIenv or SV-A-MLV-Env to produce recombinant virions, as previously described (Thali et al, 1994; Karlsson et al, 1996).
  • HeLa cells to be used as target cells were plated at 7 X 10 5 cells per 100 mm dish, cultured overnight, and then transfected by the calcium phosphate method Mg pcDNA3 expressing chemokine receptors.
  • Control HeLa cells were transfected with 10 Mg pCD4 and 25 with 10 ⁇ g pCD4 and 25 ⁇ g pCDM8 expressing the CD2 protein, which has been shown to have no effect on HIV- 1 infection (H.-R. Choe and J. Sodroski, unpublished observations).
  • the pCDM8 plasmid expressing CD2 was a gift from Dr. Ellis Reinherz.
  • the HeLa target cells were detached from the tissue culture dish by treatment with phosphate-buffered saline and 5mM EDTA. The cell suspension was diluted in medium, with one aliquot used for FACS analysis and the remaining aliquots replated into 6-well plates for infection.
  • the level of CD4 expressed on the cell surface was measured by flow cytometry, using the FITC-conjugated OKT4 antibody reactive with CD4 domain 3 (McDougal et al, 1986). Approximately six hours after replanting, cells were infected by incubation with recombinant virions (20,000 cpm of reverse transcriptase activity) in 1 ml of medium. After overnight incubation at 37°C, additional medium was added to the cells. After a total of approximately 60 hours of incubation of the virus-cell mixture at 37°C, the cells were lysed and used for determination of CAT activity.
  • the level of chemokine receptor expression on the transfected HeLa cells was measured by FACS analysis 60 hours following transfection.
  • the antibody anti-FLAG M2, Kodak
  • the epitope tag FLAG tag
  • Monoclonal antibodies were used to detect surface expression of CCR2, I18R-A and IL8R-B, respectively.
  • Cf2Th canine thymocytes were used as target cells.
  • the Cf2Th cells were transfected by the calcium phosphate technique with 10 ⁇ g of the pCD4 plasmid and 25 ⁇ g of the pcDNA3 plasmid expressing chemokine receptors or, as a control, with 10 ⁇ g of the pCD4 plasmid and 25 ⁇ g of the pCDM8 plasmid expressing CD2 (see above).
  • the Cf2Th cells were incubated with recombinant HIV- 1 and used for measurement of CAT as described above. Eotaxin Inhibition of HIV- 1 Infectivity
  • Recombinant HIV- 1 containing the YU2 and A-MuLV envelope glycoproteins were produced in HeLa cells as described above.
  • HeLa-CD4 (clone 1022) cells transfected either with the pCDM8 plasmid expressing CD2 or with the pCDNA3 plasmid expressing chemokine receptors, were used as target cells.
  • the target cells in 1 ml medium, were incubated with different concentrations (0-60 nM) of eotaxin (Jose et al., 1994; Ponath et al., 1996a and which is commercially available) for 90 minutes at 37°C.
  • Envelope glycoprotein-expressing HeLa cells were derived by transfection of HeLa cells with psVIIIenv plasmids expressing HIV- 1 envelope glycoproteins (Helseth et al, 1990).
  • Target cells were derived by transfection of HeLa-CD4 (clone 1022) cells with plasmids expressing either CCR1,CCR3 or CCR5. Forty-eight hours after transfection, the envelope glycoprotein expressing and target HeLa cells were detached from the tissue culture plates using 5 mM EDTA. Cells were replated at a ratio of ten target cells to one envelope glycoprotein-expressing cell, and incubated at 37°C in 5% CO 2 .
  • HXBc2 laboratory-adapted viral envelope glycoproteins
  • HXBc2 laboratory-adapted viral envelope glycoproteins
  • A-MuLV amphotropic murine leukemia virus
  • Recombinant viruses containing the A- MuLV envelope glycoproteins were able to infect the Cf2Th cells at a high level of efficiency. This was expected since all of the Cf2Th cells in the culture were potentially susceptible to infection by the viruses with A-MuLV envelope glycoproteins. By contrast, only the fraction of cells successfully transfected were potentially infectible by the viruses with HIV- 1 envelope glycoproteins.
  • Expression of HUMSTSR in addition to CD4 facilitated infection by the HXBc2 and 89.6 recombinant viruses but did not affect infection by viruses with ADA or YU2 envelope glycoproteins. A small positive effect of HUMSTSR expression was seen on infection by the ELI recombinant virus.
  • Table 3 shows that recombinant viruses containing the chimeric glycoproteins with the ADA and YU2 V3 loops, in contrast to those containing the parental HXBc2 envelope glycoproteins, were able to infect HeLa-CD4 cells more efficiently when either CCR3 or CCR5 was expressed on the target cell.
  • Substitution of the YU VI /V2 variable loops into the HXBc2 envelope glycoproteins did not increase the efficiency of infection of HeLa-CD4 target cells expressing CCR3 or CCR5, compared with the cells expressing the CD2 control protein.
  • Figure 4 shows that the number of syncytia observed when no envelope glycoproteins were expressed in the HeLa cells was minimal.
  • HeLa cells expressing the HXBc2 envelope glycoproteins formed syncytia with HeLa-CD4 cells (data not shown), consistent with the expression of endogenous HUMSTSR in this cell line (Feng et al, 1996).
  • the ADA and YU2 envelope glycoproteins did not mediate the formation of syncytia with the CD4-positive HeLa cells expressing the CCRl protein.
  • Syncytium formation directed by the chimeric HXBc2 (ADA-V3) and HXBc2 (YU2-V3) envelope glycoproteins was also inefficient with CCRl -expressing HeLa-CD4 target cells.
  • Expression of CCR3 in addition to CD4 on the HeLa cells resulted in syncytium formation directed by the YU2 envelope glycoproteins.
  • the expression of CCR5 on the HeLa-CD4 cells allowed the formation of syncytia with cells expressing the ADA, YU2 and chimeric envelope glycoproteins.
  • the number of syncytia formed by the HXBc2 envelope glycoproteins was not affected by CCR3 or CCR5 expression on the CD4-positive HeLa cells (data not shown).
  • Syncytium formation in this assay was dependent upon gpl20 binding to CD4, since the OKT4a anti- CD4 monoclonal antibody, which blocks gp l20-CD4 interaction (McDougal et al, 1986), inhibited the formation of syncytia.
  • results indicate that expression of CCR3 and CCR5 on CD4-positive target cells can enhance fusion events mediated by macrophage-tropic primary virus envelope glycoproteins.
  • the results also indicate that the HIV- 1 gpl20 V3 loop sequence determines the ability of the envelope glycoproteins to utilize CCR5 as a fusion cofactor.
  • results presented herein indicate that, in addition to CD4, members of the chemokine receptor family play critical roles in early events in HIV- 1 infection.
  • the particular chemokine receptors utilized by HIV- 1 variants differ, depending upon previously characterized differences in target cell preference.
  • Our results confirm the involvement of CXCR4 (HUMSTSR) in infection by laboratory-adapted HIV- 1 (Feng et al, 1996) and demonstrate a role for this molecule in infection by some primary, T-cell line-tropic HIV- 1 isolates.
  • Our results indicate that the clinically-relevant, macrophage-tropic HIV- 1 can use other members of the chemokine receptor family, such as CCR3 and CCR5, to facilitate infection.
  • CCR3 and CCR5 are not completely characterized, current data are consistent with the hypothesis that these molecules contribute to in vi ⁇ o infection by macrophage-tropic primary HIV- 1 variants.
  • the tissue distribution of CCR5 has been reported to be restricted to KG- 1 A promyeloblastic cells (Samson et al, 1996), but more recent data suggest that it is expressed in both CD4- positive and CD8-positive human PBMC as well as in cells of the myeloid lineage (C.G., unpublished observations and Raport et al, 1996). The latter distribution is consistent with that expected based upon the known host cell range of primary HIV- 1 isolates.
  • CCR3 appears to be more restricted, with high levels of expression in eosinophils and little expression in peripheral blood T lymphocytes (Daugherty et al, 1996; Kitaura et al, 1996, Ponath 1996b). The latter observation suggests that CCR3 could not be the sole factor facilitating the infection of primary HIV- 1 isolates, all of which replicate in PBMC.
  • CCR3 may play an important role in a limited number of cell types, eosinophils, which express high levels of CCR3 (Daugherty et al, 1996; Kitaura et al, 1996; Ponath et al, 1996b), also express CD4 and have been reported to be infectible by HIV- 1 (Freedman et al, 1991 ; Weller et al, 1995). In one of these studies (Freedman et al, 1991), two primary HIV- 1 isolates but not a laboratory-adapted virus were able to replicate on bone marrow- derived eosinophil cultures.
  • CCR3 significantly enhanced infection by a smaller subset of primary HIV- 1 than did CCR5 expression. Although infection mediated by several of the primary HIV- 1 envelope glycoproteins was not detectably affected by CCR3 expression, significant CCR3 effects were observed for the YU2 and ADA envelope glycoproteins. This result suggests that heterogeneity in chemokine receptor utilization may occur even among macrophage-tropic primary HIV- 1 isolates.
  • CCR3 The contribution of CCR3 to primary HIV- 1 infection of different target cells in vi ⁇ o and the relationship between CCR3 use and resistance to neutralizing antibodies will be evaluated in future studies.
  • the involvement of receptors for the ⁇ chemokines in HIV- 1 infection explains the sensitivity of macrophage-tropic primary HIV- 1 isolates, but not laboratory-adapted isolates, to inhibition by RANTES, MIP- l ⁇ AND MlP- l ⁇ (Cocchi et al, 1995). Both CCR3 and CCR5 have been shown to be responsive to RANTES (Daugherty et al, 1996; Samson et al, 1996; Ponath et al, 1996b).
  • the structure of the gp l20 V3 loop previously shown to specify target cell- dependent membrane fusion efficiency (Cheng-Mayer et al, 1990; O'Brien et al, 1990; Hwang et al, Westervelt et al, Ivanoff et al, 1991; Westervelt et al, 1992; Bergeron et al, 1992; Chesebro et al, 1992), determined the ability of the viral envelope glycoproteins to utilize CCR3 and CCR5 as accessory factors for entry.
  • the simplest model for post-CD4 binding events in HIV- 1 entry would involve a direct interaction between the viral envelope glycoproteins and the chemokine receptors.
  • CCR3 and CCR5 are closely related among the chemokine receptors (Daugherty et al, 1996; Ponath et al, 1996b; Raport et al, 1996; Samson et al, 1996), the relationship of either of these molecules to CCRl, which did not affect HIV- 1 infection in our hands, is even greater. Again, simple inspection of primary sequences does not reveal determinants unique to CCR3 and CCR5 that might be targets for HIV- 1 interaction.
  • An alternative model is that the chemokine receptors affect the target membrane and/ or CD4 in ways conducive to entry by viruses with particular envelope glycoprotein configurations, without directly contacting viral components. It is also possible that G protein- mediated signaling plays a role in HIV- 1 infection. Additional studies should distinguish among these possibilities.
  • the involvement of G protein-coupled receptors in two other instances of infection with pathogens has been reported.
  • the Duffy antigen receptor which binds both ⁇ and ⁇ chemokines, facilitates invasion by the malarial parasite, Plasmodium ⁇ i ⁇ ax (Horuk et al, 1993; Chaudhuri et al, 1993). Similarly, the progression from colonization to infection with Streptococcus pneumonia is facilitated by expression of the platelet- activating factor receptor (Cundel et al, 1995).
  • the ⁇ chemokine receptors identified here may represent important host components that specify susceptibility to HIV- 1 infection or, in already infected individuals, determine viral burden and rate of disease progression.
  • Endogenous levels of RANTES, MIP- l ⁇ and MlP- l ⁇ expression in CD4- positive lymphocytes were higher in some individuals that remained uninfected despite multiple sexual exposures to HIV- 1 infected partners (Paxton et al, 1996). If direct interaction between viral components and ⁇ chemokine receptors occur, inappropriate signaling events may be initiated that contribute to pathogenesis.
  • a better understanding of the interaction of HIV- 1 , ⁇ chemokines and their receptors may clarify the contribution of these elements to virus transmission and pathogenic outcome, and may suggest approaches for intervention.
  • CCR1F CCR3F and CCR5F proteins were directly compared by using a monoclonal antibody reactive with the epitope tag (FLAG tag) on the amino terminus of each of these molecules.
  • FLAG tag epitope tag
  • HeLa cells expressing CD4 and chemokine receptors were incubated with recombinant viruses containing the designated envelope glycoproteins, and CAT activity measured.
  • the ADA and YU2 envelope glycoproteins were derived from macrophage-tropic primary HIV- 1 viruses from North America (clade B).
  • Tn243 envelope glycoproteins were derived from macrophage-tropic primary HIV- 1 viruses. The phylogenetic classification and geographic origin of these viruses are as follows: Br20-4 (clade B, Brazil), Br25-9 (clade C, Brazil), Rw20-5 (clade A, Philippine), Th966 (clade E, Thailand) and TN243 (clade E, Thailand) (Gao et al, 1996; Karlsson et al, 1996).
  • the HXBc2 envelope glycoproteins were derived from a highly laboratory-adapted clade B HIV- 1 isolate.
  • the A-MuLV envelope glycoproteins were derived from the amphotropic murine leukemia virus (Landau et al, 1991).
  • HXBc2 ADA-V3
  • HXBc2 YU2-V3
  • the HXBc2 (YU2-V1/V2) chimeric envelope glycoprotein contains a substitution of the VI /V2 loops from the YU2 virus into the HXBc2 g ⁇ l20 glycoprotein.
  • ND not determined.
  • CD4 (+CD2 control) 0.29 1.5 1.1 0.79 53.5
  • ADA YU2 HXBc2 HXBc2 HXBc2 HXBc2 ADA-V3 (YU2-V3) (YU2-V1/V2)
  • Recombinant GFP or luciferase reporter viruses were generated by cotransfection of COS-7 or 293 cells with 20 ⁇ g of pNL4-3env GFP or pNL4-3envLUC and 4 ⁇ g of pSVIIIenv plasmids encoding different HIV- 1 Envs or pSVMLVenv using the calcium phosphate method (He, J., et al., 1995).
  • the ⁇ NL4-3env GFP plasmid which encodes full- length NL4-3 HIV- 1 proviral DNA with a frameshift in en ⁇ and expresses GFP in place of nef, was constructed by replacing the alkaline phosphatase (AP) gene in pHIV-AP (He, J., et al., 1995) with the GFP gene using NotI and Xhol restriction sites introduced into pEGFP- 1 (Clontech) by PCR amplification.
  • AP alkaline phosphatase
  • pNL4-3env LUC which encodes full-length env-defective NL4-3 HIV- 1 proviral DNA and expresses the luciferase enzyme
  • pGEM-luc Promega.
  • the pSVMLV-env plasmid which expresses the amphotropic murine leukemia virus envelope glycoproteins have been described (Choe, H., et al., 1996).
  • HIV- 1 Entry and Chemokine Inhibition Assays Primary brain cultures were infected by incubation with recombinant virus (5,000 RT units) pseudotyped with the different Envs in 1 ml of medium. After 16 h of incubation at 37° C, the medium was removed and the cultures were washed three times prior to addition of fresh medium. After an additional 72 h at 37° C, the cells were fixed in 4% paraformaldehyde in PBS for visualization of GFP and immunofluorescence staining or lysed for determination of luciferase activity using commercially available reagents (Promega) after normalization for the same protein concentration. For chemokine or antibody inhibition experiments, cultures were preincubated for 60 min at 37° C in 0.5 ml medium with chemokine
  • C£2Th canine thymocytes were transfected with plasmids expressing CD4 and either CCR3, CCR5, or CXCR4 as described (Choe, H., et al., 1996). The transfected cells were incubated either in the absence of presence of the 7B11 antibody (3 ⁇ g/ml) for 1 h at 37° C.
  • the cells were infected by HIV-1 CAT reporter viruses pseudotyped with an HIV- 1 Env (YU2 Env for the CCR3 and CCR5 transfectants, HXB2 Env for the CXCR4 transfectants) and CAT activity was measured in the transfected cells 60 h after infection (Choe, H., et al., 1996).
  • HIV-1 CAT reporter viruses pseudotyped with an HIV- 1 Env (YU2 Env for the CCR3 and CCR5 transfectants, HXB2 Env for the CXCR4 transfectants) and CAT activity was measured in the transfected cells 60 h after infection (Choe, H., et al., 1996).
  • Immunofluorescence staining of fixed cultures with the indicated primary antibodies followed by FITC- or rhodamine-conjugated secondary antibodies was performed as described (Shi, B., et al, 1996).
  • the dilutions for the primary antibodies were: mouse anti-CD68 monoclonal (EBM11) (Dako), 1: 10; rabbit anti-GFAP (sigma), 1: 100; mouse anti-CCR3 monoclonal (7B1 1) (Heath, H., et al., In Press), 1:50.
  • Labeling with RCA- 1 conjugated to rhodamine (10 ⁇ g/ml) was performed for 1 h at room temperature.
  • the anti- CCR3 monoclonal antibody was detected by incubation with biotinylated horse anti-mouse IgG (5 ⁇ g/ml) for 30 min followed by avidin-FITC (20 ⁇ g/ml) for 15 min (Vector Laboratories).
  • biotinylated horse anti-mouse IgG 5 ⁇ g/ml
  • avidin-FITC 20 ⁇ g/ml
  • RT-PCR Reverse Transcription-polvmerase Chain Reaction
  • One tenth of this reaction was used as a template for PCR amplification with AmpliTaq DNA polymerase (Perkin- Elmer) (CXCR4 primers (SEQ ID NO:6), 5'-GACCGCTACCTGGCCATT-3; and (SEQ ID NO:7) 5'-GTTGTAGGGCAGCCAGCA-3'; CCR5 primers, (SEQ ID NO:8) 5 AATCTTCTTCATCATCCTCC-3' and (SEQ ID NO:9) 5'- TCTCTGTCACCTGCATAGC-3'; CCR3 primers, (SEQ ID NO: 10) 5'- TCCTTCTCTCTTCCTATCAATC-3' and (SEQ ID NO: 11) 5'GGCAATTTTCTGCATCTG-3') for 30 cycles
  • CCR5, CXCR4, and CCR3 transcripts were detected in primary brain cultures (Fig. 6A). The identity of these transcripts was confirmed by DNA sequencing of the PCR products (data not shown). CCR3 transcripts were relatively abundant. Based on this finding, we examined whether CCR3 is expressed in microglia by double immunofluorence staining with an anti-CCR3 monoclonal antibody (Heath, H., et al.) and the microglial cell marker Ricinus communis agglutinin I (RCA- 1). CCR3 was expressed in >95% of microglia in primary brain cultures and was not detected in other brain cell types (Fig 6B).
  • astrocytes A minor fraction of astrocytes (2-4%) was infected by viruses with YU2, ADA, 89.6, Br20-4, or HXB2 Envs, indicating that a low efficiency of HIV- 1 entry into astrocytes can be mediated by either M-tropic or T-tropic Envs.
  • the level of GFP expression in astrocytes was generally lower than that seen in microglia, consistent with previous studies demonstrating that HIV- 1 gene expression in astrocytes is restricted (Takahashi, K., et al., 1996).
  • the natural ligands for the chemokine receptors have been shown to inhibit the entry of particular HIV- 1 isolates that use these receptors (Feng, Y., et al., 1996; Deng, H.K., et al., 1996; Dragic, T., et al., 1996; Alkhatib, G., et al., 1996; Choe, H., et al., 1996; Cocchi, F., et al., 1995; Bleul, CC, et al., 1996; Oberlin, E., et al., 1996), MIP- l , MlP- l ⁇ , and RANTES, chemokines (Harouse, J.M., et al., 1989; Tornatore C, et al., 1991)that bind CCR5, inhibit infection by M-tropic but not T-tropic HIV- 1 isolates (Deng, H.K., et al., 1996; Dragic, T.
  • stromal cell-derived factor- 1 inhibits entry by T- tropic or dual-tropic HIV-1 isolates that use CXCR4 (Bleul, CC, et al., 1996; Oberlin, E., et al., 1996).
  • CXCR4 Bleul, CC, et al., 1996; Oberlin, E., et al., 1996.
  • Infection by the subset of M-tropic isolates that utilize CCR3 as a co-receptor is inhibited by eotaxin (Choe, H., et al., 1996), the major CCR3 ligand (Ponath, P., et al., 1996; Daugherty, B.L., et al., 1996; Heath, H., et al., in press).
  • chemokines were tested the ability of chemokines to inhibit HIV- 1 infection of primary brain cultures. Chemokine blocking experiments were performed using erii -defective HIV- 1 luciferase reporter viruses pseudotyped with different HIV-1 Envs. The amphotropic murine leukemia virus (MLV) Env was used to control for possible nonspecific inhibitory effects. The efficiency of the early phase of virus replication during a single-cycle infection was determined by measuring luciferase activity in the primary brain cultures at 72 h after infection (Fig. 8). The relative efficiency of virus entry mediated by the different HIV- 1 Envs was similar to that observed using the HIV- 1 GFP reporter viruses.
  • MLV amphotropic murine leukemia virus
  • MlP-l ⁇ , eotaxin, and SDF-1 since these chemokines show selective binding to either CCR5, CCR3, or CXCR4, respectively.
  • MlP- l ⁇ and eotaxin inhibited infection with HIV- 1 luciferase reporter viruses containing the YU2 or ADA Envs by 70-80% (Fig. 8A).
  • MlP-l ⁇ In contrast, infection with viruses containing the Br20-4 or 89.6 Env was inhibited by MlP-l ⁇ , but was not significantly inhibited by eotaxin. MlP-l ⁇ and eotaxin had no inhibitory effect on the virus with the HXB2 Env. SDF-1 inhibited infection by viruses with the 89.6 or HXB2 Env, but not the YU2, ADA, or Br20-4 Env. MCP-1 which does not bind CCR5, CC3, or CXCR4, had no inhibitory effect on any of the viruses tested. None of the chemokines tested inhibited infection by virus with the MLV Env (Fig. 8B).
  • Cf2Th canine thymocytes transfected with plasmids expressing CD4 and either CCR3 or CCR5 were infected with recombinant HIV- 1 CAT reporter viruses containing the YU2 Env in the absence or presence of the antibody.
  • Cf2Th cells transfected with plasmids expressing CD4 and CXCR4 were infected with the HIV- 1 CAT reporter virus containing the HXB2 Env in parallel experiments.
  • the 7Bl l monoclonal antibody blocked infection of CCR3-expressing cells, but not CCR5- or CXCR4-expressing cells, as determined by inhibition of CAT activity in the target cells 60 h after infection (Fig. 8D).
  • the anti-CCR3 antibody inhibited infection by HIV- 1 luciferase reporter viruses with the YU2 or ADA Env with an efficiency comparable to that of eotaxin (Fig. 8C).
  • ligands that bind to both CCR3 and CCR5 show greater blocking activity than ligands selective for either CCR3 or CCR5
  • the chemokines and the 7B11 antibody had no inhibitory effect on viruses with the MLV Env (Fig 8B).
  • the OKT4A antibody inhibited infection by HIV- 1 luciferase viruses containing the YU2, ADA, Br20-4, or 89.6 Envs by 70-80%, while infection by virus with the HXB2 Env was inhibited by 50% (data not shown).
  • the infection of the CD4+ Jurkat T cell line was inhibited by >95% using the same assay conditions.
  • CCR3 and CCR5 are used for HIV- 1 infection of primary brain cultures.
  • the inhibitory effects of eotaxin, MlP- l ⁇ , and the 7B1 1 anti-CCR3 antibody were examined by direct visualization of infected target cells using HIV- 1 -GFP reporter viruses.
  • Double labeling with cell-specific markers showed that eotaxin, MlP- l ⁇ , and anti-CCR3 inhibited 70-80% of microglial infection by HIV- 1 GFP reporter viruses with the YU2 Env, as determined by counting the percentage of GFP-positive, CD68 positive cells.
  • CCR3 and CCR5 both serve as major co-receptors with CD4 for HIV- 1 infection of brain microglia.
  • the M-tropic primary HIV- 1 isolates infect peripheral blood monoctye/ macrophages and brain-derived microglia more efficiently than T- tropic isolates.
  • the use of both CCR3 and CCR5 for microglial entry differs from infection of blood-derived monocyte /macrophages, which express CCR5 [Deng, H.K., et al., Nature 381:667-673 (1996); Alkhatib, G., et al., Science 262: 1955- 1958 ( 1996)] but do not express CCR3.
  • the efficiency of HIV- 1 entry was determined by single-cycle infections using HIV-l -GFP reporter viruses pseudotyped with different HIV- 1 Envs.
  • the chemokine receptor utilization is derived from the method of Choe, et al.
  • the efficiency of HIV- l entry for each Env is expressed as the percentage of CD-68-positive (microglia) or GFAP-positive (astrocytes) cells labeled with GFP, as determined by counting ten random fields (magnification: 200X). Data shown are the mean ⁇ s.d. of duplicates from a representative experiment. Results were comparable in four independent experiments, although the maximum HIV- 1 entry ranged from 30-45% for microglia and from 2-5% for astrocytes between different donors.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • AIDS & HIV (AREA)
  • Physiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention se rapporte à un site de liaison conformationnel de la glycoprotéine gp120, formé par la liaison de gp120 et CD4 qui permet la liaison du complexe aux récepteurs de la chimiokine. L'invention se rapporte également à des techniques de liaison qui permettent un criblage aisé de molécules qui modifient la liaison de gp120 et de la chimiokine, ainsi qu'à des cibles spécifiques destinées à modifier la liaison.
PCT/US1997/018397 1996-10-09 1997-10-08 Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih WO1998015569A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002267662A CA2267662A1 (fr) 1996-10-09 1997-10-08 Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih
EP97913670A EP0956290A1 (fr) 1996-10-09 1997-10-08 Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih
AU50800/98A AU731975B2 (en) 1996-10-09 1997-10-08 GP120 polypeptides having conformational discontinuous chemokine receptor binding sites and methods of inhibiting HIV infection

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2793196P 1996-10-09 1996-10-09
US60/027,931 1996-10-09

Publications (2)

Publication Number Publication Date
WO1998015569A1 true WO1998015569A1 (fr) 1998-04-16
WO1998015569A9 WO1998015569A9 (fr) 1998-06-25

Family

ID=21840588

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/018397 WO1998015569A1 (fr) 1996-10-09 1997-10-08 Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih

Country Status (4)

Country Link
EP (1) EP0956290A1 (fr)
AU (1) AU731975B2 (fr)
CA (1) CA2267662A1 (fr)
WO (1) WO1998015569A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001016182A2 (fr) * 1999-08-27 2001-03-08 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Polypeptides de liaison a la glycoproteine gp120 du virus de l'immunodeficience humaine (vih), acides nucleiques, anticorps et compositions associes, et methodes d'utilisation
WO2001043779A2 (fr) * 1999-12-16 2001-06-21 Tanox, Inc. Conjugues anti-hiv1 pour le traitement du vih
EP1130089A1 (fr) * 2000-02-17 2001-09-05 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Composition comprenant des particules sous-virales cibles et fusions de virus enveloppés, et vaccin la contenant
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7304127B2 (en) 1999-08-27 2007-12-04 United States Of America As Represented By The Secretary, Department Of Health And Human Services Polypeptides that bind HIV gp120 and related nucleic acids, antibodies, compositions, and methods of use
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CELL, 04 July 1986, Vol. 46, COFFIN J.M., "Genetic Variation in AIDS Viruses", pages 1-4. *
HORUK R., "Molecular Properties of the Chemokine Receptor Family", 1994, pages 159-165. *
JOURNAL OF VIROLOGY, January 1988, Vol. 62, No. 1, WILLEY R.L. et al., "In Vitro Mutagenesis Identifies A Region Within the Envelope Gene of the Human Immunodeficiency Virus that is Critical for Infectivity", pages 139-147. *
JOURNAL OF VIROLOGY, June 1987, Vol. 61, No. 6, HO D. et al., "Human Immunodeficiency Virus Neutralizing Antibodies Recognize Several Conserved Domains on the Envelope Glycoproteins", pages 2024-2028. *
JOURNAL OF VIROLOGY, September 1989, Vol. 63, No. 9, WILLEY R.L. et al., "Functional Interaction of Constant and Variable Domains of Human Immunodeficiency Virus Type 1 gp120", pages 3595-3600. *
SCIENCE, 15 December 1995, Vol. 270, COCCHI F. et al., "Identification of RANTES, MIP-Alpha and MIP-Beta as the Major HIV- Suppressive Factors Produced by CD8+ T Cells", pages 1811-1815. *
SCIENCE, 26 February 1988, Vol. 239, HO D. et al., "Second Conserved Domain of GP120 is Important for HIV Infectivity and Antibody Neutralization", pages 1021-1023. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7160546B2 (en) 1995-06-06 2007-01-09 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6800729B2 (en) 1995-06-06 2004-10-05 Human Genome Sciences, Inc. Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor)
US6759519B2 (en) 1995-06-06 2004-07-06 Human Genome Sciences, Inc. Antibodies to human G-protein chemokine receptor HDGNR10 (CCR5receptor)
WO2001016182A3 (fr) * 1999-08-27 2002-08-15 Us Health Polypeptides de liaison a la glycoproteine gp120 du virus de l'immunodeficience humaine (vih), acides nucleiques, anticorps et compositions associes, et methodes d'utilisation
WO2001016182A2 (fr) * 1999-08-27 2001-03-08 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Polypeptides de liaison a la glycoproteine gp120 du virus de l'immunodeficience humaine (vih), acides nucleiques, anticorps et compositions associes, et methodes d'utilisation
US7304127B2 (en) 1999-08-27 2007-12-04 United States Of America As Represented By The Secretary, Department Of Health And Human Services Polypeptides that bind HIV gp120 and related nucleic acids, antibodies, compositions, and methods of use
WO2001043779A2 (fr) * 1999-12-16 2001-06-21 Tanox, Inc. Conjugues anti-hiv1 pour le traitement du vih
WO2001043779A3 (fr) * 1999-12-16 2002-05-10 Tanox Inc Conjugues anti-hiv1 pour le traitement du vih
EP1130089A1 (fr) * 2000-02-17 2001-09-05 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Composition comprenant des particules sous-virales cibles et fusions de virus enveloppés, et vaccin la contenant
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7862818B2 (en) 2001-02-09 2011-01-04 Human Genome Sciences, Inc. Method of inhibiting human G-protein chemokine receptor (CCR5) HDGNR10
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10

Also Published As

Publication number Publication date
CA2267662A1 (fr) 1998-04-16
AU5080098A (en) 1998-05-05
AU731975B2 (en) 2001-04-12
EP0956290A1 (fr) 1999-11-17

Similar Documents

Publication Publication Date Title
Koch et al. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition
Willett et al. Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses
Bieniasz et al. Chemokine receptors and human immunodeficiency virus infection
Cooley et al. HIV-1 cell entry and advances in viral entry inhibitor therapy
US7371846B2 (en) CD4-independent HIV envelope proteins as vaccines and therapeutics
US10973906B2 (en) Therapeutic peptides and vaccines
WO1997047318A1 (fr) Utilisations d'un recepteur de chemokines en vue de l'inhibition de l'infection a vih-1
WO1998000535A2 (fr) Procede pour inhiber l'infection par le vih-1, dosages de medicaments et procedes de diagnostic et pronostic de la susceptibilite d'infection par le vih
AU731975B2 (en) GP120 polypeptides having conformational discontinuous chemokine receptor binding sites and methods of inhibiting HIV infection
Borkow et al. Multi-targeting the entrance door to block HIV-1
WO1998015569A9 (fr) Polypeptides de gp120 possedant des sites de liaison aux recepteurs de la chimiokine a discontinuite de conformation et procedes d'inhibition des infections a vih
US20040086528A1 (en) Uses of a chemokine receptor for inhibiting HIV-1 infection
EP1161455A1 (fr) Nouvelle proteine chimere permettant de prevenir et de traiter l'infection due au vih
WO1999019501A1 (fr) Vecteur d'origine retrovirale d'adn non integrant a forte expression de proteines et antigenes immunogenes secretes
EP1212363A2 (fr) Antagonistes de l'interleukine-16
Dittmar et al. A recombinant virus assay using full-length envelope sequences to detect changes in HIV-1 co-receptor usage
Howie et al. A functional, discontinuous HIV‐1 gp120 C3/C4 domain‐derived, branched, synthetic peptide that binds to CD4 and inhibits MIP‐1α chemokine binding
Doranz The use of Envelope for HIV therapeutics: from vaccines to co-receptors
Berson How human immunodeficiency virus type 1 uses alternative receptors and coreceptors to enter cells
Hung The role of HIV-1 V3 loop in CCR5 tropism and macrophage infection
Bukrinsky HIV life cycle and inherited co-receptors
Borgucci Role of Monocytes in the Pathogenesis of HIV-1 Infection
Murray Identification of residues in CD4 required for efficient HIV-1 viral entry, and a binding domain for the entry inhibitor T140
Chuang et al. Conferral of an antiviral state to CD4+ cells by a zipper motif envelope mutant of the human immunodeficiency virus type 1 transmembrane protein gp41
Holm Mechanisms of bystander T cell apoptosis in HIV-1 infection

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
COP Corrected version of pamphlet

Free format text: PAGE 58, DESCRIPTION, REPLACED BY A NEW PAGE 58; PAGES 1/26-26/26, DRAWINGS, REPLACED BY NEW PAGES 1/26-26/26; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2267662

Country of ref document: CA

Ref country code: CA

Ref document number: 2267662

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 50800/98

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1997913670

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1997913670

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 50800/98

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1997913670

Country of ref document: EP