THERAPEUTIC VACCINE COMPRISING A SPECIFIC ANTIGEN OF A DISEASE THAT DOES NOT AFFECT THE CENTRAL NERVOUS SYSTEM AND NANOPARTICLES, AND USE OF THE VACCINE

Abstract

A therapeutic vaccine for treating individuals who are carriers of a disease or pathogen that does not affect the brain, such as leishmaniasis, cancer or any pathogenic infection, comprises at least one cationic nanoparticle comprising a cationic polysaccharide core and a specific antigen, wherein the antigen is specific to the pathogen or disease.

Claims

1. A therapeutic vaccine for treating an individual carrying a disease or an intracellular pathogen that does not affect the central nervous system, comprising at least one cationic nanoparticle including a cationic polysaccharide core and a specific antigen, wherein the antigen is specific to the pathogen or disease.

2. The therapeutic vaccine of claim 1, wherein the specific antigen is from a pathogen responsible for leishmaniasis.

3. The therapeutic vaccine of claim 2, wherein the antigen is specific to a strain Leishmania infantum.

4. The therapeutic vaccine of claim 1, wherein the specific antigen is a tumor antigen.

5. The therapeutic vaccine of claim 1, wherein the cationic polysaccharide core is crosslinked.

6. The therapeutic vaccine of claim 1, wherein the cationic polysaccharide core is loaded with an anionic phospholipid.

7. The therapeutic vaccine of claim 6, wherein the anionic phospholipid is selected from diacyl phosphatidylglycerol, diacyl phosphatidylserine or diacyl phosphatidylinositol.

8. The therapeutic vaccine of claim 7, wherein the anionic phospholipid is dipalmitoylphosphatidylglycerol.

9. A vaccine composition comprising a cationic nanoparticle consisting of a cationic polysaccharide core and a pathogen-specific antigen that cannot pass a hematoencephalic barrier or a peripheral disease, for use in the treatment of diseases stemming from the pathogen or the disease.

10. The vaccine composition of claim 9, wherein the antigen is from the species Leishmania.

11. The vaccine composition of claim 10, wherein the antigen is from the genus Leishmania Infantum.

12. The vaccine composition of claim 9, wherein the antigen is from a parasite.

13. The vaccine composition of claim 9, wherein the antigen is from a virus.

14. The vaccine composition of claim 9, wherein the antigen is from a bacterium.

15. The vaccine composition of claim 9, wherein the antigen is from a fungus.

16. The vaccine composition, wherein the vaccine composition is in a form suitable for mucosal or injectable administration.

17. A method of treating a patient infected with a disease or an intracellular pathogen that does not affect the central nervous system, comprising administering to the patient a therapeutically effective amount of a substance comprising at least one cationic nanoparticle including a cationic polysaccharide core and a specific antigen, wherein the antigen is specific to the pathogen or disease, thereby treating the disease or pathogen in the patient.

18. The method of claim 17, wherein the antigen is from the species Leishmania.

19. The method of claim 18, wherein the antigen is from the genus Leishmania Infantum.

20. The method of claim 17, wherein the cationic polysaccharide core is crosslinked

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 represents a Protocol for the study of Example 1.

[0023] FIG. 2 represents a SDS-PAGE (left) and PAGE in native conditions (right) of the TEL/NPL formulations carried out, after 120 h of formulation.

[0024] FIG. 3 represents a measurement of the parasitic load in the liver, spleen and bone marrow of mice, 60 days after infection of the mice.

[0025] FIG. 4 represents a protocol for the study of Example 2.

[0026] FIG. 5 represents a SDS-PAGE (left) and PAGE in native conditions (right) of the TEL/NPL formulations carried out, after 96 h of formulation.

[0027] FIG. 6 represents a representation of the parasitic load in the liver, spleen and bone marrow of mice, 90 days after infection.

[0028] FIG. 7 represents a assay of the total IgGs (on the left) and of the IgG 1 and IgG 2a (on the right) in the mice not treated and treated by chemotherapies or by s.c. and i.n. administration of the vaccine formulations.

[0029] FIG. 8 represents a re-stimulation of the splenocytes of the mice not treated and treated by chemotherapies or by s.c. and i.n. administration of the vaccine formulations, and assays of the cytokines secreted in the supernatant.

[0030] FIG. 9 represents a protocol for the study of example 3.

[0031] FIG. 10 represents a PAGE in native conditions of the TEL/NPL formulations.

[0032] FIG. 11 represents a measurement of the parasitic load in the mouse serum, not vaccinated and vaccinated by the TEL or TEL/NPL formulations, by nasal (A) or subcutaneous (B) formulations. Measurement of the parasitic load in the liver (C) and the spleen (D) of mice, not vaccinated and vaccinated by the TEL or TEL/NPL formulations.

[0033] FIG. 12 represents a protocol for the study of Example 4.

[0034] FIG. 13 represents the analysis of the parasite load of L. infantum by qPCR in the organs of the infected mice (n=5), then treated by different conditions. One-way ANOVA #p<0.05, **p<0.01, ****p<0.0001.

[0035] FIG. 14 represents the analysis of the L. infantum parasitic load by qPCR in the bone marrow of the infected mice (n=5), treated by different conditions then reinfected for 30 days. One-way ANOVA *p<0.05.

[0036] FIG. 15 represents a protocol for the study of Example 5.

[0037] FIG. 16 represents the analysis of the parasitic load of L. donovani by qPCR in the spleens of the infected mice (n=10), treated by chemotherapy. One-way ANOVA, **p<***p<0.001.

[0038] FIG. 17 represents a protocol for the study of Example 6.

[0039] FIG. 18 represents the course of the cutaneous infection in dogs treated with miltefosine and/or vaccinated, after one month.

[0040] FIG. 19 represents the course of the parasitic load in the bone marrow of dogs treated with miltefosine and/or vaccinated, after one month.

[0041] FIG. 20 represents the course of the clinical score of dogs treated with miltefosine and/or vaccinated.

DETAILED DESCRIPTION

[0042] A first object of the present disclosure relates to a therapeutic vaccine intended for the treatment of an individual carrying a peripheral disease or an intracellular pathogen that cannot pass the blood-brain barrier, comprising at least one cationic nanoparticle consisting of a cationic polysaccharide core and at least one specific antigen, wherein the antigen is specific to a pathogen or disease that does not affect the central nervous system.

[0043] Therapeutic vaccine means an immunostimulating composition intended to stimulate the immune system to generate a specific immune response capable of treating individuals already suffering from the disease. This embodiment opposes that of prophylactic vaccines that are used in people who have not yet contracted the disease. The immunostimulating composition may also be called an immunotherapeutic composition.

[0044] Infected individual means a human or animal carrying a pathogen responsible for an infection not reaching the central nervous system.

[0045] Peripheral disease means a disease that does not affect (does not harm) the brain of the infected individual. The disease affects only metabolic systems, that is to say all organs with the exception of the brain (central nervous system). Within the meaning of the present disclosure, the diseases may be cancers that do not affect the brain or any other peripheral disease such as an autoimmune disease.

[0046] Pathogen that cannot pass the blood-brain barrier means a pathogen that does not infect the central nervous system, the infection therefore not affecting the brain of the infected individual.

[0047] Within the meaning of the present disclosure, the pathogen can be: [0048] A virus not having central tropism, such as, for example: herpes simplex virus (HSV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein-Barr virus (EBV). [0049] An intra-cellular bacterium, such as: Salmonella typhi, Neisseria gonorrhoeae, Legionella, Rickettsia, Chlamydia, Chlamydophila spp, Bartonella henselae, Francisella tularensis, Listeria monocytogenes, Brucella, mycobacterium, Nocardia, Rhodococcus equi, Yersinia. [0050] An intra-cellular parasite such as: Apicomplexans (Plasmodium spp., Cryptosporidium parvum), Trypanosomatidae (Trypanosoma cruzi, Leishmania sp.). [0051] A fungus, such as: Histoplasma capsulatum, Cryptococcus neoformans, Sporothrix spp, etc.

[0052] Cationic nanoparticle consisting of a cationic polysaccharide core means a solid nanoparticle (NP) comprising a cationic polysaccharide core. The NP may or may not be crosslinked. Its core may or may not be loaded with an anionic phospholipid. This NP is not surrounded by any phospholipid layer.

[0053] Antigen means an antigenic protein, a mixture of antigenic proteins, or a partial or total extract of pathogen. The pathogen extract may contain proteins, polysaccharides and lipids and nucleic acids. The protein may be hydrophilic or lipophilic. The antigens can be purified, alone or in combination.

[0054] In a preferred embodiment, the mixture of antigenic proteins is composed of one or more purified antigens or a pathogen extract. The pathogen extract may be a total extract or a partial extract. In a fully preferred embodiment, the antigen is a complex protein extract obtained from an entire pathogen.

[0055] In a particular embodiment, the antigen is specific for leishmaniasis.

[0056] In a particular embodiment, the antigen is specific to the genus Leishmania, such as Leishmania infantum, Leishmania donovani or Leishmania major. In a preferred embodiment, the antigen is specific to the strain Leishmania infantum.

[0057] In the case of Leishmaniasis, it is considered that immunity that is triggered by the therapeutic vaccine may be cross-immunity by inducing a memory T response capable of recognizing different species of leishmania.

[0058] In this context, individuals treated with the therapeutic vaccine produced with the strain Leishmania infantum are immunized against all other strains of Leishmania. In addition, there is species cross-immunity, namely that the vaccine prepared from a pathogen infecting a species may potentially be used to protect other species of mammals, for example. This approach therefore allows cross-vaccination, that is to say a wide spectrum, since the vaccine is effective on all the leishmaniasis strains, whether they are cutaneous, visceral or mucosal strains.

[0059] One feature of the vaccine according to the present disclosure is based on combining the antigen with cationic NP.

[0060] In a first particular embodiment, the cationic polysaccharide forming the core of the NP is a crosslinked polymer obtained by the reaction between a polysaccharide selected from starch, dextran, dextrin, and maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalactomannans (guar gum) and at least one cationic ligand selected from a primary, secondary, tertiary or quaternary ammonium, then the addition of a crosslinking agent. The crosslinking agent is chosen from epichlorohydrin, a dicarboxylic acid or an acyl chloride, such as sebacic acid. The core is not loaded with lipids.

[0061] In a preferred embodiment, the cationic polysaccharide is obtained by the reaction between maltodextrin and glycidyltrimethylammonium.

[0062] In a third particular embodiment, the cationic polysaccharide forming the nucleus of the NP is loaded with an anionic phospholipid. This anionic phospholipid can be chosen from diacyl phosphatidylglycerol, diacyl phosphatidylserine or diacyl phosphatidylinositol. In another preferred embodiment, the anionic phospholipid is dipalmitoylphosphatidylglycerol (DPPG).

[0063] In an entirely preferred embodiment, NP is a nanoparticle of maltodextrin loaded with DPPG.

[0064] In a preferred embodiment, the antigen is a complex extract obtained from a partial or total extract of a parasite responsible for a disease linked to a parasite, the infection of which does not reach the CNS. In a particular embodiment, it involves a chemoresistant parasite.

[0065] In a particular embodiment, the therapeutic vaccine induces cross-immunity of species between humans and animals. In an even more particular embodiment, it induces protective cross-immunity in humans or non-human mammals such as: canines; felines; leporidae; bovines; rodents; non-human primates; equines.

[0066] A second object of the present disclosure relates to a vaccine composition comprising a cationic nanoparticle consisting of a cationic polysaccharide core and an antigen specific to a pathogen that cannot pass the blood-brain barrier or a peripheral disease for use in the treatment of diseases stemming from a pathogen that cannot pass the blood-brain barrier or a peripheral disease.

[0067] The animals infected with Leishmaniasis are mammals such as, for example, canines, rodents, leporidae, equines, bovines, primates.

[0068] A third subject of the present disclosure relates to a composition comprising a cationic nanoparticle consisting of a cationic polysaccharide core and a specific antigen of the genus Leishmania infantum for use as a therapeutic vaccine in the treatment of cutaneous, visceral or mucosal Leishmaniasis.

[0069] In a preferred embodiment, the composition used as a therapeutic vaccine is administered nasally.

[0070] Without departing from the scope of the present disclosure, the mode of administration can also be administered mucosally (orally) or intra-dermally, subcutaneously, intravenously.

EXAMPLES

Abbreviations

[0071] NPL: Lipid maltodextrin nanoparticles; [0072] TEL: total extracts of leishmania; [0073] Glu+Allo: Glucantime+Allopurinol; [0074] i.d: Intradermal; [0075] i.n.: Intranasal; [0076] i.p.: Intraperitoneal; [0077] i.v.: Intravenous; [0078] PRO: promastigote; [0079] AMA: amastigote; [0080] W: week

[0081] Example 1: Study of the feasibility of a therapeutic vaccine via a nasal route, using total extracts of Leishmania and maltodextrin nanoparticles, compared with the reference antiparasitic treatment (Glucantime+Allopurinol).

[0082] 1-A Materials and Methods

[0083] a) Experimental Model:

[0084] Female Balb/c mice, 8 weeks old, were infected with 1.2.10.sup.7 amastigotes of the strain L. infantum injected i.v. (FIG. 1). The first vaccinal and antiparasitic treatments were carried out 10 days later (D11). The drug treatment lasted 10 days (until D21). The vaccine booster was carried out 15 days after the primary (D26). Finally, the mice were euthanized 35 days later (D61).

[0085] Liver cells, spleen and bone marrow were lysed to extract DNA. The cytochrome b gene of the parasite was used to estimate the parasitic load by qPCR. Each picogram of parasitic DNA corresponds to 10 parasites. The number of parasites is estimated in 50 ng of total DNA. This protocol is shown in FIG. 1.

[0086] b) Groups and Doses Used:

[0087] Each group consisted of 8 mice, distributed as follows: [0088] Group 1: Untreated infected control [0089] Group 2: Chemotherapy (Glucantime+Allopurinol) [0090] Group 3: NPL/TEL vaccine, s.c. [0091] Group 4: NPL/TEL vaccine, i.n.

[0092] The doses administered were: [0093] NPL/TEL s.c.: 20 g TEL+60 g NPL in 100 L [0094] NPL/TEL i.n.: 20 g TEL+60 g NPL in 20 L [0095] Chemotherapy: GLUCANTIME 100 mgSb/kg/d by i.p. injection+Allopurinol 10 mg/kg/d orally, for 10 successive days, in 100 L.

[0096] c) Vaccine Formulations

[0097] The formulations were produced, from total extract derived from the amastigote and promastigote forms, of a mixture of canine and human strains of L. infantum, and NPL.

[0098] 1-B Results

[0099] a) Characterization of the combination of antigens with NPL

[0100] The NPLs were characterized by SDS-PAGE and native PAGE analysis. It can be seen that, in the ratio 1/3 (weight/weight), 93% of the protein antigens are combined in the nanoparticles (FIG. 2). The results are shown in FIG. 3.

[0101] b) Parasitic load at D61

[0102] It is observed at D61, an identical decrease in the parasite load in the groups immunized with intravenous NPL TEL and chemotherapy (FIG. 3). In the liver, all the parasites are eliminated (it is observed that the value is under the detection threshold). On the other hand, no improvement could be observed in the NPL TEL subcutaneous groups.

[0103] Conclusion:

[0104] These results suggest that the nasal therapeutic vaccine is as effective in reducing the parasite load as the reference treatment in this study.

[0105] Example 2: Study of the efficacy of a therapeutic nasal vaccine directed against the Leishmaniosis visceral in mice, composed of total extract of Leishmania and maltodextrin nanoparticles, and associated immune response.

[0106] 2-A Materials and Methods

[0107] a) Experimental Model:

[0108] Balb/c mice, 8 weeks old, were infected with 1,108 amastigotes of the strain L. infantum injected i.d. (FIG. 4). The first vaccinal and antiparasitic treatments were carried out 10 days later (D11). The drug treatment lasted 10 days (until D21). The vaccine booster was carried out 15 days after the primary (D26). Finally, the mice were euthanized 64 days later (D90).

[0109] Liver cells, spleen and bone marrow were lysed to extract DNA. The cytochrome b gene of the parasite was used to estimate the parasitic load by qPCR. Each picogram of parasitic DNA corresponds to 10 parasites. The number of parasites is estimated in 50 ng of total DNA.

[0110] b) Groups and Doses Used:

[0111] Each group consisted of 8 mice, distributed as follows: [0112] Group 1: Untreated infected control [0113] Group 2: Chemotherapy (GLUCANTIME+Allopurinol) [0114] Group 3: NPL/TEL vaccine, s.c. [0115] Group 4: NPL/TEL vaccine, i.n.

[0116] The doses administered were: [0117] NPL/TEL s.c.: 20 g TEL+60 g NPL in 100 L [0118] NPL/TEL i.n.: 20 g TEL+60 g NPL in 20 L [0119] Chemotherapy: GLUCANTIME 100 mgSb/kg/d by i.p. injection+Allopurinol 10 mg/kg/d orally, for 10 successive days, in 100 L.

[0120] c) Vaccine Formulations

[0121] The formulations were prepared from total extract from amastigotes of canine strains of L. infantum and NPL.

[0122] In this study, the test was carried out intradermally (i.d.) to mimic biting by phlebotomine flies, the natural vectors of the infection.

[0123] 2-B Results

[0124] a) Characterization of the Combination of Antigens with NPL:

[0125] The NPLs were characterized by SDS-PAGE and native PAGE analysis. It can be seen that, in the ratio 1/3 (w/w), 100% of the protein antigens were combined in the nanoparticles (FIG. 5). The results are shown in FIGS. 6, 7, and 8.

[0126] b) Parasitic Load at D90

[0127] It was observed that after 90 days (FIG. 6), the parasitic load had decreased in the liver, the spleen and the MO of mice treated with the vaccine subcutaneously, and in the liver and the MO of mice treated by the nasal vaccine.

[0128] c) Humoral and Cellular Response:

[0129] After serum assaying of the total IgGs, it was observed that the administration of the vaccine formulations via the nasal route improves the humoral response relative to the subcutaneous route and reference drug treatment (FIG. 7). Furthermore, only the nasal route allowed a ratio IgG1/IgG2a<1, showing a Th1 response necessary to combat the infection of Leishmania.

[0130] After restimulating the splenocytes, a significant secretion of the cytokines of the Th1 (INF-y) and Th17 (IL-17a and IL-17f) routes were observed from the nasally vaccinated mice (FIG. 8).

[0131] Conclusion:

[0132] The therapeutic vaccinal treatment makes it possible to reduce the parasitic load of the infected animals, in a manner similar to that of the antiparasitic treatment.

[0133] This decrease observed with the vaccine treatment is directly correlated with the induction of a Th1 immune response, both humoral and cellular.

[0134] This memory response should also protect the animals from a future reinfection in the parasite, which makes this treatment highly attractive to treat the infected animals.

[0135] Example 3: Study of the feasibility of a prophylactic vaccine using the total extracts of Leishmania and maltodextrin nanoparticles.

[0136] 3-A Materials and Methods

[0137] a) Experimental Model:

[0138] Female Balb/c mice, 8 weeks old, were vaccinated 3 times nasally or subcutaneously, 20 days apart (D1, D22 and D41). They were subsequently infected, 9 days after the last administration (D50), by 106 promastigotes of the strain L. donovani injected i.v., then euthanized 135 days later (D185, FIG. 9).

[0139] b) Groups and Doses Used:

[0140] Each group consisted of 8 to 10 mice, distributed as follows: [0141] Group 1: Saline solution i.n. (20 L) [0142] Group 2: Saline solution s.c. (50 L) [0143] Group 3: TEL i.n (10 g TEL in 20 L) [0144] Group 4: TEL s.c. (10 g TEL in 50 L) [0145] Group 5: NPL/TEL i.n (10 g TEL+30 g NPL in 20 L) [0146] Group 6: NPL/TEL s.c. (10 g TEL+30 g NPL in 50 L)

[0147] Blood samples were taken at D64, D110, D149, and D184 to analyze the parasite load in the blood. This was measured by qPCR on the DNA of the kinetoplast of the parasites.

[0148] Mice were sacrificed by cervical dislocation after 184 days. Upon euthanasia of the mice, the livers and the spleens were collected in order to determine the parasitic load in each of these organs.

[0149] c) Vaccine Formulations

[0150] The formulations were prepared from total extract of L. donovani LV9 and NPL.

[0151] 3-B Results

[0152] a) Characterization of the Combination of Antigens with NPL:

[0153] The NPL were characterized by PAGE analysis in native conditions. It can be seen that when the ratio is 1/3 (weight/weight), 100% of the protein antigens are combined in the nanoparticles (FIG. 10).

[0154] b) Parasitic Load at D185

[0155] The administration of vaccine formulations substantially reduces parasitic load in blood, liver and mouse spleen (FIG. 11). The latter however remains high (>108 parasites in the organs), despite 3 prophylactic administrations of total extract combined or not combined with NPL, suggesting that this strategy does not make it possible to protect against infection.

[0156] Conclusion:

[0157] The CANILEISH prophylactic vaccine currently on the market remains more competitive, with significant results obtained with a single administration.

[0158] Example 4: Evaluation of the efficacy of a therapeutic vaccine against infection with a L. infantum in a mouse model using antigens derived from promastigote or amastigote form of L. infantum.

[0159] 4-A Experimental Protocol:

[0160] 60 Balb/c mice were infected with 2.107 L. infantum by subcutaneous injection (s.c.), in promastigote or amastigote form. After 10 days, the mice were then distributed in groups of 10, and treated as follows: [0161] Group 1: Untreated [0162] Group 2: Reference chemotherapy (Glucantime+Allopurinol)from D11 to D21 [0163] Group 3: NPL/TEL PRO vaccine s.c.Primary D11 and booster D26-20 g per dose [0164] Group 4: NPL/TEL PRO vaccine i.n.Primary D11 and booster D26-20 g per dose [0165] Group 5: NPL/TEL AMA vaccine s.c.Primary D11 and booster D26-20 g per dose [0166] Group 6: NPL/TEL AMA vaccine i.n.Primary D11 and booster D26-20 g per dose

[0167] After 60 days, 5 mice were euthanized and the parasitic load was measured in the liver, spleen and bone marrow (FIG. 1). The remaining 5 mice were reinfected with 2.107 parasites, and their parasitic load were measured 30 days later.

[0168] This protocol is summarized in FIG. 12.

[0169] 4-B Results

[0170] The results are presented in FIGS. 13 and 15.

[0171] The parasitic load analyses at D60 revealed a significant decrease in parasitic load in the liver of the mice treated by chemotherapy, as well as by vaccines, administered via i.n. and s.c., and regardless of the antigen source (promastigotes or amastigotes, FIG. 13). The NPL/TEL formulation made from promastigotes and administered via i.n. reduced was significantly more effective than the reference chemotherapeutic treatment.

[0172] Furthermore, a significant reduction in the parasitic load in the bone marrow was observed, only in the groups of mice treated with the NPL/TEL formulation made from promastigotes and administered i.n.

[0173] No significant variation was observed in the animal spleen.

[0174] After 90 days, a significant decrease in the parasitic load was observed, in the bone marrow of the mice treated with the NPL/TEL formulation made from promastigotes and administered i.n. (FIG. 14). No significant variation could be observed in the livers and spleens of the animals.

[0175] This study makes it possible to conclude as to the effectiveness of the NP/TEL formulations in the development of a therapeutic vaccine against Leishmaniasis. In addition, the use of promastigotes as an antigen source seems to be an interesting strategy for efficiently treating animals.

[0176] Example 5: Evaluation of the efficacy of a therapeutic vaccine against infection with a L. donovani in a mouse model using antigens derived from amastigote form of L. infantum (cross-vaccination)

[0177] 5-A Experimental Protocol:

[0178] 40 Balb/c mice were infected with 2.107 L. donovani, by subcutaneous injection (s.c.). After 10 days, the mice were then distributed in groups of 10, and treated as follows: [0179] Group 1: Untreated [0180] Group 2: Reference chemotherapy (Glucantime+Allopurinol)from D11 to D21 [0181] Group 3: NPL/TEL vaccine s.c. Primary D11 and booster D26-20 g per dose [0182] Group 4: NPL/TEL vaccine i.n. Primary D11 and booster D26-20 g per dose

[0183] After 90 days, the mice were euthanized and the parasitic load was measured in the liver, spleen and bone marrow.

[0184] 5-B Results

[0185] The results are shown in FIG. 16.

[0186] After 90 days, a significant decrease in the parasitic load of L. donovani was observed, in the spleen of the mice treated with the NPL/TEL formulation, made from amastigotes of L. infantum and administered i.n. and s.c. (FIG. 5).

[0187] This makes it possible to conclude as to the effectiveness of the formulations NP/TEL in the development of a therapeutic vaccine, via the nasal and/or subcutaneous route, targeting different strains of the parasite. These results are promising from the perspective of developing a broad spectrum therapeutic vaccine against this parasite.

[0188] Example 6: Evaluation of the efficacy of a therapeutic vaccine against infection with a L. infantum using antigens derived from the killed target parasite, in dogs naturally infected by the parasite and compared with dogs treated by the reference chemotherapy.

[0189] 6-A Experimental Protocol:

[0190] 30 mixed-breed dogs naturally infected by (at least) L. infantum, and at stage II of the infection (Leishvet.org) were selected. The dogs were distributed randomly in 3 groups of 10 dogs, according to their treatment: [0191] Group 1: Miltefosine (2 mg/kg/day for 28 days) [0192] Group 2: NPL/TEL vaccine i.n. Primary W0 and booster W4-100 g per dose [0193] Group 3: Combination of Miltefosine+Vaccine NPL/TEL i.n.

[0194] The skin infection was evaluated by microscopic analysis of skin biopsies, at W0 and W4. The presence of a parasite confirms the cutaneous infection.

[0195] The clinical score of the animals was evaluated at W0, W2, W4, W6 by taking into account the systemic signs (lymphadenopathy, apathy, diarrhea), cutaneous and mucosal signs (alopecia, hyperkeratosis, pyoderma, ulcer, vasculitis, onychogryphosis, nodules) and ocular signs (conjunctivitis, keratitis). For each parameter, a score is assigned (from 0 to 4), depending on the severity of the observed condition. The clinical score is the sum of these different scores.

[0196] The parasitic load was evaluated at W0 and W4, by qPCR analysis of a bone marrow sample at the sternum.

[0197] 6-B Results

[0198] The results are presented in FIGS. 18, 19 and 20.

[0199] In group 1 (Miltefosine)10/10 dogs had visceral leishmaniasis (VL), and 6/10 had skin damage (CL).

[0200] In group 2 (Nasal vaccine)10/10 dogs had visceral leishmaniasis (VL), and 9/10 had skin damage (CL).

[0201] In group 3 (Miltefosine+nasal vaccine)10/10 dogs had visceral leishmaniasis (VL), and 6/10 had skin damage (CL).

[0202] a) Skin Infection

[0203] In group 1, 5 of the 6 dogs infected and treated with Miltefosine no longer have parasites detected in the skin; in group 2, 7 of the 9 infected and vaccinated dogs no longer parasites detected in the skin; in the group 3, 4 of the 6 infected, treated and vaccinated dogs are no longer detected in the skin. The nasal vaccine seems at this stage to also be effective for treating cutaneous leishmaniasis than chemotherapy (FIG. 18).

[0204] b) Parasitic Filler

[0205] In the group treated with Miltefosine, 3 dogs have a reduction in the parasitic load, while 5 dogs have an increase, and one dog died due to the toxicity of the treatment (renal failure). In the vaccinated group, 8 dogs have a decrease in the parasite load (4 of which appear to have achieved parasite clearance), while 2 dogs have a slight increase. Finally, in the treated and vaccinated group, 6 dogs have a reduction in the parasitic load (one seems to have achieved clearance), while one dog has an increase in infection, and 2 do not show any sensitive variation (FIG. 19).

[0206] At this stage, the nasal vaccine seems to be more effective than the chemotherapy in order to reduce the parasitic load in the bone marrow.

[0207] c) Clinical Score

[0208] In the 3 groups studied, a notable decrease in the overall clinical score may be observed. The vaccine is as effective as chemotherapy in the improvement of the general state of health of the animals (FIG. 20).

[0209] This study demonstrates that vaccination is at least as effective as Miltefosine to treat dogs infected with L. infantum in terms of cutaneous and visceral Leishmaniasis. Furthermore, the two nasal administrations did not lead to side effects and are well tolerated. On the other hand, the 28 oral administrations of Miltefosine caused significant side effects (diarrhea, vomiting, renal failure).