WO2010040853A1

WO2010040853A1 - A method for the screening of candidate substances active against the infection of a subject by a hiv virus and kits for performing the said method

Info

Publication number: WO2010040853A1
Application number: PCT/EP2009/063244
Authority: WO
Inventors: Patrice Debre; Vincent Vieillard
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2008-10-10
Filing date: 2009-10-12
Publication date: 2010-04-15

Abstract

The invention consists in an in vitro method for the screening of candidate substances active against the infection of a subject by a HIV virus comprising the steps of : a) providing cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, b) incubating the cells provided at step a) with a candidate substance, c) measuring the expression level of NKp44L at the cell membrane, d) comparing the NKp44L expression value obtained at step c) with a reference NKp44L expression value determined in the absence of the said candidate substance, and e) selecting positively the said candidate substance when the NKp44L expression value measured at step c) is higher than the said reference NKp44L expression value.

Description

A method for the screening of candidate substances active against the infection of a subject by a HIV virus and kits for performing the said method.

FIELD OF THE INVENTION The present invention relates to the field of the screening of substances useful as active ingredients for treating subjects that have been infected by a virus of the HIV family.

BACKGROUND OF THE INVENTION

AIDS disease, which is primarily caused by infection of individuals with a HIV retrovirus, is now the most devastating disease in the whole world, since the number of individuals which are, to date, infected with HIV viruses is estimated to about 40 millions of individuals.

During the sole year 2001 , 5 millions of individuals were infected with HIV while 3 millions of individuals have deceased in the same time. Since the discovery of the AIDS causative agent in 1983, the HIV virus, extensive efforts have been made in order to understand the mechanism of action of this virus and to develop accurate methods for (i) reproducibly diagnosing the infection, as well as (ii) carrying out a prognosis of the progression of the disease in a given patient. When AIDS first surfaced in the United States, there were no medicines to combat the underlying immune deficiency and few treatments existed for the opportunistic diseases that resulted. During the past 10 years, however, researchers have developed drugs to fight both HIV infection and its associated infections and cancers. The U.S. Food and Drug Administration (FDA) has approved a number of drugs for treating HIV infection. The first group of drugs used to treat HIV infection, called nucleoside reverse transcriptase (RT) inhibitors, interrupts an early stage of the virus making copies of itself. Included in this class of drugs (called nucleoside analogs) are AZT, ddC (zalcitabine), ddl (dideoxyinosine), d4T (stavudine), 3TC (lamivudine), abacavir (ziagen), and tenofovir (viread). These drugs may slow the spread of HIV in the body and delay the start of opportunistic infections. Health care providers can prescribe non-nucleoside reverse transcriptase inhibitors (NNRTIs), such as delvaridine (Rescriptor), nevirapine (Viramune), and efravirenz (Sustiva), in combination with other antiretroviral drugs. More recently, FDA has approved a second class of drugs for treating HIV infection. These drugs, called protease inhibitors, interrupt virus replication at a later step in its life cycle. They include Ritonavir (Norvir), Saquinivir (Invirase), etc.

Because HIV can become resistant to any of these drugs, there is a need in the art for novel compounds of pharmaceutical interest that are biologically active in HIV-infected patients. Today, health care providers must use a combination treatment to effectively suppress the virus. When RT inhibitors and protease inhibitors are used in combination, it is referred to as highly active antiretroviral therapy, or HAART, and can be used by people who are newly infected with HIV as well as people with AIDS. Researchers have credited HAART as being a major factor in significantly reducing the number of deaths from AIDS in this country. While HAART is not a cure for AIDS, it has greatly improved the health of many people with AIDS and it reduces the amount of virus circulating in the blood to nearly undetectable levels. Researchers, however, have shown that HIV remains present in hiding places, such as the lymph nodes, brain, testes, and retina of the eye, even in patients who have been treated.

An increasing number of studies were aimed at identifying new target molecules for designing alternative therapies against an infection with an HIV virus. Illustratively, the NKp44L protein which is expressed by the CD4⁺ cells from HIV-infected individuals had been described in the art as a potential new medical target. Further, it had been shown in the art that a peptide derived from the gp41 structural protein from HIV enhanced the expression of the NKp44L protein at the membrane surface of CD4⁺ T cells. The gp41 -derived peptide was described as an NKp44L expression enhancer that increased the lysis of CD4+ T cells by the endogenous NK cells (See PCT Application n ° WO/2005/076001 ).

There is a need in the art for novel therapeutically useful compounds for preventing individuals from the occurrence of AIDS upon infection with a HIV virus or, more generally, for treating patients infected with a HIV virus. Particularly, in the definition of HAART, there is a need to include novel pharmaceutically active compounds that will specifically be directed against other target molecules than the HIV protease and the HIV retrotranscriptase and which will act on targets involved in distinct stages of the disease.

SUMMARY OF THE INVENTION

The present invention relates to a method for the screening of candidate substances active against the infection of a subject by a HIV virus comprising the steps of : a) providing cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, b) incubating the cells provided at step a) with a candidate substance, c) measuring the expression level of NKp44L at the cell membrane, d) comparing the NKp44L expression value obtained at step c) with a reference NKp44L expression value determined in the absence of the said candidate substance, and e) selecting positively the said candidate substance when the NKp44L expression value measured at step c) is higher than the said reference NKp44L expression value.

This invention also pertains to kits for the in vitro screening of candidate substances active against the infection of a subject by a HIV virus comprising :

- cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, and

- a detectable ligand that binds specifically to NKp44L

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Down modulation of cell-surface NKp44L in CD4+ T cells expressing Nef protein.

Figure 1 A : CD4+ T cells were uninfected (Ul), treated with 3S peptide (3S) or infected with AD8 or NL HIV-1 viral strains. Cell-surface expression of NKp44L on the CD4 cell gate was analyzed before intracellular staining of HIV -infected cells by anti-p24 mAb flow. Regions used to determine the proportion of NKp44L expression of HIV-p24 cells are shown. Abscissa : HIV-p24 expression; Ordinate : NKp44L expression

Figure 1 B : Absence of co-expression of NKp44L and p24 antigen in CD4+ T cells from 4 HIV- infected patients. CD4 cells count per ul, and viral load (VL) were shown for each patient. Abscissa : HIV-p24 expression; Ordinate : NKp44L expression

Figure 1 C :Down-modulation of NKp44L expression on CD4+ T cells infected with recombinant vaccinia virus expressing Nef. CD4+ T cells were infected with recombinant vaccinia virus expressing HIV protein and then stimulated with 3S peptide. These were stained with anti- NKp44L mAb, without 3S peptide stimulation (thick line), stained with anti-NKp44L mAb after stimulation by 3S peptide (black line), or stained with IgM isotype control (dotted line). Ul, uninfected; WT, CD4+ T cell infected with a wild type vaccinia virus. Abscissa : NKp44L expression.

Figure 2. Nef protein protects CD4+ T cells from NK cell-mediated cytotoxicity. CD4+ T cells were uninfected (Ul), or infected with wild type HIV-1 AD8 virus (WT), or Nef deficient virus (Δnef).

Figure 2A : Cell-surface expression of NKp44L on the CD4 cell gate was analyzed before intracellular staining of HIV -infected cells by anti-p24 mAb flow. Regions used to determine the proportion of NKp44L expression of HIV-p24 cells are shown. Abscissa : NKp44L expression; Ordinate : HIV-p24 expression.

Figure 2B : Cytolytic activity of autologous NK cells after IL2 activation. NK cytolytic activity was tested in the presence of anti-lgM isotype control (black lines) or anti-NKp44L mAb (dotted lines). Square: uninfected cells; circle: cells infected with wild type virus; triangle: cells infected with Nef-deficient virus. Abscissa : NKp44 expression; Ordinate : CD107a expression. Figure 2C : Degranulation responses was determined by CD107a expression on CD3-CD56+ NK cells expressing NKp44. Region used to determine the proportion of degranulating NKp44+ NK cells are shown. Abscissa : NKp44 expression; Ordinate : CD107a expression. Fig 2D and Fig 2E Nef protein protects CD4+ T cells from NK cell-mediated cytotoxicity. CD4+ T cells were uninfected (Ul), or infected with wild type HIV-1 AD8 virus (WT), or Nef deficient virus (Δnef). Cytolytic activity of autologous (Figure 2D) or allogenic (Figure 2E) NK cells after IL2 activation. NK cytolytic activity was tested in the presence of anti-lgM isotype control (black lines) or anti-NKp44L mAb (dotted lines). Square: uninfected cells; Triangle: cells infected with wild type virus; Circle: cells infected with Nef-deficient virus.

Figure 3. Myristoylation of Nef protects CD4+ T cells from NK cell-mediated cytotoxicity. CD4+ T cells were infected with a panel of HIV-1 variants for Nef protein. ). Figure 3A : Cell-surface expression of NKp44L on the CD4 cell gate was analyzed before intracellular staining of HIV -infected cells by anti-p24 mAb flow. Regions used to determine the proportion of NKp44L expression of HIV-p24 cells are shown. Ul : Uninfected cells; WT cells infected with wild type virus; Δnef: cells infected with Nef-deficiency virus; mG2A, mp76/79A and ml_168/69A: cells infected with HIV Nef variants. Ordinate : HIV-p24 expression. Figure 3B : Cytolytic activity of autologous NK cells after IL2 activation. NK cytolytic activity was tested in the presence of anti-lgM isotype control (black lines) or anti-NKp44L mAb (dotted lines). Square: uninfected cells; circle: cells infected with mG2A variant virus; diamond: infected with mp76/79A variant virus; triangle: cells infected with ml_168/169A variant virus. Ordinate : CD107a expression.

Fig 3C. Nef protects CD4+ T cells from NK mediated cytotoxicity.

NK cytolytic activity was tested by 51 Cr in the presence of anti-lgM isotype control (black lines) or anti-NKp44L mAb (dotted lines). DB: WT infected cells; AΔ Nef deficient virus; O+: cells infected with mG2A variant virus; • O : infected with mP76/79A variant virus; *+: cells infected with ml_168/169A variant virus.

DETAILED DESCRIPTION OF THE INVENTION It has been shown according to the invention that the Nef protein from HIV induces a down-regulated expression of the NKp44L protein at the membrane surface of HIV virus- infected cells.

More precisely, it has been shown herein that the expression of NKp44L is practically abolished in CD4+ T cells infected with vaccinia virus expressing Nef protein. It has also been shown that, among the population of CD4+ T cells from patients infected with HIV, the down-regulation of the NKp44L expression exclusively affect those of the CD4+ T cells that are infected with the HIV virus. In contrast, the non-infected CD4+ T cells from the HIV-infected patients have a normal or a high NKp44L expression.

It has also been found herein that, because of the down regulation of the NKp44L expression that is induced by Nef protein, the HIV-infected CD4+ T cells were no more susceptible to lysis by NK cells.

Further, as shown in the examples herein, the conserved ability of patient-derived Nef proteins to act on NKp44L cell-surface expression demonstrates that this activity of Nef is conserved in vivo and is not restricted to laboratory-grown HIV strains. Taken altogether, the invention's findings show that Nef protein from HIV effectively prevents recognition of HIV-infected cells by cells of the innate immune system, namely the NK cells. Thus, the HIV-infected cells expressing Nef protein are escaping control by the immune system from the host.

More precisely, It is thus shown herein that HIV-1 induced simultaneously the destruction of non-infected cells, including non-infected CD4+ T cells, which over-express NKp44L, and also induces an increased capacity of the HIV-infected cells, including the HIV-infected CD4+ T cells, to prolong viral production and long-term persistence of the virus, by causing the Nef- induced down-regulation of NKp44L. The invention's findings are all the more surprising that it was previously known in the art that other proteins from HIV, like gp41 protein, induced an over-expression of NKp44L (see notably PCT Application n °WO/2005/076001 ).

It flows from the invention's findings that substances having the ability to restore the Nef- down-regulated expression of NKp44L in HIV-infected cells would be valuable active ingredients against an infection by HIV. Further, such substances, that would render HIV-infected CD4+T cells susceptible to NK cell lysis, would actually represent novel therapeutic tools for treating subjects infected with HIV, since these substances will act as immune system restoration agents and not as antiviral substances like most of the anti-HIV active ingredients that are presently used in conventional therapy.

This is why it has been designed herein screening methods aimed at selecting substances that possess the capacity of restoring NKp44L expression at the surface of cells expressing Nef protein from HIV.

An object of the present invention consists of an in vitro method for the screening of candidate substances active against the infection of a subject by a HIV virus comprising the steps of : a) providing cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, b) incubating the cells provided at step a) with a candidate substance, c) measuring the expression level of NKp44L at the cell membrane, d) comparing the NKp44L expression value obtained at step c) with a reference NKp44L expression value determined in the absence of the said candidate substance, and e) selecting positively the said candidate substance when the NKp44L expression value measured at step c) is higher than the said reference NKp44L expression value. As intended herein, a "subject" encompasses a mammal, including a human individual.

Most preferably, a subject consists of a human individual.

As used herein; a HIV virus encompasses HIV-1 and HIV-2 viruses. However, preferably, an HIV virus consists of an HIV-1 virus.

As intended herein, cells expressing a Nef protein from an HIV virus encompass notably (i) cells infected with wild-type HIV virus particles and which express Nef, (ii) cells infected with variant HIV virus particles and which express Nef, (iii) cells infected with recombinant virus particles comprising all or part of the HIV genome and which express Nef protein and (iv) cells transfected with a recombinant vector encoding Nef protein and which express Nef protein and (v) cells transfected with recombinant virus particles or a recombinant vector and which express a Nef-derived peptide or a protein derived from Nef which comprises one or more amino acid substitution(s), and/or one or more amino acid addition(s) and/or one or more amino acid deletion(s) as compared to the corresponding wild-type Nef protein..

Illustrative embodiments of HIV Nef-expressing cells that are found in the examples herein include (i) purified CD4+ T cells originating from human patients infected with HIV, (ii) cell lines infected by HIV proviral plasmids prepared from various natural HIV virus isolates and (iii) recombinant vaccinia virus comprising a DNA insert encoding Nef protein.

In certain embodiments, HIV Nef-expressing cells may be obtained by transducing recipient cells with lentiviral vectors comprising an expression cassette encoding a complete Nef protein, as disclosed notably by Le gall et al. (Le Gall et al., 1998, Immunity, Vol. 8 : 483- 495), Wang et al. (Wang et al., 2000, Proc. Natl. Acad. Sci. USA, Vol. 97 : 394-399), Sol-Foulon et al. (Sol-Foulon et al., 2004, J Biol Chem, Vol. 279 (n °30) : 31398-31408) or Dorival et al. (Dorival et al., 2008, Virology, Vol. 377 : 207-215). Illustratively, these lentiviral vectors may be used for transducing NKp44L-expressing cells selected from the group consisting of purified CD4+ T cells or Jurkat T cells, e.g. by performing cell transduction methods disclosed by Wang et al. (2000, cited above), Sol-Foulon et al. (Sol-Foulon et al., 2002, Immunity, Vol. 16 : 145- 155) or Percherancier et al. (Percherancier et al., 2003, J Biol Chem, Vol. 278 : 3153-3161 ).

In certain other embodiments, HIV Nef-expressing cells may be obtained by infecting or transfecting recipient cells with adenoviral vectors comprising an expression cassette encoding a complete Nef protein, as disclosed notably by Yamamoto et al. (Yamamoto et al., 2006, Microbes and Infection, Vol. 8 : 2522-2530). Again, the recipient cells may be NKp44L- expressing cells selected from the group consisting of purified CD4+ T cells or Jurkat T cells, e.g. by performing infection or transfection methods disclosed by Yamamoto et al. (2008, cited above). As specified above, those Nef-expressing cells also express NKp44L protein. NKp44L- expressing cells that may be used for performing the in vitro screening method according to the invention encompass (i) cells that express NKp44L constitutively, as well as (ii) cells that transiently express NKp44L.

As intended herein, "NKp44L-expressing cells" consist of cells that actually produce the NKp44L protein, either intracellular^, or at the cell membrane, or both intracellular^ and at the cell membrane. In other words, "NKp44L-expressing cells" encompass those cells that produce the NKp44L protein mainly intracellular^, or mainly at the cell membrane.

For the purpose of the screening method according to the invention, the essential cell marker that is measured is the level of NKp44L protein expression at the cell membrane, preferably irrespective of the level of NKp44L protein expression in the intracellular compartments.

Thus, as it is shown in the examples herein, the CD4+ T cells expressing Nef from a HIV virus remain "NKp-44L-expressing cells" although the NKp44L protein expression at their cell membrane is down-regulated or even abolished. One illustrative embodiment of NKp44L-expressing cells includes CD4+ T cell lines selected from the group consisting of CCRF-CEM (ATCC n ⁰ CCL-1 19), MOLT-3 (ATCC n ⁰ CRL-1552), MOLT-4 (ATCC n ⁰ CRL-1582), SUP-T1 [VB] (ATCC n ⁰ CRL-1942), and HH (ATCC n° CRL-2105).

Another illustrative embodiment of NKp44L-expressing cells includes Jurkat cell lines that express NKp44L constitutively (e.g. ATCC n ⁰ TIB-152). A further illustrative embodiment of NKp44L-expressing cells encompasses monocyte/macrophage-derived cell lines, including the U937 cell line (ATCC n ⁰ CRL-1593.2).

As already mentioned, the cells provided at step a) of the in vitro screening method according to the invention also encompass purified CD4+ T cells originating form an HIV- infected subject, as disclosed in the examples herein.

As intended herein, NKp44L protein encompasses the NKp44L protein of SEQ ID N⁰ L

As intended herein a "Nef" protein encompasses any Nef protein originating from an HIV virus, including from an HIV-1 or an HIV-2 virus, preferably from an HIV-1 virus, and wherein the said Nef protein comprises a N-terminal region bearing an intact myristoylation site. An illustrative embodiment of a HIV-2 Nef protein consists of the Nef protein of SEQ ID NO 2.

At step b) of the screening method, the cells are cultured in a suitable culture medium and in a suitable environment, e.g. in a humidified cell culture incubator at 37⁰C in a 5% CO₂ atmosphere.

At step b) the candidate substance to be tested is added to the cell culture at an appropriate final concentration and during an appropriate time period before measuring the NKp44L expression at step c).

Generally, a plurality of cell cultures are used for assaying the same candidate substance, so that a given cell culture, or alternatively a given subset of cell cultures, contains a determined final concentration of the substance to be tested, and that a serial of final concentrations of the candidate substance are assayed.

Also, for a given final concentration of a candidate substance to be tested, one or more incubation time periods before measuring NKp44L expression are assayed.

For a given candidate substance, final concentration in the cell cultures may range from

1 pg/mL to 10 mg/mL, In certain embodiments, the final concentration of the candidate substance in the cell culture is of at least 10 pg/mL, 100 pg/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL,

1 μg/mL, or 10 μg/mL. In certain embodiments, the final concentration of the candidate substance in the cell culture is of up to 1 mg/mL, or 100 μg/mL.

In certain embodiments of the screening method, the candidate substances may be selected from a library of compounds previously synthesised. In other embodiments of the screening method above, the candidate substances are selected from compounds, the chemical structure of which is defined in a database, for example an electronic database.

In further embodiments of the screening method above, the candidate substances are conceived de novo. The candidate substances may be selected from the group consisting of (a) proteins or peptides, (b) nucleic acids, and (c) organic or mineral chemical compounds.

The expression level of the NKp44L protein at the membrane surface may be measured by any method known in the art.

Whatever the measuring method which is used at step c), step c) preferably includes a step of incubating the cell cultures with a detectable ligand compound that specifically binds to NKp44L, so that step c) includes a step of measuring the detectable compound bound to the NKp44L in the population of cells contained in the cell cultures.

In certain embodiments of the ligand compound used in the screening methods above, said ligand compound consists of an antibody directed to the NKp44L protein of SEQ ID N°1 or of an antibody directed to the extracellular domain portion thereof. According to this first preferred embodiment, said ligand compound consists of a ligand compound produced by partial or complete biological synthesis, as defined previously.

Said anti-NKp44L antibody may consist of a polyclonal antibody which may be obtained by (i) administering an immunologically effective amount of the purified NKp44L protein to an animal, preferably in combination with an adjuvant of immunity, such as the Freund's complete adjuvant, (ii) then collecting the whole blood of the immunised animal and (iii) purifying the anti-

NKp44L polyclonal antibodies, such as for example by using an immunoaffinity chromatographic substrate onto which has previously been immobilised the purified NKp44L protein. These techniques for obtaining purified polyclonal antibodies are well known from the one skilled in the art.

Said anti-NKp44L antibody may consist of a polyclonal antibody, in which case said antiNKp44L antibody may be prepared from hybridomas obtained after fusion of B cells of animals immunised against the purified NKp44L protein with myeloma cells, according to the well known technique described by Kohler and Milstein in 1975 (Kohler G. and Milstein C, 1975, Nature, 256 : 495).

Said anti-NKp44L antibody may also consist of an antibody which has been produced by the trioma technique or by the human B-cell hybridoma technique described by Kozbor et al. in 1983 (Kozbor et al., 1983, Hybridoma, 2(1 ) : 7-16).

Said anti-NKp44L antibody may also consist of single chain Fv antibody fragments (United States Patent n⁰ US 4,946,778; Martineau et al., 1998), of antibody fragments obtained through phage display libraries (Ridder R., Scmitz R., Legay F., Gram H., 1995, Biotechnology

(N Y), 13(3) : 255-260) or of humanised antibodies (Reinmann KA et al., 1997, AIDS Res Hum

Retroviruses, 13(1 1 ) : 933-943; Leger OJ et al., 1997, Hum Antibodies , 8(1 ) : 3-16).

Most preferably, the anti-NKp44L antibody consists of a monoclonal antibody which is obtained by the following steps :

(i) preparing a batch of purified recombinant NKp44L protein of SEQ ID N°1 ; (ii) immunising mice, for example BALB/c mice, with an effective amount of the purified NKp44L protein provided at step (ii), for example through three successive injection of said purified protein, each spaced by a one month time period (iii) preparing hybridoma cell lines by fusion of the purified B cells of the mice immunised at step (ii), for example using the ClonaCell-HY hybridoma cloning kit according to the manufacturer's instructions (StemCell Technologies Inc., Vancouver, BC, Canada); (iv) culturing clones of the hybridoma cell lines prepared at step (iii) and selecting the clone(s) which secrete a monoclonal antibody directed against the NKp44L protein; and (v) purifying the monoclonal antibodies produced by the hybridoma cell clones which have been selected at step (iv).

A most preferred hybridoma cell clone producing an anti-NKp44L monoclonal antibody consists of the hybridoma cell line NKp44L # 7.1 . Anti-NKp44 and anti-NKp44L monoclonal antibodies are preferably prepared such as taught in the examples.

For preparing a batch of purified recombinant NKp44L protein of SEQ ID N °1 at step (i) of the method above, the one skilled in the art may perform a method comprising the following steps :

(i) transfecting a recipient cell host, preferably a mammal cell line such as COS-7 cells, with an expression vector into which has been inserted a nucleic acid encoding the Nkp44L protein of SEQ ID N °1 , or a polypeptide comprising the extracellular domain thereof, and wherein said nucleic acid is operably linked to expression signals comprising at least a promoter which is functional in said recipient cell host, so that the resulting transfected cell host actually produces the NKp44L protein, when place in appropriate culture conditions; (ii) culturing the transfected cell host in an appropriate culture medium, so that the NKp44L protein, or the extracellular portion thereof, is produced;

(iii) collecting the NKp44L protein, or the extracellular portion thereof, from the cell culture supernatant or from the cell lysate of the cultured transfected cell host; (iv) purifying the NKp44L protein, or the extracellular portion thereof, collected at step (iii), for example through immunoaffinity chromatographic substrate onto which anti-NKp44L antibodies, or alternatively purified NKp44 proteins, have previously been immobilised.

Conventional methods for preparing purified NKp44L are well known by the one skilled in the art.

In a second embodiment of the ligand compound used in any one of the methods above, said ligand compound consists of the purified NKp44 protein of SEQ ID N °3, or a polypeptide comprising the extracellular domain portion thereof. This second embodiment illustrates a further embodiment wherein the ligand compound is produced through complete biological synthesis.

As used herein, the extracellular domain portion of the NKp44 protein is located in the N- terminal part of the NKp44 protein of SEQ ID N °3 and consists of the amino acid sequence starting from the amino acid residue in position 22 and ending at the amino acid residue in position 169 of SEQ ID N °3.

For producing the NKp44 protein, or the extracellular domain portion thereof, under a purified form, the one skilled in the art will advantageously refer to the methods disclosed by Cantoni et al. (Cantoni C, Bottino C, Vitale M, Pessino A, Augugliaro R, Malaspina A, Parolini S, Moretta L, Moretta A, Biassoni R., 1999, NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily, J Exp Med 1999 Mar 1 ;189(5):787-96). For example, the NKp44 recombinant protein may be prepared under a purified form through the following steps : (i) transfecting a recipient cell host, preferably a mammal cell line such as COS-7 cells, with an expression vector into which has been inserted a nucleic acid encoding the Nkp44 protein of SEQ ID N°3, or a polypeptide comprising the extracellular domain thereof, and wherein said nucleic acid is operably linked to expression signals comprising at least a promoter which is functional in said recipient cell host, so that the resulting transfected cell host actually produces the NKp44 protein, when place in appropriate culture conditions; (ii) culturing the transfected cell host in an appropriate culture medium, so that the NKp44 protein, or the extracellular portion thereof, is produced;

(iii) collecting the NKp44 protein, or the extracellular portion thereof, from the cell culture supernatant or from the cell lysate of the cultured transfected cell host;

(iv) purifying the NKp44 protein, or the extracellular portion thereof, collected at step (iii), for example through immunoaffinity chromatographic substrate onto which anti-NKp44 antibodies, or alternatively purified NKp44L proteins, have previously been immobilised.

For performing the measure of the amount of said ligand compound that has bound to the cells, at step (c) of the screening method described above, it is most preferred that said ligand compound is labelled with a detectable molecule, so that the measure consists of detecting a physical signal produced by said detectable molecule, and wherein the value of said physical signal which is obtained reflects the amount of said ligand compound which is bound to the NKp44L protein expressed by the cells contained in the cell cultures that are assayed. In certain embodiments, the said detectable molecule consists of a radioactive molecule, for example when the ligand compound is itself radioactively labelled, through conventional techniques or also when the ligand compound also binds to a radioactively labelled detectable molecule. The said radioactive molecule may be labelled with a radioactive isotope selected from the group consisting of [³²P], [³H] and [³⁵S]. In other embodiments, the said detectable molecule consists of a fluorescent molecule.

The said fluorescent molecule is most preferably selected from the group consisting of Green Fluorescent protein (GFP) and the Yellow Fluorescent Protein (YFP), which are both well known from the one skilled in the art. Illustratively said fluorescent molecule consists of the fluoreporter FITC protein and for labelling, it may be used for FITC labelling kit which is marketed by Molecular Probes Inc. (U.S.A.).

In further embodiments, the said detectable molecule consists of a luminescent molecule. The said luminescent molecule is most preferably selected from the group consisting of luciferase.

In still further embodiments, the said detectable molecule consists of a receptor that is selectively recognised by a ligand molecule. In these still further embodiments, the said detectable molecule consists of a biotin, most preferably under the form of a biotinylated ligand compound, in which case the corresponding ligand molecule consists of a molecule containing an avidin or a straptavidin, said ligand molecule being either (i) radioactively labelled, (ii) fluorescent or (iii) luminescent, so that the physical signal which is detected for measuring the expression level of the NKp44L protein by the cultured cells under assay can be produced. At step (c) of the screening method according to the invention, the measure of the expression level of NKp44L and thus the measure of the amount of the ligand compound that is bound to the NKp44L protein expressed by the assayed cells contained in the cell cultures can be carried out using any one of the various techniques allowing the measure of the binding of a compound, and especially of a detectable compound, onto the membrane surface of cells, which are already available to the one skilled in the art.

Most preferably, step (c) of the screening method above is carried out by performing a flow cytometry analysis of the biological sample, using various detectable markers, most preferably various fluorescent markers, including the detectable ligand compound that specifically binds to the NKp44L protein, as it is fully disclosed in the examples herein.

In certain preferred embodiments, the flow cytometry analysis is performed with a detectable marker, most preferably a fluorescent marker, namely the detectable ligand compound that specifically binds onto the NKp44L protein, or onto the extracellular domain portion thereof. Typically, the detectable ligand compound is fluorescently labelled so as to emit a fluorescent signal at a given wavelength upon appropriate light excitation

When using the detectable markers above, the expression level of the NKp44L at the cell membrane of the cultured cells is determined by counting, by flow cytometric analysis, the number of cells that emit light at the selected given wavelength corresponding to the fluorescently labelled ligand compound that specifically binds to the NKp44L protein, as well as the corresponding fluorescence signal intensity.

As already described in the present specification, the cells provided at step a) of the screening method may consist of cells expressing NKp44L constitutively, e.g. cells from a Jurkat cell line.

In other embodiments, the cells provided at step a) of the screening method may consist of cells infected with a HIV virus expressing a complete Nef protein, e.g. purified CD4+ T cells originating from an HIV-infected individual.

In further embodiments, the cells provided at step a) of the screening method may consist of cell transfected with a vector encoding a complete Nef protein, e.g. primary culture CD4+ T cells or CD4+ T cell lines that have been infected with a vector selected from the group consisting of a HIV-1 proviral clone expressing a complete Nef protein, a recombinant vaccinia virus expressing a complete Nef protein, or any other recombinant vector that is known in the art for being functional in mammal cells; especially in cells of human origin, and which comprises inserted therein an expression cassette encoding a complete Nef protein.

In the most preferred embodiments of the screening method, the cells provided at step a) express a mirystoyl-Nef protein. In fact, any HIV Nef protein that is produced in a mammal cell, especially in a human cell, consists of a mirystoyl-Nef protein, provided that the said Nef protein comprises an intact N-terminal amino acid residue, and thus more generally an intact N- terminal protein region, which is the case for any one of the complete Nef proteins. It is recalled that the expression level of NKp44L is measured at step c) by incubating the said cells with detectable ligand that binds specifically to NKp44L, and performing the NKp44L expression level measure, e.g. by flow cytometry, immunofluorescence or immuno-chemistry methods, as it has already been described in detail previously in the present specification, and as it is fully shown in the examples herein.

In most embodiments of the in vitro screening method according to the invention, The NKp44L expression value is expressed as arbitrary units that simply reflect the number of NKP44L protein molecules that are exposed at the membrane surface of the cultured cells used. Illustratively, in the embodiments wherein the NKp44L expression value is measured at step c) by flow cytometry techniques, then the NKp44L expression value is expressed as arbitrary fluorescence signal units, or alternatively as the ratio of the cultured cells expressing NKp44L at their membrane, as shown in the examples herein.

At step d) of the screening method according to the invention, the expression level value of NKp44L at the cell membrane that is previously measured at step c) is compared to a determined reference expression value of NKp44L at the cell membrane of cells that have been cultured in the absence of the said candidate substance being tested. In certain embodiments of step d), the said reference value has been determined prior to performing the in vitro screening method with the candidate substance being tested. Illustratively, the said reference NKp44L expression value has been determined previously by performing the said screening method by omitting step b) of incubating the cells with a candidate substance. According to these embodiments, the said reference NKp44L expression value consists of the NKp44L expression value that is expected for the cells provided at step a) in the absence of any candidate substance. Thus, most preferably, the reference NKp44L expression value that is used at step d) is obtained by performing the method (at the exclusion of step b)) with the same kind of cells or with the same cell line as that which is provided at step a) of the in vitro screening method which is being performed. In other embodiments of step d), the said reference NKp44L expression value is determined at the same time when performing step c), by performing simultaneously to step c) a measure of the expression level of NKp44L at the cell membrane of one or more cell cultures that have not been incubated with the said candidate substance that is being tested.

At step e), an NKp44L expression value is considered as being "higher" than the reference NKp44L expression value when there is a statistically significant detectable increase of the membrane NKp44L expression measure in the cell cultures incubated with the candidate substance, as compared with the reference NKp44L expression value.

In most embodiments, the NKp44L expression value which is measured at step c) is considered as being higher than the reference NKp44L expression value when the said measured value is 0.5 times or more greater than the said reference NKp44L expression value, and preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 times greater than the said reference NKp44L expression value.

In certain embodiments of the screening method according to the invention, the said method comprises a further step f) of measuring the lysis activity of NK cells against cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein at their membrane, which Net- and NKp44L-expressing cells have been incubated with a compound that has been positively selected at step e).

In certain embodiments of the screening method herein, step f) above is followed by an additional step g) of selecting positively the candidate compound when the NK cell lysis activity against the Nef- and NKp44L-expressing cells that have been incubated with the said candidate compound is higher than the NK cell lysis activity against the Nef- and NKp44L-expressing cells cultured in the absence of the said candidate compound.

At step f) of the screening method, the measure of the NK cell lysis activity against the

Nef- and NKp44L-expressing cells, although it is performed in vitro, may directly reflect the therapeutical potential of the candidate compound by directly evidencing the biological activity of the said candidate compound, as regards inducing or enhancing HIV-infected cell lysis in a subject infected with a HIV virus.

The activated NK cells may consist of cells from a NK cell line, such as the NK92 cell line described by Gong et al. (Gong et al., 1994, Leukemia, vol.8 (4):652-658) or may consist of a primary culture of normal human purified NK cells.

In a specific embodiment of the screening method above, the activated NK cells and the

CD4⁺ T-cells are autologous in that they both come from the same HIV-infected patient.

In some embodiments, NK cell lines may be generated from Peripheral blood mononuclear cells (PBMC), and then purified using the StemSep® cell separation system and the NK cell enrichment antibody cocktail (StemCell technologies). NK purified cells were cultured in MyeloCult® 5100 medium (StemCell technologies) supplemented with 100 units of rhlL-2 (e.g. from Boehringer). The purity of these NK cell preparations may be evaluated by flow cytometry after staining with anti-CD3 (BD), anti-CD56 (BD), anti-NKp44, and anti-NKp46 antibodies. Preferably, the cytolysis measure consists of the conventional technique wherein the

Nef- and NKp44L-expressing cells, which are the target cells, are initially rendered radioactive with ⁵¹Cr, and wherein the cytolysis value consists of the percentage of cell lysis, as measured by the amount of ⁵¹Cr that is released in the cell culture medium by the lysed Nef- and NKp44L- expressing cells. Most preferably, the cytolysis value is obtained by assaying the cytolytic activity of the

NK cells at increasing effector (NK cells) to target (Nef- and NKp44L-expressing cells) ratios, for example from 1 :1 to 50:1 effector : target cell ratios.

Another object of the present invention consists of an in vitro method for the screening of candidate substances active against the infection of a subject by a HIV virus comprising the steps of : a) providing cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, b) incubating the cells provided at step a) with a candidate substance, c) measuring the lysis activity of NK cells against the cells obtained at step b), d) comparing the lysis activity of NK cells measured at step c) with a reference NK cell activity determined in the absence of the said candidate substance, and e) selecting positively the said candidate substance when the lysis activity of NK cells measured at step c) is higher than the said reference NK cell activity. All details for performing steps a) to e) of the screening method above are provided to the one skilled in the art in the present specification, including the examples herein, as well as in the corresponding references cited.

A further object of the present invention consists of a kit for the in vitro screening of candidate substances active against the infection of a subject by a HIV virus comprising : - cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, and

- a detectable ligand that binds specifically to NKp44L, and

- one or more reagents that are necessary for detecting the binding of the said ligand to the said cells.

In some embodiments, the said reagents necessary for detecting the binding of the said ligand to the said cells consist of antibodies directed against the said ligand that are labeled by a detectable marker, which encompasses a chromogenic marker, a fluorescent marker, an enzyme marker and a radioactive marker.

In the kit above, the cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein at their membrane encompass the various embodiments that have been previously described in the present specification.

Notably, the cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein at their membrane encompass those selected from the group consisting of CD4⁺ T cell lines and Jurkat cell lines.

In the kit above, the ligand compound encompasses the various embodiments that have been previously described in the present specification.

Most preferably, the detectable ligand compound consists of a labelled monoclonal antibody that specifically binds to the NKp44L protein of SEQ ID N°1 , or to the extracellular domain portion thereof, and the reagents necessary for detecting the binding of the said ligand to the said cells encompass are those which are useful for detecting the presence of a chromogenic marker, a fluorescent marker, an enzyme marker or a radioactive marker, that are well known in the art.

This invention also relates to the use of a kit comprising :

- a detectable ligand that binds specifically to NKp44L, for the screening of candidate substances active against the infection of a subject by a HIV virus.

Generally, the said kit consists of a kit as defined above.

In some embodiments, the said cells are selected from the group consisting of CD4⁺ T cell lines and Jurkat cell lines. In some embodiments, the said kit further comprises one or more reagents that are necessary for detecting the binding of the said ligand to the said cells, such as those described previously herein.

The instant invention is further illustrated, without in any way being limited to, the examples below.

EXAMPLES

A. Materials and Methods of the examples A.1. HIV-infected patients.

Peripheral blood from untreated HIV-infected patients were obtained after approval by the relevant institutional review board. All subjects provided written informed consent. Routine laboratory analysis of these patents included viral load determination, and complete blood count.

A.2. CD4⁺ T cells purification and culture

Leukocytes from healthy donors were obtained by buffy coat from the hospital blood bank (Hόpital Pitie-Salpetriere, Paris, France). CD4+ T cells were purified using CD4 microbeads (Mitenyi Biotechs). Flow cytometric analysis demonstrated a purity of >95% CD4⁺ cells. Purified CD4⁺ T cells were activated with 1 μgml-1 PHA-L in RPMI-1640 medium supplemented with 10% FCS, and then cultured with 100 IUml-1 IL2 (Roche), for an additional 2 days.

A3. peptides and antibodies The synthetic NH2-PWNASWSNKSLDDIW-COOH 15-mer peptide of SEQ ID N⁰ 4, called 3S, from gp41 HIV-1 protein was purchased from Covalabs. Purity was more than 80%, as verified by HPLC profile. Monoclonal anti-NKp44L antibody (IgM; #7.1 ) was previously described (Vieillard et al., 2005, Proc Natl Acad Sci USA 102(31 ): 10981 -10986).

A.4. Viral stock generation and HIV infection

Virus stocks were generated by transfection of proviral plasmids into 293T cells by the

Ca-phosphate method. Two days after transfection, culture supernatants were harvested and concentration of virus stocks determined as described (Fackler et al., 2006, Virology 351 :322-

339). HIV-1 infection of CD4⁺ T cells was carried out with 50ng of virus per 10⁶ cells for 2h30 at 37^<€. After incubation, cells were extensively washed and then cultured in presence of

10IUmM IL2 for 7 days.

A.5. HIV-1 Nef mutants Isogenic HIV-1 proviral clones encoding for established Nef mutants as previously described (Fackler et al., 2006, Virology 351 :322-339-). Briefly, the prototype chimera containing the HIV-1 NL4-3 provirus with the HIV-1 SF2 nef gene was designated wild type (WT), the HIV-1 NL4-3 provirus deleted to the nef gene was designated Δnef, and the respective nef mutants (m) were all designated according to their amino acid changes in the encoded Nef protein; including G2A (mutation G2A), AxxA (mutation P76/79A), and LLAA (mutation L168/169A), as previously described (Fackler et al., 2006, Virology 351 :322-339).

A.6. Recombinant Vaccinia virus expressing HIV-1 proteins CD4⁺ T cells were infected with wild-type (WT) vaccinia virus or with recombinant vaccinia viruses expressing various HIV-1 proteins at a multiplicity of infection of 20 plaque- forming units (pfu)cell ¹ , as previously described (Vieillard et al., 2005, Proc Natl Acad Sci USA 102(31 ): 10981 -10986-). Recombinant vaccinia viruses for HIV-BRU proteins provided from Transgene (Strasbourg, France). The efficacy of vaccinia infection was verified with polyclonal anti-vac Ab (AB technologies).

A.7. Flow cytometric analysis

FACS analysis was performed on purified CD4⁺ T cells. Isotype-matched immunoglobulin served as the negative control. Cells were incubated with 2 μg anti-NKp44L mAb (#7.1 ) for 1 h at A⁰O, washed in PBS/1 % BSA and then incubated in 100 μl of 1 :100 PE conjugated anti-mouse IgM mAb (BD) plus 5 μl APC-conjugated anti human CD4 mAb (Beckman Coulter) for 30 min at 4⁰C. These cells were fixed and permeabilized using cytofix/Cytoperm kit (BD), according to the manufacturer's instructions and then stained using anti-HIV-1 p24 mAb (KC57, Beckman Coulter) for 1 -hr at 4⁰C. At least 20 000 leukocytes were detected on a FACScalibur. Results were analyzed with cellQuest software and expressed as percentage of CD4⁺ T cells in the mononuclear cell gate.

A8. Cytolytic and deqranulation assays

NK cells were culture in the presence of 100IU/ml IL2 The cytolytic activity of NK cells was assayed in a standard 4-hr ⁵¹Cr-release assay. The role of NKp44L was analyzed by adding an anti-NKp44L (#7.1 ) mAb or its IgM isotype control (BD Biosciences), at a final concentration of 20 μgml-1 , and incubating them with the effector cells for 30 min at 37°C.

The degranulation assay was performed by CD107a detection, according to methods previously described. NK cells were incubated overnight with IL2 and then resuspended at 10⁶ cellml^'1 with target cells at a 1 :1 ratio in the presence of anti-CD107a mAb (BD Biosciences).

After 1 h of incubation, monensin (Sigma) was added at 6 μgml^"1 for an additional 4-h incubation.

Expression of CD107a was detected by flow cytometry following NK cell straining with anti-CD3, anti-CD56, and anti-NKp44 mAb (Coulter). Results were expressed as percentage of CD3^"

CD56⁺ NK cells. Example 1 : Expression of NKp44L on HIV-infected CD4⁺ T cells

In the present study, we wanted to determine whether HIV-infected CD4⁺ T cells integrating HIV provirus expressed NKp44L. For this purpose, purified CD4⁺ T cells were in vitro infected with two different strains of HIV-1 (AD8 and NL4-3). To detect cells integrating virus, we stained for intracellular HIV-1 p24 Ag.

As shown in Figure 1 A, NKp44L was absent on CD4⁺ T cells before HIV-1 infection. However, when infected with either NL4-3 or AD8, 30 to 48.5% of CD4⁺ T cells expressed NKp44L. More interestingly, the expression of NKp44L was strikingly observed on cells, which are not infected by HIV-1. Similar results were observed using NKp44-lg fusion protein to detect NKp44 ligand (data not shown). To avoid the possible bias due to the in vitro experiments with a limited number of HIV-1 isolates, expression of NKp44L was next performed on CD4⁺ T cells from HIV-infected patients. In line with the in vitro experiments, NKp44L was highly expressed on uninfected CD4⁺ T cells from HIV-infected patients, contrasting with the very little expression on p24⁺ H I V- infected cells, whatever the patient tested (Figure 1 B). Together, these data demonstrate that HIV-infected cells express any NKp44 ligand at the cell-surface level suggesting a viral escape mechanism to protect HIV-infected cells from NK cells attacks.

Example 2 : Nef expression protects HIV-infected CD4⁺ T cells from NK cell-mediated cytotoxicity

To obtain further insight in the role of HIV-1 proteins in the modulation of cell-surface expression of NKp44L on p24⁺ H I V- infected cells, experiments were performed using a panel of recombinant vaccinia virus expressing HIV-1 viral proteins. As Figure 1 C shows, the expression of NKp44L is practically abolished in CD4⁺ T cells infected with vaccinia virus expressing Nef protein. Interestingly, none of the other HIV proteins tested appeared to influence the expression of NKp44L, with an exception for gp41 , which induced an over-expression of NKp44L, as previously described (Vieillard et al., 2005, Proc Natl Acad Sci USA 102(31 ): 10981 -10986). This suggests that the HIV-1 Nef protein blocks cell surface expression of NKp44L. To confirm the involvement of Nef on NKp44L expression, CD4⁺ T cells were infected with WT and Δnef viruses and stained for NKp44L and intracellular HIV p24 antigen. As Figure 2A shows, similar level of NKp44L expression was obtained in cells infected either with WT or Δnef HIV infectious particles. However, quantification of cells which co-expressed NKp44L and HIV p24 antigen, revealed that more than 46% of Δnef-infected cell population were positive, as compared to less than 3% for the WT-infected cell population (Figure 2A).

The susceptibility of CD4⁺ T cells infected with either WT or Δnef viruses against autologous NK cells were compared. ⁵¹Cr release assay shown that NK cells were non efficient against uninfected as well as WT-infected CD4⁺ T cells (Figure 2B). By contrast, after infection with Δnef-HIV particles, CD4⁺ T cells were highly more susceptible against autologous IL2- activated NK cells. Interestingly, treatment of CD4⁺ T cells with the anti-NKp44L mAb significantly reduced their cytolytic potentiality (Figure 2B). To confirm the cytolytic activity of autologous NK cells, against WT and Δnef-infected cell populations, we tested their degranulation capacities. As expected, the cell surface mobilization of CD107a was around 2- times more frequent on NK cells in presence of Δnef-infected-CD4⁺ T cells than in presence of WT- infected-CD4⁺ T cells (Figure 2C).

Example 3 : Myristoylation of Nef is required to modulate NKp44L expression and cytolytic activity

In order to gain insights into the escape mechanism mediated by Nef from NK-cell recognition, a panel of isogenic HIV-1 NL4-3 proviral clones encoding established Nef variants with mutation at various amino acid residues was tested. It is well documented that to down modulate HLA class-l and CD4 receptors, Nef uses different domains. However an N-terminal myristoylation signal, required for localization at the plasma membrane, has been shown to be critical for all Nef activities. Viruses including Nef gene with mutations that abolish N-terminal myristoylation (G2A), the capacity to down modulate HLA class-l (P76/79A) or CD4 molecules (L168/169A) were tested. When compared with WT Nef , both P76/79A and L168/169A Nef mutants induce similar profile concerning the specific expression of NKp44L on non infected CD4⁺ T cells (Figure 3A). By contrast, in presence of G2A mutant, which abolished the myristoylation capacities of Nef, a significant proportion of p24⁺-infected CD4⁺ T cells expressed NKp44L, at a level previously observed with Δnef HIV-1 particles (Figure 3A).

After having shown a differential expression of NKp44Lin presence of Nef variants, it was important to assess if this could affect their sensitivity to NK lysis. For this purpose, we evaluated the ability of IL2-actived NK cells to destroy autologous CD4⁺ T cells infected with the panel of Nef variants in a 4-hour ⁵¹Cr release assay in the presence of interfering antibodies to NKp44L. As shown in Figure 3B, a significant increase of G2A-infected CD4⁺ T target cell destruction was detected, as compared to WT-infected cells, at a level previously detected with CD4⁺ T cells infected with Δnef HIV-1 particles. This functional effect was virtually abrogated when the target cells were selectively neutralized with the NKp44L mAb. By contrast, with CD4⁺ T cells infected with either P76/79A or L168/169A Nef mutants, the level of cytotoxicity remained closed to the WT-infected control cells, and was not significantly decreased following anti-NKp44L treatment (Figure 3B). In line with these data, the mobilization of CD107a on autologous IL2-activated NK cells expressing NKp44, was more frequent against both Δnef- and G2A-infected CD4⁺ T cells than against cells infected with WT, P76/79A and L168/169A viral particles (Figure 3C). Taken together, these results suggested that conserved residues and motif of Nef that modulate expression of either HLA class-l or CD4 molecules are dispensable, but that the myristoylation of Nef protein could play a key role for the down-modulation of NKp44L on p24⁺- infected CD4⁺ T cells and then for their protection against NK cell- lysis. Table 1 : Sequence references

Claims

1. An in vitro method for the screening of candidate substances active against the infection of a subject by a HIV virus comprising the steps of : a) providing cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, b) incubating the cells provided at step a) with a candidate substance, c) measuring the expression level of NKp44L at the cell membrane, d) comparing the NKp44L expression value obtained at step c) with a reference NKp44L expression value determined in the absence of the said candidate substance, and e) selecting positively the said candidate substance when the NKp44L expression value measured at step c) is higher than the said reference NKp44L expression value.

2. The method according to claim 1 , wherein the cells provided at step a) consist of cells expressing NKp44L constitutively.

3. The method according to claim 1 , wherein the cells provided at step a) consist of cells infected with a HIV virus expressing a Nef protein.

4. The method according to claim 1 , wherein the cells provided at step a) consist of cells transfected with a vector encoding a complete Nef protein.

5. The method according to claim 1 , wherein the cells provided at step a) express a myristoyl-Nef protein.

6. The method according to any one of claims 1 to 5, wherein the expression level of NKp44L is measured at step c) by incubating the said cells with detectable ligand that binds specifically to NKp44L.

7. The method according to any one of claims 1 to 6, wherein the expression level of NKp44L is measured at step c) by flow cytometry, immuno-fluorescence, or immuno-histochemistry.

8. The method according to any one of claims 1 to 7 further comprising step f) of measuring the lysis activity of NK cells against cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein at their membrane, which Nef- and NKp44L-expressing cells have been incubated with a compound that has been positively selected at step e).

9. The method according to claim 8 further comprising step g) of selecting positively the candidate compound when the NK cell lysis activity against the Nef- and NKp44L-expressing cells that have been incubated with the said candidate compound is higher than the NK cell lysis activity against the Net- and NKp44L-expressing cells cultured in the absence of the said candidate compound.

10. An in vitro method for the screening of candidate substances active against the infection of a subject by a HIV virus comprising the steps of : a) providing cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein, b) incubating the cells provided at step a) with a candidate substance, c) measuring the lysis activity of NK cells against the cells obtained at step b), d) comparing the lysis activity of NK cells measured at step c) with a reference NK cell activity determined in the absence of the said candidate substance, and e) selecting positively the said candidate substance when the lysis activity of NK cells measured at step c) is higher than the said reference NK cell activity.

1 1. A kit for the in vitro screening of candidate substances active against the infection of a subject by a HIV virus comprising :

- cells expressing a Nef protein from an HIV virus, and expressing NKp44L protein,

- a detectable ligand that binds specifically to NKp44L, and

12. The kit according to claim 1 1 , wherein the said cells are selected from the group consisting of CD4⁺ T cell lines and Jurkat cell lines.

13. The use of a kit comprising :

14. The use according to claim 13, wherein the said cells are selected from the group consisting of CD4⁺ T cell lines and Jurkat cell lines.

15. The use of a kit according to any one of claims 13 and 14, wherein the said kit further comprises one or more reagents that are necessary for detecting the binding of the said ligand to the said cells.