FR3014103B1 - VACCINE COMPOSITION FOR THE PREVENTION AND / OR TREATMENT OF LEISHMANIOSES, IMMUNOGENIC PEPTIDES AND METHOD OF OBTAINING - Google Patents

Abstract

The invention relates to the use, as a vaccine molecule in a mammal, of a PSA, such as leishmania, or a part of this PSA having immunogenic properties, said PSA designated hereinafter by ES PSAr, presenting in soluble, native, recombinant form. It also relates to a vaccine composition for the prevention and / or treatment of leishmaniases comprising an ES PSAr protein.

Description

The invention relates to a vaccine composition for the prevention and / or treatment of leishmaniases in mammals. It also relates to immunogenic peptides and their production process.

Large parasite endemics are by far the most important causes of morbidity and mortality not only for the human species, but for all other domestic and wild animal species.

Leishmaniases are among the most serious parasitic infections affecting humans in the world. Three hundred and fifty million people are exposed in eighty-eight countries spread over four of the five continents, and sixteen million of them carry the parasite. They are responsible for a broad spectrum of clinical manifestations: cutaneous, mucocutaneous and visceral.

Visceral leishmaniasis (VL) is caused by parasites of the Leishmania donovani complex (L. infantum, L. chagasi and L. donovani) which are intracellular parasites of the macrophage. Unlike other Leishmania species causing the integumentary forms of the disease (L. major, L. brasiliensisr ...) that affect the dermis or mucous membranes, leishmanias donovani complex have an ability to spread to all organs deep (liver, spleen, ganglion and bone marrow) where they multiply. Patients with LV then present a clinical picture associating a moderate but persistent fever, hepatosplenomegaly and pancytopenia generally leading to death if no specific treatment is initiated sufficiently quickly or in case of treatment failure. Even though the LV at L. infantum / chagasi is a major problem in Southern Europe with the spread of the AIDS pandemic and the emergence of an increasing number of Leishmania / HIV co-infections, it is far from controlled and becomes very worrisome in terms of public health problem in South American countries. In other parts of the world, particularly in the endemic areas of L. donovanir, deadly epidemics have claimed hundreds of thousands of lives in recent decades, particularly in Sudan and India. Moreover, the situation is becoming critical in India with the emergence of strains resistant to pentavalent derivatives of antimony (first-line drugs). The annual incidence of human leishmaniasis is estimated at between 2 and 2.5 million new cases, of which more than 500 000 are visceral leishmaniasis and 1.5 to 2 million integumentary leishmaniases, some of which are very disfiguring and mutilating, such as mucocutaneous leishmaniasis (CML). This results in an overall morbidity of more than two million DALYs (Disability Adjusted Life Years) and the deaths of nearly sixty thousand people each year. These diseases are still a public health problem in Latin America, Asia, Africa, the Near East and the Middle East, and southern Europe.

Visceral leishmaniasis also affects the canine population, which is a reservoir of parasites supplying the cycle of transmission continuously. It is due to L. infantum / chagasi, a species responsible for a zoonosis of domestic and wild canids widespread throughout the world. It affects millions of dogs in Europe, Asia, North Africa and South America. Symptomatic but asymptomatic dogs are a source of parasites for transmission of LV in humans.

Prevention measures against leishmaniasis are numerous. For a long time, they were based on the fight against the insect vector and the main objective was to control their multiplication by the use of insecticides and to avoid the contact between the vector (sandfly) and their hosts (dog, Man , ...). Control of leishmaniases, once diagnosed, also involves chemotherapeutic treatment. The latter are unfortunately endangered by an old and very limited therapeutic arsenal, long, toxic and expensive treatments accompanied by numerous cases of relapse and by the emergence of chemoresistance phenomena. In the absence of effective treatments and the scale of the problem, it is therefore necessary to provide the most severely affected populations with prevention methods that are applicable on a large scale, the most appropriate of which would be vaccination.

Several arguments are in favor of the feasibility of a vaccine in humans.

However, in humans, no vaccine is currently available against leishmaniasis.

In fact, only a pragmatic approach using whole parasites killed has been made possible to date.

Historically, first-generation vaccines, based on the use of whole attenuated or dead parasites (including autoclaved), combined or not with adjuvants, were intended primarily to protect against cutaneous leishmaniases by trying to reproduce the levels of protection obtained with live parasites.

However, the numerous clinical trials conducted on thousands of individuals in South America, but also in Iran, have yielded more than mixed or even disappointing results.

Numerous subunit vaccines consisting of purified (first-generation vaccine) or recombinant (second-generation vaccine) native proteins have been tested. Some vaccine candidates have conferred a good level of protection against an experimental infection. the mouse.

More recently, so-called third-generation DNA vaccines have been developed with the nucleotide sequences encoding the proteins used in second generation vaccines (References 1, 2 and 3).

Finally, other vaccine tracks have been successfully explored in mice, such as the antigens based on antigens present in sandfly saliva (References 4 and 5), which have made it possible to obtain relative resistance to experimental infection. at Leishmania major.

However, to date, few candidate vaccines have passed the experimental stage (mice, hamsters) because of the difficulty of developing an appropriate animal model reproducing the characteristics of the human disease and to implement clinical trials in natural hosts of infection that require large cohorts, heavy infrastructure and that can take years to give a definitive result. Moreover, it is difficult to directly extrapolate results obtained in mice to natural hosts of the infection, such as dogs or humans. Finally, protective immune responses controlling a challenge infection may not reflect those required to prevent leishmaniasis in endemic areas.

Two vaccine candidates are under clinical development: • The Leish 111 f MPL SE of the Infections Disease Research Institute (IRDI) in Seattle is a chimeric vaccine called LEISH-F1 consisting of 3 recombinant fusion proteins (TSA-LmSTIl-LelF) formulated with a monophosphorylated lipid and squalene in a stable emulsion (MPL-SE). However, while protection against cutaneous leishmaniasis appears to be well established, studies have shown that Leish-111 f MPL-SE does not protect dogs exposed to natural infection with L. infantum. • HASPB + KMP-11 from the University of York (UK) on which the results of clinical trials are not known to date.

In Dogs, there is an effective solution with CaniLeish®, which is a product composed of excretion excretion antigens (AES) of L. infantum promastigotes (based on WO 9426899 and its extensions, on behalf of IRD) . Canine leishmaniasis thus sees its preventive measures profoundly modified and completed.

Vaccination not only controls the development of the disease, but also significantly decreases the parasite load in the dog and thus contributes to the interruption of the transmission cycle of LV in sandflies, dogs and humans.

However, the production yields of AES do not allow an industrial scale production of a human vaccine.

In this context, there is an urgent need to invest in new avenues offering a more effective alternative to existing vaccines, in order to allow the prophylactic and / or therapeutic vaccination of patients against leishmanias, particularly patients resistant to therapies. existing, and to provide a reproducible, heat-stable and easy-to-produce vaccine molecule in endemic areas.

The solution provided by the invention is based on the work carried out by some of the co-inventors on the major immunogen naturally secreted excreted antigens (AES).

This work, concerning the biochemical, immunological and more recently molecular characterization of the AESs of L. amazonensis or L. infantum, has clearly demonstrated that they contain major immunogens constituted by proteins called PSA (for "Promastigote Surface Antigen").

In the context of the study of these proteins as vaccine candidates, a new production strategy has been developed by the inventors, making it possible to obtain highly immunogenic excreted / secreted products and to obtain them in native, recombinant form, with high yields. thus making it possible to exploit them on an industrial scale.

In addition, it has been found that parts of such molecules unexpectedly also have great interest in the intended prophylactic and therapeutic applications. The invention therefore aims to use, as a vaccine molecule, a PSA, as obtained by implementing said strategy and / or the use of a part of this molecule, and their applications. prophylactic and / or immunotherapeutic agents in mammals.

It also aims at a method of production in a completely defined environment of a PSA, assuring its reproducible production devoid of any foreign proteins and at a cost allowing exploitation on an industrial scale, as well as the production of a part of this PSA. The invention also aims, as new molecules, with major immunogens of AES, and a portion of these immunogens, as produced according to the method of the invention.

According to yet another aspect, the invention is further directed to a preventive and / or therapeutic vaccine composition comprising such an immunogenic molecule and / or a part of this molecule and the antibodies directed against this molecule. The invention thus relates to the use, as a vaccine molecule in a mammal, of a PSA such as leishmania, or a part of this PSA, hereinafter referred to as ES PSAR, having immunogenic properties, said PSA is presenting in soluble, native, recombinant form.

In the specification and claims, "soluble PSA" refers to soluble PSA excreted / secreted in the Leishmania culture supernatant. "Native PSA" refers to a PSA as produced in a culture supernatant by a leishmania, comprising the co- and post-translational modifications of a PSA such as leishmania, or a portion of that PSA having immunogenic properties, said PSA hereinafter referred to as ES PSAr which is in soluble form, native in its conformation, recombinant. "Recombinant PSA" refers to a PSA as expressed by genetic recombination, integrated into the genome of the host cells of the expression system. - "ES PSAr" generally means a PSA of leishmania, in soluble, native, recombinant form or a part of such a PSA.

It is recalled that the PSA sequence comprises a signal peptide involved in the secretory pathways located at the end of the amino-terminal part of the molecule, a variable number of repeat domains rich in leucine (LRR motifs, for "Leucine"). Rich Repeats ") and a carboxy-terminal portion of the proline-rich molecule, threonine and cysteine (see Figure 1). The end of the carboxy-terminal portion has a GPI anchor or hydrophobic GPI anchor signal (Glycosyl Phosphatidyl Inositol).

PSA also has the particularity of being present in all species of leishmania (integumentary and visceral), which represents a real advantage in terms of cross-vaccination vis-à-vis the different clinical expressions of leishmaniasis.

According to one embodiment, the invention relates to the use of the entire ES PSAr molecule. The term "whole PSAr PS" means that the protein possesses the co- and post-translational modifications of the native molecule, as produced by the parasite and is not truncated.

In a variant, the invention aims to use an ES PSAr that does not include all the co- and post-translational modifications. In particular, the invention relates to the use of a deglycosylated ES PSAr.

According to another variant, the use according to the invention comprises a glycosylated PSAR PS.

In another embodiment, the invention relates to the use of an ES PSAr devoid of the GPI anchor of the hydrophobic part in the C-terminal end.

In another embodiment, the invention is directed to the use of a PS PS ES devoid of the carboxy-terminal portion.

According to yet another embodiment, the invention aims at the use of the C-terminal part of a PSA or ES PSAr molecule (hereinafter referred to as C-ter PSA or C-ter ES PSAr) such that isolated from a leishmaniasis culture excretion / secretion supernatant.It is a non-native molecule, as encoded by a truncated DNA sequence of PSA or ES PSAr

Advantageously, the invention relates to the use as a vacant molecule of an ES PSAr such as obtained from the PSA L.amazonensis or a leishmania complex donovani, including L. infantum or L. donovanirou of the complex braziliensis and tropica. The invention thus aims in particular at using an ES PSAr of sequence SEQ ID No. 1. This ES PSAr is obtained more particularly from the PSA produced by L. amazonensis deleted from its hydrophobic membrane anchoring peptide.

In a variant, the PSAR ES is more particularly obtained from L. infantum PSA of sequence SEQ ID No. 2 or L.donovani of sequence SEQ ID No. 3, these sequences also being deleted at the carboxy-terminal end. of their hydrophobic membrane anchoring peptide.

The vaccine molecule used may correspond to a portion of the ES PSAr, particularly at the C-terminus. Such a molecule has in particular the sequence SEQ ID No. 4.

More specifically, the invention is directed to the use, as a vaccine molecule, of an ES PSAr in soluble form, as produced by insertion of a vector comprising the gene encoding PSAR ES in a recombinant eukaryotic expression system. Leishmania.

Preferably, the recombinant expression system used is that of a nonpathogenic leishmania in mammals. The invention is especially directed to the use, as a vaccine molecule, of ES PSAr in soluble form, as produced by insertion of the gene encoding PSAR ES, into a eukaryotic recombinant expression system REV13 consisting of Leishmania tarantolae or variant of the gene encoding part of ES PSAr, in particular a part devoid of its hydrophobic part as indicated above.

Advantageously, this system allows the integration of the expression vector containing the gene coding for ES PSAr in the 18S rRNA locus of the chromosome of Leishmania tarentolae which has a very high transcription rate and thus the obtaining of an abundant amount of the Recombinant PSA.

The protein can be produced with all or part of the co- and post-translational modifications of Leishmania proteins. The invention also relates to a method for producing ES PSAr in soluble form, comprising the characteristics defined above.

Thus, this method comprises the integration of an expression vector comprising the gene encoding ES PSAr in a non-pathogenic eukaryotic recombinant system for humans or animals.

Advantageously, the transfection by the vector is carried out in the locus of a chromosome of a non-pathogenic leishmania having a high level of transcription.

A particularly suitable leishmania is L. tarentolae, at the promastigote stage. The insertion in the 18 s rRNA locus of the chromosome whose transcription rate is high makes it possible to obtain high production yields.

The expression vector used is advantageously a plasmid. As illustrated in the examples, pF4X1.4 is particularly suitable for the implementation of the invention. The sequences necessary for the post-translational modifications in L. tarentolae are introduced on both sides of the cloning cassette.

The ES PSAr protein thus produced is a soluble protein in leishmania culture supernatants, possessing the properties of the native protein, in particular one or more of the co- and post-translational modifications of Leishmania proteins, such as acetylation, Hydroxylation, disulfide bridges, glycylation, glycosylation, phosphorylation, lipid anchoring and the like.

Thus, deglycosylated ES PSAr, in one variant of the invention, is produced from the culture of the promastigote forms of L. tarentolae in the presence of tunicamicyne. Tunicamycin is an antibiotic that inhibits the GlcNAc phosphotransferase (GPT) enzyme. It thus inhibits the N-glycosylation necessary for binding the precursors of N-glycosides to dolichol diphosphate (see FIG. 3).

The culture of L. tarentolae promastigote transgenes is advantageously carried out in the fully defined culture medium as described in the aforementioned international application in the name of the IRD and its extensions, which makes it possible to recover the PSA produced without serum contaminants. and cell provided by conventionally used culture media.

A purification step can be carried out for example by gel affinity chromatography.

The production of the C-ter portion of PSA is advantageously carried out in a recombinant bacterial system, followed preferably by a purification step.

The expression vector used, which comprises the cDNA of the gene coding for the carboxy-terminal part of PSA, is in particular a plasmid.

A plasmid construct suitable for carrying out the invention comprises the elements for producing a recombinant protein with a 6-Histidine tag in the NH 2 -terminal position and a tag cleavage site.

The colonies producing the desired recombinant proteins are selected and after amplification in culture are advantageously purified.

The recombinant protein, in native form, advantageously with one or more of the co- and post-translational modifications described above, constitutes a novel product and as such is within the scope of the invention. The invention is directed in particular to a glycosylated PSAR PS or alternatively a deglycosylated PSAr ES.

It also relates to a deleted ES PSAr, in particular devoid of the GPI anchor and therefore the hydrophobic peptide in the C-terminal position, which makes it possible to avoid aggregation phenomena and the formation of inclusion bodies during production. from ES PSAr.

Similarly, the invention aims as a new product, the molecule consisting of the C-ter PSA portion of the protein as defined above.

The production methods described above are illustrated in the examples starting from the PS PSAr L.amazonensis and with C-ter PSA L.amazonensis.

As also shown by the examples, the ES PSAR protein as well as its carboxy-terminal part (C-ter PSA), in combination with an adjuvant, confer in the dog a very high level of protection against an experimental infection. to L. infantum.

This protection is accompanied by: the production of specific antibodies mainly IgG2 isotype which appear to play a significant role in the protection (leishmanicidal and neutralizing effects); the polarization of the cell-mediated response towards a Th1-type pathway revealed by an increase in the production by IFN-gamma T lymphocytes, leading to the activation by the classical macrophage pathway, characterized by the increased synthesis of NO and thus the elimination of intracellular leishmanias (leishmanicidal activity of canine macrophages).

The remarkable properties of ES PSAr as well as its C-ter part are also revealed by: the inhibitory effect of a monospecific polyclonal rabbit hyperimmune serum directed against C-ter PSA on the growth of promastigote forms of L. infantum ; the neutralizing effect of monospecific polyclonal rabbit sera anti-ES PSAr and anti-C-ter PSA (non-native) on the ability of promastigotes to infect ex vivo macrophages, - the "growth factor" effect of ES PSAr on proliferation of promastigote forms.

The examples also show a protective and induced immuno-reaction on human cells, which confirms the results obtained in dogs and makes it possible to envisage the development of a vaccine for human purposes. This immunoreactivity is accompanied by the production of cytokines. TH1 type and a T-cytotoxic response demonstrated by the production of granzyme B. The invention therefore also relates to a vaccine composition for the prevention and / or treatment of leishmaniases in humans or animals, characterized in that it comprises: an ES PSAr protein, in soluble form, of leishmania and / or a truncated PSAr ES, in particular a deprive of the GPI anchor, and / or the C-ter peptide part of the PSA such that from leishmania, or from ES PSAR and one or more adjuvants and / or one or more immunomodulators. The adjuvant is advantageously chosen from those capable of inducing a cell-mediated response leading to the elimination of the parasite.

Suitable adjuvants include those of the TLR3, TLR4, TLR5, TLR7, TLR8, TLR9 classes, saponins and their derivatives, oil-in-water or water-in-oil emulsions, polysaccharides, cationic liposomes, virosomes or polyelectrolytes.

Suitable immunomodulators include phlebotomy saliva proteins, cytokines and heat shock protein (HSP).

The vaccine composition of the invention is particularly in a form allowing administration by different routes, such as the subcutaneous, intradermal, intramuscular, parenteral, oral, endonasal or mucosal route.

The vaccine composition defined above is especially suitable for the manufacture of a drug or a vaccine, an in vivo or in vitro diagnostic reagent for the induction or diagnosis in mammals of cell-mediated immunity of Th1-type lymphocytes and / or humoral effector immunity.

This composition is suitable for induction or diagnosis in the mammal of the passage from a Th2-type immune state to a Th1-type immune state, or for the induction or diagnosis in the mammal of isotype-specific antibodies. Neutralizing and / or lytic IgG2 that recognize the antigenic determinants of parasites.

Such antibodies and the antisera containing them also form part of the invention. Other features and advantages of the invention are given in the examples which follow, with reference to FIGS. 1 to 18, which represent, respectively, FIG. 1: a schematic representation of the Leishmania PSA protein, FIG. polypeptide analysis of purified ES PSAr in SDS-PAGE gel (reducing and non-reducing conditions,

3: electrophoretic profiles obtained after staining of the proteins (A) and sugars (B) of the ES PSARs produced in the presence (lane 3) or in the absence (lane 2) of tunicamycin, FIG. 4: the polypeptide analysis of purified C-ter PSA in SDS-PAGE gel (reducing and non-reducing conditions, - Figure 5: Electrophoretic analysis of soluble glycoproteins ES PSAr deglycosylated (1 and 3) ES native PSAR (2 and 4), - Figure 6: ELISA IgG2 versus AES expressed in optical density (OD), FIG. 7: IgG2 ELISA versus PSAR ES expressed in optical density (OD), FIG. 8: IgG2 versus C-ter PSA ELISA expressed in optical density (OD), FIG. 9 : Effect of sera from vaccinated dogs (CaniLeish®, ES PSAr and C-ter PSA) on the viability and proliferation of L.infantum promastigotes, compared to those of placebo sera: TO: preimmune sera; T5: sera taken two months after three doses of the vaccine,

Figure 10: Neutralizing effect of anti-ES PSAr and anti-C-ter PSA monospecific polyclonal rabbit sera (used at different dilutions (1/10, 1/50 and 1/100),

Figure 11: Effect of a monospecific polyclonal rabbit serum directed against the C-ter PSA (carboxy-terminal portion of PSA) used at different dilutions (1/25 and 1/50) on the growth of promastigote forms of L infantum,

FIG. 12: Evaluation, compared to a control, of the number of promastigotes / mL with concentrations of ES PSAR of 17.8 μΜ and 0.22 μΜ, at different addition times,

Figure 13: Leishmanicidal activities of canine macrophages in the placebo group versus the group vaccinated before (T0), one month after the administration of 2 doses of vaccine (T3) and two months after the injection of 3 doses (T5),

Figure 14: Determination of interferon (IFN) -y levels present in the supernatants of co-culture of cells from placebo dogs and vaccinated dogs (ES PSAr or C-ter PSA). T0: pre-immune sera; T5: sera taken two months after the administration of three doses of the vaccine,

Figure 15: Evaluation of nitric oxide (NO) levels produced in co-culture supernatants of cells from placebo dogs and vaccinated dogs (ES PSAr or C-ter PSA). T0: pre-immune sera; T5: sera taken two months after the administration of three doses of the vaccine,

Figure 16: Determination of Percentages of Parasitologically Positive Dogs in Placebo Dog Groups and Groups of Dogs Vaccinated with ES PSAr,

Figure 17: Determination of Percentages of Parasitologically Positive Dogs in Placebos and Groups of Dogs Vaccinated with C-ter PSA,

Figure 18: Determination of parasite loads by RT-PCR in dogs of placebo and vaccinated groups.

Example 1: Obtaining, producing and purifying recombinant proteins

1. Cloning and sequencing of PSA genes

A hyperimmersum generated in the rabbit and directed against the exosproting secretion antigens (AES) of L. amazonensis promastigote and a monoclonal antibody (E2) directed specifically against the major immunogen of parasitic AES were used to screen a bank cDNA of promastigote and amastigote forms (produced in 1ZAPII) of L. amazonensis. Sequence analysis of the inserts contained in the recombinant plasmids made it possible to identify mainly cDNA clones, selectively located on chromosome 12 and encoding proteins belonging to the family of PSAs.

The sequence SEQ ID No. 1 is derived from the LaPSA38 gene coding for the entire 45 kDa (372 aa) protein which corresponds to WO 2005/051989 in the name of IRD and the sequence SEQ ID No. 4 is derived from the truncated gene. PSA19 which codes for the carboxy terminal portion of LaPSA38. This is the C-ter PSA part, 19kDa (119 aa).

The sequence SEQ ID No. 2 is derived from the LiJll gene.

The LiJll gene was obtained by using the C-ter PSA sequence (radiolabeled probe) to screen a genomic library of genomic DNA of L. infantum promastigotes, which made it possible to characterize a homologous PSA in Leishmania infantum (PSA IJ11). 463 aa (50 kDa). The protein sequence of L. infantum PSAIJ11 exhibits strong sequence homology with that of LAPSA38s of L. amazonensis in its amino- (73.8%) and carboxy- (77.5%) terminal parts with a number of motifs. higher leucine-rich repeat (9 against 6) (in the aforementioned international application WO 2005/051989, said gene coding for the non-recombinant protein corresponds to SEQ ID No. 11 and the protein encoded in SEQ ID No. 12). 2. Production of LaES PSAr deleted from the GPI anchor in a recombinant eukaryotic system and purification

This step is carried out with a gene expression system in L. tarentolae marketed by Jena Bioscience: "Leishmania tarentolae Gene Expression Starter Kit".

This expression system uses the parasite itself to produce its own protein. The latter has many advantages for the production of therapeutic proteins:

Leishmania tarantolae is a non-pathogenic lizard parasite for humans. Its manipulation does not require any restrictive security measures; L. tarentolae is easily grown in cell suspension in completely defined culture media (no contamination with other foreign proteins), at 26 ° C, with a good yield (of the order of 108 cells per ml). doubling of population by 4 hours);

This expression system makes it possible to produce proteins native to Leishmania. The parasite contains the machinery necessary for the expression of native eukaryotic proteins;

Thus, the protein produced possesses all post-translational modifications of Leishmania proteins such as glycosylation and disulfide bond formation;

The vector used in this system makes it possible to clone the gene of interest in a bacterial system and then to express the protein in Leishmania tarentolae;

It allows the integration of the expression cassette into the 18S rRNA locus of the chromosome which has a very high transcription level and thus the stable obtaining of an abundant quantity of recombinant protein.

The LaPSA38s gene was amplified, then modified by site-directed mutagenesis to allow the production of a protein with a poly-histidine tag (5 histidines) partially carboxyterminal, and remove the anchor site to the membrane.

Directional cloning of the ES PSAr gene was performed in the pF4X1.4sat expression vector. The plasmids were specifically constructed to allow the production of the recombinant protein in the culture medium supernatant from which it can be easily isolated. This construct was transfected into Leishmania tarentolae, where it was integrated into the genome of the parasite, at the promastigote stage, by homologous recombination, conferring at the same time a resistance to nseothorin (NTC), making it possible to select the recombinant individuals. Overexpression is achieved by integrating the gene within the 18S rRNA locus, taking advantage of the high level of transcription of host RNA polymerase I. This plasmid also contains around the cloning cassette the sequences necessary for the co- and post-translational modifications in Leishmania tarentolae, making it possible to obtain a protein in its native form.

The promastigote transgenes of L. tarentolae have been adapted in the completely defined medium developed by the IRD (WO application cited above and its extensions) so as to easily purify the parasite protein from the culture supernatants and to produce it in the absence of any foreign protein (molecules of serum and cellular origin).

The purification from the culture medium supernatants of the ES PSAr thus produced was carried out by Agarose-Nickel gel affinity chromatography.

Under these experimental conditions, only LAPSA38snr of L. amazonensis was produced with a production yield of 0.2 to 0.3 mg of purified protein per liter of culture, sufficient to allow its use in the development of new analytical methods and its evaluation as a vaccine candidate. It corresponds to an apparent molecular weight of 45 kDa and a theoretical molecular weight of 37 kDa. It has the sequence SEQ ID No. 1 referred to above. This protein comprises 349 amino acids; his ft is 5.07. The polypeptide analysis of the purified ES PSAr in SDS-PAGE gel (reducing and non-reducing conditions) is given in FIG.

The electrophoretic profiles obtained after staining the proteins (A) and sugars (B) of PSAR ES produced in the presence (lane 3) or in the absence (lane 2) of tunicamycin are given in FIG.

3 - Production in a recombinant bacterial system and purification of C-ter PSA

The cDNAs of the LaPSAl9s gene (C-ter PSA) were inserted into the plasmid pQE-31. The plasmids were specifically constructed to allow the production of a recombinant protein having a 6-His-tag in the amino-terminal position and a tag cleavage site. The transformation with pQE-31-6 (His) -COOH LaPSA19s was carried out in chemiocompetent E. coli M15 bacterial cells. The recombinant colonies were selected to produce the recombinant proteins. The most productive clone was amplified in culture. The corresponding C-ter PSA protein was purified on Agarose-Nickel gel. A yield of about 1 mg of recombinant protein per liter of culture was obtained. It comprises 119 amino acids and has a molecular weight of 18 kDa under reducing conditions and in dimer form, under non-reducing conditions, a molecular weight of 36 kDa (see FIG. 4).

The results of the electrophoretic analysis of soluble glycoproteins are reported in Figure 5: ES PSAr deglycosylated (1 and 3) and ES PSAR native (2 and 4)

EXAMPLE 2 Immunoprotective responses induced by ES PSAr and C-ter PSA in dogs

This clinical trial reported hereinafter aims to compare in the same vaccination trial in dogs "Beagle" the protective effects of the two recombinant proteins ES PSAr and C-ter PSA on groups of 9 dogs and 5 dogs respectively. A placebo group of 5 dogs is the unvaccinated control group. An experimental challenge infection is performed 2 months after the immunization protocol. Clinical and parasitological follow-up are carried out every 2 months until 8 months after the experimental infection.

QA21 is used at 60 μg per dose of vaccine, an amount of adjuvant equivalent to that contained in the CaniLeish vaccine. Used alone and at this concentration, it has no significant effect on the immune system of the dog and mouse.

1 - IMMUNIZATION PROTOCOL The study was conducted in double blind, randomized and controlled. It has been approved by the Ethics Committee of the Ecole Nationale Vétérinaire de Lyon.

The immunization protocol includes 3 injections by the subcutaneous route performed at one month intervals. Three groups of dogs were selected as follows: * Group 1: Placebo (Physiological Injection for Injection); * Group 2: Injection of 25 μg of LaES PSAr adjuvanted with 60 μg of QA-21; Group 3: Injection of 25 μg of C-ter PSA (protein of sequence SEQ ID No. 4) adjuvated with 60 μg of QA-21.

RANDOMIZATION

Vaccinated Vaccinated Witnesses Total ES PSAR C-ter

PSA

Number of Dogs 9 5 5 19

Number of females 523 10

Number of males 432 9

Average age (years) 2, 69 2, 93 2.87 2.89

Standard Deviation of Age (year) 1.12 1.10 1.14 1.08

Average Weight (Kg) 15.2 15.4 14.8 14.9

Standard Deviation of Weight (Kg) 2.5 2.2 2.4 2.3

2 - PROTOCOL OF THE LEVIES

Different samples before the infectious challenge were made at: TO: before any T3 immunization: one month after the administration of two T5 vaccine doses: two months after the administration of three doses of the vaccine. 3 - CLINICAL FOLLOW-UP:

These follow-ups are carried out - at the reception of the animals - once a month during the entire study daily for 5 days after the experimental challenge 4 -SUIVIS IMMUNOLOGICAL AND PARASITOLOGICAL:

Immunological: - serology specific to ΤΟ, T3 and T5 leishmanicidal effect at TO and T5 - nitric oxide (NO) assay and determination of IFN-γ production in TO and T5 co-culture supernatants.

Parasitological:

An experimental challenge infection (injection of 109 metacyclic promastigotes of L. infantum by the intravenous route) was performed 2 months after the immunization protocol. Clinical and parasitological follow-ups were conducted every 2 months for 8 months after the experimental infection.

PCR and culture of bone marrow samples at TO, 2 months post-challenge (T6), 4 months postchallenge (T7), and 6 months post-challenge (T8) were performed to highlight the presence of the parasite or its DNA.

5 - RESULTS 5.1 - Humoral Mediated Responses a- IgG2 Isotype Specific Antibody Response Analysis ELISA techniques were used to analyze the antibody responses in the dogs in the study. The average titers of IgG2 anti-AES, anti-ES PSAr and anti-Cter PSA antibodies were compared by ELISA between placebo dogs and vaccinated dogs before immunization (TO) and at different post-immunization times: - one month after administration of two vaccine doses (T3) and / or - two months after three injections (T5).

All dogs in the study were serologically negative for TO (Figures 5, 6 and 7). All the dogs belonging to the placebo group have IgG2 anti-AES, anti-ES PSAr and anti-C-ter PSA antibody titers lower than the corresponding off off, whatever the sampling period (T3 and T5) ( Figures 6,7 and 8).

The determined anti-Leishmania IgG antibody responses were negative in all dogs in the pre-immunization study and in all dogs in the T5 placebo group.

Only a significantly high production of IgG2 isotype antibodies specifically directed against AES (FIG. 6), PSAR ES (FIG. 7) and C-ter PSA (FIG. 8) is demonstrated in all dogs vaccinated first month after two doses of vaccine (T3) and two months after three doses of the vaccine (T5).

Mean IgG2 antibody titers were significantly higher in vaccinated dogs than in placebo dogs (P <0.05) at post-immunization T3 and T5 times (Figures 6, 7, and 8).

Vaccine candidates do not induce anti-Leishmania IgG antibody (Figurative Antigen) in the dog by the indirect immunofluorescence technique (IFI, standard technique for serological diagnosis) in all vaccinated dogs (T3-negative IFI titre). and T5).

The detection of IgG2 isotype antibodies strongly demonstrates the induction of Th1-type cellular immunity (protective immunity). b - Functional biological role of antibodies produced bl- Effect of sera from vaccinated dogs (CaniLeish®, ES PSAr and C-ter PSA) on the viability and proliferation of Leishmania

The L. infantum transgene promastigotes (MON1, C1, NEO / Luc) transfected with the luciferase gene are incubated for 5 days in the absence or in the presence of serum (decomplemented) of placebos and vaccinated dogs. The number of parasites is determined by luminometry (RLU). The results are expressed as percentage inhibition of the proliferation of promastigotes.

As shown in Figure 9 a significant leishmanicidal effect (p <0.01) is observed on the promastigote forms of L. infantum when the parasites are incubated for 30 min with undiluted serum of vaccinated dogs (ES PSAr and C-ter PSA), while viability percentages remain high with placebo serum (less than 10% reduction in parasite growth). Inhibitions of 60%, 70% and 85% of the parasite proliferation are respectively observed with the sera of dogs vaccinated with CaniLeish®, ES PSAr and C-ter PSA (p <0.01). This leishmanicidal effect is less pronounced at the dilution of U and fades at a dilution of 1/8.

The proliferation of untreated parasites was very similar to that of parasites treated with sera from placebo dogs regardless of the dilution of the serum used. Less than 10% reduction in growth was observed.

On the other hand, the sera of vaccinated dogs have no effect on the differentiation of promastigotes into amastigotes).

b2- Neutralizing effect of monospecific polyclonal rabbit sera anti-ES PSAr and anti-Cter PSA

Promatigative forms were incubated in the presence of different dilutions (1/10, 1/50 and 1/100) of rabbit sera healthy or previously immunized with PSAR ES (or C-ter PSA and then washed three times by centrifugation for eliminate excess serum Their ability to infect macrophages derived from canine monocytes was studied by determining the percent reduction in Parasite Index (PI) after 72 hours of interaction.

As shown in Figure 10, promastigotes treated with healthy rabbit serum were found to be more infectious to macrophages than untreated ones. In contrast, parasite exposure to monospecific anti-ES PSAr or anti-C-ter PSA polyclonal rabbit sera inhibits their ability to infect ex vivo macrophages derived from canine monocytes. The effect of the anti-C-ter PSA serum at the 1/100 dilution is more pronounced than that of the anti-ES PSAr serum at the same dilution (Figure 10). The effect of monospecific polyclonal rabbit serum directed against the C-ter PSA portion was also evaluated on the in vitro growth of promastigote forms of L. infantum. As shown in Figure 11, almost complete inhibition of promastigote multiplication is observed when the serum is added at the 1/25 dilution every other day to the parasite culture. Used at 1/50 dilution, this inhibition is partial.

On the other hand, the addition every other day of different concentrations of PSAR ES (0.22 μΜ and 17.8 μΜ) to the culture of L. infantum promastigotes significantly activates the proliferation of parasites (Figure 12). 5.2 Cell-mediated responses a- Leishmanicidal activities of canine macrophages

The leishmanicidal activities revealed by the Placébo group versus the Vaccinated group cells were evaluated before (T0) and one month after two doses of vaccine (T3) and two months after the injection of three doses ( T5). The incubation of autologous lymphocytes with infected macrophages from pre-immune dogs (T0), but also placebo dogs at different post-immunization times (T3 and T5) induces leishmanicidal activities less than 20% reduction of the index parasitic (Figure 13).

Remarkably, the leishmanicidal effects revealed by the co-culture of cells isolated from dogs vaccinated with ES PSAr or C-ter PSA increase significantly at T3 (60% and 40% reduction of the parasite index, respectively, P < 0.05) and still rise to T5 (90% and 60% reduction respectively, P <0.01) (Figure 13).

These leishmanicidal effects are associated with T5 selective and significant production of nitric oxide (NO) (Figure 15) and interferon (IFN) -γ (Figure 14) in dogs vaccinated with ES PSAr or C-ter PSA. 5.3 Parasitological follow-ups of dogs after infectious challenge

Parasitological monitoring was performed in dogs using two methods of amplification: - that aimed at detecting the presence of parasites in the bone marrow after culturing bone marrow samples in NNN medium; - that aimed at detecting parasite DNA by a gene amplification technique (PCR) which, coupled with PCR-quantitative, has the advantage of quantitatively evaluating the parasite load.

Parasitological monitoring performed at T0 shows that all the dogs in the study were free from leishmania before the experimental infectious test (Figures 16 and 17).

According to Figures 16, 17 and 18 and the statistical study, the parasitological monitoring results are as follows: - 2 months after the infectious test

No significant difference in placebo and vaccinated groups is highlighted. All dogs were parasitologically negative. - 4 months after the infectious test

The results are not very significant between the group of placebo dogs and the group of dogs vaccinated with Cter PSA (p = 0.04852). Placebos, on the other hand, had significantly more infected dogs than those vaccinated with PSAR (p = 0.0256) - 6 months after the infectious test.

All dogs in the placebo group are infected whereas only 22% and 20% of the dogs vaccinated respectively with ES PSAr and C-ter PSA are infected. The differences between placebo and vaccinated groups are therefore very significant (p <0.01). 5.4 Conclusions:

The recombinant PSA protein (ES PSAr) as well as its carboxy-terminal part (C-ter PSA) combined with QA-21 confer in the dog an excellent level of protection against an experimental infection with L. infantum. This protection is accompanied by: the production of specific antibodies mainly IgG2 isotype which seems to play a significant role in the protection (leishmanicidal and neutralizing effects); the polarization of the cell-mediated response towards a Th1-type pathway revealed by an increase in the production by IFN-γ T lymphocytes leading to the activation by the classical macrophage pathway characterized by the increased synthesis of NO and therefore the elimination of intracellular leishmanias (leishmanicidal activity of canine macrophages).

The remarkable properties of ES PSAr as well as its C-ter portion are also revealed by: the inhibitory effect of a monospecific polyclonal rabbit serum directed against C-ter PSA on the growth of promastigote forms of L. infantum; the neutralizing effect of anti-rPSA monospecific polyclonal rabbit sera and anti-C-ter PSA on the ability of promastigotes to infect ex vivo macrophages; - the "growth factor" effect of ES PSAr on the proliferation of promastigote forms.

Example 3: Assays on Human Cells The immunogenicity of native recombinant PSA (ES PSAr) was evaluated on human cells by analyzing the profile of the cellular immune response generated ex-vivo in humans.

The ability of ES PSAr to induce, ex-vivo, on human cells, a specific cell-mediated immune response by activating Thl-producing cytokine-producing T lymphocytes and / or Granzyme B from healthy donors as compared to individuals immune or healed exposed to visceral leishmaniasis L. infantum in the south of France, was evaluated. To this end, a set of immunological and parasitological methods, associated with clinical monitoring, has been set up.

The results are reported in the following Tables 1 and 2:

Table 1

IFN-gamma (μg / ml) in supernatants co-culturing human cells from healthy individuals (B, IND 8 to 15) and asymptomatic or cured immune individuals (A, IND there 7) exposed in southern France to visceral leishmaniasis L. infantum.

AT

B ____

TSLA Ldd8: total parasite extract of L. donovani promastigotes TSLA Ldi: total parasite extract of L. infantum promastigotes.

Table 2

Production of Granzyme B (μg / ml) in coculture supernatants of human cells from healthy individuals (B, IND 8-15) and asymptomatic or cured immune individuals (A, IND 1, 3, 5 and 6) exposed in southern France to visceral leishmaniasis L. infantum.

AT

B

TSLA Ldd8: total parasite extract of L. donovani promastigotes TSLA Ldi: total parasite extract of L. infantum promastigotes The analysis of the cell test results shows that the ES PSAr constituting the vaccine candidate is capable of inducing a specific immune response of Th1 type, generating significant levels of IFN-gamma (Table 1), and / or a T-cytotoxic response determined by a significant production of Granzyme B (Tabeau 2) in asymptomatic or cured individuals with respect to individuals healthy. The results of the immunogenicity tests of the vaccine candidate ES PSAr confirm the interest of this recombinant protein to be selected to enter the composition of a vaccine against human leishmaniases.

BIBLIOGRAPHIC REFERENCES 1 - Dumonteil E. et al., Vaccine, 2003 May 16; 21 (17-18): 2161-8. ; 2 - Rafati S. et al, Vaccine, 2005, May 25; 23 (28): 3716-5; 3 - Rodriguez-Cortes A. et al., Vaccine, 2007, Nov. 14; 25 (46): 7962-71; Morris R.V. et al., J. Immunol., 2001, Nov. 1, 167 (9): 5226-30; Valenzuela J.G. et al., J. Exp. Biol., 2004, Oct 207 (Pt21): 3717-29.

Claims (27)

claims 1 - Excreted / secreted soluble PSA protein, such as leishmania, or a part of this PSA having immunogenic properties, said PSA, hereinafter referred to as ES PSAr, in native or recombinant form, for its use as a molecule vaccine in a mammal. 2 - soluble PSA protein excreted / secreted for use according to claim 1, characterized in that the ES PSAr molecule is whole. 3 - soluble PSA protein secreted / secreted for its use according to claim 1 or 2, wherein an ES PSAr comprises one or more co- and post-translational modifications of the group comprising acetylation, hydroxylation, disulfide bridges, glycylation, glycosylation, phosphorylation, lipid anchorage, and / or devoid of the GPI anchor. 4 - soluble PSA protein excreted / secreted for use according to any one of claims 1 to 3, characterized in that it is a glycosylated PSAr ES or a deglycosylated PSAr ES. 5. Protein soluble PSA excreted / secreted for use according to claim 3 or 4, characterized in that the ES PSAr is denuded of GPI anchor. 6. Protein soluble PSA excreted / secreted for its use according to any one of claims 1, 3 or 4, characterized in that ES PSAr is devoid of the carboxy-terminal portion. 7 - soluble PSA protein excreted / secreted for its use according to claim 1, characterized in that the part of ES PSAr is the C-terminal part, hereinafter referred to as C-ter PSA, or the C-terminal part of the PSA as isolated from a leishmania excretion / secretion culture supernatant. 8- soluble PSA protein excreted / secreted for its use according to any one of claims 1 to 7, characterized in that the ES PSAr or the part of this PSA is as obtained from the PSA L.amazonensis or a leishmania of the donovani complex, especially L. infantum or L.donovani, or the braziliensis and tropica complex. 9- soluble PSA protein excreted / secreted for use according to claim 8, characterized in that said ES PSAr has the sequence SED ID No. 1. 10 - soluble PSA protein excreted / secreted for use according to claim 7 or 8, characterized in that the C-terminal portion of the PSA has the sequence SEQ ID No. 4. 11 - Process for producing soluble PS PSAR excreted / secreted for its use according to any one of claims 1 to 6, 8 or 9, wherein the ES PSAr is produced by insertion of a vector comprising the gene encoding the ES PSAr or a portion of ES PSAr, particularly a deleted portion of the GPI anchor, in a eukaryotic recombinant leishmanian expression system. 12 - Process for producing soluble PS PSAR excreted / secreted for its use according to claim 11, characterized in that the recombinant expression system used is that of a non-pathogenic leishmania in mammals. 13 - Process for producing soluble PS PSAR excreted / secreted for use according to claim 11 or 12, characterized in that the eukaryotic recombinant expression system consists of that of promastigotes Leishmania tarentolae. 14 - Process for producing soluble PS PSAR excreted / secreted for its use according to claim 13, characterized in that the expression vector containing the gene encoding ES PSAr PSA is integrated in the 18S rRNA locus of the chromosome of Leishmania tarentolae. 15 - Process for the production of ES PSAr in soluble form, characterized in that it comprises the steps defined in any one of Claims 11 to 14. 16 - Process for producing the C-ter PSA part. according to one of claims 1, 7, S or 10, characterized in that it is carried out in a recombinant bacterial system. 17 - soluble recombinant protein excreted / secreted, characterized in that it is an ES PSAr in native or recombinant form, including where appropriate one or more of the co- and post-translational modifications, such as acetylation , Hydroxylation, disulfide bridges, glycylation, glycosylation, phosphorylation, lipid anchoring, and / or lacking the GPI anchor. 18- soluble recombinant protein excreted / secreted according to claim 17, characterized in that it is a glycosylated PSAr PS or deglycosylated ES PSAr. 19 - soluble recombinant protein excreted / secreted according to claim 17 or 18, characterized in that it is deleted from the GPI anchor. Peptide, characterized in that it is the C-ter part of the ES PSAr according to claim 7, 8 or 10. 21 - Vaccinal composition for the prevention and / or treatment of leishmaniasis, characterized in that it comprises: a soluble PSCH protein PSCH excreted / secreted from leishmania and / or the C-ter part of the leishmania PSA, as used according to any one of claims 1 to 14, or as defined in one of claims 17 to 20 and one or more adjuvants and / or one or more immunomodulators. 22 - vaccine composition according to claim 21, characterized in that the adjuvant is advantageously selected from those capable of inducing a cell-mediated response leading to the elimination of the parasite. 23 - The vaccine composition according to claim 22, characterized in that the adjuvants are chosen from those of the classes TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, the saponins and their derivatives, the oil-in-water or water-in-oil emulsions, the polysaccharides, cationic liposomes, virosomes or polyelectrolytes and immunomodulators are selected from phlebotomy saliva proteins, cytokines and HSP20 thermal thing proteins. 24 - vaccine composition according to any one of claims 21 to 23, characterized in that it is in a form allowing administration by different routes, such as subcutaneous, intradermal, intramuscular, parenteral, endonasal, mucosal , or oral. Vaccine composition according to any one of Claims 21 to 24, for the manufacture of a medicament or a vaccine, an in vivo or in vitro diagnostic reagent for the induction or diagnosis in mammalian an activation of cell-mediated immunity of Th1-type lymphocytes and / or humoral effector immunity. 26 - The composition according to claim 24, for the induction or the diagnosis in the mammal of the passage from a Th2-type immune state to a Th1-type immune state, or for induction or diagnosis in mammalian animals. neutralizing and / or lytic IgG2 isotype-specific antibodies that recognize the antigenic determinants of parasites. 27 - IgG2 specific antibodies and antiserums containing them directed against ES PSAr or part of ES PSAr according to any one of claims 17 to 20.