WO1993001820A2 - Inhibition of non-cd4 mediated hiv infection - Google Patents

Abstract

A specific nonCD4-gp120 receptor has been isolated which has specific binding affinity for the gp120 surface protein of human immunodeficiency virus (HIV). Methods of treating HIV infection of CD4 negative cells, such as colon and brain, are disclosed together with methods of detecting HIV and diagnostic kits.

Description

INHIBITION OF NON-CD4 MEDIATED HIV INFECTION

TECHNICAL FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Two types of human retroviruses 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. The human retroviruses first discovered, Human T Lymphotropic Virus Type 1 (HTLV-1) and Human T Lymphotropic Virus Type H (HTLV-II), have a preferential tropism for T4 cells and some T8 cells, share significant sequence homology, and are mainly associated with T cell leukemias and lymphomas. The other group of human retroviruses, generally called Human Immunodeficiency Viruses (HIV), is discussed in greater detail below. There are two major differences between the two types of human retroviruses: (1) there is substantial genomic variability among various HTV isolates, whereas the genomes of HTLV-I and HTLV-π are stable; and (2) HIV entered human populations much more recently than HTLV-I or HTLV-π.

The human immunodeficiency virus (HTV) is a cytopathic retrovirus and the causative agent of the acquired immunodeficiency syndrome (AIDS). Two forms of HIV have now been identified. The prototype virus, HIV-1, previously termed lymphadenopathy-associated virus (LAV) and Human T Lymphotropic Virus Type HI (HTLV-πi), is responsible for the vast majority of reported AIDS cases worldwide. 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.

Over half of the people that have contracted AIDS in the United States have already died. As many as three million persons in this country may be asymptomatic carriers of HIV and are capable of transmitting the virus. It had been estimated in 1986 that 270,000 cases of AIDS will have occurred in the United States by 1991 (U.S. Public Health Service, (1986), Public Health Rep. 1HL:341). The mortality rate from AIDS is disturbingly high, exceeding 80% within three years of diagnosis and possibly reaching 100% over a longer period.

Worldwide, the AIDS epidemic may involve some five to ten million presently infected persons. Particularly troublesome are statistics from the African continent where millions of individuals are believed infected with HIV, deaths range in the hundreds of thousands, and heterosexual transmission predominates. To date, there is neither a known cure for ALDS nor an effective vaccine against HIV infection.

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.

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.

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).

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. However, it has been observed that 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).

The symptoms of ADC include cognitive impairment, apathy and motor dysfunctions, and may affect as many as 90% of AIDS victims. 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).

An early study suggested that the cellular attachment site for HIV in brain might be CD4 (Pert et al., (1986) Proc. Nail. Acad. Sci. USA £3_:9254-9258) but attempts to replicate these findings were not successful (Kozlowski et al., (1989) Neurosci. Abstr. 15_:671). It now appears unlikely that the CD4 antigen is involved in the infection of brain-derived cells by HIV. Susceptibility of brain cells to infection with HIV-1 does not correlate with the level of expression of CD4 (Chang-Mayer et al., (1987) Proc. Natl. Acad. Sci. USA £4:3526-3530; Srinivasan et al., (1988) Arch. Virol. 9_£: 135-141), and infection of brain-derived cells by HIV-1 is not blocked by anti-CD4 antibodies (Clapham et al. , (1989) Nature 3J7:368-370; Li et al. , (1990) J. Virol. £4: 1383-1387).

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.

SUMMARY OF THE INVENTION

Many cells that are susceptible to HTV infection appear to bind gpl20 through a non-CD4 surface protein. The present invention has identified this non-CD4 gpl20 receptor (gpl20r) and has recombinantly expressed and characterized gpl20r. In this invention a specific non-CD4 gpl20r has been isolated which has specific binding activity for gpl20 present on Human Immunodeficiency Virus-1 (HTV). 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. 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. i one embodiment of the present invention, 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. In this method, 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. Specific inhibitors 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. In this method, the binding of HTV to gpl20r is detected by an indicating means such as a labelled antibody capable of binding to the HTV-gpl20τ reaction product. It is also contemplated that the gpl20r can be affixed to a solid matrix to form a solid support that is useful in this method and/or kit.

DESCRIPTION OF THE FIGURES

In the drawings:

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 [¹²⁵T]vgpl20; A, gpl20τ; 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 [¹²⁵T]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 [¹²⁵TJvgpl20 binding to gpl20r and CD4. A-F gpl20r and G-

L CD4. A+G, HTV antisera (1:20; Trimar); B+H, D-galactose (100 mM); C+I, D-mannose (100 mM); D+J, L-fiicose (100 mM); E+K, Concanavalin A (1 mg/ml); F+L, pradimicin A (100 xg/ml). C: gpl20r binding of HIV. A, HIV; B, HIV with 200 nM bgpl20.

FIGURE 2 illustrates the characterization of the gpl20r.

125 A: Scatchard analysis of [ TJgpl20 binding. A - ▲, vgpl20 binding to placenta, Kd 1.3 nM, Bma 19 fmol/mg protein; ■ with μg/ml G17-2; • - #, vgρl20 binding to gpl20r COS cells, Kd 1.7 nM, Bmax 150,000 receptors/cell (R/C); O, ngpl20, Kd 1.8 nM, 149,000 R/C. B: Inhibition of [¹²⁵IJgpl20 binding to gpl20r COS cells. Open symbols ngpl20, filled symbols vgpl20. The relative values were the same with both forms of gpl20. Mannan expressed as mg/ml. □, mannan (IC50 6 μg/ml); •, L-fucose (K . 6 mM); Δ, α-methyl D-mannoside (K. 15 mM),

O, D-mannose (K. 23 mM); , N-acetylglucosamine (K. 70 mM), ■,

EGTA (K . 0.3 mM). C: Intemalization of gpl20 by gpl20r COS cells. Points represent the mean of two experiments with vgpl20 and ngpl20. • - #, suface; O - O internal.

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 μg A+) 14, B cell (TS-1; 16 μg A+); 15, macrophage (U937; 8 μg A⁺); 16, cervical carcinoma (HeLa; 16 μg 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.

C: Aminoacid alignment of the gpl20r C-type lectin domain.

DESCRIPTION OF PREFERRED EMBODIMENTS

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. ϋ>4:2101-2106; Ezekowitz, R.A.B. et al., (1989) J. Exp. Med. 161:185-196; Larkin M. et al., (1989) AIDS 2: 793-798; Tanabe-Tochikura A. et al., (1990) Virology 176:473-476), suggest a potential role for the gpl20τ in HTV infection or pathology. The present invention demonstrates that the 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 tτansplacental transport of HTV (Zacher, V. et al., (1991) J. Virol. £5_:2102-2107) and colon infection. (Barnett, S.W. et al. (1991) Virol. 181:802-809). Gρl20 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. i the present invention, 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 cloned gpl20r of the present invention binds gpl20 with an affinity (Kd) of about 1 to 2 nM, which is considerably greater than the affinity of CD4 for gpl20 (about Kd = 4 nM).

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 gpl20τ 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 gpl20τ 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.

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.

In the present invention a cDNA which encodes gpl20 was isolated and cloned. 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. 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.

As used herein, the phrases "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.

For therapeutic use, 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. In these compositions, the agent is typically dissolved or dispersed in a physiologically tolerable carrier.

As an example, 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. Particularly well suited for the present purposes are 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. As is known in the art, 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.

Methods to form liposomes are known in the art. See, for example, Prescott, Ed., "Methods in Cell Biology". Volume XIV, Academic Press, New York, N.Y. (1976) p 33 et seq.

The present compounds can also be used in compositions such as tablets or pills, preferably contøining a unit dose of the compound. To this end, the agent (active ingredient) is mixed with 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.

It should be understood that in addition to the aforementioned carrier ingredients 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.

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. A variety of materials can be used for such 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. Those skilled in the art will recognize that there are well known clinical diagnostic procedures that can be utilized for the formulation and detection of such reaction products. Thus, while exemplary assay methods are described herein, the invention is not intended to be so limited.

Various heterogeneous and homogeneous assay protocols can be employed for detecting the presence, and preferably the amount, of HTV in a fluid sample. For example, the present invention contemplates a method for assaying a sample, such as a body fluid, for the presence of HTV comprising the steps of:

(a) admixing a fluid sample with gpl20r, either in solution or affixed to a solid matrix;

(b) maintaining the admixture for a predetermined time period such as about 10 minutes to about 16 - 20 hours and under biological assay conditions at a temperature of about 4°C to about 45°C that is sufficient for any HTV present in the sample to react with (bind) the gpl20r to form a reaction product; and

(c) determining the presence of any reaction product that is formed, and thereby the presence of any HTV in the admixture. Preferably, the fluid sample is a body fluid sample, such as blood, plasma, serum, urine, saliva, semen or cerebrospinal fluid (CSF).

The determination of the presence of a reaction product, either directly or indirectly, can be accomplished by assay techniques well known in the art such as by the use of an indicating or labelling means, as discussed hereinbelow. In a preferred embodiment, a labelled indicating means, such as a fluorescein-labelled antibody, 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. In particularly preferred embodiments, 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.

In a preferred embodiment, the gpl20r is affixed on a solid matrix to form a solid phase support. In that embodiment, 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.

As used herein, the term "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. As used herein, 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. As used herein, the term "corresponds", and its various grammatical modifications, means "is similar or in agreement with".

A diagnostic system in kit form for assaying a fluid sample for the presence of HIV is also contemplated by the present invention. Such a kit includes, in an amount sufficient for at least one assay, gpl20τ 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.

As used herein, 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.

The 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.

As used herein, 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. In one embodiment, 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.

As used herein, the terms "label" "indicating means" and "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. 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 & Sons, Ltd., pp. 189-231, which is incorporated herein by reference.

Other preferred 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. Elements

124 125 128 132 51 which emit 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.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

EXAMPLE 1

Cloning and Isolation of Non-CD4 Gpl40 Receptor Protein

Human placental membranes were found to be able to bind vaccinia derived recombinant gpl20 (vgpl20) with a Kd of 1.3 nM. At nM (concentrations) of gpl20 none of this binding was inhibited by an antibody (G17-2) which has been reported to efficiently block gpl20 binding to CD4 (Linsley et al. (1988) J. Virol. £2:3695-3702), as shown in HGURE 2A. Approximately 50 - 90% of the total placental gpl20 binding was not due to CD4.

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.

This protein, designated as gpl20 receptor (gpl20r), also bound native gpl20 (ngpl20), and the binding component was precipitated in the presence of an antibody directed against gpl20.

EXAMPLE 2

Characterization

The binding of radiolabelled gpl20 to gpl20r expressed in COS-7 cells was studied. Pools of 90,000 cDNA molecules, obtained from a placental pCDM8 library, were transfected by electroporation into COS-7 cells. Cells which expressed gpl20r on the surface was identified by screening with either 1 nM of 125 I-labelled vgpl20 ( 125 I- vgρl20) or ^JI-ngpl20 by the method described in Kozlowski et al., (1990) Antivir.

Chem. Chemother. 1:175-182, incorporated herein by reference. The results of binding studies utilizing the transfected COS-7 cells are shown in 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-gρl20 monoclonal antibody 110.1 (25 μg/ml), D-mannose (100 mM), D- galactose (100 mM), L-fucose (100 mM), concanavalin A (1 mg/ml) or pradimicin A (100 ug/ml). The cells were monitored after autoradiography (3 days). The results seen in FIGURES 1 (A and B) illustrate that gρl20 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 π H3MCD4 by the method of Peterson et al. (1988) Cell 54:65-72. Control studies of the binding of 125 I-labelled psoralen-UV inactivated HTV-BRU to the gpl20r expressing COS-7 cells demonstrated binding of HIV to gpl20r and blockage by excess bgpl20 (FIGURE IC). A tabular compilation quantitating the amount of bound material to the cells in FIGURE 1 is shown in Table. 1

Scatchard plots of gpl20 binding to placental membranes and to COS cells expressing the gpl20r were carried out in the presence and absence of a 200 fold excess of bgpl20 or ngpl20. The results, shown in FIGURE 2A, demonstrate a specific binding of vgpl20 to gρl20r with a Kd of 1.7 nM ± 0.4 (n=4) and of ngpl20 to gpl20r with Kd of 1.8 nM ± 0.2 (n=4), with 150,000 and 149,000 receptors per cell, respectively. Concurrent analysis of gpl20 binding to CD4 expressed on COS cells gave a Kd of 4-5 nM in agreement with previous reports (Linsley, P.S. et al. (1988) J. Virol 62:3695-3702; Schnittman, et al. (1988) J. Immunol. 141:4181-4186). Calculations from the association and dissociation rate constants gave a similar comparative result. The expressed gpl20r has a relative molecular mass (Mr) of "48,500 and a protein of similar size was also partially purified from placental membranes (FIGURE 2D).

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.

Northern analysis of the expression of the gpl20r RNA indicated a major species of

"5 kb and a minor species of ^"1.7 kb which may represent an alternatively processed transcript and is more consistent with the size of the gpl20r cDNA. RNA was denatured, separated in an agarose gel, transferred to nitrocellulose, hybridized to gpl20r cDNA and autoradiographed for 3 days.

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 π 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 π 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. 174:671-678') for the sequence within fifteen residues of the transmembrane region predicts a cytoplasmic amino terminus in agreement with the homology to membrane associated C-type lectins with similar membrane orientation (FIGURE 3C) (Spiess, M. (1990) Biochemistry 29_: 10009-10018). This region, Met 1 to Ala 76, represents the first domain of the gpl20r sequence.

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 α-helices interrupted by β-turns. Circular Dichroism spectra in 40% trifluoroethanol of a consensus repeat peptide beginning with the β-tum, PEKSKLQEIYQELTQLKAAVGEL (single-letter amino-acid code), demonstrated an all α- 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 π 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).

The most closely related sequences were the group of Type π 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. 262:7487-7492). The most similar 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).

The inability to crosslink gpl20 to the non-CD4 sites on placenta and brain cell lines (not shown) was consistent with an interaction of the gpl20r with carbohydrate, and polyclonal HTV antisera added to gpl20 blocked binding to CD4 but not to the gpl20r (FIGUKE IB). Galactose and N-acetylgalactosamine did not block gpl20 binding, but mannose and fucose completely blocked binding to the gpl20r without an effect on CD4 (FIGURE IB). Inhibition by a series of sugars is shown in FIGURE 2B. Human IgE (10 μg/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. Biol. Chem. 262:11760-11767; Mizuochi, T. et al. (1990) J. Biol. Chem. 265:8519-8524). The affinity of the gpl20r for all three forms was similar (FIGURE 2A ) suggesting that the teπninal mannose of high mannose chains are the primary determinants of binding. As expected for a C-type lectin the gpl20r required calcium and binding was blocked by EGTA (FIGURE 2B). The gpl20r carbohydrate specificity is more closely related to semm mannose binding proteins and to the Mr 175,000 mannose-specific endocytosis receptor found in macrophages and placenta (Taylor, M.E. et al. (1990) J. Biol. Chem. 265:12156-12162; Ezekowitz, R.A.B. et al. (1990) J. Exp. Med. 122:1785-1794) (FIGURE 3C). Low (1 nM) concentrations of gpl20 did not purify a Mr 175,000 band from placental membranes (FIGURE 2D) consistent with a reported concentration of 150- 300 nM for gpl20 saturation of the macrophage receptor (Larkin, M. et al. (1989) AIDS 3, 793-798).

The importance of 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 gpl20τ 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. The observation the the gpl20r rapidly internalized its bound ligand gpl20 (FIGURE 2C), and also binds radiolabelled HTV in a gpl20 dependent fashion (HGURE IC) also support this conclusion. The foregoing description and Examples are intended as illustrative of the present invention, but not as limiting. Numerous variations and modifications may be effected without departing from the true spirit and scope of the present invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Curtis, Benson

(ii) TITLE OF INVENTION: INHIBITION OF NON-CD4 MEDIATED

HIV INFECTION

(iii) NUMBER OF SEQUENCES: 9

(iv) CORRESPONDENCE ADDRESS:

(v)

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US UNKNOWN

(B) FILING DATE: ll-JUL-1991

(C) CLASSIFICATION:

(viii) ATTORNEY/ AGENT INFORMATION:

(A) NAME: Sorrentino, Joseph M.

(B) REGISTRATION NUMBER: 32,598

(C) REFERENCE/DOCKET NUMBER: ON0086-

(ix) TELECOMMUNICATION INFORMAΗON:

(A) TELEPHONE: (206) 728-4800

(B) TELEFAX: (206) 448-4775

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1312 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human immunodeficiency vims type 1

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 42..1253

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CTAAAGCAGG AGTTCTGGAC ACTGGGGGAG AGTGGGGTGA C ATG AGT GAC TCC 53

Met Ser Asp Ser

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

AGA GGC CTT GGA TTC CGA CAG ACT CGA GGA TAC AAG AGC TTA GCA GGG 149 Arg Gly Leu Gly Phe Arg Gin Thr Arg Gly Tyr Lys Ser Leu Ala Gly 25 30 35

TGT CTT GGC CAT GGT CCC CTG GTG CTG CAA CTC CTC TCC TTC ACG CTC 197 Cys Leu Gly His Gly Pro Leu Val Leu Gin Leu Leu Ser Phe Thr Leu 40 45 50

TTG GCT GGG CTC CTT GTC CAA GTG TCC AAG GTC CCC AGC TCC ATA AGT 245 Leu Ala Gly Leu Leu Val Gin Val Ser Lys Val Pro Ser Ser lie Ser 55 60 65

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

AAA GCT GCA GTG GGT GAG CTC TCA GAG AAA TCC AAG CTG CAG GAG ATC 341 Lys Ala Ala Val Gly Glu Leu Ser Glu Lys Ser Lys Leu Gin Glu lie 85 90 95 100

SUBSTITUTE SHEET TAC CAG GAG CTG ACC CAG CTG AAG GCT GCA GTG GGT GAG CTT CCA GAG 389 Tyr Gin Glu Leu Thr Gin Leu Lys Ala Ala Val Gly Glu Leu Pro Glu 105 110 115

AAA TCT AAG CTG CAG GAG ATC TAC CAG GAG CTG ACC CGG CTG AAG GCT 437 Lys Ser Lys Leu Gin Glu lie Tyr Gin Glu Leu Thr Arg Leu Lys Ala 120 125 130

GCA GTG GGT GAG CTT CCA GAG AAA TCT AAG CTG CAG GAG ATC TAC CAG 485 Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gin Glu lie Tyr Gin 135 140 145

GAG CTG ACC TGG CTG AAG GCT GCA GTG GGT GAG CTT CCA GAG AAA TCT 533 Glu Leu Thr Trp Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser 150 155 160

AAG ATG CAG GAG ATC TAC CAG GAG CTG ACT CGG CTG AAG GCT GCA GTG 581 Lys Met Gin Glu lie Tyr Gin Glu Leu Thr Arg Leu Lys Ala Ala Val 165 170 175 180

GGT GAG CTT CCA GAG AAA TCT AAG CAG CAG GAG ATC TAC CAG GAG CTG 629 Gly Glu Leu Pro Glu Lys Ser Lys Gin Gin Glu lie Tyr Gin Glu Leu 185 190 195

ACC CGG CTG AAG GCT GCA GTG GGT GAG CTT CCA GAG AAA TCT AAG CAG 677 Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gin 200 205 210

CAG GAG ATC TAC CAG GAG CTG ACC CGG CTG AAG GCT GCA GTG GGT GAG 725 Gin Glu lie Tyr Gin Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu 215 220 225

CTT CCA GAG AAA TCT AAG CAG CAG GAG ATC TAC CAG GAG CTG ACC CAG 773 Leu Pro Glu Lys Ser Lys Gin Gin Glu lie Tyr Gin Glu Leu Thr Gin 230 235 240

CTG AAG GCT GCA GTG GAA CGC CTG TGC CAC CCC TGT CCC TGG GAA TGG 821 Leu Lys Ala Ala Val Glu Arg Leu Cys His Pro Cys Pro Trp Glu Trp 245 250 255 260

ACA TTC TTC CAA GGA AAC TGT TAC TTC ATG TCT AAC TCC CAG CGG AAC 869 Thr Phe Phe Gin Gly Asn Cys Tyr Phe Met Ser Asn Ser Gin Arg Asn 265 270 275

TGG CAC GAC TCC ATC ACC GCC TGC AAA GAA GTG GGG GCC CAG CTC GTC 917 Trp His Asp Ser lie Thr Ala Cys Lys Glu Val Gly Ala Gin Leu Val 280 285 290

GTA ATC AAA AGT GCT GAG GAG CAG AAC TTC CTA CAG CTG CAG TCT TCC 965 Val lie Lys Ser Ala Glu Glu Gin Asn Phe Leu Gin Leu Gin Ser Ser 295 300 305

SUBSTITUTE SHEET AGA AGT AAC CGC TTC ACC TGG ATG GGA CTT TCA GAT CTA AAT CAG GAA 1013 Arg Ser Asn Arg Phe Thr Trp Met Gly Leu Ser Asp Leu Asn Gin Glu 310 315 320

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

CAG TAT TGG AAC AGA GGA GAG CCCAAC AAC GTT GGG GAG GAA GAC TGC 1109 Gin Tyr Trp Asn Arg Gly Glu Pro Asn Asn Val Gly Glu Glu Asp Cys 345 350 355

GCG GAA TTT AGT GGC AAT GGC TGG AAC GAC GAC AAA TGT AAT CTT GCC 1157 Ala Glu Phe Ser Gly Asn Gly Trp Asn Asp Asp Lys Cys Asn Leu Ala 360 365 370

AAA TTC TGG ATC TGC AAA AAG TCC GCA GCC TCC TGC TCC AGG GAT GAA 1205 Lys Phe Trp lie Cys Lys Lys Ser Ala Ala Ser Cys Ser Arg Asp Glu 375 380 385

GAA CAG TTT CTT TCT CCA GCC CCT GCC ACC CCA AAC CCC CCT CCT GCG 1253 Glu Gin Phe Leu Ser Pro Ala Pro Ala Thr Pro Asn Pro Pro Pro Ala 390 395 400

TAGCAGAACT TCACCCCCTT TTAAGCTACA GTTCCTTCTC TCCATCCTTC GACCTTTAG 1312

(2) INFORMAΗON FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 404 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ser Asp Ser Lys Glu Pro Arg Leu Gin Gin Leu Gly Leu Leu Glu 1 5 10 15

Glu Glu Gin Leu Arg Gly Leu Gly Phe Arg Gin Thr Arg Gly Tyr Lys 20 25 30

Ser Leu Ala Gly Cys Leu Gly His Gly Pro Leu Val Leu Gin Leu Leu 35 40 45

Ser Phe Thr Leu Leu Ala Gly Leu Leu Val Gin Val Ser Lys Val Pro 50 55 60

SUBSTITUTE SHEET Ser Ser lie Ser Gin Glu Gin Ser Arg Gin Asp Ala lie Tyr Gin Asn 65 70 75 80

Leu Thr Gin Leu Lys Ala Ala Val Gly Glu Leu Ser Glu Lys Ser Lys 85 90 95

Leu Gin Glu lie Tyr Gin Glu Leu Thr Gin Leu Lys Ala Ala Val Gly 100 105 110

Glu Leu Pro Glu Lys Ser Lys Leu Gin Glu lie Tyr Gin Glu Leu Thr 115 120 125

Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gin 130 135 140

Glu lie Tyr Gin Glu Leu Thr Trp Leu Lys Ala Ala Val Gly Glu Leu 145 150 155 160

Pro Glu Lys Ser Lys Met Gin Glu lie Tyr Gin Glu Leu Thr Arg Leu 165 170 175

Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gin Gin Glu lie 180 185 190

Tyr Gin Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu 195 200 205

Lys Ser Lys Gin Gin Glu lie Tyr Gin Glu Leu Thr Arg Leu Lys Ala 210 215 220

Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Gin Gin Glu lie Tyr Gin 225 230 235 240

Glu Leu Thr Gin Leu Lys Ala Ala Val Glu Arg Leu Cys His Pro Cys 245 250 255

Pro Trp Glu Trp Thr Phe Phe Gin Gly Asn Cys Tyr Phe Met Ser Asn 260 265 270

Ser Gin Arg Asn Trp His Asp Ser lie Thr Ala Cys Lys Glu Val Gly 275 280 285

Ala Gin Leu Val Val lie Lys Ser Ala Glu Glu Gin Asn Phe Leu Gin 290 295 300

Leu Gin Ser Ser Arg Ser Asn Arg Phe Thr Trp Met Gly Leu Ser Asp 305 310 315 320

Leu Asn Gin Glu Gly Thr Trp Gin Trp Val Asp Gly Ser Pro Leu Leu 325 330 335

Pro Ser Phe Lys Gin Tyr Trp Asn Arg Gly Glu Pro Asn Asn Val Gly 340 345 350

SUBSTITUTE SHEET Glu Glu Asp Cys Ala Glu Phe Ser Gly Asn Gly Trp Asn Asp Asp Lys 355 360 365

Cys Asn Leu Ala Lys Phe Trp lie Cys Lys Lys Ser Ala Ala Ser Cys 370 375 380

Ser Arg Asp Glu Glu Gin Phe Leu Ser Pro Ala Pro Ala Thr Pro Asn 385 390 395 400

Pro Pro Pro Ala

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 127 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human immunodeficiency vims type 1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Cys His Pro Cys Pro Trp Glu Trp Thr Phe Phe Gin Gly Asn Cys Tyr 1 5 10 15

Phe Met Ser Asn Ser Gin Arg Asn Trp His Asp Ser lie Thr Ala Cys 20 25 30

Lys Glu Val Gly Ala Gin Leu Val Val lie Lys Ser Ala Glu Glu Gin 35 40 45

Asn Phe Leu Gin Leu Gin Ser Ser Arg Ser Asn Arg Phe Thr Trp Met 50 55 60

Gly Leu Ser Asp Leu Asn Gin Glu Gly Thr Trp Gin Trp Val Asp Gly 65 70 75 80

Ser Pro Leu Leu Pro Ser Phe Lys Gin Tyr Trp Asn Arg Gly Glu Pro 85 90 95

SUBSTITUTE SHEET Asn Asn Val Gly Glu Glu Asp Cys Ala Glu Phe Ser Gly Asn Gly Trp 100 105 110

Asn Asp Asp Lys Cys Asn Leu Ala Lys Phe Trp lie Cys Lys Lys 115 120 125

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Cys Gly Ala Gin Ser Arg Gin Trp Glu Tyr Phe Glu Gly Arg Cys Tyr 1 5 10 15

Tyr Phe Ser Leu Ser Arg Met Ser Trp His Lys Ala Lys Ala Glu Cys 20 25 30

Glu Glu Met His Ser His Leu lie lie lie Asp Ser Tyr Ala Lys Gin 35 40 45

Asn Phe Val Met Phe Arg Thr Arg Asn Glu Arg Phe Trp lie Gly Leu 50 55 60

Thr Asp Glu Asn Gin Glu Gly Glu Trp Gin Trp Val Asp Gly Thr Asp 65 70 75 80

Thr Arg Ser Ser Phe Thr Phe Trp Lys Glu Gly Glu Pro Asn Asn Arg 85 90 95

Gly Phe Asn Glu Asp Cys Ala His Val Trp Thr Ser Gly Gin Trp Asn 100 105 110

Asp Val Tyr Cys Thr Tyr Glu Cys Tyr Tyr Val Cys Glu Lys 115 120 125

(2) INFORMATION FOR SEQ ID NO: 5:

SUBSTITUTE SHEET (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 125 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Cys Asn Thr Cys Pro Glu Lys Trp lie Asn Phe Gin Arg Lys Cys Tyr 1 5 10 15

Tyr Phe Gly Lys Gly Thr Lys Gin Trp Val His Ala Arg Tyr Ala Cys 20 25 30

Asp Asp Met Glu Gly Gin Leu Val Ser lie His Ser Pro Glu Glu Gin 35 40 45

Asp Phe Leu Thr Lys His Ala Ser His Thr Gly Ser Trp lie Gly Leu 50 55 60

Arg Asn Leu Asp Leu Lys Gly Glu Phe lie Trp Val Asp Gly Ser His 65 70 75 80

Val Asp Tyr Ser Asn Trp Ala Pro Gly Glu Pro Thr Ser Arg Ser Gin 85 90 95

Gly Glu Asp Cys Val Met Met Arg Gly Ser Gly Arg Trp Asn Asp Ala 100 105 110

Phe Cys Asp Arg Lys Leu Gly Ala Trp Val Cys Asp Arg 115 120 125

(2) INFORMAΗON FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

SUBSTITUTE SHEET (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Arg Thr Cys Cys Pro Val Asn Trp Val Glu His Glu Arg Ser Cys Tyr 1 5 10 15

Trp Phe Ser Arg Ser Gly Lys Ala Trp Ala Asp Ala Asp Asn Tyr Cys 20 25 30

Arg Leu Glu Asp Ala His Leu Val Val Val Thr Ser Trp Glu Glu Gin 35 40 45

Lys Phe Val Gin His His lie Gly Pro Val Asn Thr Trp Met Gly Leu 50 55 60

His Asp Gin Asn Gly Pro Trp Lys Trp Val Asp Gly Thr Asp Tyr Glu 65 70 75 80

Thr Gly Phe Lys Asn Trp Arg Pro Glu Gin Pro Asp Asp Trp Tyr Gly 85 90 95

His Gly Leu Gly Gly Gly Glu Asp Cys Ala His Phe Thr Asp Asp Gly 100 105 110

Arg Trp Asn Asp Asp Val Cys Gin Arg Pro Tyr Arg Trp Val Cys Glu 115 120 125

Thr

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 129 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

SUBSTITUTE SHEET (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Arg Thr Cys Cys Pro Val Asn Trp Val Glu His Gin Gly Ser Cys Tyr 1 5 10 15

Trp Phe Ser His Ser Gly Lys Ala Trp Ala Glu Ala Glu Lys Tyr Cys 20 25 30

Gin Leu Glu Asn Ala His Leu Val Val lie Asn Ser Trp Glu Glu Gin 35 40 45

Lys Phe lie Val Gin His Thr Asn Pro Phe Asn Thr Trp lie Gly Leu 50 55 60

Thr Asp Ser Asp Gly Ser Trp Lys Trp Val Asp Gly Thr Asp Tyr Arg 65 70 75 80

His Asn Tyr Lys Asn Trp Ala Val Thr Gin Pro Asp Asn Trp His Gly 85 90 95

His Glu Leu Gly Gly Ser Glu Asp Cys Val Glu Val Gin Pro Asp Gly 100 105 110

Arg Trp Asn Asp Asp Phe Cys Leu Gin Val Tyr Arg Trp Val Cys Glu 115 120 125

Lys

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 130 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Leu Gin Leu lie Met Gin Asp Trp Lys Tyr Phe Asn Gly Lys Phe Tyr 1 5 10 15

SUBSTITUTE SHEET Tyr Phe Ser Arg Asp Lys Lys Ser Trp His Glu Ala Glu Asn Phe Cys 20 25 30

Val Ser Gin Gly Ala His Leu Ala Ser Val Thr Ser Gin Glu Glu Gin 35 40 45

Ala Phe Leu Val Gin He Thr Asn Ala Val Asp His Trp He Gly Leu 50 55 60

Thr Asp Gin Gly Thr Glu Gly Asn Trp Arg Trp Val Asp Gly Thr Pro 65 70 75 80

Phe Asp Tyr Val Gin Ser Arg Arg Phe Trp Arg Lys Gly Gin Pro Asp 85 90 95

Asn Trp Arg His Gly Asn Gly Glu Arg Glu Asp Cys Val His Leu Gin 100 105 110

Arg Met Trp Asn Asp Met Ala Cys Gly Thr Ala Tyr Asn Trp Val Cys 115 120 125

Lys Lys 130

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 130 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Pro Thr His Cys Pro Ser Gin Trp Trp Pro Tyr Ala Gly His Cys Tyr 1 5 10 15

Lys He His Arg Asp Glu Lys Lys He Gin Arg Asp Ala Leu Thr Thr 20 25 30

Cys Arg Lys Glu Gly Gly Asp Leu Thr Ser He His Thr He Glu Glu

35 40 45

SUBSTITUTE SHEET Leu Asp Phe He He Ser Gin Leu Gly Leu Glu Pro Asn Asp Glu Leu ^• 50 55 60

Trp He Gly Leu Asn Asp He Lys He Gin Met Tyr Phe Glu Trp Ser 65 70 75^' 80

Asp Gly Thr Pro Val Thr Phe Thr Lys Trp Leu Arg Gly Glu Pro Ser 85 90 95

His Glu Asn Asn Arg Gin Glu Asp Cys Val Val Met Lys Gly Lys Asp 100 105 110

Gly Tyr Trp Ala Asp Arg Gly Cys Glu Trp Pro Leu Gly Tyr He Cys 115 120 125

Lys Met 130

SUBSTITUTE SHEET

Claims

We claim:

1. A method of inhibiting HIV infection of mammalian cells comprising contacting the cells with an effective amount of a compound selected from the group consisting of a mannose carbohydrate, a fucose carbohydrate, a lectin and a dmg, for a time period sufficient to significantly inhibit the binding of HIV to a non-CD4 cell surface protein.

2. The method of Claim 1, wherein the non-CD4 cell surface protein is a gpl20 receptor having a specific binding affinity for gpl20 of about Kd — 1.3 nM to about Kd = 2.0 nM.

3. The method of Claim 2, wherein the gpl20 receptor is present on placental cells.

4. The method of Claim 2, wherein the gpl20 receptor is present on muscle cells.

5. The method of Claim 2, wherein the gpl20 receptor is present on neural cells.

6. The method of Claim 5, wherein the neural cells are brain cells.

7. The method of Claim 5, wherein the neural cells are dendritic cells.

8. The method of Claim 2, wherein the gpl20 receptor is present on mucosal cells.

10. The method of Claim 1, wherein the compound is mannose.

10. The method of Claim 1, wherein the compound is fucose.

11. The method of Claim 1, wherein the compound is a mannose-containing carbohydrate.

12. The method of Claim 11, where the carbohydrate is mannan.

13. The method of Claim 1, wherein the compound is a pradimicin A antibiotic.

14. A substantially purified non-CD4 gpl20 receptor protein comprising a protein substantially corresponding to a non-CD4 mammalian cell surface protein that has a

SUBSTITUTE SHEET specific binding affinity for gpl20, said protein containing about 400 amino acid residues, having a molecular weight of about 45,000 daltons and having a binding affinity for gpl20 characterized by a Kd of about 1.3 nM to about 2 nM.

15. The gpl20 receptor protein of Claim 14, wherein the binding of the gpl20 receptor protein to gpl20 is inhibited by a compound selected from the group consisting of a mannose carbohydrate, a fucose carbohydrate, a lectin and a dmg.

16. The gpl20 receptor of Claim 15, wherein the compound is mannose.

17. The gpl20 receptor protein of Claim 15, wherein the compound is a pradimicin A antibiotic.

18. The gpl20 receptor protein of Claim 14, wherein the protein is produced by recombinant means.

19. The gpl20 receptor protein of Claim 18, wherein said recombinant means comprises the cloning of a cDNA isolated from a library of recombinant placental genes.

20. A DNA molecule encoding the gpl20 receptor protein of Claim 14, wherein the DNA is a complementary DNA that transcribes an mRNA found in cells selected from the group consisting of placental cells, brain cells, muscle cells and colon cells.

21. A method of detecting the presence of HTV in a sample comprising:

(a) admixing in an aqueous medium a sample to be assayed with a non- CD4 gpl20 receptor protein having a specific binding affinity for gpl20 characterized by a Kd of about 1.3 nM to about 2.0 nM in an amount sufficient to carry out at least one assay;

(b) maintaining the admixture for a time period sufficient for the gpl20 receptor protein to bind to any HIV present in the sample and form a reaction product; and

(c) determining the presence of the HIV containing reaction product.

SUBSTITUTE SHEET

22. The method of Claim 21, wherein the gpl20 receptor protein contains about 400 amino acid residues and has a molecular weight of about 45,000 daltons.

23. The method of Claim 21, wherein the gpl20 receptor protein is affixed to a solid matrix to form a solid support.

24. The method of Claim 21, wherein the presence of the reaction product is determined by contacting the sample with a reagent capable of detecting the bound gpl20 receptor protein.

25. The method of Claim 24, wherein the reagent is a labelled antibody directed against the gpl20 receptor protein.

26. A diagnostic system in kit form, for assaying for the presence of HIV in a fluid sample, comprising a package containing a non-CD4 receptor protein having a specific affinity for gpl20 characterized by a Kd of about 1.3 nM to about 2.0 nM, and instructions for use.

27. The diagnostic system of Claim 26, wherein the non-CD4 gpl20 receptor protein is affixed to a solid matrix to form a solid support.

SUBSTITUTE SHEET