WO2003014155A2

WO2003014155A2 - Labelled conjugates of p17 protein and their use in aids diagnosis

Info

Publication number: WO2003014155A2
Application number: PCT/IB2002/003092
Authority: WO
Inventors: Arnaldo Caruso; Simona Fiorentini; Josè Sebastian FRANZONE
Original assignee: Medestea Internazionale S.R.L.
Priority date: 2001-08-07
Filing date: 2002-08-05
Publication date: 2003-02-20
Also published as: WO2003014155A3; AU2002355369A1

Abstract

The invention relates to the protein p17 of HIV-1 in isolated form conjugate with a fluorescent substance or with a primary reagent capable of being specifically bound by a secondary reagent conjugated with a detectable molecule selected from a fluorochrome and an enzyme. The protein p17 of the invention is particularly suitable for use as a specific reagent in a procedure for the identification of the cells which express on their surface a receptor for p17 of HIV-1.

Description

Method for the identification of cells which express on their surface a receptor for the p!7 protein of HIV-1, and p!7 protein in an isolated and labelled form suitable for use in said method

The present invention relates to a process for identifying and purifying cells expressing on their surface a receptor for the protein pl7 of HIV-l, as well as the protein pl7 of HIV-1 in an isolated and labelled form suitable for use in the said process.

The pl7 protein of the viral matrix is a structurally important protein in the life cycle of HIV. It has a role in the early phases of the viral replication and participates in the initial integration of proviral DNA in the nucleus of the host cell. Moreover, pl7 binds viral RNA and mediates its transport to the plasma membrane, and is also involved in the processes of cointegration of the antigens into the envelope and in the assembly of the viral particles.

pl7 shows a structural similarity with the cytokine INF-γ (Matthews S, et al . (1994) Nature, 370, 666) and recently the capacity of pl7 to act as a viral cytokine has been shown. It is, in fact, capable if increasing the proliferation of the mononucleated blood cells (PBMCs) stimulated with interleukin-2 (IL-2) or with phytohemagglutinin and to increase, in these cells, replication of HIV (De Francesco M, et al. 1998, AIDS 12, 245). More recent data has further shown that pl7 could promote viral replication in that it increases the production of inflammatory cytokines (Th-1) with pro-HIV action, such as INF-γ and Tumour Necrosis Factor α (TNF-α) . This activity develops on PBMCs stimulated with itogens and is dose-dependent , with a maximum increase at doses of pl7 equal to 50 ng/ml . The present inventors have also found that pl7 is capable of counteracting the inhibition of release of INF-γ and TNF-α induced by the treatment of the activated lymphocytes with the anti inflammatory cytokine (Th-2) interleukin-4 (IL-4) , leading to the production of the two pro-inflammatory cytokines at normal values (A. Caruso et al, data not published) .

It has moreover been reported that the pl7 protein is the target of antibodies which neutralise the replication of HIV and that high levels of anti-pl7 antibodies are correlated with a slower progression of AIDS. The discovery of a protective role of the humoral anti-pl7 immune response and the presence on pl7 of epitopes neutralising the replication of HIV is rather surprising since electron microscopy studies suggest that pl7 is localised exclusively within the viral particle. It is therefore probable that pl7 can act as an independent free-standing entity released in the cellular microenvironment , which promotes viral replication.

In the prior art there is described a study (De Francesco M, et al. 1998 AIDS 12, 245) in which the pl7 protein of HIV labelled with radioactive isotopes (¹²⁵I) was utilised as a specific reagent for verifying if the biological activity of this viral protein would be mediated by the interaction with a specific cellular receptor. The results obtained with the radiolabelled protein led to exclude the existence of a cellular receptor for the protein pl7, and it was therefore hypothesised that the activity of the viral protein developed through the interaction with a molecule released by the human monocyte and lymphocyte cells into the cell culture supernatant . The present inventors have now surprisingly found that by utilising a fluorescent or enzyme label it is on the other hand possible to demonstrate the existence of cells expressing a specific cell receptor for the protein pl7.

As will be illustrated in more detail in the section relating to the examples, by making use of this labelling it has been possible to verify that the activity of pl7 is effectively mediated by the interaction with a high affinity specific receptor.

A first object of the invention is therefore the protein pl7 of HIV-1 in isolated form conjugated with a fluorescent substance or with a primary reagent capable of being specifically bound by a secondary reagent conjugated with a detectable molecule selected from a fluorochrome and an enzyme .

The pl7 protein conjugated according to the present invention is found to be particularly suitable for use as a reagent in an essay for detecting the expression of a receptor for the pl7 protein of HIV-1.

Therefore, a second object of the invention is the pl7 protein of HIV-1 in isolated form conjugated with a fluorescent substance or conjugated with a primary reagent capable of. being specifically bound by a secondary reagent conjugated with a detectable molecule selected from a fluorochrome and an enzyme, for use in an assay for detecting the expression on the cell surface of a receptor for the pl7 protein of HIV-1. The pl7 protein utilised for this purpose is a protein in isolated form, that is to say substantially purified from its natural environment or in any event produced through any technical procedure which makes it possible to obtain this protein in a substantially isolated form, such as, the recombinant DNA technology.

As has been indicated above, pl7 according to the invention can be directly labelled with a fluorescent substance; for example it can be conjugated with the fluorescent protein Green Fluorescent Protein (GFP) of Aeguorea victoria . In this form the protein pl7 is produced by recombinant DNA technology as a pl7/GFP fusion protein.

Alternatively, pl7 according to the invention can be conjugated with a primary reagent which is specifically recognised by a secondary reagent conjugated with a detectable molecule.

For example, the pl7 can be conjugated with biotin, which is bound with high affinity by avidin and by streptavidin (secondary reagents) . Other primary and secondary reagents suitable for this purpose and known in the art can however be utilised.

In order to be able to verify the formation of the primary reagent - secondary reagent complex, the secondary reagent is in turn conjugated with a detectable molecule, such as, for example, a fluorochrome, that is to say a molecule capable of emitting radiation in the form of visible light. Fluorochromes suitable for this purpose have been widely described in the prior art . Alternatively, the secondary reagent is conjugated with an enzyme, such as, for example, peroxidase or beta galactosidase, capable of reacting with a chromogenic substrate, that is to say a substrate which initially is uncoloured but following enzymatic reaction changes colour with an intensity proportional to the quantity of enzyme present .

Another object of the invention is therefore a kit for detecting the expression of a receptor for the protein pl7 of HIV-1 on the cell surface, comprising the protein pl7 of HIV- 1 in isolated form conjugated with a primary reagent, and a secondary reagent conjugated with a detectable molecule selected from a fluorochrome and an enzyme, said secondary reagent being able to specifically bind the said primary reagent. In the specific embodiment in which the detectable molecule is an enzyme, the kit further comprises a chromogenic substrate of the enzyme .

Preferably, the primary reagent is biotin and the secondary reagent is avidin or streptavidin.

The availability of a specific reagent able to detect the expression of a receptor for the protein pl7 on a cell surface, makes it possible to identify within a cell population, of the presence of cells which express this receptor. The cell population can for example be a mixed cell population. The test can be carried out both on a sample of cells in culture and on a sample of tissue fixed with histochemical techniques.

A further object of the invention is therefore a method for the identification, within a cell sample, of the presence of cells which express on their surface the receptor for the protein pl7 of HIV-1.

According to a first embodiment, in which the protein pl7 directly conjugated with a fluorescent substance is utilised as the specific reagent, the method comprises the steps of: a) incubating the said cell sample with the protein pl7 of HIV-1 in isolated form conjugated with a fluorescent substance; b) exposing the said cell sample to electromagnetic radiation having a wavelength capable of exciting the said fluorescent substance; c) analysing the fluorescence emitted by the cells of the said sample, thus identifying the presence of cells on the surface of which the said protein pl7 conjugated with the said fluorescent substance is selectively bound, the said binding being indicative of the expression of the specific receptor for the protein pl7 of HIV-1.

For this purpose the protein pl7 produced by recombinant DNA technology such as, for example, the pl7/GFP fusion protein, can be utilised.

According to another embodiment, in which the protein pl7 conjugated with a primary reagent capable of being specifically bound by a secondary reagent conjugated with a fluorochrome is utilised as the specific reagent, the method comprises the steps of: a) incubating the said cell sample with the protein pl7 of HIV-1 in isolated form conjugated with a primary reagent capable of being specifically bound by a secondary reagent conjugated with a fluorochrome; b) adding to the said cell sample the said secondary reagent conjugated with a fluorochrome, thus obtaining a pl7/primary reagent/secondary reagent/fluorochrome complex; c) exposing the said cell sample to electromagnetic radiation having a wavelength capable of exciting the molecules of the said fluorochrome; d) analysing the fluorescence emitted by the cells of the said sample, thus identifying the presence of the cells on the surface of which the said pl7/primary reagent/secondary reagent/fluorochrome complex is selectively bound, the said binding being indicative of the expression of the specific receptor for the protein pl7 of HIV-1.

According to another embodiment, in which the protein pl7 conjugated with a primary reagent capable of being specifically bound by a secondary reagent conjugated with an enzyme is utilised as the specific reagent, the method comprises the steps of : a) incubating the said cell sample with the protein pl7 of HIV-1 in an isolated form conjugated with a primary reagent capable of being specifically bound by a secondary reagent conjugated with an enzyme; b) adding the said secondary reagent conjugated with an enzyme to the said cell sample thus obtaining a pl7/primary reagent/secondary reagent/enzyme complex; c) contacting the said cell sample with a chromogenic substrate of the said enzyme; d) verifying the appearance of colour in the said cell sample, the said appearance of colour being indicative of the presence of cells on the surface of which the pl7/primary reagent/secondary reagent/enzyme complex is selectively bound, the said binding being indicative of the expression of the specific receptor for the protein pl7 of HIV-1. The appearance of the colour in the sample subjected to the assay can be verified for example by observation with an optical microscope.

Alternatively, when the pl7 is fluorescently labelled, the method can be put into practice by flow cytofluorimetry which, as is known, is based on the use of a flow cytofluorimeter, that is to say a device within which the cells pass one at a time through an aperture or capillary.

The use of the flow cytofluorimetry technique makes it possible therefore to discern, within the sample subjected to the assay, the individual cells which express the receptor for pl7 from those which do not express it.

The identified cells can then be separated from the rest of the sample. For this purpose methods known in the art can be utilised such as, for example, the technique of cell separation activated by fluorescence in which a device called a cell sorter is utilised located downstream of the analyser of the flow cytofluorimeter, or the technique of magnetic immune sorting which is based on the use of a specific secondary reagent conjugated with magnetic beads. These methods are in any event widely known to the man skilled in the art and their application in the present invention lies within his capability.

By utilising the protein pl7 produced by recombinant techniques and conjugated with biotin as the primary reagent, and by studying its interaction with the cell surface by means of flow cytofluorimetry, there have been obtained results illustrated in the attached figures, in which: Figure 1 illustrates the interaction between pl7 and peripheral blood mononuclear cells (PBMCs) a) freshly obtained, or b) stimulated for 72 hours with IL-2. The visualisation of the binding of pl7 to its receptor is obtained by utilising streptavidin conjugated with phycoerythrin (PE) as the specific reagent. The data are shown as dot plots. The percentage of cells is indicated in the top right hand corner.

Figure 2 illustrates the interaction between pl7 and the cell receptor expressed on lymphocyte sub-populations. The recombinant pl7 conjugated with biotin (400ng/ml) is reacted with freshly obtained peripheral blood mononuclear cells (A, B, C) or PBMCs stimulated for 72 hours with IL-2 (D, E, F) . The visualisation of the binding of pl7 to its receptor is obtained by utilising streptavidin conjugated with phycoerythrin (PE) as the specific reagent. The cells were then stained with a mixture of monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) . This staining showed the presence of the specific receptor for the protein pl7 of HIV on B lymphocytes (identified by the monoclonal anti-CD19) and on CD4⁺ and CD8⁺ lymphocyte cells (respectively identified by monoclonal anti-CD4 and anti- CD8) . The data are shown as bivariate dot plots. The percentage of cells in each quadrant is indicated in the top right hand corner of each panel .

Figure 3 shows that binding of pl7 to its receptor is dose- dependent. Peripheral blood mononuclear cells were incubated with different doses of pl7 conjugated with biotin. The binding of pl7 to the cell receptor was evidenced by utilising streptavidin conjugated with PE. The cells expressing the receptors for pl7 were analysed by means of flow cytofluorimetry. 100% of positive cells corresponds to a concentration of pl7 capable of demonstrating the maximum level of receptor expression. The insert shows a Scatchard plot of the pl7/receptor binding data generated by staining of the cells (at numbers lying between 1.25 x IO⁵ and lxlO⁶) with different concentrations of pl7 comprised between lOng/ml and 1.6μg/ml).

Figure 4 shows the kinetics of the expression of the receptor for pl7 on T lymphocyte sub populations. CD4⁺ and CD8⁺ lymphocytes were cultivated in the presence or absence of different mytogenic stimuli. The cells were collected at the times indicated and reacted with pl7 conjugated with biotin at concentrations of 400ng/ml. The visualisation of the binding of pl7 to its receptor was obtained by utilising streptavidin conjugated with PE as the specific reagent.

Figure 5 shows the expression of the receptor for pl7 on activated T lymphocytes. Peripheral blood mononuclear cells were stimulated for 48 hours with PHA and reacted with pl7 conjugated with biotin at concentrations of 400ng/ml. The visualisation of the binding of pl7 to its receptor was obtained by utilising streptavidin conjugated with PE as the specific reagent. The cells were then stained with antibodies specific for the lymphocyte activation marker (conjugated with FITC) and with antibodies directed towards the phenotypic markers CD4 (conjugated with Per-CP) and CD8 (conjugated with APC) . A window was generated for analysing only the CD4⁺ and CD8⁺ lymphocytes (top right quadrants respectively) . The expression of the activation marker CD69 and the receptor for pl7 relative to the cells CD4⁺ and CD8⁺ is plotted in the lower left and right quadrants respectively. The data are shown as monovariated dot plots (upper quadrants) and bi variated dot plots (lower quadrants) . The percentage of cells positive for CD69 and/or for the pl7 receptor is indicated in the top right corner of each panel .

Figure 6 shows the expression of the pl7 receptor on cell lines. Cells H9 were stimulated (b) or not (a) for 48 hours with PHA and reacted with pl7 conjugated with biotin at concentrations of 400ng/ml. The determination of the binding to its receptor was obtained by utilising streptavidin conjugated with PE as specific reagent. The data are shown as dot plots. The percentage of positive cells is indicated in the top right hand corner .

Figure 7 shows the inhibition of the interaction between pl7 and its receptor with antibodies anti-pl7. The pl7 conjugated biotin was reacted with freshly obtained peripheral blood mononuclear cells (A,B,C) or PBMCs simulated for 72 hours with IL -2 (D,E,F) in the absence (A,D) or in the presence of anti-pl7 neutralising (B,E) monoclonal antibodies (MBS-3) and non neutralising antibodies (MK-1) (C,F). The visualisation of the binding of the pl7 to its receptor is obtained by utilising streptavidin conjugated with PE as the specific reagent . The data are shown as dot plots . The percentage of cells is indicated in the top right hand corner.

The data shown in the Figures demonstrate that the viral protein interacts with a receptor which is present on a low percentage (9.6%) of mononuclear cells freshly isolated from healthy subjects (Figure la) . The percentage of positive cells for the pl7 receptor increased considerably (30.2%) when the cells are incubated with biotinylated pl7 before stimulation with substances having mitogenic activity (IL-2, Figure lb) .

The results obtained with multi parametric analysis on mononuclear cells freshly isolated from healthy volunteers show that the pl7 does not bind the CD4⁺ and CD8⁺ T lymphocytes but is present on the surface of the majority of the B lymphocytes (Figure 2a, b, c) . On the contrary, pl7 binds to the CD4⁺ and CD8⁺ T lymphocytes activated with mitogenic stimuli (Figure 2d, e, f) . The data demonstrate that a receptor exists for pl7 of HIV on circulating lymphocytes and that it is constitutively expressed on B lymphocytes, whilst it is inducible by means of appropriate mitogenic stimuli on the surface of T lymphocytes.

The affinity constant (K_d) between pl7 and the specific receptor was determined to be equal to 2.5 x IO^"8 M, by Scatchard analysis (Figure 3) .

On CD4⁺ and CD8⁺ lymphocytes purified by magnetic immunosorting the stimulation with different molecules having mitogenic action also induces the expression of the receptor for pl7 in a high percentage of cells (Figure 4) .

Analysis of the activated lymphocytes, characterised by the expression of the phenotypic marker CD69, provides evidence that a consistent portion of activated cells, but not the entirety thereof, express the receptor for pl7 (Figure 5) . This suggests the presence of more or less sensitive cells, or cells which are entirely non-sensitive to the biological activity of pl7. The receptor for pl7 is also expressed by human cell lines of lymphocytic origin both B (Raji, for example) and T (H9, for example) . These latter express the receptor for pl7 on the totality of the cells only after stimulation with PHA (Figure 6) -

Incubation of the cells with biotinylated pl7 in the presence of an anti-pl7 monoclonal antibody having neutralising activity inhibits the interaction of the viral protein with its specific cell receptor (Figure 7) . This data, together with the high affinity constant of the pl7/receptor binding, confirms the specificity of the binding of pl7 to its receptor.

Production of recombinant p!7 of HIV-1 and Conjugation with biotin

pl7 of HIV 1 (amino acids 1 to 132) was amplified from the viral isolate BH-10 by means of polymerase chain reaction (PCR) utilising specific primers which allow cloning of the pl7 gene in the BamHI site of the prokaryotic expression vector pGEX-2T (Pharmacia, Uppsala, Sweden) .

The pl7 and Glutathione Transferase fusion protein (pl7/GST) was expressed in Escherichia coli and affinity purified utilising glutathione bound to a 4B sepharose resin (Pharmacia) . The viral protein was cut from GST when the complex was still bound to the glutathione column by the addition of thrombin as described in the literature (Gearing D, et al. (1989), Biotechnology 7, 1157). The protein pl7 was further purified (>98% of purity) by means of reverse phase of FPLC . The absence of bacterial endotoxin contamination in the preparation of recombinant pl7 (<0.l units of endotoxin/ml) was confirmed with the Limulus test (Whittaker Bioproducts Inc., alkersville, Maryland, United States of America) . The purified pl7 was conjugated with biotin using the AH-NHS-biotin reagent (SPA, Milan, Italy) with a method modified with respect to that described in the literature (Imunological Techniques Made Easy (1998) Eds., Cochet) , Teilland J-L and Sutes C. (Wiley & Sons, Chichester) pp. 230-231) . In particular, a) the concentration of pl7 was equal to 0.5 mg/ml diluted in bicarbonate buffer 0.1 M, pH 8.0, containing NaCl at concentrations of 0.3 M; b) the concentration of biotin was equal to 2mg/ml in DMSO (dimethylsulphoxide) ; c) the volume of biotin to add to the pl7 for conjugation was equal to 450μl; d) omission of the step of blocking the conjugation reaction by means of NH₄C1 (ammonium chloride) and insertion of a dialysis step of the biotin/pl7 solution against PBS (phosphate buffered saline) .

Construction of the p!7/GFP Fusion Protein.

The sequence coding for the Green Fluorescent Protein was isolated from the plasmid pEGFP/C2 by gene amplification reaction. The restriction sites upstream and downstream of the sequence coding for the protein were created by utilising mutagenic primers suitably designated (GFP_X0 i for: 5' -GTC TCG AGC ATG GTG AGC AAG GGC GA-3'; GFPχ_{ba τ} rev: 5'-CGT CTA GAG CTT GTA CAG CTC CTC CA-3' . 20 ng of plasmid pEGFP/C2 were utilised directly as template in the PCR reaction, in a final volume of 200μl. The conditions utilised for the PCR reaction were the following: 94°C, 30 seconds; 50°C, 30 seconds; 72° C; 30 seconds, for a total of 35 cycles.

The PCR product obtained was cloned in the multiple cloning site of the vector pVAX-1. The gene sequence of the protein pl7 was isolated from the cloning construct containing the pl7 gene by a gene amplification reaction. The restriction sites upstream and downstream of the sequence coding for the protein were created utilising mutagenic primers suitably designated (pl7mut _{xho τ} for : 5' - GTC GCT CGA GAG TAT GGG TGC GAG A -3;' pl7mut o i rev: 5'- CTA GCT CGA GTA ATT TTG GCT GAC -3'). The sense primer makes it possible to insert, upstream of the gene to express, the Kozak sequence (Kozak M. , Proc Natl Acad Aci USA 87, 8301-05, 1990) necessary for an optimal expression in eukaryotic systems. The antisense primer was designated in such a manner as to create the restriction site downstream of the sequence coding for pl7 and to permit its cloning in frame with the sequence coding for GFP. The resulting construct generated an autofluorescent pl7/GFP fusion protein usable in cytofluorometric analysis.

Cytofluorometric Analysis of the Binding of p!7 to the surface of Fixed Cells.

Fresh peripheral blood mononuclear cells, or PBMCs stimulated in vitro for 72 hours with IL-2, were washed three times with PBS and fixed in suspension utilising a cold solution of paraformaldehyde at 4%. After 5' of incubation the cells were stained with biotinylated pl7 according to the protocol already described. The experimental data showed that the fixation of the cells did not alter the capacity of pl7 to interact with its receptor (data not shown) . These results make the staining with pl7 compatible with methods which envisage the fixation of cells and/or tissue fragments, such as, for example, histochemical methods.

PBMCs Cell Culture Peripheral blood mononuclear cells (PBMC) were obtained from healthy subjects, which had provided their informed consent to the research in accordance with the Helsinki declaration. The cells were seeded in plates with 96 wells at a density of IO⁶ cells/ml of RPMI-1640 medium supplemented with 10% of heat-inactivated human AB serum (Sigma, St. Louis, MO), 100 U/ml of penicillin and 100 μg/ml of streptomycin.

Purification of CD4⁺ and CD8⁺ T Lymphocytes

The CD4⁺ and CD8⁺ T lymphocytes were purified from the peripheral blood mononuclear cells of healthy individuals by positive selection utilising anti-CD4 and anti-CD8 antibodies conjugated with magnetic beads (MiniMacs, Milteny Biotec, Bergish Gladbach, Germany) . The purity was always greater than 96 %.

Cytofluorometric Analysis of the Binding of p!7 to the Cell Surface.

Fresh peripheral blood mononuclear cells, or PBMCs stimulated in vitro for different time periods with phytohemagglutinin

(PHA) , staphylococcal enterotoxin B(SEB), interleukin-2 (IL- 2) , anti-CD3 antibodies (0KT3) or anti-CD28 antibodies, were incubated for 30 mins at 4° C with different concentrations of biotinylated pl7 comprised between 10 ng/ml and 1.6 μg/ml. After two washes with PBS containing 2% of foetal calf serum

(FCS) the cells were incubated for 30 mins at 4°C with streptavidin conjugated with phycoerythrin (PE) (Becton Dickinson, San Jose, CA) . In some experiments the cell were incubated also with antibodies directed towards other cell surface activation or phenotypic markers, conjugated with emission fluorochromes different from PE, such as FITC, PerCP or APC. The acquisition and multi parametric analysis of the data was carried out with a Becton Dickinson FACScalibur cytofluorometer. The data obtained was utilised to construct a Scatchard plot which allowed the binding affinity of pl7 to its receptor to be defined (Campbell Am. (1991) . Laboratory techniques in biochemistry and molecular biology, Ed., Van Der Vliet, PC (Elsevier, Amsterdam) , BP. 303-342) . In some experiments the peripheral blood mononuclear cells were incubated for 30 minutes at 4°C with biotinylated pl7 in the presence or in the absence of an anti pl7 antibody having neutralising activity at concentrations comprised between 0.5 and 10 μg/ml.

Claims

1. A protein pl7 of HIV-1 in isolated form conjugated with a fluorescent substance or with a primary reagent capable of being specifically bound by a secondary reagent conjugated with a detectable molecule selected from a fluorochrome and an enzyme .

2. A protein pl7 of HIV-1 according to Claim 1, which is fused with the Green Fluorescent Protein (GFP) .

3. A protein pl7 of HIV-1 according to Claim 1, which is conjugated with biotin.

4. A protein according to any of Claims 1 to 3 for use in an assay for detecting the expression of the receptor for the protein pl7 of HIV-1 on a cell surface.

5. Kit for detecting the expression of the receptor for the protein pl7 of HIV-1 on a cell surface, comprising:

(i) the protein pl7 of HIV-1 in isolated form conjugated with a primary reagent; and

(ii) a secondary reagent conjugated with a fluorochrome, said secondary reagent capable of specifically binding the said primary reagent .

6. Kit for detecting the expression of the receptor for the protein pl7 of HIV-1 on a cell surface comprising:

(i) the protein pl7 of HIV-1 in isolated form conjugated with a primary reagent;

(ii) a_ secondary reagent conjugated with an enzyme, the said secondary reagent capable of specifically binding the said primary reagent; and (iii) a chromogenic substrate of the said enzyme.

7. Kit according to Claim 5 or Claim 6, in which the said primary reagent is biotin and the said secondary reagent is avidin or streptavidin.

8. A process for the identification, within a cell sample, of the presence of cells which express on their surface the receptor for the protein pl7 of HIV-1, comprising the steps of: a) incubating the said cell sample with the protein pl7 of HIV-1 in isolated form conjugated with a fluorescent substance; b) exposing the said cell sample to electromagnetic radiation having a wavelength capable of exciting the said fluorescent substance; c) analysing the fluorescence emitted from the cells of the said sample, so as to identify the presence of the cells on the surface of which the said protein pl7 conjugated with the fluorescent substance is selectively bound, the said binding being indicative of the expression of the specific receptor for the protein pl7 of HIV-1.

9. A process according to Claim 8, in which the said protein pl7 in isolated form is fused with the Green Fluorescent Protein (GFP) .

10. A process for the identification, within a cell sample, of the presence of cells which express on their surface the receptor for the protein pl7 of HIV-1 comprising the steps of: a) incubating the said cell sample with the protein pl7 of HIV-1 in isolated form conjugated with a primary reagent capable of being specifically bound by a secondary reagent conjugated with a fluorochrome; b) adding to the said cell sample the said secondary reagent conjugated with a fluorochrome, thus obtaining a pl7/primary reagent /secondary reagent/fluorochrome complex; c) exposing the said cell sample to electromagnetic radiation having a wavelength capable of exciting the molecules of the said fluorochrome; d) analysing the fluorescence emitted by the cells of the said sample, thus identifying the presence of the cells on the surface of which the said pl7/primary reagent/secondary reagent/fluorochrome complex is selectively bound, the said binding being indicative of the expression of the specific receptor for the protein pl7 of HIV-1.

11. A process for the identification, within a cell sample, of the presence of cells which express on their surface the receptor for the protein pl7 of HIV-1, comprising the steps of: a) incubating the said cell sample with the protein pl7 of HIV-1 in isolated form conjugated with a primary reagent capable of being specifically bound by a secondary reagent conjugated with an enzyme; b) adding to the said cell sample the said secondary reagent conjugated with an enzyme, thus obtaining a pl7/ primary reagent/secondary reagent/ enzyme complex; c) contacting the said cell sample with a chromogenic substrate of the said enzyme; d) verifying the appearance of colour in the said cell sample, the said appearance of colour being indicative of the presence of cells on the surface of which the pl7/primary reagent/ secondary reagent/ enzyme complex is selectively bound, the said binding being indicative of the expression of the specific receptor for the protein pl7 of HIV-1.

12. A process according to Claim 10 or Claim 11, in which the said primary reagent is biotin and the said secondary reagent is avidin or streptavidin.

13. A process according to any of Claims 8 to 12, further comprising the final step of separating the said cells which express on their surface the receptor for the protein pl7 of HIV-1 from the rest of the cell samples.

14. A process according to any of claims 8 to 13, in which the said cell sample is a cell culture.

15. A process according to any of Claims 8 to 13, in which the said cell sample is a tissue fixed by histochemical methods .