WO1998029452A1 - Antibodies specifically recognising a nitrosylated protein, method of preparation, therapeutic and diagnostic use - Google Patents

Abstract

The invention concerns a purified antibody specifically recognising a nitrosylated protein and more particularly a NO carrier such as albumin. These antibodies may be polyclonal-or monoclonal. The invention also concerns immunogens for preparing said antibodies and the pharmaceutical compositions containing them. The invention further concerns a method using said antibodies for detecting in vitro nitrosylated proteins in a biological sample.

Description

 ANTIBODIES SPECIFICALLY RECOGNIZING A NITROSYLATED PROTEIN, THEIR PREPARATION METHOD, THEIR THERAPEUTIC AND DIAGNOSTIC USE.

The present invention relates to antibodies specifically recognizing a nitrosylated protein and advantageously the compounds resulting from the conjugation of NO with its transporters, such as albumin. The invention also relates to immunogenic compositions for the preparation of such polyclonal or monoclonal antibodies. The subject of the invention is also pharmaceutical compositions comprising these antibodies as active principle, and their use for the treatment of pathologies involving nitric oxide, its derivatives and conjugates, in particular in situations where there is a hyperproduction of NO. Finally, the invention relates to the use of these antibodies for the detection of nitrosylated compounds and the diagnosis of pathologies involving these compounds.

Nitric oxide, also referred to below as NO, is described as the smallest molecule produced by cells. First assimilated to EDRF, it was then recognized as a neuromediator, and would be the first neurotransmitter with retrograde activity, as well as as a cytotoxic / cytostatic molecule. Due to its high reactivity, nitric oxide is capable of reacting with a large number of molecules to form conjugates with multiple functions and thus participate in numerous physiological and pathophysiological mechanisms.

Nitric oxide, produced in large quantities or inadequately, is involved in a large number of pathophysiological mechanisms, such as infections, septic shock, degenerative and autoimmune diseases, transplant rejection, and its action is often relayed by that of its derivatives and conjugates. Thus, nitrosylated albumin has a hypotensive activity, explaining the duration of action of NO. Likewise, nitrohemoglobin has recently been described as acting on vasodilation. In order to overcome the harmful effects of the production in large quantities or inadequately of NO or its conjugates, it has been proposed in the prior art to use inhibitors of the enzyme NO-synthase. However, it has been observed that these inhibitors are not active on already formed molecules and that, furthermore, their distribution in the body and their toxicity limit their use.

The present invention aims precisely to overcome these drawbacks by proposing biological molecules which act selectively on the molecules transporting nitrogen monoxide.

In addition, the detection of nytrosylated substances would make it possible to reveal target molecules or tissues involved in the pathologies mentioned above. It has thus been proposed in the prior art to use anti-nitrotyrosine antibodies to demonstrate nitro tyrosine, which is the marker for the formation of peroxynitrite, resulting from the reaction between the superoxide anion-02 ^~ with NO, in many pathologies.

The present invention also aims to provide new means of detecting nitrosylated molecules useful for the diagnosis of pathologies involving these.

These objects are achieved according to the invention thanks to purified antibodies specifically recognizing a nitrosylated protein, and advantageously compounds resulting from the conjugation of NO with its transporters. Among these, the invention particularly relates to purified antibodies directed against nitrosylated albumins. In the following is meant by protein nitrosylated, also nitrosylated peptides and polypeptides and more generally any nitrosilated antigenic conjugate.

The antibodies of the invention have numerous advantages, in particular as regards their rapidity of action and their effectiveness on already formed and active nytrosylated molecules. In addition, their diffusion capacity in an organism is limited to the physicochemical properties of immunoglobulins. In addition to their effectiveness, the antibodies of the invention have the advantage of being very specific and non-toxic.

Antibodies are macromolecules developed by organisms in response to the presence of a foreign substance. Antibodies of animal origin have long been used in human therapy. For example, horse serum has been used as a tetanus antibody after it has been immunized with tetanus toxin. The research work which led to the present invention began with the induction of polyclonal antibodies directed against a nitrosylated protein, normally more stable in vivo than a nitrosylated amino acid. In addition, the various immune responses have been studied by modifying not only the epitopes and their presentation but also the animal species. The invention also relates to a set of preceding polyclonal antibodies obtained by injecting an animal with an immunogen described below. Then, in a second step, monoclonal antibodies were prepared according to conventional methods of cell fusion.

NO has a great affinity for and binding to cysteine and tyrosine. It is involved in multiple functions which could be linked to transport, storage, and ultimately NO release processes at its action sites. Polyclonal and monoclonal antibodies have been made using different nitrosylated antigenic molecules. To make them immunogenic, the amino acids (haptens) were coupled to carrier proteins via different coupling agents. Thus, albumin, which is the most abundant protein in the plasma, stores and transports NO, and this nitrosylated albumin has been shown to have significant vasodilatory activity and also cytotoxic and cytostatic. This property of albumin is due to the presence of a cysteine whose affinity for NO is high. Research carried out by the inventors and reported below, made it possible to prepare poly and mono-clonal antibodies directed against conjugates of NO and amino acid residues, cysteine, tyrosine and tryptophan, themselves same coupled to a carrier protein, human or bovine serum albumin. Then, antibodies specific to nitrosylated serum albumin were selected and were used to recognize and neutralize some of the properties of this conjugate in vitro.

By synthesizing several forms of immunogens, the inventors have shown that these compounds are sufficiently stable to induce an immune response and that this is specifically directed against the corresponding nitrosylated epitope. The possibility of inducing these antibodies and the intervention of nitrosothiols and cysteine in the vasodilation mechanisms (Ignarro et al., 1981), led the inventors to search for the existence of nitrosyl derivatives in vitro and in vivo and to block their effects.

The invention also relates to the immunogens for the preparation of the antibodies defined above. These immunogens consist of a nitrosylated amino acid coupled to a carrier protein. The synthesis of these immunogens is carried out for example by coupling the hapten L-tyrosine (Tyr) or L-cysteine-N-acetylated (Cys) to a carrier protein such as bovine serum albumin (SAB) or human serum albumin (SAH) by carbodiimide. Then, the conjugates obtained are nitrosylated by an NO donor such as sodium nitrite (NaNθ2) in an acid medium. The use of different coupling agents such as carbodiimide, glutaraldehyde (G) or succinic anhydride (AS) and of distinct carrier proteins, peptides or polypeptides, makes it possible to obtain epitopes associated with nitrosylation of different conformation.

These immunogens can also consist of a protein whose sequence has an amino acid residue capable of being nitrosylated like albumin which has a cysteine.

In the immunogens of the above invention, the term “carrier protein” also means peptides and polypeptides.

The following nitrosyl conjugates were prepared in the context of the invention: NO-Tyr-SAB; NO-Cys (acetylated) -SAB; NO-Cys (non-acetylated) -SAB; NO-Tyr-G- SAB; NO-Cys-G-SAB; NO-Tyr-SA-SAB; NO-Cys - SA-SAB; NO-Tryp-SAB; NO-Tryp-G-SAB; NO-SAB. The preparation of these nitrosyl antigens has made it possible to develop polyclonal and monoclonal antibodies which have been used for the detection of nitrosylation of molecules in the body (SAB) or pathogens and for the neutralization of their activity. The production of Ac specifically directed against immunogens carrying a nitrosylated epitope is delicate, since numerous couplings of different small molecules with carrier proteins have proved effective in one animal species and not in another. This was noticed when changing the presentation of the antigen. In the rabbit, for example, two immunogens have been shown to be effective, NO-Cys (acetylated) -SAB and NO-Tyr-SAB, where the coupling agent used is carbodiimide. The development of these sera rabbit immune systems allowed the development of murine monoclonal antibodies to be undertaken. The realization of these first required the production of polyclonal Ab in mice, where only the coupling with glutaraldehyde allowed to obtain good results. The latter were obtained only when using the immunogen NO-Cys-G-SAB. For the other conjugates, the responses obtained were not specific for the nitrosylated epitope. Consequently, the presentation of the immunogen in vivo is a decisive element for stimulating the immune response, and obtaining a successful approach against small nitrosylated molecules.

The research carried out by the inventors has made it possible to highlight activities carried by nitrosylated molecules and to neutralize them thanks to the production of antibodies directed against nitrosyle epitopes. Consequently, the invention relates to pharmaceutical compositions comprising as active ingredient an antibody of the invention advantageously dispersed in a pharmaceutically acceptable vehicle or excipients. The invention also relates to the use of the antibodies of the invention for the preparation of medicaments intended to treat or prevent pathologies in which NO, its derivatives or conjugates are involved, such as infections by microorganisms and parasites, autoimmune diseases and inflammatory, septic shock, cancers, transplant rejection, neurotoxicity, ... The overproduction of "NO-bound" is said to be responsible for the deleterious effect of NO in certain pathologies, and could therefore be neutralized by antibodies of the invention advantageously and previously humanized.

The inventors have in fact demonstrated in vi vo the role of NO in the development of autoimmune and inflammatory diseases. Until now, NO synthase inhibitors have been the molecules most used for blocking the activity of NO in given pathological conditions. The work carried out within the framework of the present on two experimental diseases: autoimmune encephalitis and experimental inflammatory arthritis in the Lewis rat, have made it possible to demonstrate the usefulness of the antibodies of the invention as inhibitors of the harmful effect of NO or its derivatives

The antibodies of the invention are extremely useful physiologically and pathophysiologically for both therapeutic and diagnostic applications. They indeed make it possible to detect the synthesis of molecules carrying NO-cysteine epitopes and therefore to elucidate new mechanisms and to treat conditions involving nitrogen monoxide, its derivatives and conjugates, especially in situations where one is confronted with an overproduction of NO, such as infections, shocks, acute or chronic inflammations (systemic or localized), transplants, degenerative diseases, diabetes, autoimmune diseases, cancers, etc.

Also, the invention relates to a method for in vitro detection of nitrosylated proteins in a biological sample, such as a fluid or a tissue, comprising at least the following steps:

- bringing this sample into contact with at least one antibody of the invention or a set of these antibodies, optionally labeled, under conditions allowing the formation of immunological complexes;

- the detection of an antigen-antibody immunological complex by physical or chemical methods.

The invention also relates to a kit for the in vitro detection of nitrosylated proteins in a biological sample, in particular for the diagnosis of pathology involving NO, its derivatives and conjugates. This kit includes:

- at least one antibody of the invention or a set of these optionally labeled antibodies; - reagents for the constitution of a medium suitable for the immunological reaction between said antibody and the nitrosylated proteins possibly present in a biological sample;

- optionally one or more detection reagents, optionally labeled, capable of reacting with the immunological complexes possibly formed;

- possibly one or more biological reference and control reagents.

Other advantages and characteristics of

1 invention will appear in the examples which follow concerning:

- the characterization and functions of NO;

- the preparation of polyclonal antibodies directed against nitrosyle conjugates;

- detection and neutralization of no derivatives by polyclonal antibodies;

- the preparation of monoclonal antibodies directed against NO-cys-glutaraldehyde conjugates coupled to a carrier protein;

- The detection of nitrosyl antigens and the protective role of monoclonal antibodies.

I - CHARACTERIZATION AND FUNCTIONS OF NITROGEN MONOXIDE.

Nitric oxide or nitric oxide is a radical diatomic gas. It is produced enzymatically by several isoforms of the enzyme NO-synthase. NO synthesis takes place in many cell types. This partly explains its involvement in a very wide variety of biological functions (Moncada et al., 1991; Knowles and Moncada, 1992; Lowenstein & Snyder, 1992; Nathan, 1992; Sta ler et al., 1992a).

a) The synthesis of NO:

NO is formed by oxidation of L-arginine at the terminal nitrogen of the guanidine function. The production of NO from arginine initiates the simultaneous formation of L-citrulline (Marletta et al., 1988). The oxidation of L-arginine is controlled by NOS and requires the presence of molecular oxygen and cofactors such as flavin adenine dinucleotide (FAD), flavin mononucleotide (FM), tetrahydrobiopterin (BH4), nicotinamide adenine dinucleotide phosphate (NADPH) (Marletta, 1993; 1994).

b) The different forms of NO synthase (NOS): Several types of NOS have been cloned and classified into two distinct families: the so-called constitutive NOS (cNOS) and the inducible NOS (iNOS). A fundamental difference between these isoforms concerns the NOsynthase activity in situ (Bredt et al., 1991; Moncada et al., 1991; Hevel and Marletta, 1992; Lamas et al., 1992; Nathan, 1992): cNOS are calcium / calmodulin dependent and produce small amounts of NO (picomoles / min / mg protein) over short periods in response to activation of receptors under physiological conditions (Stuehr and Griffith, 1992). These enzymes are generally found in endothelial cells, neurons, astrocytes, platelets, polymorphonuclear neutrophils and the adrenal glands (Moncada et al., 1991; Lacaze-Masmonteil, 1992; Nussler et al., 1995). The iNOS are independent calcium / calmodulin. They are induced by immunocompetent cells, mainly via cytokines, and many other molecules, in particular derived from microorganisms (Moncada et al., 1991). The stimulation of iNOS leads to the synthesis of large amounts of NO (nanomoles / min / mg protein) for long periods (Nathan and Xie, 1994). These NO discharges are responsible for cytostatic and cytotoxic phenomena. These iNOS are found in activated macrophages, muscle cells, hepatocytes, fibroblas, astrocytes, polymorphonuclear neutrophils and endothelial cells (Stuehr and Griffith, 1992; Marletta, 1993; Anggard, 1994). In humans, iNOS has been demonstrated in hepatocytes and smooth muscle cells of the aorta (Geller et al., 1993), in chondrocytes (Palmer et al., 1993), keratinocytes (Heck and al., 1992), astrocytes (Lee et al., 1993) and islets of Langerhans where induction of NOS by interleukin 1 (IL-1) could participate in the destruction of these cells (Corbett et al ., 1993). More recently, production of NO has been demonstrated in human monocytes by a transduction mechanism involving CD23 (Dugas et al., 1995).

c) NOS inhibitors:

Many N-substituted arginines have been synthesized as specific inhibitors of NOS, including: Nco monomethyl-Larginine (NMMA) which is a good inhibitor of the three isoforms and Nco-nitro L-arginine which is more active on cNOS (Stuehr and Griffith, 1992). Aminoguanidine is more active on iNOS (Misko et al., 1993). Other inhibitors of the various NOS isoforms have recently been demonstrated, such as thiocitrulline (Southan et al., 1995) and 7-nitro-indazole (Mayer et al., 1994).

d) The chemistry and biochemistry of NO:

Nitric oxide, unlike most free radicals, does not dimerize and does not disproportionate.

Its reactivity depends essentially on its capacities to yield its celibate electron to other radicals (ion superoxide, tyrosyl radical, etc. ) or to species likely to intervene in radical reactions

(molecular oxygen, thiols, phenols, etc.) or even transition metals eager for electrons (iron, copper, etc.) (Stamler et al., 1992a). NO is either complexed or transformed into nitrosonium ion N0 +. The oxidation-reduction potential of the surrounding medium is decisive for the fate of NO into N0 ⁺ and N0 ^~ . However, the existence of these two ions has never been demonstrated in physiological media.

- Reaction of NO with oxygen:

NO is oxidized to nitrite (Nθ2 ^~ ) in the presence of oxygen (Farkas and Menzel, 1995). Its lifespan in vivo depends essentially on this reactivity with oxygen. In the gaseous state, it forms nitrogen dioxide o, radical (N02), which dimerizes into N2O4 and which dissolves in water giving nitrous and nitric acids. In the dissolved state and at pH 7.4, the mixture of N0 ° and oxygen generates only N02 ^~ , but not nitrate, with the following stoichiometry:

4NO + 02 + 2H20 -> 4HN02 -> 4H ⁺ + 4N02

- Reaction with the superoxide anion: Oxygen derivatives, the superoxide ion

(O2), the hydroxyl radical (HO) and hydrogen peroxide (H2O2), are generated in normal and pathological metabolic processes. Simultaneously with NO, the superoxide ion O ^ -, produced by NADPH oxidase, is secreted by various tissues, in particular during inflammation or septic shock. The coupling of the radicals NO and O ^ - gives peroxynitrite with a reaction constant of 6.7 x 10 ° mol ^~ l L s ^" .

In fact, the reaction constant of SOD with O2 ^" being (2 x 10 ⁹ mol ^-1 L s ^-1 ) NO and superoxide dismutase (SOD) compete to trap O2 (Koppenol et al., 1992) Peroxynitrite can also be form by the reaction of N02 ^~ with H2O2 or the nitroxyl anion (N0 ^~ ) with oxygen (Fontecave and Pierre, 1994; Butler et al., 1995):

NO + 02 ^" N02 ^~ + H2O2 =======> 0 = N-0-0> NO3 ^""

NO- + 02

- Reaction with hydrogen peroxide: H2O2 can react directly with NO. Its concentration in biological media is higher than that of 02 ^~ (Fontecave and Pierre, 1994). The reaction does not give 0N00 ^" but singlet oxygen (- ^L θ2) detectable by chemiluminescence:

2N0 + H2O2> ¹ θ2 + H2O + N2O This singlet oxygen formed is very reactive and can participate in cell destruction and inflammatory processes during activation of macrophages.

Reaction with thiols:

Thionitrites, such as S-nitrosocysteine or nitrosoalbumin, with a lifespan greater than that of NO, would be the compounds capable of transporting it from NOS to the target of NO. Thionitrites can be formed by nitrosylation from free thiols present in the cell cytosol and blood (Girard and Potier, 1993). The formation of thionitrites following activation of NOS in vivo has often been described: it could play a role in the modulation of certain enzymatic activities as well as in the transport of NO.

Numerous studies have demonstrated the nitrosylation of the free SH grouping of free cysteine or bovine serum albumin (Cys 34). Recently, it has been shown that the NO released in a biological system reacts in the presence of thiols to form S-nitrosoprotein derivatives. Indeed, plasma proteins serve as a reservoir of NO produced by endothelial cells (Stamler et al., 1992b).

In humans, the plasma contains approximately 7 M S-ni trosothiols, 96% of which are in the form of S-nitrosoproteins, of which 82% is S- or troso-serum albumin (Stamler et al., 1992b) . The plasma concentration of thiols is 0.5 mM and the plasma NO concentration is 3 nM (Stamler et al., 1992b). Free NO has a half-life of a few seconds to a few minutes (Kelm and Schrader, 1990). In plasma, in the SNO-Cys or S-NO-protein form, it has a higher half-life, 10 and 40 min respectively (Ignarro et al., 1981; Ignarro, 1989; Stamler et al., 1992c) . In a phosphate buffer at pH 7.4 at 25 ° C., NO-SAB has a half-life of approximately 24 hours (Stamler et al., 1981, 1992c, d) which changes to 12 hours at 37 ° C. (Stamler et al ., 1992c). Under these same conditions, NO and S-nitrosocysteine respectively have a half-life of 0.1-1 s (Kelm and Schrader, 1990) and 15-30 s (Ignarro et al., 1981). In vi tro, the treatment of thiols in an acid medium with a nitrosating agent such as NaNO2 gives the following reaction (Fontecave and Pierre, 1994):

NaNθ2,

RSH> RiSNO (nitrosothiol) HCl

The nitrosothiol formed can transfer NO to a second thiol or another nucleophile by a transnitrosylation mechanism (Butler et al., 1995):

RiSNO + R ² S> RiS ^" + R SNO

- Reaction with serum albumin:

At pH 7, NO does not react directly with thiols to give nitrosothiols but the formation of the latter is observed in oxygenated medium. Recent data show that for low molecular weight thiols (N-acetyl-Cys, glutathione, etc.), the essential nitrosating agent is N2O3 (Kharitovov et al., 1995) according to the following reactions: 2N0 + 02> N02

N02 + NO> N2O3

N2O3 + H2O> 2H ⁺ + 2N02 ^"

N2O3 + RSH> H + + N02 ^" + RSNO

On the other hand, in the case of the two proteins serum albumin human and bovine the situation is different because the speed of the reaction is 10 times slower than that of thiols of low molecular weight (3-1.5 x 10 ⁵ M ^-1 s ^{- 1} , against 0.3-0.06 x 10 ⁵ M ^"1 s ^-1 ). Their nitrosylation by N2O3 is therefore not significant.

Keaney et al., (1993) suggested the possibility of nitrosylation of SAH by its reaction with NO ⁺ which could be formed following the reaction of NO with Fe ²⁺ of hemoproteins such as hemoglobin

(Kharitonov et al., 1995):

Hb ⁺ + NO> Hb + NO ⁺

- Reaction with tyrosine: Since tyrosine has an aromatic group, nitrosylation is carried out by charge transfer between NO ⁺ and this aromatic group which donates an electron:

Ar -NO ⁺ <= = = = = = = => Ar + NO (Stamler et al.,

1992a). This reaction can take place at the level of a protein containing a radical amino acid at its active site. This is the case with ribonucleotide reductase (Fontecave and Pierre, 1994). At the R2 subunit of this enzyme, there is a stable tyrosyl radical which is essential for enzymatic activity. The reversible reaction of this radical with NO may partly explain the regulatory mechanism of the latter at the level of the enzyme.

- Reaction with transition metals:

Many NO targets have been shown to be metalloproteins. Indeed, NO can be attached to all transition metals. Therefore, it is used as an inhibitor of transport proteins and oxygen metabolism: hemoglobin, myoglobin, oxidases, oxygenases, etc. The affinity of NO is generally much stronger for Fe ²⁺ than for Fe ^ ⁺ . NO also binds to Co ²⁺ in several of its oxidation states, and to Mn ²⁺ and Cu ²⁺ . NO can also act as a reducing agent with respect to certain metalloproteins (Henry et al., 1991; Stamler et al., 1992a; Traylor and Sharma, 1992; Henry et al., 1993).

e) The physiological and pathological roles of NO:

- Role of NO in the cardiovascular system:

As early as 1980, Furchgott and Zawadski observed that the relaxation of isolated arteries subjected to the action of

Acetylcholine depends on the endothelium (Furchgott and

Zawadski, 1980). They deduced therefrom the existence of a fleeting factor called "endothelium derived relaxing factor" (EDRF), secreted by endothelial cells treated with acetylcholine or bradykinin, causing

1 elongation of adjacent smooth muscle cells.

Its chemical nature was only recognized in 1987 by Palmer and Moncada (Palmer et al., 1987, 1988; Ignarro et al., 1987). In addition to the EDRF action, radical NO has an effect on platelets: the increase in cGMP under its control causes a decrease in platelet aggregation and adhesion (Radomski et al., 1987). In humans, NO is used therapeutically: molsidomine, for example, constitutes an NO donor used as a vasodilator in the treatment of a number of cardiovascular conditions.

NO is also directly used in gas form inhalation at a dose of 10 to 40 ppm in certain intensive care units, with the aim of causing vasodilation of the pulmonary circulation, and therefore improving respiratory gas exchanges, in the framework for the treatment of patients with severe pulmonary arterial hypertension or adult acute respiratory distress syndrome (ARDS) (Pepke-Zaba et al., 1991; Falke et al., 1991). . Massive NO production:

An excess of NO production can have harmful consequences for the cardiovascular system.

In septic shock, for example, there is massive secretion of NO not only by endothelial cells but also by mast cells, smooth muscle fibers, leukocytes and kidney cells. This significant release of NO is due to the induction of iNOS (Anggard, 1994). The excess NO synthesized is responsible for hypotension, vascular hyporeactivity and myocardial depression (Lancaster,

1992) in experimental septic shock to endotoxin, or cytokines (Kilbourn et al., 1990; Reed et al., 1990;

Thiemermann and Vane, 1990; Gray et al., 1991; Vallance and

Moncada, 1993) and can cause tissue destruction (Palmer et al., 1992).

In humans, the use of NMMA in patients with septic shock has been shown to cause a dose-dependent increase in blood pressure (Petros et al., 1994). These results indicate the contribution of NO in cardiovascular changes and suggest a possible role for NOS inhibitors in septic shock therapy. . Decreased NO production

A defect in NO production could be the cause of certain blood pressure increases. Thus, in genetically hypertensive, salt-sensitive rats (Dahl rats), administration of L-arginine lowers blood pressure (Chen and Sanders, 1991). This decrease in blood pressure may be the result of the stimulation of NO production.

In experimental models of atherosclerosis, it has been shown to reduce NO release by 1 vascular endothelium in rabbits (Coene et al., 1985). It is possible that the destruction of the endothelium, in particular by atherosclerosis, leads to an inability to correctly synthesize NO, with as a consequence a reduction in vasorelaxation and, perhaps, a permissive effect on cell proliferation.

Thus, hypertension or other blood pressure disorders could result from an abnormal production of NO. It now turns out that the endothelium-dependent vasodilatory response is decreased in animal models of hypertension, as well as in atherosclerosis and coronary artery disease in humans.

- Role of NO in the nervous system: Produced under the influence of stimulation of NMDA-type presynaptic receptors by glutamate, and acting through the increase in the postsynaptic intraneuronal level of cGMP (Green et al., 1991 ), NO exerts, both in the mouse and in humans, a signal action in the nervous system, not only central (Bredt and Snyder, 1989), but also peripheral.

In neurophysiology NO is now considered a biological messenger (Garthwaite, 1991) or a retrograde messenger (Barinaga, 1991; Feldman et al., 1993). It could have a role in long-term potentiation (PLT) in the hippocampus. PLT is a phenomenon which corresponds to a particular increase in the efficiency of synaptic transmission, thus promoting memorization. This indicates an implication of NO production in certain forms of hippocampal dependent learning (Bredt and Snyder, 1990; Bredt et al., 1991; Zhuo et al., 1993).

It constitutes a non-cholinergic, non-adrenergic neuro-transmitter and mediator of the relaxation of the digestive tract (Desai et al., 1991); and participates in both neuronal control of cerebral vascular tone (Beckman, 1991), or erection (Rajfer et al., 1992), that of the secretion of insulin by the β cells of the islets of Langerhans

(Schmidt et al., 1992).

Furthermore, NO is involved in brain destruction processes after acute shock, such as ischemia-hypoxia (Moncada et al., 1991; Snyder and Bredt, 1992; Choi, 1993). NO formed during ischemia is thought to be neurotoxic by several mechanisms. NO reacts with superoxide ions to form peroxynitrites which initiate lipid peroxidation (Beckman, 1990). NO is able to block mitochondrial respiration (Drapier et al., 1988) and to inhibit DNA synthesis (Kwon et al., 1993). In addition to these direct cytotoxic effects, NO exerts an effect by promoting the release of glutamate occurring during ischemia (Buisson et al., 1993) which triggers the entry of calcium ions which bind to calmodulin, leading upon activation of the NOS. The NO produced diffuses through cell membranes and reaches target cells where it binds to the iron atom of the active site of soluble guanylate cyclase. This then produces the cGMP. In addition, NO released can act at the presynaptic level where it facilitates the release of glutamate.

However, it has been demonstrated in vi tro that the direct addition of NO or NO releasing molecules to neuron cultures blocks the activation of NMDA receptors (Manzoni et al., 1992). These observations led the authors to suggest that the endogenous NO synthesized by the neurons exerts a retro-inhibition activity with respect to the NMDA receptors. This retro-control would be exercised by reducing the frequency of opening of the channels associated with the receptors and consequently by reducing the calcium influx. This effect is believed to be due to NO blocking of the NMDA receptor modulator site (Lipton et al., 1993). - Role of NO in the immune system:

The first studies of NO mainly showed its toxicity. It was once thought that nitrates excreted by humans were only from food. However, the Tannenbaum team observed in 1981 that individuals and rats who consumed little food nitrates nevertheless excreted large quantities.

Food was therefore not the only source of nitrates. In addition, a subject with infectious diarrhea excreted a lot of urinary nitrates: the formation of these nitrates seemed to be linked to the inflammatory process. More recently, a team has observed that the injection of bacterial endotoxin stimulates the excretion of nitrates in rats (Green et al., 1981; Snyder and Bredt, 1992).

Indeed, NO intervenes in the antitumor defense, in particular in the mechanisms of cytotoxicity mediated by macrophages (Stuehr and

Marletta, 1985; Hibbs et al., 1987a, b; Drapier et al., 1988; Stuehr and Nathan, 1989; Connect et al. ,1990).

The activation of murine peritoneal macrophages, whether in vi vo by cell multiplication pathogens such as MycoJacterium bovis, or in vi tro by cytokines in particular the interferon-gamma (1 'IFN-γ), makes them cytotoxic for target tumor cells, the cytotoxicity mechanisms involved do not pass through phagocytosis processes. It was then demonstrated that these activated macrophages express iNOS and synthesize NO3 ^"" (Stuehr and Marletta, 1987a) and Nθ2 ^~ in response to stimulation by IFN-γ, alone or in combination with lipopolysaccharide (LPS ) or with tumor necrosis factor-alpha (TNF-α) (Stuehr and Marletta

1987a, b; Ding et al., 1988). Thus, the cytotoxicity of macrophages activated against tumor cells has been established (Hibbs et al., 1987b) as dependent on the concentration of L-arginine. These authors also show that activated macrophages synthesize L-citrulline and Nθ2 from arginine and that L-NMMA inhibits the synthesis of these two products as well as the expression of cytotoxicity (Hibbs et al., 1987b).

The role of NO as mediator of cytotoxicity was initially demonstrated (Hibbs et al., 1988), and the metabolic pathway of L-arginine was considered as an important defense mechanism against intra- and extracellular microorganisms, parasites, bacteria, and fungi (Hibbs et al., 1990). NO acts on these targets in particular by binding to the iron-sulfur centers of proteins (Drapier et al., 1991; Feldman et al., 1993). We also note that a massive loss of intracellular iron is a possible cause of the lysis of tumor cells induced by activated macrophages. NO seems to act on the active site of different enzymes involved in both DNA replication (ribonucleotides reductase, RNR) (Lepoivre et al., 1990) and in the citric acid cycle (aconitase) (Drapier and Hibbs, 1986 ) or in mitochondrial respiration (complexes I and II of the electron transport chain) (Granger and Lehninger, 1982; Drapier and Hibbs, 1988). Electronic paramagnetic resonance (EPR) studies have shown that NO disrupts the spatial configuration of iron-sulfur structures of certain enzymes, forming iron-nitrosyl complexes (NO-Fe-SR), which would inhibit enzyme activity (Drapier and Hibbs, 1988; Lancaster, 1990; Pellat, 1990; Drapier, 1991). This phenomenon occurs for high amounts of NO, produced over several hours. It is noted that the production of NO can also be induced in the target cells themselves, for example, at the time of a self-destruction of the tumor cells, or autotoxicity of bacteria (Heiss et al., 1994). In the mechanism of action of the antiinfectious effect, it has also been shown that NO can react with oxygen and produce various toxicants such as hydroxyl radical (OH) or nitrogen dioxide (NO2) (Stamler et al., 1992a). The peroxynitrite ion, one of the most oxidizing and cytotoxic derivatives of NO, is now proposed as the main mediator of the cytotoxic activity of NO. It is thought to be involved in the nitration of tyrosine residues of certain cellular proteins (Beckman et al., 1994a). It is also known that certain S-nitrosylated compounds (S-ni trosocys teine, S-nitrosoglutathion), and NO-0 donating thionitrites can be powerfully microbicides, antiviral or anticancer (Maul, 1993; Roy et al., 1995).

NO appears to have been recently identified in rats and mice as a mediator of macrophage immunosuppression. A high concentration of L-arginine is involved in the acquisition of the suppressor character (Albina, 1989a, b; Hoffman et al., 1990; Mills, 1991). In addition, the addition of NMMA increases the proliferation of lymphocytes (Mills, 1991; Nussler et al., 1995). This NO immunomodulatory role has been confirmed by other teams, using Hb (Albina and Henry,

1991; Mills, 1991), or the anti-IFNγ monoclonal antibody in macrophage-spleen cell co-cultures (Albina et al., 1991) in order to induce lymphocyte proliferation. NO would pre erectively inhibit the proliferation of Thl lymphocytes, and exercise a feedback control over its production.

In some t rypano s o o s e s, where

Immunosuppression is very marked, macrophages exert a powerful suppressive activity by) mechanisms involving prostaglandins and NO

(Schleifer, & Mansfield, 1993).

- NO and Hemoproteins:

NO binds to many hemoproteins (Fe 5 II) in vitro. It also sometimes attaches to their ferric form (Henry et al., 1991). On the other hand, when inhaled, NO is a harmful gas due to its binding to iron ferrous hemoproteins (Boucher et al., 1993; Rossaint et al., 1993). The best known example is that of hemoglobin. NO is able to quickly bind to deoxyhemoglobin (Hb [FeII]), to form the relatively stable Hb [FeII] NO complex (tι / 2 = 12 min). Indeed, the affinity of NO for deoxyhemoglobin is 106 times greater than that of oxygen. As for oxyhemoglobin (Hb [FeII] 02), it can be converted by NO into methe oglobin (tι / 2 = 20h) and into nitrate (Ducrocq et al., 1994).

Furthermore, hemoglobin could play a role as a transporter or oxidizer of NO (Henry et al., 1991; Traylor and Shar a, 1992; Boucher et al., 1993). In fact, in humans, each hemoglobin subunit contains a hemin group, and the β subunits contain very reactive thiol residues (Cys β93), forming S-nitrosohemoglobin. This nitrosylated form of hemoglobin has recently been identified not only as a factor in the regulation of vascular, pulmonary and systemic activities, but also as an element of transnitrosylation (Jia et al., 1996).

Other hemoproteins are activated by NO, such as guanylate cyclase where the NO receptor is the iron atom. The fixation of NO deforms the heme group (which contains the iron atom), and activates the production of the second messenger the cGMP from GTP, which induces vasodilation and an inhibition of platelet aggregation (Ignarro, 1991; Moncada et al., 1991; Schrrtidt, 1992; Snyder and Bredt, 1992). We also mention prostaglandin H synthase (Salvemini et al., 1993; Stadler et al., 1993), and others which are more or less reversibly inhibited (Rogers and Ignarro, 1992; Assreuy et al., 1993; Rengasamy and Johns , 1993), such as the cytochromes P-450 (Adams et al., 1992; Ducrocq et al., 1994) and the NOS themselves (Rogers and Ignarro, 1992; Assreuy et al., 1993; Rengasamy and Johns, 1993). In conclusion, NO comes in different forms of transport; the enzymatic mechanisms of synthesis, storage and transport are carried out according to its roles of intermediary and effector (Stamler et al., 1992a, 1994).

- NO and carcinogenesis:

In different situations, NO seems to exercise several roles, sometimes even antagonistic. This is true with regard to carcinogenesis (Calmels and Ohshima, 1995). In contrast to its role in the body's defenses (bactericidal and cytotoxic), NO is likely to lead to alterations in DNA, thus promoting the development of cancer. This, however, has most often been highlighted in in vitro work often far removed from physiological conditions.

Several distinct roles of NO at the various stages of carcinogenesis have been observed: (i) the formation of peroxynitrite, a naturally cytotoxic product, which after protonation, can also decompose into OH and NO2 (Beckman et al., 1990). These radicals are said to be partly responsible for oxidative damage to DNA, thus allowing subsequent neoplastic transformation of the affected cells;

(ii) the genotoxicity of NO and the deamination of DNA: NO can induce mutations in human lymphoblastoid cell lines (Nguyen et al., 1992);

(iii) the formation of nitrosamines: NO after oxidation is capable of reacting with a nitrosatable substrate to form nitrosamines. The latter are potent carcinogens in animals and are also suspected of causing certain cancers in humans (Loser et al., 1989; Jacob and Belleville, 1991). NO can also promote the progression of cancer through immunosuppression (Hoffman et al., 1990; Albina et al., 1991; Mills, 1991). Studies have shown, for example, that its presence appears to favor the process of neoplastic transformation of murine fibroblasts in vitro (Werner-Felmayer et al., 1993). However, recent work indicates a more or less controversial protective effect of NO vis-à-vis the cytotoxicity induced by oxygenated species (ink et al., 1993) and vis-à-vis the progression of skin tumors induced in mice (Yuin et al., 1993; Calmels and Ohshima, 1995). Finally, despite its role in carcinogenesis, NO is also an important effector vis-à-vis tumor cells (Hibbs et al., 1990). In addition, NO can also mediate apoptosis of different cell types (Sarih et al., 1993; Me, Bmer et al., 1994; Fehsel et al., 1995). All these data highlight the various influences of NO.

- NO and autoimmune inflammatory diseases:

It has recently been discovered that NO is involved in certain inflammatory pathologies (Schmidt and alter, 1994; Moilanen and Vapaatalo, 1995). Among these, multiple sclerosis (MS) (Sherman et al., 1992) and rheumatoid arthritis (RA) (Grabowski et al., 1996) are the most studied.

The inventors are interested in these last two pathologies to show the effect of NO and its nitro or nitrosyl derivatives in their clinical evolution. The studies are based on experimental models for MS (experimental autoimmune encephalitis) and for RA (experimental inflammatory arthritis).

The mechanisms of action of NO, direct or via derivatives, are still poorly understood. Due to its nature, NO can diffuse across the membranes of cells neighboring those where it has been synthesized. This diffusion of NO can explain an effect local at very short distance because of its great reactivity. It is probably this mechanism which activates the guanylate cyclase of the smooth muscle cells of the vessels by the NO synthesized by the endothelial cells. However, action at a distance from the NO can also be envisaged. This action at a distance can be linked to the existence of molecules capable of fixing NO and then releasing it. Among the compounds capable of playing this role, the S-nitrosothiols compounds play an important participating role. Thus S-nitrosothiols have been demonstrated in vasodilation phenomena since 1981 (Ignarro et al.).

More recently, ni trosothiols have also been demonstrated in the immune system cells (Dias-Da-Motta et al., 1996; Wagner et al., 1996; Zhao et al., 1996). The mechanisms of NO release by these molecules are poorly understood and in particular the reactions leading to the passage of NO over other molecules. In addition, is it still the NO form or can the NO ⁺ forms be involved in nitrosylation mechanisms? Can there also be intervention of other radicals in particular derivatives of 1 '02? Whatever the modes of action of NO locally or remotely, there may therefore be intermediate molecules endowed with properties and greater stability than NO whose function is not yet known.

The antibodies of the invention first made it possible to study the stability and the properties of nitrosyle compounds, then they were used to mask nitrosyle epitopes which appear in certain pathological states where an overproduction of NO has been objectified. A high production of these nitrosyl derivatives could be responsible, at least in part, for the disorders observed. II- PREPARATION OF POLYCLONAL ANTIBODIES DIRECTED AGAINST NITROSYL CONJUGATES.

1) The immune response. The destruction of infectious agents is produced by a set of elements belonging to the immune system. This protection is obtained thanks to an immune response of the humoral and / or cellular type, the essential actors of which are the lymphocyte and macrophagic cells (Oberg et al., 1993). B lymphocytes promote the humoral response via the production of antibodies specifically recognizing a given antigen, accompanied in certain cases by the neutralization of the harmful effect of the latter. T helper lymphocytes (Thl) are involved in the activation of macrophages by secreting cytokines such as 1 IFN-γ and TNF-α / β. The activated macrophages release different so-called effector molecules which exert a cytostatic or cytotoxic effect with respect to intra- or extracellular microorganisms. Among these molecules, there are free radicals and in particular NO (Liew, 1991; Ahvazi et al., 1995; Blasi et al. 1995; Juretic et al., 1995; Lin et al. 1995) which, by binding on molecular targets, generates in vivo nitrosyle neoantigens which can induce a humoral response.

To best reproduce the structure of these neoantigens, the inventors synthesized artificial conjugates by coupling NO to two amino acids, tyrosine and cysteine. With the aid of these conjugates, they induced polyclonal antibodies directed against NO-Tyr and NO-Cys epitopes in rabbits.

2) The immunochemistry of haptens. The immunochemistry of small molecules or haptens, is based on the specific recognition of a particular antigen by an antibody (Ac), and this in a way highly selective (Landsteiner, 1945; Kabat, 1968; Goodman, 1975). Haptens such as amino acids (cysteine, tyrosine, tryptophan, ...) have a low molecular weight (75-300 daltons) and are therefore not immunogenic by themselves. The development of specific Acs is based on a number of methodological principles and concepts. So :

- a substance is immunogenic when it has the capacity to stimulate the production of Ac; its size must be greater than 1000 daltons.

- a compound is antigenic when it interacts specifically with an Ac site. For this, it must discriminate structurally close conjugates, different by the position of a chemical group on the hapten (Landsteiner, 1945; Geffard et al., 1985a);

- the antigen-Ac bond is established thanks to bonds of different types: hydrogen, Van Der Walls, ionic, hydrophobic. To achieve this approach, the inventors first synthesized nitrosyl conjugates, then conjugates having a structural analogy with the former. The study focused exclusively on nitrosyle conjugates and their non-nitrosyle correspondents as well as nitro conjugates. Using these tools, polyclonal anti-hapten nitrosyl Ab coupled to carrier proteins were produced and characterized using carbodiimide as a coupling agent.

3) Materials and methods. a) Synthesis of the NO-tyrosine and NO-cvstein immunogens coupled to a carrier protein by a “carbodiimide” type coupling:

The synthesis of immunogens is carried out initially by coupling the hapten L-tyrosine

(Tyr) or L-cysteine-N-acetylated (Cys) to a protein carrier such as bovine serum albumin (BSA) or human albumin serum (BSA) by carbodiimide according to the method indicated below (Harlow and Lane, 1988). Secondly, the conjugates obtained are nitrosylated by an NO donor.

. 10 g of Tyr (Fluka), or 20 mg of Cys (Sigma), are dissolved in MES buffer (2 [N-Morpholino] ethanesulfonic acid, Sigma) (0.2 M) pH 5.4, then a microliter of the radioactive isotope [ ³ H-Tyr] (NEN, specific activity: 31.0 ci / mmol) is added. Then, 20 mg of SAB or SAH dissolved in the same MES buffer are then added to conjugate the hapten via an amide bond. Activation of the carboxylic group of Tyr or Cys is initiated by the addition of carbodiimide [1-ethyl-3 (3-dimethylamino propyl) carbodiimide, Sigma]. Unbound Tyr and Cys molecules are removed by dialysis against distilled water for 24 hours (h) at 4 C.

. The radioactive marker makes it possible to determine the concentration of conjugated hapten as follows:

Concentration (M) of coupled hapten = X mg hap x CPM after / CPM before x Vol av x PM hap where X mg is the quantity of hapten used to make the coupling; CPM av is radioactivity before dialysis; CPM after is radioactivity after dialysis; Vol av is the volume before dialysis; PM hap is the molecular weight of hapten.

. To calculate the protein concentration according to the Beer-Lambert law DO = ε c 1 (in this work, cell length 1 = 1 cm), the absorbance at wavelengths 280 nm and 300 nm was used (Geffard et al., 1985a):

Protein concentration (M) = DO 280 nm - DO 300 nm / ε protein where ε is the molar extinction coefficient of the protein at 280 nm; it is 34,500 for SAB and SAH; For the haptens which absorb at 280 nm (Tyrosine, Tryptophan), the inventors used the following formula to determine the concentration of the protein: D280 = C ap ^{x ε} hap + Cp _ro tein ^{x ε} protein

. The coupling ratio is the number of moles of hapten coupled by one mole of protein:

Ratio (R) = hapten concentration / protein concentration. The conjugate weight is determined using the following relationship:

Weight of the conjugate = [(R x PMhap} + PM prot] x conc protein where the concentration (conc) is expressed in M. The weight is in mg / ml.

- Nitrosylation of neosynthesized conjugates:

Nitrosylation is carried out using a chemical NO donor, sodium nitrite (NaNO2) in an acid medium (Stamler et al., 1992d). Thus, the formation of the NO-hapten bond was evaluated by spectrophotometry by measuring the OD between 320 and 500 nm. In addition, the

S-nitrosylation has been proven by the method of (1958) using mercury chloride HgCl2 - Indeed, HgCl2 in the presence of the S-nitrosylated compound causes an immediate release of the nitrogen bound to sulfur from the nitrosothiol residue.

This rapid hydrolysis can be explained by the affinity of Hg ²⁺ ions for sulfur.

Table 1 below shows the structure of the two nitrosyl haptens linked to the SAB carrier protein. Table 1

b) Synthesis of other nitrosyl and n very conjugates

The use of different coupling agents such as carbodiimide, glutaraldehyde (G) or succinic anhydride (AS), makes it possible to obtain epitopes of distinct conformation. - "Carbodiimide" type:

Synthesis of NO-Tryptophan - SAB: Carbodiimide, tryptophan (Tryp, Sigma) and SAB were used for the synthesis of the Tryp-SAB conjugate. This conjugate was therefore then nitrosylated using NaNO2.

- Type "glutaraldehyde":

Synthesis of NO-Tyr-G-SAB, NO-Cys-G-SAB, and NO-Tryp-G-SAB: 5 mg of each hapten: Tyr, Cys (non-acetylated) or Tryp were dissolved in acetate buffer ( 1.5 M), pH 8.3. To follow the coupling reaction, a small amount of tritiated hapten is added. Then an aqueous solution of glutaraldehyde (G) 5% (Merck) is mixed with the haptenic solution. After a few seconds, the protein (10 mg) diluted in the same buffer is mixed with the haptenic solution until a yellow color appears. The reaction is then stopped by the addition of 200 μl of a NaBH4 solution (10 mM); when the mixture has become translucent, each solution of the various conjugates is dialyzed against distilled water for 24 hours at 4 ° C. (Atassi, 1984).

- Type "succinicrue anhydride":

. Syntheses of NO-Tyr-AS-SAB and NO-Cys-AS-SAB: 20 mg of Tyr or Cys (not acetylated) are taken up in 200 μl of Dimethyl-sulfoxide (DMSO, Merck) and 800 μl of distilled water . Succinylation is carried out by adding 17 mg of succinic anhydride (AS) (Sigma), then an amount of NaOH IN is added to neutralize the mixture. It is then put to lyophilize.

5 mg of succinylated hapten are dissolved in 1 ml of anhydrous pure dimethylformamide (DMF, Merck), containing 50 μl of pure, anhydrous triethylamine (TEA, Merck). The free COOH groups of the succinylated hapten are activated by adding 200 μl of Ethyl chloroformate (ECF, Prolabo) prediluted to 1/6. Then, the solution containing 10 mg of SAB is added. The reaction mixture is dialyzed against distilled water for 24 h at 4 ° C with stirring.

The nitrosylation method of the two types of coupling (G and AS) is identical to that described above for the coupling of carbodiimide type. . Synthesis of NO2-tyrosine-SAB: The synthesis of this conjugate requires 20 mg of the hapten NO2-Tyr (Sigma) and 20 mg of SAB. The coupling is carried out using carbodiimide according to the same protocol described above.

. Synthesis of NO-protein conjugates: This form of nitrosylated SAB or S-nitroso-SAB was prepared from SAB and sodium nitrite. The same amount of protein and NaNO2 is weighed to make the NOSAB coupling according to the method of Stamler (1992d).

c) Obtaining and characterizing anti-NO conjugated polyclonal Acs:

- Rabbit immunization: From the NO-Tyr and NO-Cys conjugates, the inventors have immunized subcutaneously (Vaitukaitis et al., 1971) rabbits: the "T" rabbit by the NO-Tyr-protein conjugates (SAB or SAH), and rabbit "C" by the NO-Cys-protein conjugates. Immunization was done by alternating these two carrier proteins (Geffard et al., 1984a, b; 1985a). Immunization was carried out by injecting an emulsion containing 200 μg of the conjugate, complete Freund's adjuvant (ACF, DIFCO) and phosphate buffered saline (TPS). Antisera collection was done between 12-21 days after each injection.

- Purification of immunoqlobulins:

An immune response has been induced. To enrich the seru s with immunoglobulins (Ig) specifically directed against coupled haptens, the following purification methods were used:

. Adsorption of polyclonal sera on carrier proteins: The polyclonal sera were adsorbed on the corresponding non-nitrosyl conjugates: Tyr-SAB / SAH for rabbit "T" and Cys-SAB / SAH for rabbit "C". (Geffard et al., 1984a; Geffard et al., 1985b; Campistron et al., 1986). The adsorption was carried out in the proportions of 5 mg of conjugate per ml of native serum. The mixture was incubated for 16 h at 4 ° C. with shaking and the immunoprecipitates were removed by centrifugation for 15 minutes at 10 000 g. The supernatant is enriched in specific Ig, while the pellet contains the immune proteins-rabbit Ig complexes.

. Purification of Ig by precipitation with ammonium sulphate:

To a volume of polyclonal rabbit serum is added an equal volume of a saturated solution of ammonium sulfate (NH4) 2SO4. The mixture is incubated for 1 h at 4 ° C., then centrifuged for 15 minutes at 10 000 g. The pellet (containing the precipitated Ig) is taken up in a minimum volume of TPS buffer then dialysis for 3 days against TPS buffer (Na2HP04, 0.01 M, 0.15 M NaCl).

. Purification of Ig by exclusion chromatography: After precipitation with ammonium sulphate, the

Ig were separated according to their molecular weight (PM) by the G 200 column exclusion chromatography technique (Sephadex) (Mons and Geffard, 1987). The determination of the Ig concentration was deduced from the spectrophotometric analysis of a diluted aliquot, it was then estimated as follows:

1.4 OD units at 280 nm corresponds to an amount of 1 g / ml of Ig.

The specific Ig are mainly Ig G. The supernatants purified according to one of these methods were used to study the titer, the avidity and the specificity using the ELISA test.

d) Immunoenzymatic test (ELISA method): Four main steps are necessary for carrying out the immunoenzymatic test (Engvall et al., 1972):

1st step: Coating of the microtiter plates. This is the step of adsorption of the conjugates: NO-Tyr-SAB and Tyr SAB for the rabbit "T" or NO-Cys-SAB and

Cys-SAB for rabbit "C", in microtiter plate wells (Maxisorp certified, NUNC). This coating step was also carried out with the coupled conjugates "G", "AS" and for SAB and NO-SAB. 2nd stage: Saturation. It is necessary that

"saturate" the wells, to avoid nonspecific snagging. This step is done with the following buffer: TPS-tween-glycerol-SAB. Tween 0.05% pH = 7.2, glycerol 10%. The concentration of SAB has been optimized to 5 g / l. 3rd step: Formation of primary complexes.

It corresponds to the fixation of the primary Ab potentially contained in the serum to be tested at a optimal dilution of 1/20 000 (for the rabbit "T" and "C") on the conjugates deposited at the bottom of the wells.

4th step: Formation of secondary complexes. It corresponds to the addition of secondary anti-rabbit Ig goat Acs (Diagnosis

Pasteur) labeled with peroxidase diluted 1/8000 in the TPS -Tween- SAB buffer.

5th step: Visualization of secondary complexes. It is done by the use of the specific substrate for peroxidase (H2O2 / Prolabo), in the presence of a chromogen, 1 orthophenylene diamine (OPD, Sigma). The visualization is carried out by the oxidation of the OPD which passes from the reduced colorless state to the yellow oxidized state. The reaction is then stopped by adding a solution of sulfuric acid (H2SO4, 4N) to each well. The ODs are read at 492 nm using a computerized multiscan spectrophotometer (EL 312e, Bio-Tek Instruments). The experimental value obtained is that measured on the wells having the conjugate (hapten-nitrosylated-protein) or nitrosylated protein subtracted from that read on the control wells containing the non-nitrosylated conjugate or BSA.

- Immunochemical characterization of Ac sites: It is based on two properties of Ac: avidity and specificity:

. The avidity of each antiserum was assessed as follows: Dilutions of NO-Tyr-SAB conjugates for the anti-NO-Tyr antiserum or NO-Cys-SAB for the anti-NO-Cys antiserum, ranging from 2 × 10 ^{~ 5} M to 2x10 ^" - ¹² M in. TPS-Tween-SAB glycerol have been prepared. These competitions are made by incubating the antiserum (" T "OR" C ") in the presence of the immunogen overnight. 4 ° C. The test is then carried out as described above using these dilutions as primary Ab.

For specificity: the compounds used for the competition experiments are the following for the two types of Ac: NO-Tyr-SAB; Tyr-SAB; NO-Cys-SAB Cys-SAB; NO-Cys (non-acetylated) -SAB; Cys (non-acetylated) -SAB NO-Tyr-G-SAB; Tyr-G-SAB; NO-Cys -G- SAB; Cys-G-SAB NO-Tyr-SA-SAB; Tyr-SASAB ;. NO-Cys-SA-SAB; Cys-SA-SAB N02 -Tyr-SAB; NO-Tryp-SAB; Tryp-SAB; NO-Tryp-G-SAB Tryp-G-SAB; NO-SAB.

At the end of the competitions, displacement curves for the different compounds tested were established.

4) Results. a) Analysis of the nitrosyl conjugates:

- Spectrophotometric analysis of conjugates: A spectrophotometric study was used to follow the different stages of the synthesis of immunogens. This is done between the wavelengths 240 and 500 nm. By comparison of the spectra obtained before and after nitrosylation, the inventors determined the absorbance zone of bound NO. As shown respectively in the curves of FIGS. 1 and 2, the absorbance zone of

NO-Tyr-SAB was observed between 320 and 440 nm (Figure 1) and that of NO-Cys-SAB was detected between 320 and 400 nm

(figure 2). An absence of absorbance between the wavelengths 320 and 500 nm was noticed in the spectra of the conjugates: Tyr-SAB and Cys-SAB;

The absorbance band of the same NO-Cys-SAB conjugate was not found after the treatment of the latter with a HMCl2 ImM solution. This shows that the "S-NO" covalent bond has been abolished.

b) Immunochemical analysis of Ac sites.

- Evaluation of the titer in Ab directed against the NO-Tyr and NO-Cys epitopes:

. For the "T" rabbit immunized with conjugated NO-Tyr, the specific Ac titer evolved after each immunization and the optimal response was obtained after the third immunization. So, as shown by the curve of figure 3, two types of answers are visualized for the rabbits "T". The first corresponds to a response directed against the NO-Tyr-SAB conjugate which remained during the higher immunization than the second which is directed against the Tyr-SAB conjugate. In FIG. 3, the white arrow indicates the sample before immunization and the black arrows indicate the days of the samples after each immunization.

For rabbit "C" immunized with conjugated NO-Cys, no specific response was obtained before the 5th immunization. Before this immunization, the inventors observed a greater increase in the anti-Cys-SAB response compared to the anti-NO-Cys-SAB. On the other hand, following the 5th immunization, the inventors detected a drop in the non-specific response. FIG. 4 shows the evolution of the two types of response in this rabbit: that corresponding to a response on the NO-Cys-SAB conjugate which is a global response comprising the antibodies directed against the carrier protein and a second directed against the Cys conjugate -SAB. In FIG. 4, the white arrow indicates the sample before immunization and the black arrows indicate the days of the samples after each immunization.

The sample from the "T" rabbit after the third immunization was used for the immunochemical characterization of the Ab specifically directed against the nitrosylated epitope. Likewise, the inventors used the sample after the 5th immunization for the rabbit "C".

- Determination of greediness and specificity of developed Ac:

The avidity of the conjugated anti-NO-Tyr and anti-NO-Cys antibodies was determined by competitive tests. Inhibition of the binding of anti-seru s "T" and "C" was obtained by incubating each of these two Ab with the nitrosylated conjugate which served as immunogen. The decrease in OD (B) indicates the presence of competition between the conjugated hapten which is adsorbed on the microtiter plate and the hapten preincubated with the corresponding antiserum. Bo is the OD corresponding to the response obtained with the antiserum in the absence of the competitor. A dilution of the antiserum (1/20 000) giving an OD of about 1.0 at 492 nm was chosen for the adjustment of the value of Bo; the B / Bo ratio was used to plot the competition curves of Figure 5 obtained with the competitors.

- For the rabbit "T", the avidity determined at half-displacement is 1.1 x 10 ^~ ^ M (Figure 1, curve 1) and for the rabbit "C", the avidity is 4.5xl0 ^~ 8 M.

The specificity of each antiserum was also evaluated by competition experiments between the conjugate NO-Tyr-SAB (for the antiserum "T"), or NO-Cys-SAB (for the antiserum "C") and the analogues structural conjugates. The displacement curves were obtained from the results of competition tests with conjugated haptens which were not used in the immunization. Using the B / Bo ratio for each of the competitors, the Inventors were able to determine the corresponding specificities:

. for the rabbit "T" the most recognized conjugate is the immunogen which gave an avidity of 1.1 × 10 ^-8 M. The other recognized conjugates are the following: NO2-Tyr-SAB (ICso = 1.4 x 10 ^"7 M); NO-Tyr-SA-SAB (ICso = 5 x 10 ^-5 M); and NO-Tryp-G-SAB (ICso = 3 x 10 ^{" 6} M). Curve 2 in FIG. 5 also shows the absence of recognition of Tyr-SAB by this antiserum.

- For the "C" rabbit 1 immunogen NO-Cys-BSA conjugate and the NO-Cys (non-acetylated) -BSA were recognized with an avidity of 4.5 x 10 ^-8 M and a specificity of 10 ^-6 ~ M. All the other conjugates, nitrosyles (for example NO-SAB) or not, are not recognized by Ac "T" or Ac "C". (IC50 is the competitor's molar concentration giving 50% inhibition).

It should also be noted that, indirect ELISA tests have shown a discrimination between NO-SAB and SAB by the polyclonal Ab "T" and "C". The ODs obtained with these antisera are respectively around 0.34 ± 0.062 and 0.27 ± 0.05. On the other hand, in the liquid phase, NO-SAB has not been recognized, which could be explained by the conformational modification of this molecules in solid phase (indirect test) and liquid phase (competition test).

5) Discussion.

Based on the work of Landsteiner, (1945); Goodman, (1975); Geffard et al. , (1985b); and Mons and Geffard, (1987) for the induction of antisera against small molecules, the development of anti-NO-conjugated Ac was carried out. The reactivity of NO with thiol groups could partly explain its intervention in different biological processes (Girard and Potier, 1993). In fact, it has been postulated that NO is stabilized in the form of S-nitrosothiol which can retain its biological reactivity. The present immunological study is based on the hypothesis of the stability of the NO derivatives. The inventors have used various nitrosyl conjugates presumed to be NO transporters (aryls nitrosyl residues and S-nitroso-Cys). The immunochemical characterization of the anti-NO-Tyr and anti-NO-Cys developed shows a different discrimination power from these Ab for the corresponding immunogens. In addition, the optimal specific anti-nitrosylated Tyr titre was observed after the 3rd immunization, after 1-2 months (FIG. 3), while the nitrosylated anti-Cys did not show any change in titre before a 2-3 month period (Figure 4), and gives an optimal titer after the 5th immunization.

Regarding the specificity of these preincubated Ab with other analogous conjugates structural with the compound used as immunogen, there is also a difference in the responses obtained with these two Ac. Thus, the Ac "T" recognizes two conjugates which have not been used in immunization, Nθ2-Tyr-SAB and NO-Tryp-SAB, which is not the case with Ac "VS". None of the other non-nitrosyl conjugates were recognized by these two Abs. The different responses observed with these two types of Ab could also be explained by the differences in molecular structures of the immunogens, their stability in the biological medium, their metabolism, or by the modifications favored by the cells presenting the antigen. All these factors can play a fundamental role in stimulating the immune system and in the greed of the induced Ab.

Finally, the recognition of NO-SAB chemically synthesized by polyclonal antibodies, led the inventors to use this form of NO transport in multiple applications in vitro, using activated macrophages as biological donors of NO.

III - DETECTION AND NEUTRALIZATION OF NO DERIVATIVES BY POLYCLONAL ANTIBODIES.

Macrophagic cells play an essential role in the immune response. Indeed, from the appearance of an infection, they can intervene in the destruction of the microorganism, by phagocytating it. In addition, macrophages activated during a specific immune response exert a cytotoxic microbicidal effect on intracellular microorganisms (Ruskin and Remington, 1968). One of the metabolic pathways of arginine is essential in the activation of macrophages and in their cytotoxic effect on tumor cells (Hibbs et al., 1987a) and cytostatic on fungal species such as Cryptosoc eus neofor ans (Granger et al., 1988). This metabolic pathway leads to the synthesis of L-citrulline and NO from arginine (Hibbs et al., 1987b; Hibbs et al., 1988), thanks to iNOS. The structural analogue of arginine, NMMA, was then characterized as a competitive inhibitor of this synthesis from activated macrophages (Hibbs et al., 1987a, b; Drapier and Hibbs 1988; Hibbs et al., 1988).

This cytotoxicity phenomenon has been studied essentially in vitro in murine macrophages and there is now a broad consensus to affirm that the biosynthetic pathway L-arginine -> NO is the basis

(Drapier, 1993).

The cytotoxic role of NO has also been interpreted as a result of the formation of iron-nitrosyl complexes at the level of enzymes in the oxidative metabolism of target cells (Stuehr and Nathan 1989;

Stuehr et al. , 1989).

In conclusion, an infection induces stimulation of T lymphocytes via the presentation of the antigen. T cells secrete IFN-γ in response to their activation and successive stimulation is therefore maintained with the persistent presence of the antigen or reinfection. IFN-γ then activates macrophages which produce other cytokines such as TNF-α and 1 'IL-1 which, in synergy with IFN-γ can stimulate macrophages. Macrophagic activation allows the elimination of certain tumor cells and many pathogens. Among the various mechanisms developed by macrophages, one of the most effective is the synthesis of NO. The destruction of leishmanias and schistosomes can be completely inhibited by NMMA (James and Glaven, 1989; Green et al., 1990; Liew et al., 1990). This seems to indicate that in these cases NO is necessary, and could even be sufficient, to explain the microbicidal role of macrophages. NO is therefore an essential element of the body's defense. The work reported below aims to highlight the form in which NO is transported to act at the level of its targets. In order to approach physiological conditions, the inventors used a biological source of NO (macrophages activated by BCG), SAB as a transporter of NO and of extracellular trypanosomes. The objective is to detect nitrosyle epitopes formed in vitro having a cytotoxic role and to obtain their blocking by using our polyclonal anti-NO-Tyr and anti-NO-Cys antibodies.

1) Materials and methods. a) Collection and culture of macrophages:

Peritoneal macrophages from Swiss mice previously infected with BCG (Institut Pasteur) or normal macrophages were cultured at 1.5 x 10 ^ cells / ml / petri dish for 8 h at 37 ° C in a oven containing 5% C02. The culture medium used is HBSS (Hank's Balanced Sait Solution, GIBCO). The culture was carried out in the presence or not of NMMA at 0.5 mM.

b) Detection of the formation of the NO-NO and NO-SAB conjugates by the ELISA test: - Competition tests between the nitrosylated conjugate formed in the culture medium and the neosvnthetized NO-Tyr-SAB or NO-Cvs-SAB conjugates:

The supernatants from the culture media of activated macrophages containing the nitrosyl conjugates (NO-Tyr-SAB or NO-Cys-SAB), were incubated in the presence of antiserum "T" or "C" ulitized at 1: 20,000 These nitrosyl conjugates were formed from NO produced by the macrophages and the Tyr-SAB or Cys-SAB conjugates added to the medium. After incubation these supernatants were then used as primary Ab for the ELISA test. The latter was carried out on microtiter plates adsorbed by the NO-Tyr-SAB or NO-Cys-SAB conjugates. The rest of the test is identical to that described above.

The same protocol was used for the supernatant from normal macrophage culture media (control). For this work, 5 experiments were made and good reproducibility was obtained with

1 'anti-NO-Cys.

- Detection of the nitrosylation of the epitopes carried by the SAB using anti-NO-Tyr and anti-NO-Cvs Ab:

This application required the use of defatted SAB (4 mg / ml), to avoid lipoperoxidation (NO + 02 ^~ °) of the fatty acids adsorbed on SAB. This lipoperoxidation gives compounds leading to non-specific reactions. The protein was added to the culture medium (HBSS) of the macrophages activated or not for 8 h. The microtiter plates were then adsorbed for 6 h by the supernatants from the culture media of activated macrophages (containing NO-SAB), non-activated macrophages or the culture medium alone. After saturation the primary Acs "T" or "C" were used at the dilution of 1/1000. . Neutralization of the trypanostatic effect of

NO-SAB:

The Partinico II strain of T. muscul i was used. The trypanosomes were purified by passage through a diethyl aminoethyl cellulose column (Lanham, 1968). The trypanosomes were then co-cultured for 8 h in the presence of macrophages from normal or BCG-infested mice in RPMI medium containing the delipidated BSA (4 mg / ml). Polyclonal Acs (1/100) or NMMA

(0.5 mM) were added to a few wells in order to compare their inhibitory effects. Normal rabbit serum and an anti-BSA polyclonal Ab were used as controls at the same dilution as the "T" and "C" antisera. Neutralization of the transfer of this trypanostaticrue effect:

The objective of this work was to confirm the trypanostatic effect of NO-SAB. For this, the supernatant of cultures containing SAB and macrophages activated by BCG in the presence or not of NMMA, is transferred after 8 h of incubation, to a coculture of normal macrophages / T. muscul i according to the same conditions. The polyclonal Ab "T", "C", normal rabbit serum or anti SAB diluted to 1/100 are applied with the supernatant.

By these two methods, the inventors first studied the cytostatic trypanostatic effect of NO-SAB either directly or transferred. In a second step, they compared the neutralization of this effect by polyclonal Ab by two slightly different methods. The fact of inhibiting the trypanostatic effect by these Ab according to these two conditions proves on the one hand the synthesis of NO and therefore the formation of NO-SAB and, on the other hand that it is an effect a priori specific to NO-SAB and not to other oxygen derivatives. . Measurement of the amount of nitrite produced: The production of NO is given by the formation of Nθ2 ^~ accumulated in the culture medium using a colorimetric reaction: the Griess method (Hageman and Reed, 1980; Lepoivre et al., 1989) . The photosensitive Griess reagent is obtained by mixing volume to volume suifanilamide (1% in 1.2 N HCl; Sigma) and N- (1-naphthyl) ethylenediamine dihydrochlorate (3% in water; Sigma); a volume of 200 μl of reagent is added to 100 μl of sample, incubated for 30 minutes in the dark, then the absorbance at 550 nm is measured using a plate reader. A standard range is carried out for each assay using a solution of NaNO2. 2) Results. a) Detection of the formation of the NO-Cvs-SAB conjugate in the macrophage culture medium:

The NO produced in vi tro by macrophages activated by BCG, induces the formation of NO-Cys-SAB from the Cys-SAB conjugate previously added to the medium. The latter plays the role of a target, reservoir and a stable form of NO produced. The nitrosylated derivative formed is detectable by competition experiments. In the presence of NMMA, inhibition of NO synthesis and the absence of a signal were noted.

FIG. 6 represents the kinetics of formation and the concentration of NO-Cys-SAB formed in the culture supernatant of the activated macrophages determined at different incubation times: 0, 3, 4, 6, 8, 11, 14, 18, and 20 h using the antiserum "C". The same process was applied to the non-activated cells and to the culture medium alone in the presence or absence of NMMA. The concentration (μM) of NO-Cys-SAB formed from the avidity curve of the antiserum "C" was then calculated.

Culture supernatants from normal macrophages were tested in the same way; no formation of nitrosyl conjugates was found.

b) Detection of nitrosyl epitopes by the ELISA test:

Nitrosylated BSA could be detected by the test

ELISA using the two Ac "T" and "C" diluted to 1/1000. These tests show recognition by these Ab of epitopes such as NO-Tyr and NO-Cys, formed at the level of NO-SAB.

The two Acs gave values of the same order with the activated and normal macrophages, in the presence or in the absence of NMMA. In Table 2 below the values represent the mean and the standard deviation of 5 experiments. Table 2

In the presence of NMMA, the OD obtained with activated macrophages are reduced by about 2.5 compared to those obtained in the absence of NMMA. On the other hand, with normal macrophages, there is no great difference in the ODs obtained in the absence or in the presence of NMMA, and to postulate while these values do not correspond to an Ac-epitope-NO bond.

The production of θ2 was also determined to follow the synthesis of NO, so when using NMMA the concentration of θ2 ^~ increased from ~ 26 μM to a value of 1 'order of 3. With normal macrophages no difference concentration has not been found.

c) Neutralization of the cytostatic and cytotoxic effect of NO by polyclonal Ab: The cytostatic and / or cytotoxic role of

NO-SAB formed in the co-cultures of activated macrophages / T. musculi was followed for several days. We observed that NO-SAB inhibited the multiplication of parasites and that the use of the Ab of the invention blocked this effect. The same results are obtained when the supernatants containing NO-SAB were transferred into normal macrophage / T. musculi co-culture wells.

Figures 7 and 8 represent the number of parasites counted every day for 4 days in the different culture wells:

- After incubation of the trypanosomes with NO-SAB according to the two methods cited in "Materials and Methods" in the absence or in the presence of normal rabbit serum or anti-SAB, the number of parasites remained almost stable and then decreased around the 4th day. These results show the trypanostatic effect of NO transported in the form of "NO-protein" and the cutostatic and / or cytotoxic effect which was revealed by the reduction in the number of parasites. - On the other hand, when the Ac "T", "C" were added to the culture media, the number of parasites increased from 104 to 106 parasites / ml between the 2nd and 4th day of incubation. This same effect was also observed when NMMA was added to the culture media of activated macrophages. This shows an inhibitory role of these molecules on the NO dependent cytostatic and cytotoxic effect which could be due to NO-SAB or even to nitrosyl epitopes (NO-Cys or NO-Tyr) formed at the level of nitrosylated SAB. FIG. 7 represents the inhibition of the cytostatic effect of the BCG macrophages on T. musculi in vi tro in the presence of NMMA (0.5 mM), of the antiserum ("T") or ("C") used in 1 / 100. The cocultures of trypanosomes and of BCG macrophages in normal medium and in medium supplemented with normal rabbit serum or anti-BSA Ab were used as controls.

FIG. 8 represents the cytostatic effect of the supernatants containing NO-SAB originating from the activated macrophages and added to normal macrophages containing T. muscul i. Inhibition of this effect in the presence of NMMA (0.5 mM), antiserum ("T") or ("C") used at 1/100. Absence of the inhibitory effect in the presence of a normal rabbit serum or anti-BSA Ab (1/100).

Each point on the curves in Figures 7 and 8 is the result of 4 experiments.

3) Discussion.

The above results show the cytostatic and / or cytotoxic effect of NO (in the form of NO-SAB) on extracellular trypanosomes. They also reveal that the nitrosyl derivatives formed in the culture media of activated macrophages, in the presence of SAB, are sufficiently stable to be detected immunochemically.

The high specificity of the polyclonal antibodies of the invention made it possible to block the effect of NO

"transported" to the level of the trypanosomes. We can conclude that one of the forms of NO transport during its cy to t tic actions could be (NO-protein) or (NO-amino acid). Let us also add that the immunochemical signal disappears when NMMA is used or when the supernatant comes from normal macrophages with or without NMMA.

This work made it possible to demonstrate in vitro nitrosylation of SAB during its exposure to NO produced at pH 7, from macrophages activated by the

BCG. This nitrosylation results in a modification of certain molecules. These are cytotoxic for parasites and carry nitrosyl epitopes, which are tyrosine and nitrosylated cysteine, recognized by the polyclonal Ab of the invention.

IV - PREPARATION OF MONOCLONAL ANTIBODIES DIRECTED AGAINST NO-CYS-GLUTARALDEHYDE CONJUGATES COUPLED TO A CARRIER PROTEIN.

As already indicated, nitrosyl derivatives are involved in a very large number of mechanisms physiological and physiopathological (inflammation, septic shock, autoimmune diseases, ... Moncada et al., 1991; Nussler and Billiar, 1993; Stevanovic-Racic et al., 1993; Anggard, 1994) of the organism. It is therefore essential to obtain molecules capable of detecting and blocking these NO derivatives, which can destroy pathogens and block a major biological function of the organism.

The previously reported polyclonal approach has shown the possibility of blocking the cytostatic and cytotoxic effect of NO on T. muscul i. However, the heterogeneity of the immune response does not allow these immuns to be efficient enough for complex studies where attention is paid to a single epitope. To confirm the effect of NO on trypanosomes and to avoid nonspecific clashes, a monoclonal approach has also been developed. The inventors have therefore induced monoclonal Ab directed against the NO-Cys-G-protein conjugate. This monoclonal Ab first served as a tool for in vi tro detection of certain NO binding sites (such as cysteine) at the level of parasites, and secondly for in vivo inhibition of the development of inflammatory diseases such as as autoimmune encephalitis and experimental inflammatory arthritis in Lewis rats.

1) Materials and methods. a) Synthesis of the immunogen:

The immunogen used in the production of the monoclonal antibody is Cys coupled by (G) on SAB or SAH then nitrosylated by NaNθ2 - in accordance with the protocol described in detail above.

b) Route of immunization: - Intraperitoneal route (IP):

5-week-old mice from the Balb / c co-bloodline (IFFA CREDO) were immunized by IP route with 100 μg of immunogen in complete Freund's adjuvant, for the first immunization. The other injections were made using Freund's incomplete adjuvant.

- Lymphatic route: plantar pad (PC):

From a 1 mg / ml solution (in the TPS) of conjugate, Balb / c mice were injected into the (CP) every three days for 10 days (3 injections). Some received a fourth injection to increase the titer and greed of the Ac response.

c) Obtaining monoclonal Ab directed against NO-Cys-G conjugated by the technique of lymphocyte hybridization and monoclonal selection:

The mice were immunized alternately with the immunogens NOCys-G-SAB and NO-Cys-G-SAH; when the titer, the avidity and the specificity of the Ac produced were considered satisfactory, the selected mice were left to rest for one month before receiving a final booster intravenously, 4 days before the lymphocyte hybridization.

The spleen cells (5 x 10 ⁷ ) and the myeloma SP2 / 0 / Ag (2.5 x 10 ⁷ ) (Shulman et al., 1978) were fused with polyethylene glycol 1500 according to a proven fusion protocol. The technique used is derived from that proposed by Kohler and Milstein (1975), improved by Lane (1985), and adapted to the laboratory for small molecules (Chagnaud et al., 1987; 1989a, b; 1990). The fused cells are then cultured in a selective medium: RPMI 1640 enriched in L-glutamine (2 mM), in penicillin / streptomycin antibiotics and supplemented with fetal calf serum (10%) containing 50 μM of hypoxanthine and 10 μM of azaserine . After ten days of culture, the wells containing hybridomas were counted: The specific Ac producing clones were then screened using the ELISA test and subcloned according to the following steps:

- For the first screening, all the clones were tested on the following conjugates: NO-Cys-G-SAB, Cys-G-SAB and SAB-G. Goat anti-mouse Ig antibodies are used at a dilution of 1/5000 for revealing positive and particularly specific clones of the immunogen (NO-Cys-G-BSA).

Then, the positive clones were subcloned (Sub-Cloning 1) to obtain a cell / well. After one week of culture, the clones were retested on ELISA plates adsorbed with the conjugates mentioned above;

- After the first subcloning we carried out studies of the specificity of positive clones.

Thus, a study of the dilution was carried out in order to obtain an OD ~ 1 to carry out the competition tests according to the same method as that described above; - The clones which have shown specificity for the NO-Cys-GSAB conjugate have been subcloned for a second time (Subcloning 2) and retested on nitrosylated or non-diferent conjugates: (NO-Cys-G-BSA; Cys-G-BSA; NO-Cys-SAB; Cys-SAB; NO-Tyr-G-SAB; Tyr-G-SAB; NO-Tyr-SAB; Tyr-SAB; SAB-G; as well as NO-SAB ). The results of indirect ELISA tests, as well as competitive tests allowed the selection of the best clones;

- After these two subcloning which made it possible to ensure the monoclonality of the hybridomas, the clones chosen were kept in liquid nitrogen. Mass production of the Ac was carried out in ascites liquid (Potter, 1976) and the Ig were then purified by precipitation with ammonium sulphate.

2) Results. a) Spectrophotometric analysis of the immunogen used for the monoclonal approach: The nitrosylation of the SH group of the coupled Cys was followed by spectrophotometry. FIG. 9 represents the spectrometric analysis of the immunogen NO-Cys-G-SAB and of its structural counterpart Cys-G-SAB as a function of the wavelength. The NO absorbance band for the NO-CysG-SAB conjugate was detected between the wavelengths 340 and 640 nm. The spectrum of the Cys-G-SAB conjugate which was obtained before nitrosylation shows an absence of absorbance between these two wavelengths.

It should also be noted that the absorbance zone obtained with NO-Cys-GSAB is greater than that of the NO-Cys-SAB conjugate despite the same coupling ratio of the two conjugates. This could be due to the type of coupling and therefore to the exposure of the accessible hapten to NO.

Table 3 below gives the molecular structure of the immunogen NO-Cys-G-SAB and Cys-G-SAB:

Table 3

b) Evolution of the immunization of mice:

- Mouse immunized by route (IP):

The optimal dilution of primary Ac is

1/8000. The evolution of two immune responses is noted: one is significant, directed against NO-Cys-G-SAB was obtained after the third immunization. It is followed by a second which is weaker. It is the latter which has been directed against hapten and the carrier protein.

(Cys-G- SAB). FIG. 10 represents the evolution during the immunization of the antibody response in the mouse immunized intraperitoneally. The same dilution of primary Ac was used for the study of avidity and specificity.

- Mouse immunized in (CP): After the third immunization, it was noted the appearance of the response directed against NO-Cys-G-SAB, and the lack of recognition of Cys-G-SAB. FIG. 11 represents the evolution during the immunization of the antibody response in the mouse immunized in the plantar pads (PC). The titer obtained after this immunization being insufficient for the characterization of the Ac (OD <0.5), a 4th immunization was carried out to improve the titer and the avidity.

c) Immunochemical characterization of mouse immune sera:

- Study of the avidity and the specificity of the responses obtained in the two mice injected by two different immunization routes: The avidity and the specificity of the anti-NO-Cys-G-SAB response were evaluated relative to 1 immunogen and its structural analogs, nitrosyles or not.

. Mouse immunized by route (IP): Figure 12 represents the avidity and the specificity of anti-NO-Cys-G antibodies of the mouse (IP). The displacement curves are obtained after competition tests in the presence of the antibody and each of the conjugates indicated. After incubation of the immunogen with the anti-NO-Cys-G-conjugate diluted to 1/8000, the avidity of this antiserum was evaluated at half-displacement. It is 1.1 x 10 ^~ 8 M. When the other competitors used (Cys-G-SAB; NO-Cys-SAB; Cys-SAB; and SAB-G) very low recognition was found for Cys -SAB and SAB-G with respectively an IC50 of 10 ^{~ 5} M and 5.6 x 10 ^~ 6 M_ Despite these cross-reactivities NO-Cys-G-SAB is the best recognized conjugate. . Immunized mouse vOar pathway (CP): Figure 13 represents the avidity and the specificity of anti-NO-Cys-G antibodies of the mouse (CP). The displacement curves are obtained after competition tests in the presence of the antibody and each of the conjugates indicated. After the fourth immunization, the inventors noticed, in the serum of this mouse, an increase in the titer (now 1 / 70,000) and in the avidity of the anti-NO-Cys-G-conjugate. The avidity of the Ac is calculated from the displacement curve obtained after incubation of the serum and the immunogen of FIG. 13. It is equal to 4 x 10-9 M.

Regarding the study of specificity, the molecules used (Cys-GSAB; NO-Cys-SAB; Cys-SAB and SAB-G) did not show a shift in the OD values which means the absence non-specific attachment with this Ac at the level of these conjugates.

By comparison of these two types of polyclonal antibodies directed against the same epitope (NO-Cys-G), differing only by the route of administration of the immunogen, different titer and avidity values were obtained. In addition, in the mouse immunized by the CP route, the response was earlier (after 10-15 days) (FIG. 11) and more specific than in the mouse immunized by the IP route. For the latter, (Figure 10) the immune response was found within a period well recognized for this classic route of immunization and which varies between 20 and 30 days.

d) Characterization of the monoclonal antibodies:

The blood of the mice chosen for carrying out this monoclonal approach was collected when the spleen was removed. The signal on the corresponding nitrosylated conjugate was analyzed. After 10 days of culture in a selective medium, the fusion yield (number of wells having one or more clones) was 25% for the fusion of mouse lymphocytes immunized in ([P). On the other hand, for the fusion of lymphocytes from the spleen of immunized mice in (CP), the yield was

97%. All wells have been tested and yield

(number of positive wells after an ELISA test) close to

85%. All selected hybridomas have recognized

1 immunogen. The background noise of the response (immunological signal on the non-nitrosylated conjugate / immunological signal on the nitrosylated conjugate) was in all cases less than 10%.

The clones which recognized the different conjugates were not selected.

- Avidity of conjugated anti-NO-Cvs-G monoclonal Ab:

The supernatant containing the anti-NO-Cys-G monoclonal antibodies was diluted to 1/5, and the ascites liquid to 1/30 000. These dilutions were chosen to give an optimal OD i 1.0 at 492 nm. Figure 14 reports the avidity and specificity of the anti-NO-Cys-G monoclonal Ab. The curves are obtained after competition tests in the presence of the Ac and each of the conjugates indicated. These competition tests carried out by incubating the NO-Cys-G-SAB immunogen in the presence of the supernatant, show an avidity of 4 × 10-9 M. The ascites liquid used at 1/30 000 gave an avidity of 2 , 5 x 10 ^~ 8 M_ This difference in avidity may be due to the presence in the liquid of ascites taken from mice, factors, proteins or immune complexes which inhibit specific responses.

- Specificity of the monoclonal anti-NO-Cys-G conjugated Ab: The specificity of the monoclonal Ab was studied by incubation of the culture supernatant of the hybridomas at a final dilution of 1/5 with conjugates having a structural analogy with the immunogen: Cys-G-SAB; NO-Cys-SAB; Cys-SAB; NO-Tyr-G-SAB; Tyr-G-SAB; NO-Tyr-SAB; TyrSAB; and SAB-G. At half-displacement, only one Cys-SAB conjugate was recognized with a specificity of 4 x 10-6 M (Figure 14). These results show that the NO-Cys-G-SAB conjugate is best recognized by these monoclonal Abs.

Despite the absence of competition with NO-SAB, indirect ELISA tests have shown that the monoclonal Ab recognizes the nitrosylated protein. The supernatant (1/5) and the ascites liquid (1/30 000) gave respectively OD of 0.36 ± 0.085 and 0.54 ± 0.12. The results represent the mean and the standard deviation of three experiments.

- Determination of the isotvpie of the monoclonal Ab produced:

Using a mouse Ig detection kit (Mouse monoclonal antibody, isotyping kit, Sigma), it was possible to determine the isotype of the clone obtained: Ig G 2b.

3) Conclusion.

Analysis of the immunochemical characteristics of the Ac of conjugated antiNO-Cys-G mice showed that: - the immunodominant part recognized by the Ac is always the NO epitope; a slight structural difference between the immunogen and its structural analogs is taken into account by the Ac sites, confirming the high discriminating power of these immunoglobulins.

These monoclonal Ab have high avidity and specificity; these immunochemical characteristics as well as the recognition of NO-SAB allow their use in well defined biological models. V - DETECTION OF NITROSYL ANTIGENS AND PROTECTIVE ROLE OF MONOCLONAL ANTIBODIES.

During the immune response, the appearance of very reactive effector molecules (oxygen derivatives, nitrogen derivatives, etc.) is likely to modify the antigenicity of the elements recognized by this response. Thus, the antigens carried by the microorganisms against which the organism develops an immune response of the Thl type with production of nitrogen derivatives, can lead to nitrosyl and nitro derivatives. These nitrosyl antigens can therefore lead to the synthesis of antibodies, their presence and their possible role are therefore to be studied. The fact that nitrates are relatively good known immunogens (Kofler et al., 1992; Maeji et al., 1992; Yuhasz et al., 1995), and the proven existence of anti-nitrotyrosine antibodies (Beckman and al., 1994a) led us to postulate the existence of such antibodies in vivo. The inventors therefore first sought the presence of nitrosyl antigens on the parasitic targets, by an immunocytochemical technique, using anti-nitroso-cysteine (poly- and monoclonal) and anti-nitroso-tyrosine (polyclonal) antibodies. . Secondly, these nitrosyl antigens having been detected, they looked for the presence of the corresponding antibodies in the serum of the trypanosome animals. Trypanosomes represent a privileged target of NO. In trypanosomiasis, there is indeed a considerable increase in the number and activity of macrophages. The cytostatic role in vi tro and the induction of NO synthase observed in macrophages of infested mice indicate that NO could be involved in the effector mechanisms against parasites. In addition, the trypanosomes circulating in their final host (trypomastigote form) have a non-functional mitochondria, their energy dependent on glycolysis. They represent therefore much less complex NO targets than other pathogens or tumor cells, hence the interest of their study.

If the cytostatic effect of NO on the natural trypanosome of mice (T. musculi) and on the trypanosomes of the brucei group (T. b. Gambiense, T. b. Rhodesienεe, T. b. Brucei), responsible for human trypanosomiasis and animal has been well established in vi tro, in vivo the effect of NO is much more complex. NO indeed involved in mechanisms ¹ immunosuppression by inhibiting the transformation and proliferation of lymphocytes, and furthermore it induces apoptosis. The immunosuppressive role of NO is important in trypanosomiasis due to the large amount of NO produced. This is why it is difficult with polyclonal and monoclonal antibodies to grasp the study of nitrosylated molecules and of a possibly protective effect linked to the injection of these antibodies. In a third step, a possible role of poly- and monoclonal antibodies of the invention, in vivo was therefore sought in less complex experimental models, where the deleterious role of NO has already been strongly incriminated.

A - Immunocytochemical approach. 1) Materials and methods.

Peritoneal macrophages from mice activated by BCG (source of NO), and trypanosomes placed in co-culture, were used. The parasitic strain used is T. brucei gambiense (T. b. Gambiense strain: "Féo" ITMAP / 1893). These parasites were maintained after injection by route (PI) (5 x 10-Vsouris) in normal mice (Noireau et al., 1989).

- The trypanosomes were grown in Petri dishes (35 mm) (Falcon Plastics) at 2 x 10 ⁶ parasites / ml / dish in the modified Me Coy 5A culture medium (GIBCO) supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, 25 mM Hepes, 2 mM sodium pyruvate, 0.2 mM L-Cysteine in the presence of activated or not activated peritoneal macrophages (Albright and Albright 1980; Hirumi and Hirumi 1989; Vincendeau et al., 1985; Oka et al., 1988). NMMA was added to some dishes at a final dilution of 0.5 mM. After 12 h, the survival of the parasites is evaluated in the wells in the presence of normal or activated macrophages, with or without NMMA. The delipidated SAB was added to the culture medium at 4 mg / ml as NO carrier. The supernatant containing the parasites is recovered after 12 h to fix the parasites for the immunocytochemical test.

- The trypanosomes are fixed on slides degreased with organic solvents (Ether-Alcohol). The most appropriate fixation for this protocol is carried out by cytocentrifugation (Shandon Elliott cytospin) at 100 g for 10 min. After fixing, the slides are dried, then after a saturation step with the TBS-SAB buffer (1 g / 1) the various primary antibodies are added. Incubation takes place for 1 h in a humid chamber at room temperature (Harlow and Lane, 1988). The anti-NO-Cys-G monoclonal antibody (supernatant) was used at 1:50; normal mouse serum used at the same dilution; polyclonal anti-sera (rabbit "T" and "C") and a non-immunized rabbit serum were used at a final dilution of 1/10000 to compare their labeling with that of the monoclonal antibody. The buffer alone was used on a few slides as a negative control.

The incubation of the primary antibodies is followed by the treatment of the slides with the secondary Ac labeled with peroxidase at a dilution of 1/5000 in TPS-SAB. The revelation is then carried out with 3-3'-Diaminobenzidine tetrahydrochloride (DAB; Sigma) at 60%, H2O2 (0.03%) in TBS. 2) Results.

The immunochemical labeling at high magnification (x 100) is shown in Figure 15:

- the anti-NO-Cys-G monoclonal antibody:. gave clear markings (immunoreactivity) in the trypanosomes co-cultured in the presence of activated macrophages (Photo 1);

. much weaker labeling was obtained in co-culture of activated macrophages / trypanosomes, in the presence of NMMA (0.5 mM) (Photo 2); no labeling was obtained on trypanosomes co-cultivated with normal macrophages.

A complete absence of labeling of the trypanosomes was obtained when using a normal mouse serum (Photo 3).

- No labeling of the trypanosomes was obtained during the use of the secondary antibody (anti-mouse Ig labeled with peroxidase) in the absence of a primary antibody. These results confirm the positivity of the marking obtained in photo 1.

- Polyclonal antibodies "T" and "C":

. the antibody "C": photo 4 of FIG. 15 shows an intensity very close to that of monoclonal Ab;

. Anti-NO-Tyr ("T"), gives a positive labeling of a lower intensity than the two types of antibodies (mono- and polyclonal) directed against the NO-Cys epitope (photo 5); . the use of Ac "T" and "C" with normal macrophages, gave a very weak labeling of trypanosomes, compared to those obtained in the presence of activated macrophages.

- The primary Ac of a normal rabbit gave an absence of labeling (Photo 6). - When the anti-rabbit Ig Ab labeled with peroxidase were used in the absence of the primary antibodies, no labeling was observed.

3) Discussion.

The work carried out shows the cytostatic and cytotoxic effect of NO on extracellular parasites such as T. b. brucei, T. b. gambi Ens and T. musculi (Vincendeau and Daulouède, 1991; Vincendeau et al., 1992; Daulouède et al., 1994), as well as the cytostatic / cytotoxic effect of NO on T. musculi by nitrosylated BSA. This effect is amplified in the presence of a protein carrier in the culture medium and inhibited in the presence of an antibody "C" or of NMMA, which demonstrates the possible microbicidal role of NO.

This model for studying the cytotoxicity of NO is a simple model which is easy to demonstrate by comparison with the use of other models such as those of tumor cells. The latter have much more complex metabolisms than that of trypanosomes, and their separation from macrophages is more

- difficult than that of trypanosomes which can be taken from supernatants of co-cultures

(macrophages / parasites). In addition, T. b. gambiense was used instead of T. musculi because the second strain is much more sensitive than the first to the action of NO or its derivatives. T. musculi requires 48 hours for NO, produced by BCG-activated macrophages, to have a cytotoxic effect of around 90%. Other molecules, such as suramin, exerting an effect on trypanosomes, require a carrier (SAB) to bind to the level of the parasites (Collins et al., 1986; Lopez et al., 1992; Vansterkenburg et al., 1989 ). NO being much more reactive than suramin, it requires the use of a carrier ensuring transport, stability, salting-out and transnitrosylation. This can take place by NO exchange between carrier protein and proteins and / or amino acids at the level of the trypanosomes, as well as at other parasitic sites not yet detected. The stability of the NO bond at these sites can vary from a few "seconds" or even a few "hours" before it is oxidized to Nθ2 ^~ in the presence of O2-

All these data encourage the use of the gambiense strain as the target of NO and the delipidated SAB as the carrier of NO. For the detection of nitrosyl derivatives such as NO-SAB or parasitic targets which have fixed NO, the anti-NO-Cys-G monoclonal antibodies and the polyclonal Ab "T" and "C" were used. To broaden the detection spectrum of nitrosyle products, the use of antibodies directed against different NO epitopes and having different avidities makes it possible to be more precise in solving the problem posed. The immunocytochemical markings obtained with poly- and monoclonal antibodies confirm the stability of the nitrosyl derivatives at the level of the trypanosomes. These results open up new perspectives in vivo and in vitro, for pathologies involving the role of NO, such as neurodegenerative diseases, chronic and acute inflammatory diseases, as well as inflammatory / autoimmune diseases such as multiple sclerosis and rheumatoid arthritis.

These new perspectives are based on:

- the detection in parasitized mice of antibodies developed against nitrosyl epitopes;

- the development in Lewis rats of experimental models (experimental autoimmune encephalitis and adjuvant polyarthritis) which are respectively models of human pathologies: multiple sclerosis and rheumatoid arthritis. The aim is to demonstrate in vivo the role of NO in the development of these two pathologies and to neutralize these harmful effects by using the monoclonal antibodies of the invention. B - Detection of antibodies directed against nitrosylated neoantigens in the serum of parasitized mice.

1) Materials and methods. Swiss mice are infested with three strains of parasites: T. b. gambi Ens, T. b. brucei (strain: ANTAT 1.1) and T. musculi. Each mouse receives 50,000 parasites intraperitoneally. Then the parasitaemia is evaluated from the 3rd day after infestation. The blood of the mice is then taken on the 5th day (time limit for the survival of mice parasitized by T. b. Gambiense and T. b. Brucei and on the 21st day for mice infested with T. musculi.

Several nitrosyl conjugates were prepared for this immunoenzymatic study: NO-SAB; NO-SAB defatted; NO-Cys-SAB; NO-Tyr-SAB; NO-Cys-G-SAB; NO-Tyr-G-SAB and NO-Tryp-G-SAB. Non-nitrosyl structural analogs were also used under the same conditions. The sera were diluted 1/1000 and the secondary Ab were used at a final dilution of 1/5000.

2) Results. - Immunological response on nitrosyl conjugates:

The search for Ac directed against nitrosyl epitopes in the sera of trypanosome mice was carried out on a population made up of: 15 mice infested with T. musculi (group 1), 15 mice infested with T. b. brucei (group 2), and 15 mice infested with T. b. gambi teach (group 3). Sera from 15 normal mice were used as controls.

The averages of the ODs obtained on different nitrosyl epitopes were calculated for each group of mice mentioned above. FIGS. 16 to 22 show the OD values for the sera of normal and parasitized mice. The ODs obtained on the molecules were corrected by the subtraction of the ODs obtained on the non-nitrosylated structural analogues which served as blank in the ELISA test. These figures 16 to 22 correspond respectively to the responses on the following epitopes: NO-SAB; NO-SAB defatted; NO-Cys-SAB; NO-Cys-G-SAB; NO-Tyr-SAB; NO-Tyr-G-SAB and NO-Tryp-G-SAB. Each OD value is an average of 2 determinations, the bars in the figures show the means (calculated by the Mann-Whitney test) of the OD of each group of mice.

- NO-SAB and NO-SAB defatted:

These nitrosylated proteins were recognized only by mice infested with the T. b. gambiense. The means and standard deviations obtained are 0.25 ± 0.029 (NO-SAB) and 1.1 ± 0.006 (NO-SAB defatted).

- NO-Cvs-SAB and NO-Cvs-G-SAB:

A statistically significant immunological signal (p <0.0001) was found only in trypanosome mice. The means and standard deviations obtained are as follows: Group 1: 0.75 + 0.39 (NO-Cys-SAB); 0.87 ± 0.37

(NO-Cys-G-SAB);

Group 2: 0.60 ± 0.30 (NO-Cys-SAB); 0.30 ± 0.15 (NO-Cys-G-SAB);

Group 3: 0.90 ± 0.34 (NO-Cys-SAB); 1.10 ± 0.41 (NO-Cys-G-SAB);

- NO-Tyr-SAB, NO-Tyr-G-SAB and NO-Tryp-G-SAB: Also an important and statically significant immunological signal (p <0.0001) is observed on these conjugates in the serum of trypanosome mice, and absent in the serum of the control mice: Group 1: 0.72 ± 0.53 (NO-Tyr-SAB) 0.73 ± 0.27 (NO-Tyr-G-SAB); 0.60 ± 0.28 (NO-Tryp-G-SAB);

Group 2: 0.62 ± 0.30 (NO-Tyr-SAB) 0.60 ± 0.15 (NO-Tyr-G-SAB); 0.95 ± 0.33 (NO-Tryp-G-SAB);

Group 3: 0.50 ± 0.26 (NO-Tyr-SAB) 0.93 ± 0.43 (NO-Tyr-G-SAB); 0.71 ± 0.22 (NO-Tryp-G-SAB);

For the control mice the means and the standard deviations obtained on the various conjugates are presented in table 4 below.

Table 4

3) Discussion. The results obtained show:

- A difference in immunological signals between the control sera and those of parasitized mice.

- Differences between the groups of parasitized mice as well as between the different epitopes used. This could be due to the type of coupling which plays a role in the presentation of the nitrosylated epitope coupled to a protein by a peptide bond (carbodiimide) or by a bond of type "primary amine"(G); - These data confirm the implication of NO in these parasitoses, which can be summarized as the result of an activation of macrophages in the presence of parasites followed by an expression of iNOS which releases NO in large quantities for hours, even days. NO will therefore exert its cytostatic or cytotoxic effect on parasites by binding to their protein targets and / or at the level of the cells of the self, thus forming different nitrosyl neoepitopes. These antigens will stimulate the immune system which will produce antibodies against these nitrosyl epitopes. After obtaining these results in animals and because of the possible role of NO in the cytostatic and cytotoxic effect on the intra and extracellular parasites, the inventors studied in patients suffering from different parasitic pathologies (trypanosomiasis, toxoplasmosis, amebiasis, hydatidosis , malaria) the evolution of immunological signals against nitrosyl epitopes. The results obtained showed a significant difference between the sera of patients with trypanosomiasis or malaria and those of other patients and controls. For the former, the best immunological signals were obtained on the NO-Tyr-G and NO-Tryp-G epitopes. On the other hand, for the others, the immunological signals were very weak even on these two epitopes. The tests were carried out on 20 sera from each pathology.

C - Protective role of monoclonal antibodies in experimental autoimmune encephalitis and inflammatory arthritis

1) Experimental autoimmune encephalitis. The objective of the experimental autoimmune encephalitis (EAE) model is to reproduce the demyelination observed in multiple sclerosis (MS). It is a demyelinating pathology of the central nervous system (CNS) affecting the white matter. Myelin, the protective sheath of nerve fibers, is the target of the pathological process. The degradation of this is responsible for the appearance of plaques. The origin of MS is certainly multifactorial: genetics, environment, autoimmunity and emotional stress (Antel and Cashman, 1991; Poser, 1992; Talbot, 1995). However, the exact etiopathogenesis remains unknown. Several anomalies of the cellular and humoral mediated immune response have been described (Brochet and Orgogozo, 1987; Olsson, 1995). Assumptions have been made regarding the autoimmune responses that have been found during illness. They would cause an increase in the production of NO by macrophages / microglia, smooth muscle cells and / or the CNS endothelium. Two mechanisms in cell destruction due to NO have been proposed (Sherman et al., 1992):

- Direct cytotoxicity by NO.

- Lesions due to the formation of peroxynitrite from the superoxide anion and NO. Other studies have indicated the role of NO in MS (Offner at al., 1989; Villas et al., 1991; Koprowski at al., 1993; Van Dam et al., 1995; einberg et al., 1994) , and the presence of auto-Ab against the NOCys-G epitope in patients (Boullerne et al., 1995). To confirm all these data postulating the role of NO in MS and especially in EAE, the model was induced in Lewis rats by immunizing them with an encephalitogenic peptide of the myelin basic protein (PBM) supplemented with Mycobacterium tuberculosis in adjuvant. The objective of this approach is to show the involvement of NO in the creation of neoepitopes therefore in the symptomatology of the disease, by the blocking of these epitopes by our monoclonal Ab which can have a preventive role. In parallel, the evolution of immune responses against nitrosyl and nitro neoantigens in rat sera was studied.

a) Materials and methods. Models of EAE in the Lewis spleen have been proposed by different teams (Panitch and Ciccone, 1981; Feurer et al., 1985; Levine, 1986; Polman et al., 1988). According to these authors, this induced model has as essential advantage a high incidence of relapses and a remarkable predictability.

- Encephalitogenic antigen: The synthetic encephalitogenic peptide (Peninsula laboratories) with a molecular weight of 1730 daltons was administered to the animals, the sequence of which reproduces that of the fragment of the guinea pig PBM between amino acids 68-84, the chemical structure of which is as follows:

Tyr-Gly-Ser-Leu-Pro-Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn 69 75 80 83

- Animals. The work was done on Lewis rats,

(IFFA CREDO), genetically susceptible to EAE. The rats were divided into homogeneous batches of age between 6 to 7 weeks. Three groups of Lewis rats (average weight: 200 g) were formed. Each animal received subcutaneously in the plantar pads of each hind leg 0.05 ml of a complete Freund's adjuvant emulsion (ACP H37Ra, DIFCO) containing: 100 μg of PBM guinea pig peptide 68-84 in 0.05 ml of 0.9% NaCl, plus 0.05 ml of ACF supplemented with 1 mg Mycoba ct eri um tuberculosis (DIFCO).

"Witness" group: it consists of 7 rats. Each animal received only the emulsion indicated above.

"Aminoguanidine" group: it consists of 5 rats. This group received, in addition to the emulsion, and after 4 days, a subcutaneous injection of a specific inhibitor of iNOS which is aminoguanidine (25 g / kg), once a day for two days. successive (Reimers et al., 1994). The iNOS inhibitor was used to compare its activity to that of the anti-NO-Cys-G monoclonal antibody.

"Monoclonal Ac" group: it consists of 5 rats. Each spleen received in addition to the emulsion, and after 4 days, subcutaneously, 5 mg / kg of monoclonal Ac purified by precipitation with ammonium sulfate (Mrabet et al., 1991; Lagier et al. , 1992). Clinical assessment of rheumatological and neurological symptoms:

The animals are observed clinically every day after administration of the emulsion. The clinical tests were evaluated using criteria relating to:

. The appearance of arthritis which is characterized by the following score: 0 = normal legs; 1 = moderate edema; 2 = more advanced edema; 3 = inflammatory arthritis; 4 - inflammatory arthritis + difficulty walking; 5 = inflammatory arthritis + difficulty walking + ulceration; 6 = inflammatory and purulent arthritis + great difficulty in walking + ulceration; 7 = inflammatory, purulent, and bloody arthritis + great difficulty in walking + ulceration.

. The behavior and tone of each animal. The following clinical score was assigned: O - normal; 1 = hypotonia of the tail; 2 = paralysis; 3 - paraplegia; 4 = quadriplegia. A score equal to or greater than 2 is required to confirm the neurological handicap (increase in EAE). If this established score decreases, the animals are in remission phase. On the other hand, when this clinical score returns to a value at least equal to 2, the animals relapse (second flare).

- Immunoenzyme test:

Blood samples are taken once a week for 5 weeks after administration of the emulsion. The sera are tested at a final dilution of 1/500 under optimal test conditions. The revelation of the signals was done using rabbit anti-rat Ig antibodies labeled with peroxidase.

(Sigma) diluted to 1/5000. These sera have been tested on the following conjugates:. NO-Cys-G-SAB; Cys-G-SAB; and SAB-G: the last two will be used for the correction of the OD obtained on the nitrosylated conjugate. The choice of NO-Cys-G-SAB is based on the fact that the monoclonal antibody used to block clinical signs is directed against this epitope. The possibility of an in vivo appearance of a neoantigen with the same spatial conformation and identical behavior to NO-Cys-G-SAB has been investigated.

. Recent work has demonstrated the formation of nitrotyrosines in inflammatory sites (Kaur and Halliwell, 1994). To detect the presence of the immunological responses directed against these epitopes in the sera of rats, Nθ2-Tyr-SAB and conjugated nitrosotyrorine (NO-Tyr-SAB) were used. Tyr-SAB was used for the correction of the OD obtained on: N02-Tyr-SAB and NO-Tyr-SAB.

b) Results.

- Development of the EAE model in Lewis rats by administration of the guinea pig encephalitogenic peptide:

Each animal in the 3 groups in this study was explored clinically every day for 40 days.

. "Witness" group (PBM only):

Three days after immunization, there is the appearance of edema of the hind legs. A few days later the rheumatological signs progress to an ulceration. It is therefore an inflammatory arthritis with an average score of 5.

Around the tenth-twelfth day: appearance of the first neurological signs: hypotonia of the tail with difficulty in walking progressing to paralysis of the hind legs. This state is followed by a phase of remission and then relapse.

. Aminoguanidine group:

Three out of five rats developed the disease.

The same neurological signs and progressive arthritis as in the previous group were observed in these rats.

The other two rats only developed signs of arthritis. These results would indicate that Aminoguanidine at a concentration of 25 mg / kg is relatively ineffective.

. "Monoclonal Ac" group:

The main objective of this model is to highlight the role of NO in EAE. The monoclonal antibody of the invention developed against the NO-Cys-G epitope has shown a protective role in the development of the disease, and has shown that NO is involved in this animal model. The neutralization of the effect of NO by Acs during the onset of outbreaks is manifested by the following observations:

- complete absence of neurological signs: no tail hypotonia, difficulty walking or posterior paralysis; - lack of development of signs of arthritis

: there appears a moderate edema (score 1) at the immunization sites, without inflammation or ulceration.

The follow-up of this group showed an absence of disease in these animals even after 40 days. It is therefore not a simple delay in the onset of the disease but a real protection. The results of this work open up a new therapeutic path in MS. The detection of NO or certain epitopes induced during human disease will serve, for example, to understand the biological and pathological mechanisms that can trigger MS.

- Study of the evolution of Ab against nitrosyl and nitro neoepitopes in the serum of rats: Immunological signals directed against the NO-Cys-GSAB conjugates; NO-Tyr-SAB; N02 -Tyr-SAB were searched. FIGS. 23 to 25 represent the study of the evolution of the antibodies directed against the conjugates NO-Cys-G-SAB, NO-Tyr-SAB and NO2-Tyr-SAB, in the serum of the rats (EAE). Figures 23 to 25 respectively show the evolution of three types of immune responses in three groups "Control", "Aminoguanidine" and "Monoclonal Ab".

The tests were carried out twice and the results (OD) obtained are very similar between the rats of the same group with non significant differences. We therefore plotted all these curves after calculating the means and standard deviations of the ODs obtained in all the rats in the same group and on the same conjugate. . A group of witnesses" :

For this group of animals a correlation between the immune responses and the clinic over time is found. In addition, the immunological signal evolves identically for the three conjugates NO-Cys-G-SAB, NO-Tyr-SAB and NO2 -Tyr-SAB. The DO obtained by the ELISA test on these conjugates are not very large (of the order of 0.125) but they cannot be considered as background noise because they are indicative of the presence of circulating Ab, the rate of which increases at the time of pushes and on the contrary decreases at the time of remissions (figure 23).

. "Aminoguanidine" group:

The anti-NO-Tyr-SAB and anti-NO2-Tyr-SAB responses are higher than those obtained in the previous group (with OD between 0.25 and 0.50 from the third week after immunization. For the anti-NO-Cys-G-SAB response, the titer did not increase but remained almost stable between the 1st and 4th week (Figure 24). The 3 rats having developed some neurological signs and the other two showed very similar results.

. "Monoclonal Ac" group:

The immunological signals of this group are different from those of the first two groups. Figure -25 represents the evolution over time of the anti-NO-Cys-G, anti-NO-Tyr and anti-NO2-Tyr responses. N02-Tyr-SAB is the best recognized conjugate among the three (DO between 0.20 and 0.30 after the 3rd week). In addition, a difference in recognition between the two epitopes Tyr-nitrate and Tyr-ni trosylated, took place by these induced Ab. The anti-NO-Cys and anti-NO-Tyr signals remained stable between the 1st and 5th week (OD - 0.125) except for a slight increase (after 1 week) in anti-NO-Tyr, followed by a decrease.

These immunoenzymatic tests made it possible to study in rats developing or not developing EAE, the evolution of the antibody titers potentially induced in vitro after the appearance of neoantigens, nitrosyls or nitrates. Serum collection of rats every week was limited. The number of tests and conjugates used was also used. It should be noted that in other tests

ELISA performed an absence of discrimination of these Ab was noticed between the NO-Cys-SAB conjugate and the Cys-SAB conjugate (carbodiimide coupling). The ODs were very identical on these two conjugates, which implies a non-specific bond and that the presentation of NO-Cys at the end of a carbon chain (G coupling), probably gives this conjugate a conformation close to that of 1 ' epitope in vivo.

These results show that:. Nitrosyl derivatives (particularly

The NO-Cys or nitrated epitope are responsible for the development of EAE in Lewis rats. These derivatives have in vi vo spatial conformations identical to the NO-Cys-G-SAB conjugates; NO-Tyr-SAB; or N02-Tyr-SAB adsorbed on microtiter plates. This allows the Ab induced in vivo to show these artificial conjugates as the derivatives against which they are directed. monoclonal Ac has a more important protective role than that of aminoguanidine.

. These molecules exert their effects on the one hand by neutralizing the harmful neoepitopes and on the other hand stimulating the immune system. The latter therefore induces in vivo, antibodies directed against these neoantigens generated after immunization of the animals by an emulsion of "immunogenic" character. This may explain the permanent increase in certain immunological signals in groups of rats receiving aminoguanidine or monoclonal Ab.

2) Experimental inflammatory arthritis. The objective of this model is to reproduce the inflammatory and rheumatological aspects present in rheumatoid arthritis (RA). It is a common autoimmune disease that begins in the form of inflammatory oligoarthritis of the wrists or metacarpophalangeal joints (Bach, 1993). RA progresses by relapses occurring more or less frequent and interspersed with usually incomplete remissions. There are several promoting factors such as genetic, environmental, and immune factors (Brostoff et al., 1993).

NO has recently been discovered as an effector in this pathology (Stefanovic-Racic et al., 1993; Moilanen and Vapaatalo, 1995) and in other joint disorders (Stadler et al., 1991; Stef novic-Racic et al. ., 1992). Excess production can cause cytotoxic and cytostatic effects (Nussler and Billiard, 1993; Stefanovic-Racic et al., 1993; Anggard, 1994).

The presence in biological fluids of aromatic groups and amino acids nitrated by ONOO- can be a marker of cell destruction in vi vo (Kaur and Halliwell, 1994). In chronic inflammatory diseases such as RA (Halliwell et al., 1992), the excessive production of free oxygen radicals contributes to cell destruction. In addition, NO is known to participate in joint destruction (Farrell et al., 1992; McCartney-Francis and al., 1993; Stefanovic-Racic et al., 1993). Inhibition of NOS suppresses arthritis in mice (McCartney-Francis et al., 1993).

Recently, NO production has been indirectly measured as nitrites in the sera and synovial fluids of RA patients (Grabowski et al., 1996). High levels of nitrites in patients compared to healthy controls show the role of NO as a possible mediator in rheumatic diseases.

Taking into account the bibliographic data indicating the role of NO in inflammatory diseases, in particular rheumatoid arthritis with adjuvant (Ialenti et al., 1993; Oyanagui, 1994; Cannon et al., 1995), the inventors studied the model of adjuvant arthritis, in order to determine whether the monoclonal Ab directed against conjugated NO-Cys-G are capable of inhibiting the effect of NO in the induction of this pathology, and of investigating whether the epitopes or the derivatives responsible for development of RA will be related to those of EAE.

a) Materials and methods. - Adjuvant arthritis: All of the experiments are carried out on the Lewis rat strain. Each animal received subcutaneously in the pads of each hind paw 0.05 ml of an H37Ra ACF emulsion containing: 0.05 ml 0.9% NaCl, plus 0.05 ml supplemented ACF per 1 mg of Mycobacterium tuberculosis.

Homogeneous batches of rats were formed.

They were subject to the same conditions as those used for the EAE model. Four groups of 5 rats, 7 weeks old (average weight: 200 g) were formed.

. "Control" group: each spleen in this group received only the emulsion indicated above. . Non-immune serum group of "SNI" mice: each spleen received the emulsion, and after 4 days an injection subcutaneously, of 5 mg / kg of immunoglobulins (precipitated with ammonium sulphate) from normal mice. The injection was given once a day for two days.

. "Aminognanidine" group: each spleen received the emulsion, and after 4 days an injection, subcutaneously, of aminoguanidine (25 mg / kg) once a day for two days.

. "Monoclonal Ab" group: each spleen received in addition to the emulsion, and, after 4 days, 5 mg / kg of anti-NO-Cys monoclonal Ac (precipitated with ammonium sulphate) subcutaneously.

Clinical evaluation of arthritis symptoms:

Particular attention was paid to detect arthritic joint damage. The inflammatory condition of the legs was assessed as follows: edema of the legs (not interfering with walking); big paw; edema of the leg hindering walking or "inflammatory" leg.

Based on the arthritis score already mentioned above, it was possible to follow in rats of these 4 groups the evolution of clinical signs over time for 40 days after immunization.

- Enzyme-linked immunosorbent assay: The inventors searched the rat serum for the presence of Ab directed against nitrosyl or nitrated epitopes which may appear in vivo during the development of this model. We therefore tested the presence of immunological signals on the following Nθ2-Tyr-SAB conjugates; NO-Tyr-SAB; Tyr-SAB; NO-Cys-SAB; Cys-SAB and SAB-G for the same reasons cited in the previous model. The conditions for the ELISA test are also the same.

b) Results. - Comparative study of clinical results:

"Control" group and "SNI" group: The rats in these two groups developed the typical signs of joint arthritis which results in: an appearance of edema in the plantar pads of the hind legs two days after immunization ;

. these rheumatological signs have evolved over time, towards large legs with purulent ulceration;

. after about 10 days, difficulty walking;

. some rats showed joint deformities in the hind legs.

In conclusion, these two groups show no difference. The clinical scores in most of these ten rats increased to scores 5, 6 and 7. The serum of non-immune mice injected 4 days after administration of the antigen, had no effect on the aggravation disease or protective effect.

"Aminoguanidine" group: Three rats developed edema without difficulty walking, then moved to scores 4 and 5. The other two rats reached scores 6 and 7.

"Monoclonal Ab" group: the results obtained with these rats can be described as follows:

. appearance at the end of the first week only of moderate edema in all the rats; . abs enc e of the evolution to the east of ulceration; . no difficulty walking: the rats kept their ability to lean normally on the hind legs; a single spleen presented an inflammatory stage without difficulty in walking, or a presence of bloody or purulent arthritis which was observed in the "Control" group and the "SNI" group.

In conclusion, NO or one of its derivatives which may be NO-Cys, peroxynitrite, nitrotyrosine or other oxygenated reagents have a role in the development of the model of articular polyarthritis and therefore in inflammatory processes.

- Study of the evolution of the Ac developed against nitrosyl and nitro neoititopes in the serum of rats:

FIGS. 26 to 29 report the study of the evolution of the antibodies directed against the conjugates N0-Cys-G-SAB, NO-Tyr-SAB and Nθ2 ~ Tyr-SAB in the sera of rats (PR), FIG. 26 for the "Control" group, FIG. 27 for the "SNI" group, FIG. 28 for the "Aminoguanidine" group and FIG. 29 for the "Monoclonal antibody" group. Each point represents the mean of the standard deviation of OD obtained in all the rats.

. "Witness" and "SNI" groups:

The sera of the ten rats collected during 5 weeks were tested on the NO-Cys-G-SAB conjugates; NO-Tyr-SAB; NO2 Tyr-SAB, and on the corresponding non-nitrosyl conjugates. Figures 26 and 27 show in the "Control" and "SNI" groups, respectively, the evolution over time of the antibodies induced against two epitopes: NO2-Tyr-SAB and NO-Tyr-SAB. These results represent the average of two tests. The ODs obtained in each group are equivalent on all the conjugates tested, each point represents the mean and the standard deviation of the OD obtained with the 5 rats of the same group for the same conjugate.

For these two conjugates the OD obtained vary between 1 and 2 between the 2nd and 5th weeks. For the NO-Cys-G-SAB conjugate the signals are very weak (OD ~ 0.1).

. The "Aminoguanidine and Monoclonal Ab" groups:

The immunological signals revealed in the rats of these two groups are equivalent between the rats of the same group and on all the conjugates tested.

The curves represent the average of two tests and the results are analyzed identically to the previous groups. As in the other groups, for the NO-Cys-G-SAB conjugate the signals remain very weak.

For the "Aminoguanidine" group: (FIG. 28) the anti-NO-Tyr is the highest response, the ODs are between 1.5 and 2 (between the 2nd and 5th weeks). The anti-NO2-Tyr response is also important, there is an increase in signals between the 1st and 2nd week. They stabilize until the 3rd week and then increase slightly around the 4th week.

For the “Monoclonal Ab” group (FIGS. 29): it is noted that in these animals also the anti-NO-Tyr response is greater than that of anti-NO2-Tyr. But, we also note that a plateau was found for the two types of Ac and this between the 3rd and 5th weeks. This may be due to a cross-reactivity between these Acs since the two responses evolved and then stabilized at the same periods. In addition, the level of anti-NO-Tyr and anti-NO2-Tyr in the sera of these rats are slightly lower than those obtained in the rats of the "SNI" and "Aminoguanidine" groups. On the other hand, the curve representing the anti-NO-Tyr shows the same appearance and the same amplitude as that of the "Control" group. Due to the importance of the anti-NO-Tyr and anti-NO2-Tyr responses, other tests for anti-NO-Tyr-G-SAB Ab were carried out. Thus, ODs obtained under the same ELISA test conditions were almost negligible in the four groups. These results mean that the presentation of the epitope is essential. A difference in the spatial conformation between the different nitrosyl epitopes is surely present in vi vo. It is essential to induce an immune response.

In conclusion, these immunoenzymatic tests revealed no significant difference between the four groups. An increase in the titer of antibodies induced against nitrosyl and nitro epitopes was found in each sample. For the “monoclonal Ab” group, effective but partial protection of the rats was observed, on the other hand, the immunological responses obtained in the animals of this group were identical to those of the other three groups.

c) Discussion.

The production of circulating antibodies was found in animal serum by the ELISA test in both models. For EAE, recent work has demonstrated the presence of anti-NO-Cys-G antibodies in the serum of rats which have developed this pathology (Boullerne, 1996, University thesis). The above work confirms the involvement of the neoepitope (NO-Cys), among other factors triggering neurological signs, in this experimental disease and therefore the presence in vivo of antibodies recognizing this epitope. The use of monoclonal Ab directed against this same epitope is therefore essential to capture the latter.

For RA, no work has demonstrated the involvement of the NO-Cys epitope in the development of this disease. But cysteine (Stamler et al., 1992b) is a target of nitrosylation with the formation of thionitrites. In addition, nitration of tyrosine with 0N00 ^" for example (Beckman et al., 1994b; Van Der Vliet et al., 1994) gives nitrotyrosine which has been demonstrated in inflammatory processes. Transnitrosylation phenomena can also take place in vivo, between different molecules carrying thiol residues accessible to NO (Scharfstein et al., 1994) which gives rise to several nitrosyle products. In the adjuvanted PR model, NO and the superoxide anion are considered as factors of induction and development of this pathology (Ialenti et al., 1993; Oyanagui, 1994; Cannon et al., 1995) .This experimental model was induced in rats injected with mycobacterium in the presence or not of aminoguanidine or monoclonal Ab directed against an NO-conjugated epitope in order to compare their protective role.

The protection of rats by Ac against the development of disease in EAE rats is 100%. Monoclonal Ab has a less important inhibitory role in the PR model, suggesting the intervention of factors other than NO-Cys in triggering the disease. It is also worth noting the good tolerance of mouse monoclonal Ab by rats, due to the short immunological distance between mouse and rat.

Furthermore, the immunological responses found in the serum of all animals against the NO-Cys-G-SAB conjugates; NO-Tyr-G-SAB and NO2-Tyr-SAB allow to monitor and compare not only clinical signs, but also the appearance and increase of these signals.

The monoclonal Ab of the invention made it possible to demonstrate the role of NO in the development of EAE where the results obtained were very satisfactory. On the other hand, the use of these Ac in rats developing adjuvant RA is less effective, which limits the use of these monoclonal Ab to the epitope against which they are directed.

VI - CONCLUSIONS.

The possibility of obtaining the induction of a specific immune response directed against nitrosyl epitopes, by using synthesized immunugens, has led to research the possibility of this induction under "natural" conditions. The presence of Ab directed against nitrosyle epitopes in parasitic affections where on the one hand the quantities of Ab produced are considerable and on the other hand the implication of nitrogen monoxide was highlighted, was sought . In these conditions, there is indeed an increase in the level of IFN-γ, the demonstration of different roles of NO and of a production of nitrites by peritoneal macrophages. In the case of work showing in vi tro the cytotoxic role of NO on intracellular parasites such as Leishmania, activated macrophages have been identified as cytotoxic towards these parasites (Murray et al., 1983; Hall and Titus , 1995). More recently, nitrogen oxides, and in particular NO released by activated macrophages, have been considered essential products for their intracellular cytotoxicity against Leishmanias (Liew et al., 1990; Reiner and Locksley, 1992). In humans Vouldoukis et al. have also demonstrated this effect in the presence of activated human monocytes (1995). They showed that the ligation of CD23 by the IgE-anti-IgE complex, or by the anti-CD23 monoclonal antibody induces the synthesis of NO. In addition, there was a generation of different cytokines by human monocytes / macrophages. Thus, ligation (IgE-anti-IgE) / CD23 induces a cytotoxic effect with respect to intracellular parasites: Leishmania major in human macrophages via the induction of the pathway. L-arginine / NO. This has been demonstrated by an increase in NO2-, and by the blocking of this effect by NMMA. Also the activation of these human macrophages by IFN ^Λ y / TNFa induces the same leishmanicidal activity inhibited by an anti-TNFa.

On the other hand, the inventors have shown the cytostatic effect of human monocytes against extracellular parasites (T. gambiense). In the presence of monoclonal Ac (anti-NO-Cys-G) or NMMA this trypanostatic effect was inhibited, indicating the role of

NO. All these results show the possibility of activating human monocytes to induce a cytostatic and / or cytotoxic effect on intracellular or extracellular microorganisms. In vivo studies have also shown the role of NO in the defense of the host against leishmaniasis. Injection of NMMA into mice resistant to this parasitosis inhibits the endogenous production of NO and leads to exacerbation of the lesions (Evans et al., 1993). More recently, it has been shown that a mutation in the iNOS gene in resistant mice makes them susceptible (Wei et al., 1995). During trypanosomiasis, there is a "Thl" type response directed against parasitic antigens (Schleifer et al., 1993). The presence of parasitic products triggering the production of IFN-γ, TNF-α, and iNOS leads to an overproduction of NO (Olsson et al., 1991; Sternberg and Mabott, 1996). In addition to its anti-parasitic role, the NO produced participates in the immunosuppression observed during trypanosomiasis.

The chronology and place of production of NO can be decisive in the overall effect linked to NO observed in trypanosomiasis. In mice (C57 BL / 6) resistant to Plasmodium chabaudi AS, there is an early expression of iNOS in the spleen, correlated with resistance to the disease. In susceptible mice, there is late expression of iNOS at liver level Uacobs et al., 1995). The same mouse strains are "resistant" (C57 BL / 6) and susceptible (BALB / c) to Leishmania major (Evans et al., 1993; ei et al, 1995) and to T.-musculi (Albright and Albright, nineteen eighty one). Protection against Leishmania major is correlated with NO production.

In trypanosomiasis caused by trypanosomes of the bru cei group, very marked immunosuppression is observed. Inhibition of NO synthesis prolongs the survival of trypanosome animals (Sternberg et al, 1994). The cytostatic and / or cytotoxic role of NO produced by macrophages on T. m u s cu l i was thus highlighted. Using different nitrosyl conjugates, it was possible to detect Ab in the serum of trypanosome mice against several nitrosyl epitopes. The presence of these Ac deserves to be emphasized for several reasons. First of all, they appear naturally during an Thl type immune response which leads to the production of NO, the binding of which to the antigens causes conformational changes recognized by the immune system.

In different parasitic diseases the "induced" Ab have a role of "marker" of the parasitosis, and little effectiveness against the pathogenic agent; in some cases, they mask the parasites or block the action of other ACs, which are more effective than them. The characterization of these Ac is therefore interesting. Also, the highlighting of these Ac at the different stages of a disease, can provide information on the successive types of immune responses involved. Thus, in certain affections, protection is ensured by the success of a response type "Thl "and a" Th2 "response. The appearance of antigens modified by NO and its derivatives could trigger the induction of a protective immune response? Whatever their role, the detection of nitrosyl antigens can be very interesting. Polyclonal and monoclonal Ab can greatly assist in this detection and are of capital interest. Thus, antinitrotyrosine Ab allow the detection of nitrotyrosine, the appearance of which is linked to the presence of peroxynitrite (Van Der Vliet et al., 1994; Ischiropoulos and Al-Mehdi, 1995; Crow, 1996). This Ac therefore makes it possible to know the situations where peroxynitrite is synthesized and to detect the targets of the affection. Nitrotyrosine has been visualized using immunological techniques at the level of the coronary vessels during atherosclerosis (Beckman et al., 1994a). In addition, in RA patients, it has been detected in synovial fluids (Farrell et al., 1992). But, despite a correlation between nitration and pathological evolution, it remains to be investigated whether the nitration of tyrosines at the protein level causes the primary lesions which trigger inflammation, or whether it is secondary to inflammatory processes. In fact, peroxynitrite reacts in a complex way with the different biological molecules resulting in hydroxylation, DNA fragmentation, oxidation of the iron-sulfur centers of proteins, etc. and especially the formation of nitrotyrosine which constitutes the most striking trace of peroxynitrite. This nitrotyrosine thus formed, induces tissue damage via several mechanisms (Beckman et al., 1994a): 1) alteration of the tyrosine phosphorylation; 2) alteration of protein functions by introduction of negative charges on the hydrophobic sites of tyrosine, which could alter the protein conformation; 3) initiation of autoimmune processes by the presence of nitrophenols such as nitrotyrosine, recognized as very antigenic (Kofler et al., 1992; Maeji et al., 1992; Mizutani et al., 1995; Yuhasz et al., 1995). From these data showing the role of nitrotyrosine in inflammatory and destructive processes, it is possible to deduce that the Ab directed against the various elements derived from NO can also allow their characterization and the knowledge of their properties. For example, the well-known role of thiols, S-nitrosothiols and Snitrosoalbumin in vasodilation in vivo and in vi tro (Stamler et al., 1992c, d; Keaney et al., 1993; Simon et al., 1993). Thanks to the anti-NO-Cyε Ab, an immune function carried by albumin has been established. Let us recall here that NO-SAB can serve as a reservoir of NO in plasma, from which NO can be transported to the intracellular medium by a transnitrosylation mechanism. After its transport, NO has access to its intracellular targets such as guanylate cyclase or hemoproteins, which results in physiological activity which can become pathological in the event of alteration in transnitrosylation.

The NOSAB form formed from a biological source (macrophages) was widely used, but, initially, other nitrosylated molecules synthesized at acid pH from an NO donor were used. This made it possible to develop poly- and monoclonal Abs. Secondly, the presence of nitrosylated molecules was detected using these Acs. These nitrosyl derivatives were synthesized in co-cultures: activated macrophages / SAB or activated macrophages / SAB / parasites. The detection was carried out not only by the immunoenzymatic technique but also by the revelation of the cytotoxic role of NO on extracellular parasites. This NO effect is amplified during the presence of the carrier protein, SAB, and has been neutralized in the presence of the Antibodies of the invention. Activated macrophages produce NO, but can also produce other oxygenated derivatives such as HOCl (De Violet et al. , 1984; Vincendeau et al. 1989), H2O2 (Vincendeau et al., 1981), and above 02 ° ^~ The question is whether the presence of this anion is necessary for the formation of albumin nitrosylated. 02 ^~ ° or another oxygen derivative (H2O2,

HOCl) may be the element reacting with NO. It is known in fact that murine neutrophils and macrophages simultaneously produce NO and H2O2 as well as other reactive oxygen species which participate in cytotoxicity (Pacelli et al., 1995).

It is in fact known that the nitrogen and oxygen derivatives can also interact to form derivatives having different functions. The neutralization of these functions by a selective blocking of such or such molecule can prove to be very instructive. In addition, it should be noted that, when using a chemical NO donor (NaNO2), an acidic pH was necessary to carry out the nitrosylation. On the other hand, in the culture medium the pH is neutral. We can answer this question taking into account the data of

Kharitonov which shows that nitrosylation can take place in vitro, in an oxygenated medium and at pH 7 through N2O3 (Kharitonov et al., 1995), even if this nitrosylation is less important than that of Cys or Glutathione, for example. However, to answer this question, it will be necessary to use scavengers of oxygenated derivatives such as SOD, catalase, taurine, which will make it possible to study nitrosylation in vitro, at neutral pH.

The nitrosylation of SAB in vitro and the corresponding cytotoxic effect, prompt us to clarify an important point concerning the experiments carried out under slightly different conditions:

In the first conditions, the trypanosomes and the SAB were incubated together. The activated macrophages will produce NO, but also many other free radicals. Thus, oxygenated derivatives, toxic or not, will be formed. This allows us to postulate that, during incubation, these derivatives could act "in competition" with NO on the trypanosomes to mask and / or interfere with its toxic effect. - Hence the interest of the second experimental conditions where the SAB was placed in the presence of macrophages activated to be nitrosylated. Then, the NOSAB formed was transferred to normal parasite / macrophage co-culture media which produce little or no free radicals. Under these conditions, we can admit that the effect of NO-SAB added in these cultures, poor in free radicals, is due to NO and not to other oxygenated derivatives.

The results obtained during these experiments were confirmed using an anti-SAB Ab. When using this antiserum, the trypanocidal effect of NO-SAB persisted in the first and second conditions. This indicates that the neutralizing effect, observed in the presence of Ac "T" or "C", is specific to the latter despite their polyclonal character.

It should also be added that the second conditions have also made it possible to show the absence of a "stimulating" or "inhibiting" effect on the immune complexes which could be formed at the macrophage level. Indeed, the use of normal macrophages (not expressing iNOS), in the presence of polyclonal Ab, trypanosomes, and the supernatant containing nitrosylated SAB, has shown results equivalent to those obtained under the first conditions. This shows that whatever the influence of immune complexes on the activated macrophages, the production of NO from these cells (expressing iNOS) has not been modified. Thus, we deduce that according to these two conditions, the cytotoxic effect of NO on T. us culi is particularly due to the formation of NO-SAB. The latter therefore exerted this effect either by the release of NO which is fixed on its targets, or by an exchange mechanism NO between SAB and amino acids or parasitic proteins.

The demonstration of the formation of nitrosyl derivatives in vitro by immunoenzymatic tests, or by the cytotoxic effect has led to the search for NO binding sites at the level of these parasites. This research began with the use of the immunocytochemical technique:

- The best results were obtained with activated macrophages. The poly and monoclonal Ab served as tools for marking the NO fixation sites at the level of the parasites. These results were very reproducible, which was not the case when using NaNO2 or NO-SAB synthesized chemically. With these molecules, the half-life of trypanosomes did not exceed 10 min and their shape was not preserved. We explained this fact by the presence of excess NO released by these chemical donors, which had an intense action on the parasites resulting in their lysis.

The use of controls gave weak markings or a complete lack of immunoreactivity. This confirmed the positivity of the markings obtained in the presence of activated macrophages (without NMMA) with the poly and monoclonal Ab used as primary Ab.

These specific Abs of nitrosyl epitopes, have shown the presence of at least cysteine residues and nitrosyl tyrosines. These have been recognized by their corresponding Ac. This immunoreactivity could be explained either by the binding of nitrosylated SAB to the parasitic surface, or by mechanisms of transnitrosylation between this molecule and the parasitic antigens carrying a thiol residue. Their nitrosylation could therefore be at the origin of the cytostatic effect mediated by NO-SAB. Indeed, numerous studies have highlighted the role of NO in the modulation of the activity of certain proteins by fixing themselves on their thiol residue (Stamler et al., 1994) which allows the regulation of certain cellular mechanisms. Among these we cite soluble guanylate cyclase (Stamler et al., 1992a); the NMDA receptor (Lipton et al. 1993); protein kinase C (Gopalakrishna et al., 1994>; p21raS (Lander et al., 1995); GADPH (Mohr et al., 1996) and the transcription factor OxyR (Hausladen et al., 1996). Transnitrosylation of NO from SAB to thiols in biological molecules, depends on the reactivity of these molecules, that of SAB as well as the stability of S-nitrosothiol formed (Scharfstein et al., 1994). these nitrosylated molecules detected at the level of the trypanosomes, immunoblotting experiments using NO gas or NaNO2 for nitrosylation of parasitic proteins are possible These experiments allow the detection of protein sites having been accessible to the phenomenon of nitrosylation in vitro. are very preliminary and not very satisfactory, since nitrosylation under well-defined conditions could lead to better accessibility to the corresponding Abs. tones that this hypothesis has already been cited for nitro derivatives

(Crow, 1996). Optimization of this protocol can be achieved by using activated macrophages as NO donors. Mechanisms intervening during the parasite / activated cell contact, aiding in nitrosylation and in the structural modification of protein targets can be envisaged. After nitrosylation from NO chemical donors, proteins may be better recognized by Ab than when chemically nitrosylated. It is important to note here that a significant difference between the nitrosylation produced by enzymatically synthesized NO and that performed by chemical NO donors has been demonstrated (Ignarro et al., 1993). Finally, there are probably other nitrosylation sites on parasites, other amino acids (lysine, tryptophan, etc.), iron-sulfur centers of certain enzymes, etc.

After this work in vi tro, the pathological role of NO was studied in vivo. NO has been identified in many pathologies (Bagasra et al., 1995; Moilanen and Vapaatalo, 1995; Grabowski et al., 1996). For example, in the case of septic shock, the expression of iNOS in the vascular system results in vasodilation, a decrease in tricatory vasocon activity, hypotension, and tissue destruction (Petros et al., 1994) . However, these changes in the vascular system are not strictly due to free NO. Numerous studies in vivo and in vi tro have shown that S-nitrosylated derivatives are vasodilator compounds, inhibitors of platelet aggregation, and regulators of blood flow and blood pressure. The most studied are S-nitrosocysteines, S-nitrosoalbumin, and even hemoglobin-S-nitrosylated (Stamler et al., 1992b, c, d; Keaney et al., 1993; Jia et al., 1996). These molecules, characterized by a longer half-life than that of NO (Stamler et al., 1992c), will participate in the aggravation and the evolution of pathological processes in septic shock. These are probably due to the increased production of cGMP from guanylate cyclase, and to the NO exchange phenomenon between protein thiols and low molecular weight thiols. This induces a cascade of events prolonging the harmful effect of NO, especially at the vascular level. It should also be recalled that in other pathological conditions the excessive production of NO in vivo can cause various neurological disorders. For example, studies have established that certain neuronal populations are resistant to the toxic effects induced by the release of excitatory amino acids such as glutamate, while they produce toxic amounts of NO (Beal et al., 1986; Koh et al., 1986; Koh and Choi, 1988). These neurons also seem to be preserved in pathologies such as Alzheimer's disease or Huntington's chorea where the role of NO has been highlighted (Ferrante et al., 1985; Meldrum and Garthwaite, 1990; Olney, 1991) . More recently, NO has been identified as an effector of neurological disorders observed in patients with acquired immunodeficiency syndrome (Bukrinsky et al., 1995; Lipton and Gendelman, 1995). NO is considered necessary but not sufficient to induce cell death. It can react with other free radicals such as superoxides, giving rise to the formation of peroxynitrite, a particularly toxic chemical species (Beckman et al., 1990; Radi et al., 1991).

NO is also one of the mediators involved in different "autoimmune" diseases. Thus, for example, it would participate in the destruction of the β cells of the islets of Langerhans and would cause the appearance of diabetes (elsh et al., 1994; Lindsay et al., 1995; McDaniel et al., 1996). In addition, synthesized NO can bind to amino acids (especially cysteine and tyrosine), causing nitrosylation and modification of proteins or enzymes at the tissue level. These self proteins can therefore become, under pathological conditions, molecules foreign to the organism inducing in humans autoantibodies, as in the case of multiple sclerosis and inflammatory polyarthritis. The nitrosylation of antigens and the appearance of corresponding Ac would be a natural phenomenon, possibly increased by an overproduction of NO. The role of these antigenic nitrosyl derivatives could be the destruction of the target cells which carry them. Thus, the nitrosylation of microbial antigens could be accompanied by those of self antigens. Ac produced in addition to their various roles on exogenous antigens, could also act on the self. According to quantity produced, the biological properties linked to the isotype of Ac, and its affinity, various results could be observed: destruction of cells, masking of epitopes resulting in protection, etc. From an experimental point of view, the NO intervention in the inflammatory and immune pathologies induced in the Lewis spleen is now well established. Several authors have reported by different approaches that NO could have an important role in these degenerative processes, one affecting the central myelin, the other the joint and its synovium.

Two animal models have been developed in the context of the invention. The first which mimics the neurological evolution of MS and the second which consists of an adjuvant inflammatory arthritis model. The role of NO and nitrates in inflammatory processes and tissue destruction has been demonstrated (Halliwell et al., 1992; Winyard et al., 1992; Morris et al., 1995). Next, circulating Acs of early onset after induction of the experimental models were highlighted. These immunoglobulins recognize three antigenic targets carrying NO or N02 NO-Cys-G, NO-Tyr and Nθ2 ~ Tyr. Their presence could sign an antigenic stimulation linked to the hyperproduction of NO. On the other hand, the analysis of the pathophysiological, clinical and immunological processes confirms an increase in the production of NO. This could be mainly localized at the level of lesions and probably in connection with macrophagic stimulation. But endogenous targets: Are Cys and Tyr potential NO transporters key elements in pathophysiology? The therapeutic effects of the anti-NO-Cys-G monoclonal Ac underline the potential role played by the NO-Cys epitope. In vivo recognition of this target leads to the abolition of EAE symptoms and considerably reduces inflammatory arthritis. The NO-Cys antigen therefore appears in these conditions as a key step leading to myelin destruction for EAE and synovial inflammation in arthritis.

The presence of Ab in the serum of experimental animals and the protective role of the injected Ab indicate that the neutralization of the NO-CysG epitope plays an important protective role. The absence of protection in animals not injected, despite the presence of Ab, indicates that (i) the Ab produced have too low an affinity, or are in insufficient quantity, or produced too late, (ii) The Ab produced do not have the same functions or may be of a different isotype than the injected Ab. The isotypic determination, the immunochemical study of the Ac produced, their purification and then their injection into experimental animals will help answer these questions.

Finally, the two animal models used have made it possible to obtain significant results. By taking EAE (experimental model of MS in humans), for which almost total protection has been noted, the results obtained constitute a therapeutic approach to MS.

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Claims

Claims

1) Purified antibody specifically recognizing a nitrosylated protein.

2) Antibody according to claim 1, characterized in that said protein is a NO transporter.

3) Antibody according to claims 1 or 2, specifically recognizing a nitrosylated albumin.

4) Antibody according to any one of claims 1 to 3, characterized in that it is a polyclonal antibody.

5) Antibody according to any one of claims 1 to 3, characterized in that it is a monoclonal antibody.

6) set of antibodies according to claim 4 or 5

7) Immunogen for the preparation of antibodies according to any one of the preceding claims, characterized in that it consists of a nitrosylated carrier protein whose sequence has a nitrosylation site, or by a nitrosylated amino acid coupled to a protein carrier.

8) Immunogen according to claim 7, characterized in that the nitrosylation site or the amino acid is chosen from tyrosine, optionally acetylated cysteine or tryptophan. 9) Immunogen according to one of claims 7 or 8, characterized in that the carrier protein is an albumin.

10) Process for preparing an immunogen according to any one of claims 8 to 9, characterized in that an amino acid is coupled to a carrier protein, then in that the conjugate obtained is nitrosylated with a compound NO donor.

11) Pharmaceutical composition, characterized in that it comprises, as active principle, an antibody according to any one of claims 1 to 5 or a set of antibodies according to claim 6 advantageously dispersed in a pharmaceutically acceptable vehicle or excipients.

12) Use of an antibody according to any one of claims 1 to 5 or of a set of antibodies according to claim 6 or of a composition according to claim 11, for the preparation of a medicament intended to treat or prevent a pathology in which NO, its derivatives or conjugates are involved.

13) Method for in vitro detection of nitrosylated proteins in a biological sample comprising at least the following steps:

- bringing this sample into contact with at least one antibody according to any one of claims 1 to 5 or a set of antibodies according to claim 6, optionally labeled, under conditions allowing the formation of immunological complexes; - the detection of an antigen-antibody immunological complex by physical or chemical methods. 14) kit for implementing a method according to claim 13, characterized in that it comprises: at least one antibody according to any one of claims 1 to 5 or a set of antibodies according to claim 6, possibly marked,

- reagents for the constitution of a medium suitable for the immunological reaction between said antibody and the nitrosylated proteins possibly present in a biological sample;

- optionally one or more detection reagents, optionally labeled, capable of reacting with the immunological complexes possibly formed;

- possibly one or more biological reference and control reagents.