FR2844511A1 - Therapeutic vaccinating complex, useful for treating or preventing diseases for which immunity requires Th1 lymphocytes, comprises a peptide from Leishmania and an adjuvant - Google Patents

Abstract

Therapeutic vaccinating peptide complex (A) for treating or preventing diseases in mammals where protective immunity depends on stimulation of Th1 lymphocytes, particularly delayed hypersensitivity, is new. Therapeutic vaccinating peptide complex (A) for treating or preventing diseases in mammals (preferably humans, dogs, cats and horses) where protective immunity depends on stimulation of Th1 lymphocytes, particularly delayed hypersensitivity. (A) comprises at least one of the peptides A16E (where Leu is optionally replaced by Ile, and Ser by Cys) and/or A16G (where Cys may be replaced by Ser or vice versa), and an adjuvant that preferentially induces a cell-mediated response. Ala-(Ala-Arg-Ser)2-Arg-Glu-Gly-Tyr-Ser-Leu-Thr-Asp-Glu (A16E) Ala-Ala-Ser-Ser-Thr-Pro-Ser-Pro-Gly-Ser-Gly-Cys-Glu-Val-Asp-Gly (A16G). An Independent claim is also included for a kit for in vitro diagnosis comprising (A) in which A16E/G or their derivatives are bound to large molecules of the biotin or polylysine types to increase antigenicity, linked through glutaraldehyde, attached to a solid carrier, or attached to nitrocellulose, or other polymeric, membranes, latex or other polymeric materials.

Description

Peptide compounds for prevention and treatment

of affections at

the mammals.

  Peptide compounds intended for the prevention and the treatment of affections in mammals and in particular in humans, canids, felines and equines whose protective immunity depends on the stimulation of T lymphocytes of

  Thl type and in particular of a delayed type hypersensitivity state.

  Peptide compounds also intended for the diagnosis of cell-mediated immunity dependent on Thl-type T cells using various

  "in vivo" and "in vitro" methods.

  During the immune response, the T cell is an important source of cytokines. Their production is induced after stimulation in a specific manner in the presence of antigens or non-specifically in the presence of mitogens

  (concanavilin A, phytohemagglutinin).

  If it is clearly established that T lymphocytes, and among them especially CD4 + lymphocytes, represent the main source of cytokines, the T subpopulations involved in this phenomenon are diverse and seem to vary depending on the stimulus.

  Immune responses can be very schematically divided into two broad, qualitatively distinct categories, humoral responses which involve the production of antibodies by B lymphocytes and cellular responses (delayed hypersensitivity reaction, cytotoxic reactions) for which effector cells are T lymphocytes. It appears that in the majority of experimental models and clinical situations studied, cytokines of lymphocytic origin are essentially produced by T helper CD4 + cells (or helper) whose role is to modulate or regulate humoral and cellular immunity and which recognize the antigen in association with the class II molecules of the major histocompatibility complex. A major concept emerged in 1985 when T. Mosmann and R.Coffman proposed that CD4 + T lymphocyte cells expressing helper functions were in fact heterogeneous. Thus, thanks to the study of mouse CD4 + T lymphocyte clones -2, cultivated in the long term, these authors described the existence of two major subpopulations which can be distinguished by their profile of cytokine secretion to know Th1 cells (for T helper's

  type 1) and Th2 cells (for T helper type 2).

  Take for example leishmaniasis, which is an endemic or even epidemic parasitic infection of tropical and subtropical regions of the world.

  Leishmania, flagellated protozoa of the family Trypanosomatidae and

  genus Leishmania, are the pathogens responsible for these diseases.

  Numerous studies on the immune responses during experimental murine leishmaniasis have led to the demonstration of the predominant role of cell-mediated immunity and the existence of a duality of the immunological response. There are basically two types of responses against leishmanias: one described as "sensitivity", the other as "resistance". It is through lymphokines that they secrete that the different subpopulations of T lymphocytes (CD4 +) limit or exacerbate the infection. It has thus been demonstrated that the subpopulation of helper T lymphocytes of the Thl type (producer of interferon gamma and interleukin 2) was capable of eliminating the intracellular amastigote forms through the activation of macrophages (Reiner SL et aI., Annu Rev Immunol, 1995, 13, 151-177. Review). Conversely, the Th2 helper T cell subpopulation (interleukin-4 producer) is responsible for the exacerbation of the disease. In humans, certain facts are of a comparable nature. In dogs (natural "reservoir" host receptive to the evolutionary cycle of L. infantum), the duality of the immunological response is likely. Only a study by Pinelli and aI. (Infect. Immun., 62: 229, 1994) on animals experimentally and 30 naturally infected with L. infantum, made it possible to show that the asymptomatism of the dog (clinical condition frequently encountered) was accompanied by the absence of a humoral response and the development of cell-mediated immunity of the Thl type with a positive delayed-type hypersensitivity reaction -3 and high levels of interleukin 2 and

  of TNF-cL circulating in body fluids.

  A good vaccine candidate must therefore correspond to one or more highly immunogenic parasitic antigens capable of either blocking the differentiation of Th2 lymphocytes (Gurunathan S et al., J. Exp Med, 1997 Oct 6, 186, 1137- 1147) (mode of intervention comparable to "desensitization" treatments, commonly practiced in allergy cases), that is to promote the emergence of Thl lymphocytes ensuring the establishment of protective immunity.

  Considering to vaccinate against leishmanias remains problematic today. There are many attempts, but the results are weak and / or contradictory. We can cite the use of live parasites, 15 irradiated parasites and whole killed parasites (Moreau Y et al, 1994, Médecine et Armées, 22, 1, 89-93) which have given varying levels of protection in

mice and humans.

  In the 1980s, purified extracts of parasitic antigens were used in dogs by inducing an exacerbation of the disease: LIF2 fraction and anti-idiotypic vaccine from the team of Dr Montjour. (CHAUVY, J "Immunotherapy trials on a canine population in a Leishmanian endemic area" thesis n036.1993-OGUNKOLADE BW et al. Vet Parasitol.), GP63 or lipophosphoglucane (MOREAU Y et al, Médecine et Armées, 1994, 22.1, 8925 93) did not give a satisfactory result. Currently, several molecules

  are on trial and awaiting final results. Let us cite the Leishmania major heat shock protein HSP83 which stimulates the Thl pathway and the DP72 protein (JAFFE.C et al, J of Immunol, 1990, 144, 699-706). However, none of the current immunization protocols provides a sufficient level of protection or is in any case reproducible.

  To date, no work has been done with synthetic peptides.

  The present invention consists of an immunomodulatory complex using one or more peptides with an adjuvant inducing either immunostimulation of the Thl type T lymphocyte system in a reproducible manner, or a

  immunomodulation of a Th2 type response towards that of Th1 type.

  It is important to highlight two essential facts. First, the Th1 and Th2 cells exert, through some of the cytokines they produce, a reciprocal counterregulation effect, which explains why in the majority of cases we observe an alternation of responses mediated by cell and humoral. Thus, the immune responses which predilege a reaction of 10 T cells of the Thl type, that is to say with organisms inducing immunity dependent on T lymphocytes, are frequently associated with a weak humoral response; this is the case for example with mycobacterial infections. Conversely, a response which favors the production of antibodies will most often be associated either with the participation of T lymphocytes of the Th2 type or with a relative deficit in specific cellular immunity; this is the case with visceral leishmaniasis. Second fundamental fact, lymphocytes fully differentiated into Thl and Th2 type cells do not preexist in the non-sensitized naf host. During a physiological immune response following which, in most cases, the antigens are rapidly eliminated, the CD4 + cells activated, rather than being expressed either by a Th1 type phenotype or by a Th2 type phenotype usually secrete specific Th1 and Th2 type cytokines. This explains why, in humans, the demonstration of the existence of a Th1 / Th2 dichotomy required the study of a cytokine network from lymphocytes not from normal normally immunized subjects but from patients with chronic diseases

  such as parasitic infections, allergies or autoimmune diseases.

  The polarization of immune responses to a Th1 or Th2 phenotype has been associated with many pathological situations.

  For leishmania, Trypanozoma, Candida, and other intracellular organisms such as Mycobacterium and Listeria infections, a Th1 response correlates with resistance to the pathogen. On the other hand, certain conditions such as canine atopic dermatitis, allergies and asthma lead the exacerbation of a Th2 response. The use of immunostimulants allowing the passage from a Th2 response to a Thl response, therefore the transition from a state of immediate hypersensitivity to a state of delayed type hypersensitivity should induce a cure.

  Let us take as an example the asthma for which the medical community worries in front of the increasing number of individuals who suffer from it: approximately 200 million people in the world and according to the WHO 180 000 died of it in 10 1997. In France 2000 people die from asthma every year.

  Asthma is a chronic inflammatory condition of the airways o

  many cells of the immune system are involved.

  The disease becomes chronic due to the almost permanent accumulation of 15 mediators of inflammation, as well as the almost incessant recruitment of destructive eosinophils: these cells accumulate in the bronchial mucosa and release basic proteins which destroy the epithelium. bronchial. The damage caused by these basic proteins leads to hyperactivity of the bronchi: the nerve endings which innervate the bronchi are exposed and are no longer protected from external aggressions. Let us recall that the activated T lymphocytes in particular release interleukin 4, which triggers the production of immunoglobulins E. This "allergic orientation" also called Th2 pathway, opposes the Th1 pathway anti-infectious reactions: in the context In the fight against a bacterium (the bacillus of tuberculosis, for example), activated T lymphocytes release interleukin 2 and gamma interferon. Interferon gamma activates the antigen presenting cells which, in turn, produce interleukin 12. Thus, interleukin 4 30 directs immune reactions towards allergic reactions, while

  gamma interferon appears to promote defense-like reactions.

  However, this orientation of the immune system would be imprinted very early, in very young children. In an infant exposed to a pathogen, the immune system turns towards the defensive pathway: it is the T lymphocytes which produce interleukin 2 and gamma interferon that are activated first.

  and who will remain so. These children will have fewer allergic risks.

  On the other hand, infants raised in a sanitized environment are at risk of being confronted with an environment rich in allergens and poor in pathogenic microorganisms.

  If this is the case, the still immature immune system will activate the T lymphocytes which secrete interleukin 4, giving the immune system 10 an allergic orientation, which promotes asthma. This hypothesis, stemming from the work of Tim Mosman and Robert Coffann, from the DNAX molecular and cellular biology institute, in Palo Alto, would explain the increase in the prevalence of

  asthmatic disease in industrialized countries.

  A reorientation of the immune reactions of sensitive people by limiting the Th2 allergic pathway of the immune response towards activation of the pathway

  Thl would be a possibility of asthma treatment.

  We can extend this phenomenon to other forms of immediate hypersensitivity.

  Immediate hypersensitivity is the most common form of allergy and results from the synthesis of immunoglobulins E (IgE), antibodies specific to environmental allergens. These immunoglobulins E bind to cells of which mast cells are the key elements. Contact of the allergen and immunoglobulins E activates the mast cells which release the mediators of inflammation: this is the degranulation of the mast cells. We group under the name of anaphylaxis and atopic diseases all the clinical manifestations linked to immediate hypersensitivity phenomena, which result from the production of immunoglobulins E, the main culprits of allergy. However, anaphylaxis corresponds to a pathophysiological mechanism independent of any hereditary factor (for example, anaphylactic shock due to venoms of hymenoptera), while atopy expresses a particular genetic ability to produce excess immunoglobulins E directed against various natural substances of the atmospheric environment (pollens, molds, pollutants), domestic (mites, cockroaches, mammals) or professional, and also against food; this results in respiratory allergies (asthma, rhinitis), skin (hives, atopic eczema),

ophthalmic and digestive.

  In these pathologies, two points must be mentioned 1- the cascading events triggered by the binding of the allergen-IgE complex on different cell populations and in particular on mast cells 2- the role of cytokines which, through the synthesis of IgE , 10 participate in the awareness phase, on the one hand, and in the sustainability of the

allergic reaction on the other hand.

  Today, the treatments that stem allergic syndromes are part of the anti-inflammatory arsenal (antihistamines and corticodes). The preponderant place taken by cytokines in the fundamental mechanisms of

  immediate hypersensitivity opens perspectives for new targeted therapies: we hope in particular to shift the Thl / Th2 balance in favor of the Th1 pathway and modulate the cellular signaling induced by cytokines when they bind to their receptors located on lymphocytes, but also on mast cells and on eosinophils.

  The present invention relates to specific derivatives of 2 peptides of the Leishmania parasite, as well as to the use of said fragments as reagents for in vitro and in vivo diagnostics, and as reagents inducing stimulation of Thl type T lymphocytes ensuring a inhibition of the development of Th2 type lymphocytes, in particular for the prevention and treatment of conditions linked to an immediate hypersensitivity state, therefore an immune state

of type Th2.

  The two peptides named A16E and A16G have the following amino acid sequences: -8 A16E: (16 amino acids):

A-A-R-C-A-R-C-R-E-G-Y-S-L-T-D-E

A16G: (16 amino acids):

A-A-S-S-T-P-S-P-G-S-G-C-E-V-D-G

  In peptide A16E, C can be replaced by S, and L by I. In peptide A16G, C can be replaced by S, and S by C. The peptide compound according to the invention consists of possible peptide derivatives A16E and A16G, these derivatives comprising at least five contiguous amino acids taken from the sequences A16E, sequences identified in the appendix in SEQ ID NO 1 to 44, or A16G, sequences identified in the appendix in SEQ ID NO 45 to 88, and preferably in minus 8 contiguous amino acids, for example: 15 AARCARCR or

A-A-S-S-T-P-S-P

  The peptides obtained by synthesis and their derivatives are made immunogenic by binding to carriers (large molecules like KLH), and are administered to mammals in the presence of an adjuvant, preferably muramyl dipeptide.

(CDM).

  Preferably, the protein / adjuvant ratio is between 1 / 0.1 and 1/6. In the case of an infection with leishmanias, studies carried out in dogs have made it possible to determine the optimal vaccine dose at 100 μg of muramyl dipeptide per 50 gag of proteins injected. However, there is a beginning of

  response from 30 pg of protein injected.

  The specific mechanism of action of the peptide compound obtained according to the invention is verified using conventional methods allowing the assay of the peptides, their identification and using more specific methods demonstrating that the innovative peptide compound acts either by immunostimulation of the Thl-type lymphocyte system, either by immunomodulation from a Th2-type to a Th1-type.

  For each mammal studied (dog for example), a serological analysis is

  carried out with peptides A16E and / or A1 6G.

  A parasitological examination is carried out using a sample taken directly

  on the candidate studied, dog for example.

  A smear from the bone marrow puncture is performed on a slide. This 15 smear, once fixed with methanol is stained with May Gr mwald Giemsa and observed

  under an immersion microscope (x 1000).

  Bone marrow samples are cultured in the NNN biphasic culture medium (Novy and Mac Neal, 1904, 4. Infec. Dis., 1: 1-30) including 20 from RPMI 1640 supplemented with 20% calf serum decomplemented fetal constitutes the liquid phase. Blind transplants are performed every four to six days. Cultures are regularly observed under microscopy

photonics (x 400) for 20 min.

  Parasitemias are quantified as follows: +/-: elongated, refractive, immobile forms +: 1 to 5 mobile promastigote forms / fields ++:> 5 mobile promastigote forms / culture fields at confluence Evidence of the implication of immunity to Thl-type cell mediation: Leishmanias of promastigote forms are cultivated in standard culture media. Parasites at the end of the exponential phase (6-7 days) are collected. The parasitic pellet is washed three times by centrifugation (2500g, 15mn,

C) in PBS buffer.

  After checking the viability of the parasites using a vital dye (trypan blue), a suspension containing 2 x 108 parasites per ml is inactivated in PBS buffer containing 0.01% merthiolate (Pinelli et al., 1994, Infect. Immun. 62: 229-235). This constitutes the leishmanines for the intradermoreaction test

(I DR).

  The following Thl type immune response study is performed on dogs.

  Dogs are placed in the lateral decubitus position and a delicate and non-irritating clipping 15 is carried out on the thoracic zone of approximately 5 cm by 10 cm behind the elbow.

  Four circles of 1 Omm in diameter are delimited using a marker.

  0.1 ml of solution is injected into the center of the intradermoinjection solution. Two circles receive leishmanin solution and the other 2 circles receive merthiolated saline solution in negative control. The intra-dermo reaction (IDR) reading is made 48 hours later using an allergist slide. The test is considered positive if the average of two observed induration diameters is greater than or equal to 5 mm. The observation of an erythema without

  induration will be considered a negative test (Pinelli et al., 1994, Infect.

  Immun., 62: 229-335; Marty et al., 1994, trans. Roy. Soc. Too much. Med. Hyg., 88,

658-659).

  IDRs can also be carried out with a solution of A16E 30 or A16G peptides or their derivatives and of MDP, for example in proportions of

pg of peptides for 20 pg of CDM.

  The present invention therefore also relates to an in vivo diagnostic product demonstrating a state of delayed immune hypersensitivity of the Thl-il1 type by the use of peptides A16E and / or A16G and their derivatives intra-dermo

reaction in the mammal.

  Nitrogen monoxide (NO) can also be assayed for the destructive activity of Leishmania monocytes. The synthesis of NO by monocytes is indeed a sign of destruction of leishmanias by monocytes having been activated by cytokines of the gamma interferon type.

(I FNy).

  NO has a high chemical reactivity. In the presence of water and oxygen, this molecule is rapidly oxidized stochiometrically and thus forms nitrites (NO2-) according to the reaction: 4 NO + 02 + 2H20 - 4 NO2- + 4 H + Nitrites accumulate in media and are easily detectable

  chemically by the Griess method.

  To 50 μl of supernatant of monocyte cultures to be tested, 60 μl of Griess B (N- (1-Naphtyl) ethyl-enediamine 0.3%) is added. The colorimetric reaction develops away from light for 2 minutes. The optical densities obtained at 540 nm are corrected by the subtraction of the ODs obtained on the 20 wells containing the culture medium alone.

  The values obtained are plotted on a standard curve (DO = f (NO2-)

  made from known concentrations of NO2-.

  For this test, monocytes and lymphocytes are isolated from venous blood from dogs. The monocytes are cultured for 3 days at a rate of 105 cells per well in the culture chambers (Labtek) in a complete RPMI 1640 medium (containing 25 mM of HEPES; 2 mM of L-glutamine, 100 U of penicillin per mi ), at 37 C in a humid atmosphere containing 5% CO2. After 3 days of culture, the macrophages are washed in complete RPMI 30 medium, supplemented with fresh medium. The cells are incubated either alone, or in the presence of 5 g of peptides, or in the presence of autologous lymphocytes. - 12 When used, the lymphocytes cultured separately are washed, counted and added to the macrophages in the proportion of 2 lymphocytes per macrophage.

  A lymphocyte proliferation test is also performed.

  The peripheral blood mononuclear cells (PBMC) of the dogs are separated on a Ficoll gradient (density 1.078) by centrifugation at 800 g for 20 min at room temperature. These cells are cultured in a 96-well plate at a concentration of 2 × 10 5 cells per well in the presence of 2 μg per ml of Concanavalin A (Sigma), of 5 μg per ml of ES P (excretion - promastigote secretion) or 20 ml. culture supernatants harvested in the stationary phase of promastigote growth (SP) per well, and in the absence of any additive in a volume of 200 ml of RPMI 1640 medium containing 5% of decomplemented fetal calf serum, 2 mM of L- glutamine, 100 U of penicillin 15 per ml, 100 mg of streptomycin per ml. Optimal concentrations

  antigens and mitogens have been determined in previous experiments. PBMCs are incubated for 72 hours in a humid atmosphere at 370C in the presence of 5% CO 2 and then for 20 hours with 0.5 pCi of 3H thymidine. The cells are harvested on a filter and the incorporation of the radioactivity is determined by counting in scintillating liquid (counter y).

  All tests are carried out in triplicate.

  A faster and more sensitive immunohistochemical method using BrdU (5-bromo-2'-deoxyuridine), a structural analogue of thymidine is also used to measure cell proliferation (BrdU, cell proliferation detection kit 111, Boehringer Mannheim, Germany ). In our experiments BrdU is added for 18 hours after 72 hours of incubation. Cells which have incorporated BrdU into their DNA are easily detectable in the presence of a monoclonal antibody directed against BrdU. 30 The proliferative responses are expressed in stimulation indices which represent the ratio of the average proliferation after stimulation over the

  average proliferation in the absence of antigen.

  - 13 Lymphocyte proliferation was also estimated by visual readings under light microscopy (-: negative; +/-: slight proliferation; +: small proliferation in less than 5 points per microscopic field; ++: proliferation

  average in addition to 5 points; +++: strong proliferation).

  In parallel with the study of the specific activation of Thl type T lymphocytes by intradermoreaction, NO assay (NO is synthesized by monocytes activated by Thl type T cell cytokines) and lymphocyte proliferation, serological monitoring by immunofluorescence classic using 10 slides coated with promastigotes (serological reference method

  for canine leishmaniasis) is performed.

  For the conduct of the studies, other specific techniques were useful.

  Infectious test method: The infectious test consists of intravenously injecting 106 promastigotes in the metacyclic phase treated with a healthy dog supplement and 5.106

  healthy dog peritoneal macrophages, infected in vitro with amastigotes.

  The infected promastigotes and macrophages are diluted in sterile saline to a final volume of 1.5 ml. This mixing is done just

before injection.

  Detection of G2 type immunoglobulins (IgG2) from dogs, specific for the A16E and A16G peptides and their derivatives:

  This detection is carried out by ELISA method according to the microtitration technique of Kweider et al (J. mmunol, 1987, 138, 299) using an anti IgG2 conjugate.

  For this method, the peptides are biotinylated before coating on microplates.

  This binding to large biotin-type molecules has the particular effect of making the A16E or A16G peptides or their derivatives more antigenic.

  Peptides can also be linked by glutaraldehyde or associated

  to polylysine bodies (presentation of the OCTOPUS type for example).

  The innovative character of the peptide compound according to the invention does not only lie in the induction of a specific Th1 type cellular response but also in the production of low levels of IgG2 type antibodies. These IgG2s are detectable by various in vitro methods, for example: ELISA, DOT 5 BLOT, WESTERN BLOT, IMMUNOCHROMATOGRAPHY, LATEX and any other in vitro method involving a conjugate system or other systems for visualizing the Ag - Ac reaction. It can for example be used an ELISA system on a plastic support, and a WESTERN BLOT system on nitrocellulose membranes or other polymers involving an enzymatic conjugate. Latex carriers can also be used. The peptides A16E, A16G and their derivatives can also be conjugated for example to radioisotopes, fluorescent molecules, molecules

  luminescent or colored particles.

  Indeed, some preliminary work in humans (KAWANO.P et ai, Parasite Immunol, 1995, 17, 451-458) and in dogs (NIETO CG et ai, Vet Immunol and Immunopathology, 199, 67, 117-130 ) demonstrate that the IgG isotypes are markers of the Thl / Th2 immune dichotomy. More specifically, a dog suffering from leishmaniasis with convincing clinical signs has a high level of antibodies mainly of isotype IgG1 while an asymptomatic dog has specific antibodies of isotype IgG2. Dogs that have received our peptide compound have low levels of IgG2 specific for the A16E and / or A16G peptides, which is consistent with the preferential expansion of Thl-type T cells. 25 The detection of the presence of IgG2 specific for the peptides A1 6E and A16G and their derivatives makes it possible in particular: - to demonstrate a humoral response dependent on Thl lymphocytes and therefore to demonstrate an immune state of Thl type 30 - highlight a state of delayed hypersensitivity, - monitor the immune response in vaccinated or treated mammals, - monitor the effectiveness of chemotherapeutic and / or immunotherapeutic treatment.

- 15 - 2844511

  The peptide compound comprising A16E and / or A16G peptides and their derivatives and the adjuvant, according to the invention can be administered in various ways. It is however preferably administered by four routes: - either by subcutaneous injection 5 - or by intra dermal injection - or by intramuscular injection - or by oral route

  Other modes of administration can be employed such as the parenteral or intravenous route.

  In general, a vaccine is in an injectable form composed of a lyophilized fraction which is taken up in a liquid or diluent fraction. The doses used for prevention and immunotherapy are different, and are also different depending on the mode of injection: - subcutaneously and intramuscularly injection of a dose (50 μg of peptides and 1 μg of adjuvant) in patients dogs regardless of breed and sex for a preventive effect injection of half doses (25pg of secreted excreted protein and 50pg

  adjuvant) for immunotherapy of Leishmanian dogs.

  - intra dermo: - half dose injection in Leishmanian dogs for a preventive effect - quarter dose injection in Leishmanian dogs for a therapeutic effect. The injection methods are included in the examples of immunotherapy and

vaccination.

IMMUNOTHERAPY RESULTS

  - 16 According to specialists such as PINELLI (PINELLI, E et ai, Infect Immun, 1994, 62.229-235) Leishmanian dogs corresponding to the activation of the system

  Th2-type lymphocytes have a high antibody response.

  This increased antibody production corresponds to hyper proteinemia and induces the appearance of complex immune systems resulting in renal damage (increase in

  blood creatinine and urea).

  We therefore tried to modulate towards a Thl state by administering to perfectly leishmanian dogs intra dermo doses of the peptide compound. Monitoring of the immune status as well as clinical observation are

  performed before and after treatment.

  Example 1: MANON bitch A 4-year-old Breton Epagneul bitch belonging to Mr P has numerous skin lesions accompanied by a state of fatigue

  general and lean appearance, all evoking canine leishmaniasis.

  The veterinarian, Dr. LM diagnoses leishmaniasis. This diagnosis is confirmed by direct observation under a microscope of leishmanias from a skin layer and a serological analysis which gives a titer in

  1/3200 positive leishmaniasis immunofluorescence.

  The analysis of the immune state before any injection makes it possible to confirm that the dog is indeed in a Th2 type immune state with a high antibody titer as well as IDR tests and negative NO assays. We set up an immunotherapy which consists in performing intradermo 2 injections of pg of peptides (1/2 A16E,% A16G) each injection being spaced 3 weeks apart.

  A week after the second injection, the MANON dog regains appetite and a certain vitality. Dr. LM begins to observe a slight improvement in the skin.

  One month after the last injection, MANON regained a normal clinical appearance, with an increase in weight of 1 kg and an 80% disappearance of all skin lesions. The analysis of the immune state makes it possible to confirm a drop in the titer of anti leishmania antibodies which drops to 1/400 in - 1 7

  immunofluorescence. At the same time, IDR (IDR carried out with leishmanines and also with peptides A16E and A16G), the NO determination and the lymphoblastic proliferation were positive. In addition, after treatment, the MANON dog presents IgG2 specific for the 2 peptides, IgG2 assayed by the 5 ELISA and Western Blot method. These specific IgG2 were absent before any treatment.

  A search for parasites by culture on NNN medium is negative. 8 months after treatment, the MANON dog does not show any changes. Biological analyzes confirm that MANON is still in a Thl immune state.

  Example 2: JUP dog A male Rottweiler breed dog, JUP aged 5 years belonging to Mr D 15 has specific clinical signs of leishmaniasis. According to Dr. GX, presence of numerous shiny scales, depilation of the right periocular, ulcerative lesions in the 2 anterior elbows and a pronounced state of fatigue. Biological analyzes with in particular a leishmaniasis serology

  positive at 1/400 in immunofluorescence confirm the clinical diagnosis.

  Immunotherapy consisting in performing intradermo 3 injections of

  pg of peptide vaccine compound (peptide A16E) (1/2 dose), each injection being spaced 10 days apart, is started. Analysis of the immune state before any injection demonstrates that the JUP dog develops a Th2 type immune system with a strongly positive parasitaemia from the bone marrow.

  One month after the last injection, the Leishmanian clinical signs of JUP retrocede with, in particular, scarring of ulcerative lesions, a significant disappearance of scales and almost non-existent periocular depilation. Serology always has an immunofluorescence titer equal to 1/400. On the other hand, the analysis of the cellular response makes it possible to affirm that JUP presents an active Th1 state with an IDR (IDR carried out with leishmanines and also with the peptide A16E), an assay of NO and a proliferation

positive lymphoblastic.

  - 18 At the same time, the parasitaemia was negatived (culture of bone marrow in

NNN medium).

  Humorally, the JUP dog has specific IgG2 peptide A16E

  after treatment, IgG2 assayed in ELISA and Western Blot.

  The present invention therefore indeed consists of a therapeutic peptide compound which induces the passage of a Th2 type immune state with significant production of antibodies which exacerbates clinical manifestations towards

  a Thl-type immune state resulting in healing.

VACCINATION RESULTS

  In order to evaluate the efficacy of the vaccine peptide compound according to the invention, the vaccine compound is tested on 5 perfectly healthy dogs. These 5 dogs 15 have negative leishmaniasis serology, negative parasitaemia as well as completely negative Leishmania specific cellular response tests. In addition, none of the 5 dogs has IgG2 specific for one or

of the 2 peptides A16E and / or A16G.

  These 5 dogs live in a place free of any sandfly. We define 3 groups of dogs: 1- control group (placebo) - negative control: LILI dog of the Pointer breed, female gender, age: 3 years old - Control alone adjuvant: MIMI bitch of the Epagneul Breton breed, female gender. 25 Age: 6 years old 2- Group of dogs vaccinated with peptides alone - MAMA bitch of Weymar dog breed, female. Age: 2.5 years => peptide A16E dog NUNU, Pointer breed, male. Age: 2 and a half years => peptide 30 A16G 3- dog group vaccinated with peptides A16E and A16G

  - LEON dog, Epagneul Breton breed, male sex. Age: 4 years old.

  The vaccine injection schedule is as follows:

- 19 - 2844511

  OJ 1st injection 1 dose subcutaneously

D28 J84

  -> 4 weeks + 2nd injection - 8 weeks + infectious test 1 subcutaneous dose Clinical monitoring of the 5 dogs is carried out every two weeks. The biological analyzes are schematized as follows: JOlère injection _J282 nde injection J84 J84 +2 months test + 56 infectious J14 Biological analyzes J biological analyzes biological analyzes The biological analyzes consist of: - biochemical analyzes: urea, creatines, transaminases - hematological analyzes : count, formula - leishmaniasis serology: quantitative anti leishmania immunofluorescence, ELISA assay of IgG2 specific for peptides Al6E and / or Al6G - cell response tests: IDR (Intra Dermo Reaction) carried out with leishmanines and also with peptides A16E and Al6G, determination of NO and lymphoblastic proliferation To these analyzes must be added the search for Leishmania by direct observation under a microscope and culture on NNN medium from bone marrow after

the infectious test.

RESULTS:

Clinical follow-up:

  No significant clinical manifestation appeared during this entire study. It should be noted a slight weight loss and the appearance of some scales in the LILI dog, 4 months after the infectious test.

  -20 Biological monitoring 1- The biochemical and hematological parameters remained normal throughout

throughout this study.

  2- Leishmaniasis and parasitaemia serology: Before any injection, the 5 dogs have negative serologies and parasitaemias. The table below shows the serological responses obtained during our experiments and the monitoring of parasitaemia (analyzes carried out 2 months and

  12 months after the infectious test).

PARASITEMIC SEROLOGY

  (on bone marrow puncture) IF ELISA dogs Culture examination on quantitative direct IgG2 NNN medium + LILI dogs + + MIMI controls + ++ MAMA dogs (A16E) + (0.700) immunized NUNU (A16G) - + (0.520)

LEON (A1 6E + A16G) - + (0.780)

  Legend: IF: immunofluorescence (considered positive if the titer is> 1/100) ELISA: Cutt off = 0.300 in OD (optical density) 15 Parasitaemia: culture on NNN medium - = absence ++ = more than 5 mobile promastigote forms / fields Only immunized dogs present specific antibodies of isotype IgG2 20 (ELISA against the corresponding peptides) as well as negative parasitaemias. There should be a slight appearance of total antibodies (1/200 in IF) in

  all dogs after the infectious test.

  Only the control dogs (LILI and MIMI) show positive parasitaemias as well as an absence of specific anti-peptide IgG2 antibodies.

  -21 - * Cell-mediated response Before any injection, the 5 dogs exhibit a perfectly negative cell-mediated response to Leishmania infantum. According to the following table, only immunized dogs have lymphoblastic proliferation tests, positive intramacrophagic leismanicidal activities associated with

production of NO by monocytes.

  The following table shows the responses obtained of cell type (analyzes carried out 2 months after the infectious test).

  IDR Assay Proliferation test Lymphoblastic NO DOGS Leishmanines A16E A16G (in pM) Dogs LILI + - - 0.3 - 1.1 (3) MIMI controls - 0.2 - 1.2 (3.1) LAM (A16E) + + limit 2.6 + 2.1 (3.1) NUNU dogs (A16G) + limit + 2.8 ++ 3.1 (3.6) immunized LEON (A16E + + + 4.2 +++ 3 , 7 (3,8) A16G) Legend: * IDR: The Intra Dermo Reaction test is considered positive (+) if the induration is

  > 5 mm 48 hours after the intradermoinjection.

  * NO determination: * Lymphoblastic proliferation test: the results are expressed by reading in light microscopy and in stimulation indices (in brackets,

  stimulation index + Concanavalin A).

- 22 - 2844511

  Through this analysis, the peptide compounds with the adjuvant do indeed induce cell-mediated immunity of the protective Th1 type to which must be added an induction of antibodies of isotype IgG2. The mixture of the two peptides at equal concentration gives a more marked cellular response (high level of NO synthesized by activated monocytes) - 23

Claims (12)

  1- Composition intended for the prevention or the treatment of affections in mammals and in particular in humans, canids, felines and equines whose protective immunity depends on the stimulation of lymphocytes of the Thl type and in particular of '' a state of delayed hypersensitivity characterized in that it contains: - derivatives of the following 16 amino acid sequence (Al6E):   A - A - R - C - A - R - C - R - E - G - Y - S - L - T - D - E.   in which C can be replaced by S, and L by 1, these peptide derivatives consisting of at least 8 contiguous amino acids taken from said sequence A16E (SEQ ID NO 1 to 44) - and derivatives of the sequence of 16 acids next amino (Al6G)   A - A - S - S - T - P - S - P - G - S - G - C - E - V - D - G.   in which C can be replaced by S, and S by C, these peptide derivatives consisting of at least 8 contiguous amino acids taken from said sequence A16G (SEQ ID NO 45 to 88) - and an adjuvant which preferably induces a response cell-mediated, 2- Composition according to claim 1 characterized in that the derivatives of the sequences A16E and A16G are preferably associated with the same concentration   3- Composition according to any one of claims 1 to 2, characterized   in that the adjuvant is muramyl dipeptide.   4- Composition according to Claim 3, characterized in that the muramyl dipeptide is associated with the derivatives of the peptide sequences Al6E and A16G,   in a peptide to adjuvant weight ratio of 1 / 0.1 to 1/6. -24   - Composition according to Claims 3 or 4, characterized in that the   muramyl dipeptide is associated with derivatives of the peptide sequences A16E and A16G, in a ratio of 50 μg of proteins to 1OOpg of muramyl dipeptide.   6- Use of the composition according to any one of Claims 1 to 5,   for the manufacture of a medicament, or a vaccine, or a reagent for in vitro and in vivo diagnosis, for the induction or diagnosis in mammals of the transition from an immune state of type Th2 to a state 10 Thl type immune system.   7- Use of the composition according to any one of Claims 1 to 5,   for the manufacture of a medicament, or a vaccine, or an in vitro and in vivo diagnostic reagent, for the induction or diagnosis in mammals of specific antibodies and more particularly of the IgG2 isotype.   8- Composition according to any one of claims 1 to 5, characterized   in that the derivatives of the peptide sequences Al6E and Al 6G are linked to   large KLH-type molecules to make them immunogenic.   9- Composition according to any one of claims 1 to 5, or 8,   packaged in a form such that it can be administered by different routes: subcutaneous, intradermal, intramuscular, intravenous, parental and oral.   -In vivo diagnostic product characterized in that it contains the composition   according to any one of claims 1 to 5 or 8, for the detection of a delayed hypersensitivity reaction by intradermal injection of the said product in mammals and in particular in humans, canids, felines and equines .   11- In vitro diagnostic product characterized in that it contains the composition according to   any one of claims 1 to 5 or 8, for the detection of a humoral response and particularly of IgG2, allowing in particular the   -25 evidence of a Thl-type immune state and a delayed hypersensitivity state, monitoring of the immune response in vaccinated or treated mammals, and monitoring of the effectiveness of a treatment   chemotherapeutic and / or immunotherapeutic.   12- In vitro diagnostic kit comprising the diagnostic product according to claim i1, characterized in that the derivatives of the peptide sequences A16E or A16G are linked to large molecules of the type   biotin or polylysine to make them more antigenic.   13- In vitro diagnostic kit comprising the diagnostic product according to claim i1, characterized in that the derivatives of the sequences   peptides AI 6E or Ai 6G, are linked by glutaraldehyde.   1 5 14- In vitro diagnostic kit containing the diagnostic product according to the   claim 11, or according to one of claims 12 and 13, characterized in that the derivatives of the peptide sequences A16E or A16G, are linked to a solid support.   15- In vitro diagnostic kit according to claim 14 characterized in that the derivatives of the peptide sequences A16E or A16G are linked to nitrocellulose membranes or other polymers, latex supports and   various plastic (polymer) materials.   16- In vitro diagnostic kit according to claim 12, characterized in that the derivatives of the peptide sequences A16E or A16G, are conjugated to radioisotopes, fluorescent molecules, luminescent molecules,   enzymes and colored particles.