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WO1993001820A2 - Inhibition de l'infection hiv a mediation par la proteine non-cd4 - Google Patents

Inhibition de l'infection hiv a mediation par la proteine non-cd4 Download PDF

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Publication number
WO1993001820A2
WO1993001820A2 PCT/US1992/005985 US9205985W WO9301820A2 WO 1993001820 A2 WO1993001820 A2 WO 1993001820A2 US 9205985 W US9205985 W US 9205985W WO 9301820 A2 WO9301820 A2 WO 9301820A2
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WIPO (PCT)
Prior art keywords
gpl20
cells
glu
leu
gin
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PCT/US1992/005985
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English (en)
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WO1993001820A3 (fr
Inventor
Benson M. Curtis
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Bristol-Myers Squibb Company
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Publication of WO1993001820A2 publication Critical patent/WO1993001820A2/fr
Publication of WO1993001820A3 publication Critical patent/WO1993001820A3/fr

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    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • 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

Definitions

  • the present invention is directed to a non-CD4 cell surface receptor for gpl20.
  • This gpl20 receptor (gpl20r) has been isolated and cloned and is utilized in the present invention in methods and kits for the inhibition and detection of HIV infection.
  • human retroviruses Two types have been identified, leukemia viruses and ALDS- related viruses.
  • the primary targets of the human retroviruses are T lymphocytes and cells of the central nervous system. All human retroviruses are transmitted by intimate contact, blood contamination, and infection in utero or after birth by milk. It is likely that all human retroviruses originated in Africa and that they encountered the human species via interspecies infection, possibly from African green monkeys or a related species.
  • Human T Lymphotropic Virus Type 1 (HTLV-1) and Human T Lymphotropic Virus Type H (HTLV-II)
  • HTLV-II Human T Lymphotropic Virus Type 1
  • H Human Immunodeficiency Viruses
  • the human immunodeficiency virus is a cytopathic retrovirus and the causative agent of the acquired immunodeficiency syndrome (AIDS).
  • HIV-1 lymphadenopathy-associated virus
  • HTLV- ⁇ i Human T Lymphotropic Virus Type HI
  • Another retrovirus, HTV-2 has been isolated primarily from West African patients with AIDS and is pathogenically related to HTV-1. On the genetic level, HIV-2 is actually more closely related to the simian immunodeficiency virus (SIV), a retrovirus infecting monkeys.
  • SIV simian immunodeficiency virus
  • HTV is a member of the nontransforming, cytopathic lentivirus family of retroviruses. HTV causes a typically fatal disease characterized by severe immunodeficiency or neurodegenerative disease, or both.
  • the primary basis for HIV induced immunosuppression is the depletion of the helper/inducer subset of T lymphocytes expressing the CD4 molecule (T4 or CD4 cells), which serves as a high affinity cell surface receptor for the virus.
  • T4 lymphocytes are involved directly or indirectly in the induction of nearly every immunologic function in the body, and their depletion results in susceptibility to a wide range of opportunistic infections and neoplasms.
  • HTV infection In addition to the T4 lymphocyte, other cells expressing the CD4 molecule are targets of HTV infection, especially monocyte-macrophages. HTV infection also results in serious B cell abnormalities including polyclonal activation, hypergammaglobulinemia, elevated levels of circulating immune complexes, and autoantibodies. A decreased number of functional natural killer (NK) cells have also been observed in AIDS patients.
  • NK natural killer
  • Infection of CD4 cells is initiated by the interaction of the CD4 molecule with the major HIV envelope glycoprotein gpl20, an event which is followed by intemalization and uncoaring of the virion, transcription of genomic RNA to DNA by virus-encoded reverse transcriptase, and integration of the resulting proviral DNA into host cell chromosomal DNA. Also, unintegrated proviral DNA accumulates in large amounts within infected cells and is probably a significant factor in HIV cytopathology (Shaw et al., (1984) Science 22&1165).
  • CD4 T cells The depletion of CD4 T cells appears to contribute significantly to the immunosuppression associated with AIDS.
  • a primary cytopathic effect of the virus in vitro is HIV-induced syncytium formation.
  • CD4 through its interaction with gpl20 plays an important role in syncytium formation.
  • molecules on the cell surface of uninfected cells other than CD4 are also involved in HIV-induced cell fusion (Hildreth et al. (1989) Science 244:1075-1078).
  • Infection by HIV produces, in addition to AIDS, a set of neuropsychiatric disorders which are called the ADDS dementia complex (ADC) (Price et al., (19881 239:586-592).
  • ADC ADDS dementia complex
  • ADC cognitive impairment
  • apathy and motor dysfunctions
  • the underlying cause of ADC appears to be the death of brain cells and HIV-1 can be isolated from the brains of infected individuals (Ho et al, (1987) N. Eng. J. Med. 217:278-286).
  • the present invention demonstrates the presence of a non-CD4 receptor for gpl20 and a method for the inhibition of HIV infection of cells such as brain and muscle which do not express high levels of CD4.
  • the present invention has identified this non-CD4 gpl20 receptor (gpl20r) and has recombinantly expressed and characterized gpl20r.
  • gpl20r non-CD4 gpl20 receptor
  • a specific non-CD4 gpl20r has been isolated which has specific binding activity for gpl20 present on Human Immunodeficiency Virus-1 (HTV).
  • HTV Human Immunodeficiency Virus-1
  • This gpl20r has a molecular weight of about 45, 000 daltons, contains about 400 amino acid residues and is characterized by a Kd for gpl20 of about 1.3 nM to about 2.0 nM.
  • gpl20 to gpl20r The binding of gpl20 to gpl20r is inhibited by specific carbohydrates, such as mannose and fucose, plant lectins such as concanavalin A and specific antibiotics, such as pradimicin A.
  • specific carbohydrates such as mannose and fucose
  • plant lectins such as concanavalin A
  • specific antibiotics such as pradimicin A.
  • a cDNA molecule that transcribes an mRNA encoding for gpl20r is cloned and expressed to produce gpl20r.
  • the DNA is selected from a gene library obtained from tissue such as placenta, brain, muscle and colon.
  • a method of inhibiting HIV infection of mammalian cells, such as brain, muscle and neural cells, is contemplated by the present invention.
  • cells are contacted with an effective amount of an appropriate inhibitor of gpl20r binding for a time period sufficient to significantly inhibit the binding of HIV to the non-CD4 protein, gpl20r.
  • an appropriate inhibitor of gpl20r binding include mannose carbohydrates, fucose carbohydrates, plant lectins, and antibiotics such as pradimicin A.
  • the g l20r of the present invention can also be utilized in a method and a kit for the detection of the presence of HTV in a fluid sample.
  • the binding of HTV to gpl20r is detected by an indicating means such as a labelled antibody capable of binding to the HTV-gpl20 ⁇ reaction product.
  • the gpl20r can be affixed to a solid matrix to form a solid support that is useful in this method and/or kit.
  • FIGURE 1 illustrates expression cloning of the gpl20r cDNA and comparison to CD4.
  • A Autoradiography of gpl20 binding to gpl20r and CD4 expressed in COS cells.
  • A-F [ 125 T]vgpl20; A, gpl20 ⁇ ; B, gpl20r with G17-2; C, gpl20r with 200 nM unlabelled bgpl20; D, CD4; E, CD4 with G17-2; F, CD4 with bgpl20.
  • G-L [ 125 T]ngpl20; G, gpl02r; H, gpl20r with 110.1; I, gpl20r with bgpl20; J, CD4: k, CD4 with 110.1; L, CD4 with bgpl20.
  • B Inhibition of [ 125 TJvgpl20 binding to gpl20r and CD4.
  • FIGURE 2 illustrates the characterization of the gpl20r.
  • 125 A Scatchard analysis of [ TJgpl20 binding.
  • B Inhibition of [ 125 IJgpl20 binding to gpl20r COS cells. Open symbols ngpl20, filled symbols vgpl20.
  • D Placenta control sera; 2, placenta HTV sera; 3, gpl20r COS control sera; 4, gpl20r COS HTV sera.
  • E Northern blot of gpl20r expression. Polyadenylated (A+); 2, placenta; 3, thymus; 4+12, forebrain; 5, skeletal muscle; 6, heart; 7, liver; 8, kidney; 9, colon; 10 medulla; 11, cerebellum; 13, T cell (CEM; 16 ⁇ g A+) 14, B cell (TS-1; 16 ⁇ g A+); 15, macrophage (U937; 8 ⁇ g A + ); 16, cervical carcinoma (HeLa; 16 ⁇ g A + ).
  • the different apparent size of the " 5 kb band is an artifact of displacement by 28S rRNA.
  • FIGURE 3 illustrates the sequence analysis of the gpl20r.
  • A Nucleotide and deduced protein sequence of gpl20r cDNA.
  • B Hydropathicity plot of the gpl20r. The predicted transmembrane segment and the start of the eight amphipathic repeats are indicated by arrows.
  • HIV infection of brain and muscle cell lines is not blocked by soluble CD4 or anti-
  • CD4 antibodies (Clapham, P.R. et al., (1989) Nature 227:368-370; Harouse, J.M. et al., (1989) J. Virol. 61:2527-2533; Weber, J. et al., (1989) J. Gen. Virol. 7Q:2653-2660). This is consistent with the existence of a second gpl20 receptor. Binding studies indicated that human placenta was another source for a non-CD4 gpl20 receptor, and a cDNA for a second gpl20 receptor (gpl20r) was isolated by the present invention from a placental library.
  • the gpl20r has a higher binding affinity for gpl20 than CD4. Sequence analysis revealed homology to membrane associated C-type lectins, and inhibition studies have shown that the receptor binds gpl20 through a mannose or fucose containing carbohydrate. The gpl20r rapidly internalizes gpl20, and is expressed in placenta, thymus, muscle, and colon. These results, when considered with previous studies on the role of gpl20 carbohydrate in HTV infection (Lifson, J. et al., (1986) J. Exp. Med. ⁇ >4:2101-2106; Ezekowitz, R.A.B. et al., (1989) J. Exp. Med.
  • gpl20r participates in cellular binding of HTV by a non-CD4 pathway in muscle and brain, as well as, facilitating virus attachment in CD4 positive cell types. It is likely that the gpl20r plays a significant role in t ⁇ ansplacental transport of HTV (Zacher, V. et al., (1991) J. Virol. £5_:2102-2107) and colon infection.
  • G ⁇ l20 produces an increase in intracellular calcium in rat retinal ganglion cells (Dreyer, E.B. et al., (1990) Science 248_:364-367) suggesting that the gpl20r or a homologous protein may have signaling functions in the nervous system dismpted by gpl20 leading to HTV neurotoxicity.
  • a new non-CD4 binding protein, or receptor, for gpl20 was isolated.
  • the HIV surface protein gpl20 was found to bind to a receptor on human placental membranes that was not blocked by antibodies directed against CD4, such as G17-2 and OKT4a, and which interfere with gpl20 binding to CD4.
  • a cDNA encoding this receptor was isolated from a placental cDNA library in a mammalian expression vector (pCDM8). The gene products were expressed in COS cells and were screened by 125 I- labelled gpl20 binding. From a pool of 90,000 cDNA molecules, a single clone was isolated that encoded a protein which bound gpl20, even in the presence of concentrations of anti-CD4 antibody (G17-2) which completely blocked gpl20 binding to CD4. Sequence studies were carried out and indicated that the 1.5 kilobase cDNA clone encoded a previously unknown member of a family of Type H membrane proteins with an extracellular C type lectin domain.
  • the binding of gpl20 to gpl20r is not blocked by polyclonal HTV antisera, but is inhibited by mannose carbohydrates, fucose carbohydrates, plant lectins such as concanavalin A and pradimicin A antibiotics. Other sugars such as N-acetyM-glucosamine and galactose are less potent inhibitors.
  • the gpl20 ⁇ is expressed on many mammalian cells which do not exhibit high levels of CD4, such as placenta, skeletal muscle, brain, and mucosal cells.
  • Other tissue and cells displaying gpl20r include colon, thymus, heart, T cells, B cells and macrophages. The distribution of tissue having gpl20 ⁇ parallels that for binding of gpl20 which is not blocked by CD4 antibodies, and for HTV infection which is not neutralized by soluble CD4. This observation suggests a role for gpl20r in viral infection.
  • gpl20r In gpl20r expressing transfected COS cells, gpl20 is rapidly internalized following binding to gpl20r. This binding and intemalization of gpl20 is inhibited by compounds such as mannan, concanavalin A and pradimicin A.
  • a DNA molecule of the present invention corresponds to a complementary DNA molecule which transcribes a messenger RNA (mRNA) molecule which, when translated, encodes gpl20r.
  • mRNA messenger RNA
  • the cDNA molecules were obtained by reverse-transcribing mRNA molecules isolated from mammalian tissue such as placenta, colon, brain or thymus.
  • the transcription and cloning of cDNA molecules and isolation of gene products are techniques well known in the art and, for example, are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual”. 2d edition, Cold Spring Harbor Lab., Cold Spring Harbor, NY (1989), which is incorporated herein by reference.
  • physiologically tolerable and “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a mammal.
  • the physiologically tolerable carrier may take a wide variety of forms depending upon the preparation desired for administration and the intended route of administration.
  • a carrier is a material useful for administering the active compound and must be "acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • the pharmaceutical compositions are prepared by any of the methods well known in the art of pharmacy all of which involve bringing into association the active compound and the carrier therefor.
  • the agent utilized in the present invention can be administered in the form of conventional pharmaceutical compositions.
  • Such compositions can be formulated so as to be suitable for oral or parenteral administration, or as suppositories.
  • the agent is typically dissolved or dispersed in a physiologically tolerable carrier.
  • the compounds of the present invention can be utilized in liquid compositions such as sterile suspensions or solutions, or as isotonic preparations containing suitable preservatives.
  • liquid compositions such as sterile suspensions or solutions, or as isotonic preparations containing suitable preservatives.
  • injectable media constituted by aqueous injectable isotonic and sterile saline or glucose solutions.
  • Additional liquid forms in which the present compounds may be incorporated for administration include flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar pharmaceutical vehicles.
  • the present agents can also be administered in the form of liposomes.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to the agent of the present invention, stabilizers, preservatives, expedients, and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
  • the present compounds can also be used in compositions such as tablets or pills, preferably cont ⁇ ining a unit dose of the compound.
  • the agent active ingredient
  • conventional tabletting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, gums or similar materials as non-toxic, physiologically tolerable carriers.
  • the tablets or pills of the present compositions can be laminated or otherwise compounded to provide unit dosage forms affording prolonged or delayed action.
  • the pharmaceutical formulation described herein can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
  • the tablets or pills can also be provided with an enteric layer in the form of an envelope that serves to resist disintegration in the stomach and permits the active ingredient to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings including polymeric acids or mixtures of such acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate, and the like.
  • a particularly suitable enteric coating comprises a styrene-maleic acid copolymer together with known materials that contribute to the enteric properties of the coating.
  • a method of inhibiting HTV infection of mammalian cells is disclosed in the present invention.
  • a pharmaceutical composition containing a compound which effectively inhibits the binding of gpl20r to HTV is contacted with cells either in vitro or in vivo for a time period sufficient to significantly inhibit the binding of HTV to the cell surface.
  • Compounds effective in this method include mannose carbohydrates, fucose carbohydrates, plant lectins and pradimicin A antibiotics. Specifically preferred compounds are mannose, fucose, mannan, concanavalin A and pradimicin A.
  • the pharmaceutical composition of the present invention includes a compound which effectively inhibits gpl20r binding to HTV and may also include a physiologically tolerable carrier.
  • the method of the present invention is preferably utilized to inhibit HTV infection of placental, brain, muscle, neural and colon cells.
  • a diagnostic method is also described in the present invention for detecting the presence, and preferably the amount, of HTV present in a fluid sample by producing a reaction product containing HIV bound to gpl20r.
  • a reaction product containing HIV bound to gpl20r there are well known clinical diagnostic procedures that can be utilized for the formulation and detection of such reaction products.
  • exemplary assay methods are described herein, the invention is not intended to be so limited.
  • heterogeneous and homogeneous assay protocols can be employed for detecting the presence, and preferably the amount, of HTV in a fluid sample.
  • the present invention contemplates a method for assaying a sample, such as a body fluid, for the presence of HTV comprising the steps of:
  • the fluid sample is a body fluid sample, such as blood, plasma, serum, urine, saliva, semen or cerebrospinal fluid (CSF).
  • body fluid sample such as blood, plasma, serum, urine, saliva, semen or cerebrospinal fluid (CSF).
  • a labelled indicating means such as a fluorescein-labelled antibody
  • a labelled complex is capable of binding to the gpl20r present in the reaction product to form a labelled complex. Determining the presence of the labelled complex provides an assay for the presence of HTV in the sample.
  • the amount of labelled indicating means bound as part of the complex is determined, and thereby the amount of HTV present in the sample is determined. When that amount is zero, no HTV is present in the sample, within the limits of detection.
  • Methods for assaying the presence and amount of a labelled indicating means depend on the label used, such labels and assay methods being well known in the art.
  • the gpl20r is affixed on a solid matrix to form a solid phase support.
  • the assay is heterogeneous, solid/liquid phase assay and, as such, has its own preferred manipulations. For example, following admixing of a liquid sample with a solid support containing gpl20r affixed thereto, the admixture is ma tained under biological assay conditions for a time period sufficient for any HIV present in the sample to bind to gpl20r and form a solid phase bound reaction product. The solid and liquid phases are then separated to remove any material in the sample that did not react with the solid support, such as by rinsing. This removes any material present in the sample that could interfere with the detection of the reaction product.
  • a labelled indicating means is then admixed with the separated solid phase in an aqueous medium to form a solid/liquid phase labelling-reaction admixture which is maintained for a time period sufficient for the indicating means to bind to the solid bound reaction product forming a labelled complex.
  • the solid phase is then separated from the liquid phase, rinsed and the presence, and preferably amount, of the indicating means present is determined.
  • biological assay conditions refers to parameters that maintain the biological activity of the molecules and organisms in the present invention, and include a temperature range of about 4°C to about 45°C, a pH value range of about 5 to about 9, and an ionic strength varying from that of distilled water to that of about one molar sodium chloride. Methods for optimizing such conditions are well known in the art.
  • the term “about” refers to a range of values both greater than and/or less than the listed value by 10% or less. For example, a temperature of about 20° C will include temperature values of from 18° C to 22° C.
  • the term “corresponds”, and its various grammatical modifications, means “is similar or in agreement with”.
  • kits form for assaying a fluid sample for the presence of HIV are also contemplated by the present invention.
  • a kit includes, in an amount sufficient for at least one assay, gpl20 ⁇ as a packaged reagent, together with instructions for use.
  • An indicating means capable of detecting or signalling the presence of a reaction product formed between gpl20r and HTV may also be present in the kit as a separately packaged reagent.
  • the term "instractions for use” typically includes a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for admixtures, temperature, buffer conditions and the like.
  • packaging materials discussed herein in relation to diagnostic systems are those customarily utilized. Such materials include glass and plastic (e.g. polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes and the like.
  • glass and plastic e.g. polyethylene, polypropylene and polycarbonate
  • vials plastic and plastic-foil laminated envelopes and the like.
  • the term "package” refers to a solid material such as glass, plastic, paper, foil and the like capable of holding within fixed limits the gpl20r, and preferably also a detection means.
  • the package can contain a microtiter plate well to which microgram quantities of gpl20r have been operatively affixed, ie., linked so as to be capable of reacting with and bind HTV and/or gpl20.
  • label indicating means
  • labelled indicating means in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate or detect the presence of a reaction product. Such labels are themselves well known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel methods and/or systems.
  • the indicating means can be a fluorescent labelling agent that chemically binds to antibodies or protein antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
  • Suitable fluorescent labelling agents are fluorochrome, such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine- 1-naphthalene sulfonyl chloride (DANSC), tetramethylrhodamine isocyanate (TRTTC), lissamine and the like.
  • FIC fluorescein isocyanate
  • FITC fluorescein isothiocyanate
  • DANSC 5-dimethylamine- 1-naphthalene sulfonyl chloride
  • TRTTC tetramethylrhodamine isocyanate
  • lissamine lissamine and the like.
  • Immunofiuorescence analysis techniques are well known in the art, and for example, is described in DeLuca, "Immunofiuorescence Analysis” in Immunofiuorescence Analysis. Marchalonis et al., (1982) eds., John Wiley &
  • indicating means are colorimetric agents and enzymes, such as horseradish peroxidase, glucose oxidase or the like, linked as described above, as well as radioactive elements, preferably an element that produces gamma ray emissions.
  • gamma rays such as I, I, I, I, and Cr represent one class of radioactive indicating groups.
  • Another group of useful labelling means are those elements such as 11 C, 18 F, 15 O and 13 N which emit positrons. The positrons so emitted produce gamma rays upon interaction with electrons present.
  • a placental cDNA library was obtained in the mammalian expression vector pCBM8 and was screened.
  • a cDNA was isolated which expressed protein that exhibited high affinity binding for vgpl20 in the presence of G17-2.
  • gpl20 receptor gpl20r
  • ngpl20 native gpl20
  • FIGURE 1 Binding of labelled gpl20 (1 nM) to the cells was carried out following a 1 hour preincubation of the cells or GP120 at 22°C with one or more of the following: anti-CD4 antibody G17-2 (5 ug/ml), baculoviras-derived gpl20 (bgpl20, American Biotechnologies, 200 nM), anti-g ⁇ l20 monoclonal antibody 110.1 (25 ⁇ g/ml), D-mannose (100 mM), D- galactose (100 mM), L-fucose (100 mM), concanavalin A (1 mg/ml) or pradimicin A (100 ug/ml).
  • anti-CD4 antibody G17-2 5 ug/ml
  • baculoviras-derived gpl20 bgpl20, American Biotechnologies, 200 nM
  • anti-g ⁇ l20 monoclonal antibody 110.1 25 ⁇ g
  • FIGURES 1 (A and B) illustrate that g ⁇ l20 binding to the gpl20r expressed on the cells was blocked by excess bgpl20, mannose, fucose, pradimicin A, Concanavalin A, and preincubation with antibody 110.1 but not by CD4, antibody G17-2, galactose, or HTV antisera. Studies were also carried out on gpl20 binding to CD4 expressing COS cells, transfected with ⁇ H3MCD4 by the method of Peterson et al. (1988) Cell 54:65-72.
  • the placental membranes and COS cells were surface iodinated, and treated with 1 nM unlabelled vgpl20, then washed with Blotto RPMI, 5% BSA, 1% Non-fat dry milk, 0.2% sodium azide solubilized in Triton X-100 (1% in PBS with a protein inhibitor cocktail, PMSF, Pepstatin A, orthophenathroline and leupeptin) and immunoprecipitated with HTV or control human sera, according to the method described in Curtis et al. (1990) J. Immunol. 144:1295-1303.
  • RNA was denatured, separated in an agarose gel, transferred to nitrocellulose, hybridized to gpl20r cDNA and autoradiographed for 3 days.
  • gpl20r RNA Expression of gpl20r RNA was highest in colon followed by thymus, placenta, heart, skeletal muscle, and was not detected in liver or kidney. Low levels of expression in brain, T cell, B cell, and macrophage (FIGURE 2E) require verification by polymerase chain reaction (PCR). Full length CD4 RNA was highest in thymus, T cell, and macrophage followed by placenta and colon (not shown).
  • the gpl20r cDNA encodes a protein of 404 amino acids with a calculated Mr of 45,775 (FIGURE 3A). Sequencing of both strands of gpl20r cDNA was carried out by the dideoxy chain termination method. The nucleotide sequence preceeding the first ATG agrees with the Kozak consensus. The predicted cytoplasmic domain has a similar length and shows some sequence homology to other type ⁇ membrane protein C-type lectins (Spiess, M. (1990) Biochemistry 22:10009-10018). The membrane spanning sequence is underlined and was predicted in part by homology to related sequences in FIGURE 3C. The potential N- linked glycosylation site is marked by an asterisk.
  • the start of the seven complete and eighth partial tandem repeats are indicated (R1-R8).
  • the consensus repeat sequence is TYQELT(R/Q) LKAAVGELPEKSKLQE.
  • the beginning of the lectin domains is also indicated (L).
  • No signal sequence was apparent but instead demonstrated homology to a family of Type ⁇ membrane proteins which utilize a " 20 residue hydrophobic stop-transfer sequence for membrane translocation.
  • the "positive inside rale” von Heijne, G. et al. (1988) Eur. J. Biochem.
  • the second domain (He 77 to Val 249) consists of tandem repeats of nearly identical sequence (FIGURE 3A). This region was predicted to consist of a series of amphipathic ⁇ -helices interrupted by ⁇ -turns. Circular Dichroism spectra in 40% trifluoroethanol of a consensus repeat peptide beginning with the ⁇ -tum, PEKSKLQEIYQELTQLKAAVGEL (single-letter amino-acid code), demonstrated an all ⁇ - helical structure (not shown). Homology to other repeat domains suggested three possible tertiary structures, (1) antiparallel helix bundles, (2) a multimeric parallel helix bundle, and (3) a membrane pore with a hydrophobic exterior and a negatively charged interior.
  • the first two models would function as spacers to separate the lectin domain from the membrane, while the third could generate a transmembrane signal after ligand binding.
  • the third domain (Cys 253 to Ala 404) is homologous to the other known C-type lectins which are type ⁇ membrane proteins (FIGURE 3C). With the exception of the IgEr, these lectins bind terminal D-galactose and D-N-acetylgalactosamine of glycoproteins (Spiess, M. (1990) Biochemistry 22: 10009-10018).
  • Type ⁇ membrane protein C- type lectins Chick hepatic lectin (CHL) (Drickamer, KJ. (1981) Biol. Chem. 256:5827- 5839), low affinity IgE receptor (IgEr) (Kikutani, H. et al. (1986) Cell 47: 657-665), the asialoglycoptorein receptors (human HI and H2 (Spiess, M. et al. (1985) Proc. Natl. Acad. Sci. USA £2:6465-6569) are shown), and the rat Kupffer cell receptor (Hoyle, G.W. et al. (1988) J. Biol. Chem.
  • Mannose binding lectin was one of the eight carbohydrate recognition domains of the human macrophage mannose receptor (Mannr) (Taylor, M.E. et al. (1990) J. Biol. Chem. 265:12156-12162: Ezekowitz, R.A.B. et al. (1990) J. Exp. Med. 172:1785-1794). Residues identical to the gpl20r are boxed. ALIGN scores indicate significant sequence similarity if greater than 3.0. The complete gpl20r sequence was most homologous to the Kupffer cell receptor which has a similar tandem repeat (Hoyle, G.W. et al. (1988) J. Biol. Chem. 262:7487- 7492).
  • gpl20 Human IgE (10 ⁇ g/ml), sialic acid (100 mM), and mannose-6-phosphate (100 mM) had no effect on binding to the gpl20r.
  • the three forms of gpl20 used have different oligosaccaride structures.
  • Bgpl20 contains only high mannose structures (Hsieh, P. et al. (1984) J. Biol. Chem. 252:2375-2382).
  • Vgpl20 has equal proportions of high mannose and complex (Mizuochi, T. et al. (1988) Biochem. J. 254:599-603) similar to ngpl20 which has a greater structural diversity in the complex chains (Geyer, H. et al. (1988) J.
  • gpl20 carbohydrate in HIV infection has been suggested by the ability of plant lectins (Lifson, J. et al. (1986) E. J. Exp. Med. 164:2101-2106) and semm mannose-binding protein (Ezekowitz, R.A.B. et al. (1989) J. Exp. Med. 162:185-196) to block infection, and a proposed role for the macrophage endocytosis receptor in viral attachment (Larking M. et al. (1989) AIDS 3, 793-798).
  • Concanavalin A treatment of gpl20 blocked binding to the gpl20r and CD4 (FIGURE IB), consistent with a steric hindrance of receptor interaction.
  • the antibiotic pradimicin A blocks HTV infection of CD4 positive T cells and this inhibitory effect is prevented by mannan and EGTA (Tanabe- Tochikura. A. et al. (1990) Virology 176:476-473).
  • Pradamicin blocked gpl20 binding to the gpl20 ⁇ and CD4, while mannan and EGTA only inhibited binding to the gpl20r (FIGURE2B).
  • Mannan inhibited "10% of high affinity (nM) gpl20 binding to T cells and macrophages, consistent with gpl20r expression OFIGURE 2E), suggesting that in addition to CD4 the gpl20r may be important for HTV binding and infection.
  • ORGANISM Human immunodeficiency vims type 1
  • AAG GAA CCA AGA CTG CAG CAG CTG GGC CTC CTG GAG GAG GAA CAG CTG 101 Lys Glu Pro Arg Leu Gin Gin Leu Gly Leu Leu Glu Glu Glu Gin Leu 5 10 15 20
  • CAG GAA CAA TCC AGG CAA GAC GCG ATC TAC CAG AAC CTG ACC CAG CTT 293 Gin Glu Gin Ser Arg Gin Asp Ala lie Tyr Gin Asn Leu Thr Gin Leu 70 75 80
  • GGC ACG TGG CAA TGG GTG GAC GGC TCA CCT CTG TTG CCC AGC TTC AAG 1061 Gly Thr Trp Gin Trp Val Asp Gly Ser Pro Leu Leu Pro Ser Phe Lys 325 330 335 340
  • Glu lie Tyr Gin Glu Leu Thr Trp Leu Lys Ala Ala Val Gly Glu Leu 145 150 155 160
  • Lys Ser Lys Gin Gin Glu lie Tyr Gin Glu Leu Thr Arg Leu Lys Ala 210 215 220
  • Cys Asn Leu Ala Lys Phe Trp lie Cys Lys Lys Ser Ala Ala Ser Cys 370 375 380
  • ORGANISM Human immunodeficiency vims type 1
  • Lys Glu Val Gly Ala Gin Leu Val Val lie Lys Ser Ala Glu Glu Gin 35 40 45
  • Cys Asn Thr Cys Pro Glu Lys Trp lie Asn Phe Gin Arg Lys Cys Tyr 1 5 10 15
  • Lys Phe lie Val Gin His Thr Asn Pro Phe Asn Thr Trp lie Gly Leu 50 55 60
  • Trp He Gly Leu Asn Asp He Lys He Gin Met Tyr Phe Glu Trp Ser 65 70 75 ' 80

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Abstract

On a isolé un récepteur non CD4-gp120 spécifique possédant une affinité de liaison spécifique envers la protéine de surface gp120 présente dans le virus du syndrome immunodéficitaire acquis (HIV). On décrit des méthodes de traitement de l'infection HIV dans les cellules négatives CD4, telles que celles du côlon et du cerveau, ainsi que des procédés de détection de l'HIV et des trousses diagnostic.
PCT/US1992/005985 1991-07-16 1992-07-16 Inhibition de l'infection hiv a mediation par la proteine non-cd4 WO1993001820A2 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041884A1 (fr) * 1995-06-09 1996-12-27 Institut Français De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) MOYEN POUR DETECTER ET PREVENIR UNE INFECTION A VIH, FAISANT APPEL A DES RECEPTEURS OU A DES SITES DE FIXATION CAPABLES D'INTERAGIR AVEC gp120
FR2748483A1 (fr) * 1996-05-07 1997-11-14 Orstom Moyens pour la lutte ou le diagnostic d'une infection par hiv
EP1046651A1 (fr) * 1999-04-19 2000-10-25 Koninklijke Universiteit Nijmegen Composition et méthode pour moduler l'interaction des cellules dendritiques et les cellules T
WO2001019869A1 (fr) * 1999-09-13 2001-03-22 The Council Of The Queensland Institute Of Medical Research Proteine cire de membrane de cellules dendritiques
WO2004041299A1 (fr) * 2002-11-05 2004-05-21 Institut Pasteur Inhibiteurs de dc-sign et leur utilisation dans la prevention ou le traitement d'infections virales
WO2006055711A1 (fr) * 2004-11-18 2006-05-26 Kent Hann Composition contenant un isolat d'aloe vera et un prebiotique synergique et leur application therapeutique
US7427469B2 (en) 2002-11-05 2008-09-23 Institut Pasteur Method of treating cytomegalovirus with DC-SIGN blockers
WO2008095905A3 (fr) * 2007-02-05 2008-10-30 Iti Scotland Ltd Liaison d'agents pathogènes
US7541032B2 (en) 2002-09-20 2009-06-02 Stichting Katholieke Universiteit Antigen uptake receptor for Candida albicans on dendritic cells
US7691591B2 (en) 2002-09-20 2010-04-06 Stichting Katholieke Universiteit Methods of identifying and isolating cells expressing DC-sign
EP2241331A2 (fr) 2003-12-15 2010-10-20 Alexion Pharmaceuticals, Inc. Nouveaux anticorps anti-signe DC

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AIDS vol. 3, no. 12, 1989, pages 793 - 798 LARKIN. M. ET AL.; 'Oligosaccharide-mediated interactions of the envelope glycoprotein gp120 of HIV-1 that are independent of CD4 recognition"' cited in the application *
BULL. INST. PASTEUR vol. 89, no. 2, June 1991, IZRACHI, Y. ET AL.; 'Efficient binding , fusion and entry of HIV1 into CD4 negative neural cells : a mechanism for neuropathogenesis in Aids.' *
JOURNAL OF MEDICAL VIROLOGY vol. 35, no. 3, November 1991, pages 187 - 191 MORRISSON, N.K. ET AL.; 'Growth of human immunodeficiency virus 1 in cultured cells in the absence of the CD4 antigen."' *
NATURE. vol. 337, 1989, LONDON GB pages 368 - 370 CLAPHAM, P.R. ET AL.; 'Soluble CD4 blocks the infectivity of diverse strains of HIV and SIV for T cells and monocytes but not for brain and muscle cells.' cited in the application *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA. vol. 89, September 1992, WASHINGTON US pages 8356 - 8360 Curtis, Benson M.; Scharnowske, S.; Watson, A.J.; 'Sequence and expression of a membrane-associated C-type lectin that exhibits CD4-independent binding of human immunodeficiency virus envelope glycoprotein gp120.' *
THE JOURNAL OF EXPERIMENTAL MEDECINE vol. 169, January 1989, THE ROCKEFELLER UNIV. PRESS pages 185 - 196 EZEKOWITZ, R.A.B. ET AL.; 'A human serum mannose binding protein inhibits in vitro infection by the human immunodeficeincy virus.' *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041884A1 (fr) * 1995-06-09 1996-12-27 Institut Français De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) MOYEN POUR DETECTER ET PREVENIR UNE INFECTION A VIH, FAISANT APPEL A DES RECEPTEURS OU A DES SITES DE FIXATION CAPABLES D'INTERAGIR AVEC gp120
AU714902B2 (en) * 1995-06-09 2000-01-13 Institut Francais De Recherche Scientifique Pour Le Developpement En Cooperation (Orstom) Means for detecting and preventing HIV infection involving use of receptors or binding sites capable of interacting with GP120
FR2748483A1 (fr) * 1996-05-07 1997-11-14 Orstom Moyens pour la lutte ou le diagnostic d'une infection par hiv
EP1516881A2 (fr) 1999-04-19 2005-03-23 Katholieke Universiteit Nijmegen Composition et méthode pour moduler l'interaction des cellules dendritiques et les cellules T
EP1046651A1 (fr) * 1999-04-19 2000-10-25 Koninklijke Universiteit Nijmegen Composition et méthode pour moduler l'interaction des cellules dendritiques et les cellules T
US8058400B2 (en) 1999-04-19 2011-11-15 Katholieke Universiteit Nijmegen Composition and method for modulating dendritic cell-t cell interaction
US8105599B2 (en) 1999-04-19 2012-01-31 Katholieke Universiteit Nijmegen Composition and method for modulating dendritic cell-T cell interaction
AU776317B2 (en) * 1999-04-19 2004-09-02 Katholieke Universiteit Nijmegen Composition and method for modulating dendritic cell-t cell interaction
US7285642B2 (en) 1999-04-19 2007-10-23 Katholieke Universiteit Nijmegen Composition and method for modulating dendritic cell-T cell interaction
EP1516881A3 (fr) * 1999-04-19 2005-08-31 Katholieke Universiteit Nijmegen Composition et méthode pour moduler l'interaction des cellules dendritiques et les cellules T
WO2000063251A1 (fr) * 1999-04-19 2000-10-26 Katholieke Universiteit Nijmegen Composition et methode permettant de moduler l'interaction entre cellules dendritiques et lymphocytes t
US7148329B1 (en) 1999-04-19 2006-12-12 Katholieke Universiteit Nijmegen Composition and method for modulating dendritic cell-t cell interaction
WO2001019869A1 (fr) * 1999-09-13 2001-03-22 The Council Of The Queensland Institute Of Medical Research Proteine cire de membrane de cellules dendritiques
US7541032B2 (en) 2002-09-20 2009-06-02 Stichting Katholieke Universiteit Antigen uptake receptor for Candida albicans on dendritic cells
US7691591B2 (en) 2002-09-20 2010-04-06 Stichting Katholieke Universiteit Methods of identifying and isolating cells expressing DC-sign
US7419789B2 (en) 2002-11-05 2008-09-02 Institut Pasteur Method of inhibiting binding of Dengue virus to a human cell with DC-SIGN blockers
US7427469B2 (en) 2002-11-05 2008-09-23 Institut Pasteur Method of treating cytomegalovirus with DC-SIGN blockers
WO2004041299A1 (fr) * 2002-11-05 2004-05-21 Institut Pasteur Inhibiteurs de dc-sign et leur utilisation dans la prevention ou le traitement d'infections virales
EP2241331A2 (fr) 2003-12-15 2010-10-20 Alexion Pharmaceuticals, Inc. Nouveaux anticorps anti-signe DC
WO2006055711A1 (fr) * 2004-11-18 2006-05-26 Kent Hann Composition contenant un isolat d'aloe vera et un prebiotique synergique et leur application therapeutique
JP2010518046A (ja) * 2007-02-05 2010-05-27 アイティーアイ・スコットランド・リミテッド 病原体の結合
WO2008095905A3 (fr) * 2007-02-05 2008-10-30 Iti Scotland Ltd Liaison d'agents pathogènes

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IE922308A1 (en) 1993-01-27
AU2373792A (en) 1993-02-23
ZA925305B (en) 1993-06-14
MX9204131A (es) 1993-04-01

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