Therapeutic vaccine comprising a specific antigen for a disease not affecting the central nervous system and nanoparticles, and its use

Abstract

The invention relates to the field of therapeutic vaccines. More particularly, it concerns a therapeutic vaccine for treating individuals carrying a disease or pathogen that does not affect the brain, such as Leishmaniasis. Figure from the abstract: 1

Description

Title of the invention: THERAPEUTIC VACCINE COMPRISING AN ANTIGEN SPECIFIC TO A DISEASE NOT AFFECTING THE CENTRAL NERVOUS SYSTEM AND NANOPARTICLES AND USE THEREOF

[0001] The invention relates to the field of therapeutic vaccines. It relates more particularly to a therapeutic vaccine for treating diseases not affecting the brain, such as leishmaniasis. Field of invention

[0002] Therapeutic vaccination consists of inducing an immune response when the latter is insufficient to allow spontaneous healing. Activation of the immune system requires, on the one hand, that it recognizes the intruder and, on the other hand, that it considers it dangerous, and then that an appropriate response is induced.

[0003] The therapeutic strategy is very different from that of prophylactic vaccination. This therapeutic solution is based on the fact that the pathogen is already present in the body. During an infection, the immune defenses are often overwhelmed by infectious proliferation or slowed down by internal control mechanisms.

[0004] The main therapeutic vaccines developed to date are aimed at treating cancer. They generally consist of an injection of the tumor antigen and / or injection of differentiated dendritic cells and / or T lymphocytes.

[0005] Therapeutic vaccines are interesting because they constitute an effective therapeutic solution for individuals already carrying the disease. They also make it possible, in the case of certain pathologies, to generate immune responses not induced by classic prophylactic vaccines.

[0006] Leishmaniasis is a parasitic disease affecting many species of mammals, including humans and dogs. It is estimated that 2.5 million domestic dogs are affected by canine leishmaniasis, particularly in the Mediterranean basin but more generally distributed in 70 countries worldwide, and located on all continents. The incidence of this disease is also high in humans with approximately 2 million new cases per year, recorded in more than 80 countries worldwide. This disease is the result of an infection by an intracellular parasite of the genus Leishmania. This parasite is transmitted by a dipterous insect vector, the sandfly. The geographical distribution of the disease therefore depends on the distribution of sandflies and its reservoirs: mammals. Leishmania infantum is an intramacrophagic parasite responsible for the disease in Europe, in the Mediterranean basin and South America. Contamination occurs through the bite of the insect on the individual. The disease can also be transmitted from mother to child.

[0007] Parasites of the genus Leishmania infect host cells and hijack the intracellular trafficking mechanism in order to maintain their parasitophorous vacuole in which they proliferate.

[0008] The prior art reports developments of therapeutic vaccine formulas for the treatment and / or diagnosis of leishmaniasis. For example, document ES2205059 discloses a pharmaceutical composition comprising an immunogenic portion of a Leishmania antigen. However, none of the therapeutic vaccine trials against leishmaniasis have shown efficacy to date.

[0009] Furthermore, the compositions proposed in the prior art all include vaccine adjuvants, necessary for their effectiveness, but whose side effects are widely documented.

[0010] Finally, the most widespread current treatment for leishmaniasis is chemotherapy. This treatment is expensive, non-specific, toxic and can lead to the development of chemoresistance. The molecules known for the treatment of leishmaniasis in dogs are meglumine antimoniate (Glucantime®) and allopurinol, often used in combination.

[0011] In this context, there is a need for an effective treatment and not inducing resistance for infectious diseases caused by a pathogen not infecting the central nervous system such as leishmaniasis, but also to improve the efficacy of therapeutic vaccines in general, particularly in the treatment of cancer. Statement of the invention

[0012] The inventors have developed a therapeutic vaccine, without adjuvants, intended for administration to an individual carrying a disease or pathogen not affecting the central nervous system. In particular, the therapeutic vaccine can be used as a treatment against leishmaniasis or cancer.

[0013] Thus, the invention relates to a therapeutic vaccine intended for the treatment of an individual carrying a disease or a pathogen not affecting the central nervous system comprising at least one cationic nanoparticle consisting of a cationic polysaccharide core and at least one antigen specific to said disease or said pathogen not affecting the central nervous system.

[0014] In a particular embodiment, the invention relates to a therapeutic vaccine for the treatment of leishmaniasis in which the antigen is specific to the Leishmania Infantum strain. Advantage of the invention

[0015] Quite surprisingly, the inventors have demonstrated that administering to an individual already carrying a disease or pathogen inducing damage to the metabolic systems, such as leishmaniasis, a vaccine comprising nanoparticles and an antigen specific to said pathogen or said disease makes it possible to treat the individual. In the event of infection by a pathogen, this type of treatment is also likely to induce specific long-term immunity, making it possible to avoid subsequent reinfection by the same pathogen. Administering the vaccine when the individual is already carrying the disease makes it possible to effectively solicit the individual's immune defenses and to guarantee a cure by elimination of the pathogen as well as long-term immunity by the establishment of memory T cells.

[0016] It is interesting to note that this therapeutic vaccine approach constitutes a novel therapeutic solution for treating an infection due to a chemoresistant pathogen.

[0017] In the case of leishmaniasis, the inventors have shown that this therapeutic strategy proves to be as effective as the current treatment, namely the administration of antiparasitic drugs (see Examples 1 and 2). Quite advantageously, the vaccination approach does not induce any resistance mechanism, unlike antibiotics. Thus, either immunity is preserved over the long term and prevents reinfection, or it is possible to vaccinate the individual again in the event of reinfection.

[0018] The composition of the therapeutic vaccine does not contain any adjuvant (other than the nanoparticles themselves), which avoids the undesirable effects associated with this type of molecule. This is advantageous since mineral adjuvants (namely mineral salts such as aluminum salts) remain in the body for a very long time (several decades).

[0019] The antigens associate with the nanoparticles in solution and are delivered to the immune cells after administration, while the nanoparticles are eliminated rapidly (in less than 72 hours after nasal administration). The nanoparticles therefore play a triple role: stabilizing agent, antigen transport vector, and vaccine adjuvant.

[0020] The vaccine can be administered orally, nasally, intradermally, subcutaneously or intravenously and allows leishmaniasis to be treated with a single formulation regardless of the species of Leishmania that has infected the individual. In addition, the same vaccine formulation can be administered to humans and animals, particularly dogs. DETAILED DESCRIPTION OF THE INVENTION

[0021] A first subject of the present invention relates to a therapeutic vaccine intended for the treatment of an individual carrying a disease or an intracellular pathogen which does not affect the central nervous system comprising at least one cationic nanoparticle consisting of a cationic polysaccharide core and at least one specific antigen, characterized in that said antigen is specific for a pathogen or a disease which does not affect the central nervous system.

[0022] By "therapeutic vaccine" is meant a vaccine intended to stimulate the immune system to generate a specific immune response capable of treating individuals already suffering from the disease. This mode of use is opposed to that of prophylactic vaccines which are used in people who have not yet contracted the disease.

[0023] By "infected individual" is meant a human or animal carrying a pathogen responsible for an infection not affecting the central nervous system.

[0024] By "disease which does not affect the central nervous system" is meant a disease which does not affect the brain of the infected individual. The disease affects only the metabolic systems, i.e. all organs except the brain. For the purposes of the invention, the diseases may be cancers which do not affect the brain or any other peripheral disease such as an autoimmune disease.

[0025] By "pathogen not infecting the central nervous system" is meant a pathogen which cannot cross the blood-brain barrier, the infection not reaching the brain of the infected individual.

[0026] For the purposes of the invention, the pathogen may be: • A virus that does not have a central tropism, such as for example: herpes simplex virus (HSV), human immunodeficiency virus (HIV), cytomegalovirus (CMV), Epstein-Barr virus (EBV). • An intracellular bacteria, such as: Salmonella typhi, Neisseria gonorrhoeae, Legionella, Rickettsia, Chlamydia, Chlamydophila spp, Bartonella henselae, Francisella tularensis, Listeria monocytogenes, Brucella, mycobacterium, Nocardia, Rhodococcus equi, Yersinia • An intracellular parasite such as: Apicomplexans (Plasmodium spp., Cryptosporidium parvum), Trypanosomatidae (Trypanosoma cruzi, Leishmania sp.) • A fungus, such as: Histoplasma capsulatum, Cryptococcus neoformans

[0027] By "cationic nanoparticle consisting of a cationic polysaccharide core" is meant a solid nanoparticle (NP) comprising a cationic polysaccharide core. The NP may or may not be crosslinked. Its core may or may not be charged with a Anionic phospholipid. The polysaccharide core may be porous. This NP is not surrounded by any phospholipid layer.

[0028] By "antigen" is meant an antigenic protein, a mixture of antigenic proteins, or a partial or total extract of a pathogen. The pathogen extract may contain proteins, polysaccharides and lipids and nucleic acids. The protein may be hydrophilic or lipophilic. The antigens may be purified, alone or in combination. Said antigen may be a tumor antigen.

[0029] 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 most preferred embodiment, the antigen is a complex protein extract obtained from a whole pathogen.

[0030] In a particular embodiment, the antigen is specific for leishmaniasis.

[0031] In a particular embodiment, the antigen is genus specific Leishmania, such as Leishmania infantum or Leishmania donovani. In a preferred embodiment, the antigen is specific to the Leishmania infantum strain.

[0032] In the case of Leishmaniasis, the inventors consider that the immunity which is triggered by the therapeutic vaccine can be a cross-immunity by the induction of a memory T response capable of recognizing different species of Leishmania.

[0033] In this context, individuals treated with the therapeutic vaccine developed with the Leishmania infantum strain are immune to all other Leishmania strains. In addition, there is cross-species immunity, i.e. the vaccine prepared from a pathogen infecting one species can potentially be used to protect other mammalian species, for example.

[0034] The vaccine is effective against all forms of leishmaniasis, whether cutaneous, visceral or mucocutaneous.

[0035] A characteristic of the vaccine according to the invention is based on the combination of the antigen with a cationic NP.

[0036] 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 chosen from starch, dextran, dextrin, and maltodextrin, polyfructoses (inulin), polymannoses, polygalactoses, polygalactomannans (guar gum) and at least one cationic ligand chosen from a primary, secondary, tertiary amine or quaternary ammoniums, then the addition of a crosslinking agent. The crosslinking agent is chosen from epichloridrine, a dicarboxylic acid or an acid chloride, such as sebacic acid. The core is not loaded with lipids.

[0037] In a preferred embodiment, the cationic polysaccharide is obtained by the reaction between maltodextrin and glycidyltrimethylammonium.

[0038] In a third particular embodiment, the cationic polysaccharide forming the core of the NP is loaded with an anionic phospholipid. This anionic phospholipid may be chosen from diacylphosphatidyl glycerol, diacylphosphatidyl serine or diacylphosphatidyl inositol. In another preferred embodiment, the anionic phospholipid is dipalmitoylphosphatidylglycerol (DPPG).

[0039] In a highly preferred embodiment, the NP is a maltodextrin nanoparticle loaded with DPPG.

[0040] 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 whose infection does not reach the CNS. In a particular embodiment, it is a chemoresistant parasite.

[0041] In a particular embodiment, the therapeutic vaccine induces cross-species immunity between humans and animals. In an even more particular embodiment, it induces protective cross-immunity in humans or non-human mammals such as: canids; felines; leporids; bovines; rodents; non-human primates; equines.

[0042] A second subject of the present invention relates to a vaccine composition comprising a cationic nanoparticle consisting of a cationic polysaccharide core and an antigen specific to a pathogen or a disease which does not affect the central nervous system for use in the treatment of diseases linked to a pathogen or a disease which does not affect the central nervous system.

[0043] Animals infected with Leishmaniasis are mammals such as, for example, canids, rodents, leporids, equids, bovids, primates.

[0044] A third subject of the present invention relates to a composition comprising a cationic nanoparticle consisting of a cationic polysaccharide core and an antigen specific to the genus Leishmania Infantum for use as a therapeutic vaccine in the treatment of cutaneous, visceral or mucocutaneous Leishmaniasis.

[0045] In a preferred embodiment, the composition used as a therapeutic vaccine is administered nasally.

[0046] Without departing from the scope of the invention, the method of administration may also be by mucosal (oral) or intradermal, subcutaneous, intravenous route. DESCRIPTION OF FIGURES

[0047] [Fig.l] [Fig.l] represents a Protocol of the study of Example 1

[0048] [Fig.2] [Fig.2] represents an SDS-PAGE (left) and PAGE under native conditions (right) ETL / NPL formulations carried out, after 120 hours of formulation.

[0049] [Fig.3] [Fig.3] represents a measurement of the parasite load in the liver, spleen and bone marrow of mice, 60 days after infection of the mice.

[0050] [Fig.4] [Fig.4] represents a protocol of the study of Example 2

[0051] [Fig.5] [Fig.5] represents an SDS-PAGE (left) and PAGE in native conditions (right) ETL / NPL formulations carried out, after 96 hours of formulation.

[0052] [Fig.6] [Fig.6] represents a representation of the parasitic load in the liver, the spleen and bone marrow of treated mice, 90 days after infection.

[0053] [Fig.7] [Fig.7] represents a dosage of total IgG (left) and IgG 1 and IgG 2a (right) in untreated mice and mice treated with chemotherapy or sc and in administration of vaccine formulations.

[0054] [Fig.8] [Fig.8] represents a re-stimulation of the splenocytes of non-mouse treated and treated by chemotherapy or by sc and in administration of vaccine formulations, and dosages of cytokines secreted in the supernatant.

[0055] [Fig.9] [Fig.9] represents a protocol of the study of example 3

[0056] [Fig. 10] [Fig. 10] represents a PAGE under native conditions of the ETL / NPL formulations.

[0057] [Fig. 11] [Fig. 11] represents a measurement of the parasite load in the serum of mice, unvaccinated and vaccinated with ETL or ETL / NPL formulations, by nasal (A) or subcutaneous (B) route. Measurement of the parasite load in the liver (C) and spleen (D) of mice, unvaccinated and vaccinated with ETL or ETL / NPL formulations. EXAMPLES Abbreviations:

[0058] - NPL#: Lipidated maltodextrin nanoparticles#; - ETL: total leishmania extracts#; - Glu + Allô#: Glucantime + Allopurinol#; - id#: Intradermal#; - in#: Intranasal#; - ip#: Intraperitoneal#; - iv#: Intravenous

[0059] EXAMPLE 1: Study of the feasibility of a therapeutic vaccine by nasal route, using total extracts of Leishmania and maltodextrin nanoparticles, in comparison with the reference antiparasitic treatment (Glucantime + Allopurinol).

[0060] 1-A Materials and methods

[0061] a) Experimental model#:

[0062] Eight-week-old female Balb / c mice were infected with 1.2.107 L. infantum strain amastigotes injected iv ([Fig.l]). The first vaccination and antiparasitic treatments were carried out 10 days later (D11). The drug treatment lasted 10 days (until D21). The vaccination boost was carried out 15 days after the prime (D26). Finally, the mice were euthanized 35 days later (D61).

[0063] Liver, spleen, and bone marrow cells were lysed to extract DNA. The parasite cytochrome b gene was used to estimate parasite load by qPCR. Each picogram of parasite DNA corresponds to # 10 parasites. The number of parasites is estimated in 50ng of total DNA. This protocol is shown in [Fig.l].

[0064] b) Groups and doses used#:

[0065] Each group consisted of 8 mice, distributed as follows: - Group 1#: Untreated infected control - Group 2#: Chemotherapy (Glucantime + Allopurinol) - Group 3#: NPL / ETL sc vaccine - Group 4#: NPL / ETL vaccine in

[0066] The doses administered were: NPL / ETL sc: 20pg ETL + 60pg NPL in 100pL NPL / ETL in: 20pg ETL + 60pg NPL in 20pL - Chemotherapy#: Glucantime® 100mgSb / kg / day by ip injection + Allopurinol 10mg / kg / day# orally, for 10 successive days, in 100pL.

[0067] c) Vaccine formulations

[0068] The formulations were made from total extracts from the amastigote and promastigote forms, from a mixture of canine and human strains of L. infantum, and from NPL.

[0069] 1-B Results

[0070] a) Characterization of the association of antigens with NPLs

[0071] The NPLs were characterized by SDS-PAGE and native PAGE analysis. It is observed that at the 1 / 3 (weight / weight) ratio, 93% of the protein antigens are associated in the nanoparticles ([Fig.2]). The results are presented in [Fig.3].

[0072] b) Parasitic load at D 61

[0073] At D61, an identical decrease in the parasite load is observed in the groups immunized with intravenous ETL NPLs and chemotherapy ([Fig.3]). In the liver, all parasites are eliminated (we observe that the value is below the threshold of detection). In contrast, no improvement could be observed in the subcutaneous NPL ETL groups. Conclusion :

[0074] These results suggest that the nasal therapeutic vaccine is as effective in reducing the parasite load as the reference treatment in this study.

[0075] EXAMPLE 2: Study of the efficacy of a therapeutic nasal vaccine directed against canine visceral leishmaniasis in mice, composed of total Leishmania extract and maltodextrin nanoparticles, and of the associated immune response.

[0076] 2-A Materials and methods

[0077] a) Experimental model#:

[0078] Eight-week-old Balb / c mice were infected with 1,108 amastigotes of the L. infantum strain, injected id ([Fig.4]). The first vaccination and antiparasitic treatments were carried out 10 days later (D11). The drug treatment lasted 10 days (until D21). The vaccination boost was carried out 15 days after the prime (D26). Finally, the mice were euthanized 64 days later (D90).

[0079] Liver, spleen, and bone marrow cells were lysed to extract DNA. The parasite cytochrome b gene was used to estimate parasite load by qPCR. Each picogram of parasite DNA corresponds to # 10 parasites. The number of parasites is estimated in 50ng of total DNA.

[0080] b) Groups and doses used:

[0081] Each group consisted of 8 mice, distributed as follows: - Group 1#: Untreated infected control - Group 2#: Chemotherapy (Glucantime® + Allopurinol) - Group 3#: NPL / ETL sc vaccine - Group 4#: NPL / ETL vaccine in

[0082] The doses administered were: NPL / ETL sc: 20pg ETL + 60pg NPL in 100pL NPL / ETL in: 20pg ETL + 60pg NPL in 20pL - Chemotherapy#: Glucantime® 100mgSb / kg / day by ip injection + Allopurinol 10mg / kg / day# orally, for 10 successive days, in 100pL.

[0083] c) Vaccine formulations

[0084] The formulations were made from total extracts from amastigotes of canine strains of L. infantum and NPL.

[0085] In this study, the test was performed intradermally (id) to mimic the bite by sandfly flies, natural vectors of the infection.

[0086] 2-B Results

[0087] a) Characterization of the association of antigens with NPLs:

[0088] The NPLs were characterized by SDS-PAGE and native PAGE analysis. It is observed that at the ratio 1 / 3 (w / w), 100% of the protein antigens were associated in the nanoparticles ([Fig.5]). The results are presented in [Fig.6], 7 and 8.

[0089] b) Parasitic load at D90

[0090] We observed that after 90 days ([Fig.6]), the parasite load had decreased in the liver, spleen and BM of mice treated with the vaccine subcutaneously, and in the liver and BM of mice treated with the vaccine nasally.

[0091] c) Humoral and cellular response#:

[0092] After serum dosage of total IgG, we observed that the administration of the vaccine formulations by nasal route improved the humoral response compared to the subcutaneous route and the reference drug treatment ([Fig.7]). Furthermore, only the nasal route allowed an IgG1 / IgC2a ratio <1, indicating a Thl-type response necessary to combat Leishmania infection.

[0093] After restimulation of splenocytes, we observed a significant secretion of cytokines of the Thl (INF-y) and Thl7 (IL-17a and IL-17f) pathways from mice vaccinated via the nasal route ([Fig.8]). Conclusion#:

[0094] Therapeutic vaccine treatment makes it possible to reduce the parasitic load of infected animals, in a manner similar to that of antiparasitic treatment.

[0095] This decrease observed with vaccine treatment is directly correlated with the induction of a Thl-type immune response, both humoral and cellular.

[0096] This memory response should also protect animals from future reinfection with the parasite, which makes this treatment highly attractive for treating infected animals.

[0097] EXAMPLE 3: Study of the feasibility of a prophylactic vaccine by nasal route, using total extracts of Leishmania and maltodextrin nanoparticles.

[0098] 3-A Materials and methods

[0099] a) Experimental model#:

[0100] Eight-week-old female Balb / c mice 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), with 106 promastigotes of the L. donovani strain injected intravenously, and then euthanized 135 days later (D185, [Fig.9]).

[0101] b) Groups and doses used#:

[0102] Each group consisted of 8 to 10 mice, distributed as follows: - Group 1#: Physiological serum in (20pL) - Group 2#: Physiological serum sc (50pL) - Group 3#: ETL in (lOpg ETL in 20pL) - Group 4#: ETL sc (lOpg ETL in 50pL) Group 5#: NPL / ETL in (lOpg ETL + 30pg NPL in 20pL) Group 6#: NPL / ETL sc (lOpg ETL + 30pg NPL in 50pL)

[0103]

[0104] 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 kinetoplast DNA of the parasites.

[0105] The mice were sacrificed by cervical dislocation after 184 days. When the mice were euthanized, the livers and spleens were removed to determine the parasite load in each of these organs.

[0106] c) Vaccine formulations

[0107] The formulations were made from total extracts of L. donovani LV9 and NPL.

[0108] 3-B Results

[0109] a) Characterization of the association of antigens with NPLs:

[0110] The NPLs were characterized by PAGE analysis under native conditions. It is observed that when the ratio is 1 / 3 (weight / weight), 100% of the protein antigens are associated in the nanoparticles ([Fig. 10]).

[0111] b) Parasitic load at D 185

[0112] Administration of the vaccine formulations significantly reduces the parasite load in the blood, liver and spleen of mice ([Fig. 11]). However, this remains high (>108 parasites in the organs), despite 3 prophylactic administrations of total extracts associated or not with NPLs, suggesting that this strategy does not protect against infection. Conclusion#:

[0113] The CaniLeish® prophylactic vaccine on the market today remains more competitive, with significant results obtained with a single administration.

Claims

Claims

1. Vaccine composition comprising a cationic nanoparticle consisting of a cationic polysaccharide core and an antigen specific to a pathogen or disease that does not affect the central nervous system for use as a therapeutic vaccine in the treatment of diseases related to said pathogen or disease.

2. Vaccine composition according to claim 1 for its use according to claim 1, wherein said specific antigen originates from a pathogen responsible for leishmaniasis.

3. Vaccine composition according to claim 1 for its use according to claim 1, wherein said specific antigen is selected from: a virus having no central tropism, an intracellular bacterium, an intracellular parasite and a fungus.

4. Vaccine composition according to claim 1 or 2 for its use according to claim 1 or 2, wherein said antigen is specific for one of the strains chosen from: Leishmania infantum or Leishmania donovani.

5. Vaccine composition according to claim 1 for use according to claim 1, wherein said specific antigen is a tumor antigen.

6. Vaccine composition according to one of the preceding claims for its use according to one of the preceding claims in which said porous cationic polysaccharide core is crosslinked.

7. Vaccine composition according to one of the claims for its use according to one of the preceding claims, in which said porous cationic polysaccharide core is loaded with an anionic phospholipid.

8. Vaccine composition according to one of the preceding claims for its use according to one of the preceding claims, in which said anionic phospholipid is chosen from diacylphosphatidyl glycerol, diacylphosphatidyl serine or diacylphosphatidyl inositol.

9. Vaccine composition according to claim 7 for use according to claim 7, wherein said anionic phospholipid is dipalmitoylphosphatidylglycerol.

10. Vaccine composition according to one of the preceding claims for use according to one of the claims in a form suitable for nasal administration.

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