The present invention relates to a pharmaceutical composition that may be used in the preventive treatment of infections caused by an intracellular pathogen like, for instance, a virus, a bacterium or a parasite and more particularly Toxoplasma gondii.
Toxoplasma gondii (hereinafter T. gondii) is a species of parasite protozoa in the genus Toxoplasma. The definitive host of T. gondii is the felids, but the parasite can be carried by many warm-blooded animals (birds, cattle, sheep and also humans). The life cycle of T. gondii has two phases. The sexual part of the life cycle (coccidia like) takes place only in cats, both domestic and wild, which makes cats the parasite's primary host. The second phase, the asexual part of the life cycle, can take place in another warm-blooded animal called intermediate host.
T. gondii exists in three forms:
Toxoplasmosis, the disease of which T. gondii is the causative agent, is usually minor and self-limiting but can have serious or even fatal effects on fetus whose mother first contracts the disease during pregnancy or on an immune-deficient patient. In cattle and sheep, for example, toxoplasmosis is a main cause of abortion and thus generates great losses.
Recently it has been postulated that Toxoplasma has some degree of causal relation to schizophrenia. This postulate rests on the positive relationships between the prevalence of Toxoplasma antibodies and the development of schizophrenia. This study provides one example of the value of integrating behavioral effects of Toxoplasma in models of emotional and psychiatric conditions (Yolken R H, Bachmann S, Rouslanova I, Lillehoj E, Ford G, Fuller Torrey E et al. Antibodies to Toxoplasma gondii in individuals with first-episode schizophrenia. Clin. Infect. Dis. 2001; 32: 842-44.).
Accordingly, one purpose of the present invention is to provide a pharmaceutical composition enabling a vaccine for patient against an intracellular pathogen, more particularly against an intracellular parasite like, for instance, a protozoa and more particularly, T. gondii.
The present invention relates to a pharmaceutical composition comprising, as an active ingredient, the mixture of:
More particularly, the invention relates to the afore-mentioned pharmaceutical composition for its use in the prophylactic treatment against an intracellular pathogenic agent in a patient or in the prophylactic treatment of a disease or infection caused by an intracellular pathogenic agent.
According to the invention, the term “treatment” refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the disease condition more tolerable to the patient, slowing in the rate of degeneration or decline, or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of an examination by a physician.
The term “treatment” also refers to any decrease of the amount of the pathogenic agent in the patient's body or to any alteration of the pathogenic agent, even through a short time period. When the pathogenic agent has severals forms including extracellular forms, a decrease or a modification of at least one pathogenic agent's form is considered as a treatment effect. If the pathogenic agent may be present in several organs of the patient's body, a decrease of the amount of pathogenic agent or a modification of the pathogenic agent in at least one organ is considered as a treatment effect.
Accordingly, the term “treating” includes the administration of the composition of the invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a disease, condition or disorder as described herein. The term “therapeutic” refers at least to the partial reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
The term “prophylactic” means that the treatment is able to prevent or delay the onset of the disease, or is able to prevent the manifestation of clinical or subclinical symptoms thereof.
The terms “pharmaceutically acceptable” refer to those solvent, compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.
According to one aspect of the invention, the invention relates to the before-mentionned composition for its use as a medicament and particularly as an immunogenic medicament enabling a cellular immune response in a patient. However, the present invention is not limited to a Th1 immune response (cellular response). The pharmaceutical composition may also induce the production of specific antibodies (humoral response) or induce both responses. In the present invention, “immune response” means any reaction by the immune system, a cellular immune response and/or an humoral immune response. These reactions include the alteration in the activity of an organism's immune system in response to an antigen and can involve, for example, antibody production, induction of cell-mediated immunity, complement activation, or development of immunological tolerance. The immune response may be a Th1 response or a Th2 response or a mixture thereof. The term “immunogenic” refers to the capacity of inducing an immune response, directly or not.
According to the present invention, the nanoparticle core is charged with at least a phospholipid. Accordingly, the solid core is porous and the pores thereof are filled with a phospholipid or a mixture of at least two lipids or phospholipids. The nanoparticle core is made of a cationic polysaccharide or a mixture of at least two cationic-polysaccharides. It may also be made of any other compound that are co-polymerized or not with said cationic polysaccharide(s).
According to the invention, the nanoparticle core is not surrounded by any phospholipidic layer. A phospholipidic layer is defined as a layer comprising at least one phospholipid.
The cationic polysaccharide may be a crosslinked polymer and may be obtained by the reaction between a polysaccharide chosen among starch, dextran, dextrin, and maltodextrin, derivatized with cationic ligands such as quaternary ammonium salt. Primary, secondary and tertiary amines may also be used. More particularly, the cationic polysaccharide can be obtained from the reaction between maltodextrin and glycidyl-trimethylammonium.
The anionic phospholipid may be chosen among glycerol phospholipids and may be, for instance, diacylphosphatidyl glycerol such as diacylphosphatidyl serine, diacylphosphatidyl inositol and dipalmitoylphosphatidylglycerol.
The following experimental results show that at least in the case of T. gondii, the composition of the invention induces the production of specific IFN-γ against T. gondii. In the case of toxoplamosis, it is known that host resistance seems to occur via synthesis of IFN-γ by NK cells and adaptive T lymphocytes. Following infection, antigen-presenting cells synthesize TNF-α and IL-12 which induce NK cells to secrete IFN-γ. The combined action of IL-12 and IFN-γ induce a strong differentiation of T helper precursors into Th1 lymphocytes. These CD4+ T cells then synthesize large amounts of IFN-γ and IL-2. These two cytokines finally induce CD8+ T lymphocytes proliferation and IFN-secretion. Thus protection against T. gondii infection is mainly attributed to cell-mediated immunity. Therefore, the composition of the invention can also protect the fetus when the mother has been treated with the composition/vaccine of the invention.
According to the invention, the intracellular pathogenic agent is an intracellular pathogen that may be a virus, a bacteria, a mycobacteria, a fungus or a parasite. An intracellular pathogenic agent is defined as any micro or macro organism having at least one intracellular form. The term “pathogenic” refers to an agent able to involve physiological and/or psychological changes in the patient or in the patient's progeny or offsprings.
Herpes simplex virus 1 and 2, human papillomavirus, Epstein-Barr virus, cytomegalovirus, Mycobacterium tuberculosis, dengue fever virus, HIV, Human respiratory syncytial virus (RSV), hepatitis A, virus, hepatitis B virus and hepatitis C virus may be cited as examples of intracellular pathogenic agent.
A “parasite” means, according to the invention, any micro or macro organism which benefits at the expense of an other called “the host”. According to the invention, the term parasite includes macroparasites (typically helminths) and microparasites (typically protozoa) and and all of the forms that may take during their life cycle.
When the pathogenic agent is a parasite having an intracellular form during its life-cycle, it may be chosen among apicomplexan intracellular parasites, like, for instance Toxoplasma gondii, Emeria spp, Neospora caninum, Sarcocystis spp, Plasmodium spp (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi for instance) and Cryptosporidium spp. It may also be chosen among Acanthamoeba spp., Babesia spp., Balantidium coli, Blastocystis, Dientamoebafragilis, Entamoebahistolytica, Giardia lamblia, Isospora belli, Leishmania spp., Naegleriafowleri, Rhinosporidiumseeberi, Trichomonasvaginalis, Trypanosomabrucei, and Trypanosomacruzi. These parasites are known as at least human and veterinary parasites.
According to the present invention, the terms “antigen obtained from the pathogenic agent” may be any antigen or mixture thereof known and already used to obtain an immune response in a patient against said pathogenic agent. These terms refer to native antigen(s) or mixture thereof as well as recombinant antigen(s) or mixture thereof and mixture(s) of native and recombinant antigens. The antigen may be a protein, particularly a surface protein. The term “antigen” refers to any substance able to induce by itself (directly) an immune response and/or to any substance able to induce an immune response by combining with some products of an immune response once they are made.
According to the invention, the antigen may be comprised in any form of the pathogenic agent. The pathogenic agent itself may be used alive, killed or attenuated for providing said antigen or mixture thereof.
The antigen or antigen mixture may be obtained from infected cells. The intracellular pathogen contained in these infected cells is then extracted, for example by destroying the cell membrane. Depending on the technique used for lysing cell membrane, the pathogenic agent may be killed in the same time.
According to an aspect of the invention, the antigen is obtained from a previously killed pathogenic agent. When the pathogenic agent is a parasite and particularly a protozoa that can be a tachyzoite, the antigen may be obtained from said tachizoite and particularly to a previously killed tachyzoite.
The inventors have found that the tachyzoite form of T. gondii, which is intracellular but not contaminant, provides at least one efficient antigen for the prophylaxy of toxoplasmosis. The antigen(s) may be present in a mixture obtained from infected cells containing tachyzoites.
The term “patient” refers to a warm-blooded animal such as a mammal which is afflicted with, or has the potential to be afflicted with one or more diseases and conditions described herein. The term “patient” as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, ovines (sheep, goat . . . ), canines, felines, murines, bovines, equines, and porcines. The patient may also be a bird.
The present invention also relates to a vaccine against an intracellular pathogen comprising the pharmaceutical composition of the invention and further comprising a suitable excipient and/or a suitable carrier and/or a suitable vehicle.
Carriers, excipients, diluents and/or adjuvants are chosen according to the administration way. They may be chosen among lactose, dextrose sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof. The pharmaceutical compositions can optionally contain other substances that are commonly used in pharmaceutical formulations, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, disintegrating agents, stabilizing agents, isotonic agents, bulking agents, fillers, preserving agents, sweetening agents, flavoring agents, perfuming agents, coloring agents, antibacterial agents and/or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, dispensing agents, flow regulators, release agents, etc.
The term “vaccine” relates to a product able to prevent infection caused by an intracellular pathogenic agent upon future exposure thereto.
The compositon and/or the vaccine of the invention can be variously administrated. For example, the composition/vaccine may be formulated to be orally or topically or enterally or non-parenterally administered. The terms “oral”, “enteral”, “enterally”, “orally”, “non-parenteral”, “non-parenterally”, and the like, refer to administration of a composition to an individual by a route or mode along the alimentary canal. Examples of “oral” routes of administration of a composition include, without limitation, swallowing liquid or solid forms of a vaccine composition from the mouth, administration of a vaccine composition through a nasojejunal or gastrostomy tube, intraduodenal administration of a vaccine composition, and rectal administration, e.g. using suppositories.
The term “topically administrated” refers to the application of a pharmaceutical agent to the external surface of the skin or the mucous membranes (including the surface membranes of the nose, lungs and mouth), such that the agent crosses the external surface of the skin or mucous membrane and enters the underlying tissues. Topical administration can result in a limited distribution of the agent to the skin and surrounding tissues or, when the agent is removed from the treatment area by the bloodstream, systemic distribution of the agent.
The composition and/or the vaccine according to the present invention may be formulated for intranasal administration. The term “intranasal administration” refers to any form of administration whereby an active ingredient is propelled or otherwise introduced into the nasal passages of a subject so that it contacts the respiratory epithelium of the nasal cavity, from which it is absorbed into the systemic circulation. Nasal administration can also involve contacting the olfactory epithelium, which is located at the top of the nasal cavity between the central nasal septum and the lateral wall of each main nasal passage.
The present invention also relates to the use of a nanoparticle comprising a cationic-polysaccharide solid core which is porous, loaded with an anionic phospholipid without being surrounded by any phospholipidic layer, as a vaccine adjuvant. As explained hereinafter, the nanoparticles used in the composition of the invention can be used as vaccine adjuvant and are at least as efficient as cholera toxin.
The polysaccharide and/or the phospholipid(s) may be chosen as described above as regards to the pharmaceutical composition of the invention.
The present invention also relates to a method for eliciting an immune response against an intracellular pathogen in a patient, said method comprising administrating to a patient a therapeutic amount of a composition of the invention or a vaccine of the invention.
According to one aspect of the method of the invention, the immune response comprises a Th1 response.
The present invention also relates to the use of a pharmaceutical composition as previously described for the fabrication of a vaccine against an intracellular pathogenic agent.
According to the present invention, the amount of antigen(s) contained in the vaccine of the invention is not limited to a determined value. For instance, the composition may comprise from 5 μg to 1 mg of antigen(s), more particularly from 10 μg to 1 mg of antigen(s) and for example from 15 μg to 200 μg of antigen(s).
The present invention also relates to a kit comprising a vaccine of the invention and a leaflet.
Polysaccharide particles are prepared from US Pharmacopoeia maltodextrin, as described previously (Paillard et al, 2010). Briefly, 100 g of maltodextrin were dissolved in 2N sodium hydroxide with magnetic stirring at room temperature. Further, 1-chloro-2,3-epoxy propane (epichlorhydrin) and glycidyl-trimethylammonium chloride (hydroxycholine, cationic ligand) is added to make cationic polysaccharide gel. The gel is then neutralized with acetic acid and sheared under high pressure in a Minilab homogenisor (Rannie; APV Baker, Evreux, France). The 60 nm polysaccharide nanoparticles obtained are ultra-filtered on an SGI Hi-flow system (hollow fiber module: 30 UFIB/1 S.6/40 kDa; Setric Génie Industriel, France) to remove low molecular weight reagents and salts. The obtained nanoparticles are hereinafter called NPS.
Some of the previous NPS are loaded with anionic phospholipids. Anionic phospholipids are loaded into these porous NPS by injecting an ethanol solution of dipalmitoyl-phosphatidyl glycerol (DPPG). The porous nanoparticles containing a phospholipid are hereinafter called DGNP. The core of these nanoparticles is not surrounded by any phospholipid layer.
Tachyzoites are obtained from successive splitting of infected HFF cells (Human Foreskin Fibroblasts). About 1.108 tachyzoites derive from one 225 cm2 culture flask corresponding to 200 μg of TE. Lysis of tachyzoites is then performed by freeze/thaw cycles, pooled, sonicated (2×10 mn, 60 W in ice) and protein amount is evaluated by micro BCA method.
TE refers to the product obtained from tachyzoites according to the process as above described. TE is used for mice immunization in combination with nanoparticles (NPS and DGNP), Elisa coating and cellular restimulation test. TE is a mixture of several antigens.
The most effective nanoparticles as antigen carriers were determined on the basis of intensity of humoral and cellular responses and protection.
Adult females Swiss and CBA/J mice of 20-25 g and 6-8 weeks were purchased from Janvier (France). The animal experiments comply with the French Government's ethical and animal experiment regulations.
Swiss mice received an intranasal treatment, three times at 15-day intervals, with TE (10 μg) and DGNP nanoparticles (30 μg) alone (defined as control groups) or with the combination TE+NPS, TE+DGNP (10 μg of TE and 30 μg of NPS or DGNP).
Each dose of the above-mentioned total extract (TE), nanoparticles and mixtures thereof was diluted to a final volume of 10 μl in phosphate-buffered saline (10 mM phosphate, 140 mM NaCl [PBS]) and instilled into the nostrils of non-anesthetized mice with a micropipettor (5 μl/nostril). Treated mice were infected per os with 50 cysts of 76 K Toxoplasma strain 1 month post-treatment and followed up by clinical examination for a further period of 6 weeks.
Specific Toxoplasma IgG were quantified in the sera of treated mice by ELISA. IgG synthesis against Toxoplasma antigens was monitored sequentially in sera. The results are shown in
As shown in
To investigate cellular immune responses, splenocyte cytokines, a strong immunogenicity biomarker of vaccine efficacy, were analyzed in supernatants of Toxoplasma-stimulated splenocytes from the before-mentioned treated mice, 3 weeks after the third intranasal administration. Cytokines (IFN-γ, IL-12, IL-10, IL-13, TNF-α, IL-5) were quantified by ELISA.
As T-cell-derived IFN-γ was also a valuable hallmark of protective immunity in toxoplasmosis, IFN-γ will be determined by ELI spot analysis after Toxoplasma antigen stimulation.
To investigate the cellular immune response induced after treatment with TE-NPS, TE-DGNP, TE alone or nanoparticles alone, the supernatants of cultured cells from spleen of 2 mice from the different groups were evaluated for the production of IFN-γ, IL-10 and IL-12 in response to TE restimulation (10 μg·ml−1).
Each column referred as DGNP corresponds to a mouse treated with DGNP alone. Each column referred as TE corresponds to a mouse treated with TE alone. Each column referred as TE-NPS corresponds to a mouse treated with a mixture of TE and NPS. Each column referred as TE-DGNP corresponds to a mouse treated with a mixture of TE and DGNP.
As shown in
Six weeks after T. gondii infection, mice treated with a mixture of TE and DGNP and then infected with T. gondii were killed and their brains were collected.
Brains were harvested 6 weeks after infection from surviving mice and homogenized in 5 mL of RPMI 1640 with a pestle and mortar. The cysts in each brain homogenate were counted under a microscope (10 counts, each on 10 μl). The results are expressed as means±SEM for each group. The data were statistically analysed using the Mann-Whitney U test (GraphPad prism software), (P<0.05). The results are shown in
As shown in
According to the obtained results, DGNP nanoparticles were used for the following experiments of vaccination and to compare the vaccination protocol with DGNP to vaccination protocol with cholera toxin (CT).
Six groups of 10 CBA/J mice were treated by three intranasal administrations at 15-day intervals with TE (10 μg), nanoparticles (DGNP—30 μg), Cholera Toxin (CT—0.5 μg) alone (defined as control groups), with the combination TE+DGNP, TE+CT (10 μg of TE and 30 μg of DGNP or 0.5 μg of CT), respectively. The experimental design includes a group of untreated mice. Each dose of the above-mentioned toxin, nanoparticles and combinations (mixtures) was diluted to a final volume of 10 μl in phosphate-buffered saline (10 mM phosphate, 140 mM NaCl [PBS]) and instilled into the nostrils of non-anesthetized mice with a micropipettor (5 μl/nostril). Five independent experiments have been done.
IgG synthesis against Toxoplasma antigens was monitored in sera 14 days after the third intranasal administration. The experimental protocol is described in reference to
As shown in
To investigate the cellular immune response induced after treatment as above-mentioned, the supernatants of cultured cells from spleen of 2 mice from the hereinbefore different groups were evaluated for the production of IFN-γ, IL-10, IL-13, TNF-α, IL-5 and IL-12 in response to TE restimulation with an ELISA analysis and IFN-γ with an Elispot. The results are shown in
As shown in
Moreover, the IFN-γ production by splenocytes was measured two weeks after the third intra-nasal administration. The results are shown in
Mice treated with three intranasal administrations of DGNP alone, TE alone, the mixture of TE and DGNP, CT alone and the mixture of CT and DGNP, respectively, were orally infected with cysts of the 76K strain of T. gondii. A group of non-treated mice was also infected as a control. Percentage of survival
The survival of each group was followed up during 30 days after the infection with 80 cysts. The total number of tested animals in each group is n=8.
Except one mouse in the group control, all mice rapidly show clinical symptoms of disease, lost body weight and were dead within 11 days after infection, while 100% of the mice vaccinated with the mixture of TE and DGNP survived over the experimental period of 30 days.
Mice of each treated group were orally infected with 50 cysts and sacrificed one month after the oral infection. Protection against T. gondii was evaluated by measuring mouse brain cyst number (three experiments with 50 cysts). The total number of tested animals in each group is n=8.The protocol is as described in reference of
Number | Date | Country | Kind |
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12370002.3 | Sep 2012 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/IB2013/002372 | 9/13/2013 | WO | 00 |