Patent Application
20240009289
The present disclosure relates to the field of immunotherapeutic compositions used as therapeutic vaccines. It relates more particularly to a therapeutic vaccine for treating diseases that do not affect the brain, such as leishmaniasis.
Therapeutic vaccination consists of inducing an immune response, when such response is insufficient to allow spontaneous healing. The activation of the immune system requires on the one hand that it recognize the intruder and, on the other hand, that it consider the intruder to be dangerous, and then that an appropriate response is induced, both in terms of specificity and intensity.
The therapeutic strategy is very different from that of prophylactic vaccination. Prophylactic vaccination consists of presenting an antigen to the immune system so as to induce a rapid, strong response when the organism comes in contact with the same antigen again (memory response). Therapeutic vaccination is based on the fact that the pathogen is already present in the organism. This is because during an infection, the immune defenses are often overwhelmed by infectious proliferation or hindered by internal control mechanisms, which prevents an effective immune response against the pathogen. The therapeutic vaccine will allow amplification of the specific immune response.
The main therapeutic vaccines developed to date have the objective of treating cancer. They generally consist of injecting the tumor antigen and/or injecting differentiated dendritic cells and/or T lymphocytes.
Therapeutic vaccines are of interest since they constitute an effective therapeutic solution for individuals who already carry the disease. They also make it possible in the case of certain pathologies to generate immune responses not induced by conventional prophylactic vaccines.
Leishmaniasis is a parasitic disease that affects many species of mammals and, in particular, humans and dogs. The number of household dogs affected by canine leishmaniasis is estimated at 2.5 million, but more generally distributed in 70 countries worldwide, and located on all continents. The incidence of this disease is also strong in humans, with about 2 million new cases per year, found 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 vector diptera insect, phlebotomines. The geographical distribution of the disease therefore depends on the distribution of phlebotomines and its reservoirs: mammals. Leishmania infantum is an intra-macrophage parasite responsible for the disease in Europe, in the Mediterranean basin and in South America. Contamination travels through an insect bite on the individual. The disease can also be transmitted from the mother to the child.
Parasites of the Leishmania genus infect host cells and divert the intracellular traffic mechanism in order to maintain their parasitophorous vacuole wherein they proliferate.
The prior art reports developments of therapeutic formulae for the treatment and/or diagnosis of leishmaniasis. For example, ES2205059 discloses a pharmaceutical composition comprising an immunogenic part of a Leishmania antigen. However, none of the therapeutic vaccine tests against leishmaniasis has proved effective to date.
In addition, the compositions proposed in the prior art comprise all vaccinal adjuvants, which are necessary for their effectiveness, but whose side effects are widely documented.
Finally, the most widespread current treatment of leishmaniasis is carried out by chemotherapy. This treatment is expensive, nonspecific, toxic and can induce the appearance of chemoresistance. The molecules known for the treatment of leishmaniasis in dogs are meglumine antimoniate (GLUCANTIME®) and allopurinol, often used in combination.
In this context, there is a need to have an effective treatment that does not induce any resistance for infectious diseases due to a pathogen that does not infect the central nervous system such as leishmaniasis, but also improves the efficacy of therapeutic vaccines in general, in particular, in the treatment of cancer.
A therapeutic vaccine, without adjuvants, intended for administration in an individual carrying a disease or a pathogen that does not reach the central nervous system has been developed. In particular, the therapeutic vaccine may be used as treatment against leishmaniasis or cancer.
Thus, the present disclosure relates to a therapeutic vaccine for treating a living being who carries a disease or pathogen that does not affect the central nervous system, comprising at least one cationic nanoparticle consisting of a cationic polysaccharide nucleus and at least one antigen specific to the disease or the pathogen that does not affect the central nervous system.
In a particular embodiment, the present disclosure relates to a therapeutic vaccine intended for the treatment of leishmaniasis wherein the antigen is specific to the species Leishmania.
Quite surprisingly, it has been demonstrated that administration to an organism, already carrying a disease or pathogen that induces harm to metabolic systems, such as leishmaniasis, from an immunotherapeutic composition comprising nanoparticles and an antigen specific to the pathogen or the disease makes it possible to treat the individual. During 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 while the individual is already carrying the disease makes it possible to effectively enlist the immune defenses of the individual and to guarantee healing by eliminating the pathogen and long-term immunity by putting memory T cells in place.
It is interesting to note that this therapeutic vaccine approach constitutes a novel therapeutic solution for treating an infection caused by a chemoresistant pathogen.
In the case of leishmaniasis, it has been shown that this therapeutic strategy proves to be effective as the current treatment, namely the administration of antiparasitics. These results are validated not only a mouse model, but also by a clinical study carried out in a naturally infected dog. Quite advantageously, the vaccine approach does not induce any resistance mechanism, unlike antibiotics. Thus, either immunity is retained over the long term and prevents reinfection, or it is possible to vaccinate the individual in the event of reinfection.
The composition of the therapeutic vaccine contains no adjuvant (other than the nanoparticles themselves), which prevents the undesirable effects associated with this type of molecule. This is advantageous since the mineral adjuvants (namely mineral salts such as aluminum salts) remain in the body for very long (several decades).
The antigens combine with the nanoparticles in solution and are delivered to the immune cells after administration, while the nanoparticles are rapidly removed (in less than 72 hours after nasal administration). The nanoparticles therefore play a dual role: stabilizing agent and antigen delivery vector.
The vaccine can be administered by oral, nasal, intra-dermal, subcutaneous or intravenous route and makes it possible to treat, with a single formulation, leishmaniasis regardless of the species of Leishmania that infected the individual. In addition, the same vaccine formulation can be administered in humans and in animals, in particular, in dogs.
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.
“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.”
“Infected individual” means a human or animal carrying a pathogen responsible for an infection not reaching the central nervous system.
“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.
“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.
Within the meaning of the present disclosure, the pathogen can be:
“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.
“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.
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.
In a particular embodiment, the antigen is specific for leishmaniasis.
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.
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.
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.
One feature of the vaccine according to the present disclosure is based on combining the antigen with cationic NP.
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.
In a preferred embodiment, the cationic polysaccharide is obtained by the reaction between maltodextrin and glycidyltrimethylammonium.
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).
In an entirely preferred embodiment, NP is a nanoparticle of maltodextrin loaded with DPPG.
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.
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.
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.
The animals infected with Leishmaniasis are mammals such as, for example, canines, rodents, leporidae, equines, bovines, primates.
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.
In a preferred embodiment, the composition used as a therapeutic vaccine is administered nasally.
Without departing from the scope of the present disclosure, the mode of administration can also be administered mucosally (orally) or intra-dermally, subcutaneously, intravenously.
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).
1-A Materials and Methods
a) Experimental Model:
Female Balb/c mice, 8 weeks old, were infected with 1.2.107 amastigotes of the strain L. infantum injected i.v. (
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
b) Groups and Doses Used:
Each group consisted of 8 mice, distributed as follows:
The doses administered were:
c) Vaccine Formulations
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.
1-B Results
a) Characterization of the combination of antigens with NPL
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 (
b) Parasitic load at D61
It is observed at D61, an identical decrease in the parasite load in the groups immunized with intravenous NPL TEL and chemotherapy (
Conclusion:
These results suggest that the nasal therapeutic vaccine is as effective in reducing the parasite load as the reference treatment in this study.
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.
2-A Materials and Methods
a) Experimental Model:
Balb/c mice, 8 weeks old, were infected with 1,108 amastigotes of the strain L. infantum injected i.d. (
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.
b) Groups and Doses Used:
Each group consisted of 8 mice, distributed as follows:
The doses administered were:
c) Vaccine Formulations
The formulations were prepared from total extract from amastigotes of canine strains of L. infantum and NPL.
In this study, the test was carried out intradermally (i.d.) to mimic biting by phlebotomine flies, the natural vectors of the infection.
2-B Results
a) Characterization of the Combination of Antigens with NPL:
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 (
b) Parasitic Load at D90
It was observed that after 90 days (
c) Humoral and Cellular Response:
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 (
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 (
Conclusion:
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.
This decrease observed with the vaccine treatment is directly correlated with the induction of a Th1 immune response, both humoral and cellular.
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.
Example 3: Study of the feasibility of a prophylactic vaccine using the total extracts of Leishmania and maltodextrin nanoparticles.
3-A Materials and Methods
a) Experimental Model:
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,
b) Groups and Doses Used:
Each group consisted of 8 to 10 mice, distributed as follows:
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.
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.
c) Vaccine Formulations
The formulations were prepared from total extract of L. donovani LV9 and NPL.
3-B Results
a) Characterization of the Combination of Antigens with NPL:
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 (
b) Parasitic Load at D185
The administration of vaccine formulations substantially reduces parasitic load in blood, liver and mouse spleen (
Conclusion:
The CANILEISH® prophylactic vaccine currently on the market remains more competitive, with significant results obtained with a single administration.
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.
4-A Experimental Protocol:
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:
After 60 days, 5 mice were euthanized and the parasitic load was measured in the liver, spleen and bone marrow (
This protocol is summarized in
4-B Results
The results are presented in
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,
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.
No significant variation was observed in the animal spleen.
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. (
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.
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)
5-A Experimental Protocol:
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:
After 90 days, the mice were euthanized and the parasitic load was measured in the liver, spleen and bone marrow.
5-B Results
The results are shown in
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. (
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.
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.
6-A Experimental Protocol:
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:
The skin infection was evaluated by microscopic analysis of skin biopsies, at W0 and W4. The presence of a parasite confirms the cutaneous infection.
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.
The parasitic load was evaluated at W0 and W4, by qPCR analysis of a bone marrow sample at the sternum.
6-B Results
The results are presented in
In group 1 (Miltefosine)—10/10 dogs had visceral leishmaniasis (VL), and 6/10 had skin damage (CL).
In group 2 (Nasal vaccine)—10/10 dogs had visceral leishmaniasis (VL), and 9/10 had skin damage (CL).
In group 3 (Miltefosine+nasal vaccine)—10/10 dogs had visceral leishmaniasis (VL), and 6/10 had skin damage (CL).
a) Skin Infection
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 (
b) Parasitic Filler
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 (
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.
c) Clinical Score
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 (
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).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011304 | Nov 2020 | FR | national |
This application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/FR2021/051655, filed Sep. 24, 2021, designating the United States of America and published as International Patent Publication WO 2022/096792 A1 on May 12, 2022, which claims the benefit under Article 8 of the Patent Cooperation Treaty to French Patent Application Serial No. 2011304, filed Nov. 4, 2020.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2021/051655 | 9/24/2021 | WO |
