NZ721311B2 - Therapeutic methods and compositions - Google Patents

Abstract

The present invention relates to uses and products based on the finding that secreted Phospholipase A2 group IB (GIBsPLA2) is a factor in CD4 T-cell immunodeficiency, particularly in subjects with HIV. The claimed invention relates to use of inhibitors of GIBsPLA2 in preparing a medicament for treating a CD4 T-cell immunodeficiency where the inhibitor is anti-GIBsPLA2 antibody or derivative, soluble GIBsPLA2 receptor, inhibitory nucleic acid based on the GIBsPLA2 gene sequence, or a peptide of 3 to 20 amino acids obtained by selecting test peptides that inhibit GIBsPLA2. The claimed invention also relates to injectable pharmaceutical compositions comprising an inhibitory anti-GIBsPLA2 antibody or derivative and a carrier or excipient. ing a CD4 T-cell immunodeficiency where the inhibitor is anti-GIBsPLA2 antibody or derivative, soluble GIBsPLA2 receptor, inhibitory nucleic acid based on the GIBsPLA2 gene sequence, or a peptide of 3 to 20 amino acids obtained by selecting test peptides that inhibit GIBsPLA2. The claimed invention also relates to injectable pharmaceutical compositions comprising an inhibitory anti-GIBsPLA2 antibody or derivative and a carrier or excipient.

Description

THERAPEUTIC METHODS AND COMPOSITIONS FIELD OF THE INVENTION The present invention relates to compositions and methods for modulating the immune system in a subject in need thereof. The invention more particularly discloses the existence and characterization of a key endogenous factor of the immune response and provides novel therapeutic and diagnostic s and compositions based on a modulation of this factor. The invention particularly provides itions and methods suitable to stimulate or inhibit CD4 T cell-mediated immune responses in a subject, as well as methods and compositions for monitoring immunodeficiency, including immunodeficiency associated with human immunodeficiency virus (HIV) infection. Also provided are methods and compositions to diagnose and assay CD4 T-cell-defects that t after antiretroviral therapy, as well as methods to develop drugs able to specifically treat this immunodeficiency.

INTRODUCTION CD4 T lymphocytes play a inent role in controlling the immune system (both ar and humoral responses) and are critical in various disease conditions.

During the immunological e associated with HIV pathogenesis, less than 0.5% of all CD4 T cells are actually infected (as measured in the peripheral blood), but the great majority of CD4 T cells shows major regulatory dysfunction. Uninfected CD4 T lymphocytes progressively loose their function, become anergic, and their s se ing in CD4 lymphopenia. Anergy and lymphopenia are the hallmarks of the deficiency characterizing HIV-infected patients. The mechanisms behind these phenomena have never been fully elucidated (l ).

WO 97140 Immune activation and inflammation also play a critical role in HIV pathogenesis (2, 3). The inventors have previously demonstrated that a decrease in responsiveness to interleukin-2 (IL-2), leading to CD4 anergy (4), and a ion in responsiveness to interleukin-7 (IL-7) which, by disrupting the D4 regulatory loop, participates in the mechanisms leading to CD4 lymphopenia (5). The mechanisms involved have been uted to defects in the Janus kinase (Jak) / Signal Tranducer and Activator of Transcription (STAT) y (6, 7). Similar results have been obtained by other laboratories (8, 9). In this regard, compartmentalization of the IL-7 receptor (IL-7R) is required to initiate normal CD4 T cell responses (10). Upon IL-7 binding, the two chains of the IL-7R (IL-7R alpha and gamma-c) are first driven into membrane microdomains (MMD). These are cellular compartments which, like lipid rafts, are rich in terol and sphingomyelin, but they also contain very significant amounts of structural and functional proteins (11). IL-7R complexes induce a reorganization of the cytoskeleton which then interacts with its meshwork. These two successive steps would be required for initiation of the AT pathway (12).

The present inventors have investigated the mechanisms behind the unresponsiveness of CD4 T lymphocytes in viremic HIV-infected patients (VP). The experiments provided herein demonstrate that chronic activation of CD4 T lymphocytes drives them into an aberrant state of activation/differentiation which s them refractory to certain physiological s such as those delivered by interleukin-7.

Furthermore, the present invention reports the identification, isolation and characterization, from human plasma, of the protein responsible for this aberrant state of CD4 T cell activation. For the first time, the invention thus discloses that immunosuppression can be mediated by an endogenous ating protein which, upon expression, is able to induce tion and inactivation of CD4-T cells and, upon inhibition, can stimulate the immune system in a t.

Based in part on these remarkable findings, the invention now provides new methods, compositions and compounds for modulating the immune system, particularly for modulating the immune system in subjects having altered ty (e.g.; immuno- depressed or pathologic immune reactions). The invention further provides novel methods for treating immune diorders by ting CD4 T cells. The invention is particularly suited for treating immunodeficiencies linked to CD4 T cell impairement, such as immunodeficience syndrome ated with HIV-infection. The invention also provides reagents and methods for characterizing the aberrant activation state, veness to 1L7 and/or for monitoring immunoresponse impaired in HIV infected patients. Response of CD4 T cells can be evaluated in untreated or treated patients with antiretroviral drugs, and qualify their response to treatment and evaluate the competency of their CD4 T cells.

SUMMARY OF THE INVENTION An object of the invention relates to a method for modulating an immune response in a subject, sing exposing the t to a compound that modulates the amount (e.g., sion) or ty of GIBsPLA2.

A further object of the invention relates to a method of treatment of an immune disorder in a subject, comprising exposing the subject to a compound that modulates the amount (e.g., expression) or activity of GIBsPLA2.

A further object of the invention relates to a method of treatment of an immune disorder in a subject, comprising modulating the amount (e.g., expression) or ty of A2 in the subject.

[0010] Another object of the invention relates to the use of a compound that modulates the amount (e.g., expression) or activity of GIBsPLA2 for the cture of a medicament for modulating an immune response or for treating an immune disorder in a subject.

Another object of the ion relates to a GIBsPLA2 modulator for use in a method of modulating an immune response or of treating an immune disorder in a subject.

Another object of the invention relates to a GIBsPLA2 modulator for use to modulate White blood cells in a subject.

In a first embodiment, the invention is used to induce or stimulate an immune response in the t, and comprises inhibiting GIBsPLA2 in said t, or exposing the subject to a GIBsPLA2 inhibitor. Such embodiment is particularly suited to treat immuno-deflcient subjects or subject in need of stimulated immunity (e.g., infectious diseases, , etc.).

A particular object of the invention thus resides in a method of stimulating an immune response in a subject, comprising inhibiting A2 in said subject or exposing the subject to a GIBsPLA2 inhibitor.

A further object of the invention relates to a method of treating an infectious disease in a subject, comprising inhibiting GIBsPLA2 in said subject or exposing the subject to a GIBsPLA2 inhibitor.

A more particular embodiment of the invention relates to a method of treating AIDS in a HIV-infected subject, comprising inhibiting GIBsPLA2 in said subject or exposing the t to a A2 inhibitor.

In a particular embodiment, exposing the subject to an inhibitor comprises administering the inhibitor to the subject. In another embodiment, exposing the subject to an inhibitor ses ating the subject against GIBsPLA2.

[0018] In this regard, in a particular embodiment, the invention relates to a method for stimulating the immune system of a subject in need thereof, the method comprising ating the subject against GIBsPLA2.

In another particular ment, the invention relates to a GIBsPLA2 antigen for use to vaccinate a subject in need thereof.

In another aspect, the invention is used to reduce or suppress an unwanted or deletorious immune se in the subject, and comprises causing or increasing GIBsPLA2 in said subject, or exposing the subject to a GIBsPLA2 t or activator.

Such ment is particularly suited to treat subjects having abnormal and/or pathologic immune responses (e.g., auto-immune diseases, inflammation, urticaria, eczema, allergies, asthma, etc.).

In a further aspect, the invention provides methods for diagnosing human immunodeficiency associated with CD4 T cell tion. In some ments the methods comprise (a) providing a sample containing a body fluid, preferably plasma from a subject, and (b) detecting the presence of GIBsPLA2 in the sample. In some embodiments of the methods the immunodeficiency is immunodeficiency associated with human immunodeficiency virus (HIV) infection. In some ments the method ses contacting the sample with an antibody specific for GIBsPLA2. In some embodiments of the methods the presence of GIBsPLA2 in the sample is detected by an enzyme-linked immunosorbent assay (ELISA).

In another aspect, the invention provides methods for identifying candidate immunodeficiency therapeutic agents. In some embodiments the deficiency is associated with CD4 T cell alteration. In some embodiments of the methods, the human immunodeficiency ated with CD4 T cell alteration is caused by viral ion, particularly human immunodeficiency virus (HIV) infection. In some embodiments the methods comprise: (a) contacting CD4 T lymphocytes with GIBsPLA2 in the presence of an agent, (b) measuring GIBsPLA2-induced CD4 T cell activation, and (c) comparing the level of GIBsPLA2-induced CD4 T cell activation in the presence of the agent with the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent. In some embodiments of the methods, if the level of A2-induced CD4 T cell activation in the presence of the agent is lower than the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent, then the agent is identified as a candidate immunodeficiency therapeutic agent. In some embodiments of the s, if the level of GIBsPLA2-induced CD4 T cell activation in the presence of the agent is not lower than the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent, then the agent is identified as a candidate immunosuppressing therapeutic agent. In some ments the methods comprise measuring A2-induced CD4 T cell activation by determining the number of MMD per CD4 T cell. In some embodiments the methods comprise measuring GIBsPLA2-induced CD4 T cell activation by determining the mean diameter ofMMD on CD4 T cells. In some embodiments the methods comprise measuring GIBsPLA2-induced CD4 T cell activation by determining the IL-7 responsiveness of CD4 T cells.

In another aspect, the invention relates to a ceutical composition comprising a GIBsPLA2 modulator and a pharmaceutically acceptable carrier or excipient.

In a preferred embodiment, the GIBsPLA2 modulator is a GIBsPLA2 inhibitor, more preferably selected from an antibody or a fragment or derivative thereof, an inhibitory nucleic acid, a peptide or a small drug. In another ular embodiment, the GIBsPLA2 tor is a GIBsPLA2 t or tor, more particularly a GIBsPLA2 protein.

[0024] In another aspect, the invention relates to a vaccine composition comprising a GIBsPLA2 antigen (e.g., an immunogenic GIBsPLA2 protein or an epitope-containing fragment thereof), a pharmaceutically acceptable carrier or excipient and, optionally, an adjuvant. In a preferred ment, the GIBsPLA2 antigen is a GIBsPLA2 protein or a fragment thereof treated to (i) increase its immunogenicity in human ts and/or to (ii) reduce its biological activity.

The invention may be used in any mammal. It is particularly suited for use in human subjects. It may be used to increase the immune response in any mammal, and it is ularly adapted to induce potent CD4-T cell activity in immuno-depressed subjects.

BRIEF DESCRIPTION OF THE DRAWWGS Figures 1a to 1e shows that, before any stimulation, CD4 T cells from VP show an aberrant state of activation with many large membrane microdomains that are unaffected by IL-7. (a) Membrane omains (MMD) were labelled with a toxin subunit B (CtXB-AF48 8) and analyzed by STED microscopy. From top to bottom, purified CD4 T cells from HD, VP and PHA-activated (40 ug/ml, 30 min) HD T cells. For each group the top half of a representative CD4 T-cell before and after IL-7 stimulation (2 nM, min) is shown from Z-stack image series. CD4 T lymphocytes were also d with cholesterol oxidase (COase, 31uM, 25min) plus sphyngomyelinase , 2.7uM, 5min) before stimulation by IL-7. (b, c) MMD were counted on the entire surface of the purified CD4 T cells.

An average of 50 cells were examined. (b) HD cells before (HDc: NS) and after IL-7 stimulation (HDc: IL—7). (0) VP cells before (VPc: NS) and after IL-7 stimulation VP (VPc: IL-7), PHA-activated HD cells before (HDc: PHA) and after IL-7 stimulation (HDc: PHA/IL-7). (d, e) MMD size was measured at the surface ofpurified CD4 T cells (d) 1L- ulated HD cells (HDc: IL-7), (e) ILstimulated VP cells (VPc: IL-7) and IL ated PHA- pre-activated HD cells (HDc: IL-7).

[0030] Figures 2a to 2c show that IL-7R chains from VP CD4 T-cells are embedded in detergent-resistant microdomains (DRM) that are unaffected by IL-7.

Purified CD4 T lymphocytes were lysed (0.5% Triton X-100) and 200ul of the lysate was loaded on a 5-40% sucrose gradient. After 16h of centrifiigation (50k1pm) at 4°C, 18 fractions were collected (#1 left = tube top = 5% sucrose; #18 right = tube bottom = 40% e). Each fraction was analyzed on SDS-PAGE (7% acrylamide-bis). Flottilin, IL-7R alpha and gamma-c were detected by immunoblotting (10). (a) Flottilin was used as a marker to indicate low density fractions corresponding to DRM and high-density fractions outside rafts. (b) IL-7Ralpha and (c) gamma-c bands are shown for purified non- stimulated HD CD4 T-cells (HDc: NS), ILstimulated HD cells (HDczlL-7), non stimulated VP cells (VPczNS) and PHA-activated HD cells (HDc:PHA).

Figures 3a to 3e show that IL-7R fiinction is altered in membrane microdomains of VP CD4 T-cells. (a) mensional effective diffiJsion rates Defy for IL-7Ralpha were measured as ped in Figure 7. ion rates were also measured after adding various drugs: COase (3luM, 30min) plus SMase (2.7uM, 5min) (CO/SM), Col (lOuM, 30min) plus CytD (20uM, 30min) (CytD/Col), or in the presence of all these inhibitors (all). CD4 T cells from HD (HDc) and VP (VPc) were studied, as were FHA-activated HD CD4 T cells (HDc: PHA). Bars indicate SEM from 5 independent experiments. More mental data are given in Figure 8.

[0035] (b) IL—7-induced phosphorylation and nuclear translocation of STATS were ed using rabbit phospho-STATS labelled with goat abbit-Atto642 and analyzed by pulsed-STED microscopy (0.5um slices). The ments involved purified non stimulated HD CD4 T cells (HDc: NS), ILstimulated HD CD4 T cells (HDc: IL-7), non stimulated VP CD4 T cells (VPc: NS), ILstimulated VP CD4 T cells (VPc:]L-7), PHA- activated HD CD4 T cells (HDc:PHA) and PHA-activated HD CD4 T cells stimulated by IL-7 (HDc:PHA/IL-7). The effects of colchicin plus cytochalasin D are shown in the left panel. (c, d, e) After IL-7 stimulation, the kinetics of phospho-STATS appearance in the cytoplasm and accumulation in the nucleus were measured using ImageJ software. (c) HD CD4 T cells (black line) and HD CD4 T cells treated with C01 plus CytD (blue line), ((1) VP CD4 T cells (red line) and (e) PHA-activated HD CD4 T cells (green line). 2014/078969 Figures 4a to 4d show that plasma from VP induces an aberrant activation pattern in HD CD4 T cells as measured by the number of MMD. (a) Representative images of HD CD4 T cells treated with plasma (10%) from VP (HDc: VPp), HIC (HDc: HICp) or ART patients (HDc: ARTp) are shown. MMD were stained with cholera toxin (CtXB-AF488). For each group the top half of a representative CD4 T-cell from Z-stack images before (left) and after IL-7 stimulation (2nM, 15min) (right) is shown. (b) MMD induced at the surface of CD4 T-cells (HDc) by plasmas (10%) from 5 ent VP (VPpl to VPp5). Results were obtained from the analysis of 50 cells before (white) and after (blue) IL-7 stimulation. Mean values and quartiles are shown. (c) Comparison of the effects of plasmas from HD (HDp), VP (VPp), HIC (HICp) and ART patients (ARTp) afier (blue) and before (white) IL-7 stimulation. ((1) Dose (0.01% to esponse obtained with the plasmas described in c.

The number of MMD induced at the surface of HDc CD4 T-cells is shown. The effect of VP plasma is shown as a plain red line.

Figures 5a to 5d show that plasma from VP inhibits ILinduced STATS phosphorylation and nuclear translocation ofphospho-STATS in HD CD4 T lymphocytes. (a) Before IL-7 stimulation, purified HD CD4 T cells were pre-incubated with plasma (10%). lLinduced phosphorylation and nuclear translocation of phospho- STATS were followed by -STED copy (0.5um slice). The following plasmas (10%) were studied: control (HDc: NS), VP (HDc: VPp), HIC (HDc: HICp) and ART patients (HDc: ARTp). (b) is of phospho-STATS recovered in the cytoplasm (blue) and nucleus (red) of ILstimulated HD CD4 T-cells pre-treated with plasmas from 5 different VP (10%). 2014/078969 (c) Comparison of the effects of plasma (10%) pre-incubation on IL stimulated HD CD4 T cells. Plasma were from HD (HDp), VP (VPp), HIC (HICp) and ART patients (ARTp) ((1) Dose -10%)-response obtained with the plasmas as measured by the inhibition ofphospho-STAT5 nuclear translocation in lLstimulated HD CD4 T-cells.

The effect ofVP plasma is shown as a plain red line.

Figures 6a to 6d show molecular characterization of the Refractory state Inducing Factor (RIF) recovered from VP plasma. (a) Treatment of VP plasma by trypsin, DNase, RNase and . RIF activity was followed by measuring the number of MMD and effects on ILinduced nuclear phospho-STAT5 in HD CD4 s. (b) RIF MW was measured by gel filtration on a ex G100 column.

RIF activity on HD CD4 T-cells was followed by measuring the numbers ofMMD induced by the different fractions of the column (thick red curve). Each fraction was also tested for the presence of viral ns by dot blot using onal antibodies from VP plasma.

Background obtained with HD plasma has been cted. Experiments were repeated three times. (c) RIF MW was also measured after gel filtration on a Sephadex G100 column and its activity followed by inhibition of ILinduced phospho—STAT5 as measured by FACS. Percentages of maximum ILinduced phospho-STAT5 were recorded. The amount of protein in each fraction is also reported. Experiments were repeated twice. (d) Isoelectric point was measured as follows. RIF eluted from the Sephadex G100 column was loaded onto an anion (MonoQ) or cation (MonoS) exchange column.

RIF activity was eluted by pH-step buffers. The number of MMD on HD CD4 T-cells was d against pH.

Figures 7a to 7c show a 2D gel analysis of the IL-7 signalosome in purified CD4 T cells from HD, VP and ILstimulated HD cells. (a) non-stimulated (NS) HD CD4 T-cells. (b) VP CD4 T-cells. (c) ILstimulated HD CD4 T-cells. s 8a to 8g show an analysis of the diffusion rate of lpha at the surface of purified CD4 T cells from HD, VP and imulated HD cells. (a, d) at the surface of HD CD4 s, (b, e) at the surface of VP CD4 T cells, (c, i) at the surface of HD CD4 T cells pre-activated with FHA (l ug/ml). (g) Scheme of the mechanism of IL- 7Ralpha diffusion embedded in MMD before and after treatment by MMD inhibitors or cytoskeleton inhibitors.

[0054] Figures 9a to 9d showd a schematic representation ofthe hypothetical mode of action of RIF on HD CD4 T cells and mechanism of IL-7 unresponsiveness. RIF induces abnormal MMD which are non onal. The IL-7 signalosome is therefore altered and the cells remain unresponsive to the cytokine, as in VP CD4 T cells. Aberrant tion patterns and signalling defects in RIF-induced HD CD4 T cells and in VP CD4 T cells are undistinguishable. The left part of the scheme illustrates the different steps in the mechanisms of lL-7 signal transduction in HD (10, 12). (a) In resting CD4 T cells, before IL-7 recognition, the IL-7R chains are associated but their ytoplasmic domains are far apart and the signaling molecules Jakl and Jak3 are not interacting.

[0056] (b) In ILactivated CD4 T cells, the IL-7R is compartmentalized in normal MMD (90 nm in diameter) and the signalosome becomes functional. After eleton organization, STAT5A and STAT5B are phosphorylated in contact With the IL- 71VJakl/Jak3 complexes then migrate to the nucleus by moving along the microtubules as previously discussed (12).

[0057] The right part ofthe scheme illustrates the etical mechanism of action of RIF. The proposed mechanism of action is derived from preliminary data and comparison of RIF-induced defects with the alterations characterized in purified CD4 T cells from VP (unpublished data). (c) RIF induces many large abnormal MMDs. IL-7Rs are embedded in abnormal MMDs and their ability to induce a functional signalosome is altered. (d) RIF-treated HD CD4 T cells are unresponsive to lL-7. Jakl and Jak3 phosphorylate STATS, gh with reduced kinetics, but phospho-STATS do not migrate into the s due to the lack of cytoskeleton and microtubules organization.

Panels a, b, c and (1 show STED microscopy images of MMD labelled with Cth: AF488 (half pile of Z-stack from CW-STED). Panels b and (1 show tubulin d with rabbit anti-tubulin/goat anti-rabbit-Atto642, actin stained with mouse anti-actin/goat- anti-mouse-Chr494 and o-STATS stained with rabbit anti-phospho-STATS/goat- anti-rabbit-Atto642. Pulsed-STED microscopy shows a 0.5um slice of methanol- permeabilized CD4 s. After IL-7 stimulation, actin in the MMD cytoplasmic area of RIF-treated HD CD4 T lymphocytes fails to trate as structured pads and does not form a cortex surrounding the nucleus, unlike in HD. Furthermore, the tubulin in these RIF- treated HD CD4 T cells, like in VP CD4 T cells, fails to form microtubules which have been hypothesized as being critical rods bridging the cytoplasm and nuclear membrane and thereby essential for STATS nuclear translocation.

Summary ofthe defects: Circled numbers 1, 2, 3 and 4 indicate the different defective steps related to the aberrant activation pattern and IL-7 unresponsiveness in RIF- treated HD T cells: (1) abnormal protein n of signalling complexes as described by 2D-gels, (2) abnormal membrane structures such as large MMD as seen by STED microscopy, (3) abnormal cytoskeleton organization as measured by ion kinetics and STED microscopy, and (4) al signalling intermediate and tion of phospho- STATS nuclear translocation as shown by STED microscopy.

Figure 10: PLAZsGIB inhibits IL-2 d PStat5 nuclear translocation in CD4 T cells of healthy donors (HD): Resting CD4 T cells purified from 4 healthy donors were WO 97140 treated for 30 minutes at 37°C with 3% or 1% of plasma from 5 VP (VP63, VP68, VP69, VP74 and VP75) and from 3 HD used as control. When indicated they were stimulated with 2nM IL-2 for 15 minutes at 37°C. The percentage of cells positive for nuclear PStat5, with mean and SD, in whole CD4 T cells (a) and in CD4+ CD25+ T cells (b), before (blue points) and after IL-2 stimulation (red points) are shown. Intracellular localisation of PStat5 was observed using Laser Scanning Confocal Microscopy (LSM 700, Zeiss) after indirect staining with rabbit anti human PStat5 (pY694) followed by donkey anti rabbit IgG—Die light 405. Total CD4 T cells were stained with goat anti human b-Tubulin followed by donkey anti goat IgG-AF555. CD25+ CD4 T cells were targeted with a mouse anti human CD25 followed by donkey anti mouse IgG-AF488.

Figure 11: IB ts IL-4 induced PStat6 nuclear translocation in CD4 T cells ofhealthy donors (HD): Resting CD4 T cells purified from 4 y donors were treated for 30 minutes at 37°C with 3% or 1% of plasma from 5 VP (VP63, VP68, VP69, VP74 and VP75) and from 3 HD used as control. When indicated they were stimulated with 2nM IL-4 for 15 minutes at 37°C. The percentage of cells positive for nuclear PStat6, with mean and SD, in whole CD4 T cells, before (blue points) and after IL-2 stimulation (red points) are shown. Intracellular localisation of PStat6 was observed using Laser Scanning Confocal copy (LSM 700, Zeiss) after indirect staining with rabbit anti human PStat6 (pY694) followed by goat anti rabbit 488. Total CD4 T cells were stained with mouse anti human a-Tubulin followed by goat anti mouse 647.

Figure 12: Absence of activity of mutant pPLAZGIB H48Q.

Figure 13: Comparison of the activity of wild type cloned porcine PLA2 G18 and of its mutant H48Q. A: induction of abnormal Membrane Microdomains (aMMD); B: effect on the IL-7 induced Nuclear Translocation ofphosphoSTAT5 (NT of pSTAT5).

Figure 14 shows the treatment of plasma from Viremic patients with goat anti-PLA2 GlB antibodies coupled to sepharose beads. Green: VP68; pink: VP69; blue: VP LJT. After ent (30 min at room temperature) the plasmas were tested: a. The tage ofCD4 T cells showing abnormal MMD/cell was measured after staining with Cholera toxin B (Cth-AF48 8) b. The nuclear translocation of pSTAT5 was measured after IL-7 stimulation and the percentage of positive nucleus counted.

Figure 15: Effect of anti-PLA2 GIB dies on the induction of aMMD and inhibition ofNT pSTATS.

Figure 16 : Soluble PLAZGIB mouse receptor (sMR) inhibits the activity of human PLA2G1B 2GlB) on the response to lL-7 of CD4 T cells from healthy donors, expressed as the percent of cells positive for nuclear translocation of PStatS. The restoration of the response is calculated as: 100 x (%Pos cell huG1B+sMR - %Pos cell humB) / (%Pos cell culture medium - %Pos cell huGlB) Figure 17 shows the plasma from CD4 non-responder R) patients induce aberrant MMD in HD CD4 T cells — (a) Images of HD CD4 T cells treated with plasma (1%) from CD4-NR patient obtained using Structured Illumination Microscopy (SIM).

MMD were stained with cholera toxin B F488). Projection of Z-stacks images of a representative CD4 T cell is shown. After IL-7 stimulation (2nM, 15min) there is no modification of the image (right). (b) Dose curve response (0.0001% to 1%) obtained with plasmas from 5 CD4-NR patients (blue curve, mean and SD) and from a representative viremic patient (red . The number of abnormal MMD induced at the surface of HD CD4 T cells.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to compositions and methods for modulating the immune system in a subject in need thereof. The invention more particularly discloses the fication of GIBSPLAZ as a key endogenous factor of the immune response and provides novel therapeutic and diagnostic methods and compositions based on a modulation of this factor.

A hypothesis of the present invention was that chronic activation of the immune system in HIV-infected patients is abnormal and drives CD4 T cells into an aberrant state of activation/differentiation that is unresponsive to the gamma-c cytokines involved in controlling many s of immune es and homeostasis of the CD4 compartment, despite the fact that more than 99.5% of CD4 T cells from the peripheral compartment are uninfected. This hypothesis was evaluated by the inventors and the present invention dates the nature and significance of this aberrant state of activation.

[0064] More specifically, in a first aspect, the t invention demonstrates that the teristics of this state may be summarized as follows: 1) before any stimulation, all the CD4 T cells in Viremic fected patients (VP) possess numerous large MMD on their surface, 2) these abnormal MMD sequester all the cell's IL-7Ralpha and gamma-c chains and 3) this sequestering ofthe chains in abnormal MMD alters their ability to induce the formation of a functional signalosome, 4) leading to a slowdown and a reduction of STATS phosphorylation and 5) a reduction of phospho-STATS nuclear import. This abnormal n of pre-existing MMD on the surface of VP CD4 T lymphocytes has multiple consequences and is a basic mechanism explaining the various manifestations of the immunodeficiency in HIV-infected patients. Loss of IL-7 responsiveness is an important factor that partly explains the CD4 penia observed. The persistent loss of these cells in VP - due to their sensitivity to apoptosis and their destruction by low-level but continuous Virus proliferation - cannot be compensated e increased levels of IL—7. In addition, since abnormal MMD sequester all the gamma-c chains in a non functional state, this blocks the function of the other nes in this .

[0065] The t invention further discloses the identification of the key endogenous factor responsible for this abnormal state of the immune system in infected subjects and, more generally, responsible for a drastic modulation of the immune response in various pathophysiological conditions. Plasma samples from VP were indeed shown to contain an activity - termed RIF — which is able to induce aberrant activation of Healthly Donors (HD) CD4 T lymphocytes. RIF was found in all the plasma samples of the VP examined. The pathophysiological significance of this activity was demonstrated by its absence in HIV Controller (HIC) patients where the IL-7/IL-7R system is normal and immune activation is beneficial. RIF is also absent in the plasma of ART ts who have diminished their immune activation, restored IL-7R filnction and recovered CD4 counts > 500/ mm3 (5).

RIF thus ents a major factor that ls the immune response, particularly through a tion ofCD4 T lymphocytes. It is remarkable that RIF induces an aberrant n of activation in HD CD4 T cells that is undistinguishable from that observed directly ex vivo in purified VP CD4 T cells. The invention further shows that RIF is the secreted phospholipase A2 from Group I B (“PLA2 GIB”). The results disclosed in this application show that (i) over expression of PLA2 GIB leads to a potent immunosuppression and that (ii) inhibition of PLA2 GIB leads to a remarkable se or stimulation of immune function. GIBsPLA2 inhibitors were able to correct the inappropriate state of the immune cells in plasma from subjects and can thus be used to treat (e.g., prevent, t) immunodeficiency or immune disorders in mammals.

GIBsPLA2 inhibition can also , stimulate, or help maintaining CD4 T cell counts and function, and thereby help stimulate efficient immune responses in patients. In particular, in HIV-infected patients, ART might be spared, or could be ded, were an equilibrium to be reached between patient immune defenses and the virus. Were ART, given very early after infection as suggested by recent s, to be combined with RIF inhibitors, this would prevent any RIF-induced alteration ofthe immune . In addition, in the context of some t failures of ART, patients with low CD4 counts after prolonged ART may benefit from these inhibitors. Accordingly, the invention provides methods for treating a subject by modulating GIBsPLA2 expression or activity in the subject. More particularly, the invention provides a method for modulating an immune response in a subject in need thereof, comprising modulating GIBsPLA2 activity or expression in said subject.

WO 97140 The data provided in the examples also demonstrate that the presence ofRIF in the plasma of a subject tes the HIV-induced pathogenesis state of CD4 T cells.

Accordingly, this ion provides methods of monitoring and/or sing HIV infection in a subject by detecting the level of RIF in the plasma of the subject, among other things.

The data provided in the examples further demonstrate that the number and/or size of membrane microdomains (MMD) on the T-cells of a t indicates the HIV-induced pathogenesis state ofCD4 T cells. Accordingly, this sure also provides provides methods of monitoring and/or diagnosing HIV infection in a subject by measuring the number and/or size of membrane microdomains (MMD) on the T-cells of the subject, among other things.

The data provided in the examples also indicate a role for RIF in creating and/or maintaining the CD4 T cell disease state in HIV infected subjects. Accordingly, this sure also provides methods for identifying a candidate HIV eutic agent that include measuring RIF-induced CD4 T cell activation in the presence of an agent. In some embodiments the methods comprise comparing the level of RIF-induced CD4 T cell activation in the ce of the agent with the level of RIF-induced CD4 T cell activation in the absence of the agent.

Definitions The term “sequence identity” as applied to nucleic acid or protein sequences, refers to the quantification (usually percentage) of nucleotide or amino acid residue matches between at least two sequences aligned using a standardized algorithm such as Smith-Waterman ent (Smith and Waterman (1981) J Mol Biol 1471195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389—3402). BLAST2 may be used in a standardized and WO 97140 reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningfiil comparison between them.

As used herein, "treatment" or “treat” refers to al intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for tive or curative purpose. ble effects of treatment include, but are not d to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or ct pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, ration or palliation of the disease state, and remission or improved prognosis. In some embodiments, compositions and methods of the invention are used to delay development of a disease or disorder or to slow the progression of a disease or disorder.

The term "isolated", as used herein, refers to molecules (e.g., nucleic or amino acid) that are removed from a component of their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An "isolated" polypeptide (or protein) is for instance a polypeptide separated from a component of its natural environment and, preferably purified to greater than 90% or 95% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric ng (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) ion. An ted" nucleic acid refers to a nucleic acid le separated from a component of its natural environment and/or assembled in a different construct (e.g., a vector, expression cassette, recombinant host, etc.).

"Nucleic acid encoding an anti-GIBsPLA2 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

A "subject" refers to a mammal. Examples of mammals include humans and non-human animals such as, without limitation, icated s (e.g., cows, sheep, cats, dogs, and horses), non-human primates (such as monkeys), rabbits, and rodents (e.g., mice and rats).

The “modulation ofan immune response” designates, within the context of the invention, any modification of the amount or activity or ratio of immune cells, preferably White blood cells (e.g., T cytes, B lymphocytes, NK, NKT cells, macrophages, dendritic cells).

In a particular embodiment, modulating an immune response includes modulating the amount or activity of T cytes, preferably of CD4-T lymphocytes.

Refractory State Inducing Factor (RIF) 0r Phospholipase A2 group [B The term RIF is used hangeably with Phospholipase A2 group IB, GIBsPLA2 (or PLA2 GIB). Phospholipase A2 group IB is a secreted protein having a MW of from about 15 kDa and an isoelectric point of from about 6.5 to about 8.0.

Within the context of the present invention, the term "GIBsPLA2" or “phospholipase A2 group IB” designates any native GIBsPLA2 protein from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed A2, as well as any form of GIBsPLA2 that results from sing inside or outside a cell. The term also encompasses naturally-occurring variants of GIBsPLA2, e.g., splice variants or allelic variants.

The amino acid ce of an exemplary human GIBsPLA2 is shown below (SEQ ID NO: 2).

MKLLVLAVLL SGIS PRAVWQFRKM IKCVIPGSDP FLEYNNYGCY GTPV CQTH DNCYDQAKKL DSCKFLLDNP YTHTYSYSCS GSAITCSSKN KECEAFICNC DRNAAICFSK APYNKAHKNL DTKKYCQS Amino acids 1 to 15 of SEQ ID NO: 2 (underlined) are a signal sequence, and amino acids 16 to 22 ofSEQ ID NO: 2 (in bold) are a propeptide sequence. The mature protein corresponds to amino acid residues 23-148 of SEQ ID NO: 2, which is an exemplary processed human GIBsPLA2 protein.

Naturally-occurring variants include any protein comprising the sequence of SEQ ID NO: 2, or the sequence of amino acid residues 23-148 of SEQ ID NO: 2, with one or more amino acid substitution, addition and/or deletion of one or several (typically 1, 2 or 3) amino acid residues, preferably not more than 10 distinct amino acid substitution(s), addition(s), and/or deletion(s) of one or several (typically 1, 2 or 3) amino acid es.

Typical naturally-occurring variants retain a biological activity of SEQ ID NO: 2.

In this regard, in some embodiments, GIBsPLA2 has at least one activity selected from induction of formation of membrane microdomains (MMD) in CD4 T cells from healthy subjects, or rendering CD4 T cells of healthy subjects refractory to interleukin ing, such as refractory to IL-2 signaling or refractory to IL-7 signaling.

In some embodiments inducing ion ofMMD ses increasing the number of MMD on CD4 T cells of healthy subjects to at least about 80 per cell, at least about 90 per cell, at least about 100 per cell, at least about 110 per cell, or at least about 120 per cell. In a non-limiting prefered embodiment, inducing formation of MMD comprises increasing the number of MMD on CD4 T cells of healthy subjects to more than 100 MMD per cell.

In some embodiments inducing formation of MMD comprises stimulating formation of larger MMD than would ise be present on the CD4 T cells. In some embodiments inducing formation of larger MMD comprises ating formation MMD having a diameter of at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, or at least 140 nm. In a non-limiting prefered ment, inducing formation of larger MMD comprises stimulating formation ofMMD having a diameter larger than 120 mm.

In some embodiments rendering CD4 T cells of healthy ts refractory to interleukin-7 signaling comprises a reduction of STATSA and/or B phosphorylation in said cells by at least about 10%, at least about 20%, at least about 30%, or at least about 40%. In some embodiments rendering CD4 T cells of healthy ts refractory to interleukin-7 signaling comprises reducing the rate of nuclear translocation of phospho- STATSA and/or phospho-STATSB by at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

[0079] GIBSPLA2 activity may be measured by any suitable method known in the art, as illustrated in the es, or later developed. GIBsPLA2 activity may be measured in a plasma sample such as for example a fractionated plasma sample, using e.g., ligand recruitment assays, immunoassays and/or enzymatic assays.

In a particular embodiment, the term GIBsPLA2 designates a human protein, particularly a protein comprising or having SEQ ID NO: 2, or a naturally-occurring variant thereof.

GIBsPLA2 according to this disclosure may be isolated, purified, and/or recombinant. In certain embodiments, the ion may use, instead or in addition to a GIBsPLA2 protein, a nucleic acid encoding GIBsPLA2. The c acid may be DNA or RNA, single— or double-stranded.

An ary nucleic acid sequence ng a GIBsPLA2 is shown in SEQ ID NO: 1 below.

ATGAAACTCCTTGTGCTAGCTGTGCTGCTCACAGTGGCCGCCGCCGACAGCGGCATCAGC CCTCGGGCCGTGTGGCAGTTCCGCAAAATGATCAAGTGCGTGATCCCGGGGAGTGACCCC TTCTTGGAATACAACAACT‘ACGGCTGCTACTGTGGCTTGGGGGGCTCAGGCACCCCCGTG GATGAACTGGACAAGTGCTGCCAGACACATGACAAC[TGCTACGACCAGGCCAAGAAGCTG GACAGCTGTAAATTTCTGCTGGACAACCCGTACACCCACACCTATTCATACTCGTGCTCT GGCTCGGCAATCACCrTGTAGCAGCAAAAACAAAGAGrTGTGAGGCCTTCATTTGCAACTGC GACCGCAACGCTGCCATCTGCTTrl".1CAAAAGCTCCAFTAFTAACAAGGCACACAAGAACCTG AAGAAG‘I‘ATrTGTCAGAG‘I‘r_1GA

[0082] Alternative nucleic acid molecules encoding a GIBsPLA2 include any variant of SEQ ID NO:1 resulting from the degeneracy of the c code, as well as any sequence which hybridizes to SEQ ID NO: 1 under stringent conditions, more preferably having at least 80%, 85%, 90% 95% or more sequence identity to SEQ ID NO; , 1, and ng a GIBsPLA2 protein.

Method of production of GIBsPLA2 GIBsPLA2 can be produced by any conventionally known protein expression method and purification method. For example: (i) a method for synthesizing peptides; (ii) a method for purifying and isolating them from the liVing body or cultured cells; or (iii) a method for producing them with the use of genetic recombination techniques; and combinations thereof and the like (for e, the standard techniques described for example in Molecular Cloning (Sambrook, J., Fritsch, E. F., is, T., Cold Spring Harbor Laboratory Press) (1989) and Current Protocols in Molecular Biology (Ausubel, F. M., John Wiley and Sons, Inc. (1989)).

[0084] In a particular embodiment, the invention relates to a method for producing GIBsPLA2 by expression of a coding nucleic acid in a host cell, and collection or purification of A2. In this regard, the invention also described recombinant host cells comprising a nucleic acid encoding a GIBsPLA2. Such cells may be prokaryotic (such as bacteria) or eukaryotic (such as yeast cells, insect cells, plant cells or mammalian cells).

The nucleic acid may be placed under the control of any suitable tory sequence, such as a er, terminator, and the like. Alternatively, the c acid may be inserted in WO 97140 2014/078969 the host cell in a location where expression is driven by an endogenous promoter.

Techniques for ing nucleic acids in cells are well known in the art.

GIBsPLA2 modulation The invention provides novel s which comprise a modulation of GIBsPLA2 in a subject in need thereof. The term “modulation” designates any modification of the level (e.g., expression) or activity of A2 in a subject. Also, modulation designates either an increase or a decrease GIBsPLA2 level or activity. A modulation more ably designates a change by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more as compared to non-modulated situation. As a result, ting A2 designates reducing by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more GIBsPLA2 level or activity, as well as completely blocking or suppressing GIBsPLA2 level or activity.

Conversely, stimulating GIBsPLA2 designates increasing by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more GIBsPLA2 level or activity. Depending on the situation, the modulation may be transient, sustained or permanent. Also modulating the activity includes modulating the amount of GIBsPLA2 in the subject, particularly in body fluids, modulating the potency ofthe protein (for ce by modulating the level of co-factors or ate in the subject), and modulating the level or activity of degradation products produced by GIBsPLA2.

GIBsPLA2 inhibition In a particular embodiment, the invention provides compositions and methods for inhibiting GIBsPLA2 in a subject. GIBsPLA2 inhibition may be obtained by the use of GIBsPLA2 inhibitors, i.e., any compound that inhibit the expression or activity of GIBsPLA2. GIBsPLA2 inhibitors include expression inhibitors, antagonists, sequestrators, or target masking compounds. Preferred types of GIBsPLA2 inhibitors include GIBsPLA2 ligands (covalent or non-covalent), IBsPLA2 antibodies (and fragments and derivatives thereof), c acids encoding anti-GIBsPLA2 antibodies (or fragments and derivatives thereof), inhibitory nucleic acids, peptides, or small drugs, or c0mbination(s) thereof. atively, or in addition, GIBsPLA2 tion can be obtained by vaccinating a subject against a GIBsPLA2 n, so that antibodies are produced by the subject in need of PLA2-GIB inhibition.

Antibodies against GIBsPLAZ Specific examples of GIBsPLA2 inhibitors are antibodies that specifically bind to A2.

Antibodies can be synthetic, monoclonal, or polyclonal and can be made by techniques well known in the art. Such dies specifically bind Via the antigen-binding sites of the antibody (as opposed to non-specific binding). GIBsPLA2 polypeptides, fragments, variants, fusion proteins, etc., can be employed as immunogens in producing antibodies immunoreactive therewith. More cally, the polypeptides, fragments, variants, fusion proteins, etc. contain nic determinants or epitopes that elicit the formation of antibodies.

[0087] These antigenic inants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids ns from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of es available is quite numerous; however, due to the conformation of the protein and steric hinderances, the number of antibodies that actually bind to the epitopes is less than the number of ble epitopes (C. A. y, Jr. and P.

Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any of the methods known in the art. Both polyclonal and monoclonal antibodies can be prepared by conventional techniques. red antibodies of the invention are directed to a GIBsPLA2 e, and/or have been generated by immunization with a polypeptide comprising a GIBsPLA2 epitope selected from: the mature GIBsPLA2 protein, a fragment of A2 comprising at least 8 consecutive amino acid residues of SEQ ID NO: 2 (or the corresponding residues of a natural variant of SEQ ID NO: 2), said nt comprising at least amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof. Preferred antibodies of the invention bind an epitope comprised between amino acid residues 50-71 of SEQ ID NO: 2 or the corresponding residues of a natural variant of SEQ ID NO: 2.

[0089] The term “antibodies” is meant to include polyclonal dies, monoclonal dies, fragments thereof, such as F(ab')2 and Fab fragments, single-chain variable fragments (scFvs), -domain antibody fragments (VHHs or Nanobodies), bivalent antibody fragments (diabodies), as well as any recombinantly and synthetically produced binding partners, human dies or humanized antibodies.

[0090] Antibodies are defined to be specifically binding preferably if they bind to GIBsPLA2 with a Ka of r than or equal to about 107 M-l. Affinities of antibodies can be readily determined using conventional techniques, for example those described by ard et al., Ann. NY. Acad. Sci., 51 :660 (1949).

Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, donkeys, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures that are well known in the art. In l, purified GIBsPLA2 or a peptide based on the amino acid sequence of GIBsPLA2 that is appropriately conjugated is administered to the host animal typically through parenteral injection. The immunogenicity of GIBsPLA2 can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following r immunizations, small samples of serum are collected and tested for reactivity to GIBsPLA2 polypeptide. Examples of s assays useful for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as rcurrent immuno-electrophoresis , radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies can be readily prepared using well known procedures. See, for example, the procedures bed in US. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal dies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980.

[0093] For example, the host animals, such as mice, can be injected intraperitoneally at least once and preferably at least twice at about 3 week intervals with isolated and purified wild-type or mutant GIBsPLA2 protein or conjugated GIBsPLA2 peptide, optionally in the presence of nt. Mouse sera are then assayed by tional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of n or peptide. Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following ished protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one a cell. The fiJsing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the filSCd cells. The filsed cells can then be allowed to grow for approximately eight days.

Supematants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse lg. Following washes, a label, such as a labeled GIBsPLA2 polypeptide, is added to each well followed by incubation. Positive wells can be subsequently detected. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).

The monoclonal antibodies of the disclosure can be produced using ative techniques, such as those described by Alting-Mees et al., "Monoclonal Antibody Expression Libraries: A Rapid ative to Hybridomas", Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7:394 ( 1989).

Antigen-binding fragments of such antibodies, which can be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')2 nts. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

The onal antibodies of the present disclosure include chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are stered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.

Alternatively, a humanized antibody nt can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the tion of chimeric and further engineered onal antibodies include those bed in Riechmann et al. (Nature 3, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993). ures to generate antibodies transgenically can be found in GB 2,272,440, US. Pat. Nos. 5,569,825 and 5,545,806.

[0097] Antibodies produced by genetic ering methods, such as ic and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, can be used. Such chimeric and humanized monoclonal antibodies can be produced by c ering using standard DNA techniques known in the art, for example using methods described in Robinson et al. ational Publication No. WO 87/02671; Akira, et a1. European Patent ation 0184187; Taniguchi, M., an Patent Application 0171496; Morrison et al. European Patent Application 0173494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. US. Pat. No. 4,816,567; Cabilly et al. European Patent ation 0125023; Better eta1., Science 240:1041 1043, 1988; Liu et al., PNAS 84:3439 3443, 1987; Liu et al., J. Immunol. 139:3521 3526, 1987; Sun et al. PNAS 842214 218, 1987; Nishimura et al., Canc. Res. 47:999 1005, 1987; Wood et al., Nature 314:446 449, 1985; and Shaw et al., J. Natl. Cancer Inst. 8021553 1559, 1988); Morrison, S. L., Science 229:1202 1207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter US. Pat. No. 5,225,539; Jones et al., Nature 321:552 525, 1986; Verhoeyan et al., Science 34, 1988; and r et al., J.

Immunol. 14114053 4060, 1988.

In connection with synthetic and semi-synthetic antibodies, such terms are intended to cover but are not limited to dy fragments, isotype switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, antibodies having plural specificities, and fully synthetic antibody-like molecules.

For therapeutic applications, "human" monoclonal dies having human constant and variable regions are often preferred so as to minimize the immune response of a patient against the dy. Such antibodies can be generated by immunizing transgenic animals which contain human immunoglobulin genes. See Jakobovits et al. Ann NY Acad Sci 5-535 (1995).

Human monoclonal antibodies against GIBsPLA2 polypeptides can also be prepared by constructing a combinatorial immunoglobulin library, such as a Fab phage display library or a scFv phage display y, using immunoglobulin light chain and heavy chain cDNAs prepared from mRNA derived from lymphocytes of a subject. See, e.g., McCafferty et a1. PCT publication WO 92/01047; Marks et al. (1991) J. Mol. Biol. 222581 597; and Griffths et al. (1993) EMBO J 12:725 734. In addition, a atorial library of antibody variable regions can be generated by mutating a known human antibody. For example, a variable region of a human antibody known to bind A2, can be mutated by, for e, using randomly altered mutagenized oligonucleotides, to generate a library of mutated variable regions which can then be screened to bind to GIBsPLA2. Methods of inducing random nesis within the CDR regions of immunoglobin heavy and/or light , methods of crossing ized heavy and light chains to form pairings and screening methods can be found in, for example, Barbas et al.

PCT publication WO 96/07754; Barbas et al. (1992) Proc. Nat'l Acad. Sci. USA 89:4457 4461.

An immunoglobulin library can be expressed by a tion of display packages, preferably derived from filamentous phage, to form an antibody display library.

Examples of methods and reagents particularly amenable for use in generating antibody display library can be found in, for example, Ladner et al. US. Pat. No. 5,223,409; Kang et al. PCT publication WO 92/18619; Dower et al. PCT publication WO 91/17271; Winter et a1. PCT publication WO 92/20791; Markland et a1. PCT publication W0 92/15679; Breitling et al. PCT ation WO 93/01288; McCafferty et al. PCT publication WO 92/01047; Garrard et al. PCT publication WO 90; Ladner et a1. PCT publication WO 90/02809; Fuchs et a1. (1991) Bio/Technology 921370 1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81 85; Huse et al. (1989) Science 246:1275 1281; Griffths et a1. (1993) supra; Hawkins et a1. (1992) J Mol Biol 226:889 896; Clackson et al. (1991) Nature 352:624 628; Gram et al. (1992) PNAS 89:3576 3580; Garrad et a1. (1991) chnology 9:1373 1377; boom et al. (1991) Nuc Acid Res 19:4133 4137; and Barbas et a1. (1991) PNAS 88:7978 7982. Once displayed on the surface of a display package (e.g., filamentous phage), the antibody library is screened to identify and isolate packages that express an antibody that binds a GIBsPLA2 polypeptide. In a preferred embodiment, the primary screening of the library involves panning with an immobilized GIBsPLA2 polypeptide and y packages expressing dies that bind immobilized GIBsPLA2 polypeptide are selected.

In a ular embodiment, the invention relates to a composition comprising an anti-GIBsPLA2 antibody (or a fragment or derivative f) and a pharmaceutically acceptable excipient.

Existing anti—Phospholipase A2-GIB monoclonal antibodies include Mab CPI-7 (Labome), MABSOI8 (Labome), EPR5186 (Genetex); LS-Cl38332 (Lifespan) or CABT-17153MH ive t). es of polyelonal antibodies include for instance N1C3 from GeneTex. As indicated above, preferred anti-GIBsPLA2 antibodies of the invention bind mature GIBsPLA2, even more preferably an e comprised in a domain of GIBsPLA2 sing an amino acid selected from amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof.

Preferred antibodies of the invention bind an epitope sed between amino acid residues 50-71 of SEQ ID NO: 2 or the corresponding residues of a natural variant of SEQ ID NO: 2.

In an alternative embodiment, the invention relates to and uses a composition comprising a nucleic acid encoding an anti-GIBsPLA2 antibody (or a fragment or derivative thereof) and a pharmaceutically acceptable excipient.

Inhibitory Nucleic acids In an alternative embodiment, the GIBsPLA2 inhibitor is an inhibitory nucleic acid, i.e., any nucleic acid molecule which inhibits GIBsPLA2 gene or protein expression. Preferred tory nucleic acids include antisense nucleic acids, short interfering RNAs (siRNAs), small hairpin RNAs (shRNA), microRNAs, aptamers, or mes. In a particular embodiment, the inhibitory nucleic acid is a small interfering RNA that prevents translation of GIBsPLA2 mRNA. In another ular embodiment, the inhibitory nucleic acid is an nse oligonucleotide that prevents translation of GIBsPLA2 mRNA. In another particular embodiment, the inhibitory nucleic acid is a small hairpin RNA that prevents translation of GIBsPLA2 mRNA. siRNA comprise a sense nucleic acid sequence and an anti-sense nucleic acid sequence of the polynucleotide of interest. siRNA are constructed such that a single transcript (double stranded RNA) have both the sense and complementary antisense sequences from the target gene. The nucleotide sequence of siRNAs may be ed using an siRNA design er program available from, for e, the Ambion website on the world wide web.

In some embodiments, the length of the antisense oligonucleotide or siRNAs is less than or equal to 10 nucleotides. In some ments, the length of the antisense ucleotides and siRNAs is as long as the naturally occurring transcript. In some embodiments, the antisense oligonucleotides and siRNAs have 18-30 nucleotides. In some embodiments, the antisense oligonucleotides and siRNAs are less than 25 nucleotides in length.

Preferred inhibitory nucleic acid molecules comprise a domain having a nucleotide sequence that is perfectly complementary to a region of a GIBsPLA2 gene or RNA. Such a domain contains typically from 4 to 20 nucleotides, allowing specific hybridization and optimal inhibition the the gene transcription or RNA ation. The sequence of the inhibitory c acids may be derived directly from the sequence of a gene encoding GIBsPLA2, such as SEQ ID NO: 1. atively, or in addition, inhibitory nucleic acids may hybridize to a regulatory element in a GIBsPLA2 gene or RNA, such as a promoter, a splicing site, etc., and prevent effective regulation thereof.

Specific examples of tory nucleic acid molecules of the present invention include isolated single strand nucleic acid molecules consisting of from 10 to 50 consecutive nucleotides of SEQ ID NO: 1. Specific examples of inhibitory c acid molecules of the invention are antisense nucleic acids consisting ofthe following nucleoitide sequence or the perfectly complementary strand thereof: ATGAAACTCCTTGTGCTAG (SEQ ID NO: 3) GCATCAGC (SEQ ID NO: 4) TTCCGCAAAATGATCAA (SEQ ID NO: 5) 2014/078969 CCCGGGGAGTGACCCC (SfiQ 3 NO: 6) TACGGCTGCTACTGTGGCTT (SEQ ID NO: 7) GACACATGACAACTGCTACGACC (SEQ ID NO: 8) ACCCACACCTATTCATACTCGT (SEQ ID NO: 9) ATCACCTGTAGCAGCA (SEQ ID NO: 10) AGCTCCAEATAACAAGGCA (SEQ ID NO: 11) CAAGAAGEATTGTCAGAG (SfiQ D NO: 12) Peptide and Small Drugs In an alternative embodiment, the GIBsPLA2 inhibitor is a e or small drug that inhibits the activity of GIBSPLAZ. The peptide or small drug is typically a molecule that selectively binds GIBsPLA2, or a substrate of GIBsPLA2, or a co-factor of GIBsPLA2, or a degradation t or metabolite of A2 pathway.

Peptides preferably contain from 3 to 20 amino acid residues, and their sequence may be identical to a domain of GIBsPLA2 (bait peptide) or to a domain of a GIBsPLA2 substrate, co-factor, degradation product or lite. Preferred peptides of the invention contain from 4 to 30 consecutive amino acid residues of SEQ ID 0: 2 (or of a corresponding sequence of a l t of SEQ ID NO: 2). Most preferred peptides of the invention comprise from 5 to 25 consecutive amino acid residues of SEQ ID 0: 2 (or of a corresponding sequence of a natural variant of SEQ ID NO: 2) and further comprise at least one of the following amino acid residues of SEQ 1D 0: 2 (or of a corresponding sequence of a natural variant of SEQ ID NO: 2): amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof. Specific examples of peptides of the invention are peptides of less than 25 amino acids comprising anyone of the following sequences: NNYGCY (SEQ ID NO: 13) CYCGLG (SEQ ID NO: 14) YCGLGGSG (SEQ ID NO: 15) FLEYNNYGCYCGLGGSGTPV (SEQ ID NO: 16) QTHDN (SEQ ID NO: 17) CQTHDNC (SEQ ID NO: 18) ECEAFICNC (SEQ ID NO: 19) DRNAAI (SEQ ID NO: 20) DRNAAICFSKAPYNKAHKNL (SEQ ID NO: 21) The peptides of the invention can se peptide, non-peptide and/or modified peptide bonds. In a particular embodiment, the peptides comprise at least one peptidomimetic bond selected from intercalation of a methylene (-CH2-) or phosphate (- P02—) group, secondary amine (-NH—) or oxygen (-O-), alpha—azapeptides, alpha- eptides, N-alkylpeptides, phosphonamidates, depsipeptides, hydroxymethylenes, hydroxyethylenes, dihydroxyethylenes, hydroxyethylamines, retro-inverso peptides, methyleneoxy, cetomethylene, , phosphinates, phosphinics, or phosphonamides. Also, the peptides may comprise a protected N—ter and/or C-ter fiinction, for example, by acylation, and/or amidation and/or esterification.

The peptides of the invention may be ed by techniques known per se in the art such as chemical, biological, and/or genetic synthesis.

Each of these es, in isolated form, represents a particular object of the present invention.

Preferred small drugs are hydrocarbon compounds that selectively bind GIBsPLA2.

Small drugs and peptides are preferably obtainable by a method comprising: (i) contacting a test nd with GIBsPLA2 or a fragment thereof, (ii) selecting a test compound which binds GIBsPLA2 or said fragment thereof, and (iii) selecting a compound of (ii) which inhibits an activity of GIBsPLA2. Such a method represents a particular object of the invention.

Small drugs and es are also obtainable by a method comprising: (i) contacting a test nd with a GIBsPLA2 substrate, co-factor, or degradation product, or a nt thereof, (ii) selecting a test compound which binds to said GIBsPLAZ substrate, co-factor, or degradation t, or a nt thereof, and (iii) selecting a compound of (ii) which inhibits an activity of GIBsPLA2. Such a method ents a particular object of the invention.

GIBsPLA2 soluble receptors In an alternative embodiment, the GIBsPLA2 inhibitor is a soluble form of a GIBsPLA2 receptor. Such soluble or compounds are able to bind GIBsPLA2, thereby ting its activity by acting as a bait or g agent.

A specific embodiment of such inhibitors is a soluble form of a human or murine GIBsPLA2 receptor, or a GIBsPLA2-binding fragment thereof.

The amino acid sequences of murine and human soluble receptors are depicted in SEQ ID NOs: 22 and 23, respectively. The term soluble receptor thus encompasses any GIBsPLA2- binding polypeptide comprising all or a fragment of the sequence of SEQ ID NO: 22 or 23.

A GIBsPLAZ-binding fragment designates any nt of such a polypeptide sing preferably at least 5 consecutive amino acid residues thereof, more preferably at least 8, 10, or 12, which binds PLA2GIB specifically. Specific binding of the receptor molecule indicates that the receptor molecule binds to PLA2GIB with higher affinity (e.g., by at least 5 fold) than to PLA2-IIA or IID. A fragment as defined above most preferably comprises less than 50 amino acid residues.

Examples of GIBsPLAZ-binding polypeptides are, Without limitation, polypeptides comprising at least one ofthe following amino acid sequences: LSEYECDSTLVSLRWRCNRKHITGPLQYSVQVAHDNTVVASRKYIHKW (SEQ ID NO: 24) WE<DLNSHICYQFNLLS (SEQ ID V0: 25) DCESTLPYICKKYLNHIDHEIVEK(SEQ ID V0: 26) QY{VQVKSDNTVVARKQIHRWIAYTSSGGDICE(SEQ ID NO: 27) LSYLNWSQEITPGPFVEHHCGTLEVVSA (SEQ ID NO: 28) SRFEQAFITSLISSVAEKDSYFW (SEQ ID NO: 29) WICRIPRDVRPKFPDWYQYDAPWLFYQNA (SEQ ID NO: 30) AFHQAFLTVLLSRLGHTHWIGLSTTDNGQT (SEQ ID NO: 31) SEQ ID NOs: 24-26 derive from the ce of human soluble PLA2GIB receptor, while SEQ ID NOS: 27-31 derive from the sequence of murine e PLAZGIB receptor.

Vaccination In an alternative (or cumulative) embodiment, inhibition of A2 in a subject is obtained by vaccinating (or immunizing) the subject with a GIBSPLAZ antigen. As a result of such a vaccination or immunization, the subject produces antibodies (or cells) Which inhibit GIBsPLAZ. In particular, injection(s) of a GIBsPLA2 antigen (e.g., an genic GIBSPLAZ essentially devoid of biological activity) can generate antibodies in the treated t. These antibodies will protect t an excess of GIBsPLA2 expression and can be used along as immunotherapy or a vaccine prophyllaxy.

An object of the invention thus resides in a method of vaccinating a subject comprising administering to the subject a GIBsPLA2 antigen.

A fithher object of the invention relates to a GIBsPLA2 n for use to vaccinate a subject in need thereof.

In a particular embodiment, the GIBsPLA2 antigen used for ation is an inactivated immunogenic molecule that induces an immune response against GIBsPLA2 in a subject. Inactivation may be obtained e.g., by chemically or physically altering GIBsPLA2 or by mutating or truncating the n, or both; and immunogenicity may be obtained as a result of the inactivation and/or by further conjugating the protein to a suitable carrier or hapten, such as KLH, HSA, sine, a viral anatoxin, or the like, and/or by polymerization, or the like. The antigen may thus be chemically or physically modified, e.g., to improve its immunogenicity.

In a preferred embodiment, the GIBsPLA2 antigen of the invention comprises A2 or an epitope-containing fragment or mimotope thereof.

In a particular ment, the GIBsPLA2 antigen comprises a fill] length GIBsPLA2 protein. In a r particular ment, the GIBsPLA2 antigen comprises a protein comprising SEQ ID NO: 2, or a sequence having at least 90% identity to SEQ ID NO: 2.

In an alternative embodiment, the GIBsPLA2 antigen comprises a fragment of a GIBsPLA2 protein comprising at least 6 consecutive amino acid residues and containing an immunogenic epitope, or a mimotope thereof. In a preferred embodiment, the GIBsPLA2 antigen comprises at least from 6 to 20 amino acid residues. Preferred es of the invention contain from 4 to 30 consecutive amino acid residues of SEQ 1D 0: 2 (or of a ponding sequence of a natural variant of SEQ ID NO: 2). Most preferred peptides of the invention se from 5 to 25 consecutive amino acid residues of SEQ ID 0: 2 (or of a corresponding sequence of a l variant of SEQ ID NO: 2) and further comprise at least one of the following amino acid es of SEQ 1D 0: 2 (or of a corresponding sequence of a natural variant of SEQ ID NO: 2): amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof. Specific examples of peptides of the invention are peptides of less than 50 amino acids comprising anyone of the following sequences: NNYGCY (SEQ ID NO: 13) CYCGLG (SEQ ID NO: 14) YNNYGCYCGEGGSG (SEQ ID NO: 15) FLEYNNYGCYCGLGGSGTPV (SEQ ID NO: 16) QTHDN (SEQ ID NO: 17) CQTHDNC (SEQ ID NO: 18) ECEAFICNC (SEQ ID NO: 19) DRNAAI (SEQ ID NO: 20) DRNAAICFSKAPYNKAHKNL (SEQ ID NO: 21) The A2 antigen may be in s forms such as in free form, polymerized, chemically or physically modified, and/or coupled (i.e., linked) to a carrier molecule.

Coupling to a carrier may increase the immunogenicity and (filrther) suppress the biological ty of the GIBsPLA2 ptide. In this regard, the carrier molecule may be any r molecule or protein conventionally used in logy such as for instance KLH (Keyhole limpet hemocyanin), ovalbumin, bovine serum albumin (BSA), a viral or bacterial anatoxin such as toxoid tetanos, toxoid diphteric B cholera toxin, mutants thereof such as diphtheria toxin CRM 197, an outer membrane vesicle protein, a polylysine molecule, or a Virus like le (VLP). In a preferred embodiment, the carrier is KLH or CRM197 or a VLP.

Coupling of GIBsPLA2 to a carrier may be performed by covalent try using linking chemical groups or reactions, such as for ce glutaraldehyde, biotin, etc.

Preferably, the conjugate or the GIBsPLA2 protein or fragment or mimotope is submitted to treatment with formaldehyde in order to complete inactivation of GIBSPLAZ.

In a ular embodiment, the GIBsPLA2 antigen comprises a fill] length GIBsPLA2 protein, optionally coupled to a carrier protein. In a preferred embodiment, the GIBsPLA2 antigen comprises a protein comprising SEQ ID NO: 2, or a sequence having at least 90% identity to SEQ ID NO: 2, coupled to a carrier protein. 2014/078969 In another particular embodiment, the GIBsPLA2 antigen comprises an immunogenic peptide or mimotope of GIBsPLA2, optionally d to a carrier protein. In a more preferred embodiment, the GIBsPLA2 antigen comprises a polypeptide of at least 10 amino acids long comprising at least one of the following amino acid residues of SEQ ID 0: 2 (or of a corresponding sequence of a natural t of SEQ ID NO: 2): amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof, optionally coupled to a carrier molecule.

The immunogenicity ofthe GIBsPLA2 antigen may be tested by various methods, such as by immunization of a non-human animal grafted with human immune cells, followed by verification of the presence of antibodies, or by sandwich ELISA using human or humanized antibodies. The lack ofbiological activity may be verified by any ofthe activity tests described in the application. In a preferred embodiment, the GIBsPLA2 antigen has less than 20%, more preferably less than 15%, 10%, 5% or even 1% ofthe ty of a wild-type GIBsPLA2 protein in an in vitro method of (i) induction of formation of membrane microdomains (MMD) in CD4 T cells or (ii) in rendering CD4 T cells refractory to 1L-2 signaling or refractory to IL-7 signaling.

In a particular embodiment, the invention relates to an inactivated and immunogenic GIBsPLA2.

In a further particular ment, the ion relates to a GIBsPLA2 protein or a fragment or mimotope thereof conjugated to a carrier molecule, ably to KLH.

In a fithher aspect, the invention relates to a vaccine comprising a GIBsPLA2 antigen, a suitable excipient and, optionally, a le adjuvant.

Such les and conjugates and vaccines represent potent agents for use to immunize subjects, thereby g a sustained A2 tion. Upon repetition, such methods can be used to cause a permanent GIBsPLA2 inhibition.

A further object of the invention relates to of a method for inducing the tion of antibodies that neutralize the activity of nous A2 in a subject in need thereof, the method comprising administering to said subject a therapeutically effective amount of a GIBsPLA2 antigen or vaccine.

Administration of an antigen or vaccine of the invention may be by any suitable route, such as by injection, preferably uscular, subcutaneous, transdermal, intraveinous or rterial; by nasal, oral, mucosal or rectal administration.

The GIBsPLA2 antigen or vaccine may be used for ng any disease linked to an over-production of GIBsPLA2. More cally, this invention relates to a method for treating a disease linked to an over-production of GIBsPLA2 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GIBsPLA2 antigen or of a vaccine composition comprising a GIBsPLA2 antigen.

GIBsPLA2 ts or activators The term GIBsPLA2 “agonist”, within the context of the present invention, encompasses any substance having, or mediating or up-regulating GIBsPLA2 activity such as, without limitation, a peptide, a ptide, a inant protein, a conjugate, a natural or artificial ligand, a degradation product, a homolog, a nucleic acid, DNA, RNA, an aptamer, etc., or a combination thereof. The term “agonist” encompasses both full and partial agonists. A particular example of a GIBsPLA2 agonist is a GIBsPLA2 n or a nucleic acid encoding a GIBsPLA2 protein.

In a particular embodiment, the invention relates to methods for inhibiting an immune se in a subject, comprising administering to the subject a GIBsPLA2 protein or a nucleic acid encoding a GIBsPLA2 protein.

Compositions The ion also relates to compositions comprising a GIBsPLA2 modulator or antigen as herein described as an active ient, and preferably a pharmaceutically acceptable carrier.

A “pharmaceutical composition” refers to a formulation of a compound of the invention (active ingredient) and a medium lly accepted in the art for the delivery of biologically active compounds to the subject in need thereof. Such a carrier includes all pharmaceutically acceptable rs, diluents, medium or supports therefore. Conventional pharmaceutical practice may be employed to provide suitable formulations 0r compositions to subjects, for example in unit dosage form.

The compounds or compositions according to the ion may be formulated in the form of ointment, gel, paste, liquid solutions, suspensions, tablets, gelatin capsules, capsules, suppository, powders, nasal drops, or aerosol, preferably in the form of an injectible solution or suspension. For injections, the compounds are generally packaged in the form of liquid suspensions, which may be injected via es 0r perfusions, for example. In this respect, the compounds are generally dissolved in saline, physiological, ic 0r buffered solutions, compatible with pharmaceutical use and known to the person skilled in the art. Thus, the itions may contain one or more agents or excipients ed from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or excipients that can be used in liquid and/or injectable formulations are notably methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, e, vegetable oils, acacia, etc. The carrier can also be selected for e from methyl-beta-cyclodextrin, a polymer of c acid (such as carbopol), a mixture of polyethylene glycol and polypropylene , monoetrhanol amine and hydroxymethyl cellulose.

The compositions generally comprise an effective amount of a compound of the invention, e.g., an amount that is effective to modulate GIBsPLA2. Generally, the compositions according to the invention comprise from about 1 ug to 1000 mg of a 2014/078969 GIBsPLA2 modulator, such as from 0.001-0.01, 0.01-0.l, 0.05-100, 0.05-10, 0.05-5, 0.05- 1, 01-100, 01-10, 01-5, 10-10, 5-10, 10-20, 20-50, and 50-100 mg, for example between 0.05 and 100 mg, preferably between 0.05 and 5 mg, for example 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, l, 2, 3, 4 or 5 mg. The dosage may be adjusted by the skilled person depending on the modulator and the disease.

The itions of the ion can fithher comprise one or more additional active compounds, for simultaneous or sequential use.

The ion also relates to a method for preparing a pharmaceutical composition, comprising mixing a GIBsPLA2 modulator as previously bed and a pharmaceutically acceptable excipient, and formulating the composition in any suitable form or container (syringe, apoule, flask, bottle, pouch, etc).

The invention also relates to a kit comprising (i) a composition comprising a GlBsPLA2 modulator as previously described, (ii) at least one container, and optionally (iii) written instructions for using the kit.

Diseases The compounds and compositions of the invention may be used to treat any disease d to an inappropriate (e.g., defective or improper) immune response, particularly to an inappropriate CD4 T cell activity, as well as any e where an increased immunity may ameliorate the t condition. These diseases are sometime referred to as “immune disorders” in the present application. This includes immunodefective situations (e.g., caused by Viral infection, pathogenic ion, , etc.), autoimmune diseases, grafts, diabetes, inflammatory diseases, cancers, allergies, asthma, psoriasis, urticaria, eczema and the like.

Immunodeficiencies and associated disorders In a first aspect, the invention is based on an inhibition of GlBsPLA2 in a subject, thereby increasing or ing an immune activity, ularly a CD4-T cell—mediated activity.

In a ular embodiment, the invention is ore directed to methods for stimulating an immune response in a subject in need f, comprising inhibiting GlBsPLA2 in said subject.

In a particular embodiment, the invention is directed to methods for modulating white blood cells in a subject in need thereof, comprising inhibiting GlBsPLA2 in said subject.

Examples of diseases that can benefit from GlBsPLA2 inhibitors are all diseases with an immunodeficiency such as diated immunodeficiency. In this regard, in a particular embodiment, the invention is directed to methods for treating an immunodeficiency or an associated disorder in a subject in need thereof, comprising inhibiting GlBsPLA2 in said subject.

In another ular embodiment, the invention is directed to a GIBsPLA2 inhibitor for use for treating an immunodeficiency or an associated disorder in a subject in need thereof.

Immunodeficiencies and associated disorders designate any condition or pathology characterized by and/or caused by a reduced immune function or response in a subject.

Immunodeficiencies may be caused by e.g., viral infection (e.g., HIV, hepatitis B, etc.), ial infection, cancer, or other pathological conditions. The terme “immunodeficiency- associated disorder” therefore ates any disease caused by or associated with an immunodeficiency. The invention is ularly suitable for ng immunodeficiencies related to CD4-T cells, and associated diseases. The present application indeed demonstrates that the biological effects of GIBsPLA2 are involved in CD4 T cell disease state. Accordingly, blocking the activity of GlBsPLA2 has a therapeutic t in subjects with altered response to cytokine causing immunodeficiency as often observed in patients infected With HIV.

Accordingly, in a particular embodiment, the invention relates to methods of treating HIV infection in a subject by ting GIBsPLA2 in the subject, preferably by stering a GIBsPLA2 inhibitor or vaccine to the subject. In some embodiments the subject is an early HIV patient and the methods results in increasing the probability that the patient is a HIV controller. In some embodiments the subject is a patient with low reconstitution after antiretroviral ent and/or with severe idiopatic CD4 T lymphopenia (ICL). The ion also relates to a method for increasing CD4-T cell activity in a HIV-infected subject by inhibiting GIBsPLA2 in the subject, preferably by administering a GIBsPLA2 inhibitor or vaccine to the subject.

In another embodiment, the invention s to methods of treating acute and/or chronic inflammation and sus derived from inflammatory reactions in a subject by injecting GIBsPLA2 in the subject, either directly or associated with anti- inflammatory drugs.

The invention also provides methods for treating cancer by increasing an immune response in the subject, comprising inhibiting A2 in the t, preferably by stering a GIBsPLA2 inhibitor or vaccine to the subject. The invention also provides methods of treating CD4 T cell-linked immunodeficiency associated with cancer in a subject by inhibiting GIBsPLA2 in the subject, ably by stering a GIBsPLA2 tor or vaccine to the subject.

Pathologic immune responses and associated diseases The invention may be used to treat any disease related to an inappropriate (e.g., pathologic or improper) immune response or to an undesirable (hyper)activity or (hyper)activation of the immune system, particularly to an inappropriate CD4 T cell WO 97140 activity. These diseases include, for instance, autoimmune diseases, grafts, diabetes, allergies, asthma, psoriasis, urticaria, eczema and the like.

In a further aspect, the invention is thus based on an activation or induction of GIBsPLA2 in a subject, thereby inhibiting an immune activity, particularly a CD4-T cell— mediated activity.

In a particular embodiment, the invention is therefore directed to methods for inhibiting an immune response in a subject in need thereof, comprising inducing or ting GIBsPLA2 in said subject.

In a particular embodiment, the invention is directed to methods for inhibiting white blood cells in a subject in need f, comprising inhibiting GIBsPLA2 in said subject.

In another particular embodiment, the ion is directed to methods for treating disorder caused by an undesirable immune response in a subject in need thereof, comprising inducing or activating GIBsPLA2 in said t.

Inducing or activating GIBsPLA2 in a subject preferably comprises administeriong to the subject a GIBsPLA2 agonist, for example a GIBsPLA2 protein or a filnctional fragment thereof.

In r particular ment, the invention is directed to a A2 agonist or activator for use for treating a disorder caused by an undesirable immune response in a subject in need f.

Examples of diseases that can benefit from GIBsPLA2 agonists are autoimmune ers, cancers, Viral diseases, bacterial ions, etc.

In a particular embodiment, the invention is directed to methods for treating an auto- immune disorder in a subject in need thereof, comprising stimulating or inducing GIBsPLA2 in said subject.

In another particular embodiment, the ion is directed to a compound or a composition of the invention for use in treating an auto-immune disorder in a t in need thereof.

In a particular embodiment, the invention is directed to methods for treating a cancer in a subject in need thereof, comprising stimulating or inducing GIBsPLA2 in said subject.

In r particular ment, the invention is directed to a compound or a ition of the invention for use in treating cancer in a subject in need thereof.

Another particular embodiment of the invention relates to a method for treating (e.g., reducing or preventing or inhibiting) graft rejection, or for treating graft vs host disease in a transplanted subject, comprising stimulating or inducing GIBsPLA2 in said subject. A further object of the invention is a method for ing allogeneic graft tolerance in a subject comprising stimulating or inducing GlBsPLA2 in said subject.

Anti-microbial activity The present application also provides, in a fithher aspect, a method for killing microbes using AZ. By acting directly on the membranes, GIBsPLA2 can destroy or kill bacteria, enveloped s, parasites and the like.

In acute infections or in infections, GIBsPLA2 may be used either alone or associated With antibiotics, anti-viral, anti-retroviral and anti-parasite drugs. In the case of microbes resistant to known anti-microbial drugs, GlBsPLA2 may represent an alternative therapy. It can be used in very short term treatment, e.g., in very ous and acute clinical situations.

Specific examples of diseases that can benefit from treatment by GlBsPLA2 according to the invention are all the clinical situations with an hyper activity of the immune system or a chronic inflammation suc as Multiple sclerosis, Myasthenia gravis, Autoimmune neuropathies such as Guillain-Barré, Autoimmune uveitis, Uveitis, mune tic anemia, Pemicious anemia, Autoimmune thrombocytopenia, Temporal arteritis, Anti- phospholipid syndrome, itides such as Wegener's granulomatosis, Behcet's disease, Atherosclerosis, Psoriasis, Dermatitis herpetiformis, Pemphigus vulgaris, Vitiligo, Pemphigus is, Mycosis Fungoides, Allergic Contact Dermatitis, Atopic Dermatitis, Lichen Planus, PLEVA, , Crohn‘s Disease, Ulcerative colitis, Primary biliary cirrhosis, Autoimmune hepatitis, Type 1 diabetes mellitus, Addison's Disease, Grave's Disease, Hashimoto's thyroiditis, Autoimmune itis and orchitis, Autoimmune Thyroiditis, Rheumatoid arthritis, Systemic lupus erythematosus, Scleroderrna, Polymyositis, Dermatomyositis, Spondyloarthropathies such as ankylosing spondylitis, or Sjogren's Syndrome.

The duration, dosages and frequency of administering compounds or compositions of the invention may be d ing to the subject and disease. The treatment may be used alone or in combination with other active ingredients, either simultaneously or separately or sequentially.

The nds or compositions according to the invention may be administered in various ways or routes such as, t limitation, by systemic injection, intramuscular, intravenous, intraperitoneal, cutaneous, subcutaneous, derrnic, transderrnic, intrathecal, ocular (for example l) or rectal way, or by a topic administration on an inflammation site, and ably by intramuscular or intravenous ion.

A typical regimen comprises a single or repeated administration of an effective amount of a GIBsPLA2 modulator over a period of one or several days, up to one year, and including between one week and about six months. It is understood that the dosage of a pharmaceutical compound or composition of the invention administered in viva will be dependent upon the age, health, sex, and weight of the recipient ct), kind of concurrent treatment, if any, frequency of treatment, and the nature of the pharmaceutical effect desired. The ranges of effectives doses provided herein are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one skilled in the relevant arts (see, e.g., Berkowet et al., eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodmanetna., eds., Goodman and Cilman’s The pharmacological Basis ofTherapeutics, 10th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001)).

The invention also provides methods for detecting an immune defect in a subject based on a detection of the ce or amount or absence of GIBsPLA2 in a sample from a subject. The method of the invention may be carried out using a variety of detection logies or platforms known per se in the art such as, t limitation Capture assay, Sandwich assay, Competition assay, Radio-immuno assays, Enzyme labels with substrates that generate colored, fluorescent, chemiluminescent, or electrochemically-active products, Fluorescence, fluorescent polarization, Chemiluminescence, Optical and colorimetric, Electrochemiluminescence, Time-resolved fluorescence, e n resonance, Evanescent wave, Multiwell plate (ELISA), dual assay, Multiplex assay, Latex bead — multiplex assay, Microarray (Laminar surface) — multiplex assay, Glass, Plate based assays or Strip based assays.

In a particular ment, the method comprises determining the presence, or amount, or absence of a polymorphism in the GIBsPLA2 gene, RNA or protein. Our results show that GIBsPLA2 is subject to high polymorphism and that this correlates to the physiological status of subjects. The invention thus comprises (i) determining the presence, or amount, or absence of a particular polymorphic isoform of GIBsPLA2, and/or (ii) ining the global rate of polymorphism of A2 in a subject, said data being correlated to the physiological status of the subject. In particular, specific isoforms may be characteristic of the predisposition, presence or onset in a subject of a disorder as described above. Such determination may also be used in personalized medicine, to adjust treatment.

Methods of Monitoring and/or Diagnosing Immunodeficiency Associated With CD4 T Cell Defects Comprising Detecting A2 s of monitoring and/or diagnosing immunodeficiency associated to CD4 T cell defects in particular in human immunodeficiency virus (HIV) infection in a subject, are provided by this disclosure. In some embodiments the methods comprise (a) ing a sample containing a body fluid, preferably plasma from a subject, and (b) detecting a level of GIBSPLA2 in the sample above a old. The presence of GIBsPLAZ in the sample may be detected by any method known in the art, such as for example by a method comprising an enzymatic assay, a ligand-capture assay and/or an immunoassay.

In some embodiments the method comprises obtaining a sample comprising plasma from a subject and ining Whether the plasma has at least one activity selected from inducing formation of al membrane microdomains (MMD) in CD4 T cells from healthy subjects and rendering CD4 T cells of healthy subject refractory to interleukin-7 (IL-7) signaling. If the plasma from the subject comprises such an activity then the subject is in some embodiments determined to have a CD4 T cell-linked immunodeficience as often observed in HIV-infected patients but not only. If the plasma fraction does not comprise such an activity then the subject is in some embodiments determined to have low exposure to deficiency ated to the alteration of T CD4 cells to cytokine-regulated homeostasis.

In some embodiments the subject is ined to have an HIV infection.

In contrast, if the protein fraction does not comprise such an activity then the subject is in some embodiments determined to not have an immunodeficiency associated to CD4 T cell defects as sed herein. In some embodiments the t is determined to not have an HIV infection.

In some embodiments the methods comprise contacting the sample sing a body fluid, preferably plasma, from the subject with an antibody c for GIBsPLA2 and ining the presence or absence of an immunological reaction. In some embodiments the presence or absence of an immunological reaction is determined by a method comprising an enzyme-linked immunosorbent assay (ELISA). The presence of an logical reaction between the antibody specific for GIBsPLA2 and the sample indicates the presence of GIBsPLA2 in the sample, which in turn indicates that the subject has an immunodeficiency associated to CD4 T cell s. In some embodiments the subject is determined to have an HIV infection. In contrast, the absence of an immunological reaction between the antibody specific for GIBsPLA2 and the sample indicates that the subject does not have an immunodeficiency associated to CD4 T cell defects as sed herein. In some ments the subject is determined to not have an HIV infection.

In some embodiments the assay for the presence of GIBsPLA2 in the sample is qualitative. In some embodiments the assay for the presence of GIBsPLA2 in the sample is tative.

In some embodiments the methods comprise comparing the results of the assay to the results of a similar assay of a control sample comprising plasma of a subject who does not have an immunodeficiency associated to CD4 T cell defects. In some embodiments the methods comprise comparing the results of the assay to the results of a similar assay of a sample comprising plasma of the same subject harvested earlier. s of Monitoring and/or Diagnosing Immunodeficiency Associated With CD4 T Cell Alteration Comprising Characterizing Membrane Microdomains on CD4 T Cells

[00118] The data in the examples demonstrate that HIV-infected patients present formation of distinctive ne microdomains (MMD) on the surface of CD4 T cells although very few cells are really ed by HIV. Accordingly, this disclosure also provides methods for diagnosing immunodeficiency ated with CD4 T cell alteration, such as for example immunodeficiency caused by human immunodeficiency Virus (HIV) WO 97140 infection in a subject. In some embodiments the methods comprise: (a) isolating CD4 T lymphocytes from a subject, and (b) measuring the number and/or size of membrane microdomains (MMD) on the T-cells. In some ments the methods fithher comprise at least one of (c) measuring the amount of phospho-STATS in the T-cells and (d) assaying the nuclear import fraction of phospho-STAT5 in the T-cells. In some embodiments the number and/or size of MMD on the T-cells is measured in the absence of interleukin. In some embodiments the number and/or size of MMD on the s is measured in the absence of IL-2. In some embodiments the number and/or size of MMD on the T-cells is measured in the absence of IL-7. In some embodiments the number and/or size of MMD on the T-cells is measured in the presence of a subthreshold level of interleukin.

In some embodiments if the number of MMD on the T cells isolated from the subject is at least a threshold that indicates that the subject has immunodeficiency associated with CD4 T cell alteration. In some embodiments it indicates that the t has an HIV infection. In some embodiments if the number of MMD on the T cells isolated from the subject is not at least a threshold that indicates that the subject does not have immunodeficiency associated with CD4 T cell alteration as disclosed herein. In some embodiments it means that the subject does not have an impaired CD-4 T cell response to cytokine signaling. In some embodiments it means that the subject does not have an impaired CD-4 T cell response to interleukin-7. In some embodiments it indicates that the t does not have an HIV ion. In some embodiments the threshold is at least about 80 per cell, at least about 90 per cell, at least about 100 per cell, at least about 110 per cell, or at least about 120 per cell. In a non-limiting prefered embodiment, the threshold is at about 100 per cell.In some embodiments if the MMD on the T cells ed from the subject have a er of at least a threshold that indicates that the subject has an HIV infection. In some embodiments ifthe MMD on the T cells isolated from the subject do not have diameter of at least a threshold that indicates that the subject does not have an ed response to interleukin-7 and more generally to cytokines. In some embodiments it tes that the subject does not have an HIV infection. In some embodiments the threshold is a diameter of at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, 2014/078969 or at least 140 nm. In a non-limiting prefered embodiment, the threshold is a diameter of at least about 120 nm.

Because RIF may alter the responsiveness of CD4 T cells to IL-7 by aggregating membrane receptors in abnormaly large MMD, responses to other c and cytokines may be affected as well and RIF might be also associated to other pathologies involving altered CD4 T cell response.

Methods of Identifying Candidate Therapeutic Agents This invention also provides methods for identifying a candidate eutic agent, comprising: (a) contacting CD4 T lymphocytes with GIBsPLA2 in the presence of an agent, and (b) measuring GIBsPLA2-induced CD4 T cell activation. In some embodiments the methods comprise (c) comparing the level of GIBsPLA2-induced CD4 T cell activation in the presence of the agent with the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent. In some embodiments, if the level of GIBsPLA2- induced CD4 T cell activation in the presence of the agent is lower than the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent, then the agent is fied as a ate immunodeficiency therapeutic agent. In some embodiments the agent is identified as a candidate HIV therapeutic agent. In some embodiments, if the level of GIBsPLA2-induced CD4 T cell activation in the presence of the agent is higher than the level of GIBsPLA2-induced CD4 T cell tion in the absence of the agent then the agent is fied as a ate immunosuppressive therapeutic agent.

In some embodiments, measuring GIBsPLA2-induced CD4 T cell activation ses determining the number of MMD per CD4 T cell.

In some ments, measuring GIBsPLA2-induced CD4 T cell activation comprises determining the mean diameter ofMMD on CD4 T cells.

[00124] In some embodiments, measuring GIBsPLA2-induced CD4 T cell activation comprises determining the IL-7 responsiveness of CD4 T cells assayed by STATS phosphorylation and/or nuclear import.

As used herein an “agent” may be any chemical entity under evaluation as a potential therapeutic. In some embodiments the agent is an organic molecule. In some embodiments the agent comprises from 2 to 100 carbon atoms, such as from 2 to 50 carbon atoms, 5 to 50 carbon atoms, or 10 to 50 carbon atoms. In some embodiments the agent is a peptide, a n, a glyco-protein, or a lipoprotein. In some embodiments the agent is an antibody.

In some embodiments the agent has not been previously determined to have a ical activity implying an y as a therapeutic agent for treatment of immunodeficiency, such as that often associated with HIV infection. In some embodiments the agent has been previously determined to have a biological activity implying an utility as a therapeutic agent for treatment of immunodeficiency such as that often associated with HIV infection.

As used herein, a “candidate immunodeficiency therapeutic agent” or a “candidate HIV therapeutic agent” is an agent that inhibits the ability of RIF to ate CD4 T cells in at least one assay. Consistent with the data reported herein, the ability of an agent to inhibit the ability of GIBsPLA2 to te CD4 T cells in at least one assay is a useful way to identify agents that are likely to be therapeutically useful for treating deficiencies including immunodeficiencies associated with HIV ions.

Accordingly, it is also a useful way to identify agents that are likely to be therapeutically useful for treating HIV infection. Of course, as with all therapeutic molecules further characterization will be required. However, this does not t from the utility of candidate HIV therapeutic agents of this disclosure.

Further aspects and advantages of the invention are disclosed in the following experimental section, which shall be considered as illustrative.

EXAMPLES 1. Materials and Methods 1.1. Patients

[00128] VP ed in the study had been HIV-positive for more than one year.

They had never received any antiretroviral drugs and had a viral load > 10,000 RNA copies/ml with a CD4 count > 200/ul at the time of blood collection (ANRS EP 33 and EP20 studies). All blood samples from VP were drawn at the Centre Hospitalier de Gonesse. Blood from HD was provided by the Etablissement Francais du Sang (Centre Necker—Cabanel, Paris). Plasma samples from ART patients were drawn from individuals who had been receiving treatment for at least one year. Their viral load had been undetectable for at least 6 months and their CD4 counts > 11 at the time of blood collection. Plasma samples from HIC patients were drawn from individuals with an undetectable viral load 10 years after infection. Plasma samples were collected at Centre d’Infectiologie Necker—Pasteur. 1.2. Analysis of membrane microdomains (MMD). receptor diffiJsion rates and phospho-STATS cellular tmentalization in purified CD4 T lymphocytes CD4 T-cells were purified by negative selection as already described (10) then activated with 2 nM recombinant glycosylated human IL-7 (Cytheris) or 40ug PHA (Sigma). The confocal and STED microscopy used to study cell surface microdomains (MMD) and phospho-STATS cellular compartment bution has already been described (10, 12). FCS analysis of protein ion at the e of living cells has also been described (10, 12 1.3. Preparation and analysis of detergent-resistant omains (DRM) The preparation of -X100 lysates of CD4 T lymphocytes from HD or VP, followed by centrifiJgation h sucrose gradients and Western blot analysis of the fractions collected, has been usly described (12). mAb specific for flotillin, IL- 7Ralpha and gamma c were used to detect the corresponding bands by Western blots (12). 1.4. Characterization of RIF from VP plasma 1.4.1. Bioassays The MMD induction assay was as follows: VP plasma (5 or 10%) was first incubated (20 min) in medium with ed HD CD4 T cells. The cells were then plated on polylysine-coated glass slides for 10 min then activated by 15 min IL-7 (2nM) or not for control (NS), then fixed by PFA (PFA, 1.5 %, 15 min at 37°C followed by 15 min at room temperature) equilibrated one hour in PBS/SVF 5% before being stained by cholera toxin B (Cth-AF488). MMD were counted by STED microscopy.

The assay for inhibition of STAT phosphorylation and nuclear translocation was as follows: VP plasma (5 or 10%) was first ted with purified HD CD4 T cells (20 min) before ation by IL-7 (2 nM, 15 min.). Cells were then plated on polylysine- coated glass slides for 10 min then activated by 15 min IL-7 (2nM) or not for l (NS), then fixed by PFA (PFA, 1.5 %, 15 min at 37°C ed by 15 min at room temperature) and permeabilization by methanol (90% at -20°C). Cells were equilibrated one hour in PBS/SVF 5% then phospho-STATS was then stained by rabbit anti-STATS labelled with goat anti-rabbit-Atto642 and analyzed by FACS or STED microscopy. 1.4.2. Enzyme Treatments

[00133] The effects of enzyme digestion on RIF activity were evaluated by treating VP plasma filtered on a 30 kDa membrane. Plasma compounds with MW < 10 kDa were used as negative controls. Effects of porcine n (1 U/ml for 30min at 37°C, followed by PMSF tion and buffer exchange with Millipore 5kDa-membrane centrifugal filters), or DNAse I (l U/ml for 30min at 37°C), or RNAse (1 U/ml for 30min at 37°C) or Peptide N—glycanase (1 U/ml for 30min at 37°C) were tested. All preparations were analyzed at 10% final concentration. 1.4.3. MW Determination or RIF purification Size exclusion chromatography was performed by loading 1.6ml of plasma onto a 85-ml Sephadex G100 column pre-equilibrated with ammonium carbonate (0.1M) or PBS, then collecting 0.8 ml fractions of the eluate. The column was ated using a protein set (GE-Healthcare). n concentration was measured by the Bradford method.

VP plasma previously filtered on a 100 kDa membrane and total VP plasma were tested and gave identical s. Fractions between Da were collected, which contain urifed RIF. 1.4.4 Isoelectric Point Determination Anion or cation exchange chromatography was performed on MonoQ or MonoS lml columns (GE-Healthcare) with elution by successive pH steps (ammonium carbonate/ammonium acetate buffers). The pH of each eluated on was measured and these were then adjusted to pH 7.4 before testing of their biological effects. RIF activity was measured in the ponding fractions immediately after elution. 1.4.5 MS analysis

[00136] Samples from gel filtration (G100) were lyophilized then resuspended, pooled and proteolysed with porcine trypsin, according to methods known per se in the art. lytic peptides were then separated in 12 fractions by chromatography through C18 column eluted in ammonium acetate. The 12 fractions were separated through C18 eluted in reverse phase (acetonitrile) and directly injected by ospray in an orbitrap Velos (Thermo Scientific) for MS analysis with secondary Ar—fragmentation then MS/MS for the higher-intensity peaks per MS scan.

Standard Mascot and X-Tandem programs were used. For each n of database subsets, 3 criteria were computed: - i-score: Computation oftheoretical specificity of every peptides from trypsin digestion of a single protein in the NextProt se enriched with mature proteins with signal peptide cleavage (number of unique peptides/protein): number of specific peptides overall human sequences (all), ces with a N—term signal peptide (sec) per protein - ation of the theoretical ence of es compatible with peaks from all MS scan series (theoretical peptide matching peaks/protein) - Computation of the theoretical coverage of protein sequence with peak-matching peptides For each protein a [9 score was determined as a computation of all three scores. e 1: Aberrant activation of CD4 T lymphocytes from VP as measured by the presence of al membrane microdomains (MMD).

This example describes the investigation of new molecular and cellular parameters that explain some of the abnormal responses seen in the CD4 T lymphocytes of chronically HIV-infected patients. Chronic activation of the immune system is usually measured by assessing the over expression of cell surface molecules such as CD38, HLA-DR and CD25 that are considered as the main markers of CD4 dysfiJnction (15). However, despite many efforts, these data have remained blurred, and the phenotype of the CD4 T cells cannot directly explain their immune defects.

STED microscopy and labeling with cholera toxin B (Cth-AF488) were used to detect the presence of MMD (12). Before any stimulation, the surface of CD4 T lymphocytes purified from VP was found to bear far more MMD than ent CD4 T lymphocytes purified from HD (Fig. 1a). And most antly, all the CD4 T cells from VP showed increased numbers ofMMD. This abnormal pattern was not the consequence of stimulation by IL-7 in VP plasma since average IL-7 concentrations in this plasma (0.4 pM) were only 100th the Kd of the IL-7R (13, 14). When purified CD4 T cells from HD were stimulated by IL-7, large numbers of MMD were observed. By st, the MMD pattern of CD4 T cells fiom VP was unaffected by 1L-7 (Fig. 1a). This abnormal activation coupled with the e of any response to IL-7 can be mimicked by a non physiological stimulus such as with phytohemagglutinin (PHA) (Fig. 1a).

These s abnormal MMD were then counted. Around 150-200 MMD were observed per CD4 T cell from VP, as with FHA-stimulated HD CD4 T cells (Fig. lc).

Here again, the results obtained showed that all CD4 T cells from VP expressed MMD, including all the major CD4 subpopulations (Fig. 1c). IL-7 failed to increase MMD numbers in VP. By contrast, MMD numbers in HD CD4 T cells increased from a ound level of around 10 MMD/cell to 300 after IL-7 stimulation. A study of MMD size was also conducted (Fig.1d and e). This showed that the MMD on CD4 T cells from VP and on FHA-stimulated HD CD4 T cells were far larger (250 nm) than those from HD CD4 T cells stimulated by lL-7 (90 nm).

Example 2: All IL-7R alpha and gamma-c chains are sequestered in abnormal detergent-resistant membrane microdomains (DRM) isolated from VP CD4 T cells g HD CD4 T cells were analyzed to verify that IL-7R alpha and gamma-c chains are located in ensity fractions e MMD. When these HD CD4 T cells are stimulated by lL-7, these two chains are located in low-density fractions corresponding to detergent-resistant MMD or DRM containing all the proteins sequestered in MMD (Fig. 2).

When the study was repeated on CD4 T cells purified from VP, the pattern was different (Fig. 2). Before any stimulation, all the lL-7R alpha and gamma-c chains were already tered in DRM; none were d in the high-density fractions corresponding to free receptors outside the MMD. Furthermore, pre-stimulation of the CD4 T cells by IL-7, before DRM preparation, did not affect this pattern (data not shown). Here again, pre-stimulation of HD CD4 T cells by non physiological PHA reproduced this pathological situation. This confirms the data in Fig. 1 and demonstrates that the CD4 T cells in VP are subject to aberrant activation prior to any ation. In addition, these al MMD contain all the IL-7R chains (Fig. 2).

Example 3: 2D gel analysis of the IL-7 signalosome in purified CD4 T cells from HD, VP and ILstimulated HD cells. Characterization of the aberrant state of activation by the protein pattern recovered after immunoprecipitation 2D-electrophoresis was used to demonstrate that the compostion of the IL-7 signalosome in VP was abnormal and different from that in quiescent and ILactivated HD CD4 T cells (Figures 7a, 7b and 7c).

Proteins were immunoprecipitated with anti-IL-7Ralpha (mouse mAb 40131, R&D System) immobilized on proteinG-Sepharose 4G from d CD4 T-cell lysate and separated on 2D-PAGE (IEF on pH 3-10 gel stripes followed by SDS-gel with 12% acrylamide-bis). pH and MW (kDa) scales are displayed. Gels were stained with Ruby. The gels shown are representative of 8 NS/IL-7 pairs obtained from HD and 3 gels from VP.

[00145] (Fig. 7a) non-stimulated (NS) HD CD4 T-cells.

(Fig. 7b) VP CD4 T-cells. More spots were observed in Sypro Ruby-stained 2D-gels prepared from VP than firom HD. In addition we observed that common spots were more intense when 2D-gels were prepared with VP extracts.

(Fig. 7c) ILstimulated HD CD4 T-cells. The n in HD CD4 T cells stimulated by IL-7 differs from that in VP CD4 T cells. This further supports the proposal that the aberrant tion found in VP is not the consequence of IL-7 stimulation that could take place in organs with high levels of IL-7, for example in IL—7—producing organs.

It may be concluded from this analysis that IL-7R chains in VP CD4 T cells are not only part of abnormal MMD but also that they interact with protein complexes different from those found in the normal 1L-7 signalosome.

Example 4: Diffusion rate of IL-7Ralpha at the e of purified CD4 T cells from HD, VP and PHA-stimulated HD cells. IL-7Ralpha in VP CD4 T cells is embedded in lipid-rich abnormal MMD, thus limiting its diffusion rates and precluding any ctions with the cytoskeleton and ore any y to transmit signals The two-dimensional ion of IL-7Ralpha stained with AF488-anti-1L- 7Ralpha mAb was ed by FCS at the surface of living CD4 T-cells. The results are shown in Figure 8. Diffusion times I'D (in 10'3 sec) were measured in the absence of IL-7 (0, autocorrelation) or in the presence of ILbiotin-SAF633 (O, crosscorrelation) as described (10, 12). These times were then plotted versus cell surface area 0302 (in 103 nm2) intercepted by the confocal volume. The diffiision plots are shown with and without pre- treatment with MMD inhibitors (COase l ug/ml plus SMase 0.1 ug/ml for 30 min) or cytoskeleton inhibitors (CytD 20 uM plus Col lOuM for .

Bars indicate SEM from 5 independent experiments. Slopes of the linear regression give effective ion rates Defy and y-intercepts olate confinement time 1:0 as we described previously (12). Del?" are shown in the bar graph Fig.3a.

] (Figs. 8a, 8d) at the surface of HD CD4 T-cells, (Figs. 8b, 8e) at the surface ofVP CD4 T cells, (Figs. 8c, St) at the surface of HD CD4 T cells pre-activated with PHA (lug/ml).

(Fig. 8g) Scheme of the mechanism of lL-7Ralpha diffiJsion embedded in MMD before and after treatment by MMD inhibitors or cytoskeleton tors. MMD are indicated by disks, receptors by rods, cytoskeleton is shown as a net. ion rates (fast, slow, very slow) are indicated to facilitate data interpretation. This scheme illustrates the results also ed in Fig. 3a.

Example 5: IL-7R chains sequestered in the abnormal MMD of VP CD4 T cells are non functional IL-7R alpha diffusion rates were ed at the surface of CD4 T cells as usly described (10, 12) and as detailed in Example 4. Before any stimulation, these diffusion rates were seen to be three times slower on VP than HD CD4 T cells (Fig. 3a).

This further demonstrates that IL-7R alpha chains are embedded in abnormal MMD at the surface of these CD4 T cells (Fig. 3a). COase plus SMase treatment released the receptor from its MMD constraints and therefore increased its diffusion rate (Fig. 3a). By contrast, ent with cytochalasin D (Cyt D) plus colchicin (Col) - which disorganizes the cytoskeleton - had no effect on the diffiJsion rate of the lL-7R alpha chain in VP CD4 T cells (Fig. 3a). Since cytoskeleton organization is an absolute necessity for signal transduction, this absence of any onal or ural link between IL-7R alpha and the cytoskeleton rk suggests that signaling cannot proceed when IL—7R complexes are sequestered in abnormal MMD, as is the case in VP CD4 T cells.

Pulsed-STED microscopy was then used to study STAT5 phosphorylation (phospho-STATS) and phospho-STAT5 partition in the cytoplasm and nucleus of both HD and VP CD4 T cells. Fig. 3b shows STED images of phospho-STAT5 distribution before and after 15 min of IL-7 stimulation. We noted that o-STATS accumulated in the s of HD CD4 T cells, and this phenomenon was inhibited by cytoskeleton disorganization. By contrast, no phospho-STAT5 translocation to the nucleus occurred in VP CD4 T cells or in PHA pre-stimulated HD CD4 T cells (Fig. 3b).

The cs of phospho-STAT5 appearance in the cytoplasm and s was then followed for one hour (Figs. 30, d, e). This showed that phospho-STAT5 in VP CD4 T cells mostly accumulated in the cytoplasm and did not migrate to the nucleus (Fig. 3d), as in FHA-stimulated HD CD4 T cells (Fig. 3e). This was particularly clear when the results were compared with those obtained in the five minutes following IL-7 stimulation of HD CD4 T cells where 50% ofphospho-STAT5 was found in the nucleus (Fig. 3c).

Example 6: Plasma from VP induces abnormal MMD at the surface of purified HD CD4 T cells The origin of the aberrant activation of VP CD4 T cells was then investigated. The fact that all the CD4 T cells were ed and that a non physiological signal such as PHA mimics the results led to an investigation of the plasma of VP. Purified HD CD4 T cells were incubated with 10% VP plasma for 30 min and MMD counted at the surface of the CD4 T cells as detected by labeled cholera toxin B (Cth-AF488). Fig. 4a shows the images obtained. VP plasma alone induced large numbers of MMD on HD CD4 T cells. Adding IL-7 did not affect the size or number of these MMD (Fig. 4a). These results are shown for plasma from five different VP (Fig. 4b) and were verified using many more plasma samples from these VP (> 15). The experiments were also ed using CD4 T cells from different HD (> 5). Controls consisted of testing plasma samples from HIV- llers (HIC) and antiretroviral-treated (ART) patients on purified HD CD4 T cells.

None of these induced MMD or inhibited the 1L-7 ion ofMMD (Fig. 4c).

This was further verified by testing a large number of dilutions of the various plasmas (Fig. 4d). VP plasma down to a 0.1% dilution resulted in the formation of MMD scattered across the cell surface. VP plasma diluted 50 to 100 fold gave 50% maximun activity. None of the plasma samples from HIC or ART patients induced MMD at any dilution.

Example 7: Plasma from VP ts ILinduced phospho—STATS nuclear translocation The function of the IL-7R in HD CD4 T cells treated with VP plasma was tested by following STAT5 phosphorylation and nuclear translocation. As seen in , pre-incubation of HD CD4 T cells with VP plasma (10% concentration) inhibited lL d STAT5 phosphorylation and its nuclear translocation. Fig 5b shows the results obtained with five VP plasma samples. All at a 10% dilution ted the nuclear translocation of phospho-STAT5. These results were confirmed with plasmas from ent VP ( > 15) and various sources ofHD CD4 T cells (>5).

[00161] The effect of plasma d from HIC and ART patients was also tested by pre-incubating these with purified HD CD4 T cells (Fig. 5a and 5c). Here again, only VP plasma was able to inhibit the ILinduced nuclear translocation of phospho-STAT5. It was also determined (Fig. 5d) that VP plasma was active down to a 0.1% dilution, and half maximun activity was obtained at a 50 to 100 fold dilution, thus correlating with the ability to induce abnormal MMD (Fig. 4d).

The effect of plasma derived from ART-treated patients but non-responsive (CD4-NR) to their treatment (low count of Viral RNA and low count of CD4 T-cells) was also tested by pre-incubating these with purified HD CD4 T cells. Here again, only CD4- NR plasma was able to inhibit the ILinduced nuclear ocation of o-STAT5.

It was also determined that CD4-NR plasma was active down to a 0.1% dilution, and half n activity was obtained at a 50 to 100 fold dilution, thus correlating with the ability to induce abnormal MMD as observed with VP. e 8: lar characterization of the Refractory state Inducing Factor The al nature of RIF was investigated. The studies performed (Fig. 6a) showed that RIF is a n since its actiVity was destroyed by trypsin. Treatment with peptide N—glycanase (PNGase) had no effect, indicating that N—glycolysation is not required for RIF actiVity.

The molecular weight of RIF was then measured by xclusion tography on Sephadex G—100. Induction of MMD (Fig. 6b) and inhibition of lL induced phospho-STATS nuclear translocation (Fig. 6c) was measured for all fractions eluted from the column. Two entative column profiles are given in Fig. 6. Both show that RIF is a single factor with a MW between 10 and 15 kDa.

Fig. 6b shows the densities of the Viral peptides or proteins measured by dot blot in each of the 100 fractions collected from the Sephadex G100 column. Measurements were repeated three times with different polyclonal antibodies from VP plasma samples characterized by their high activity against Viral proteins. For each experiment the signals obtained with HD plasmas were then subtracted from the values. The pattern shown in Fig. 6b demonstrates that no Viral proteins or fragments were ed in the fraction containing RIF activity while the dot blot assay was able to detect Viral proteins at higher MW (from 190 to 32 kDa).

Ten to 15 kDa active, enriched fractions from the Sephadex G100 columns were then used to frame the isoelectric point of RIF by retention on anion (MonoQ) or cation (MonoS) exchange columns followed by pH elution (pH increase with MonoS or pH decrease with MonoQ) (Fig. 6d). The MMD-inducing activity of the various pH fractions was then measured after adjusting their pH to 7.4. In all, 25 to 30% of the initial activity was recovered in two fractions, a result consistent with an isoelectric point of 6.5 to 8.0.

RIF is therefore a secreted protein, with a MW of about 15kDa, a pI around 7.5-8.0, which contains disulfide bridge. Following the above structural and flinctional features, RIF identity was directly obtained. In particular, amongst all of the 36853 known human proteins, 62 only had the above four characteristics of RIF. Semi-purified material prepared from three c patients and three HD were analyzed using mass spectrometry and standard Mascot program. Proteins recovered were ranked according to the p score described in Materials and Methods. The results shown in Table 1 below clearly and directly te that RIF is GIBsPLA2.

Table l Mnemonic ID P1 MW i s 1 _score descri otion PA21B HUMAN P04054 .14138.99 9 0.64 ...hoshoiiase A2 mu 1 0.29 Transmembrane protein 9 TMEM9_HUMAN Q9POT7 6.23 18568.37 (TM) 0.10 Endothelial cell-spe ESMl_HUMAN Q9NQ30 6.83 18122.42 molecule 1 CYTD_HUMAN P2832513858.6 3 0.08 Cystatin-D SSRB_HUMAN P4330818273.74 7 0.05 Signal sequub beta (TM) GPIX HUMAN P14770 6.14 17316.06 6 0.04 Platelet rotein IX UMAN P61769 7.67 18510.47 4 0.03 -micr0globulin EPGN_HUMAN Q6UW0914724.99 1 0.02 Epigen UMAN Q9UHDO 56 5 0.02 eukin-19 IL3_HUMAN P0870015091.38 3 0.02 Interleukin-3 7 0.02 Glycosyl-PPI—anc like GML_HUMAN Q99445 6.67 15918.41 protein CYTM_HUMAN P051 13 7 .02 13149.22 4 0.017 Cystatin-M 2014/078969 The protein found in the plasma mic patients is thus the secreted form of GIBsPLA2. The mature protein has 125 aa (MW14138), PI 7.95 and 7 de bridges.

Using commercial purified porcine GIBsPLA2, we were able to verify in vitro that this protein induces abnormal MDM, which block IL-7 pSTAT5 nuclear translocation in the plasma of Viremic patients, further confirming that RIF is GIBsPLA2, more specifically the secreted form thereof. The amino acid sequence of a human GIBsPLA2 is provided as SEQ ID NO: 2.

Example 9: IB induces onsiveness (anergy) of CD4 lymphocytes

[00170] Example 7 shows that PLA2sGIB, through induction of aMMD, s a blockade of ILinduced nuclear translocation ofphospho STATS (NT pSTAT5).

Consequently, CD4 T lymphocytes do not respond to IL-7 and despite of the high level of this cytokine in the plasma ofHIV patients, their number decreases then leading to CD4 lymphopenia the rk of HIV-infected patients.

[00171] Here we investigated the possibility that PLA2sGIB also participates to the ion of anergy, another characteristic of the CD4 lymphocytes from chronically HIV- infected patients.

Bioassay MMD induction: VP plasma containing PLA2sGIB was first incubated (20 min) in medium with purified HD CD4 T cells. The cells were then plated on polylysine-coated glass slides for an additional min. They were then fixed with paraformaldehyde (PFA, 1.5%, 15 min at 370C ed by 15 min at room temperature) before being stained by cholera toxin B (Cth-AF488), MMD were counted by CW-STED microscopy.

[00173] Inhibition ofSTAT phosphorylation and nuclear translocation: VP plasma containing PLA2sGIB was first incubated with purified HD CD4 T cells (20 min) before stimulation by IL-7 (2 nM, 15 min). Cells were then plated on polylysine coated glass slides before fixation by PFA (1.5%) and permeabilization by methanol (90% at - 20°C). pSTATS was then stained by rabbit anti-STATS labelled with goat anti-rabbit- Atto642 and analyzed by FACS or pulsed STED microscopy.

Results Figure 10a shows that after exposition to PLAZ GIB (plasma of Viremic patient), CD4 lymphocytes from healthy donors (HD) become unable to respond to IL—2, as measured by the inhibition of the ILinduced NT pSTATS. This inhibition is total with 3% plasma, and highly significant with 1% plasma (p<0.0001).

] We further d the response of CD4+ CD25+ T reg lymphocytes to PLA2 GIB. The results are presented in Figure 10b. As illustrated, while 100% of healthy cells respond to IL-2 by NT pSTATS, PLA2 GIB (1% plasma of Viremic patients) completely inhibited this signal transduction mechanism. Since CD4+ CD25+ cells represent less than 5% of total CD4 T cells, they cannot cantly influence the data ted in Figure 10a.

IL-7 and IL-2 are members of the gamma c ne family. To confirm that unresponsiveness to this cytokine may be linked to gamma c, we tested the response to IL- 4. IL-4 response was measured by ing the IL-4 induced NT of pSTAT6 (Figure 11).

Our results clearly show that IL-4 response is inhibited by PLAZ GIB (completely with 3% plasma and greatly with 1% plasma).

] These results therefore show that the signaling mechanisms induced by cytokines of the gamma c family are altered by PLA2 GIB. This is in complete agreement with our finding that gamma c or chain is found completely sequestered in aMMD spontaneously found at the e of CD4 lymphocytes from HIV-patients (data not shown).

WO 97140 Example 10: Activity of recombinant forms of PLA2 GIB In this example, the activity of various d forms of PLA2 GIB proteins was tested, to further confirm the effect of this protein in purified form on the immune system, and to further confirm its specificity.

Enzymatic assay The assay was performed with the Enz Check PLA2 assay kit from Life Technologies (ref: E102147). This assay provides a continuous rapid real-time monitoring of PLA2 enzyme activities. The PLA2 ty is followed by the intensity increase of a single wavelength at 515 nm. PLA2 is detected by changes in the emission intensity ratio at 515/575nm with excitation at 460nm. Specific activities are expressed in amount of cent substrate (U) obtained per second and per ug of enzyme in solution.

Results The results are provided in Table 2 below.

Table 2: Activity of recombinant PLA2 GIB proteins Nature Initial Final Specific PLA2 concentration concentration Quantity activity (mg/ml) (ug/ml) (ug) (U/ug/s) Purified e ppPLA2 IB pancreas 7694.31 recombinant pPLA2 1B porcine (in E. coli) 10353.57 recombinant human hPLA2 IB (in E.coli) 0.70 1.40 0.07 10694.57 recombinant human hPLA2 11A (in E. coli) 1.45 2.90 0.15 214.93 recombinant human hPLA2 IID (E. coli) 445 .21 inant human hPLA2 X (in E. coli) 0.68 1.36 0.07 3318.97 The s show that recombinant human PLA2 GIB produced in E. Coli exhibit a potent enzymatic activity. Furthermore, the results also show that inant porcine PLAZGIB produced in E. Coli has a specific ty similar to that of recombinant human B.

By contrast, recombinant PLAZGIIA and PLAZGIID are not active and PLAZGX has a very d activity.

Recombinant PLA2 GIB thus represents a potent active agent for use in the present invention.

Example 11: The effects of PLAZsGIB on CD4 lymphocytes involve its enzymatic activity In this example, we investigated whether the ty of PLAZsGIB on CD4 lymphocytes involved (e.g., was a consequence of) an enzymatic (e.g., catalytic) activity of PLAZsGIB.

Such enzymatic activity would modify the membrane structure leading to the formation of multiple aMMD at the surface ofCD4 lymphocytes.

In these experiments, we tested a mutant of PLA2sGIB where a critical histidine at position 48 was replaced by glutamine (mutant H48Q). Using the enzymatic test described in example 10, we compared the enzymatic ty of recombinant porcine PLA2 GIB produced in E. Coli with the activity of mutant H48Q also produced in E. Coli. Each protein was used at 200microM. As shown Figure 12, the mutant has lost all of its enzymatic activity, illustrating the critical role of histidine at position 48 in PLA2 GIB.

We then compared the activity of Wild type porcine PLA2 GIB with its mutant H48Q in a ay. The results presented in Figure 13 show that the mutant has lost the ability of thLA2 GIB to induce aMMD or to reduce or abrogate IL—7 induced Nuclear Translocation ofpSTATS (NT pSTATS).

These results thus demonstrate that the enzymatic activity is involved in the pathogenic effects of PL2 GIB on CD4 lymphocytes. e 12: Anti-GIBsPLA2 antibodies e CD4-T cell activity in the plasma of HIV viremic patients.

This example rates that, in the plasma of viremic patients, GIBsPLA2 transforms CD4 cytes from HD into “sick” lymphocytes comparable to those characterized in the blood of HIV—infected patients. This example further shows that anti-GIBsPLA2 antibodies do effectively suppress the pathogenic activity.

In a first series of experiments, the plasma were treated by sepharose beads coated either by goat antibodies ed against human A2 or by two control goat antibodies directed against non relevant antigens. Fig. l4(a) y shows that anti-GIBsPLA2 antibodies completely abolished or d the activity of the plasma, which became unable to induce abnormal MMD in CD4 lymphocytes from HD. Control I and control II antibodies had no effect. These experiments were repeated three times for each plasma and three different plasma from viremic patients were d.

Fig. 14(b) shows identical results. Here the plasma were d as above but were analyzed using the second bioassay. The plasma treated by sepharose beads coated with anti- GIBsPLA2 antibodies do not inhibit anymore ILinduced pSTATS nuclear translocation.

Control I and control II goats antibodies did not affect the ability of the plasma from viremic patients to inhibit IL-7 induced pSTATS nuclear translocation.

In a second series of experiments, we tested the s of neutralizing rabbit antibodies specifically directed against human GIBsPLAZ, -GIIA and -GIID. These antibodies were incubated with the plasma and the cells during the bio assays. The results obtained show that anti-GIBsPLA2 antibodies neutralize the effects of the Viremic plasma as measured by the induction of abnormal MMD and by inhibition of ILinduced pSTATS nuclear translocation. It is noteworthy that antibodies directed against secreted PLA2-GIIA or secreted PLA2-GIID, two phospholipases which are closely related to GIBsPLA2, had no effect in this test.

These results show that anti-GIBsPLA2 antibodies can revert and t the immunosppressive effect of Viremic . These results show that anti-GIBsPLA2 antibodies can prevent immunodeficiency and restimulate the immune se in immuno-defective subjects.

These results further demonstrate that the response is specific. GIBsPLA2 is the only effector ed in the pathogenic effect examined and characterizing the plasma of c patients.

Example 13: Anti-PLAZGIB antibodies inhibit PLA2 GIB effects on CD4 cells.

Cloned and purified human PLA2G1B was used to immunize rabbits. Immunoglobulin fractions of the corresponding sera were prepared. Their capacity to inhibit the enzymatic activity of B was measured on abelled E. Coli membranes. Active immunoglobulin fractions were added to the bioassay including CD4 Lymphocytes purified from the blood of y donors. Cloned and purified secreted PLA2 (GIB, GIIA, GIID and GX) were subsequently added to the cultures. As controls immunoglobulin fractions prepared from rabbits immunized with various ed PLA2 were used Figure 15 show that different concentrations of polyclonal antibody inhibit the induction of aMMD (Figure 15a) and block the nduced NT pSTATS e 15b). This activity can be obtained from 1 itng to 100 itng of Ig containing anti-PLA2 GIB antibodies.

This activity is totally specific since dies directed against PLA2 GIIA, PLAZGIID or PLAZGX showed no effect in the bioassay (Figure 15 a and b).

These results thus further trate that inhibiting PLA2G1B can be used to treat immunodeficiencies and to restore CD4 ty.

Example 14: Soluble PLAZGIB receptor inhibits PLAZ GIB effects on CD4 T cells.

As a further demonstration that inhibitors of PLAZGIB can exert therapeutic effect, we tested a soluble form of a B receptor.

In a first series of experiment, we used, the soluble murine receptor c for PLAZ GIB having the following amino acid sequence (SEQ ID NO: 22): MVQWLAHLQLLWLQQLLLLGIHQGIAQDLTHIQEPSLEWRDKGLELiQSESLKTCiQAGK SVATLEWCKQPNEHMLWKWVSDD{QFNVGGSGCPGVNTSALEQPUKTYFCDSTL"SURWH CDRKMIEGPLQY{VQVKSDNTVVARKQIHRWIAYTSSGGDICEHPSRDLYTLKGNAAGMP CV?PFQ?KGHWHHDCTRFGQKFHTTWCATTSRYEEDEKWGFCPD?TSMKVFCDATWQRNG SSQICYQFNLLSSLSWNQAHSSCU-QGGAULS ADfiDEfiDEIRKiUSKVVKfiVW GUNQL DEKAGWQWSDGTPLSYLNWSQEITPGPFVEHHCGTQEVVSAAWRSRDCESTLPY:CKRDL NHTAQGILEKDSWKYHAT{CDPDWTPFNR<CYKLK<DR<SWLGAQ{SCQSNDSVQMDVAS LAEVEFLVSLLRDENASETWIGLSSNKIPVSFEWSSGSSVIFTNWYPLEPRILPNRQQLC VSAEESDGRWKVKDCKEQLFYICKKAGQV?ADEQSGCPAGWERHG?FCYK‘DTVVRSFEF ASSGYYCSPALUT TSREfiQAE TSLLSSVAEKDSYFW"AUQDQNWTGEYTWKTVGQREP VQYTYWNTRQPSNRGGCVVVRGGSSLGRWEVKDCSDFKAUSLCKTPVKIWEKTELEERWP FHPCYMDWESATGLASCF{VFHSEKVL AFAFCfiflFGAHUASFAH"EEFNFVNE LLASKFNWTQERQFWIGFWRRNPLNAGSWAWSDGSPVVSSFLDNAYFEEDAKNCAVYKAN KTTWPSNCASKHEWICRI??DVR?KFPDWYQYDAPWLFYQWAEYLTHTHPAEWATFEFVC GWURSDFUTIYSAQEQfiE {SK <GLT<YGVKWW GLEfiGGARDQIQWSNGSPVIFQNWD KGREERVDSQRKRCVFISSITGLWGTENCSVPLPSICKRVKIWVIEKEKPPTQPGTCPKG WLY?NYKCFLVT PKD??{TKTWTGAQEFCVAKGGTLVS KSELfiQAFTTMNTFGQTTNV W:GLQSTVHEKWVNGKPLVYSNWSPSDI:NIPSYVTTEFQKHIPQCALMSSNPNFHFTGK WYFDDCGKEGYG?VCEKMQDTLEHHVVVSDTSAI?STLEYGNRTYKIIRGNMTWYAAGKS CRMARAfiUAS AFLTVLLSRLGHTHW"GUSTTDWGQTFDWSDGTKSPFTYWKDE ESAFLGDCAFADTNGRWHSTACESFLQGA-CHVVTETKAEEHPGLCSETSVPWIKFKGNC YSFSTVUDSRSF?DAHFFCKSFGSV17 QDAAfiWSFLUfifiLLAEGSSVQMVWLNAQFDW NNKTLRWFDGTPTEQSNWGLRKPDHDHLKPHPCVVLRIPEGIWHFTPCEDKKGFICKMEA GIPAVTAQ?EKGLSHSIVPVTVTTTWTTATGIFWTCFWTYKQKSD:FQRLTGSRGSYYPT LNESTAHPfiEN I SDLfiKWTNDfifiVRDAPATES<RGH<GQPICISP WO 97140 The inhibitor was tested in the ay described in example 9, at a concentration of 100nM. The results are presented in Figure 16. They show that a recombinant PLA2 soluble receptor can be used as a potent antagonist and that such molecule is able to significantly block the negative effect ofPLA2sGIB on the NT of pSTAT5 (figure 16).

Similar results can be obtained in further sets of experiments using PLA2-GIB-binding polypeptides sing the sequence of SEQ ID NO: 25 or 28.

Example 15: Overexpression of GIBsPLA2 induces immunological deficiency It has been previously shown that Highly Active Anti-Retroviral y (HAART) which reduced Viral load also induces a CD4 count increase in most ts. However, in some patients, despite the fact that HIV becomes undetectable, the CD4 counts do not increase.

We have previously studied this clinical situation and we have shown that in these patients called CD4 Non Responders (CD4-NR) a strong and persistant s of the CD4 T lymphocytes tion is found.

Figure 17 shows that the plasma of CD4-NR patients do contains more PLA2 GIB activity than plasma from a Viremic t taken as l. This was first measured by the induction of abnormal MMD per cells. These data were also confirmed by measuring the ability to inhibit ILinduced pSTAT5 nuclear translocation.

Altogether, the results show that the plasma of CD4-NR patients contains hundred times more PLA2 GIB actiVity than the plasma from Viremic patients.

Discussion Our results show that PLA2 GIB induces an immunosuppression similar to that which characterizes CD4 T cells from Viremic patients, including the inability to respond to IL-2 (anergy) and to IL-7 (central mechanism towards CD4 lymphopenia). Therefore, expression of GIBsPLA2 during HIV infection plays a central role in the pathophysiology of the immune disease that characterizes these patients. These defects are cell-type specific since CD8 T cytes from HIV patients do not t abnormal MMD and continue to respond to IL-7 (data not . The mode of action of PLA2 GIB is ly the consequence of its enzymatic activity. By attacking the membrane of CD4 lymphocyte, it modifies its fluidity and probably allows the formation ofabnormal and very large MMD.

Inflammatory reactions play an important role during HIV infection. However, their exact role in HIV pathogenesis remains to be elucidated. Taking into account our data, one can hypothesize that HIV infection induces a very peculiar type of inflammation which includes GIBsPLA2. rmore, one can speculate that after PLA2 GIB induction, its secretion escape to normal regulatory processes therefore leading to a chronic production and to the immunological disorders which are the direct consequence of the CD4 T lymphocytes dysfunction. As an indirect uence of the CD4 T cytes dysfunction, other defects can also be ed. For instance, diminished production of Interferon gamma will decrease the functions of monocytes/macrophages and of natural killers.

Correlation between the recovery of plasmatic PLA2 GIB activity and the characteristics of different groups of patients is also very informative. “HIV controllers” are very rare patients which maintain an undetectable viral load and quasi normal CD4 counts over the years. Our results show that they do not express PLA2 GIB activity in their plasma. By st, in most patients, this enzyme is expressed and represents the negative side of the inflammation which leads to the immunological disease. Altogether, this clearly establishes that PLA2 GIB is a very critical parameter in the pathophysiology of HIV infection.

HAART viral load decrease is followed by an immune restoration including CD4 counts increase. During this treatment, PLA2 GIB activity disappears in the plasma of the ts.

Since, HAART is considered to decrease the inflammatory reactions this further suggests that PLA2 GIB is part of these inflammatory processes. More importantly, we be here the case of the CD4-NR patients which remain with very low CD4 counts while HAART control their viral load. The oduction of PLA2 GIB found in these individuals may explain the persistence of the immune disease that characterizes this al status. ore, after HAART, there is a strong ation between the decrease production of PLA2 GIB leading to immune restoration or its persistent overproduction leading to the irreversibility of the immune disease.

The therapeutic consequences and utilities of this discovery are huge. Inhibition of PLA2 GIB may indeed be used to prevent or cure the immunological disease of HIV patients as well as, more generally, of immunodepressed subjects. Applied early during infection, inhibitors of PLA2 GIB lead the patients toward a HIV controller status. d later, alone or in conjunction/alternance with HAART, they accelerate the recovery of the CD4 T lymphocytes fianctions and by boosting host defenses, tors of PLA2 GIB lead to an equilibrium between the virus and the immune system like in many other Viral chronic infection. Therefore, inhibitors of PLA2 GIB represent very potent agents for use, alone or in combination, to treat disorders associated with an al immune response or activity.

They can also help in sparing HAART and could lead to the interruption of these treatments which are known for their severe detrimental effects.

Furthermore, since a lack of GIBSPLAZ expression (as in mice KO for the corresponding gene) is well tolerated, transient or permanent suppression of GIBsPLA2 using tors or through vaccination, represents a strong and valid immunotherapy of immune diseases, particularly HIV patients.

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PNAS105(50):19851-19856. (3) Armah KA, et al. (2012) HIV , burden of comorbid disease and biomarkers of inflammation, d coagulation, and monocyte activation. Clin Infec Dis 55(1)126-36. (4) David D, et al. (1998) Regulatory ction of the Interleukin-2 receptor during HIV-infection and the impact of triple combination therapy. PNAS 95:11348-11353. ] (5) Colle JH, et al. (2006) Regulatory dysfiJnction of the interleukin-7 receptor in CD4 and CD8 lymphocytes from HIV-infected patients - s of antiretroviral y. JAcquir Immune Defic Syndr 42(3):277-285. (6) Kryworuchko M, Pasquier V, Theze J (2003). Human Immunodeficiency Virus - 1 envelope roteins and anti-CD4 antibodies inhibit Interleukin-2 induced TAT signaling in human CD4 T lymphocytes. Clinical and Experimental Immunolagy 131(3):422-427. (7) Landires I, et al. (2011) HIV infection perturbs IL-7 signaling at the step of STAT5 nuclear relocalization. AIDS 25(15):1443-1453. (8) Vranjkovic A, Crawley AM, Patey A, Angel JB (2011) IL-7 dependent STAT-5 activation and CD8+ T cell proliferation are impaired in HIV infection.

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Claims (10)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. The use of an inhibitor of secreted Phospholipase A2 group IB LA2) in the manufacture of a ment for ng a CD4-T cell immunodeficiency in a 5 subject in need thereof, wherein the GIBsPLA2 inhibitor is selected from the group consisting of an anti-GIBsPLA2 antibody or a A2-binding derivative thereof, a soluble GIBsPLA2 receptor, an inhibitory nucleic acid molecule based on the gene sequence of the GIBsPLA2 and a peptide containing from 3 to 20 amino acid residues, said peptide being obtainable by a method comprising: (i) contacting 10 a test peptide with GIBsPLA2, (ii) selecting a test peptide which binds A2, and (iii) selecting a peptide of (ii) which inhibits an activity of GIBsPLA2.

2. The use of claim 1, wherein GIBsPLA2 is a protein comprising amino acid residues 23-148 of SEQ ID NO: 2 or a naturally-occurring variant thereof.

3. The use of claim 1 or 2, wherein the anti-GIBsPLA2 antibody or a GIBsPLA2- 15 binding derivative f is selected from an IBsPLA2 polyclonal antibody, an IBsPLA2 monoclonal antibody, fragments thereof selected from F(ab')2 and Fab fragments, single-chain variable fragments (scFvs) and single-domain antibody fragments (VHHs or Nanobodies), bivalent antibody fragments dies), as well as human or humanized anti-GIBsPLA2 antibodies or 20 fragments f.

4. The use of any one of claims 1 to 3, wherein the immuno-deficiency is caused by a viral or ial infection.

5. The use of any one of claims 1 to 4, wherein the subject has a viral infectious disease. 25

6. The use of any one of claims 1 to 5, for treating AIDS in a HIV-infected subject.

7. The use of claim 6, to suppress or reverse HIV-mediated immunodeficiency.

8. The use of any one of claims 1 to 3, wherein the the immunodeficiency is caused by a cancer.

9. The use of any one of claims 1 to 8, wherein the inhibitor is formulated to be administered by injection, preferably intramuscular, subcutaneous, transdermal, intraveinous or intraarterial, by nasal, oral, mucosal, rectal administration or by inhalation. 5

10. A pharmaceutical ition comprising, in the form of an injectable solution or an injectable suspension: (i) an inhibitory dy to a human GIBsPLA2 selected from anti-GIBsPLA2 polyclonal antibodies, anti-PLA2GIB onal antibodies, and fragments thereof which bind human GIBsPLA2 selected from F(ab')2 and Fab fragments, single-chain le fragments ), single-domain antibody 10 fragments (VHHs or Nanobodies), and bivalent antibody fragments (diabodies) and (ii) a pharmaceutically acceptable carrier or excipient suitable for an injectable solution or an injectable suspension. WO 97140